IL47004A - Synergistic antibiotic mixture and antibiotic and feed compositions containing it - Google Patents

Description

suns o»V»aan »-'W-uait* ©**»■* *a*tti« />" > 'J w ffJril S^ergietic antibiotic laixtws and aatibiotlc and feed compositions con &in&ig I FFIZER χυσ*.

C. 44890 P.C. 5574A The phenomenon of synergism has been extensively reported in antibiotic literature: The Journal of Antibiotics 25^ No. 6, 371 (1972); J. Chem. Soc. 19C, 1653 (1966); Bull. Soc. Chim. Belg. 6_8, 716 (1959); J. Amer. Chem. Soc. Z2_, 4414 (1960); Tetrahedron Letters 2687 (1971); J. Antibiotics, Ser. A 14, 14 (1961); Nature 187_, 598 (1960); J. Chem. Soc. 2286 (1960); Antimicrobial Agents & Chemotherapy 360-365 (1964); Tetrahedron Letters 4231-4238 (1966); Organic Mass Spectrometry 6_, 151-166 (1972); Tetrahedron Letters 369-372 (1966) and J. Chem. Soc. 19C, 1669-1676 (1966).

The new synergistic mixtures of antibiotics of the present invention join the family of other reported synergistic mixtures^ mikamycin, pristinamycin, ostreogrycin, strepto- / gramin, P.A. 114, vernamycin and virginiamycin.

This -invention is concerned with a mixture of antibiotics produced by the submerged aerobic propagation of Actinoplanes auranticolor ATCC 31011 in aqueous nutrient media. This mixture, containing macrocyclic lactones and depsipeptides, may be separated and recovered from fermentation broth by solvent extraction, counter-current distribution, column chromatography or combinations thereof. The individual antibiotic components exhibit significant antibiotic activity. The crude antibiotic mixture or combinations of a pure macrocyclic lactone and a pure depsipeptide obtained from the crude mixture demonstrate marked synergistic antibiotic activity. The crude antibiotic mixture, the pure individual antibiotic components and mixtures of pure macro-cyclic lactones and depsipeptides are effective chick and swine growth promotants and therapeutic agents in the control of swine dysentery.

The microorganism useful for the preparation of the antibiotics of this invention was isolated from a soil sample in Egypt. It was grown on potato-carrot agar and found to belong to the class of actinomycetes producing sporangia like those of the genus Actinoplanes. It was grown, therefore, on a number of media used for the study of this genus. Suspensions of the culture were prepared by crushing pieces of the culture, derived from agar slants, in small tubes each containing about 0.2 ml. sterile distilled water, rinsing out the contents and combining the contents with additional sterile water to make a volume of about 5 ml. for the culture. These suspensions were used to plant the culture in tubes, slants or petri dishes of the various media. The incubation temperature was 28°C. except where otherwise noted. Readings of results were made at intervals up to 22 days for some tests but most results were recorded after 14 days. The colors of the culture are those of Maerz and Paul, Dictionary of Colors, 2nd edition, 1950, as well as personal descriptive terms. This new culture (Pfizer F.D. 24090) was sub-mitted to the American Type Culture Collection in Rockville, Maryland on March 11, 1974 and given the designation Actinoplanes auranticolor ATCC 31011. The permanency of the deposit and ready accessibility thereto by the public are afforded in the event the patent is granted. Access to the culture is available during pendency of the application under Rule 14 and 35 USC 112. All restrictions on the availability to the public of the culture deposited will be irrevocably removed upon granting of the patent.

Identification media used for the characterization of the culture and references for their composition are as follows : 1. 2% Tap water agar. 2. Potato-Carrot Agar. M.P. Lechevalier, J. Lab. & Clin. Med. 71, 934-944 (1968). Use only 30 g. potatoes and 2.5 g. carrots but 20 g. agar. 3. Czapek-Sucrose Agar. Waksman, S.A., The Actino- mycetes 2_, 328 (1961) Medium No. 1, p. 328. 4. Glucose-Asparagine Agar. Waksman, as above, Medium No. 2, p. 328. 5. Yeast Extract-Malt Extract Agar. Antibiotics Ann. 1956/1957, pp. 947-953. 6. Hickey and Tresner Agar. J. Bact. 6_4, 891-892 (1952) . 7. Potato-Glucose Agar. Peel, cut up and steam 100 g. potatoes in 500 ml. water, filter through cheese cloth, add 10 g. glucose, 20 g. agar and enough water to make one liter. 8. Starch Agar. J. Bact. 73, 15-27 (1957). 9. Gelatin. J. Bact. 73.' 15-27 (1957). 10. Tyrosine Agar. J. Bact. 69, 147-150 (1955). 11. Difco Peptone Iron Agar. 12. Difco Skim Milk. 13. Dextrose Nitrate Broth. Waksman, S.A., The Acti- nomycetes 2 , 328 (1961) .

Medium No. 1 with 3.0 g. glucose in place of sucrose - - and without agar. 14. Organic Nitrate Broth. J. Bact., 7_3, 15-27 (1957). 15. ATCC Medium 172. American Type Culture Catalogue, 10th edition, p. 235 (1972). 16. Carbon Utilization. J. Bact., 5_6, 107-114 (1948). The description of this new culture is as follows: Tap Water Agar - growth poor, thin, flat, near 9D2 (very pale pink) ; no aerial mycelium; substrate mycelium colorless to 9D2; no soluble pigment.

Czapek-Sucrose Agar - growth moderate to good, flat, near 9G6 (pale orange) ; no aerial mycelium; substrate mycelium near 9G6; no soluble pigment.

Glucose-Asparagine Agar - growth moderate, raised, roughened, near 9L9 (light orange) ; no aerial mycelium; no soluble pigment .

Yeast Extract-Malt Extract Agar - no growth.

Hickey and Tresner Agar - growth moderate to good, slightly raised and roughened, near 9F9 (dull orange) ; faint whitish bloom on surface; substrate mycelium near 918; pale brownish soluble pigment.

Potato-Glucose Agar - growth moderate, raised, roughened, near 9L9 (light orange) ; no aerial mycelium; substrate mycelium near 9L9; no soluble pigment Tyrosine Agar - growth poor to moderate, flat, near 13A10 (dull reddish orange); no aerial mycelium; substrate mycelium near 10D11; brown soluble pigment.

Gelatin - growth moderate, flat, near 9K12 (reddish orange) ; trace of whitish bloom; substrate mycelium near 9K12; no soluble pigment.

Starch Agar - growth moderate to good, raised, near 9K10 (orange); light whitish bloom; substrate mycelium near 9K10; pale yellow soluble pigment.

Starch was weakly hydrolyzed; gelatin liquefaction was strong; nitrates were not reduced to nitrites in either nitrate medium even in 22 days (growth was very poor in dextrose nitrate broth but good in organic nitrate broth) ; I^S was weakly produced; there was no soluble pigment in peptone iron agar; there was no coagulation or hydrolysis of milk even in 22 days; tyrosine was not digested; growth on ATCC Medium 172 occurred at 21 to 37°C. with best growth at 28 and 37°C; there was no growth at 45°C. Arabinose, fructose, glucose, mannitol, raffinose, rhamnose, sucrose and xylose were utilized; inositol was not utilized. There was no odor on any medium.

Sporangia were produced only on the potato-carrot agar. They formed a palisade layer. Measurements were 5.5-11 x 4.5-8 microns in width and breadth and 9-12 microns in height. They were quite numerous, irregular in shape and set spores free by gradual softening. Sporangia from potato-carrot agar after three weeks incubation released spores in a few hours at about 21°C. when pieces of the growth were submerged in a small amount of a solution of 1 g. of glucose and 1 ml. of Tween 80 in 1 liter of water (a modification of a solution used by M.L. Higgins, J. Bact. , 9_4, 495-498, 1967). The spores were in chains of irregular shape in the sporangia but when set free were subglobose and 1.6 microns wide to broadly elliptical, 1.6-2.2 x 1.1-1.6 microns. Almost all were motile.

A tentative identification led to a comparison of this culture with A. auranticolor ATCC 15330. The new strain A. auranticolor ATCC 31011 and A. auranticolor ATCC 15330 looked essentially alike in morphological traits, color and soluble pigment on Bennett's Agar, Nutrient Agar, Yeast Extract Agar, Glucose-Asparagine Agar, Glycerol-Asparagine Agar, Calcium Malate Agar and Tyrosine Agar.

Neither culture reduced nitrate to nitrite; both produced HjS weakly and failed to produce melanin on peptone-iron agar; both hydrolyzed starch. A. auranticolor ATCC 15330 caused no change in skim milk tubes whereas the new culture caused clearing in three of the six tubes of milk used and after 21 days produced a yellow-cream soluble pigment.

A. auranticolor ATCC 31011 utilized glucose, arabi-nose, fructose, mannitol, raffinose, rhamnose, sucrose and xylose. A. auranticolor ATCC 15330 utilized all these sugars with the exception of raffinose. Sporangia and spores of the two cultures were similar with the spores of A. auranticolor 15330 more -rod shaped.

Most importantly, ,A. auranticolor ATCC 15330 did not produce any antibiotic activity under the fermentation conditions in which A. auranticolor ATCC 31011 produced the mixture of antibiotics of the present invention.

Cultivation of A. auranticolor ATCC 31011 preferably takes place in aqueous nutrient media at a temperature of 28-36°C, and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as sugars, starch and molasses; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cottonseed meal, peanut meal and wheat gluten. A source of growth substances such as distillers' solubles, fish meal and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace minerals such as iron, magnesium, zinc, cobalt and manganese may also be utilized with advantageous results.

If excessive foaming is encountered during fermentation, anti-foam agents such as vegetable oils or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1/2 to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry.

Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

Inoculum for the preparation of the antibiotic mixture may be obtained by employing growth from a slant of the culture on a medium such as ATCC Medium 172 to which previous reference was made. The growth may be used to inoculate either shake flasks or inoculum tanks, or alternatively, the inoculum tanks may be seeded from the shake flasks. In shaken flasks growth will generally have reached its maximum in about 4 days whereas inoculum in submerged inoculum tanks will usually be at the most favorable period in 2 to 3 days. Substantial antibiotic activity is obtained in the final fermentor stage in approximately 20 to 30 hours.

The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Staphylococcus aureus.

Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with the broth is used as a measure of antibiotic potency. After the fermentation broth has reached a desired level of anti- biotic potency, the products are isolated from either whole broth or filtered broth. In the latter case, the mycelium is removed by filtration or centrifugation. Various types of equipment such as filter presses, centrifuges, etc. may be employed.

Thin layer chromatography employing silica gel is a useful tool for analyzing the antibiotic mixture produced in fermentation media and the composition of crude and purified materials extracted from fermentation broths. The resolution of the components of the antibiotic mixture is importantly dependent on antibiotic loading of the system.

Too little antibiotic potency fails to reveal minor antibiotic components; too much antibiotic potency results in a dragging effect with resulting poor resolution.

The developing system for the thin layer chromatography is chloroform-ethanol (9:1). The thin layer chromato-grams, after development, may be observed under ultraviolet light at 254 mu and 366 mu. Bioautographic detection of the antibiotic components may be accomplished by means of an overlay of a thin layer of nutrient agar seeded with a sensitive strain of Staphylococcus aureus or other sensitive organism.

The primary components in the antibiotic mixture produced by A. auranticolor ATCC 31011 include a number of macrocyclic lactone and depsipeptide antibiotic components. The appearance or non-appearance or percentage composition of these antibiotic components varies from fermentation to fermentation and is a function of time, pH, media composition etc. Under sets of conditions given in the examples hereinafter, major antibiotic components in the antibiotic mixture ' are Compounds 37,277 (depsipeptide) and 36,926 (macrocyclic lactone) while the minor antibiotic components are Compounds 37,932 (depsipeptide) and 35,763 (macrocyclic lactone).

The components of the antibiotic mixture may be separated and recovered from fermentation broth by a number of different procedures including solvent extraction, Craig counter-current distribution, column chromatography or combinations thereof. Various organic solvents such as chloroform, ethyl acetate and methyl isobutyl ketone are useful in extracting the antibiotics from broth. Solvent extraction is preferably carried out by twice extracting the broth at about pH 7 with a volume of solvent approximately equal to about 1/3 to 1/2 the volume of broth from which it is desired to recover the antibiotic mixture. Depending on volumes of broth involved, various pieces of equipment such as separatory funnels, stirred tanks and mechanical extracting devices such as centrifugal separators are helpful for extraction purposes.

The preferred method of separation and recovery of the components of the antibiotic mixture is as follows: either whole or clarified broth is adjusted to about pH 7 and twice extracted with about 1/3 to 1/2 volume of methyl isobutyl ketone. The solvent extract is concentrated under vacuum and the concentrate defatted by extraction with heptane or petroleum ether. The defatted solvent concentrate is then taken to dryness under vacuum. The solids are subjected to Craig counter-current distribution (6 plates) utilizing toluene, 5 parts: ethanol, 2 parts: aqueous phosphate buffer, pH 4.5, 3 parts . The separated layers furnish the upper and lower phases of the counter-current distribution system.

After distribution, the layers are monitored by thin layer chromatography. The separated fractions are taken to dryness under vacuum.

The solids containing the depsipeptides are dis-solved in chloroform, treated with activated charcoal, filtered and evaporated in vacuo. The residue obtained on evaporation of the chloroform is dissolved in acetone. The solids precipitated by the addition of heptane are dissolved in a small amount of chloroform and applied to a column of pH 6 buffered silica gel made up in chloroform: n-propanol (99:1% v/v) . The column is developed with the same solvent system under 80 psi. The column cuts are monitored by thin layer chromatography. The cuts containing separated depsipeptides are combined, evaporated in vacuo and crystallized from acetone-heptane.

The counter-current fractions containing the macro-cyclic lactones are combined, evaporated in vacuo and the solids taken up in ethyl acetate. The solution is stirred with silica gel, filtered and the solvent removed in vacuo. The residue is taken up in ethyl acetate and precipitated with hexane. The solids are dissolved in a small amount of chloroform and chromatographed under 80 psi on a pH 6.0 buffered silica gel column made up in ethyl acetate. The develop ing system is ethyl acetate-tetrahydrofuran-hexane (80:20:20) saturated with aqueous pH 6.0 phosphate buffer. The column cuts are monitored by thin layer chromatography. The cuts containing separated macrocyclic lactones are combined and evaporated in vacuo . The individual fractions are separately worked up by further Craig counter-current distribution and/or column chromatography utilizing different developing systems.

Careful monitoring at every purification stage locates the individual macrocyclic lactones sufficiently isolated so that the solvent fractions can be taken to dryness to yield the pure compounds.

A. auranticolor ATCC 31011 produces at least four depsipeptides and at least four macrocyclic lactones. However, the primary components are the depsipeptides Compounds 37,277 (major) and 37,932 (minor) and the macrocyclic lactone Compounds 36,926 (major) and 35,763 (minor).

Crude antibiotic mixtures obtained directly from broth and purified individual components possess wide antibacterial spectra. Among the organisms failing to propagate in the presence of the antibiotics are Salmonella typhosa , Shigella dysenteriae, Escherichia coli, Klebsiella pneumoniae , Staphylococcus aureus , Streptococcus pyogenes , Streptococcus faecalis , Diplococcus pneumoniae, Bacillus subtilis , Corynebacterium diphtheriae, Clostridium septicum, Brucella abortus , Neisseria sicca, Iactobacillus acidophilus and Pasteurella multocida.

The antibiotics of this invention, either as a crude mixture or in the form of the purified individual components or mixtures thereof, may be employed in the treatment of various infections in man and animals. In general, these antibiotics are most desirably administered in daily oral doses of 0.5-1 gram or parenteral doses of 100 to 500 mg. , depending on the type and severity of the infection and weight of the subject being treated.

The compounds of this invention may be administered alone or in combination with pharmaceutically-acceptable carriers, and such administration can be carried out in both single and multiple doses.

For purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and dicalcium phosphate may be employed along with various disintegrants such as starch, alginic acid and certain complex silicates together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and gum acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting pur-poses. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules; preferred materials include lactose as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents as well together with such diluents as water, ethanol, propylene glycol, glycerol and various combinations thereof.

Solutions of these antibiotics in sesame or peanut oil or in aqueous propylene glycol may be employed for parenteral administration.

It is of interest that the individual antibiotics of this invention exhibit antimicrobial activity which is largely bacteriostatic in nature. However, crude antibiotic mixtures or mixtures of a purified depsipeptide and a purified macrocyclic lactone exhibit synergistic activity which is largely bactericidal in nature.

When major macrocyclic lactone Compound 36,926 (A) and major depsipeptide Compound 37,277 (B) were assayed in-dividually and in combination by the tube dilution method versus the following organisms, the following minimal inhibitory concentration in micrograms/ml . (M.I.C.) were found Crude Organism A B A + B Mixture Staph, aureus 005 1.56 25 0. 10 7 < 0.10 Staph, aureus 400 3.12 12.5 0. 78 > < 0.10 Strep, faecalis 100 12.5 1. 56 0.20 Neisseria sicca 0.39 50 0. 10 < 0.10 Treponema hyodysenteriae — — 0.19 Strep, pyogenes 0.39 12.5 0. 10 <^ 0.10 Bacteroides fragilis 35614 25 50 0. 78 0.78 Clostridium inocuum 6.25 yioo 0. 20 0.39 Lactobacillus casei var. casei 3.12 6.25 0. 20 0.39 Comparable results were obtained when minor macro cyclic lactone Compound 35,763 (A1) and minor depsipeptide Compound 37,932 (B1) were assayed individually or in appropriate combinations, i.e. A' A, B* B while (A* + B') (Α' + Β') (A + Β') (A + B) .

Maximum synergistic activity of combinations of purified macrocyclic lactone and depsipeptide is obtained over a range of about a ratio of 1-2:1. Approximately such ratios occur in fermentation broths of A. auranticolor ATCC 31011 and in crude antibiotic mixtures isolated therefrom. This finding is in contrast to other reported synergistic antibiotic mixtures where the synergistic factors occur in fermentation broths and crude mixtures at sub-optimal ratios.

In vivo protection data provided by oral and subcutaneous administration in mice experimentally infected with a strain of Staphylococcus aureus are shown in Table I.

Table I PD5Q Values (mg./kg.) S. aureus 01A005 Oral Subcutaneous Compound 36,926 - 200 >200 Compound 37,277 200 >200 Compounds 36,926 + 37,277 210 60 (1:1) Crude antibiotic mixture 150-200 72-120 (Lot 1, p. 21) The antibiotics of the present invention may be regarded to be of special interest as growth promotants in poultry and animals because of their wide antibacterial spectra and for the treatment of swine dysentry because of marked activity against Treponema hyodysenteriae , an anaerobic spirochete implicated in this disease.

The growth promoting activity of a crude antibiotic mixture (Lot 1, p. 21) was determined in young feeder pigs in a 40-day trial. Average daily gain, feed consumption and efficiency were significantly improved (p<0.01) over the non-medicated control (Table II) .

Table II Average Average Feed Treatment Daily Gain (kg) Daily Feed (kg) Efficiency* Non-medicated 0.35 0.91 2.57 Lot 1, (p. 21) 50 ppm 0.63 1.35 2.15 * lbs. of feed per lb. of weight gain Comparable results may be obtained with the other crude antibiotic mixtures of the compositions described on page 21 over a range of 10 to 100 ppm.

Comparable results may also be obtained by administration of the individual pure Compounds 36,926, 37,932 and 37,277 or mixtures of the pure compounds approximating in composition those of the crude antibiotic mixtures described on page 21.

Growth promoting efficacy was demonstrated in a chick battery feed trial. Significant improvements (p<-0.01) in weight gains over the non-medicated controls were observed for the chicks on an antibiotic-feed diet (Table III) .

Table III Average Average Feed Weight Gain Consumed Feed Treatment (grams) (grams) Efficiency Non-medicated 566 939 1.66 Lot 1, (p. 21) 10 ppm 608 949 1.56 Comparable results may be obtained with the other crude antibiotic mixtures of the compositions described on page 21 over a range of 10 to 100 ppm.

Comparable results may also be obtained by administration of the individual pure Compounds 36,926, 37,932 and 37,277 or mixtures of the pure compounds approximating in composition those of the crude antibiotic mixtures described on page 21.

The prophylactic efficacy of the antibiotics of this invention was determined in swine experimentally infected with infectious material causing swine dysentery.

Colonic content and mucosal scrapings were obtained from a clinically diagnosed field outbreak of swine dysentry.

Normal pigs were infected with this material by direct -1 - I inoculation. Antibiotic-containing feed was administered over a 28 day period. The results are shown in Table IV.

Table IV Average Treatment Morbidity (%) Mortality (%) Daily Gain (kg) Non-medicated 100 40 -0.13 Lot 1, (p. 21) 50 ppm 0 0 0.66 37.5 ppm 0 0 0.57 25 ppm 0 0 0.68 12.5 ppm 0 0 0.61 6.25 ppm 50 10 0.47 Comparable results may be obtained over a range of 10 to 100 ppm with the other antibiotic mixtures of page 21, the pure individual Compounds 36,926, 37,932 and 37,277, or mixtures of the pure compounds approximating in composition those of the crude antibiotic mixtures described on page '21.

EXAMPLE I A sterile aqueous medium having the following composition is prepared: Grams/liter Glucose 10.0 Soluble starch 20.0 Yeast extract 5.0 Enzymatic digest of casein 5.0 CaC03 1.0 pH - 7.0 Cells from a slant of A. auranticolor ATCC 31011 grown on ATCC medium 172 are transferred to a series of 300 ml . -Erlenmyer flasks each containing 50 ml. of this medium and shaken on a rotary shaker for 3-4 days at 28-30°C. Aliquots of 5 ml. of the grown inoculum are transferred to 300 ml. - Erlenmyer flasks each containing 100 ml. of the sterile medium described above. After shaking for 3-4 days at 28-30°C. , 5-10% v/v of the grown inoculum is transferred to a four liter fermentor containing two liters of the following sterile medium: Grams/liter Yeast extract 2.0 Glucose 10.0 Corn Steep Liquor 1 ml.

Enzymatic digest of casein 5.0 Cobalt Chloride 0.002 pH - 7.0 The fermentation is conducted for 20 to 30 hours at 28-36°C. with stirring at 1700 revolutions/minute and aeration at about one volume of air per volume of broth per minute.

The whole broth is adjusted to pH 7 if necessary, and twice extracted with 1/3 to 1/2 volume methyl isobutyl ketone. The solvent extract is concentrated under vacuum and defatted by extraction with petroleum ether. The activity in the broth, solvent extract and subsequent fractions is followed by silica gel thin layer chromatography using a developing system of chloroform-ethanol (9:1) and observa-tion under ultraviolet light at 254 and 366 mu.

The defatted solvent concentrate is taken to dryness under vacuum. The solids are subjected to a 6 plate Craig counter-current distribution utilizing toluene, 5 parts: ethanol, 2 parts: aqueous phosphate buffer, pH 4.5, 3 parts. . The separated layers furnish the upper and lower phases of the counter-current distribution system. After distributiPon/ the layers are monitored by thin layer chromatography.

The depsipeptide , Compound 37 ,277 , is concentrated in the upper layer, primarily in plate 0. A second depsi-peptide, Compound 37,932, is recovered from the upper layers of plates 1, 2 and 3. The macrocyclic lactone, Compound 35,763, is found primarily in the lower layers of plates 0 and 1. Compound 36,926 is concentrated in the lower phases of plates 2, 3, 4 and 5.

The upper phase of plate 0 containing Compound 37,277 is taken to dryness under vacuum, dissolved in chloroform and stirred for about 30 minutes with activated charcoal. The solution is filtered and evaporated in vacuo . The residue is dissolved in acetone and the solids precipitated by the addition of heptane. The precipitated solids are dissolved in a small amount of chloroform and chromatographed on a column of pH 6 buffered silica gel made up in chloroform: n-propanol (99:1% v/v) . The column is developed with the same system under 80 psi. Column cuts are monitored by thin layer chromatography. The fractions containing separated Compound 37,277 are combined, evaporated in vacuo and the compound crystallized from acetone-heptane.

Compound 37,277 does not have a definitive melting point. Decomposition commences at 140-150°C. It is insoluble in diethyl ether, hexane, heptane and water. It is slightly soluble in acetone and benzene and readily soluble in methanol, ethanol, chloroform and methylene chloride.

Analysis of Compound 37,277 gives the following average proportions: Carbon 60.91 Hydrogen 5.98 Nitrogen 10.45 Oxygen (by difference) 22.66 Compound 37,277 is optically active having a rotation of ]2D°= + 11° (c = 1.0, EtOH) . Its ultraviolet light absorption maxima in ethanol occur at 225, 274, 282, 303 1% and 355 mu with cm values of 309.3, 36.67, 45.01, 70 and 20, respectively.

The infrared spectrum of Compound 37,277, Figure 1, is attached. A chloroform solution shows characteristic absorption in the infrared region at the following wavelengths in microns: 3.05, 3.40, 5.70, 5.77, 5.93, 6.07, 6.62, 6.82 and 7.67.

The counter-current fractions containing Compounds 35,763 and 36,926 are evaporated in vacuo , the residue taken up in ethyl acetate and the solution stirred with silica gel. The filtered solution is evaporated in vacuo, the residue taken up with ethyl acetate and the solids precipitated by the addition of hexane. The precipitated solids are dissolved in a small amount of chloroform and chromatographed on a column of pH 6.0 buffered silica gel made up in ethyl acetate. The column is developed with ethyl acetate: tetra-hydrofuran: hexane (80:20:20) saturated with aqueous pH 6.0 phosphate buffer under 80 psi. The first 17 column cuts each of 20 ml. contain a yellow oil which is discarded. Cuts 26-40 are rich in Compound 36,926. Cuts 41-50 are largely a mixture of Compounds 36,926 and 35,763. Cuts 26-40 are combined, concentrated in vacuo and again chromatographed on silica gel with 20 ml. cuts being collected. The first 15 cuts contain a yellow oil which is discarded. Cuts 16-30 are primarily Compound 36,926. Cuts 31-40 contain a mixture of Compounds 36,926 and 35,763. Cuts 16-30 are evaporated in vacuo, the residue dissolved in a small amount of chloroform and applied to a column of pH 6.0 buffered silica gel made up in chloroform. The column is developed with chloroform: ethanol (95.5:4.5% v/v) under 130 psi, and 160 cuts of 6 ml. collected. The cuts 60-90 assay for only Compound 36,926. They are combined and evaporated in vacuo . The residue is dissolved in the minimum volume of ethanol and precipitated with ether to give pure, amorphous Compound 36,926.

Compound 36,926 is soluble in methanol, ethanol, chloroform and methylene chloride. It is insoluble in diethyl ether, hexane and heptane.

Compound 36,926 does not have a definitive melting point. Decomposition commences at about 100 °C. Analysis gives the following average proportions: Carbon 57.89 Hydrogen 6.78 Nitrogen 8.04 Oxygen (by difference) 27.29 The molecular weight by high resolution mass spectrum is 501, and the molecular formula C26H35N3°7* Compound 36,926 is optically active having a rotation of bQ2p° = - 130° (c = 1.0, EtOH). Its ultraviolet 1% light absorption maximum in ethanol is 214 mu with E^ cm of 723.8.

The infrared spectrum of Compound 36,926, Figure 2, is attached. A KBr pellet shows characteristic absorption in the infrared region at the following wavelengths in microns: 2.95, 3.40, 5.75, 5.98, 6.23, 6.58, 6.87, 7.45, 8.25, 8.38, 8.80, 9.08, 10.15, 10.35, 11.10 and 13.30.

This compound may be similar to or indistinguishable from antibiotic A2315B reported at the Fourteenth Interscience Conference on Antimicrobial Agents and Chemotherapy, September 11-13, 1974.

Compound 35,763 is isolated and purified in much the same way as Compound 36,926. The pure compound is soluble in methanol, ethanol, chloroform and methylene chloride. It is insoluble in diethyl ether, hexane and heptane. There is no definitive melting point. Decomposition commences at about 100°C. Analysis gives the following average proportions: Carbon 61.29 Hydrogen 6.73 Nitrogen 8.83 Oxygen (by difference) 23.15 Molecular Weight by high resolution mass spectrum is 503, and the molecular formula C2gH37N3°7* Compound 35,763 is optically active having a rotation of fo 2 light absorp of 668.9.

The infrared spectrum of Compound 35,763, Figure 3 is attached. A KBr pellet shows characteristic absorption in the infrared region at the following wavelengths in microns: 2.95, 3.38, 5.73, 6.00, 6.23, 6.60, 6.88, 7.23, 8.25, 8.38, 8.83 and 10.20.

Compound 35,763 may be similar to or indistinguish able from antibiotic A-2315 described in Dutch Patent 7,310,613.

The counter-current fractions containing Compound 37,932 were taken to dryness under vacuum, the residue triturated with petroleum ether and chromatographed on a silica gel column as described above. The appropriate fractions were combined and crystallized from acetone : heptane to afford Compound 37,932.

Compound 37,932 is soluble in methanol, ethanol, chloroform and methylene chloride. It is insoluble in di-ethyl ether, hexane, heptane and water. The compound does not have a definitive melting point. Decomposition commences at approximately 185°C. Elementary analysis gives the following average proportions: Carbon 59.41 Hydrogen 6.01 Nitrogen 10.66 Oxygen (by difference) 23.92 Compound 37,932 is optically active having a rotation of [°]2p0 = + 5.0° (c = 0.25, CHCI3). Its ultraviolet light absorption maxima in ethanol occur at 226, 276, 283, 305 and 355 mu with *m values of 304.4, 36.8, 43.49, 70.25 and 20.07, respectively.

The infrared spectrum of Compound 37,932, Figure 4, is attached. A KBr pellet shows characteristic absorp-tion in the infrared region at the following wavelengths in microns: 2.96, 3.05, 3.38, 5.68, 5.73, 5.93, 5.98, 6.13, 6.50, 6.58, 6.88, 7.40, 7.65, 8.08, 8.45, 8.60, 9.03, 9.45, 9.70, 9.98, 10.55, 11.00, 11.25, 11.60, 12.35 and 13.25.

EXAMPLE II The method of Example I may be repeated with com- - - parable results employing a fermentation medium of the following composition: Grams/liter Glucose 8.0 Tryptose 3.0 Enzymatic digest of casein 1.0 Glutamic acid 1.0 Soy flour 0.5 NaCl 8.3 K2HP04 2.4 KH2P04 2.0 MnCl0 0.02 EXAMPLE III The method of Example I may be repeated with comparable results employing a fermentation medium having the following composition: Grams/liter Glucose 10.0 Soy flour 10.0 Corn steep liquor 1 ml.

EXAMPLE IV The method of Example I may be repeated with comparable results employing a fermentation medium having the following composition: Grams/liter Molasses 10.0 Enzymatic digest of casein 1.0 Yeast extract 2.0 Corn steep liquor 1 ml.

Casein 3.0 EXAMPLE V The method of Example I was repeated. The methyl isobutyl ketone extract of the finished fermentation broth was taken to dryness under vacuum and the residue triturated with petroleum ether. The friable material was milled and the antibiotic components were quantitatively determined by high pressure liquid chromatographic assay. Representative bulk lots of crude antibiotic mixtures from separate fermentation runs assayed (per cent) as follows: Lot No. 35 763 36,926 37,932 37, 277 Misc. Components 1 8.5 32.0 8.7 19.3 1.3 2 3.1 39.5 7.8 15.9 1.2 3 6.0 38.6 10.7 25.5 2.4 4 9.4 47.8 0.9 26.9 0.5 5 8.4 31.9 2.2 20.3 1.4 Example VI An antibiotic premix to be used for incorporation in feed compositions for increasing feed efficiency in non- . ruminant animals and for the control of dysentery in swine may be prepared from a basic formulation of the following composition: Ingredients Per Cent Ground yellow corn 78.40 Soybean meal, 49% 18.50 Dicalcium phosphate 1.50 Limestone 0.50 Iodized salt 0.50 Vitamin premixa'b 0.50 Trace mineral premix Contributes the following levels of vitamins per pound of ration; Vitamin Λ, 2000 I.U. ; Vitamin D, 200 I.U.; Vitamin E 5 I.U.; Vitamin K, 1 mg.; Niacin, 12 mg.; riboflavin, 1.5 mg.; Pantothenic acid, 7 mg.; choline chloride, 500 mg.; Vitamin Bi2' 1 meg.

^Contributes the following levels of trace minerals - in parts per million: manganese, 120; iron, 40; copper, 4; iodine, 2.4; cobalt, 0.4; zinc, 100.

The amount of antibiotic contained in each pound of premix may be varied as desired. A preferred antibiotic premix contains 10 grams of antibiotic activity/lb. which may contain the antibiotic mixture comprising Compound" 35,763, 36,926, 37,277 and 37,932 produced under fermentatio conditions by Actinoplanes auranticolor ATCC 31011 or Compound 37,277 or Compound 36,926 or Compound 37,932. A pound of premix per ton of complete ration gives a final antibioti level of 11 ppm.

EXAMPLE VII A liquid antibiotic premix concentrate suitable for addition to drinking water to increase feed efficiency i non-ruminant animals and for the control of dysentery in swine may be prepared at desired concentrations by dissolving in an appropriate solvent such as ethanol or propylene glycol the antibiotic mixture comprising Compound 35 763, 36 926, 37 277 and 37,932 produced under fermentation conditions by Actinoplar.es auranticolor-'ATCC 31-011- or Compound 37,277 or- Compound 36,926 or Compound 37,932.

Claims (12)

P.C. 5574A

1. A synergistic antibiotic mixture comprising Compounds 35,763, 36,926, 37,277 and 37,932 and miscellaneous lesser antibiotic components produced by cultivating Actino-planes auranticolor ATCC 31011 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen until substantial antibiotic activity is obtained and separating said antibiotic mixture therefrom.

2. Antibiotic substance Compound 37,277 which in 25° crystalline form has an optical rotation of [a_D = + 11° at a concentration of 1% in ethanol; absorption maxima in ethanol in the ultraviolet light region of the spectrum at 225, 274, 282, 303 and 355 my with Ej*m values of 309.3, 36.67, 45.01, 70 and 20, respectively; having the average composition by weight of 60.91% carbon, 5.98% hydrogen > 10.45% nitrogen and 22.66% oxygen (by difference); and when dissolved in chloroform exhibiting characteristic absorption in the infrared region at the following wavelengths in microns: 3.05, 3.40, 5.70, 5.77, 5.93, 6.07, 6.62, 6.82 and 7.67.

3. Antibiotic substance Compound 36,926 having the average composition by weight of 57.89% carbon, 6.78% hydrogen, 8.04% nitrogen and 27.29% oxygen (by difference) and the molecular formula C26H35N3°7? having an optical rotation of [ot ] D = -130° at a concentration of 1% in ethanol; ab-sorption maxima in ethanol in the ultraviolet light region 1% of the spectrum at 214 my with El cm of 723.8; and when pelleted in KBr exhibiting characteristic absorption in the infrared region at the following wavelengths in microns: 2.95, 3.40, 5.75, 5.98, 6.23, 6.58, 6.87, 7.45, 8.25, 8.38, 8.80, 9.08, 10.15, 10.35, 11.10 and 13.30.

4. Antibiotic substance Compound 37,932 which in 25° crystalline form having an optical rotation of [ot ] D = + 5.0 at a concentration of 0.25% in chloroform; absorption maxima in ethanol in the ultraviolet light region of the spectrum at 226, 276, 283, 305 and 355 my with E % values of 304.4, lcm 36.8, 43.49, 70.25 and 20.07, respectively; having the average composition by weight of 59.41% carbon, 6.01% hydrogen, 10.66% nitrogen and 23.92% oxygen (by difference); and when pelleted in KBr exhibiting characteristic absorption in the infrared region at the following wavelengths in microns: 2.96, 3.05, 3.38, 5.68, 5.73, 5.93, 5.98, 6.13, 6.50, 6.58, 6.88, 7.40, 7.65, 8.08, 8.45, 8.60, 9.03, 9.45, 9.70, 9.98, 10.55, 11.00, 11.25, 11.60, 12.35 and 13.25.

5. A process for increasing growth and improving feed efficiency in non-ruminant animals which comprises feeding said animals a feed composition containing from about 10 to 100 ppm of the antibiotic mixture of claim 1 or Compound 37,277 or Compound 36,926 or Compound 37,932.

6. The method of controlling dysentery in swine which comprises incorporating in the diet of said swine from about 10 to 100 ppm of the antibiotic mixture of claim 1, or Compound 37,277 or Compound 36,926 or Compound 37,932. 47004/3

7. A process for preparing an antibiotic substance it or mixtures of antibiotic substances substantially as hereinbefore described.

8. A process for increasing growth and improving feed efficiency in non-rurainant animals substantially as hereinbefore described.

9. A process of controlling dysentery in swine substantially as hereinbefore described.

10. An antibiotic substance or mixture of antibiotic substances whenever prepared by the process substantially as hereinbefore described.

11. A synergistic antibiotic composition which comprises Compound 36, 926 and/o 35,763 and Compound 37,277 and/or 37,932.

12. An antibiotic composition for use in animals which comprises Compound 36,926 or Compound 37,277 or Compoun 37,932 or mixtures thereof and a pharmaceutically acceptable carrier including an animal feed, drink or premix. For the Applicants DR, Bys