HU226731B1 - Synergetic veterinay composition containing a combination of fluoroquinolone and colistin for the inhibition of fluoroquinolone-resistance - Google Patents

Abstract

A new preparation is proposed, which prevents the development of resistance to fluorochinolon and acts in synergy. It is applied through the mouth, dissolved in drinking water or neat, sprayed into the mouth by a syringe. The preparation is an aqueous solution containing active components. A new preparation is proposed, which prevents the development of resistance to fluorochinolon and acts in synergy. It is applied through the mouth, dissolved in drinking water or neat, sprayed into the mouth by a syringe. The preparation is an aqueous solution containing active components, including the following: In 5.1-30% concentration: 6-fluoro-7-piperazinyl-4-chinolon [fluorochinolon] and or their salts produced with acid, In 2.6-15% concentration: chitosan-sulphate, In 0.1-50% concentration: a hydroxyl-carbonic acid and/or amino-acid, and according to requirements, additives in concentrations of 0.1-50%.

Description

SUMMARY OF THE INVENTION The present invention relates to novel synergistic veterinary compositions which are highly inhibited against the development of pathogenic microbial resistance to fluoroquinolones. The field of application of the invention is veterinary medicine.

Fluoroquinolones (6-fluoro-7-piperazinyl-4-quinolones; such as norfloxacin, ciprofloxacin, enrofloxacin, danofloxacin, sarafloxacin, difloxacin, marbofloxacin) are fluoro-substituted, broad-spectrum quinolone, resulted in the explosive development of antibacterial chemotherapy. Well-known compounds widely used in veterinary medicine (Van Cutsem, PM et al., Cornell et al., 1990, 80, 173, Prescott, JF et al., Antimicrobial Therapy in Veterinary Medicine, 3rd Ed., Lowa / Ames, 2000), against which, however, in recent years there has been increasing resistance of several important pathogenic bacterial species. From a veterinary point of view, the increased resistance of Escherichia coli strains (Trolldenier, H., Dtsch. Tierarztl. Wschr., 1996, 103, 237) and, in human health, the decreasing fluoroquinolone et al. ., Antimicrob Agents Chemother 2000, 44, 1140). The possibility of acquired (secondary) resistance was also supported by the results of passage tests with pathogenic microbes; for example, enrofloxacin with a minimal inhibitory concentration (MIC) against E. coli increased 8-64-fold in 10 passages (Laurentie et al., Proc. 8 ^th EAVPT Congress, Jerusalem, 2000, Abstract G18).

Colistin is a bactericidal, narrow-spectrum compound belonging to the class of polypeptide antibiotics. It is a surfactant with a mode of action similar to cationic detergents. Synergistic with multiple antibiotics; Thus, synergistic effects have been described between colistin and penicillins, tetracyclines, certain macrolides and sulfonamides (Ziv, G., J. Am. Vet. Med. Assoc., 179, 711), and several combinations thereof are used in veterinary medicine. (e.g., colistin-amoxicillin, colistin-oxytetracycline, colistin-spiramycin).

Generally, the purpose of using antibacterial combinations may be to broaden the spectrum of action, increase efficacy (synergism), delay the development of resistance and break through existing resistance.

It is an object of the present invention to develop combinations having antibacterial activity which, above all, can inhibit / delay the development of resistance (which is one of the major problems of chemotherapy today) and at the same time increase the antibacterial activity.

Our studies have surprisingly shown that colistin, when used in combination with a fluoroquinolone compound, greatly inhibits (delays) the development of resistance to fluoroquinolones and, at the same time, synergistically enhances the action of fluoroquinolone against several important pathogenic microbes.

In our microbiological studies, we investigated the development of resistance to E. coli strains against enrofloxacin and norfloxacin and tested the in vitro antibacterial activity of enrofloxacin-colistin and norfloxacin-colistin combinations. The results of the tests are shown in Tables 1 and 2.

Table 1

Development of resistance to enrofloxacin and norfloxacin after 10 passages when used alone or in combination with colistin

E. coli strain passages number MIC (μ9 / ΓηΙ) enrofloxacin enrofloxacin + colistin norfloxacin norfloxacin + colistin S1 1 0,015 0.015 / 0.007 0,125 0.03 / 0.06 (Kólibacillózis, 5 0.03 0.06 / 0.03 8.0 0.03 / 0.06 pig) 10 1.0 0.125 / 0.06 32.0 0.03 / 0.06 G6 1 0.25 0.125 / 0.06 2.0 1.0 / 0.25 (Kólibacillózis, 5 2.0 0.125 / 0.06 8.0 2.0 / 0.25 pig) 10 4.0 0.125 / 0.06 16.0 4.0 / 0.25 B8 / 3 1 0.03 0.03 / 0.015 0,125 0.03 / 0.06 (Kólibacillózis, 5 0.25 0.03 / 0.015 0.5 0.03 / 0.06 broilers) 10 1.0 0.03 / 0.03 1.0 0.03 / 0.06

As the data indicate, the MICs of both fluoroquinolones increased significantly, 8-64-fold during passage, but the MIC60 values were not at all (2-2 strains) or only slightly (1-1 strain) in combination with colistin. They increased.

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Table 2

Antibacterial activity of enrofloxacin and enrofloxacin-colistin combination (2: 1)

bacterial species MIC (g / ml) enrofloxacin colistin Enrofloxacin- colistin FIC index * E. coli G6 (coli bacillosis, broiler chicken) 0.25 0.5 0.125 / 0.06 0.75 E. coli B-412 (coli bacillosis, broiler chicken) 0.06 0.5 0.03 / 0.015 0.53 P. past 32978 (poultry cholera, domestic goose) 0.25 0.25 0.03 / 0.015 0.18 P. past 13824 (poultry cholera, duck) 0.03 0.5 0.015 / 0.007 0.51

* FIC index <1 = synergism; FIC index 1-2 = indifferent effect; FIC index> 2 = antagonism

As shown by the values of the FIC index, the fixed combination of enrofloxacin and colistin in a 2: 1 ratio proved to be synergistic against pathogens isolated from practical cases. There was an even more pronounced synergistic effect when the two compounds were not combined in a fixed ratio; for example, E. coli strain B8 / 3, used for passage, was inhibited by enrofloxacin at 1/4 MIC and colistin at 1/16 MIC, i.e. a FIC index of 0.29.

The results of the in vitro studies clearly demonstrate that the development of resistance to enrofloxacin or norfloxacin can be inhibited by the very low subMIC concentrations of colistin used in combination. This effect is very important in the therapeutic control of pathogens and can greatly extend the useful life of fluoroquinolone. At the same time, colistin is synergistic with fluoroquinolones against a number of practical pathogens, such as E. coli and P. pastodd.

The favorable results of our in vitro studies were largely confirmed by data from clinical trials. In this context, the efficacy of a 2: 1 combination of enrofloxacin-colistin was investigated in broiler chickens artificially infected with E. coli bacteria and inoculated turkeys inoculated with P. pastodod. In both experiments, the combination of 10 mg / kg body weight enrofloxacin and 5 mg / kg body weight colistin sulfate for 5 days was more effective than enrofloxacin 10 mg / kg body weight alone.

Based on the foregoing, the present invention provides for the treatment of gastrointestinal and respiratory diseases of animals, preferably poultry and pigs, as well as for the treatment of various forms of colitis, salmonellosis, mycoplasmosis, staphylococci, and streptococci. veterinary medicinal products having the following composition:

a) 5.1-30% by weight of the total volume of the solution of the 6-fluoro-7-piperazinyl-4-quinolone (fluoroquinolone) derivative, preferably enrofloxacin or norfloxacin, in the form of a base and / or an acid addition salt,

b) 2.6-15% colistin sulfate,

c) hydroxycarboxylic and / or amino acids,

In a concentration of 0.1-50%, preferably 5-25%,

d) formulation adjuvants at a concentration of 0.1% to 50%, if necessary,

(e) water up to 100% solution.

According to the invention, the active ingredients of the composition are dissolved in an aqueous solution of a hydroxy carboxylic acid and / or amino acid in a concentration of 0.1-50% (based on the total volume of the composition) of a fluoroquinolone compound and / or an acid addition salt thereof and colistin sulfate, and if necessary, 0.1-50% by weight of formulation auxiliaries are added to the resulting solution and water is made up to 100% by volume.

The veterinary compositions of the present invention are administered to animals at a dose of 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight, of the active compounds, dissolved in drinking water or injected directly into the oral cavity of piglets.

According to the invention, the veterinary compositions may contain as a fluoroquinolone compound a 6-fluoro-7-piperazinyl-4-quinolone derivative, preferably enrofloxacin or norfloxacin, or acid addition salts thereof, from 5.1 to 30% (mixed percentage), preferably from 10 to 20%. solution.

Hydroxycarboxylic acids such as tartaric acid, lactic acid, malic acid, citric acid, gluconic acid, glucuronic acid, amino acids such as glutamic acid, aspartic acid or a mixture thereof may be used in a concentration of 0.1-50%, preferably 5-25%.

The solution may also contain formulation auxiliaries such as glycerol, propylene glycol, polyethylene glycol, complexing agents such as ethylenediaminetetraacetic acid or its salts, citrates, phosphates, acetates, antioxidants such as ascorbic acid, sulphites, and or benzoic acid esters.

The pH of the solution according to the invention ranges from 2 to 6, preferably from 2.5 to 4.5.

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According to the invention, the solvents are filled into a heated, acid-proof container and the active ingredients and various excipients are dissolved therein under continuous stirring. If necessary, the active compounds can be dissolved by raising the temperature to 30-60 ° C. After cooling to room temperature, the solution is disposed in plastic containers commonly used for oral solutions.

The veterinary compositions of the present invention are used for the treatment and prevention of systemic and digestive and respiratory diseases of bacterial origin in animals, preferably poultry and pigs. The formulations are administered at a dosage of 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight of the active compounds, dissolved in the drinking water of the animals, or directly into the oral cavity of piglets. The recommended duration of treatment is 3-5 days.

The use of the veterinary compositions of the present invention, through the synergism between the two active ingredients contained therein, provides effective protection against major bacterial diseases in poultry and pigs such as coli, salmonellosis, mycoplasmosis, staphylococci, streptococci, streptococci. The formulations may also be effective against microbes that are individually resistant or hypersensitive to their active ingredients. Most importantly, however, the use of the formulations greatly delays or inhibits the development of resistance to fluoroquinolones, which is one of the major concerns of chemotherapy today, and can thus significantly increase the useful life of this critical class of compounds.

The present invention provides, without limiting the scope of the invention, the veterinary preparations and methods for their use, which are described below.

Example 1

An oral solution is prepared using Enrofloxacin and Colistin Sulphate as active ingredients. The composition of the solution is as follows:

Ingredients Concentration (g / 100 ml) enrofloxacin 6.66 Colistin sulfate 3.33 Tartaric acid 2.25 Benzyl alcohol 1.00 Cleaned water ad 100.00 ml

Example 2

An oral solution is prepared using Enrofloxacin and colistin sulfate as active ingredients. The composition of the solution is as follows:

Ingredients Concentration (g / 100 ml) enrofloxacin 10.00 Colistin sulfate 5.00 Lactic acid 7.26 L-glutamic acid 2.50 propylene 10.36 Sodium metabisulfite 0.20 Cleaned water ad 100.00 ml

Example 3

An oral solution is prepared using ciprofloxacin and colistin sulfate as active ingredients. The composition of the solution is as follows:

Ingredients Concentration (g / 100 ml) ciprofloxacin 6.66 Colistin sulfate 3.33 Lactic acid 8.47 Benzyl alcohol 0.50 Cleaned water ad 100.00 ml

Example 4

Norfloxacin lactate and colistin sulfate are used as active ingredients in an oral solution. The composition of the solution is as follows:

Ingredients Concentration (g / 100 ml) Norfloxacin lactate 8.52 Colistin sulfate 3.33 Lactic acid 5.00 Benzyl alcohol 1.00 Cleaned water ad 100.00 ml

Example 5

Enrofloxacin lactate and colistin sulfate as active ingredients are prepared as an oral solution. The composition of the solution is as follows:

Ingredients Concentration (g / 100 ml) Enrofloxacin lactate 8.31 Colistin sulfate 3.33 Lactic acid 2.65 Tartaric acid 1.25 Benzyl alcohol 0.50 Cleaned water ad 100.00 ml

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Example 6

Broiler chickens showing respiratory symptoms were treated with an oral solution containing 10% enrofloxacin and 5% colistin sulfate. The formulation is dissolved in drinking water at a dose of 0.1 ml / kg body weight for 5 days.

Example 7

An oral solution containing 8.52% norfloxacin lactate and 3.33% colistin sulfate was used to treat suckling pigs with symptoms of colic. The preparation is diluted 10-fold with drinking water at a dose of 1 ml / animal (6.66 mg norfloxacin + 3.33 mg colistin / animal) directly into the oral cavity of the animal by syringe. The treatment is performed for 3 consecutive days.

Claims (7)

PATENT CLAIMS 1. A synergistic veterinary composition which inhibits the development of fluoroquinolone resistance, characterized in that it is present in a concentration of 5.1-30% of a 6-fluoro-7-piperazinyl-4-quinolone derivative (fluoroquinolone) and 2.6-15%. colistin sulfate in an aqueous solution containing 0.1-50% of a hydroxycarboxylic acid, amino acid or mixture thereof, and optionally 0.1-50% of a formulation aid. A veterinary composition according to claim 1, wherein the fluoroquinolone compound is enrofloxacin or ciprofloxacin or norfloxacin or danofloxacin or difloxacin or marbofloxacin or sarafloxacin and / or acid addition salts thereof. The veterinary composition according to claim 1, wherein the solvent is tartaric acid, lactic acid, malic acid, citric acid, gluconic acid, glucuronic acid, glutamic acid, aspartic acid or a mixture thereof. The veterinary composition according to claim 1, wherein the total weight of the solvents is preferably from 5% to 25% by weight of the total volume of the solution. The veterinary composition according to claim 1, characterized in that glyceryl, propylene glycol, polyethylene glycol, complexing agents such as ethylenediaminetetraacetic acid or its salts, citrates, phosphates, acetates, antioxidants such as ascorbic acid or salts are optionally formulated as formulation aids. and preservatives such as benzyl alcohol, ethanol or benzoic acid esters. 6. A process for the preparation of a veterinary composition according to claim 1, wherein the fluoroquinolone and colistin sulfate are dissolved in an aqueous solution containing the solvent or mixture of solvents with continuous mixing, optionally adding the formulation adjuvants and water with the desired formulation. make up to volume. Veterinary composition according to claim 1 for the treatment of bacterial diseases of animals, preferably poultry and pigs, in a dose of 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight, orally, dissolved in the drinking water of the animals or directly into the oral cavity. fed.