WO2009083521A2

WO2009083521A2 - Novel antibacterial compositions

Info

Publication number: WO2009083521A2
Application number: PCT/EP2008/068128
Authority: WO
Inventors: Pascal Butty; Laurent Mogenet; Brigitte Manco
Original assignee: Ceva Sante Animale
Priority date: 2007-12-21
Filing date: 2008-12-21
Publication date: 2009-07-09
Also published as: FR2925334A1; WO2009083521A3

Abstract

The present invention relates to novel antibacterial compositions comprising particular antibiotics and antiseptics in synergic combination. These novel compositions in particular find use as a therapeutic preparation in veterinary fields contributing to environmental clean-up.

Description

NEW ANTIBACTERIAL COMPOSITIONS

The present invention relates to novel antibacterial compositions comprising particular antibacterials and antiseptics in synergistic combination. These novel compositions find particular application as therapeutic preparations in veterinary fields and contribute to the cleansing of the environment.

These antibacterial compositions are particularly effective for the treatment of health disorders such as respiratory and digestive infections which are commonly observed in animal rearing. The etiological factors of these infections are bacteria from the external environment and / or the digestive tract of animals, which colonize the mucous membranes of the upper respiratory tract. Under breeding conditions, bacteria easily spread from one animal to another and can cause significant economic losses if respiratory infections are not treated in time.

In addition to sanitary prophylaxis measures and inactivated and adjuvanted vaccines, veterinarians and breeders use a whole range of antibiotic arsenals such as fluoroquinolones (enrofloxacin or marbofloxacin), cephalosporins (ceftiofur and cefquinome), tetracyclines (the oxytetracycline), macrolides (spiramycin) or penicillins (amoxicillin). However, the effectiveness of these prescriptions is often disappointing and does not allow to eradicate effectively and permanently the bacteria in the farms. In addition, the repeated use of these antibiotics in recent decades has led to the emergence of many resistances, rendering them ineffective in a number of cases. Moreover, the limitation of these acquired antimicrobial resistance phenomena has become an essential issue.

Unexpectedly, the Applicant has demonstrated a synergistic effect for particular combinations of antibiotics and antiseptics. Thus, the Applicant has shown that particular antiseptics such as natural aromatic alcohols of plant origin or biguanide polymers used in combination with certain antibiotics allowed to synergize the antibacterial activity of these antibiotics. The Applicant has also shown that the superior antibacterial properties of these combinations can advantageously be used to treat and / or prevent respiratory and / or digestive infections of non-human animal subjects. Thus, the subject of the present invention is novel compositions having a synergistic antibacterial activity comprising the combination of at least one antiseptic selected from natural aromatic alcohols of vegetable origin or biguanide polymers with at least one antibiotic such as a β-lactam, fosfomycin, an antibiotic glycopeptide, bacitracin, an antibiotic polypeptide, an aminoglycoside, a macrolide, a lincosamide, a streptogramin, a tetracycline, a phenicole, a fusidanine, or a quinolone.

The subject of the present invention is also a veterinary composition for the treatment of non-human animal subjects suffering from respiratory infections and / or digestive infections. Another object of the present invention is a kit for preventive and / or curative veterinary use comprising a therapeutically effective dose composition for obtaining an antibacterial synergistic effect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the kinetics over 24 hours of the antibacterial effect of florfenicol at concentrations of 4 to 32 μg / ml on an E. coli strain of serotype O ₇₈ K ₈₀ No. 05194 of avian origin (test preliminary).

FIG. 2 is a graph of the kinetics over 24 hours of the antibacterial effect of thymol at concentrations of 128 to 256 μg / ml on a strain of Eco // serotype O ₇₈ K ₈₀ # 05194 of avian origin (test preliminary).

FIG. 3 is a graph of the 24h kinetics of the antibacterial effects of florfenicol at 8 μg / ml, and thymol at 150 μg / ml, alone or in combination, on an Eco // strain of serotype O ₇₈ K ₈₀ n ° 05194 of avian origin (demonstration of synergistic effect). Figure 4: is a graph of the kinetics over 24h of the antibacterial effects of florfenicol to

8 μg / ml, and thymol at 170 μg / ml, alone or in combination, on a strain of Eco // serotype O ₇₈ K ₈₀ No. 05194 of avian origin (demonstration of the synergistic effect).

FIG. 5 is a graph of the kinetics over 24 hours of the antibacterial effects of florfenicol at 8 μg / ml, and thymol at 200 μg / ml, alone or in combination, on a strain of Eco // of serotype O ₇₈ K ₈₀ n ° 05194 of avian origin (demonstration of synergistic effect).

FIG. 6 is a graph of the kinetics over 24 hours of the antibacterial effects of florfenicol at 16 μg / ml, alone or in combination with thymol at various concentrations ranging from 8 to 256 μg / ml, on a serotype E. coli strain. O ₇₈ K ₈₀ No. 05194 of avian origin (preliminary test).

FIG. 7 is a graph of the 24h kinetics of the antibacterial effects of florfenicol at 32 μg / ml, alone or in combination with thymol at different concentrations ranging from 8 to 64 μg / ml, on a strain of E. coli άe serotype O ₇₈ K ₈₀ No. 05194 of avian origin (preliminary test).

FIG. 8 is a graph of the 24h kinetics of the antibacterial effects of florfenicol at concentrations of 4 μg / ml, 8 μg / ml, and 16 μg / ml on a non-typeable E. coli strain No. 05208 of origin avian (preliminary test). Figure 9: is a graph of the 24h kinetics of the antibacterial effects of florfenicol to

8 μg / ml and thymol at 150 μg / ml on a non-typeable E. coli strain No. 05208 of avian origin (demonstration of the synergistic effect).

FIG. 10: is a graph of the 24h kinetics of the antibacterial effects of florfenicol at 8 μg / ml and thymol at 170 μg / ml on a non-typeable E. coli strain No. 05208 of avian origin (demonstration of the synergistic effect).

Figure 11: is a graph of the kinetics of bactericide over 24 hours from fusidic acid to concentrations between 0.125 and 8 μg / ml with Staphylococcus aureus (ATCC 29213) (preliminary test).

Figure 12: is a graph of bactericidal kinetics over 24 h of PHMB at concentrations between 0.25 and 8 μg / ml with Staphylococcus aureus (ATCC 29213) (preliminary test).

FIG. 13: is a graph of the kinetics of bactericidal activity over 24 h of PHMB at concentrations of between 0.125 and 8 μg / ml in association with fusidic acid at a concentration of 0.125 μg / ml with Staphylococcus aureus (ATCC 29213) ( preliminary test).

FIG. 14 is a graph of the 24-hour bactericidal kinetics of fusidic acid at a concentration of 0.125 μg / ml and of PHMB at a concentration of 1 μg / ml or 2 μg / ml, alone or in combination with -vis Staphylococcus aureus (ATCC29213) (demonstration of the synergistic effect).

DETAILED DESCRIPTION

The compositions according to the present invention are particularly effective antibacterial compositions for the treatment and / or prevention of respiratory and / or digestive infections of non-human animal subjects. These compositions comprise a synergistic combination of at least one antiseptic chosen from a natural aromatic alcohol of vegetable origin or a biguanide polymer and at least one antibiotic such as a β-lactam, fosfomycin, an antibiotic glycopeptide, bacitracin, an antibiotic polypeptide, aminoglycoside, macrolide, lincosamide, streptogramin, tetracycline, phenicole, fusidanine, or quinolone.

By antibacterial or antibiotic properties is meant not only the bactericidal property characterized by the destruction of bacteria including mycoplasmas, but also the bacteriostatic property, characterized by the inhibition of the growth of said bacteria including mycoplasmas. The antiseptics used in synergistic combination according to the present invention may be chosen from natural antiseptic aromatic alcohols of vegetable origin or antiseptics derived from biguanide polymers.

Among the naturally occurring antiseptic aromatic alcohols of plant origin which can be used in synergistic combination with the antibiotics according to the present invention, mention may be made of thymol, carvacrol, eugenol, guaiacol, and derivatives.

Thymol, which is also designated 5-methyl-2- (1-methylethyl) phenol, is obtained from the essential oils of plants Thymus vulgaris L. and Monarda punctata L. It has been described by J.

M. Schaffer, F. W. Tilley, J. Bacteriol. 14, 259 (1927); by W. W. Richards, K. J. Hawley, J. Chem. Educ.

16, 6 (1939); by H. B. Myers, J. Am. Med. Assoc. 89, 1834 (1927); by Mr. Dersarkissian, Mr. Goodberry, Stud. Conserv. 25, 28 (1980), and in US Pat. No. 2,840,616 dating from 1958.

Carvacrol can also be used in the compositions according to the present invention, it corresponds to 2-methyl-5- (1-methylethyl) phenol and therefore has a chemical formula very close to that of thymol. It is obtained from the oils of oregano, thyme, and marjoram. Carvacrol has been described by E. Guenther, The Essential OiIs vol. 2 (Van Nostrand, New York, 1949) p 503; by Carpenter et al., Org. Chem. 20, 401 (1955); by Ritter et al., J. Am. Chem. Soc. 72, 2381 (1950); by Strubell et al., Arch. Pharm. 291, 66 (1958); by Kochmann et al., Arch. Exp. Pathol. Pharmakol. 161, 196 (1931). Eugenol is a phenol that makes up the bulk of the essence of clove. Its chemical name is 2-methoxy-4- (2-propenyl) phenol. It has been described by Priester, Rec. Trav. Chim. 48, 1272 (1929), and by Hagan et al., Toxicol. Appl. Pharmacol. 7, 18 (1965).

Guaiacol is isolated from guaiac wood and has the chemical name 2-methoxyphenol. It also has a variety of names, such as methylcatechol, o-hydroxyanisole or 1-hydroxy-2-methoxybenzene. This compound and the synthetic routes have been described by McClure et al., Org. Chem. 27, 627 (1962); by Taylor et al., Toxicol. Appl. Pharmacol. 6, 378 (1964); and in US Patent 3,057,927 of 1962 on behalf of the Foundation Ontario Research, and DE 1 148236 in the name of Hoechst.

Among the biguanide polymer antiseptics, mention may in particular be made of hexamethylene biguanide polymer (PHMB), which is also designated polyaminopropyl biguanide (PAPB) or polyhexanide and is commonly used as a preservative in contact lens cleaning solutions. The exact chemical nomenclature is poly (iminocarbonimidoylimino carbonimidoylimino-1,6-hexanediyl) hydrochloride. It has been described in US Pat. No. 4,758,595 and G.C. East et al., Polymer 38, 3973 (1997); P. Broxton et al., J. Appl. Bacteriol. 57, 1 (1984); by T. Ikeda et al., Bull. Chem. Soc. Jpn. 58, 705 (1985); by W. Khunkitti et al., J. Appl. Microbiol. 82, 107 (1997); and by T. Hattori et al., Anal. Sci. 19, 1525 (2003).

The antibiotics used in synergistic combination according to the present invention may be chosen from the group consisting of β-lactams, fosfomycin, glycopeptides or polypeptides with antibiotic activity, bacitracin, aminoglycosides, macrolides, lincosamides, streptogramins , tetracyclines, phenicolés, fusidanides, or quinolones.

Β-lactams include, for example, amoxicillin, ampicillin, piperacillin, cefpodoxime, cefotaxime, cefoperazone, ceftriaxone, cefixime, cefuroxime, and cefaclor.

Fosfomycin, whose chemical name is (2R-c / s) - (3-methyloxiranyl) phosphonic acid, has been described inter alia by Shogi et al., J. Antibiot. 39, 101 (1986); by Kahan et al., Ann. N.Y. Acad. Sci. 235, 354 (1974); by Glamkowski et al., J. Org. Chem. 35, 3510 (1970), as well as in the US Pat.

3,914,231 from Merck & Co.

Among the polypeptides, mention may be made of cyclic polypeptides such as, in particular, colistin, also known as polymyxin E. Colistin consists of a complex mixture of several components, mostly cyclic polypeptides colistin A and colistin B. This polypeptide originally described in JP 52 1546 of 1952, and by M. Barnett et al., Br. J. Pharmacol. Chemother. 23, 552 (1964); by B. Cancho-Grande et al., Chromatagraphia 54, 481 (2001); by J. Horton, GA Pankey, Med. Clin. North Am. 66, 135 (1982); by ME Evans et al, Ann. Pharmacother. 33, 960 (1999); and by J. Li et al., Int. J. Antimicrob. Ag. 25, 11 (2005).

Also, there may be mentioned bacitracin, a cyclic polypeptide isolated from Bacillus subtilis sold under the name Baciim by the company Pharma-Tek, and described in US Patents

2,915,432, US 2,498,165, and US 2,828,246, as well as Galardy et al., Biochemistry 10, 2429 (1971); by Storm, Ann. N.Y. Acad. Sci. 235, 387 (1974); by Craig et al., "Bacitracin" in G. E. W. Wolstenholme,

C. O'Connor, Ciba Foundation Symposium on Amino Acids and Peptides with Antimetabolic Activity

(Little, Brown, Boston, 1958) pp 226-246; and by D. R. Storm, W.A. Toscano, Jr. in Antibiotics vol. 5, pt. 1,

F. E. Hahn, Ed. (Springer-Verlag, New York, 1979) pp. 1-17. Bacitracin derivatives can also be used such as methylene disalicylate bacitracin or bacitracin zinc. Among the glycopeptide antibiotics, mention may be made of vancomycin, teicoplanin, ristocetin, or avorpacin.

The aminoglycoside class includes amikacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, streptomycin, and tobramycin.

Among the macrolides, mention may be made of erythromycin, spiramycin, ansamycin, oleandomycin, carbomycin and tylosin. Ketolides such as telithromycin can also be used.

Lincosamides such as lincomycin and clindamycin,

The so-called first-generation tetracyclines such as, for example, tetracycline and oxytracycline, or of the second generation, such as, for example, doxycycline and minocycline may also be used.

The class of streptogramins includes pristinamycin, quinupristin or dalfopristin.

Also, quinolones include nalidixic acid derivatives, such as enrofloxacin or marbofloxaxine may be used.

Among the phenicolated antibiotics, mention may be made of thiamphenicol, florfenicol, chloramphenicol, as well as their derivatives, or a combination of these compounds. These are well known in human and veterinary medicine. These antibiotics work by blocking the protein synthesis of bacteria including mycoplasma.

Chloramphenicol, the chemical designation of which is 2-dichloro-N - [(1H-dihydroxy-1- (hydroxymethyl) -2- (4-nitrophenyl) ethyl] acetamide, is well known in antibiotic therapy. known by other designations such as Df /? reo-Λ / -dichloroacetyl-1-p-nitrophenyl-2-amino-1,3-propanediol; D (-) - f /? reo-2-dichloroacetamido; 1-p-nitrophenyl-1,3-propanediol or D-theo-N- (1,1'-dihydroxy-1-p-nitrophenylisopropyl) dichloroacetamide Chloramphenicol has been described inter alia by Bartz, J. (Biol Chem 172, 445 (1948)), by Gottlieb et al., (J. Bacteriol 55, 409 (1948)), by Rebstock et al., (J. Am Chem Soc 71, 2458). (1949)), and US Pat. Nos. 2,483,871, 2,483,884, 2,483,892, and 2,839,577, and Hahn in Antibiotics, vol 1, D. Gottlieb, PD Shaw, Eds (Springer-Verlag, New York, 1967) pp. 308-330, chloramphenicol derivatives, chloramphenicol pantothenate, chloramphenicol tate, chloramphenicol arginine succinate, or chloramphenicol sodium succinate known as globenicol and kemicetine marketed respectively by the companies Yamanouchi and Pharmacia.

Thiamphenicol is the methyl sulfonyl analogue of chloramphenicol. It corresponds to 2,2-dichloro-Λ / - [(1 f, 2 ^/??) - 2-hyclroxy-1 - (hyclroxyméthyl) -2- [4- (methylsulfonyl) phenyl] ethyl] acetamide, or it is designated D-difluoro-2-dichloroacetamido-1- (4-methylsulfonyl) phenyl-1,3-propanediol; or dextrosulphenidol. It is known by several other names such as, for example, thiophenicol or dextrosulphenidol, and is described inter alia by Cutler et al., (J. Am., Chem., Soc., 74, 5475 (1952)); and in US Patents 2,759,927; US 2,759,970; US 2,759,971; US 2,759,972; and US 2,759,976.

Florfenicol is a fluorinated derivative of thiamphenicol. It is designated fluorothiamphenicol and corresponds to 2,2-dichloro-Λ / - [(1S, 2R) -1- (fluoromethyl) -2-hydroxy-2- [4- (methylsulfonyl) phenyl] ethyl] acetamide or D- (3-fluo-1-p-methylsulfonylphenyl-2-amino-3-fluoro-1-propanol and its preparation method is further described in US Patents 5,082,863; US 4,235,892 as well as in Budavari;

S., Merck Index. An Encyclopedia of Chemicals, Drugs, and Biologicals, 12th ed. Whitehouse Station, NJ:

Merck Research Laboratories; 1996. p.4146; and in Sams RA. Florfenicol: Chemistry; as well as D. P. Schumacher et al., J. Org. Chem. 55, 5291 (1990).

When the phenolic antibiotics of the combinations according to the invention are in the form of salts of a mineral or organic acid, it is more particularly palmitates, pantothenates, stearates, or succinates.

Among the antibiotics of the fusidanin family, mention may be made of fusidic acid, salts and derivatives such as sodium fusidate. Fusidic acid corresponds to the acidic chemical designation (3α, 4α, 8α, 9β, 11α, 13α, 14β, 16β, 17Z) -16- (acetyloxy) -3,1 1 -dihydroxy-29-nordammara-17 (20), 24-dien-21-oic. It is marketed under the name among other fucithalmic or fucidine by the Leo Pharma Company. It was originally isolated from the fermentation medium of Fusidium coccineum. Fusidic acid and synthetic processes have been described by D. Arigoni et al., Experientia 19, 521 (1963); by W. G. Dauben et al., J. Am. Chem. Soc. 94, 8593 (1972); by Tanabe et al., Tetrahedron

Lett. 17: 1481 (1977); by W. von Daehne et al., Adv. Appl. Microbiol. 25, 95 (1979); and by D. Dobie, J. Gray, Arch. Dis. Child. 89, 74 (2004).

Any other antibiotic or derivative may be used in the combinations according to the present invention insofar as it makes it possible to obtain an antibacterial synergistic effect when it is used in combination with at least one natural antiseptic aromatic alcohol of plant origin or in combination with a biguanide polymer antiseptic. This can be easily determined by those skilled in the art as described in the examples below.

When the antiseptic is selected from natural aromatic alcohols of plant origin in the synergistic compositions according to the invention, these then comprise the combination of these alcohols with an antibiotic such as a β-lactam, fosfomycin, a antibiotic glycopeptide, bacitracin, an antibiotic polypeptide, an aminoglycoside, a macrolide, a lincosamide, a streptogramin, a tetracycline, a phenicole, a fusidanine, or a quinolone, and preferably chosen among the phenicolés. Alternatively, when the chosen antiseptic is a biguanide polymer such as hexamethylene biguanide polymer, the latter is used in combination with at least one antibiotic such as a β-lactam, fosfomycin, an antibiotic glycopeptide, bacitracin, an antibiotic polypeptide, aminoglycoside, macrolide, lincosamide, streptogramin, tetracycline, phenicole, fusidanine, or quinolone.

As indicated previously, the compositions according to the invention have an antibacterial effect against a broad spectrum Gram bacteria ^"and / or Gram ⁺ and also of pathogenic bacteria wall devoid such as mycoplasma. They are among other particularly effective for the destruction of Gram bacteria ^" ie Enterobacteriaceae, such as bacteria belonging to the genera Salmonella, Shigella, and Escherichia, as well as Pasteurellaceae, such as Pasteurella, and also Haemophilus, Actinobacillus, and pathogenic mycoplasmas, and this without regrowth germs. These compositions may also be effective against certain antibiotic-resistant bacteria. Moreover, it has been discovered that these compositions, because of their synergistic antibacterial activity, are particularly effective for curative and / or preventive use in non-human animal subjects infected and / or at risk of contamination by one of these agents. bacteria and / or mycoplasma, as well as the symptoms of respiratory and digestive infections.

Preferably, the active agents used in the synergistic combinations according to the invention are on the one hand a phenolic antibiotic and a natural aromatic alcohol of vegetable origin, such as florfenicol and thymol, and on the other hand the polymer of hexamethylene biguanide and fusidic acid or any other combinations of the hexamethylene biguanide polymer with an antibiotic such as β-lactam, fosfomycin, an antibiotic glycopeptide, bacitracin, an antibiotic polypeptide, an aminoglycoside, a macrolide, a lincosamide , streptogramin, tetracycline, phenicole, fusidanine, or quinolone.

According to a preferred embodiment, the compositions according to the present invention comprise synergistic combinations of at least one antiseptic chosen from a natural aromatic alcohol of vegetable origin or a biguanide polymer and at least one phenolic antibiotic. Even more preferably, the phenolic antibiotic is florfenicol.

These are present in effective amounts to achieve a synergistic antibacterial effect in vivo. The amounts of the active agents can easily be determined by those skilled in the art. "Effective amount" means an amount of the compounds or the composition according to the invention, sufficient to allow the control and / or destruction of the aforementioned pathogenic bacterial agents, by a synergistic antibacterial effect at the level of the respiratory system and / or digestive system of treated non-human animals. Such an amount is likely to vary according to the pathogens, the animals to be treated, the rearing conditions, the mode of administration of the product and the stage of the infections. These amounts can easily be determined by systematic tests based on the examples below within the abilities of those skilled in the art.

The veterinary antibacterial compositions according to the invention may further comprise aforementioned synergistic combinations, a physiologically acceptable solvent and / or an excipient also physiologically acceptable. In general, these compositions are also called drugs.

Physiologically acceptable means a solvent or an excipient which is not harmful to animals intended to receive the composition according to the invention.

By way of examples, mention may be made of organic solvents such as N-2-methylpyrrolidone, 2-pyrrolidone, N, 5-dimethyl-2-pyrrolidone, 3,3-dimethyl-2-pyrrolidone, N ethyl-2-pyrrolidone, 1-pyrrolidone, diethylene glycol monoethyl ether (marketed under the name Transcutol®), dimethylacetamide (DMAC), polyethylene glycol, propylene glycol, glycerine, water, benzoate benzyl, isopropyl alcohol, xylenes, or a combination of these solvents.

The antibacterial compositions according to the invention may furthermore contain numerous other ingredients such as, for example, flavorings, protective colloids, adhesives, thickeners, thixotropic agents, penetration agents, stabilizers, sequestering agents, antioxidants, solubilizing agents, fluidifying agents, complexing agents, preservatives, or buffering agents. More generally, the active ingredients can be combined with any solid or liquid additives corresponding to the usual techniques of formulation.

Examples of antioxidants include, but are not limited to, sodium metabisulfite, sodium formaldehyde sulfoxylate, vitamin E acetate, vitamin C, vitamin B ₁₂ , or a combination thereof. Non-limiting solubilizing agents include crosslinked polypyrrolidone such as povidone C-15 (having 15 monomers), magnesium oxide, calcium oxide, or a combination thereof.

As examples of fluidifying agents, mention may be made of mucosal-specific fluidifiers of the respiratory tract such as bromhexine. Examples of preservatives include, but are not limited to, benzyl alcohol, parabens, such as methyl, ethyl, propyl, or butylparaben, chlorobutanol, sodium benzoate, benzoic acid, myristyl-gamma-picolinium chloride, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, chlorocresol, cresol, dehydroacetic acid, phenol, phenylethyl alcohol, potassium benzoate, sorbate potassium, sodium propionate, sorbic acid, or combinations of these agents.

The proportions and nature of the various elements used in the veterinary compositions according to the invention are determined according to the mode of administration chosen and are governed by the standard practices well known to those skilled in the art of pharmaceutical products and veterinarians. The compositions for veterinary use may be used in the various galenic forms suitable for administration in animals. The modes of administration and galenical forms used preferably allow the associations to have an action at the level of the tree respiratory tract and / or deep parts of the respiratory tract that may be infected by pathogenic mycoplasmas. Also, these combinations can be administered preferably so as to have action in the digestive tract and / or systemic absorption.

The veterinary compositions according to the invention may be administered, for example, orally, in particular by oral or nasal absorption, or by direct or indirect nebulization. All other modes of administration likely to reach the upper airways, the upper part of the digestive tract, or the digestive tract of the animals can be used according to the scenarios. Alternatively, the compositions may be administered parenterally, by intramuscular, subcutaneous or intraperitoneal injection. The present invention relates to the production of a medicament containing at least two active ingredients in synergistic combination, an antiseptic and an antibiotic, as previously described or in the form of a veterinary composition in combination with one or more compatible diluents or adjuvants. These compositions may be in liquid, solid, semi-solid or aerosol form. Liquid compositions are for example solutions, suspensions, emulsions, syrups or elixirs. Such liquid compositions include an inert diluent such as water, ethanol, glycerol, vegetable oils or paraffin oil. They may also include substances other than diluents, for example wetting, sweetening or flavoring substances.

The compositions according to the present invention may also be mixed with animal feed or diluted in the drinking water with a sufficient amount of at least two of the active ingredients of the previously described combinations. They can then be in the form of water-soluble powders to be mixed with the feed or in the form of concentrated liquid solutions that can be diluted in the animal's drinking water. The veterinary antibacterial liquid compositions retain a homogeneous and clear appearance without degradation or formation of precipitate for several months.

They comprise at least one antibiotic in combination with at least one antiseptic and an organic solvent, as previously described. According to a preferred embodiment, the compositions are also miscible in water and can therefore be diluted in animal drinking water and maintain a homogeneous and stable appearance before ingestion by the animals. These drinkable compositions may comprise, for example, therapeutically effective amounts of florfenicol and thymol in solution in the organic solvent and more specifically aprotic polar solvent, such as pyrrolidone type solvents, chosen from 2-pyrrolidone, N-methyl- 2-pyrrolidone, Nτ-5-dimethyl-2-pyrrolidone, 3,3-dimethyl-2-pyrrolidone, N-ethyl-2-pyrrolidone, and 1-pyrrolidone, dimethylacetamide (DMAC), dimethyl sulfoxide (DMSO), or ethyl acetate, a solubilizing agent such as glycerol ricinoleate in a proportion of between 20 and 80%, or between 20 and 60%, preferably about 25%, by weight relative to to the total weight of the composition, and optionally a cosolvent, such as light chain macrogols or PEG (PEG200, PEG300, or PEG400) in a proportion of between 0 and 70% by weight relative to the total weight of the composition. Preferably, these compositions comprise an effective amount of florfenicol and thymol, a proportion of aprotic polar solvent as previously described, between 0.degree. and 50%, and glycerol ricinoleate, as a solubilizing agent, in a proportion of between 20 and 80%, or between 20 and 60% and preferably about 25% by weight relative to the total weight of the composition.

They may also include a flavor which makes it possible to mask the taste, for example of certain antiseptics, in particular natural aromatic alcohols of vegetable origin, and thus to increase the palatability of the veterinary compositions in oral form.

According to another preferred embodiment, the compositions according to the invention can also take the form of aerosols. The active compounds of the synergistic combinations are then dissolved or dispersed using inert solvents in the form of pressurized solutions which, when sprayed, give aerosols which are particularly suitable for treating respiratory infections. The compositions are thus sprayed or nebulized directly on the animals and come to cover, after inhalation, the mucous membranes of the upper airways of animals, such as nasal, intranasal, and / or oral mucosa. Compositions for use in the form of liquid aerosols may be stable sterile solutions or solid compositions dissolved at the time of use in sterile pyrogen-free water, serum or any other pharmaceutically acceptable carrier. The aerosols may also be dry aerosols intended to be directly inhaled, in which the finely divided active ingredient is combined with a water-soluble solid diluent or carrier such as, for example, dextran, mannitol or lactose.

Solid compositions are for example tablets, pills, powders or granules. Semi-solid compositions are, for example, pastes or gels. Such solid or semi-solid compositions comprise an inert carrier such as, for example, starch, cellulose, sucrose, lactose or silica. These compositions may comprise substances other than diluents, for example a lubricant such as magnesium stearate.

Alternatively, the compositions according to the invention may be in a form suitable for parenteral administration. These may be aqueous or non-aqueous sterile solutions, suspensions or emulsions, and comprise at least one physiologically acceptable diluting agent such as, for example, water, saline solution, dextrose, a solubilizing agent, such as for example polyethylene glycol, or polypropylene glycol, or other conventional diluents known to those skilled in the art. Such compositions are generally sterile. Sterilization can be done in several ways, for example by ionizing filtration, by incorporation of sterilizing agents, by irradiation or by heating.

The veterinary antibacterial compositions as previously described can be used for the treatment of non-human animal subjects suffering from respiratory and / or digestive infections. These may be, for example, livestock or pets, such as for example, cattle, sheep, goats, pigs, rabbits, poultry, horses, camelids, dogs and cats.

The compositions according to the invention then comprise therapeutically effective doses of at least one natural antiseptic aromatic alcohol of vegetable origin or a biguanide polymer antiseptic with at least one antibiotic as previously described in order to obtain a local synergistic effect at level of the respiratory and / or digestive system according to the route of administration.

The compositions may further comprise a therapeutic agent, a pest control agent, an antiviral agent, an anti-inflammatory agent, an anthelmintic agent, an NSAID compound, or a macrolide antibiotic, such as avermectins and milbemycins, including ivermectin. , doramectin, milbemycin D, moxidectin, selamectin, abamectin, and eprinomectin. Preferably, the therapeutic agent is a bronchial fluidifier such as bromhexine.

Preferably, the compositions according to the invention comprise effective amounts of florfenicol, thymol and bromhexine, a proportion of polar aprotic solvent of between 20 and

50% by weight relative to the total weight of the composition and a proportion of glycerol ricinoleate as a solubilizing agent, between 20 and 80%, or between 20 and 60% by weight relative to the total weight of the composition.

In general, in animal therapy, the veterinarian or breeder can determine the dosage he considers the most appropriate based on a preventive treatment or curative, depending on age, the weight of the animal , the degree of infection and other factors specific to the subject to be treated, it being understood that the dose suitable for producing an effect may vary within fairly wide limits. The invention also relates to the use of synergistic compositions as described above for the preparation of a medicament for the treatment and / or prevention of respiratory and / or digestive infections, in particular those caused by Enterobacteriaceae, such as bacteria belonging to the genera Salmonella, Shigella, and Escherichia, as well as Pasteurellaceae, such as Pasteurella, and also Haemophilus, Actinobacillus, and mycoplasmas, of said non-human animal subjects.

In addition, the present invention relates to a kit for veterinary use comprising the active agents of the synergistic composition as previously described in therapeutically effective doses. The kit according to the invention can also present a sheet of instructions concerning the operating mode and the mode of administration of the association of the active agents. The following examples illustrate compositions according to the invention for the treatment of respiratory and / or digestive infections in a number of non-human animal subjects. EXAMPLES

Example 1 Preparation of an Antibacterial Composition According to the Invention

An example of an antibacterial composition according to the present invention comprises florfenicol as phenolic antibiotic in combination with a natural aromatic alcohol of plant origin such as crystallized thymol. To test this antibacterial combination, a stock solution of thymol is prepared by dissolving in 40% ethanol and 60% water. Solutions that are prepared from this stock solution are diluted at least 1/10 in Mueller-Hinton Broth for in vitro determination of antibacterial activity.

Florfenicol is also insoluble in water and is formulated in a stock solution containing 10% methanol and 90% water. The solutions thus obtained from the stock solutions are diluted at least 1/10 in Mueller-Hinton broth.

Example 2 Test Demonstrating a Synergistic Antibacterial Effect

The demonstration or confirmation of a synergistic antibacterial effect can be achieved by placing in contact in a tube a bacterial suspension calibrated at 10 ⁶ CFU / ml in nutrient broth with different and predefined concentrations of the florfenicol and thymol combination. Samples are then taken immediately after contact (TO), then after 1 h, 3 h, 6 h and 24h incubation to

Under slight agitation. The samples are diluted so as to allow the enumeration of the viable germs by transplanting on nutrient medium agar. The total number of surviving germs is expressed as log 10 CFU / ml.

The detection limits are log ₁₀ CFU / ml: 2.3 at the low limit and 9.3 at the high limit. After 24 hours, an increase of 3 log ₁₀ normally corresponds to the growth of the control culture. By comparing the results of the tests and subtracting the log obtained at TO from the log obtained in the test after 24 h, all results below 3 correspond to an incomplete inhibitory effect of the test product on the growth of the seeds. This effect is more or less marked and it becomes bacteriostatic when the value goes from 3 to +/- 1. When the calculated value reaches -3, the effect is frankly bactericidal. The entire protocol can thus be applied to several phenolic or other antibiotic antibiotics as mentioned above and to several antiseptics of natural aromatic alcohols of vegetable origin or biguanide polymers, at different concentrations, and / or to several types of strains. serotyped or not.

EXAMPLE 3 Demonstration of the Synergistic Effect of the Florfenicol and Thymol Association

The antibacterial activity of florfenicol and thymol alone, then thymol / florfenicol combination was tested on several strains and consistent results were observed regardless of the strain tested. The results obtained with E.coli sére serotype O ₇₈ K ₈₀ and Eco // non-typable n ° 05208 of avian origin are represented respectively in Tables 1 and 2 below. The minimum inhibitory concentrations (MIC) of florfenicol and thymol vis-à-vis E. coli. co // are respectively 8 μg / ml and 256 μg / ml. Florfenicol Concentrations in the order of 20 times the MIC at 4 times the MIC of florfenicol do not have a strong bactericidal activity with respect to the microorganisms tested (Figures 1 and 8). Indeed, for a concentration of 4 times the MIC, the bacterial population is lowered by only 2 log ₁₀ after 24 hours of contact (Figure 1). thymol

Concentrations less than or equal to 128 μg / ml of thymol have no activity on the bacteria tested (sub-inhibitory concentrations).

The concentrations of 150 and 170 μg / ml of thymol begin to cause bacteriostatic activity. A concentration of 200 μg / ml exerts a bactericidal activity up to 6 hours of contact but without preventing re-growth at 24 hours. A concentration of 225 μg / ml represents the first bactericidal concentration after 1 h of contact and without regrowth at 24 h (Figure 2). The florfenicol + thymol association

The florfenicol combinations at a concentration equal to the MIC (in this case 8 μg / ml for the 2 strains tested in the context of this example) and thymol at concentrations of 150 to 200 μg / ml are synergistic with a bactericidal effect at 24 h (Figures 3 to 4, 9 and 10). Figure 5 still shows a synergistic bactericidal effect, but this effect then results especially from the use of a high concentration of thymol (200 μg / ml) which outweighs that of florfenicol. Above 200 μg / ml of thymol, the bactericidal and non-synergistic effect is ensured only by the very high concentration of the thymol.

The combinations of florfenicol equivalent to 2 times the MIC and 4 times the MIC of florfenicol with sub-inhibitory concentrations of thymol (from 8 to 128 μg / ml) are not synergistic (Figures 6 and 7). Table 1: Kinetics of bactericidal florfenicol alone or thymol alone, and thymol and florfenicol combination vis-à-vis the E. coli serotype O ₇₈ K ₈₀ of avian origin

Table 2: Bactericidal kinetics of florfenicol or thymol alone and florofenicol and thymol associations against E. coli // non-typable no. 05208 of avian origin

EXAMPLE 4 Demonstration of the Synergistic Effect of the Combination of Fusidic Acid and Hexamethylene Biguanide Polymer

The antibacterial activity of fusidic acid alone and hexamethylene biguanide polymer (PHMB) alone, followed by fusidic acid / hexamethylene biguanide polymer, was tested on the staphylococcus aureus strain (ATCC 29213). An example of an antibacterial composition according to the present invention comprises fusidic acid as an antibacterial in combination with an artificial polymer such as PHMB. To test this antibacterial combination, a stock solution of fusidic acid is prepared by dissolving in 10% sodium hydroxide and 90% water. Solutions that are prepared from this stock solution are diluted at least 1/10 in Mueller-Hinton Broth for in vitro determination of antibacterial activity.

Water-soluble PHMB is formulated in a stock solution containing 100% water. The solutions thus obtained from the stock solutions are diluted at least 1/10 in Mueller-Hinton broth. The demonstration or confirmation of a synergistic antibacterial effect can be achieved by placing a bacterial suspension calibrated at 10 ⁶ CFU / ml in nutrient broth with different predefined concentrations of the fusidic acid and PHMB combination in a tube. . Samples are then taken immediately after contact (TO), then after 1 h, 3 h, 6 h and 24h incubation at 37 ° C with gentle shaking. The samples are diluted so as to allow the enumeration of the viable germs by transplanting on nutrient medium agar. The total number of surviving germs is expressed in log ₁₀ CFU / ml. The detection limits are log ₁₀ CFU / ml: 2.3 at the low limit and 9.3 at the high limit. After 24 hours, an increase of 3 log ₁₀ normally corresponds to the growth of the control culture. By comparing the results of the tests and subtracting the log obtained at TO from the log obtained in the test after 24 h, all the results below 3 correspond to an incomplete inhibitory effect of the product tested on the growth of the seeds. This effect is more or less marked and it becomes bacteriostatic when the value goes from 3 to +/- 1. When the calculated value reaches -3, the effect is frankly bactericidal. The entire protocol can thus be applied to several antibiotics of different families and PHMB at different concentrations, and / or to several types of bacterial strains serotyped or not.

Figure 11 shows that fusidic acid has bacteriostatic activity against Staphylococcus aureus at concentrations of 0.125 μg / ml up to concentrations of 8 μg / ml. On the other hand, the hexamethylene biguanide polymer has a bactericidal activity called the concentration dependent on Staphylococcus aureus. The results are given in Table 3 and Figure 12 with tested concentrations ranging from 0.125 to 8 μg / ml of PHMB. These results show that the concentration of 8 μg / ml of PHMB is the minimum bactericidal concentration of PHMB which makes it possible to obtain a bactericidal effect after 6 hours of contact with Staphylococcus aureus, this effect being maintained for 24 hours. Table 3: Bacterial enumeration after contacting different concentrations of PHMB with Staphylococcus aureus strain ATCC 29213

Figure 13 and Table 4 demonstrate the synergistic effect of fusidic acid at a concentration of 0.125 μg / ml in combination with different concentrations of PHMB ranging from 0.125 to 8 μg / ml. These results show that fusidic acid and PHMB concentrations of 0.125 μg / ml and 1 μg / ml, respectively, are the minimum concentrations to obtain a synergistic bactericidal activity on Staphylococcus aureus. Table 4: Bacterial enumeration after contacting a combination comprising different concentrations of PHMB and 0.125 μg / ml of fusidic acid with the strain Staphylococcus aureus ATCC 29213

As shown in the results in Table 4, the concentration of fusidic acid can be greatly reduced to 0.125 μg / ml. Moreover, this combination is particularly effective in preventing bacterial survival which persists generally 24 hours after treatment with fusidic acid alone, whatever the concentration applied.

Figure 14 and Table 5 show the antibiotic activity of fusidic acid at a concentration of 0.125 μg / ml alone or in combination with PHMB at a concentration of 1 μg / ml or 2 μg / ml. Table 5: Bacterial enumeration after contact with 0.125 μg / ml of fusidic acid and 1 μg / ml of PHMB alone or in combination

Claims

1. An antibacterial synergistic composition characterized in that it comprises an antiseptic selected from natural aromatic alcohols of vegetable origin or biguanide polymers in combination with at least one antibiotic.

2. A composition according to claim 1, characterized in that the antiseptic is a natural aromatic alcohol of vegetable origin and the antibiotic is a phenolic antibiotic.

3. Composition according to claim 1 or 2, characterized in that the phenolic antibiotic agent is selected from florfenicol, thiamphenicol, chloramphenicol and derivatives, and the natural antiseptic aromatic alcohol of plant origin is selected from thymol, carvacrol, eugenol, guaiacol, and derivatives.

4. Composition according to any one of the preceding claims, characterized in that the phenolic antibiotic agent is florfenicol and the natural antiseptic aromatic alcohol of vegetable origin is thymol.

5. Composition according to claim 1, characterized in that the antibiotic is chosen from a β-lactam, fosfomycin, a glycopeptide, bacitracin, a polypeptide, an aminoglycoside, a macrolide, a lincosamide, a streptogramin, a tetracycline, phenicole, fusidanine, or quinolone and the antiseptic is a biguanide polymer.

6. Composition according to claim 1 or 5, characterized in that the polymer antiseptic biguanide is the hexamethylene biguanide polymer.

7. Composition according to any one of the preceding claims, characterized in that said composition further comprises a pharmaceutically acceptable excipient.

8. A composition according to any one of the preceding claims as a veterinary medicament.

9. A composition according to any one of the preceding claims as a broad-spectrum bactericidal drug against pathogenic bacteria.

10. Composition according to any one of the preceding claims as a veterinary composition for the treatment of non-human animal subjects suffering from a respiratory and / or digestive infection.

11. Composition according to any one of the preceding claims, characterized in that it is administered orally, orally, nasally, parenterally or by direct or indirect vaporization.

12. Composition according to any one of the preceding claims, characterized in that it is in liquid, solid, semi-solid or aerosol form.

13. Composition according to any one of the preceding claims, characterized in that it is in the form of a liquid solution miscible with water.

An antibacterial oral composition comprising the composition as defined in claim 13 in diluted form in drinking water with or without addition of aroma.

15. Composition according to any one of the preceding claims, characterized in that it further comprises a therapeutic agent, a bronchial fluidifier, preferably bromhexine, an anti-inflammatory agent, an NSAID compound, a pest control agent or an antiviral agent. .

16. Composition according to any one of the preceding claims, characterized in that it comprises a therapeutically effective amount of florfenicol, thymol, and bromhexine.

17. Use of an antibacterial synergistic composition as defined in any one of claims 1 to 16, for the preparation of a veterinary medicament for the treatment of non-human animal subjects suffering from a respiratory and / or digestive infection.

18. A kit for veterinary use comprising an antibacterial synergistic composition as defined in any one of claims 1 to 16 in therapeutically effective doses to obtain a synergistic effect.

19. Kit according to any one of claims 18, characterized in that it further comprises a sheet of instructions concerning the procedure and the method of administration of the composition.