GB2103085A - Antimicrobial compositions containing primycin and doxycycline and/or sisomycin or a derivative thereof - Google Patents

Abstract

A pharmaceutical composition comprises (a) primycin or a derivative thereof; and (b) doxycycline or a derivative thereof and/or sisomicin or a derivative thereof; the ratio of components (a) and (b) being such that the composition exhibits a synergistic antimicrobial effect. The combination shows enhanced antimicrobial activity and is of interest for the therapy of infections, for example mastitis, caused by drug- resistant organisms.

Description

SPECIFICATION Synergistic antimicrobial compositions The present invention relates to new synergistic antimicrobial compositions.

Resistance of pathogens to the commercially available antimicrobials has increased to a great extent and thus several such substances, after a long period of effective use, cannot be used anymore.

In addition to the chromosomal resistance, the recently discovered "plasmid resistance" is responsible for the "resistance" phenomenon. The latter is the capability of pathogens to directly transfer their plasm it resistance to individuals belonging to the same species or even to other species.

Thus polyresistant pathogens may be produced within a short period.

Due to the above facts, the use of active ingredient combinations is becoming more important.

The interaction of simultaneously administered active ingredients-antagonism, synergism-has been known for a long time. Synergism means an enhanced effect of the combination relative to its components, and this effect is especially important in case of mixed infections.

The present invention concerns synergistic antimicrobial pharmaceutical compositions comprising (a) primycin or a derivative thereof; and (b) doxycyline or a derivative thereof and/or sisomicin or a derivative thereof; the ratio of components (a) and (b) being such that the composition exhibits a synergistic antimicrobial effect.

The composition may include a pharmaceutically acceptable carrier and optionally one or more adjuvants.

The synergistic pharmaceutical compositions according to the invention have the following advantages: 1. By attacking the metabolism of the pathogens simultaneously at several points a bactericidal effect may be better achieved and this effect is more advantageous than the bacteriostatic effect, which is a simple inhibition of the development of the micro-organisms.

2. By attacking several metabolic routes, the resistance against the active ingredient combination does not occur at all or occurs only after a long time.

3. As a consequence of the synergistic effect between the active components, the amount of the active ingredients may be greatly reduced, having the following advantages: a) During a long-term administration the toxicity of the components is greatly reduced, by reducing the effective amounts introduced in to the organism.

b) In some expensive compositions the reduced weight of active ingredients may have an economic advantage as well.

As further advantage one may mention that in case of some active ingredient combinations, not only are significantly lower MIC's observed, but in most cases the spectrum of activity is also broader.

Thus we have found that primycin and its derivatives [prepared e.g. from Thermomonospora galeriensis strain by fermentation (HU-PS 1 53 593)] in an amount e.g. of 5 to 50% by weight gives a synergistic effect with 95 to 50% by weight of sisomicin and/or doxycycline.

Due to the strong synergistic effect, the combinations according to the invention are effective and have a broad spectrum of activity and thus may be successfully employed against polyresistant strongly pathogenic microorganisms causing severe epidemics and diseases. Their use is particularly favourable in the case of mixed infections.

Compositions according to the invention may preferably contain primycin in the form of heterocolloidal primycin (GB-PS 1 512 604).

Compositions according to the invention e.g. contain as doxycycline derivative a doxycycline salt of a mineral acid, preferably doxycycline-hydrochloride or doxycycline-hyclate.

As a sisomicin derivative the compositions according to the invention may contain sisomicin substituted on the nitrogen atom by lower alkyl, hydroxy-lower alkyl, lower aminoalkyl or lower alkanoyl or a sisomicin salt of an acid. As sisomicin derivatives preferably N-methyl, N-hydroxyethyl, N acetyl-sisomicin or sisomicin-hydrochloride may be employed.

Doxycycline and its derivative mentioned above (GB-PS 845 649) and sisomicin and derivatives thereof mentioned above (US-PS 3907771 and HU-PS 170 513) are known compounds.

According to one preferred embodiment of the present invention, as active ingredients primycin and sisomicin are used. According to another feature of the invention, as active ingredients primycin and doxycycline are used. In the above combinations the derivatives of said antibiotics mentioned above may be employed as well.

A preferred composition according to the invention contains 30 to 35% primycin, 30 to 35% doxycycline or an equivalent amount of the hydrochloride thereof and 30 to 35% by weight of sisomicin related to the total active ingredient content.

The synergistic pharmaceutical compositions according to the invention may be formulated as solid compositions such as tablets, capsules, dragées, suppositories, semi-solid compositions such as ointments, or liquid compositions such as injectable solutions, suspensions or emulsions. As the most advantageous formulations gels, ointments, talcs, injectable solutions or suspensions, and powder ampoule-solvent-ampoule combinations may be mentioned. The compositions may be administered orally, parenterally rectally or topically e.g. ointments.

The pharmaceutical compositions may contain pharmaceutically acceptable carriers, such as magnesium carbonate, magnesium stearate, starch, talc, water etc. and optionally excipients, e.g.

fillers, disintegrating agents, lubricants, emulsifiers etc.

Orally administered compositions may be in form of tablets, capsules or dragées.

The synergistic compositions may be employed in veterinary therapy too, for example as a powder mixture added to the feed or as a solution mixed in the watering mixture of the animals.

The parenterally employed compositions may be e.g. aqueous solutions, emulsions or suspensions. For topical use one may employ ointments, aqueous or oily emulsions or suspensions or sprays. A parenterally employable composition may be prepared by filling sisomicin and doxycycline into powder ampoules, e.g. in the presence of buffer materials such as sodium acetate, and into solvent ampoules heterocolloidal primycin and surfactants, preferably quaternary ammonium salts such as cetyl trimethylammonium bromide are placed. The content of the solvent ampoule is injected into the powder ampoule directly prior to use. After dissolving, a well employable veterinary injectable composition is obtained.

A parenterally administrable composition may preferably be prepared by admixing an aqueous alcoholic solution of primycin with a suitable carrier (e.g. castor-oil) and by suspending sisomicin and/or doxycycline in the castor-oil mixture obtained after distilling off alcohol and after cooling.

The ointments may be prepared by distributing the active ingredient components homogeneously in a conventionally used ointment e.g. petrolatum.

The biological (in vitro) activity of the compositions according to the invention is demonstrated in the following Examples.

The following international resistant and/or polyresistant human pathogen and/or animal pathogen microorganisms were used in the course of the tests: 1. Vibrio parahaemolyticus CCM. 5938, 2. Pseudomonas acidovorans CCM. 283, 3. Proteus vulgaris CCM. 1 799, 4. Proteus mirabilis CCM. 1944, 5. Shigella sonnei CCM. 1373, 6. Salmonella typhimurium CCM. 5445, 7. Salmonella cholerae-suis CCM. 5435, (Inst. Pasteur Stamm), 8. Salmonella cholerae-suis CCM. 5874, 9. Salmonella cholerae-suis subsp. Kunzendoff. CCM. 5967, 10. Escherichia coli DSM. 30038, 11. Escherichia collATTC 11775, (cystitis, poultry pathogen), 1 2. Escherichia coli CCM 180, lysogenicus, colicinogenicus, 13.Escherichia coil, CCM. 5863, haemolyticus, 14. Klebsiella pneumoniae CCM. 1 848, 1 5. Serratia macerscens CCM. 303, 1 6. Staphylococcus aureus CCM. 885, 17. Staphylococcus aureus DSM. 20231, 18. Staphylococcus aureus CCM. 231 7, human mastitis, 19. Staphylococcus aureus CCM. 2326 human mastitis, 20. Staphylococcus aureus CCM. 251 5 beta haemylysis, 21. Staphylococcus aureus CCM. 2515 coagulase positive, 22. Streptococcus faecalis CCM. 885, 23. Streptococcus agalactiae CCM. 5153, from milk of cows having mastitis 24. Streptococcus agalactiae CCM. 5534, from milk of cows having mastitis, 25. Bacillus cereus CCM. 2010, 26. Listeria monocytogenis CCM. 5576.

ATCC=The American Type Culture Collection, CCM=Czechoslovak Collection of Microorganisms, DSM=Deutsche Sammlung fur Mikroorganismen.

Abbreviations: P=primycin sulphate S=sisomicin sulphate D=doxycycline sulphate yg/ml=microgramm/milliliter MlC=minimal inhibitory concentration O=no growth, complete inhibition of the microorganism ~=poor growth ++=moderate growth +++=strong growth, no inhibition.

The tests were carried out on DIFCO Bouillon medium, evaluation after incubation for 24 and 48 hours at 370C.

Table I to XIII show that the combinations of primycin [prepared from the ferment broth of Thermomonospora glariensis]--with sisomicin or doxycycline have a synergistic effect and the potentiating effect may increase the original activity by 30 to 40 times.

The pharmaceutical compositions according to the invention may contain other active ingredients such as chemotherapeutic agents as well.

Table I Primycin+Sisomicin Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus CCM. 2317 CCM. 2326 CCM. 2514 P S Incubation P S Incubation P S Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.1 0.1 0 0 0.5 0.5 0 0 0.5 0.5 0 0 0:0.75 0.1 0 0 0.25 0.25 0 0 0.25 0.25 0 0 0.075 0.075 0 0 0.1 0.25 0 0 0.1 0.25 0 0 0.05 0.1 0 0 0.075 0.25 0 0 0.075 0.25 0 0 0.05 0.075 0 0 0.1 0.1 0 +++ 0.1 0.1 0 +++ 0.025 0.075 0 0 0.1 0.075 0 +++ 0.1 0.075 0 +++ 0.05 0.05 0 + 0.075 0.1 0 +++ 0.075 0.1 0 +++ 0.025 0.05 0 + 0.075 0.075 0 +++ 0.075 0.075 + +++ 0.025 0.025 +++ +++ 0.05 0.01 +++ +++ 0.05 0.01 +++ +++ 0.01 0.01 +++ +++ 0.05 0.075 0 +++ 0.05 0.075 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.25 S=0.25 MIC : P=0.5, S=0.5 MIC: P=0.5.S=0.5 Table II Primycin+Sisomicin Staphylococcus aureus Streptococcus faecalis Streptococcus agalactiae DSM. 20231 CCM. 1875 CCM. 5534 P S Incubation P S Incubation P S Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.5 0.5 0 0 1 2.5 0 0 0.5 2.5 0 0 0.25 0.25 0 0 0.75 1 0 0 0.25 1 0 0 0.1 0.25 0 0 0.75 0.75 0 0 0.25 0.75 0 0 0.075 0.25 0 0 0.5 1 0 0 0.1 1 0 0 0.1 0.1 0 +++ 0.5 0.75 0 0 0.1 0.75 0 0 0.1 0.075 0 +++ 0.5 0.5 0 0 0.25 0.5 0 +++ 0.075 0.1 0 +++ 0.25 1 0 0 0.1 0.5 + +++ 0.075 0.075 0 +++ 0.25 0.75 0 +++ 0.1 0.25 +++ +++ 0.05 0.01 +++ +++ 0.25 0.5 +++ +++ 0.1 0.1 +++ +++ 0.05 0.075 +++ +++ 0.25 0.25 +++ +++ 0.075 0.5 + +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.5, S=0.5 MIC : P=1, S=2.5 MIC :P=0.5, S=2.5 Table III Primycin+Sisomicin Bacillus cereus Vibrio parahaemolyticus Proteus vulgarls CCM. 2010 CCM. 5938 CCM. 1799 P S Incubation P S Incubation P S Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.25 1 0 0 25 150 0 0 200 2.5 0 0 0.1 0.75 0 0 10 100 0 0 150 2.5 0 0 0.1 0.5 0 0 5 100 0 0 150 1 0 0 0.075 0.75 0 0 5 75 0 0 100 1 0 0 0.075 0.5 0 0 5 50 0 0 50 1 0 0 0.1 0.25 ++ +++ 2.5 100 0 0 50 0.75 0 0 0.1 0.1 +++ +++ 2.5 75 0 0 50 0.5 0 + 0.075 0.25 +++ +++ 2.5 50 0 0 25 1 + ++ 0.075 0.1 +++ +++ 2.5 25 ++ ++ 25 0.75 + ++ 0.05 0.1 +++ +++ 2.5 10 ++ ++ 25 0.25 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.25, S=1 MIC : P=25, S=150 MIC : P# 200, S=2.5 Table IV Primycin+Sisomicin Salmonella Cholerae-suis Salmonella Cholerae-suis subsp. Kunzendorf.Escherichia coll CCM. 5874 CCM. 5967 DSM. 30038 P S Incubation P S Incubation P S Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 75 2.5 0 0 200 2.5 0 0 200 2.5 0 0 50 1 0 0 150 2.5 0 0 150 2.5 0 0 50 0.75 0 0 100 1 0 0 150 1 0 0 25 1 0 0 50 1 0 0 100 1 0 0 25 0.75 0 0 50 0.75 0 +++ 50 1 0 0 10 1 0 0 50 0.5 0 +++ 50 0.75 0 0 25 0.5 0 + 25 1 0 # 50 0.5 0 0 10 0.75 0 ++ 25 0.75 0 +++ 25 1 # # 10 0.5 0 ++ 25 0.25 +++ +++ 25 0.75 # # 25 0.25 +++ +++ 25 0.25 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC: P=75, S=2.5 MIC: P=200, S=2.5 MIC :P=200, S=2.5 Table V Primycin+Doxycycline Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus CCM. 885 CCM. 2326 CCM. 2514 P D Incubation P D Incubation P D Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.5 0.25 0 0 0.5 0.5 0 0 0.05 1 0 0 0.25 0.1 0 0 0.225 0.25 0 0 0.25 0.75 0 0 0.25 0.075 0 0 0.25 0.25 0 0 0.25 0.5 0 0 0.1 0.1 0 0 0.25 0.25 0 0 0.25 0.25 0 0 0.075 0.1 0 0 0.1 0.1 0 0 0.1 0.5 0 0 0.1 0.075 0 0 0.1 0.075 0 0 0.1 0.25 0 0 0.075 0.075 0 ++ 0.1 0.1 0 0 0.1 0.1 0 0 0.075 0.05 0 +++ 0.075 0.075 0 0 0.075 0.5 0 0 0.05 0.075 # +++ 0.075 0.01 0 + 0.075 0.1 0 0 0.05 0.05 +++ +++ 0.05 0.075 +++ +++ 0.05 0.05 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.5, D=0.25 MIC :P=0.5, D=1 MIC:P=0.5, D=1 Table VI Primycin+Doxycycline Staphylococcus aureus Staphylococcus aureus Streptococcus agalactiae DSM. 20231 CCM. 2515 CCM. 5153 P D Incubation P D Incubation P D Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.5 0.5 0 0 0.25 0.25 0 0 0.5 0.5 0 0 0.25 0.25 0 0 0.1 0.1 0 0 0.25 0.25 0 0 0.1 0.25 0 0 0.1 0.075 0 0 0.1 0.25 0 0 0.075 0.25 0 0 0.075 0.1 0 0 0.075 0.25 0 0 0.1 0.1 0 + 0.075 0.075 0 0 0.1 0.1 0 + 0.075 0.1 0 +++ 0.075 0.05 0 +++ 0.075 0.1 0 + 0.075 0.075 + +++ 0.05 0.1 0 + 0.075 0.075 0 +++ 0.05 0.1 0 +++ 0.05 0.075 0 +++ 0.05 0.075 + +++ 0.05 0.075 +++ +++ 0.05 0.05 # +++ 0.05 0.05 ++ +++ 0.025 0.025 +++ +++ 0.01 0.01 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.5, D=0.5 MIC : P=0.25, D=0.25 MIC :P=0.5, D=0.5 Table VII Primycin+Doxycycline Bacillus cereus Listeria monocytogenes Salmonella cholerae-suis subsp.Kunzendorf.

CCM. 2010 CCM. 5576 CCM. 5967 P D Incubation P D Incubation P D Incubation g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h g./ml. g./ml. 24h 48h 0.25 1 0 0 0.25 0.25 0 0 200 2.5 0 0 0.1 0.75 0 0 0.1 0.1 0 0 150 1 0 0 0.1 0.5 0 0 0.1 0.075 0 0 100 1 0 0 0.1 0.25 0 0 0.075 0.1 0 0 100 0.75 0 0 0.075 0.75 0 0 0.075 0.075 0 0 50 1 0 + 0.075 0.5 0 0 0.075 0.05 0 0 50 0.75 0 + 0.05 0.5 0 0 0.05 0.1 0 0 50 0.5 + ++ 0.075 0.25 0 0 0.05 0.075 0 + 25 1 # ++ 0.075 0.1 0 ++ 0.05 0.05 0 + 25 0.5 +++ +++ 0.05 0.1 ++ +++ 0.025 0.025 +++ +++ 10 0.5 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC : P=0.25, D=1 MIC : P=0.25, D=0.25 MIC :P=200, D=2.5 Table VIII Primycin+Sisomicin+Doxycycline Staphylococcus aureus Staphylococcus aueus Staphylococcus aureus CCM. 2326 CCM. 5967 CCM. 2515 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h 0.1 0.1 0.25 0 0 0.25 0.25 0.25 0 0 0.1 0.1 0.1 0 0 0.1 0.1 0.1 0 0 0.25 0.1 0.25 0 0 0.1 0.075 0.1 0 0 0.1 0.075 0.1 0 0 0.25 0.1 0.1 0 0 0.1 0.075 0.075 0 0 0.11 0.075 0.075 0 0 0.1 0.1 0.1 0 0 0.075 0.075 0.075 0 0 0.075 0.075 0.075 0 0 0.1 0.075 0.1 0 0 0.075 0.05 0.075 0 0 0.05 0.075 0.075 0 0 0.1 0.075 0.075 0 0 0.05 0.05 0.05 0 0 0.05 0.05 0.075 0 0 0.075 0.075 0.075 0 +++ 0.05 0.05 0.05 0 0 0.05 0.05 0.05 0 0 0.05 0.05 0.05 # +++ 0.025 0.05 0.05 0 0 0.025 0.025 0.025 0 0 0.05 0.05 0.025 +++ +++ 0.025 0.025 0.05 +++ +++ 0.01 0.025 0.025 +++ +++ 0.05 0.025 0.025 +++ +++ 0.025 0.025 0.025 +++ +++ Control +++ +++ Control +++ +++ Contro +++ +++ MIC: P=0.5, S=0.5, D=1. MIC: P=0.5, S=0.25, D=0.25 MIC : P=0.25, S=0.25, D=0.25 Table XI Primycin+Sisomicin+Doxycycline Streptococcus agalactiae Bacillus cereus LIsteria monocytogenes CCM. 5153 CCM. 2010 CCM. 5576 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h 0.1 5 0.1 0 0 0.1 0.5 0.5 0 0 0.1 0.1 0.1 0 0 0.1 2.5 0.1 0 0 0.1 0.25 0.5 0 0 0.075 0.1 0.1 0 0 0.1 2.5 0.075 0 0 0.1 0.25 0.25 0 0 0.075 0.1 0.075 0 0 0.1 2.5 0.05 0 0 0.075 0.25 0.25 0 0 0.075 0.075 0.075 0 0 0.1 1 0.05 0 0 0.075 0.1 0.25 0 0 0.05 0.075 0.075 0 0 0.075 1 0.075 0 0 0.05 0.25 0.25 0 0 0.05 0.075 0.05 0 0 0.075 1 0.05 0 0 0.075 0.25 0.1 0 +++ 0.05 0.05 0.05 0 0 0.05 1 0.075 0 0 0.075 0.1 0.1 0 +++ 0.025 0.05 0.025 0 0 0.05 0.75 0.05 0 0 0.05 0.1 0.1 0 +++ 0.025 0.025 0.025 0 +++ 0.05 0.05 0.05 ++ +++ 0.05 0.075 0.075 ++ +++ 0.01 0.025 0.01 + +++ Control +++ +++ Control +++ +++ Contro +++ +++ MIC: P=0.5, S=50, D=0.5 MIC: P=0.25, S=1, D=1 MIC :P=0.25, S=0.75, D=0.25 Table X Primycin+Sisomicin+Doxycycline Vibrio parahaemolyticus Pseudomonas acidovorans Proteus mirabilis CCM. 5938 CCM. 283 CCM. 1944 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h 10 100 0.25 0 0 5 10 0.1 0 0 100 1 75 0 0 10 50 0.25 0 0 5 5 0.1 0 0 100 1 50 0 0 5 50 0.25 0 0 1 1 0.1 0 0 50 1 25 0 0 5 50 0.1 0 0 5 5 0.075 0 0 50 0.5 25 0 0 2.5 25 0.1 0 0 5 5 0.05 0 0 25 0.5 10 0 0 1 25 0.1 0 0 5 2.5 0.075 0 0 10 0.5 10 0 0 2.5 10 0.1 + ++ 2.5 2.5 0.075 0 0 10 0.5 5 0 +++ 2.5 10 0.075 -+ ++ 2.5 2.5 0.05 0 0 10 0.5 2.5 0 +++ 2.5 10 0.05 ++ ++ 2.5 2.5 0.025 0 0 5 0.25 2.5 +++ +++ 1 10 0.1 + ++ 1 1 0.01 ++ +++ 5 0.1 2.5 +++ +++ +++ +++ +++ +++ +++ +++ MIC: P=25, S=150, D=0.5 MIC: P=50, S=50, D=0.5 MIC :P= > 200, S=2.5, D=150 Table XI Primycin+Sisomicin+Doxycycline Shigella sonnei Salmonella typhimurium Salmonella cholerae-suis CCM. 1373 CCM. 5445 CCM. 5438 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h 100 1 1 0 0 100 0.5 2.5 0 0 50 0.5 2.5 0 0 50 1 1 0 0 50 0.5 2.5 0 0 25 0.5 2.5 0 0 25 1 1 0 0 25 0.5 2.5 0 0 25 0.25 1 0 0 10 0.5 1 0 0 25 0.25 2.5 0 0 10 0.25 1 0 0 10 0.5 0.5 0 0 10 0.1 1 0 0 5 0.25 1 0 0 10 0.5 0.25 0 0 5 0.1 2.5 0 0 5 0.1 1 0 0 5 0.5 0.5 0 0 10 0.075 2.5 0 +++ 1 0.1 1 0 0 5 0.25 0.5 0 0 5 0.075 2.5 0 +++ 2.5 0.1 0.75 0 0 5 0.1 0.5 0 0 10 0.075 1 +++ +++ 2.5 0.075 0.5 ++ +++ 5 0.1 0.25 +++ +++ 10 0.1 1 +++ +++ 2.5 0.05 0.5 +++ +++ Control +++ +++ Control +++ +++ Contro +++ +++ MIC: P=150, S=2.5, D=2.5 MIC: P=150, S=0.75, D=5 MIC :P=75, S=0.75, D=5 Table XII Primycin+Sisomicin+Doxycycline Salmonella cholera-suis Salmonella cholerae-suis Escherichia coli subsq. Kunzendorf CCM. 5874 CCM. 5967 DSM. 2515 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24h 48h 50 1 2.5 0 0 100 1 1 0 0 25 0.5 1 0 0 25 1 2.5 0 0 50 1 1 0 0 10 0.5 1 0 0 10 1 2.5 0 0 25 1 1 0 0 10 0.25 1 0 0 5 0.5 1 0 00 25 0.5 0.5 0 0 10 0.5 0.75 0 0 5 0.5 0.75 0 0 10 0.5 0.5 0 0 10 0.25 0.75 0 0 5 0.5 0.5 0 0 10 0.25 0.5 0 0 10 0.5 0.5 0 0 2.5 0.5 0.75 0 +++ 10 0.5 0.25 0 0 5 0.5 0.5 0 0 2.5 0.5 0.5 0 +++ 5 0.5 0.5 0 0 5 0.25 0.25 + +++ 2.5 0.25 0.5 +++ +++ 5 0.25 0.25 0 +++ 2.5 0.25 0.25 + +++ 2.5 0.25 0.25 +++ +++ 5 0.1 0.1 +++ +++ 1 0.25 0.25 +++ +++ Control +++ +++ Control +++ +++ Contro +++ +++ MIC: P=75, S=2.5, D=5.MIC: P=200, S=2.5, D=2.5 MIC : P=200, S=2.5, D=5 Table XIII Primycin+Sisomicin+Doxycyclne Escherichia coli Eschrichia coli Serratia Macrescens ATCC. 11775 CCM. 180 CCM. 303 P S D Incubation P S D Incubation P S D Incubation g./ml. g./ml. g./ml. 24h 48h g./ml. g./ml. g./ml. 24 48h g./ml. g./ml. g./ml. 24h 48h 25 0.5 2.5 0 0 10 0.25 1 0 0 100 5 10 0 0 10 0.5 2.5 0 0 5 0.25 1 0 0 50 5 10 0 0 10 0.5 1 0 0 5 0.1 1 0 0 25 5 10 0 0 10 0.25 1 0 0 5 0.1 0.75 0 0 25 2.5 10 0 0 5 0.5 1 0 0 2.5 0.1 1 0 0 10 2.5 10 0 0 5 0.25 1 0 0 2.5 0.1 0.75 0 0 10 2.5 5 0 0 5 0.25 0.75 0 0 2.5 0.1 0.5 0 0 5 2.5 5 0 + 1 0.25 0.5 0 + 1 0.1 0.75 0 0 5 1 5 0 ++ 1 0.1 0.5 0 +++ 1 0.075 0.75 0 0 5 1 2.5 + ++ 1 0.05 1 +++ +++ 1 0.05 0.5 +++ +++ 5 0.75 2.5 +++ +++ Control +++ +++ Control +++ +++ Control +++ +++ MIC:P= > 200, S=1, D=5 MIC : P =200, S=0.75, D=2.5 MIC :P=200, S=10, D=25 Further details of the invention are illustrated by the following Examples which serve merely for illustration and not for limitation.

Examples 1. I. Composition of a powder ampoule Sisomicin 0.10 g.

Doxycycline. HCI 0.10 g.

Sodium acetate 0.533 g.

II. Composition of a solvent ampoule Primycin heterocolloid 10.0 g.

(active ingredient content: 0.1 g.) cetyl trimethyl ammonium bromide 0.5 mg.

The composition is prepared as follows: Sisomicin, doxycycline and previously pulverized sodium acetate are homogenized, measured into a powder ampoule and sealed (I). Cetyl trimethyl ammonium bromide is dissolved in heterocolloidal primycin and filled into 10 ml. ampoules and sealed (II). Prior to use the content of the solvent ampoule is injected into a powder ampoule and after dissolution it is injected into udderquarters with a plastic needle.

The same composition may be formulated by conventional methods into a two-chamber syringe.

2. Sisomicin 0.10 g.

Doxycycline . HCI 0.10 g.

Primycin-heterocolloidal (active ingredient content: 0.1 g.) 10.00 g.

Sodium acetate 0.97 g.

cetyl-trimethyl-ammonium-bromide 0.5 mg.

The composition is prepared as follows: Sodium acetate is dissolved in primycin-heterocolloid, filled into powder ampoules and lyophilized conventionally, Sisomicin and doxycycline are added to the lyophilized product in dried state and the powder ampoule is sealed with a rubber stopper conventionally. The content of the powder ampoule is dissolved prior to use in 10 ml. of distilled water and injected into udder-quarters with a plastic needle or by other methods.

3. Primycin-heteroclloid (alcoholic) (active ingredient content: 0.1 g.) 10.00 g.

Castor-oil 10.00 g.

Cholesterol 0.25 g.

Sisomicin 0.10g.

Doxycycline. HCI 0.10g.

The composition is prepared as follows: Alcoholic solution of primycin-heterocolloid is mixed with castor-oil and the alcohol is distilled off, preferably in vacuo. Cholesterol is dissolved, preferably hot in the castor oil-primycin mixture. The dried sisomicin and doxycycline are suspended conventionally in the cooled mixture of castor oil and primycin.

The suspension thus obtained is filled into suitable plastic ampoules or an aluminium tube and equipped with a plastic needle most suitable for use.

4. 1. Composition of a powder ampoule Sisomicin 0.10g.

Doxycycline. HCI 0.10 g.

II. Composition of a solvent ampoule Primycin (active ingredient content: 0.1 g.) 10.0 g.

cetyl-trimethyl-ammonium-bromide 0.5 g.

disodium hydrogen phosphate 0.1716 g.

citric acid, cryst. 0.7596 g.

The composition is prepared as follows: Doxycycline and sisomicin are mixed together and sealed into an ampoule (I). Cetyl-trimethylammonium bromide, crystalline disodium hydrogen phosphate (Na2HPO4. 12 H20) and citric acid are dissolved in primycine-heterocolloid (C6H8O7 . H20), filled into ampoules and sealed (II).

5. Ointment Sisomicin 0.109.

Doxycycline. HCI 0.10 g.

Primycin 0.10 g.

Polyethyleneglycol 400 4.85 g.

Polyethyleneglycol 4000 4.85 g.

The composition is prepared as follows: Sisomicin, doxycycline and primycin pulverized under 50 u are used for the preparation of the ointment. Polyethyleneglycol 400 and polyethylene 4000 are homogenized and the active ingredients are mixed with a small part of the homogenized excipients and gradually mixed with the rest of the excipients. The mixture is then filled into tubes.

6. Aerosol filmformer Sisomicin 0.10 g.

Doxycycline. HCI 0.10 g.

Primycin 0.10g.

Polyvinylpyrrolidone 2.0 g.

Anhydrous ethanol 47.7 g.

Freon 11/12 5050 50.0 g.

The composition is prepared as follows: The dried and pulverized (under 50 y) Sisomicin, Doxycycline . HCI and Primycin are introduced into aerosol bottles whereafter the anhydrous ethanolic polyvinylpyrrolidone solution is added. The bottles are filled and sealed by methods known per se.

7. Aerosol talc Sisomicin 0.10 g.

Doxycycline. HCI 0.10 g.

Primycin 0.10g.

Isopropyl myristate 1.0 g.

Freon 11/12 5050 98.7 g.

The composition is prepared as follows: The previously dried and pulverized (Size < 50,u) sisomicin, doxycycline . HCI and primycin are homogenized and triturated with isopropylmyristate. Each dose is filled into aerosol bottles. The bottles are filled and sealed by method known peruse.

8. Comparative tests carried out with primycin and primycin-combinations on animals suffering from mastitis Primycin and primycin combination are examined on cows suffering clinically manifested mastitis. The tests were carried out by a thermographic method (cholesteric film set). As reference substances, active ingredients commercially available in Hungary, such as Neomaticur or Mastalone were employed.

Distribution of diseases: simple catarrhal mastitis acute contagious catarrhal mastitis chronic contagious catarrhal mastitis purulent mastitis with abscesses Treatment: 1. Primycin 100 mg/udder quarter 8 recovered out of 1 5 cases.

2. Primycin-combination Composition: I. powder ampoule sisomicin (dried) 85 mg.

doxycycline (dried) 100 mg.

sodium acetate (anhydrous) 533 mg.

prednisolone 10 mg.

ll. solvent ampoule primycin heterocolloid (active ingredient content: 0.1 g.) 20 ml.

cetyl trimethyl-ammoniumbromide 1.0 mg.

The primycin combination proved to be effective in 14 cases out of 1 6 cases. In the remaining one case the animal had to be sacrificed due to abscess-formation while the other case was a chronic contagious catarrhal mastitis.

The above results are confirmed by in vitro test-results.

Claims (14)

Claims

1. A pharmaceutical composition comprising (a) primycin or a derivative thereof; and (b) doxycycline or a derivative thereof and/or sisomicin or a derivative thereof; the ratio of components (a) and (b) being such that the composition exhibits a synergistic antimicrobial effect.

2. A composition as claimed in claim 1 wherein the mixture of components (a) and (b) comprises 5 to 50% by weight of (a) and 95 to 50% by weight of (b).

3. A composition as claimed in claim 1 or 2 comrising a doxycycline salt of a mineral acid.

4. A composition as claimed in claim 3 comprising doxycycline hydrochioride or doxycycline hyclate.

5. A composition as claimed in any of the preceding claims comprising as sisomicin derivative a sisomicin substituted on the nitrogen atom with lower alkyl, hydroxy(lower alkyl), lower aminoalkyl, lower alkanoyl, or a sisomicin salt formed with an acid.

6. A composition as claimed in claim 5 comprising as sisomicin derivative N-methyl, Nhydroxyethyl, N-acetyl-sisomicin or sisomicin-hydrochloride.

7. A composition as claimed in any of claims 1 to 6 comprising as components (a) and (b) primycin and sisomicin respectively, or derivatives thereof.

8. A composition as claimed in any of claims 1 to 6 comprising as components (a) and (b) primycin and doxycycline respectively, as derivatives thereof.

9. A composition according to any of claims 1 to 6 comprising primycin, sisomicin and doxycycline, or derivatives thereof.

10. A composition as claimed in claim 9 wherein the mixture of components (a) and (b) comprises 30 to 35% by weight of primycin, 30 to 35% by weight of doxycycline or an equivalent amount of the hydrochjoride thereof and 30 to 35% by weight of sisomicin.

11. A composition as claimed in any of the preceding claims wherein the primycin component is in the form of heterocolloidal primycin.

12. A composition as claimed in claim 11 for parenteral administration comprising heterocolloidal primycin and a surfactant in a first ampoule and component (b) in a second ampoule in the dry state, for dissolution in the contents of said first ampoule prior to use.

13. A composition as claimed in any of claims 1 to 11 including a pharmaceutically acceptable carrier and optionally one or more adjuvants.

14. A composition as claimed in claim 1 3 in the form of an ointment, talc or aerosol film former.

1 5. A composition as claimed in claim 13 in injectable form.

1 6. A composition as claimed in claim 1 5 in the form of an injectable solution, or suspension or a combination of powder and solvent ampoules.

1 7. A composition as claimed in claim 1 , substantially as described herein.

1 8. A composition as claimed in claim 1, substantially as described herein with reference to any one of Examples 1 to 7.

1 9. Mixtures of components (a) and (b) as defined in claim 1, for use as an antimicrobial agent for human or veterinary therapy.