CN101953784B

CN101953784B - Veterinary suspension containing amoxicillin, colistin sulfate and prednisolone and preparation method thereof

Info

Publication number: CN101953784B
Application number: CN2009100889984A
Authority: CN
Inventors: 肖希龙; 沈建忠; 汤树生; 何家康; 李兰; 张朝明
Original assignee: BEIJING ZHONGNONGDA ANIMAL HEALTH-CARE PRODUCT TECHNOLOGY RESEARCH INSTITUTE; China Agricultural University
Current assignee: BEIJING ZHONGNONGDA ANIMAL HEALTH-CARE PRODUCT TECHNOLOGY RESEARCH INSTITUTE; China Agricultural University
Priority date: 2009-07-16
Filing date: 2009-07-16
Publication date: 2012-02-08
Anticipated expiration: 2029-07-16
Also published as: CN101953784A

Abstract

The invention discloses a veterinary suspension containing amoxicillin, colistin sulfate and prednisolone and a preparation method thereof. The veterinary suspension adopts the amoxicillin, the colistin sulfate and the prednisolone as active ingredients. The preparation method of the veterinary suspension containing the amoxicillin, the colistin sulfate and the prednisolone comprises the following steps of: (1) dissolving or dispersing a suspending agent, an antioxidant and a preservative in a hot dispersion medium to obtain a solution (A); (2) adding 40-80 percent of dispersion medium in a formula ratio in a colloid mill, starting the colloid mill, then slowly adding the solution (A) and adding a wetting agent while stirring after the solution (A) is fully added; (3) sequentially adding the amoxicillin, the colistin sulfate and the prednisolone after fully adding all the accessories, and grinding by adopting two alternate modes, i.e. an endless grinding mode and a non-endless grinding mode; and (4) detecting the grain fineness, stopping grinding when the grain fineness accords with the requirement, adding the dispersion medium to the formula ratio, mixing, canning, sealing and sterilizing to obtain the veterinary suspension containing the amoxicillin, the colistin sulfate and the prednisolone.

Description

Contain animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone and preparation method thereof

Technical field

The invention belongs to the herding field of pharmaceutical preparations, what be specifically related to is a kind of animal compound suspension that contains amoxicillin, colistin sulfate and prednisolone and preparation method thereof.

Background technology

Fast development along with China's livestock and poultry breeding industry; Various pathogenic bacterium are more and more complicated on the veterinary clinic in recent years, and big multiple-case all is the mixed infection of many cause of diseases, wherein existing gram positive bacteria infection; Gram positive bacterial infection is arranged again, bring very big difficulty for veterinary's preventing and controlling.Owing to lack the sophisticated compound preparation that various pathogenic bacterium is all had good result; The veterinary work person often need unite the multiple folk prescription pharmaceutical preparation of use and treat mixed infectious disease, and carries out auxiliary treatment at early stage normal glucocorticoids medicine such as prednisolone, the dexamethasone etc. of using of infection.So, treat a kind of disease and often need give multiple pharmaceutical preparation simultaneously and just can reach ideal therapeutic effect, brought huge workload both for the veterinary drug staff, brought great stress to infected animal again.Therefore, researching and developing a kind ofly all has the long-acting composite preparation of good prevention effect to various pathogens, is the urgent needs of the anti-Zhiduo County of present livestock breeding industry cause of disease mixed infectious disease, significant.

Aspect the compound preparation research of anti-Zhiduo County cause of disease mixed infectious disease; Domestic still do not have a more sophisticated launch; Abroad; Though Pfizer Inc. has developed the confession breast perfusion of amoxicillin, clavulanic acid, prednisolone with sterilization oiliness compound recipe suspension solution, its main uses is treatment gram positive bacteria and negative microbial milch cow mastitis lactation period; France Virbac has also successfully developed the compound injection suspension of ampicillin, colistin sulfate and dexamethasone, but this product also is mainly used in the treatment of mammitis of cow.More than existing two kinds of compound preparations because the main treatment that is suitable for mammitis of cow, narrow application range can not satisfy the mixed infectious disease of various animals (especially pig).

Summary of the invention

What the invention provides a kind of determined curative effect is used to prevent and treat the mixed infection that animal leather gram-positive bacteria and gram negative bacteria cause and the animal compound suspension that contains amoxicillin, colistin sulfate and prednisolone of secondary infection.

For solving the problems of the technologies described above, the present invention takes following technical scheme:

A kind of animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone is to be the animal use suspensoid liquid of effective ingredient with amoxicillin, colistin sulfate and prednisolone.

Each content of effective is in the said animal use suspensoid liquid: (weight/volume percent concentration g/100ml), is preferably 10-20% (g/100ml) to amoxicillin 5-30%; Colistin sulfate 0.25-3% (g/100ml) is preferably 0.5-2.5% (g/100ml); Prednisolone 0.01-0.1% (g/100ml) is preferably 0.02-0.05% (g/100ml).

Also comprise following excipient substance in the said animal use suspensoid liquid: dispersion medium, suspending agent, wetting agent, antioxidant and antiseptic.

Said dispersion medium can be selected from one or both in injection soybean oil, ethyl oleate, glyceryl triacetate, myristic acid isopropyl ester and the injection Oleum Ricini.

Said suspending agent can be selected from one or both in aluminium stearate, polyvinylpyrrolidone, lecithin, cholesterol, vaseline, sodium carboxymethyl cellulose and the castor oil hydrogenated.

Optional a kind of in tween 80, Arlacel-60, Arlacel-80 and oxireme-40-castor oil hydrogenated of said wetting agent.

Said antioxidant can be selected from a kind of in vitamin E, thiourea, butylhydroxy anisole, sodium metabisulfite and the gallic acid third lipoprotein.

Said antiseptic can be selected from a kind of in benzyl alcohol, methyl hydroxybenzoate and the propylparaben.

Specifically, the content of each composition is in the said animal use suspensoid liquid: amoxicillin 5-30% (g/100ml) is preferably 10-20% (g/100ml); Colistin sulfate 0.25-3% (g/100ml) is preferably 0.5-2.5% (g/100ml); Prednisolone 0.01-0.1% (g/100ml) is preferably 0.02-0.05% (g/100ml), suspending agent 0.1-2% (g/100ml); Wetting agent 0.2-2% (ml/100ml); Antioxidant 0.01-1% (g/100ml), antiseptic 0.005-1% (g/100ml), all the other are dispersion medium.

Second purpose of the present invention provides a kind of above-mentioned method that contains the animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone for preparing.

Method for preparing provided by the present invention can may further comprise the steps:

1) gets suspending agent, antioxidant and antiseptic and be dissolved in or be scattered among the dissipation of heat matchmaker, obtain A liquid;

2) dispersion medium of getting formula ratio 40-80% is poured in the colloid mill, starts colloid mill, slowly adds above-mentioned A liquid then, treats all to add fully, adds wetting agent while stirring;

3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and prednisolone successively, the mode that adopts circular grinding and acyclic grinding to alternate is then ground;

4) inspection fineness of the particles stops after meeting the requirements grinding, and adds dispersion medium to formula ratio, mixing, and fill, sealing, sterilization obtains the animal use suspensoid liquid that the present invention contains amoxicillin, colistin sulfate and prednisolone.

In manufacturing method for above mentioned medicine, said dispersion medium can be selected from one or both in injection soybean oil, ethyl oleate, glyceryl triacetate, myristic acid isopropyl ester and the injection Oleum Ricini.

Optional in tween 80, Arlacel-60, Arlacel-80 and oxireme-40-castor oil hydrogenated one or both of said wetting agent.

Said antioxidant can be selected from one or both in vitamin E, thiourea, butylhydroxy anisole, sodium metabisulfite and the gallic acid third lipoprotein.

Said antiseptic can be selected from one or both in benzyl alcohol, methyl hydroxybenzoate and the propylparaben.

The usage and dosage of above-mentioned animal use suspensoid liquid is generally the 0.1-0.2mL/kg body weight, and 1 day 1 time, logotype 3-5 days.The ID and the course of treatment can suitably be adjusted according to practical situation.

The invention provides a kind of animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone and preparation method thereof.Advantage of the present invention and good effect are following:

(1) utilizes amoxicillin, these two antibiotic of colistin sulfate, unite and use the Synergistic antimicrobial advantage of having brought into play two medicines, widened antimicrobial spectrum, strengthen antibacterial efficacy, reduce, delay chemical sproof generation with different antibiotic mechanism.

(2) prednisolone administration post-absorption is rapid, and blood drug level is high, organizes affinity strong, can arrive inflammation part performance antiinflammatory action rapidly, and suspension of the present invention is respond well to mixed infectious disease, for veterinary clinic provides a kind of good preparation.

(3) amoxicillin, colistin sulfate and prednisolone are united use, widened antimicrobial spectrum, strengthened antibacterial efficacy, avoid single medicine to use the limitation of bringing, delay chemical sproof generation, and have antiinflammatory, antitoxic effect.In addition, adopt compound preparation only to need be administered once every day, both can reduce because frequent drug administration (former single medicine all need administration every day two or three times) and, save human and material resources and financial resources again the stress that animal produces.

(4) suspension of the present invention adopts the dispersion medium of oil-based solvent as preparation; Not only solved the difficult problem of amoxicillin easy degraded in aqueous solution, and in oil-based suspension, colistin sulfate discharges slowly in vivo; Blood drug level is steady, has reduced the toxic and side effects of colistin sulfate.

In addition; The animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention is to adopt dispersion method that amoxicillin, colistin sulfate and prednisolone are scattered in the solvent; And add suspending agent, wetting agent and antioxidant, and to utilize colloid mill to grind and get, production technology is simple; Reduce cost, be easy to realize industrialization.

In sum; The present invention organically combines time-dependent antibiotic amoxicillin, concentration dependent form antibiotic colistine sulfate, has both utilized the antibacterial action of single medicine well, has brought into play the Synergistic antimicrobial advantage of two medicine couplings again; Two medicine couplings have enlarged antimicrobial spectrum; Strengthened antibacterial efficacy, combined antiinflammatory, the antiinflammatory advantage of prednisolone simultaneously, the three share have infection, antiphlogistic effect.Be the epidemic characteristic of mixed infection or secondary infection to China's veterinary clinic livestock and poultry in recent years, the present invention is mainly used in clinically and prevents and treats mixed infection and the secondary infection that animal leather gram-positive bacteria and gram negative bacteria cause more.Preparation technology of the present invention is simple, is easy to realize industrialization, and cost is low, low toxicity and efficient, has high application value.

Below in conjunction with specific embodiment the present invention is explained further details.

The specific embodiment

The present invention is national Eleventh Five-Year Plan science and technology support plan problem (problem title: the development of animal compound novel formulation and application; Project number: partial content 2006BAD31B08).

The present invention is in order to provide a kind of compound injection that can generally be used for the mixed infectious disease of various animals; With isolating multiple common pathogen in the different animals clinical cases such as pig, cattle, chicken is object of study; From multiple common drugs such as enrofloxacin, amoxicillin, tylosin, colistin sulfate, filtering out synergism better and to gram positive bacteria and gram negative bacteria all has the compound recipe of extremely strong effect to make up (amoxicillin and colistin sulfate); Combine the antiinflammation of prednisolone simultaneously, successfully developed amoxicillin, colistin sulfate, the prednisolone compound injection of the mixed infectious disease that can be widely used in preventing and treating various animals.

It is to have overcome because of giving the stress that multiple medicine causes animal simultaneously that the present invention becomes the advantage of compound preparation to amoxicillin, colistin sulfate, three kinds of drug developments of prednisolone, has reduced veterinary personnel's workload.The particularly important is this compound preparation and not only overcome single preparations of ephedrine narrow antimicrobial spectrum and the little defective of the existing compound preparation scope of application, and strengthened the antibacterial efficacy of amoxicillin and colistin sulfate, and delayed the generation of these two kinds of drug resistances.

The present invention provides a kind of compound preparation that contains amoxicillin, colistin sulfate and prednisolone.In compound recipe assembly process, need the special concertedness of considering between the medicine, and through screening the preparation that appropriate pharmaceutic adjuvant forms determined curative effect and has good stability.

Institute's medicament among the present invention; Optimize to use free dependent form antibiotic amoxicillin and concentration dependent form antibiotic colistine sulfate, two kinds of antibiotic couplings can the Synergistic antimicrobial advantage; Enlarge antimicrobial spectrum, strengthen antibacterial efficacy (referring to subsequent experimental one and experiment two); Be used in combination again have antiinflammatory, the prednisolone of antiinflammatory advantage, the three share have infection, antiphlogistic effect (referring to subsequent experimental three, four---clinical effectiveness test).Wherein each content of active component in the injection: amoxicillin 5-30% (g/100ml) is preferably 10-20% (g/100ml); Colistin sulfate 0.25-3% (g/100ml) is preferably 0.5-2.5% (g/100ml); Prednisolone 0.01-0.1% (g/100ml) is preferably 0.02-0.05% (g/100ml).

The present invention utilizes compound medicine to combine suitable pharmaceutic adjuvant to process animal use suspensoid liquid, comprises excipient substances such as using dispersion medium, suspending agent, wetting agent, antioxidant and antiseptic.

Dispersion medium: can be selected from one or both compound in injection soybean oil, ethyl oleate, glyceryl triacetate, myristic acid isopropyl ester and the injection Oleum Ricini, the compound dispersion medium of preferred oil acetoacetic ester and injection soybean oil.Select the dispersion medium of oil-based solvent among the present invention especially for use as preparation; Solved the difficult problem of amoxicillin easy degraded in aqueous solution, and in oil-based suspension, colistin sulfate discharges slowly in vivo; Blood drug level is steady, has reduced the toxic and side effects of colistin sulfate.

Suspending agent: can be selected from one or both compound in aluminium stearate, polyvinylpyrrolidone, lecithin, cholesterol, vaseline, sodium carboxymethyl cellulose and the castor oil hydrogenated; The kind of suspending agent and consumption are adjusted according to drug dose and dispersion medium kind, are beneficial to form stable injection.Suspending agent content is 0.1-2% (g/100ml) in injection.

Wetting agent: optional a kind of in tween 80, Arlacel-60, Arlacel-80 and oxireme-40-castor oil hydrogenated, select one-component as far as possible for use, but do not get rid of the multiple compound mode of using in pharmacy procedure.Wetting agent content is 0.2-2% (ml/100ml) in injection.

Antioxidant: can be selected from a kind of in vitamin E, thiourea, butylhydroxy anisole, sodium metabisulfite and the gallic acid third lipoprotein, in pharmacy procedure, select one-component as far as possible for use, but not get rid of the multiple compound mode of using.Antioxidant content is 0.01-1% (g/100ml) in injection.

Antiseptic: can be selected from a kind of in benzyl alcohol, methyl hydroxybenzoate and the propylparaben, in pharmacy procedure, select one-component as far as possible for use, but not get rid of the multiple compound mode of using.The content of antiseptic is 0.005-1% (g/100ml) in injection.

In the animal use suspensoid liquid that forms according to the present invention the content of each composition can for: amoxicillin 5-30% (g/100ml) is preferably 10-20% (g/100ml); Colistin sulfate 0.25-3% (g/100ml) is preferably 0.5-2.5% (g/100ml); Prednisolone 0.01-0.1% (g/100ml) is preferably 0.02-0.05% (g/100ml), suspending agent 0.1-2% (g/100ml); Wetting agent 0.2-2% (ml/100ml); Antioxidant 0.01-1% (g/100ml), antiseptic 0.005-1% (g/100ml), all the other are dispersion medium (ml).

Below further specify the present invention with specific embodiment.Following embodiment 1-10 adopts best experimental program of the present invention, and those of ordinary skill in the art can obviously expect some identical, replacement schemes according to content disclosed by the invention, all should belong to disclosure of the present invention.Method therefor is conventional method if no special instructions among the embodiment.

Embodiment 1

It is following that the present invention contains the prescription of animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone:

Amoxicillin 20kg,

Colistin sulfate 1.5kg,

Prednisolone 0.04kg,

Aluminium stearate 1kg,

Arlacel-80 1L,

Propyl gallate 0.02kg,

Benzyl alcohol 0.5kg

The injection soybean oil adds to 100L.

Prepare the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone with the present invention, concrete grammar may further comprise the steps:

1) aluminium stearate, the propyl gallate with formula ratio adds in the 2L injection soybean oil, and heated and stirred is dissolved it fully, adds the benzyl alcohol of formula ratio, obtains A liquid;

2) get 60L injection soybean oil and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-80 of formula ratio while stirring;

3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and the prednisolone of formula ratio successively, the mode that adopts circular grinding and acyclic grinding to alternate is then ground;

4) inspection fineness of the particles, result meet the requirements (standard be particle diameter should be less than 10% greater than the granule number of 15 μ m), stop to grind; Add the injection soybean oil to 100L, mixing, fill; Sealing, sterilization obtains the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention.

Embodiment 2

Amoxicillin 20kg,

Colistin sulfate 1kg,

Prednisolone 0.03kg,

Castor oil hydrogenated 1kg,

Arlacel-60 0.5L,

Propyl gallate 0.02kg,

Methyl hydroxybenzoate 0.05kg,

The myristic acid isopropyl ester adds to 100L.

Prepare the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone with method of the present invention, concrete grammar may further comprise the steps:

1) get in castor oil hydrogenated, propyl gallate and the methyl hydroxybenzoate adding 2L myristic acid isopropyl ester of formula ratio, heated and stirred is dissolved it fully and is obtained A liquid;

2) get 40L myristic acid isopropyl ester and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-60 of formula ratio while stirring;

4) inspection fineness of the particles, the result meets the requirements, and stops to grind, and adds the myristic acid isopropyl ester to 100L, mixing, fill, sealing, sterilization obtains the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention.

Embodiment 3

Amoxicillin 10kg,

Colistin sulfate 1kg,

Prednisolone 0.05kg,

Castor oil hydrogenated 0.5kg,

Vaseline 0.25kg,

Arlacel-80 0.2L,

Thiourea 0.2kg,

Methyl hydroxybenzoate 0.05kg,

Ethyl oleate 30L,

The injection soybean oil adds to 100L.

1) get in castor oil hydrogenated, vaseline, thiourea and the methyl hydroxybenzoate adding 2L injection soybean oil of formula ratio, heated and stirred is dissolved it fully, obtains A liquid;

2) ethyl oleate and the 30L injection soybean oil of getting formula ratio are poured in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-80 of formula ratio while stirring;

4) inspection fineness of the particles, the result meets the requirements, and stops to grind, and adds the injection soybean oil to 100L, mixing, fill, sealing, sterilization obtains the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention.

Embodiment 4

Amoxicillin 15kg,

Colistin sulfate 2.5kg,

Prednisolone 0.04kg,

Lecithin 0.8kg,

Arlacel-80 1L,

Vitamin E 0.05kg,

Propylparaben 0.05kg,

The injection Oleum Ricini adds to 100L.

(1) get in lecithin, vitamin E and the propylparaben adding 2L injection Oleum Ricini of formula ratio, heated and stirred is dissolved it fully, obtains A liquid;

(2) get 80L injection Oleum Ricini and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-80 of formula ratio while stirring;

(3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and the prednisolone of formula ratio successively, the mode that adopts circular grinding and acyclic grinding to alternate is then ground;

(4) after the inspection fineness of the particles meets the requirements, stop to grind, add the injection Oleum Ricini to 100L, mixing, fill, sealing, sterilization obtains the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention.

Embodiment 5

Amoxicillin 15kg,

Colistin sulfate 2kg,

Prednisolone 0.05kg,

Lecithin 0.2kg,

Castor oil hydrogenated 0.5kg,

Arlacel-60 0.8L,

Butylhydroxy anisole 0.05kg,

Methyl hydroxybenzoate 0.05kg,

Ethyl oleate adds to 100L.

1) get in castor oil hydrogenated, lecithin, butylhydroxy anisole and the methyl hydroxybenzoate adding 2L ethyl oleate of formula ratio, heated and stirred is dissolved it fully, obtains A liquid;

2) get the 60L ethyl oleate and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-60 of formula ratio while stirring;

3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and the prednisolone of formula ratio successively, begin to grind, the mode that adopts circular grinding and acyclic grinding to alternate is ground;

4) inspection fineness of the particles, the result meets the requirements, and stops to grind, and adds ethyl oleate to 100L, mixing, fill, sealing, sterilization obtains the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone of the present invention.

Embodiment 6

Amoxicillin 20kg

Colistin sulfate 2kg

Prednisolone 0.04kg

Castor oil hydrogenated 0.3kg

Lecithin 0.5kg

Arlacel-80 0.6L

Vitamin E 0.05kg

Methyl hydroxybenzoate 0.05kg

Ethyl oleate adds to 100L

The method for preparing of the animal use suspensoid liquid of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) castor oil hydrogenated, lecithin and the methyl hydroxybenzoate of getting formula ratio are dissolved in the 2L deep fat acetoacetic ester, are A liquid;

(2) get the 60L ethyl oleate and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-80 of formula ratio while stirring;

(3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and the prednisolone of formula ratio successively, begin to grind, the mode that adopts circular grinding and acyclic grinding to alternate is ground;

(4) after the inspection fineness of the particles meets the requirements, stop to grind, add ethyl oleate to 100L, mixing, fill, sealing, sterilization promptly gets described suspension.

Embodiment 7

Amoxicillin 20kg

Colistin sulfate 2kg

Prednisolone 0.04kg

Castor oil hydrogenated 0.3kg

Lecithin 0.3kg

Arlacel-80 1.5L

Vitamin E 0.075kg

Methyl hydroxybenzoate 0.05kg

Ethyl oleate 10L

The injection soybean oil adds to 100L

(1) castor oil hydrogenated, lecithin and the methyl hydroxybenzoate of getting formula ratio are dissolved in the hot injection soybean oil of 2L, are A liquid;

(2) ethyl oleate of getting 50L injection soybean oil and formula ratio is poured in the colloid mill, starts colloid mill, slowly adds above-mentioned A liquid then, treats all to add fully, adds the Arlacel-80 of formula ratio while stirring;

(4) after the inspection fineness of the particles meets the requirements, stop to grind, add the injection soybean oil to 100L, mixing, fill, sealing, sterilization promptly gets described suspension.

Embodiment 8

Amoxicillin 15kg

Colistin sulfate 2.5kg

Prednisolone 0.05kg

Lecithin 1.5kg

Arlacel-60 0.5L

Vitamin E 0.075kg

Methyl hydroxybenzoate 0.05kg

The myristic acid isopropyl ester adds to 100L

(1) lecithin, vitamin E and the methyl hydroxybenzoate of getting formula ratio are dissolved in the hot myristic acid isopropyl ester of 2L, are A liquid;

(2) get 60L myristic acid isopropyl ester and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the Arlacel-60 of formula ratio while stirring;

(4) after the inspection fineness of the particles meets the requirements, stop to grind, add injection myristic acid isopropyl ester to 100L, mixing, fill, sealing, sterilization promptly gets described suspension.

Embodiment 9

Amoxicillin 30kg

Colistin sulfate 0.5kg

Prednisolone 0.02kg

Cholesterol 0.1kg

Oxireme-40-castor oil hydrogenated 1.0L

Butylhydroxy anisole 0.01kg

Methyl hydroxybenzoate 0.005kg

Glyceryl triacetate adds to 100L

(1) cholesterol, methyl hydroxybenzoate and the butylhydroxy anisole of getting formula ratio are dissolved in the 2L glyceryl triacetate, are A liquid;

(2) get the 60L glyceryl triacetate and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add the oxireme-40-castor oil hydrogenated of formula ratio while stirring;

(4) after the inspection fineness of the particles meets the requirements, stop to grind, add the injection glyceryl triacetate to 100L, mixing, fill, sealing, sterilization promptly gets described suspension.

Embodiment 10

Amoxicillin 10kg

Colistin sulfate 2kg

Prednisolone 0.1kg

Lecithin 0.5kg

Cholesterol 0.5kg

Methyl hydroxybenzoate 0.5kg

Ethyl oleate 50L

The injection soybean oil adds to 100L

(1) is dissolved in the 2L ethyl oleate by measuring lecithin, cholesterol, methyl hydroxybenzoate, is A liquid;

(2) get the surplus ethyl oleate and pour in the colloid mill, start colloid mill, slowly add above-mentioned A liquid then, treat all to add fully, add oxireme-40-castor oil hydrogenated while stirring according to quantity;

(3) treat that whole supplementary material adds fully, add amoxicillin, colistin sulfate and prednisolone successively, begin to grind, the mode that adopts circular grinding and acyclic grinding to alternate is ground;

Experiment one, amoxicillin and colistin sulfate associating antibacterial action

Whether can strengthen the curative effect that encountered pathogenic bacterias such as swine escherichia coli, pig pasteurella multocida, Salmonella choleraesuls, Streptococcus suis, actinobacillus pleuropneumoniae, cow breast inflammatory staphylococcus aureus, cow breast inflammatory streptococcus are infected for inquiring into amoxicillin and colistine sulfate Combined application, the present invention has carried out the external associating antibacterial tests of two medicines.

1 material and instrument

1.1 supply the reagent article

Amoxicillin (AMO), content are 99.5%, lot number 20080312, Zhuhai United Laboratories Ltd;

Colistine sulfate (COS), content are 23694IU/mg, lot number N0710003J, Foochow Fu Xing Pharmacy stock Co., Ltd of beautiful pearl group;

1.2 strain subject

Isolated strains: swine escherichia coli 17 strains; 6 strains of pig pasteurellosis bacillus; Pig, Salmonella gallinarum be totally 8 strains; 10 strains of mammitis of cow staphylococcus aureus; Pig, bargen's streptococcus be totally 16 strains; Actinobacillus pleuropneumoniae 7 strains.

Quality Control bacterial strain: ATCC25922, ATCC29213, ATCC49619 are all available from China Veterinary Drugs Supervisory Inst..

1.3 culture medium

The MH broth bouillon, lot number 20071102, MH agar culture medium, lot number 20071103, Beijing extensive and profound in meaning star biotechnology responsibility company limited.Above-mentioned various culture medium prepares according to conventional method, after the process sterility test is qualified, places 4 ℃ of refrigerators to preserve, and is used to cultivate escherichia coli, Salmonella choleraesuls and staphylococcus aureus.Be used for cultured swine pasteurella multocida, chain also the culture medium of coccus then add the top grade hyclone of 5% (V/V) in above-mentioned compound method.The culture medium that is used for the cultured swine Actinobacillus pleuropneumoniae is the PPLO culture medium.

1.4 key instrument

(1) spectrophotometer: 721 types, the highly dense analytical tool in Shandong factory.

(2) micro-chessboard dilution method associating drug sensitive test plate: Tianjin gold chapter scientific and technological development company limited.

(3) continuous micro sample adding appliance, eight road micro sample adding appliances: U.S. Eppendorf company.

2 test methods

2.1 the preparation of medicine stock solution and preservation

Amoxicillin stock solution: with 0.1mol/L phosphate buffer (pH6.0) amoxicillin is made into the medicine stock solution that concentration is 1280 μ g/mL, the 0.2 μ m aperture filter of crossing with autoclaving filters, and divides to be filled in the 2mL centrifuge tube, puts in-20 ℃ of refrigerators and preserves, and is for use.

Colistin sulfate stock solution: use distilled water that colistin sulfate is made into the medicine stock solution that concentration is 2560 μ g/mL, the 0.2 μ m aperture filter of crossing with autoclaving filters, and divides to be filled in the sterilization 2mL plastic centrifuge tube, and sealing is put in-20 ℃ of refrigerators and preserved, and is for use.

2.2 the preparation of bacterium liquid

2.2.1 the preparation of escherichia coli, Salmonella, staphylococcus aureus bacterium liquid

Dip in and get a little bacterium colony from preserving culture of strains base inclined-plane, be inoculated in the MH cultured solution of broth, cultivate 18h, will cultivate the gained original bacteria liquid with 10 at 37 ℃ ^-1Doubling dilution, respectively as follows: 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Each bacterium liquid is got 10 respectively ^-6, 10 ^-7, 10 ^-8Concentration get 100 μ L in preprepared MH agar plate with micro sample adding appliance, L type Glass rod calcination sterilization after the cooling, is uniformly coated on media surface (sterility test is qualified) with bacterium liquid; Then plating medium is put into the water isolation type constant incubator, cultivate after 24 hours and observe, calculate bacterial concentration.Confirm the concentration of former bacterium stock solution, be diluted to 10 with sterile saline then ⁶CFU/ml, it is subsequent use to put 4 ℃ of refrigerators, and the holding time is no more than 6h.

2.2.2 the preparation of pasteurella multocida, streptococcus bacterium liquid

Dip in from the inclined-plane of preserving strain and to get a little lawn, in the inoculation blood MH cultured solution of broth, cultivate 18h at 37 ℃, with original bacteria liquid with 10 ^-1Doubly dilution, respectively as follows: 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Each bacterium liquid is got 10 respectively ^-6, 10 ^-7, 10 ^-8Concentration get 100 μ L in preprepared blood MH agar plate with micro sample adding appliance, with the calcination sterilization on flame of L type Glass rod, after the cooling, bacterium liquid evenly pushed away being dispersed in media surface (sterility test is qualified); Then plating medium is put into the water isolation type constant incubator, cultivate after 24 hours and observe, calculate bacterial concentration.Confirm the concentration of bacterium stock solution, be diluted to 10 with sterile saline then ⁶CFU/ml, it is subsequent use to put 4 ℃ of refrigerators, and the holding time is no more than 6h.

2.2.3 the preparation of actinobacillus pleuropneumoniae bacterium liquid

Dip in from the inclined-plane of preserving strain and to get a little lawn, be inoculated in the PPLO culture medium, cultivate 18h at 37 ℃, with original bacteria liquid with 10 ^-1Doubly dilution, respectively as follows: 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Each bacterium liquid is got 10 respectively ^-6, 10 ^-7, 10 ^-8Concentration get 100 μ L in preprepared PPLO culture medium with micro sample adding appliance, with the calcination sterilization on flame of L type Glass rod, after the cooling, bacterium liquid evenly pushed away being dispersed in media surface (sterility test is qualified); Then plating medium is put into the water isolation type constant incubator, cultivate after 24 hours and observe, calculate bacterial concentration.Confirm the concentration of bacterium stock solution, be diluted to 10 with sterile saline then ⁶CFU/ml, it is subsequent use to put 4 ℃ of refrigerators, and the holding time is no more than 6h.

2.3MIC mensuration

2.3.1 the mensuration of escherichia coli, Salmonella and staphylococcus aureus MIC

Adopt broth microdilution antifungal susceptibility test: with sterilization MH meat soup with amoxicillin stock solution, (amoxicillin is from 0.125-512 μ g/mL for 11 concentration of colistine sulfate stock solution doubling dilution; Colistine sulfate is from 0.0625-128 μ g/mL); Getting 100 μ L respectively adds in the 96 orifice plates trace hole; Add the finite concentration bacteria suspension afterwards, making the whole bacterial concentration of every Kongzui is 5 * 10 ⁵CFU/mL, and establish bacterial growth contrast and blank cultivates 24h in 37 ℃ of constant incubators, is MIC with minimum antibiotic single medicine drug level of no bacterial growth.Escherichia coli ATCC25922 is the Quality Control bacterium that MIC measures.

2.3.2 the mensuration of pasteurellosis bacillus and streptococcus m IC

Adopt broth microdilution antifungal susceptibility test: with serum broth with amoxicillin stock solution, (amoxicillin is from 0.125-128 μ g/mL for 11 concentration of colistin sulfate stock solution doubling dilution; Colistine sulfate is from 0.031-64 μ g/mL); Getting 100 μ L respectively adds in the 96 orifice plates trace hole; It is outstanding to add the finite concentration bacterium afterwards, and making the whole bacterial concentration of every Kongzui is 5 * 10 ⁵CFU/mL, and establish bacterial growth contrast and blank cultivates 24h in 37 ℃ of constant incubators, is MIC with minimum antibiotic single medicine concentration of no bacterial growth.

2.3.3 the mensuration of actinobacillus pleuropneumoniae MIC

With the PPLO culture medium with amoxicillin stock solution, (amoxicillin is from 0.125-128 μ g/mL for 11 concentration of colistin sulfate stock solution doubling dilution; Colistine sulfate is from 0.031-64 μ g/mL); Getting 100 μ L respectively adds in the 96 orifice plates trace hole; It is outstanding to add the finite concentration bacterium afterwards, and making the whole bacterial concentration of every Kongzui is 5 * 10 ⁵CFU/mL, and establish bacterial growth contrast and blank cultivates 24h in 37 ℃ of constant incubators, is MIC with minimum antibiotic single concentration of no bacterial growth.

2.4 associating drug sensitive test

Result according to the MIC of single medicine; Amoxicillin stock solution, colistin sulfate stock solution are become 6 different working concentration with the meat soup doubling dilution respectively; By the chessboard method design, two medicines are successively joined respectively in each hole of 96 orifice plates, every kind of antibacterials are got 50 μ l in every hole; Make every kind of medicine ultimate density in each hole be respectively 1/8MIC, 1/4MIC, 1/2MIC, 1MIC, 2MIC and the 4MIC of its single medicine, and establish single medicine contrast, bacterial growth contrast and the blank of two medicines respectively.Again with 10 ⁶The bacterium liquid 100 μ L of CFU/mL add in each hole, and the final concentration that makes antibacterial in each hole is 5 * 10 ⁵CFU/mL cultivates 24h for 37 ℃, observes and the record result.Every bacterial strain repeats chessboard test 3 times.Separately MIC when selecting the best of breed effect _{The coupling of first medicine}, MIC _{The coupling of second medicine}To calculate the FIC index.

FIC Index for Calculation and interpretation standard:

FIC index≤0.5, synergism;＞0.5～1, summation action;＞1～2, irrelevant effect;＞2, antagonism.

3 result of the tests

3.1 the external drug sensitive test result to escherichia coli, golden staphylococci etc. when amoxicillin and colistin sulfate list usefulness and coupling sees table 1-1～table 1-6.

Single usefulness of table 1-1AMO and COS and coupling are to the external drug sensitive test result (MIC unit is ug/mL) of swine escherichia coli

Single usefulness of table 1-2AMO and COS and coupling are to the external drug sensitive test result (MIC unit is ug/mL) of staphylococcus aureus

Table 1-3AMO and COS singly with and coupling to streptococcic external drug sensitive test result (MIC unit is ug/ml)

Single usefulness of table 1-4AMO and COS and coupling are to the external drug sensitive test result (MIC unit is ug/ml) of Salmonella

The external drug sensitive test result (MIC unit is ug/ml) to pasteurellosis bacillus of single usefulness of table 1-5AMO and COS and coupling

Single usefulness of table 1-6AMO and COS and coupling are to the external drug sensitive test result (MIC unit is ug/ml) of actinobacillus pleuropneumoniae

Can know that according to the associating drug sensitive test behind the drug combination, the MIC after the coupling of amoxicillin is 1/16～1 of single time spent MIC, the MIC value of part bacterial strain obviously descends; Especially to the coli strain and the staphylococcus aureus of some anti-amoxicillin, the MIC value of AMO is reduced to below the drug resistance breakpoint (breakpoint) after the coupling, makes Resistant strain become sensitive strain; MIC after the colistin sulfate associating is 1/8～1 of single time spent MIC, and the MIC value also has decline in various degree.

3.2AMO FIC exponential (seeing table 1-7) with the COS Combined application; AMO and COS Combined application; The FIC exponential concentrates on 0.5～1, shows that both couplings are summation action (70%～100%) to most of antibacterials, have 30% to be synergism to staphylococcus aureus; Then have 33.33% to be irrelevant effect to pasteurellosis bacillus, but all do not have antagonism.

After table 1-7AMO and the COS coupling to the influence of antibacterial MIC

4 conclusions

Above-mentioned experimental result shows; Amoxicillin, colistin sulfate are processed the compound preparation use streptococcus, staphylococcus aureus, pasteurellosis bacillus, escherichia coli, actinobacillus pleuropneumoniae, Salmonella are all had collaborative or summation action; Widen antimicrobial spectrum, strengthened antibacterial efficacy.

Experiment two, amoxicillin and colistin sulfate are united the research to escherichia coli protrusion-dispelling degree of thickening

For inquiring into the generation whether amoxicillin and colistine sulfate Combined application can delay drug resistance; The present invention with the standard broth dilution method determination amoxicillin, colistine sulfate to colibacillary minimum inhibitory concentration (MIC); And measured the amoxicillin to colibacillary protrusion-dispelling degree of thickening (MPC) with agar dilution; Investigated unite use with colistin sulfate after, the amoxicillin is to the variation of escherichia coli protrusion-dispelling degree of thickening and drug resistance frequency.

1 material and instrument

1.1 supply the reagent article

1.2 strain subject

Strain subject 9 strains, wherein escherichia coli reference culture (ATCC25922) is available from China Veterinary Drugs Supervisory Inst.; 8 strain swine escherichia colis are clinical separation strain in addition.

1.3 culture medium

The MH broth bouillon, lot number 20071102, MH agar culture medium, lot number 20071103, Beijing extensive and profound in meaning star biotechnology responsibility company limited.Above-mentioned various culture medium prepares according to conventional method, after the process sterility test is qualified, places 4 ℃ of refrigerators to preserve.

1.4 key instrument

2 test methods

2.1 the preparation of medicine stock solution and preservation

2.2 the preparation of bacterium liquid

2.3MIC mensuration

Adopt broth microdilution antifungal susceptibility test: with sterilization MH meat soup with amoxicillin stock solution, (amoxicillin is from 0.5-512 μ g/mL for 11 concentration of colistine sulfate stock solution doubling dilution; Colistine sulfate is from 0.031-32 μ g/mL); Getting 100 μ L respectively successively adds in each micro-hole of 96 orifice plates; In each hole, add the finite concentration bacteria suspension afterwards, making the whole bacterial concentration of every Kongzui is 5 * 10 ⁵CFU/mL, and establish bacterial growth contrast and blank, cultivate 24h in 37 ℃ of constant incubators, by the standard determination MIC of Standardization Research institute of U.S. clinical laboratory (CLSI) value.Escherichia coli ATCC25922 is the Quality Control bacterium that MIC measures.

2.4 protrusion-dispelling degree of thickening (MPC) is measured:

Protrusion-dispelling degree of thickening (MPC) is meant and prevents the required minimum antibacterials concentration of being selected property of medicament-resistant mutation bacterial strain enrichment amplification.MPC is theoretical to provide new thinking and reference frame for effectively suppressing bacterial resistance and formulating the antibacterials application strategy.

Single medicine MPC measures: in 20mL MH meat soup, 37 ℃ of overnight shakings are cultivated with single colony inoculation, and after 4 ℃ of 3000r/min centrifugal enrichment, antibacterial is resuspended in the 200mL MH meat soup (10 times of stock solution), shaken cultivation 6h, and bacterial concentration reaches 6 * 10 ⁹～9 * 10 ⁹During CFU/mL, collect whole bacterium liquid, after 4 ℃ of centrifugal 30min enrichments of 3000r/min, bacterial concentration is adjusted into 3 * 10 with fresh meat soup ¹⁰CFU/mL.Getting 100 μ L bacterium liquid places the 90mm diameter to contain on the M-H flat board of variable concentrations AMO, COS respectively; With the L rod bacterium is smoothened; Cultivate 72～96h for 35 ℃; Carry out colony counting, press document (Zhao X, Drlica K.Restricting the selection of antibioticresistant mutant bacteria:measurement and potential use of the mutantselection window [J] .J Infect Dis; 2002, method 185:561-565.) is calculated the MPC of single medicine.Its average is got in all experiment repetitions 3 times.

Drug combination MPC measures: get above-mentioned bacterium liquid 100 μ L place respectively contain 1 *, 2 *, 4 *, 8 *, 16 *, the AMO of 32 * MIC and being added with in the plate well of COS 2mg/L, with the L rod bacterium is smoothened, cultivate 72～96h for 35 ℃, count bacterium colony.The drug resistance frequency computation part is pressed document (Zhanel G G; Mayer M, Laing N, et al.Mutantprevention concentrations of levofloxacin; Ciprofloxacin alone and incombination with azithromycin cef-tazidime; Colistin (polymyxin E), meropenem, piperacillin tazobactam; And tobramycin gainst Pseudomonas aeruginosa [J] .Antimicrob, Agents Chemother.2006.50 (5): 2228-2230.): with the amoxicillin of variable concentrations, on the colistin sulfate flat board clump count divided by the inoculation clump count.Its average is got in all experiment repetitions 3 times.

2.5 data analysis adopts SPSS 10.0 statistical softwares that each group data is carried out rank test.

3 results

3.1 amoxicillin and colistin sulfate list medicine are seen table 2-1 to the MIC value of swine escherichia coli.

Table 2-1AMO and COS are to the MIC value (mg/L) of swine escherichia coli

3.2 the amoxicillin single with and unite use with colistin sulfate and the MPC of swine escherichia coli is seen with the MPC/MIC value show 2-2.

Table 2-2AMO is single with reaching and MPC and the MPC/MIC value (mg/L) of COS coupling to swine escherichia coli

Annotate: the MPC/MIC of the MPC/MIC usefulness single with it of AMO and COS coupling compares, and difference all has significance (P＜0.05)

3.3 amoxicillin and colistin sulfate are united use the drug resistance frequency of swine escherichia coli is seen table 2-3.

Table 2-3AMO and COS unite the drug resistance frequency of use to swine escherichia coli

Annotate: the drug resistance frequency has significance (P＜0.05) than its single time spent difference when AMO and COS coupling

4 conclusions

Above-mentioned experimental result shows, the MPC/MIC of amoxicillin and colistin sulfate drug combination descends 2～16 times than the MPC/MIC of the independent medication in amoxicillin, and behind the drug combination, the drug resistance frequency of amoxicillin is than its single time spent obviously descend (P＜0.05).Because the antibacterial mechanisms of AMO and COS is different, and different action target spots is arranged, so antibacterial needs could survive to two kinds of medicines while drug resistances; After both couplings, dwindle drug resistance and selected window, improved the threshold value of mutant bacteria; Reduce MPC/MIC ratio, MPC/MIC and drug resistance frequency are descended greatly.Therefore, after share with colistin sulfate, amoxicillin overriding resistance ability strengthens, and has reduced the generation of Resistant strain, therefore can delay chemical sproof generation.

The animal use suspensoid liquid effect experiment of experiment three, amoxicillin, colistin sulfate and prednisolone

1. test material and method

1.1 receive the reagent thing

1. the animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone according to the invention is pressed instance 3 preparations, is called A-C-P.

2. amoxicillin sodium for injection, commodity not Sa by name, Lukang Medical Co., Ltd., Shandong; I

3. colistin sulfate water soluble parenteral solution, commodity are called XIELITING, Beijing Xiang Tan animal pharmaceutical factory of middle peasant's large animal health product group;

4. ampicillin-colistin sulfate-dexamethasone suspension, commodity are called Multibio d, pharmaceutical factory of French Virbac;

5. amoxicillin, colistin sulfate suspension are pressed instance 3 preparations and are got, and do not contain prednisolone, are called A-C.

1.2 microbionation

Actinobacillus pleuropneumoniae for the serum I type, is numbered 259 available from China Veterinery Drug Inspection Office.After PPLO agar is cultivated 24 hours, be 1.0 * 10 through the counting dilution ⁶CFU/ml is subsequent use.The pig vaccine program is summarized as follows, every pig is taked to carry on the back a mode bondage on special support, and inoculation position is coated with 1% procaine local anaesthesia, and with 35 millimeters, No. 18 syringe needles are through thrusting between trachea two cartilaginous rings, with 0.05ml/kg (1.0 * 10 ⁶CFU/ml) the dose inoculation Actinobacillus pleuropneumoniae of body weight, negative control group is injected physiological saline solution with same dose.In inoculation administration after 4 hours.

1.3 experimental animal and processing

86 long white two-way cross pigs of Du Luoke that body weight is the 20-25 kilogram, the conventional feed of the antibiotic-free additive of feeding, pig freely drinks water.The after date of resting through a week is divided into 9 groups at random.Except that the blank group, all the other respectively organize every pig by 0.05ml/kg (1.0 * 10 ⁶CFU/ml) the dosage trachea injection inoculation Actinobacillus pleuropneumoniae of body weight, negative control group is injected physiological saline solution with same dose.After inoculation 4 hours, by the test design method administration of table 3-1.All pigs via intranasal application before experiment is gathered secretions and is carried out the antibacterial separation and Culture, and the result fails in nasal secretion, to detect Actinobacillus pleuropneumoniae.Each treated animal is raised under identical conditions, and daily ration composition and trophic level are just the same, and the test period was 2 weeks.

Table 3-1 EXPERIMENTAL DESIGN

Group	Number of animals (head)	Medicine	Dosage and method
				High dose group	10	A-C-P	Intramuscular injection, AMO 20mg/kg, COS 50,000IU/kg and PRE 0.1mg/kg body weight, 1 time on the one, logotype 5 days.
Middle dose groups	10	A-C-P	Intramuscular injection, AMO 10mg/kg and COS 25,000IU/kg and PRE 0.05mg/kg body weight, 1 time on the one, logotype 5 days.
				Low dose group	10	A-C-P	Intramuscular injection, AMO 5mg/kg and COS 12,500IU/kg and PRE 0.025mg/kg body weight, 1 time on the one, logotype 5 days.
Medicine matched group I	10	Sa not	Intramuscular injection, AMO 10mg/kg body weight, 2 times on the one, logotype 5 days.
				The medicine control Group II	10	XIELITING	Intramuscular injection, COS 25,000IU/kg body weight, 2 times on the one, logotype 5 days.
Medicine control Group II I	10	A-C	Intramuscular injection, AMO 10mg/kg and COS 25,000IU/kg body weight, 1 time on the one, logotype 5 days.
				Medicine matched group IV	10	Multibio d	Intramuscular injection, ampicillin 10mg/kg and COS25,000IU/kg and dexamethasone 0.025mg/kg body weight, 1 time on the one, logotype 5 days.
Positive controls	10	Infect not administration	-
				Negative control group	6	Do not infect not administration	-

Annotate: on behalf of amoxicillin, COS, AMO represent colistin sulfate, PRE to represent prednisolone in the table.

1.4 feeding and management and observation

The body weight of each test group every pig of weighing during respectively at on-test and off-test.The clinical manifestation of every pig of close observation every day behind the clinical artificial challenges such as morning every day, observation in evening respiration, appetite, the mental status behind the artificial challenge, as breathing, rectal temperature is measured in performances such as the appetite and the mental status.Morbidity number and the death toll of pig respectively organized in record.Dead pig is cutd open inspection, observe the variation of lung, liver spleen, heart, mesentery etc.All dead pigs and off-test rear section pig are slaughtered; Get liver, spleen is coated with tryptose soya agar plate culture medium, cultivates 20～48 for 37 ℃, whether inspection has bacterial growth; To the antibacterial that detects, carry out whether a series of laboratorys detections are Actinobacillus pleuropneumoniae with inspection.

1.5 curative effect judging standard

Cure: duration of test, medication pig clinical symptom disappearance, it is normal that spirit, appetite, body temperature, breathing etc. recover, weightening finish near or above the blank group; Cuing open the visible pulmonary in inspection back has the old focus, and antibacterial separates no Actinobacillus pleuropneumoniae.Account for the ratio calculating cure rate that this group is tested a number according to curing a number.

Produce effects: duration of test, symptom obviously alleviates or disappears, and body weight is no significant change or slightly increase before and after test.Cut open and examine the back it is thus clear that there is certain pathological changes in pulmonary, antibacterial separates the back it is thus clear that carry Actinobacillus pleuropneumoniae.The ratio that accounts for this group test number according to the produce effects number is calculated obvious effective rate.

Effectively: duration of test, the mental status take an evident turn for the better, appetite increases, slight ventral breathing or cough are arranged.The ratio that accounts for this group test number according to effective number is calculated effective percentage, and effective number comprises a produce effects number and cure a number when calculating effective percentage.

Invalid: duration of test, symptoms such as test pig spirit, appetite, breathing, body temperature still do not take a turn for the better even worsen dead person.The ratio that accounts for this group test number according to invalid number is calculated inefficiency.

1.6 weightening finish

The body weight of every pig of weighing is calculated average gain in weight when on-test and off-test.

1.7 data statistic analysis

Adopt X 2 test (SPSS10.0 statistical analysis software) to carry out statistical analysis.P＜0.05 expression significant difference, P＜0.01 expression difference is extremely remarkable, and P＞0.05 expression difference is not remarkable.

2 result of the tests and brief summary

2.1 blank group pig is only good in duration of test spirit, diet is desired normally, 38 ℃～40 ℃ of body temperature belong to normal range.

2.2 positive infect the matched group swinery in the inoculation back 2～3h, begin to occur 0.5～1 ℃ of transience vomiting, rapid breathing, fervescence.Behind 4～6h, rapid breathing also is tangible ventral breathing, fervescence 1.5-2.5 ℃.Dyspnea finally occurs, be the sitting position of dog gesture, very fast dead, acute death mostly occurs at 4-24h, and the positive matched group that infects has 7 pigs dead in 24h, and other has 3 pigs dead in infecting 24～48h.

2.3 sick pig is after death flowed out a large amount of color secretions in oral cavity and nostril.The visible thoracic cavity of postmortem is long-pending to have pale red blood sample liquid, and a large amount of color liquid are flowed out in lungs kermesinus, congested enlargement.Be full of the mucus foam appearance exudate of band color in the trachea and bronchus, the pig that the course of disease is long slightly can be found lungs surface adularescent fibrinous exudate, so with thoracic cavity adhesion, the pig that the course of disease is long, spleen, liver furvous.

2.4 behind trachea inoculation Actinobacillus pleuropneumoniae bacterium liquid, typical pleuropneumonia clinical symptoms all appears in all inoculation pigs.With positive controls relatively, each organizes medicine after the medication treatment, and clinical symptoms has improvement in various degree, and the cough of the pig that promptly falls ill, dyspnea, spirit are depressed etc., and symptom is obviously slowed down, appetite is progressively recovered; The survival number average of each medication group pig is significantly higher than positive controls (P＜0.05), shows that each medication all has the certain protection effect to pig pleuropneumonia.A-C-P is high, the cure rate of middle dose groups, effective percentage, average growth rate be all apparently higher than A-C-P low dose group, not Sa group and XIELITING group (P＜0.05), shows that A-C-P is high, the curative effect of middle dose groups is superior to the A-C-P low dose group, not Sa group and XIELITING group; Cure rate, effective percentage, average growth rate between A-C-P height, middle dose groups are compared no significant difference (P＞0.05), are illustrated in the clinical treatment porcine contagious pleuropneumonia course of infection, and A-C-P is rational with middle dosage as clinical recommended drug dosage; Dose groups is compared with marketed drugs Multibio d group among the A-C-P; Its cure rate, obvious effective rate and effective percentage do not have significant difference (P＞0.05); But the cure rate of dose groups, obvious effective rate all exceed 10% among the A-C-P; Effective percentage exceeds 20%, and marketed drugs Multibio d has 20% inefficiency, shows that general curative effect is superior to marketed drugs Multibio d to A-C-P under the dosage condition waiting.Concrete test data is seen table 3-2.

Table 3-2 respectively organizes therapeutic test result (X ± SD)

*: compare significant difference (p＜0.05) with A-C-P height, middle dose groups, medicine matched group IV;

★: compare significant difference (p＜0.05) with the normal healthy controls group

2.5 breathe, in the observation process of appetite and mental status etc.; Find that A-C-P is high, middle dose groups and marketed drugs Multibio d group have 7,6,5 pigs can return to health status in back 7 days in infection respectively, and the A-C group, Sa group and XIELITING group only are not respectively 2,1,0.A-C group, Sa group and XIELITING group time of returning to health status does not concentrate on metainfective 9-14 days mostly; High, middle dose groups of long A-C-P far away recovery time and marketed drugs Multibio d organize; Reason is all to contain glucocorticoids anti-inflammatory component (containing prednisolone and dexamethasone respectively in A-C-P and the Multibio d prescription) in the pharmaceutical formulation that A-C-P organizes and marketed drugs Multibio d organizes; Glucocorticoid medicine has effects such as antiinflammatory, antiendotoxin, enhancing human body immunity power; Therefore in anti-infective therapy, play the effect of auxiliary treatment, be beneficial to the recovery of ill domestic animal health-physical fitness.From the result of weightening finish in 15 days, also can find out; The treatment group that contains glucocorticoid medicine is under suitable dosage situation; Degree and the normal healthy controls group of weightening finish do not have marked difference, this with treatment afterwards body recovery to the time length of health level certain dependency is arranged.

2.6 this test and Selection Multibio d as wherein a kind of control drug; The main component of mainly considering this medicine is comparatively similar with suspension composition of the present invention; This medicine is that French Virbac produces, and is used for the treatment (this product is not as yet in China's registration at present) of mammitis of cow in the European and American areas.Result of the test shows; Suspension product of the present invention all is superior to Multibiod from the curative effect judge index aspects such as time of cure rate, effective percentage and body recovery normal condition; Be illustrated on the medicament selection of control porcine contagious pleuropneumonia, product of the present invention is a choice drug.Both compare with the A-C product (no prednisolone) of contrast, and there were significant differences (p＜0.05) aspect time of recovering normal condition and weightening finish, thereby show that anti-inflammatory component has played important function in therapeutic process.

Test the efficacy experiment of the animal use suspensoid liquid of four amoxicillin, colistin sulfate and prednisolone to porcine contagious pleuropneumonia and colibacillosis mixed infection

Experiment purpose: investigate suspension product of the present invention and commercially available product curative effect to porcine contagious pleuropneumonia and colibacillosis mixed infection.

1. test material and method

1.1 receive the reagent thing

2. ampicillin-colistin sulfate-dexamethasone suspension, commodity are called Multibio d, pharmaceutical factory of French Virbac (unregistered in China);

3. amoxicillin, colistin sulfate suspension are pressed instance 3 preparations and are got, and do not contain prednisolone, are called A-C.

1.2 experimental animal: select the piglet at pig farm 2-3 monthly ages such as Guangxi, Hunan, Beijing, Shandong, kind be Du Luoke * length white * big York ternary is assorted, the white binary of Du Luoke * length is mixed etc.Natural infection Pulmonis Sus domestica escherichia coli and contagious pleuropneumonia.

1.3 diagnosis basis

1.3.1 clinical symptom: suffer from the pig sudden onset, spirit is depressed, and appetite reduces or useless food, 40.5～42 ℃ of hyperpyrexias, visual mucosa flushing; Cough, sneeze, accelerated breathing is breathed, and dog sits the gesture ventral breathing of dehiscing; The sick pig seriality cough that has, severe patient is breathed and is the devil, and nose stream serosity or mucus nose liquid wave on foot, ataxia, or do not rise, drive extremity and be duck swimming shape; with crouching Under head, neck, the jaw, under the abdomen, death by suffocation the sick last depletion of pig of aubergine appears and in limb end skin.

1.3.2 pathological change: the pig whole body pale asphyxia of dying of illness, mouth and nose flow out pale red or red foam appearance liquid.Trachea, bronchus are full of foam solution, and pleural space has flaxen hydrops, and pleural space serous coat and internal organs tunicle have fiber disposition thin film, normal and pleural space adhesion; Lung, heart, liver enlargement, hyperemia, hemorrhage; The thickened stomach wall of stomach bottom, gel-shaped liquid, strip ecchymosis and petechia are arranged, the intestinal mucosa edema has point-like and strip hemorrhage, and the intestinal mucosa that has has hemorrhagic inflammation.

1.3.3 pathogen separation is differentiated

1.3.3.1 the small intestinal leading portion content of gathering, liver, spleen, the heart, diseased region lymph node tissue inoculation plain agar, maconkey agar are cultivated the back and observed: 1. the plain agar flat board grows the bacterium colony of median size, circle, translucent, canescence, dewdrop shape; The maconkey agar flat board grows red bacterium colony.2. dyeing microscopic examination: the negative bacillus pumilis of gram stain microscopy.3. biochemical characteristic: this bacterium can ferment maltose, lactose, glucose, mannitol, produce indole, M-R test the positive, does not produce hydrogen sulfide, not decomposing urea.Judge that this bacterium is escherichia coli.

1.3.3.2 tissue inoculation PPLO agar plates such as the lung of gathering, the heart, spleen, kidney are cultivated the back and observed: 1. bacterium colony is the needle point size, circle, neat in edge, smooth surface, canescence dewdrop shape; 2. dyeing microscopic examination: the little coccobacillus of the negative redness of gram stain microscopy; 3. biochemical characteristic: this bacterium can ferment fructose, lactose, azymic mannitol, sorbitol, rhamnose and mannose.Indole, C.I. 13020., V-P test negative.The hydrolyzable urease.Be judged to be Actinobacillus pleuropneumoniae.

1.4 experimental animal and processing

Adopt 112 piglets of the method for a plurality of centers, a plurality of plant, STOCHASTIC CONTROL grouping, select as the case of this experiment with 6 different breeding fields.The feed conventional feed of antibiotic-free additive, pig freely drinks water.All pigs only are divided into 4 groups at random.Test design method administration by table 4-1.

Table 4-1 EXPERIMENTAL DESIGN

Medicine	Number of animals (head)	Dosage and method
			A-C-P	35	Intramuscular injection, AMO 10mg/kg, COS 25,000IU/kg and PRE 0.05mg/kg body weight, 1 time on the one, logotype 4 days.
Multibio d	32	Intramuscular injection, AMP 10mg/kg and COS 25,000IU/kg and DEX 0.025 mg/kg body weight, 1 time on the one, logotype 4 days.
			A-C	30	Intramuscular injection, AMO 10mg/kg and COS 25,000IU/kg body weight, 1 time on the one, logotype 4 days.
Ill not administration group	15	-

Annotate: on behalf of amoxicillin, AMP, AMO represent ampicillin, COS to represent colistin sulfate, DEX to represent dexamethasone, PRE to represent prednisolone in the table.

1.5 efficacy of medicine observing: finish totally 12 days from beginning to be administered into to observe, observe clinical symptoms such as the breathing of respectively organizing test pig, appetite, the mental status, and perform itemized record.

1.6 curative effect judging standard

Cure: the test pig mental status, appetite are breathed after 4 days, the body temperature each side all recovers normally in administration, clinical symptoms such as limpings, dysentery no longer occur, and nothing recurs after the drug withdrawal.Respectively after drug withdrawal the 3rd, 6 and off-test time statistics cure rate.The ratio that accounts for this group number according to a healing number is calculated cure rate.

Effectively: the test pig mental status takes an evident turn for the better after 4 days, appetite increases, slight ventral breathing or cough are arranged, and limping, dysentery phenomenon are arranged in administration.Effective number is total effective number with curing a number sum, and the ratio that accounts for this group test number according to total effective number is calculated total effective rate.

Invalid: administration after 4 days symptoms such as test pig spirit, appetite, breathing body temperature, limping, dysentery still do not take a turn for the better even worsen dead person.The ratio that accounts for this group test number according to invalid number is calculated inefficiency.

1.6 data statistic analysis

2 result of the tests and brief summary

2.1 the pig of ill not administration matched group is through observing, symptom has no significant change or increases the weight of even death in 5 days, promptly takes treatment to handle for reducing the loss.

2.2A-C-P group, Multibio d group and A-C group are respectively 82.9%, 75.0% and 73.3% to the cure rate of porcine contagious pleuropneumonia and colibacillosis mixed infection, total effective rate is respectively 91.4%, 81.3%, 80.0%; Concrete test data is seen table 4-2.

Table 4-2 respectively organizes therapeutic test result (X ± SD)

*: compare significant difference (p＜0.05) with the A-C-P group; ★: expression spontaneous recovery rate.

2.3 statistical results show: A-C-P group, Multibio d group and A-C group are compared with ill not administration group; Its cure rate, total effective rate difference is (P＜0.01) very significantly, shows that three kinds of medicines all have good curative effect to porcine contagious pleuropneumonia and colibacillosis mixed infection.The A-C-P group is compared with A-C group, marketed drugs Multibio d group; Its total effective rate difference is remarkable (P＞0.05) not; But the total effective rate of A-C-P group exceeds 11.4% and 10.1% respectively than A-C group, marketed drugs Multibiod group, shows that the general curative effect of A-C-P is superior to A-C and marketed drugs Multibio d.

This mainly investigates the investigation that index has been carried out 3 stages 2.4 this test is to cure rate; Result of the test shows, 3 kinds of medicines all had higher cure rate on the 3rd day after drug withdrawal, but effect is different; After the drug withdrawal the 3rd day; The cure rate of A-C group is compared with the A-C-P group, significant difference (P＜0.05), and the cure rate of A-C group is organized the no significant difference (P＞0.05) of comparing with marketed drugs Multibiod; But its cure rate is lower by 15.6% than marketed drugs Multibio d, other stage when off-test (after the drug withdrawal the 6th day with) no significant difference of comparing (P＞0.05).Show that the time that A-C-P group, Multibio d group make the disease pig return to normal condition is smaller than the A-C group, reason is that A-C-P group, Multibiod assembly Fang Zhongjun contain anti-inflammatory component, helps the recovery of ill domestic animal body.

2.5 take all factors into consideration from total effective rate and the length of body recovery time; The general curative effect of suspension product of the present invention all is superior to A-C and marketed drugs Multibio d, shows that suspension product of the present invention more is applicable to the treatment of porcine contagious pleuropneumonia and colibacillosis mixed infection.

Claims

1. an animal use suspensoid liquid that contains amoxicillin, colistin sulfate and prednisolone is to be the animal use suspensoid liquid of effective ingredient with amoxicillin, colistin sulfate and prednisolone; The content of each active drug composition is in the said animal use suspensoid liquid: amoxicillin 10-20%, colistin sulfate 0.5-2.5%, prednisolone 0.02-0.05%; The % implication is g/100mL.

2. animal use suspensoid liquid according to claim 1 is characterized in that: also comprise following excipient substance in the said animal use suspensoid liquid: dispersion medium, suspending agent, wetting agent, antioxidant and antiseptic.

3. animal use suspensoid liquid according to claim 2 is characterized in that: said dispersion medium is selected from one or both compound in injection soybean oil, ethyl oleate, glyceryl triacetate, myristic acid isopropyl ester and the injection Oleum Ricini.

4. animal use suspensoid liquid according to claim 2 is characterized in that: said suspending agent is selected from one or both compound in aluminium stearate, polyvinylpyrrolidone, lecithin, cholesterol, vaseline, sodium carboxymethyl cellulose and the castor oil hydrogenated; Suspending agent content is 0.2-1.5%, and the % implication is g/100mL.

5. animal use suspensoid liquid according to claim 2 is characterized in that: said wetting agent is selected from one or both in tween 80, Arlacel-60, Arlacel-80 and oxireme-40-castor oil hydrogenated; Wetting agent content is 0.2-1.3%, and the % implication is ml/100mL.

6. animal use suspensoid liquid according to claim 2 is characterized in that: said antioxidant is selected from one or both in vitamin E, thiourea, butylhydroxy anisole, sodium metabisulfite and the gallic acid third lipoprotein; Antioxidant content 0.01-1%, the % implication is g/100mL.

7. animal use suspensoid liquid according to claim 2 is characterized in that: said antiseptic is selected from one or both in benzyl alcohol, methyl hydroxybenzoate and the propylparaben; Antiseptic content 0.005-1%, the % implication is g/100mL.

8. animal use suspensoid liquid according to claim 2 is characterized in that: the content of each composition is in the said animal use suspensoid liquid: amoxicillin 10-20%, colistin sulfate 0.5-2.5%; Prednisolone 0.02-0.05%; Suspending agent 0.1-2%, wetting agent 0.2-2%, antioxidant 0.01-1%; Antiseptic 0.005-1%, all the other are dispersion medium; Wherein, wetting agent % implication is ml/100mL, and other component % implication is g/100mL.

9. one kind prepares the arbitrary said method that contains the animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone of claim 2 to 8, may further comprise the steps:

2) dispersion medium of getting formula ratio 40-80% (V/V) is poured in the colloid mill, starts colloid mill, slowly adds above-mentioned A liquid then, treats all to add fully, adds wetting agent while stirring;

4) inspection fineness of the particles stops after meeting the requirements grinding, and adds dispersion medium to formula ratio, mixing, and fill, sealing is sterilized, and obtains containing the animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone.