CA1155765A - Synergistic antimicrobial compositions - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention provides synergistic pharmaceutical compositions containing from 5 to 50% of primycin or a derivative thereof and 95 to 50% of doxycycline or a derivative thereof or sisomicin or a derivative thereof, the percentages being by weight based on the total active ingredient content of the composition. The compositions have a broad spectrum of activity and may be successfully employed against polyresistant strong pathogen microorganisms causing severe epidemic diseases. The compositions are particularly effective against mixed infections.

Description

Synergistic antimicrobial compositions The present invention relates to new synergistic antimicro-bial compositions.
Resistance of pathogens against the commercially available active ingredients has increased to a great extent and thus several long-used effective active ingredients cannot be used anymore.
In addition to the cromosomal resistance the recently discovered "plasmid resistance" is responsible for the phenomenon "resistance". Latter is the capability of the pathogens of directly transferring the plasmid resistance thereof to individuals belonging to the same species or other species as well. Thus polyresistant pathogens may be produced within a short period.
Due to the above facts the use of active ingredient com-binations is becoming more important. The interaction sf simultane-ously administeret active ingredients - antagonism, synergism - has been known for a long time. Synergism means an increased effect of the combinations related to the components, and this effect is especially important in case of mixed infections.
The present invention refers to synergistic antimicro-bial pharmaceutical compositions by mixing 5 to 50 percents by weight of primycin or a derivative thereof with 95 to S0 per-cents by weight of doxycycline or a derivative thereof and/or sisomicin or a derivative thereof and with inert solid or liquid non-toxic pharmaceutically acceptable carriers and by formulating the mixture to a pharmaceutical composit-ion.
In particular, this invention provides a synergistic pharmaceutical composition containing 5 to 50% by weight of primycin or a derivative thereof and 95 to 50~ by weight of doxycycline or a deriYative thereof and/or sisomicin or a terivative thereof, the percentages being based on the total active ingred-ient content of the composition, and an inert, solid or liquid nontoxic pharma-ceutical carrier.
In a second aspect, this invention provides a process for the prepar-ation of a synergistic antimicrobial pharmaceutical composition which process comprises mixing together, relative to the total active ingredient content, 5 to 50% by weight of primycin, or a derivative thereof, and relative to the total active ingredient content, 95 to 50% by weight of doxycycline or a derivative thereof and/or sisomicin or derivative thereof with inert solid or liquid car-riers and formulating the obtained mixture to a pharmaceutical composition.
The synergistic pharmaceutical compositions according to the invention have the following advantages: -1. By attacking the metabolism of the pathogens simultaneously atseveral points a "cid" effect may be better achieved and this effect is more ad-~antageous than the "static" effect which is a simple inhibition of the develop-ment of the micro-organisms.

2. In case of attacking several metabolism routes the resistance against the use active ingredient combination does not occur at all or occurs only after a long ti~e.

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` 11~5765 ~ . As a consequer.ce of the synergistic effect between the active components the amount of the active ingredients may be reduced to a great extent, having the follo~ing advantages:
a) In case of a long-lasting administration the toxicity of the components is greati;i reduced by reducing the effective amounts introduced to the organism.

- 2a -1~55765 b) In case of some more expensive compositions the reduced active ingredient amounts may have economic advantage as well.
As further advantage one may mention that in case of some active ingredient combinations not only the extent of the minimal inhibitory concen-trations decreases significantly, but in most of the cases the spectrum of activity is also broader.
We have now found that primycin and its derivatives prepared from Thermomonospora galeriensis strain by fermentation (HU-PS 153 593) in an amount of 5 to 50 % by weight gives a synergistic effect with 95 to 50 % by weight of sisomycin and/or doxycyclin.
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 strong pathogen microorganisms causing severe epidemics and diseases. Their use is particularly favourable in 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 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 3 907 771 and 1 ~55765 HU-PS 170 513) are known compounds.
According to one preferred embodiment of the present invention as active ingredient 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 hydro-chloride thereof and 30 to 35 % by weight of sisomicin related to the total active ingredient content.
The synergistic pharmaceut1cal compositions according to the inven-tion may be formulated to solid compositions such as tablets, capsules, dragées, suppositories, semi-solid compositions such as ointments, or liquid composi-tions 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 accep-table 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 the veterinary therapy too, for example as a powder mixture mixed to the feed or as a solution mixed to the watering mixture of the animals.

The parenterally employed compositions may be in form of 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 in the presence of sodium acetate, and into solvent ampoules hetero-colloidal primycin and surfactants, preferably quaternary ammonium salt such as cetyl trimethylammonium bromide are placed. The content of the solvent ampoule is injected to 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 the aqueous alcoholic solution of pyrimycin 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. vaseline.
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. 1799,

4. Proteus mirabilis CCM. 1944,

5. Shigella sonnei CCM. 1373,

6. Salmonella typhimurim CCM. 5445,

7. Salmonella cholerae-suis CCM, 5438 (Inst. Pasteur Stamm), 1~5576~

8. Salmonella cholerae-suis CCM. 5874,

9. Salmonella cholerae-suis subsp. Kunzendorf. CCM. 5967,

10. Escherichia coli DSM. 30038,

11. Escherichia coli ATTC 11775, cyctitis, poultry pathogen,

12. Escherichia coli CCM. 180, lysogenicus, colicinogenicus,

13. Escherichia coli, CCM. 5863, haemolyticus,

14. Klebsiella pneumoniae CCM 1848,

15. Serratia Macerscens CCM. 303,

16. Staphylococcus aureus CCM. 885,

17. Staphylococcus aureus DSM. 20231,

18. Staphylococcus aureus CCM. 2317, human mastitis,

19. Staphylococcus aureus CCM. 2326 human mastitis,

20. Staphylococcus aureus CCM. 2515 beta haemylysis,

21. Staphylococcus aureus CCM. 2515 coagulase positive,

22. Streptococcus faecalis CCM. 885,

23. Streptococcus agalactiae CCM. 5153, from cow milk of cows having mastitis,

24. Streptococcus agalactiae CCM. 5534, from cow milk of cows having mastitis,

25. Bacillus cereus CCM. 2010,

26. Listeria monocytogenws 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 ~g./ml. = microgramm/milliliter MIC = 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 resp. at 37 C.
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 ori-ginal activity by 30 to 40 times.
The pharmaceutical compositions according to the invention may contain other active ingredients such as chemotherapeutic agents as well.

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Further details of the invention are illustrated by the following Examples which serve merely for illustration and not for limitation.
E-xamples 1. I,\Composition of a powder ampoule Sisomicin 0.10 g.
Doxycycline . HCl 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 to a powder ampoule and after dissolution it is pressed to udder-quaters with a plastic needle. The thus prepared composition may be finished by methods known per se into a two-chamber syringe.
2. Sisomicin 0.10 g.
Doxycycline . HCl 0.10 g.
Primycin-heterocolloidal10.00 g.
(active ingredient content:
0.1 g.) Sodium acetate 0.97 g.
cetil-trimethyl-ammonium- 0.5 mg.
bromide 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 as usual. The content of the powder ampoule is dis-solved prior to use in 10 ml. of distilled water and pressed to udder-quarters with a plastic needle or by other methods.
3. Primycin-heterocolloid (alcoholic) 10.00 g.
(active ingredient content: 0.1 g.) Castor-oil 10.00 g.
Cholesterol 0.25 g.
Sisomicin 0.10 g.
Doxycycline . HCl 0.10 g.
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 suitablè for use.

4. I.~Composition of a powder ampoule Sisomicin 0.10 g.
Doxycycline . HCl 0.10 g.
II. Composition of a solvent ampoule Primycin 10.0 g.
(active ingredient content: 0.1 g.) cetyl-trimethyl~ammonium- 0.5 g.
bromide 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-trimethyl-ammonium bromide, crystalline disodium hydrogen phosphate (Na2HPO4 . 12 H2O) and citric acid are dissolved in primycine-heterocolloid (C6H8O7. H20), filled to ampoules and sealed (II).
5. Ointment Sisomicin 0.10 g.
Doxycycline . HCl 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 - ~ 23 -,~

1 1557~5 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 . HCl 0.10 g.
Primycin 0.10 g.
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 /u) Sisomicin, Doxycycline . HCl 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 . HCl 0.10 g.
Primycin 0.10 g.
Isopropylmiristate 1.0 g.
~reon 11/12 5050 98.7 g.
The composition is prepared as follows:
The previously dried and pulverized (under 50 /u) sisomicin, doxycycline.HCl and primycin are ho genized and triturated with isopropylmiristate. Each dose is filled into aerosol bottles. The bottles are filled and sealed by method known per se.
8. Comparative tests carried out with primycin and primycin-combination on *Trade Mark ~r `` 1 155765 patients suffering of mastitis Primycin and primycin combination are examined on clinically manifested patients suffering of mastitis. The tests were carried out by thermographic method ~cholesteric film set).
As referential substances active ingredients commercially available in Hungary, such as Neomaticur, Mastalone were employed.
Distribution of diseases:
simple catarrhal mastitis acute contagious catarrhal mastitis cronic contagious catarrhal mast~tis purulent mastitis with abscesses Treatment:
1. Primycin 100 mg./udder quarter 8 recovered out of 15 cases 2. Primycin-combination Combination~ o_d_r_amp_ule sisomicin tdried) 85 mg.
doxycycline (dried) 100 mg.
sodium acetate (anhydrous) 533 mg.
Prednisolone 10 mg.
II. solvent amEoule _ _ _ _ _ _ _ _ _ primycin heterocolloid20 ml.
(active ingredient content:
0.1 g.) cetyl~trimethyl-ammoniumbromide 1.0 mg.

_ 25 -The primycin combination proved to be effective in 14 cases out of 16 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 (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A synergistic pharmaceutical composition containing 5 to 50% by weight of primycin or a derivative thereof and 95 to 50% by weight of doxycycline or a derivative thereof and/or sisomicin or a derivative thereof, the percentages being based on the total active ingredient content of the composition, and an inert, solid or liquid nontoxic pharmaceutical carrier.

2. A composition as claimed in claim 1 which further comprises one or more additional excipients.

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

4. A composition as claimed in claim 1 which is in injectable form.

5. A composition as claimed in claim 1 which is in the form of injectable solutions, or suspensions or combinations of powder-solvent ampoules.

6. A composition as claimed in claim 1 which contains a mineral acid salt of doxycycline as doxycycline derivative.

7. A composition as claimed in claim 1 which contains doxycycline hydro-chloride or doxycycline hyclate.

8. A composition as claimed in claim 1 which contains a sisomicin sub-stituted on the nitrogen atom with lower alkyl, hydroxy (lower alkyl), lower aminoalkyl, lower alkanoyl or a sisomicin salt formed with an acid.

9. A composition as claimed in claim 1 comprising as sisomicin derivative N-methyl, N- hydroxy-ethyl, N-acetyl-sisomicin or sisomicin-hydrochloride.

10. A composition as claimed in claim l comprising an active ingredient primycin and sisomicin.

11. A composition as claimed in claim l comprising as active ingredient primycin and doxycycline .

12. A composition according to claim l comprising as active ingredient primycin, sisomicin and doxycycline.

13. A composition as claimed in claim 1 comprising, related to the total active ingredient content, 30-35% by weight of primycin, 30% by weight of doxycycline or an equivalent amount of hydrochloride thereof and 30-35% by weight of sisomicin.

14. A composition according to claim 1 wherein the primycin component is present in the form of heterocolloidal primycin.

15. A process for the preparation of a synergistic antimicrobial pharmaceu-tical composition which process comprises mixing together, relative to the total active ingredient content, 5 to 50% by weight of primycin, or a derivative there-of, and relative to the total active ingredient content, 95 to 50% by weight of doxycycline or a derivative thereof and/or sisomicin or derivative thereof with inert solid or liquid carriers and formulating the obtained mixture to a pharmaceutical composition.

16. A process as claimed in claim 15 which further comprises mixing to-gether one or more excipients.