CA1160569A - Antibiotic compositions - Google Patents

Abstract

Abstract Antibiotic compositions comprising certain novel combinations of macrolide and aminoglycoside antibiotics or their physiologically acceptable salts have been found particularly effective in the treatment of colibacillosis in pigs. They are also of value as growth promoters and in improving the feed utilization in pigs. They are also valuable in controlling the growth of mycoplasma in mammalian tissue cultures.

Description

X-4383 -'-TITLE
ANTIBIOTIC COMPOSITIONS
SUMMARY OF lHE INVENTION
This invention relates to anti~iotic COM-~inations. In particular i~ relates to antibiotic combinations comprising certain macrolide antibiotics and certain aminoglycoside antibiotics which possess synergistic activity against mycoplasma.
Antibiotic combinations of the invention comprise a macrolide antibiotic e.g., tylosin and spiromycin in com~ination wit~ one c~ the following aminoglycoside antibiotics: tobramycin, apramycin, neomycin, gentamicin, and ne~ramycin Eactor 5. ~he compositions of the invention are particularly useful in controlling the growth of resistant mycoplasma in tissue culture media.
Preferred antibiotic compositions of this invention comprise tylosin and 'obramycin or apramycin.
The preferred composition of this invention comprising tylosin and apramycin is also usef~l i~ the treatment of post we~ning coiibacillosis in pigs when admin--istered in the animals'~eed or d_inking water as dis-closed in U.S. Patent No. 4,283,388 of Earl E. Ose issued August 11, 1981. Further, the preferred tylosin-apramycin compositions enhances the growth and feed e~icien~y in weaned pigs.
~etailed Descripti~n The an~ibiotic compo~itions provided by ~his inver.tion com2rise a macrolide antibiotic and ~n ~mino1ycoside antibiotic in a synergis.ically ef-.

11~;0~6 fective concentration. Macrolide antibiotic~ which are employed in the compositions ar2 tylosin, spiromycin, magna~ycin (also kno-"n as car~omycin A~, leucomycin, and oleandomycin. A.-ninoglycoside or aminocyclitol 5 antibiotics w'nich are used in combina~ion with one of the a}~ove ~nacrolides are tobramycin, apr~mycir" nebra-mycin 5, gentamic~n, and neomycin.
The macrolide and a~ninoglycoside antibiotics comprising the compositions are all known. Tylos~
10 U.S. Patent 3,178~34l, Merck Index 9th Ed., No. 9486 is sold commercially as a veterinary antibiotic; spiromycin is disclosed in U.S. Patent 2,943.023, Merc}c Index 9tA
Ed., No. 8525; leucomycin is disclosed in Merck Index 9th., ~o. 53Q7; magnarnycin is disclosed in U~S. Patent 15 2,960,438 and Merck Index 9th Ed., No. 1812 and oleando-mycin is disclosed in U.S. Patent Nos. 2,757,123 and

2,842,481 and Merck Index 9th Ed., ~lo. 6671.
The aminoglyco~iide antibiotics tobrarnycin and apramycir. are alternatively known as nebramycin factors 20 6 and 2 respect~vely. Tobrarnycin, apramycin and nebra-mycin factor 5 are mem~ers cf the neb~amycin ccmplex of antibiotics produced by treptomyces tennibrarius and are described in U.S. Patent 3,~91,279. Tobramycin is used clinically in kuman medicine. Neo~ycin i5 25 disclosed in U.S. ?atent 2,799,620 while gentamicin, a clinically useful antibiotic ln h~lman medicine,is disclosed in U.S. Patent 3,091,572 and Merck Index 9th Ed., No, 4223.
~he antibiotic compositions of this invent on 30 are ~seful in the control of mycoplasma and in particular i'l'~Sfi9 mycoplasma that are resistant to the individual anti-~iotics, The synergism of the compositions was demo~-strated in mammalian tissue cultures where mycopla~ma are frequent and often unre~ognized contaminants. The mycoplasma contamination of tissue culture prepar~tions of various cell lines, for example in the propagation of animal virus, can ruin the culture ~reparation and in some instances after culture has been maintained for several weeks. The synergistic activity of the com-positions of this invention is best demonstrate~against resistant mycoplas~a in mammalian tiss~le culture. The term, "resistant mycoplasma," as used herein refers to mycoplasma which are resistant to both the macrolide antibiotics and the aminoglycoside antibiotics comprising the compositions of this invention at minimal inhibitory concentrations higher than those generally observed with the antibiotics against vario~ls microogranisms. A number of mycoplasma ar~ resistant to the antibiotics comprising the compositions of this in~ent on~ Prevalent among resistant mycoplasma contaminar.ts in mammalian tissue culture pre~arations are strains of M. hyorhinis. M. qallisepticum resistant strains are also frequent contaminants. The synergistic compositions of this invention inhibit the growth of resistant ~ycoplasma microorganisms at low concen-trations when the individual antibiotics are ineffecti~e alone at high concentra~ions. The foilowing TA~LE 1 lists the minimal inhibitory concentrations (MIC) in ~cg. /ml for the individual masrolide and aminoglycoside antibiotics and their combinations. T;~e ~IC values 11605.69 were obtained with mammalian tissue cultuxes contam-inated with the designated mycoplasma in experiments carried out as follows.
Suspensions of monkey kidney cells, LL~-MK2, in medium 199 (Morgan et al., Proc. Soc. Exp. Bio. Med, 73, 1 1950), were inoculated with cultures of the indicated ~ycoplasma to achieve an inoculum concen-tration of 1.5 X 107 organisms/ml of cell suspension.
Control cultures and the mycoplasma positive cultures included in each test were incubated at 37C for 3-5 days. After incubation the medium in all cultures was changed to 1% horse serum in Medium 199 containing 1. 6a g of sodium bicarbonate per liter. The tissue cultures innoculated with mycoplasma (except for mycoplasma positive control cultures) were then treated with the indicated individual antibiotics alone or with the combination of antibiotics shown in TABLE l. ~hen combinations of antibiotics were tested equal amounts of the antibiotics were used.
Solutions of the antibiotics were made up in Medium 199 containing 1.68 g of sodium bicarbonate per liter of medium. The solutions were sterile filtered through a ~'Millipore~*filter having a 0.2 micron porosity and were stored at 4C prior to use. Two rold dilutions 25 of the antibiotic solutions were made and each anti-biotic solution was added to separate cuitures to determine the minimal inhibitory concentration. The medium in each culture was chansed twice a week with fresh antibiotic solution at the same concent.ation being added at each medium change. After two weeks, * Trad~rk -, ~

il60S~;9 X-4383 ~~~

medium changes were made without antibioti-~ for two more weeks. Samples of the cultures wer2 taken before each medium change in the lzst two week3 and ~ele cultured to determine the presence of ~Ico~lasma.

~1~60~69 x-43a3 -6-MINIMAL INHIBITORY CONCENTRATIONS OF A~TIBIOTICS
vs MYCOPLASMA IN LLC-MK2 TISS~E CULTURE
~inimal Inhibitory Concentration Antibioticl Mycoplasma (mcq/ml) Tylosin M. ~yorhinis HH >250 Tobr~mycin M. Hyorhinis HH2 62.5 Combination ~. Hyorhinis ~H2 <15.6 Tylosin M. Hyorhinis 100 Tobramycin ~. Hyorhini~ 100 Combination M. Hyorhinis 3 Tylosin M. Hyorhinis HEK3 >~00 Tobramycin M. Hyorhinis HEK >500 Combination ~1. Hyorhinis HEK 3.1 Tylosin tar- M. Hyorhinis HH 200 trate Tobramycin M. Hyorhinis HH 200 sulfate Combination ~. Hyorhinis HH 1.5 Tylosin tar- M. gal~isepticum >800 trate 36 F
Tobramycin M. gal~isepticum 100 Combination M. ga~isepticum 25 Tylosin ~. gall~septicum 800 Tobramycin M.4la~j.septicum 200 Combination M. gallisepticum 12.5 llt~OS~69 X-4383 ~7~

TA~LE 1 (continue~3 Mirlimal Innibi~ory Concent ation Antibioticl Mycoplasma (mcg/m~
Tylosin Swine L3F12F76 200 Tobramycin Swine L3F12F76 50 Comblnation Swine L3Fl2F76 <1.5 Tylosin M. Hyorhinis HH 200 Apramycin M. Hyorhinis HH 600 Combination M. Hyorhinis HH 12 Tylosin M. Hyorhinis400 Gentamicin M. Hyorhinis 100 Combination M. Hyorhinis 0.78 Tylosin M. Hyorhinis HH >800 Nebramycin M. Hyorhinis HH 200 Factor 5 Combination M. Eyorhinis HY 25 Tylosin M. Hyorhinis200 Neomycin M. EyorhinislO0 Combination M. Hyorhinis 6.3 Spiromycin M. Hyorhinis HH >800 Tobramycin M. Hyorhinis HH 200 Combination M. Hyorhinis HH 1.5 11~15ti9 TABLE 1 !contin~ed) Minimal Inhi~itory Concentration Anti~ioticl Mycoplasma (mcg/ml) Magnamycin M. Hyorhinis ~H >~00 Tobramycin ~. Hyorhinis ~ 200 Combination M. Hyorhinis HH 1.5 Leucomycin M. Hyorhinis HH >400 Tobramycin M. Hyorhinis ~H 200 Combination M. ~yorhinis HH 0.78 Oleandomycin M. Hyorhinis HH 400 lS Tobramycin M. Hyorhinis HH 200 Combination ~. Hyorhinis ~H 100 1/ Combinations of the indicated anti~iotics contained equal amounts of each antibiotic.
2/ M. hyorhinis H.H. is an isolate from human heart tissue

3/ M. hyorhinis HER is an isolate from human embryonic kidney tissue

4/ M. gallisepticum 3-6F is a strain of a chicken mycoplasma

5/ M. gallisepticum 41313 is a strain of a chicken mycoplasma

6/ Swine L3F12F7 was a mycoplasma isolated from swine and from its characteristrics appears to be r~YcoPlasma arqinini fi9 X-4383 -g-As is shown in Table 1 the ~ coplasma employ~d in the test were controlled only at high concentrations by the indi~idual antibiotics. Ho~ever when one of the macrolide - aminoglycosld~ compositions of this invention was used the ~ycoplasma were controlled at greatly reduced concentratlons. Tn each com~ination the individual antibiotics were present in equal parts.
The compositions of this invention axe non toxic to mammalian tissue and can be used to maintain cell lines free of contamination by mycoplasma. The compositions can also be used in conjunction with other antibiotics which are commonly used in tissue cultures to prevent bacterial contamination but which are ineffective in controlling mycoplas~a. For example the lS synergistic compositions can be used with a penicillin such as penicillin G or with streptomycin, two anti-biotics commonly used in tissue cultures.
A preferred composition o this irvention s tylosin-tobramycir.. This composition has consistently exhibited low minimal inhibitory concentrations against mycoplasma which tylosin or tobramycin alone control only at high minimal inhibitory concentrations. Table 2 which follows contains the results obtained with numerous combinations of tylosin and tobramycin which inhibited the growth of M. hyorhinis in tissue culture preparations using monkey kidney cells LLC-MK2. The tests were conducted in the following manner.
Tissue cultures of LLC-MK2 cells wsre lnoculated with ~. hyorhir.is culture fiYe days prior to ~0 the a~dition of tylosin-tobramyrin com~inations con-1~i056~

taining varying proportions of the antibiotics and at various concentrations. Medilm changes were made at three to five-day intervals r~ith antibiotics addition at each change. Samples of the culture media were taken at each media change and assayed for mycoplasma.
The data in Table 2 illustrate various proportions of the antibiotics in combinations which inhibited the growth of M. hvorhinis.

Inhibition of M. hYorhinis in LLC-MK2 Tissue Culture by Tylosin-Tobramycin Compositions Tobram~cin Tylosin (mcg/ml)plus (mcg/ml) ~0 0.5 12 All of the combinztions of tylosin and tobra-mycin shown in Table 2 inhibited the growth of mycoplasma for from five to eight samplings covering a period of three and a half wee~s. When used alone tllosin and tobramycin exhibit much higher MIC ~-alues as shown in Table 1.
The data in Table 2 demonstrate the low concentrations at ~hich the antibiotic compositions of this invention are synergistic~lly effective. Com-positions containing from one par~ of tobramycin 11605~;~

to 3 parts of tylosin by weight ar~ preferred syner-gistic ~ompositions.
Another pre~erred conposition of this invention comprises tylosin in combinat on with apramycin. As is shown in Table 1, the solution containing 12 mcg/ml of tylosin and 12 mcg/ml of apramycin inhibited mycoplasma wh~reas the MTC for apramycin alone was 600 mcg/ml and that for tylosin alone was 200 mcg/ml.
A further preferred composition compr ses tylosin in combiration with gentamicin.
In contrast to the synergism demonstrated by the macrolide antibiotic-aminoglycoside antibiotic compositions of this invention a number of macrolide-macrolide antibiotic combinations showed little if any enhanced activi~y in the control of mycoplasma in tissue cultures.
The macrolide and aminoglycoside antibioticsforming the synergistic compositions can be in a suitable acid addition salt form or as the free bases. These antibiotics form salts with mineral acids and carboxylic acids. Examples of such salts are tylosin hydrochloride, tylosin tartrate, tylosine phosphate, spiromycln adipate, spiromycin hydrochloride, oleandomycln hydro-chloride, leucomycin hydrobromide, magnamycin tartra~e, tobramycin sulfate, apramycin hydrochloride, neomycin sulfate, neomysin h~drochloride, gentamicin hydro-chlorid~, and gentamicin sulfate.
The antibiotlc formulations of this lnvention can be prepared by mixing the dry antibiotic powders of the free base or a salt form thereof or, a~ternatively 116~.5~

X-~383 -12-solutions of the antibiotics of the appropriate COII-centration can be mixed to obtain a solution of the two antibiotics o~ the desired concentration. The individual antibiotics are appreciably soluble in aqueous ~edia and solutions of the formulations in aqueous tissue culture medium are desira~ly used in controlling ~yco-plasma contamination in mammalian tissue cultures.
The synergistic antibiotic compositions of this invention can ~e used to inhibit the growth of mycoplasma in a wide variety of mammalian tissue and thus prevent the contamination and loss thereof. For example, a composition of the invention can be e~ployed in mammalian tissue cultures used in the preparation of plasminogen activator according to the method described by Hull et al., U.S. Patent 3,904,480.
The antibiotic compositions of this invention comprise the macrolide antibiotic in the ratio by weight to the aminoglycoside antibiotic of fro~ 15:1 to 1:15. A preferred ratio by weight of the macrolide ~0 to the aminoglycoside is 3:1 to 1:3.
A preferred antibiotic composi-tion of this in~Jention, tylosin in combination with apram~cin, is also useful in enhancing the growth in past weaned pigs. The tylosin-apramycin combination demonstrates higher feed utilization and higher weight gains in post weaned pigs than does either antiblotic wh~n administered alone. The efficacy of the tylosin-apramycin combina-tion in enhancing feed utilization and ~eight ga-n in post weaned pigs was determined in animal trials carried out as follows.

llt;OS69 X ~383 -13-The trials were carried ollt in pens with 6 pigs per pen .or 28 days. The pigs were of ~ixed sex having an average initial body weight of 12.89 kg. and an average age of six weeks. The animals received water before and a'ter feeding. The animals were fed twice daily with feed having the following composition.
Inqredient Percent Wheat 21 Barley 23.5 Corn 20 Fish meal 2.5 Yeast Wheat bran 10 Alfalfa 2.5 Soybean ~eal - 14 Oat hulls 1.8 Calcium phosphate Caicium carbonate 1.1 Sodium Chloride 0.1 Premix SV12 Premix S04 0.5 _ The tylosin-apramycin combination was compared in the trials with tylosin alone and with apramycin alone. The antibiotics and the combin~tion thereof were administered in the animal .eed at a level of 100 ppm, and, in the ca~e of the combination; at 100 ~pm for each antibiotic.
The combined reslllts of two like trials are shown below in TABLE 3.

,9 ~ABLE 3 Comparative Weight Gains And Feed Utilization in Pigs Treated With Apramycin - T~losin, Tylosin And Apramycin -Negative Tylosin-Txeatment Control Tylosin Apra~ycin Apramycin Conc. in Feed (ppm) - 100 100lO0-100 10Number of animals n 70 71 72 72 Number of replic. n12 12 12 12 Initial weight x13,11 13,03 12,55 12,85 (kg) per animal +s0,81 1,04 0,58 0,71 15(Rel) (100) ( 99) ( 96) ( 98) Final weight x 25,00 25,58 25,62 26,71 (kg) per animal +s1,48 1,65 1,14 1,49 ~ 6 6 4 6 (Rel) (100) (102) (102) (107) Daily gain x 425 449 467 495 (g) per animal +s 40 62 38 44 v 9 14 8 9 (Rel)(100) (106 (110) (116) Daily feed in x 0,92 0,92 0,92 0,94 take (kg) per - s 0,06 0,06 0,04 0, 05 animal v 7 6 4 5 (Rel) (100)(100) (100) (102) Feed efficiency ~ 2, L42,07l,96 1,90 (kg feed/kg - s 0,10 0,17 0,09 0,09 gain) ~ 4 8 4 5 (Rsl) (lO0) ( 97) ( 52) ( 89) _ 1160~6~

In Table 3 the statistical symbols have the following meanings: x = means; - s - standard devia-tion; v = coefficient of variances; and (Rel.) =
comparison treatments to negative controls in per-centage.
As shown in TAB~E 3 the pigs treated withtylosin-apramycin combination showed higher weight gains and better feed efficiency on the 28 day trial than did the pigs treated with tylosin alone or with apramycin alone.
The tylosin-apramycin compositions of this invention demonstrate synergistic activity against strains of Pasteurella hemolYtica. This organism can cause pneumonia in cattle which in cases of se~ere infection can lead to death. The synergism of the tylosin-apramycin combination was demonstrated in vitro against P. hemolytica strain 41D. Tylosin alone had a minimal inh bitory concentration of 50 mcg/ml and that for apramycin alone was 25 mcgJml. The synergistic activity of the combination of the two antibiotics was determined by the n chec~erboard" titration method. In this method two antibiotics are tested in serial dilutions and in all combinations o' these dilutions together to find the concentrations of each antibiotic, both alone and in combination, that inhibit growth of the test microorganism. The nature of the interaction between the two antibiotics may be determined alge-braically or geometrically. In the algebraic method, the concentration of each antibiotic in the combination that inhibits growth is expressed as a fraction of the 116~5~;~

concentration that produces the same effect when the antibiotic is used alone. ~en the sum of these fractions is one, the combination is additive; when the sum is less than one, the combination is synergistic;
and when the sum is greater than one, the combination is antagonistic.
Below are shown the calculations using data obtained in the ir. vitro test of tylosin, apr~mycin and the combination of the two antibiotics against 2.
10 hemolYtica.
Tylosin alone : MIC = 50 mcg/ml apramycin : MIC = 25 mcg~ml Tylosin + Apramycin 25/50 + 0.8/25 = <1 12.5/50 + 6.25/25 = <1 6.25/50 1 12.5/25 = <1 The following antibiotic compositions are preferred compositions Tylosin - Tobramycin Tylosin - Apramycin Tylosin - Gentamicin Leucomycin - Tobramycin Spiromycin - Tobramycin Magnamycin - Tobramycin Especially preferred compositions are tylosin-tobra-mycin, tylosin-apramycin, and tylosin-~entamicin.
The antibiotic compositiors of this invention are, as described hereinabove, valuable n veterinary medicine in the treatmen-t of various animal diseases.

O5~9 The compositions are also valuable in controlliny mycoplasma contamination in the propagation of virus by the tissue culture method.
The following examples further illustrate the present invention.
E~ample 1 Cell suspensions of LLC MK2 (Monkey kidney cell line) in ~edium 199 containing 1% house serum of 4 ml volumes in tissue culture flasks were inoculat2d with a 3 day old culture of Mvcoplasma h~torhinls ~5 X
108 organism per ml in Eaton's broth). Approximately 2 to 3% of the mycoplasm2 culture per Jolume of the cell suspension was used. After inoculation the cell suspensions were incubated at a temperature of 37C for form three to five days. Control cultures were also made up in the same manner. After incubation the medium in the control cultures and the mycoplasma positive cultures was replaced with fresh Medium 199 containing 1.68 g of sodium bicarbonate and 1% horse serum. The cultures were then treated with solutions of tylosin, tobramycin, and wi.h the combination of tylosin and tobramycin in ~ledium 199 containing 1.68 g of sodium bicarbonate per liter. Solutions of each individual antibiotic and of the combination at a concentration of 1000 mcg/ml were prepared. The solution of the combination had a concentration of each antibiotic of 1000 mcg. By two-foid serial dilution solutions at various concentrations down to 0.39 mcg/ml were prepared. Each of the antibiotic solutions was added to a separ2te flask containing ~he 'cissue culture.

The tissue culture medium was replaced in each flask twice a week and at each medium change for two weeks a fresh solution of the antibiotic at the same concentration was added. After two weeks fresh medium replacements were made for two more weeks without the addition of antibiotic. Samples of each culture were taken before each medium replacement and were cultured to determine the presence of mycoplasma.
The minimal inhibitory concentration of the individual antibiotics and of the combination, tylosin and tobra-mycin, was the lowest concentration of the antibiotic in a given tissue culture which failed to culture for mycoplasma on sampling. The minimum inhihitory con-centration for tylosin alone was 100 mcg/ml. and for tobramycin alone was 100 mcg/m'. while for the combina-tion the MIC was 3 mcg/ml.
Example 2 When tylosin, apramycin, and the tylosin-apramycin combinations were evaluated in tissue culturecontaminated with M. hyorhinis H. H. according to the method and procedures employed as described by EYample 1 tylosin alone had an MIC of 200 mcg/ml., apramycin alone had an MIC of 600 mcg/ml. while the tylosin~
apramycin combination had an MIC of 12 mcg/ml.
Example 3 By employing M. hYorhinis in the procedure of Example 1 and substituting gentamicin for tobramycin and tylosin-gentamycin fo~ the tylosin-tobramycin combina~ion, tvlosin alone had an iMIC of 400 mcg/ml.
The combination of tylosin-gentamicin had an MIC of 0.~8 mcg/ml.

i~5~9 Exa~ple 4 The combination, leucomycin-tobra~ycin, was substituted ~or the tylosin-tobramycin combination used in Example 1. Leucomycin ~lone had an ~.IC of 400 mcg~ml., tobramycin an MIC of 200 mcg/ml. while the combination had an MIC of 0.78 mcg/ml.

Claims (10)

X-4383-Canada -20-The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An antibiotic composition comprising a macrolide antibiotic selected from the group consisting of tylosin, leucomycln, magnamycin, spiromycin and oleandomycin or a physiologically effective salt thereof and an aminoglycoside antibiotic selected from the group consisting of tobramycin, apramycin, nebramycin Factor 5, gentamicin and neomycin or a physiologically effective salt thereof.

2. An antibiotic composition as claimed in claim l, wherein the macrolide antibiotic is tylosin or a physiologically acceptable salt thereof.

3. An antibiotic compositon as claimed in claim 2, wherein the aminoglycoside antibiotic is tobramycin or a physiologically acceptable salt thereof.

4. An antibiotic composition as claimed in claim 2, wherein the aminoglycoside antibiotic is apramycin or a physiologically acceptable salt thereof.

5. An antibiotic composition as claimed in claim 2, wherein the aminoglycoside antibiotic is gentamicin or a physiologically acceptable salt thereof.

6. An antibiotic composition as claimed in claim 1, wherein the ratio by weight of the macrolide antibiotic or a physiologically acceptable salt thereof to the aminoglycoside antibiotic or a physiologically acceptable salt thereof is from about 15:1 to about

7. An antibiotic composition as claimed in claim 6, wherein tylosin is the macrolide antibiotic X-4383-Canada -21-and the aminoglycoside antibiotic is selected from the group consisting of tobamycin, apramycin and gentamicin or a physiologically acceptable salt thereof.

8. An antibiotic composition as claimed in claim 7, wherein the ratio by weight of tylosin or a physiologically acceptable salt thereof to the amino-glycoside antibiotic or a physiologically acceptable salt thereof is from about 3:1 to about 1:3.

9. An antibiotic composition as claimed in claim 8, wherein tobramycin or a physiologically acceptable salt thereof is the aminoglycoside.

10. An antibiotic composition as claimed in claim 8, wherein apramycin is the aminoglycoside.