JPH0141609B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic combination (antibacterial composition) consisting of a macrolide antibiotic and an aminoglycoside antibiotic. This combination has been found to be particularly effective in the treatment of colibacillosis in pigs, and is also useful as a growth promoter and for improving feed intake in this type of animal.
Additionally, this combination suppresses the growth of mycoplasma in mammalian tissue culture. The macrolide antibiotics and aminoglycoside antibiotics that form this combination are all known. Tylosin is described in US Pat. No. 3,178,341 and Merck Index (9th Edition) No. 9486 and is commercially available as an antibiotic for animals. Spiromycin is US patented
2943023 and Merck Index (9th edition) No.
8525. Leucomycin is listed in the Merck Index (9th edition)
Described in No.5307. Magnamycin is described in US Pat. No. 2,960,438 and Merck Index (9th Edition) No. 1812. Oleandomycin is disclosed in U.S. Patents 2757123 and 2842481 and Merck
It is listed in Index (9th edition) No.6671. The aminoglycoside antibiotics tobramycin and apramycin are also known by their other names nebramycin factor 6 and 2, respectively. Tobramycin, apramycin and nebramycin factor 5 are used in Streptomyces
It is a component of the nebramycin complex produced by P. tennibrarius and is described in US Pat. No. 3,691,279. Tobramycin is in practical clinical use as a medicine for humans. Neomycin is described in US Pat. No. 2,799,620 and gentamycin is described in US Pat. No. 3,091,572 and Merck Index (9th edition).
Described in No.4233. The antibiotic combination provided by the present invention includes (a)
Tylosin, leucomycin, magnamycin,
(b) a macrolide antibiotic selected from spiromycin and oleandomycin and physiologically acceptable salts thereof; and (b) tobramycin, apramycin, nebramycin factor 5, gentamicin and neomycin and physiologically acceptable salts thereof. and aminoglycosides selected from salts of. The present invention also provides a method for promoting the growth or feed intake of pigs by administering the antibiotic combination described above to the pigs. The present invention also provides a method for inhibiting the growth of mycoplasma in tissue by treating cultured tissue of a mammal with the above-mentioned antibiotic combination. The present invention also provides a method for treating coliosis in pigs, which comprises administering the antibiotic combination described above to the pig. The antibiotic combination of the present invention is useful for controlling mycoplasma, particularly for mycoplasma that are resistant to individual antibiotics. The synergism of this combination was demonstrated in mammalian tissue cultures, where contamination with mycoplasma frequently occurs. Tissue culture of various cell lineages (e.g.
Mycoplasma contamination (when performing animal virus propagation) can ruin tissue cultures, sometimes after several weeks of cultivation. The synergistic activity of the combination of the invention is best demonstrated against resistant mycoplasma in mammalian tissue culture. Here “resistant mycoplasma”
The term refers to resistance to the macrolide antibiotic and aminoglycoside antibiotic compounds contained in the combination of the present invention at concentrations higher than the MIC (minimum inhibitory concentration) that these antibiotics generally exhibit against various microorganisms. means mycoplasma. Many mycoplasmas are resistant to the antibiotics contained in the combination of the present invention. The main resistant mycoplasma that is a source of contamination in mammalian tissue culture is Mycoplasma hyorhinis (M.
hyorhinis). Resistant strains of M. gallisepticum are also frequent contaminants. The synergistic combination of the present invention inhibits the growth of resistant mycoplasma at low concentrations, where individual antibiotics alone are ineffective at high concentrations. Table 1 below shows the minimum inhibitory concentrations (MICs) of individual macrolide and aminoglycoside antibiotics and their combinations.
mcg/ml). MIC values were obtained by the following experimental method using cultured mammalian tissues contaminated with each mycoplasma. Monkey kidney cells LLC-MK2 were cultured in medium 199 (Morgan et al.
al, Proc.Soc.Exp.Bio.Med., 73 , 1 (1950)) and inoculated with each mycoplasma at a cell number concentration of 1.5×10 ⁷ cells/ml. Control medium and mycoplasma positive medium were used for each test and incubated at 37°C for 3-5 days, then the medium of all cultures was replaced with sodium bicarbonate.
1% horse serum in medium 199 containing 1.68 g/ml. Tissue cultures inoculated with mycoplasma (excluding mycoplasma positive control cultures) were treated with each antibiotic alone and in combination as shown in Table 1. When testing antibiotic combinations, specific doses of antibiotics were used. Antibiotic solution contains 1.68 g of sodium bicarbonate/
It was prepared using medium 199 containing. The solution was sterilized through Millipore paper (porosity: 0.2μ) and stored at 4°C until use. Antibiotic solutions were diluted multiple times and each solution was added to separate cultures to determine the minimum inhibitory concentration. The medium of each culture was changed twice a week, and fresh antibiotic solution was added each time to the same concentration. After 2 weeks, medium exchange was performed without antibiotics for an additional 2 weeks. final 2
Samples of the culture were taken at each medium change for a week and cultured to assay for the presence of mycoplasma.

【table】

[Table] Ishin

[Table] As shown in Table 1, the mycoplasma tested were inhibited only at high concentrations for the individual antibiotics. However, when the macrolide-aminoglycoside combination of the present invention was used, these mycoplasmas were inhibited at a much lower concentration. Equal amounts of each antibiotic were used in each combination. The combination of the present invention is non-toxic to mammalian tissues and can be used to preserve cell lines free of mycoplasma contamination. The combination can also be used in combination with other antibiotics commonly used to prevent bacterial contamination of cultured tissues (but not effective in controlling mycoplasma). For example, this synergistic combination can be used with penicillins (eg, penicillin G) and streptomycin commonly used in tissue culture. A preferred combination of the present invention is tylosin-tobramycin. This combination showed consistently low minimum inhibitory concentrations against mycoplasma, where tylosin and tobramycin alone are only active at high concentrations. Table 2 below shows M. in tissue culture using monkey kidney cells LLC-MK2.
Various tylosins inhibit the growth of P. hyorhinis.
The results obtained using the tobramycin combination are shown. This test was conducted as follows. LLC-MK2 cells were inoculated with M. hyorhinis 5 days before adding the tylosin-tobramycin mixture, and both antibiotics were used in various mixing ratios and at different concentrations. The medium was replaced every 3-5 days, and antibiotics were added each time. A sample of the culture medium was taken every time the medium was replaced and assayed for mycoplasma. The values in Table 2 indicate the mixing ratio of antibiotics in the combination drug that inhibited the growth of M. hyorhinis.

[Table] All tylosin-tobramycin combinations shown in Table 2 were tested in 5-8 tests conducted over 3.5 weeks.
Mycoplasma growth was inhibited in multiple samplings. When used alone, both tylosin and tobramycin are much higher as shown in Table 1.
Indicates MIC value. The values in Table 2 show that at low concentrations the antibiotic combination of the invention is synergistically effective. Combinations containing 3 or more parts by weight of tylosin to 1 or more parts by weight of tobramycin are preferred synergistic compositions. Another preferred combination of the present invention is a combination of tobramycin and apramycin. As shown in Table 1, a solution containing 12 mcg/ml of tylosin and 12 mcg/ml of apramycin inhibits mycoplasma, but the MIC of apramycin alone is
600mcg/ml, and that of Tylosin
It is 200mcg/ml. Another preferred combination is a combination of tylosin and gentamicin. Although the combination of macrolide antibiotics and aminoglycoside antibiotics of the present invention demonstrates synergistic activity, many macrolide-macrolide antibiotic combinations show some increase in mycoplasma inhibitory activity in tissue culture. Even if they do, they rarely show synergistic effects. The macrolide and aminoglycoside antibiotics that make up the synergistic combination may be the free base or the appropriate acid addition salt.
These antibiotics form salts with mineral acids and carboxylic acids. Examples of salts of this type include tylosin hydrochloride, tylosin tartrate, tylosin phosphate, spiromycin adipate, spiromycin hydrochloride, oleandmycin hydrochloride, leucomycin hydrobromide, magnamycin tartrate. salt,
These include tobramycin sulfate, apramycin hydrochloride, neomycin sulfate, neomycin hydrochloride, gentamicin hydrochloride, and gentamicin sulfate. The antibiotic formulations of the present invention are prepared by mixing dry powders of antibiotics in free base or salt form. Alternatively, solutions of antibiotics at appropriate concentrations may be mixed to obtain a solution containing two antibiotics at desired concentrations. Each antibiotic is fully soluble in aqueous media, and solution formulation using aqueous tissue culture media is suitable for controlling mycoplasma contamination in mammalian tissue culture. The synergistic antibiotic combination of the present invention inhibits the growth of mycoplasma in a wide range of mammalian tissues,
It is therefore used to prevent contamination and loss of these tissues. For example, US Patent 3904480
The combination of the invention can also be used in mammalian tissue culture in the production of plasminogen activators by the method described in (Hull et al). The antibiotic combination of the present invention has a weight ratio of macrolide antibiotic and aminoglycoside antibiotic of 15:
1 to 1:15. The preferred weight ratio of macrolide to aminoglycoside is 3:1 to 1:3. A combination of tylosin and apramycin, which is a preferred form of the antibiotic combination of the present invention, can also be used to promote the growth of pigs after weaning. Tylosin-apramycin combination produces higher feed intake and higher weight gain in post-weaned pigs than either antibiotic alone. The effects of the tylosin-apramycin combination on feed intake and weight gain in pigs after weaning were demonstrated in animal experiments using the method described below. The experiment was conducted for 28 days using multiple pens housing 6 pigs per pen. The pigs used were of mixed sexes, with an average initial weight of 12.89 kg and an average age of 6 weeks. The animals were given water before and after feeding, and fed with the following composition twice a day. Ingredients Hundred parts perheceau 21 barley 23.5 corn 2.5 fish meat 2.5 East 1 wheat fluff 10 Alf Alfua 2.5 soy flour 14 carcaxium (OAT HULLS) 1.8 calcium carbonate 1.1 calcium carbonate 1.1 Snatium chloride 0.1 Premitics SV12 1 Premitics SO4 0.5 Experience Then, Tylosin -Apramycin The combination was compared to tylosin and apramycin used alone. Each antibiotic and their combination was administered in addition to animal feed. The mixing ratio of each antibiotic was 100 ppm for each antibiotic, and 100 ppm for each antibiotic. The overall results of two similar experiments are shown in Table 3 below.

【table】

[Table] The meanings of the statistical symbols in Table 3 are as follows.
= average value; ± s = standard deviation; v = coefficient of variation; (Rel) = comparative value with negative control as 100. As can be seen from Table 3, pigs treated with the tylosin-apramycin combination showed higher weight gain and better feed efficiency than pigs treated with tylosin alone or apramycin alone during the 28-day experiment. Next, the efficacy of the tylosin-apramycin combination was demonstrated through a controlled test on weaned pigs. The results show that pigs treated with the antibiotic combination had a very significant reduction in the frequency of E. coli diarrhea compared to pigs treated with apramycin alone or tylosin alone. Additionally, pigs fed the combination showed higher daily weight gain and improved feed efficiency compared to pigs fed either antibiotic. Table 4 below shows the results obtained in 21 days using weaned pigs. Tylosin and apramycin were administered in the feed at a rate of 110 ppm each. Similarly, antibiotic combinations are
Both antibiotics were administered in the feed at a rate of 110 ppm. The results are a statistical evaluation of the values obtained in the test and are expressed as a percentage increase over the untreated control group.

[Table] As shown in Table 4, tylosin, even when used alone, was effective in improving feed efficiency, weight gain, and reducing the frequency of diarrhea. When used alone, apramycin showed known effects on each test item. Surprisingly, the combination of both antibiotics showed greater improvement in each category than apramycin alone. As mentioned above, it was not previously known that tylosin was effective against coliosis in post-weaned pigs, so the superior effect of the tylosin-apramycin combination over apramycin alone was unexpected. Ta. The method of the invention comprises administering to a weaned pig a therapeutically effective amount of tylosin and apramycin. The total amount of both antibiotics that can be administered and their ratio can vary. The effective mixing ratio of antibiotics in animal feed will vary depending on factors such as severity of disease, age and body weight of the animal. The mixing ratio of both antibiotics in the feed is 50ppm to 1000ppm,
The weight ratio of tylosin and apramycin is 5:
The ratio is 1 to 1:5. Generally, the mixing ratio of both useful antibiotics in feed is 100ppm to 300ppm.
It is. The preferred mixing ratio is about 200 ppm and the weight ratio of both antibiotics is 1:1. Tylosin and apramycin are basic substances and form salts with mineral acids, carboxylic acids and sulfonic acids. For example, hydrochloride, sulfate, phosphate, tartrate, citrate, lauryl sulfonate, hexanoate, benzene sulfonate, etc. In the method of the invention, antibiotics can be used not only in free base form but also in salt form. Preferred salts of tylosin are tartrate and phosphate. Preferred salts of apramycin are sulfate and hydrochloride. Tylosin and apramycin may be commercialized by including both in a premix, or may be made into a premix for later addition to feed. Alternatively, the two antibiotics may be mixed directly into the final feed. Antibiotics in their basic form, like their salts, are fairly soluble in water. These antibiotics may be added to the animal's drinking water, preferably in salt form, or as solutions at therapeutically effective concentrations. As with feed, the concentration of antibiotics in drinking water can also vary. A concentration of both antibiotics between 10 mcg/ml and 500 mcg/ml and a weight ratio between antibiotics of 5:1 to 1:5 is generally sufficient to produce the desired effect on the animal. The preferred antibiotic concentration in drinking water is
50mcg/ml to 300mcg/ml. The antibiotic combination consisting of tylosin and apramycin is most practically administered by mixing it with the animal's feed or drinking water, but it is also effective when administered to pigs by injection. For injectable administration, both antibiotics are mixed in a suitable pharmaceutically acceptable diluent (e.g.
water, physiological saline, dextrose). Injectable tylosin-apramycin combination is used to treat weaned pigs that are too weak from disease to consume sufficient amounts of medicated food. In this type of case, the pigs are given daily injections of antibiotic combinations. Additionally, these antibiotics can be administered in appropriate oral dosage forms (e.g.
It can also be formulated into tablets, capsules, suspensions) and administered to pigs who cannot eat a sufficient amount of drug-added feed. The therapeutic and preventive effects of the tylosin-apramycin combination on coliform bacteriosis are demonstrated by the results of a 21-day study using weaned pigs. Depending on the weight of the pig, 8
Each head was divided into treatment and control groups. Each treatment was randomly assigned to three groups, with animals receiving normal chow ad libitum, while treatment groups received chow containing 110 ppm of each antibiotic. Weight gain, feed efficiency and frequency of diarrhea were observed as response to treatment. The results obtained after 21 days are shown in Table 5.

[Table] The attached chart is a graph plotting the average diarrhea score obtained in the above test against time (days). Plots were performed on the average scores of the control group and each treatment group. As shown by the mean score plot for the group treated with the tylosin-apramycin combination, diarrhea frequency was significantly lower than that in the group treated with apramycin alone. This graph also shows
Tylosin alone has been shown to be somewhat effective in reducing diarrhea frequency. To carry out the method of the invention, pigs are treated with the antibiotic combination from weaning period for 3-4 weeks after weaning. If the condition is severe, further long-term treatment can be provided if necessary. The values displayed in Table 4 above indicate the difference in diarrhea frequency between the treated group and the control group as a percentage of the control group. The tylosin-apramycin combination of the present invention is
Strains of Pasteurella haemolyteica (Pasteurella
haemolytica strains). This microorganism causes pneumonia in cattle, and severe infections can be fatal. The synergistic effect of tylosin-apramycin combination against P. hemolytica strain 41D
Proven in vitro tests. The minimum inhibitory concentration of tylosin alone was 50 mcg/ml and that of apramycin alone was 25 mcg/ml. The synergistic activity of the combination of two antibiotics was demonstrated by the so-called "Chetzkerboard" titration method. In this method, two antibiotics are diluted sequentially, each dilution is tested, and all combinations of dilutions are assayed to determine the concentration that inhibits the growth of each antibiotic alone and in combination. Ask about.
The interaction of both antibiotics can be determined algebraically or geometrically. The algebraic method determines the concentration of each antibiotic in the growth-inhibiting combination relative to the concentration when the antibiotic is used alone to achieve the same effect. If the sum of these ratios is 1, the combination is additive; if the sum is less than 1, the combination is synergistic; and if the sum is greater than 1, the combination is antagonistic. Become. Calculation results using values obtained in in vitro tests of tylosin, apramycin, and their combination against P. hemolytica are shown below. Tylosin alone MIC = 50 mcg/ml Apramycin alone MIC = 25 mcg/ml Tylosin + apramycin 25/50 + 0.8/25 = <1 12.5/50 + 6.25/25 = <1 6.25/50 + 12.5/25 = <1 Below A preferred combination of antibiotics is Tylosin-Tobramycin Tylosin-Apramycin Tylosin-Gentamycin Leucomycin-Tobramycin Spiromycin-Tobramycin Magnamycin-Tobramycin Particularly preferred combinations are Tylosin-Tobramycin, Tylosin-Apramycin and Tylosin-Gentamycin. As mentioned above, the antibiotic combination of the present invention is valuable as a veterinary drug in the treatment of various animal diseases. This combination is also valuable for controlling mycoplasma contamination when propagating viruses by tissue culture methods. The following examples further illustrate the invention. Example 1 In a tissue culture flask, 3-day-old Mycoplasma was added to 4 ml each of LLC-MK2 (monkey kidney cell line) cells suspended in medium 199 containing 1% horse serum.
hyorhinis (Eaton's medium, 5×10 ⁸ cells/ml). Approximately 2-3% mycoplasma culture was used relative to the volume of cell suspension. After inoculation, the cell suspension was cultured at 37°C for 3-5 days. A control medium was also prepared in the same manner. After incubation, the medium of control and mycoplasma-containing cultures was replaced with fresh medium 199 containing 1% horse serum and 1.68 g of sodium bicarbonate. The culture was then treated with a solution of tylosin, tobramycin, and tylosin-apramycin combination in medium 199 containing 1.68 g of sodium bicarbonate per portion. Solutions of each antibiotic and combination drug were prepared at a concentration of 1000 mcg/ml. The concentration of each antibiotic in the mixture solution was 1000mcg/ml. 2
Solutions with various concentrations up to 0.39 mcg/ml were prepared by the double dilution method. Each antibiotic solution was added to a separate flask containing tissue culture medium. Tissue culture medium was changed twice per week for each flask, and fresh antibiotic solution at the same concentration was added after each medium change for two weeks. Two weeks later,
Fresh medium changes were continued for an additional two weeks without the addition of antibiotics. Before each medium change, a sample of the culture was taken and cultured to determine the presence or absence of mycoplasma. The minimum inhibitory concentration of each antibiotic tylosin and tobramycin and their combination was the lowest concentration of antibiotic in tissue culture that did not successfully cultivate mycoplasma for the sample. The minimum inhibitory concentration was 100 mcg/ml for tylosin alone and 100 mcg/ml for tobramycin alone, whereas the minimum inhibitory concentration for the combination was 100 mcg/ml.
It was 3mcg/ml. Example 2 When tylosin, apramycin, and the tylosin-apramycin combination were subjected to a tissue culture contamination test with M. hyorhinisHH using the method and procedure described in Example 1, it was found that tylosin alone did not
MIC200mcg/ml, apramycin alone
The MIC was 600mcg/ml, and the MIC for the tylosin-apramycin combination was 12mcg/ml. Example 3 In the method of Example 1, using M. hyorhinis and replacing tobramycin with gentamicin,
When a tylosin-gentamicin combination was used in place of the tylosin-tobramycin combination, the MIC of tylosin alone was 400 mcg/ml. And with the tylosin-gentamicin combination,
It showed a MIC of 0.78mcg/ml. Example 4 Instead of the tylosin-tobramycin mixture used in Example 1, a leucomycin-tobramycin mixture was used. Leucomycin alone
The MIC for tobramycin alone was 400 mcg/ml, and the MIC for the combination was 200 mcg/ml.
MIC was 0.78mcg/ml. Example 5 96 weaned pigs with an average weight of 6.0 kg were randomly divided into 12 barns with 8 pigs per barn based on body weight. The following four treatments were applied to this. 1) Control without drug administration; 2) Apramycin 110ppm
Ingested by mixing with feed; 3) Tylosin phosphate
110ppm mixed into feed; 4) Both apramycin sulfate and tylosin phosphate
Ingested by mixing 110ppm into feed. Each treatment was replicated in 3 pens. Pigs were fed a 20% protein corn-soybean diet with some rolled oats ad libitum for 21 days. Added vitamins and minerals necessary for a balanced diet. All pig diets differed only by the addition of the appropriate antibiotic or antibiotic combination in the medicated section. All feeds were weighed before consumption, and remaining feed was weighed and discarded at the end of the weighing period. Pigs were weighed at the start of the study and weekly thereafter. Diarrhea scoring was done on a per-pen basis. The score is 14 if the same person is scheduled twice a day for the first 13 days.
It was conducted once on the day. The rating index is in note 2 of Table 5)
The same as shown in . Pigs in one extra barn were used for bacteriological investigation. These pigs were necropsied without being fed medicated feed when they developed diarrhea after being allocated to their respective pens. The number of beta hemolytic E. coli per ml of duodenal or ileal contents is
Measurements were made in necropsied animals. As a result, the tested 2
demonstrated the presence of 10 ⁷ and 10 ⁸ E. coli in the duodenum of pigs. Salmonella culture test results were negative. The results of this test are shown in Tables 4 and 5 for each of the following items: weight gain, reduction in diarrhea frequency, and improvement in feed efficiency.

[Brief explanation of drawings]

The figure is a graph showing the effects of apramycin, tylosin, and tylosin-apramycin combination on swine diarrhea.

Claims (1)

[Scope of Claims] 1 (a) a macrolide antibiotic selected from the group consisting of tylosin, leucomycin, magnamycin, spiromycin and oleandomycin and physiologically acceptable salts thereof; (b) and an aminoglycoside antibiotic selected from the group consisting of tobramycin, apramycin, nebramycin factor 5, gentamicin and neomycin and physiologically acceptable salts thereof. 2. The antibacterial composition according to claim 1, wherein the macrolide antibiotic is tylosin or a physiologically acceptable salt thereof. 3. The antibacterial composition according to claim 1 or 2, wherein the aminoglycoside antibiotic is tobramycin or a physiologically acceptable salt thereof. 4. The antibacterial composition according to claim 1 or 2, wherein the aminoglycoside antibiotic is apramycin or a physiologically acceptable salt thereof. 5. The antibacterial composition according to claim 1, comprising tylosin or a physiologically acceptable salt thereof and tobramycin or a physiologically acceptable salt thereof. 6 (a) a macrolide antibiotic selected from the group consisting of tylosin, leucomycin, magnamycin, spiromycin and oleandomycin and physiologically acceptable salts thereof; and (b) tobramycin, apramycin, nebramycin. Promoting the growth of pigs by administering to pigs an antibacterial composition comprising mycin factor 5, gentamicin and neomycin, and an aminoglycoside antibiotic selected from the group consisting of physiologically acceptable salts thereof. methods to improve feed intake. 7 (a) a macrolide antibiotic selected from the group consisting of tylosin, leucomycin, magnamycin, spiromycin and oleandomycin and physiologically acceptable salts thereof; and (b) tobramycin, apramycin, nebramycin. and an aminoglycoside antibiotic selected from the group consisting of mycin factor 5, gentamicin and neomycin and physiologically acceptable salts thereof. A method to suppress the growth of mycoplasma. 8 (a) a macrolide antibiotic selected from the group consisting of tylosin, leucomycin, magnamycin, spiromycin and oleandomycin and physiologically acceptable salts thereof; and (b) tobramycin, apramycin, nebramycin. and an aminoglycoside antibiotic selected from the group consisting of mycin factor 5, gentamicin and neomycin, and physiologically acceptable salts thereof. How to treat. 9. The method according to claim 8, wherein the antibacterial composition contains tylosin or a physiologically acceptable salt thereof and apramycin or a physiologically acceptable salt thereof. 10. The method according to claim 8, wherein the antibacterial composition contains tylosin phosphate and apramycin sulfate.