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

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

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

technical field

The invention belongs to the field of animal husbandry pharmaceutical preparations, and in particular relates to a veterinary compound suspension containing amoxicillin, colistin sulfate and prednisolone and a preparation method thereof.

Background technique

With the rapid development of my country's livestock and poultry breeding industry, various pathogenic bacteria have become more and more complicated in veterinary clinics in recent years. Negative bacterial infection brings great difficulties to veterinary prevention and control work. Due to the lack of mature compound preparations that have good effects on various pathogenic bacteria, veterinary workers often need to use a variety of single drug preparations in combination to treat mixed infectious diseases, and often use glucocorticoids such as Splash in the early stage of infection. Nisolone, dexamethasone and other adjuvant therapy. Therefore, to treat a disease, it is often necessary to give multiple pharmaceutical preparations at the same time to achieve the desired therapeutic effect, which not only brings a huge workload to the veterinary staff, but also brings great stress to the sick animals. Therefore, it is of great significance to develop a long-acting compound preparation that has good control effects on a variety of pathogenic bacteria.

In terms of research on compound preparations for the prevention and treatment of multi-pathogen mixed infectious diseases, there is no relatively mature product on the market in China. Bacterial oily compound suspension solution, but its main purpose is to treat lactating cow mastitis caused by Gram-positive bacteria and negative bacteria; French Vic company has also successfully developed a compound of ampicillin, colistin sulfate and dexamethasone Suspension for injection, but this product is also mainly used for the treatment of cow mastitis. The above two existing compound preparations are mainly suitable for the treatment of dairy cow mastitis, and the scope of application is narrow, so they cannot satisfy the mixed infectious diseases of various animals (especially pigs).

Contents of the invention

The invention provides a veterinary medicine containing amoxicillin, colistin sulfate and prednisolone with definite curative effect for preventing mixed infection and secondary infection caused by Gram-positive bacteria and Gram-negative bacteria in animals. Compound suspension.

In order to solve the problems of the technologies described above, the present invention takes the following technical solutions:

A veterinary suspension containing amoxicillin, colistin sulfate and prednisolone is the veterinary suspension with amoxicillin, colistin sulfate and prednisolone as active ingredients.

The content of each active ingredient in the veterinary suspension is: amoxicillin 5-30% (weight/volume percentage concentration, g/100ml), preferably 10-20% (g/100ml); colistin sulfate 0.25-3% (g/100ml), preferably 0.5-2.5% (g/100ml); prednisolone 0.01-0.1% (g/100ml), preferably 0.02-0.05% (g/100ml).

The veterinary suspension also includes the following pharmaceutical adjuvants: dispersion medium, suspending agent, wetting agent, antioxidant and preservative.

The dispersion medium may be selected from one or both of soybean oil for injection, ethyl oleate, glycerol triacetate, isopropyl myristate and castor oil for injection.

The suspending agent can be selected from one or both of aluminum stearate, polyvinylpyrrolidone, lecithin, cholesterol, vaseline, sodium carboxymethylcellulose and hydrogenated castor oil.

The wetting agent may be selected from one of Tween-80, Span-60, Span-80 and epoxyethylene-40-hydrogenated castor oil.

The antioxidant may be selected from one of vitamin E, thiourea, tert-butylhydroxyanisole, sodium metabisulfite and propyl gallate.

The preservative may be selected from one of benzyl alcohol, methylparaben and propylparaben.

Specifically, the content of each component in the veterinary suspension is: amoxicillin 5-30% (g/100ml), preferably 10-20% (g/100ml); colistin sulfate 0.25-3 % (g/100ml), preferably 0.5-2.5% (g/100ml); prednisolone 0.01-0.1% (g/100ml), preferably 0.02-0.05% (g/100ml), suspending agent 0.1- 2% (g/100ml), 0.2-2% (g/100ml) wetting agent, 0.01-1% (g/100ml) antioxidant, 0.005-1% (g/100ml) preservative, and the rest is dispersing medium .

The second object of the present invention is to provide a method for preparing the above-mentioned veterinary suspension containing amoxicillin, colistin sulfate and prednisolone.

The preparation method provided by the present invention may comprise the following steps:

1) Dissolving or dispersing the suspending agent, antioxidant and preservative in the thermal dispersion medium to obtain liquid A;

2) Pour 40-80% of the dispersing medium into the colloid mill, start the colloid mill, and then slowly add the above-mentioned liquid A, and add the wetting agent while stirring;

3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in sequence, and then grind in an alternate manner of circular grinding and non-circular grinding;

4) check particle fineness, stop grinding after meeting the requirements, add dispersing medium to formula quantity, mix, fill, seal, sterilize, obtain the veterinary medicine containing amoxicillin, colistin sulfate and prednisolone of the present invention Use a suspension.

In the preparation method of the above-mentioned medicine, the dispersion medium can be selected from one or both of soybean oil for injection, ethyl oleate, glycerol triacetate, isopropyl myristate and castor oil for injection.

The suspending agent can be selected from one or both of aluminum stearate, polyvinylpyrrolidone, lecithin, cholesterol, vaseline, sodium carboxymethylcellulose and hydrogenated castor oil.

The wetting agent can be selected from one or both of Tween-80, Span-60, Span-80 and epoxyethylene-40-hydrogenated castor oil.

The antioxidant can be selected from one or two of vitamin E, thiourea, tert-butyl hydroxyanisole, sodium metabisulfite and propyl gallate.

The preservative can be selected from one or both of benzyl alcohol, methylparaben and propylparaben.

Specifically, the content of each component in the veterinary suspension is: amoxicillin 5-30% (g/100ml), preferably 10-20% (g/100ml); colistin sulfate 0.25-3 % (g/100ml), preferably 0.5-2.5% (g/100ml); prednisolone 0.01-0.1% (g/100ml), preferably 0.02-0.05% (g/100ml), suspending agent 0.1- 2% (g/100ml), 0.2-2% (g/100ml) wetting agent, 0.01-1% (g/100ml) antioxidant, 0.005-1% (g/100ml) preservative, and the rest is dispersing medium .

The usage and dosage of the above veterinary suspension is generally 0.1-0.2mL/kg body weight, once a day, for 3-5 consecutive days. The injection dose and course of treatment can be adjusted appropriately according to the actual situation.

The invention provides a veterinary suspension containing amoxicillin, colistin sulfate and prednisolone and a preparation method thereof. Advantage of the present invention and positive effect are as follows:

(1) Using amoxicillin and colistin sulfate, two antibiotics with different antibacterial mechanisms, the combined use of the two drugs has brought into play the synergistic antibacterial advantages of the two drugs, broadened the antibacterial spectrum, enhanced antibacterial efficacy, and reduced and delayed the emergence of drug resistance .

(2) Prednisolone is absorbed rapidly after administration, and the blood drug concentration is high, and tissue affinity is strong, can arrive at inflammatory site rapidly and play anti-inflammatory effect, and suspension of the present invention is effective to mixed infectious disease, provides for veterinary clinic a good formulation.

(3) The combined use of amoxicillin, colistin sulfate and prednisolone broadens the antibacterial spectrum, enhances the antibacterial efficacy, avoids the limitations brought about by single drug use, delays the emergence of drug resistance, and has antibacterial properties. Inflammation, anti-toxin effect. In addition, the compound preparation only needs to be administered once a day, which can not only reduce the stress response to animals caused by frequent administration (the original single drug needs to be administered two or three times a day), but also save manpower, material resources and financial resources.

(4) Suspension of the present invention adopts oily solvent as the dispersion medium of preparation, not only solves the difficult problem that amoxicillin is easy to degrade in aqueous solution, and in oily suspension, colistin sulfate is released slowly in vivo, The blood drug concentration is stable, and the toxic and side effects of colistin sulfate are reduced.

In addition, the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone of the present invention adopts dispersion method to disperse amoxicillin, colistin sulfate and prednisolone in the solvent, and add The suspending agent, wetting agent and antioxidant are obtained by colloid milling, the production process is simple, the cost is reduced, and industrialization is easy to realize.

In summary, the present invention organically combines the time-dependent antibiotic amoxicillin and the concentration-dependent antibiotic colistin sulfate, which not only makes good use of the antibacterial effect of a single drug, but also brings into play the synergistic antibacterial advantages of the combination of the two drugs , The combination of the two drugs expands the antibacterial spectrum and enhances the antibacterial efficacy. At the same time, combined with the anti-inflammatory and anti-inflammatory advantages of prednisolone, the combination of the three has anti-infection and anti-inflammatory effects. Aiming at the popular characteristics of mixed infection or secondary infection in veterinary clinical livestock and poultry diseases in my country in recent years, the present invention is clinically mainly used for preventing mixed infection and secondary infection caused by Gram-positive bacteria and Gram-negative bacteria in animals. The preparation process of the invention is simple, easy to realize industrialization, low in cost, low in toxicity and high in efficiency, and has extremely high popularization and application value.

The present invention will be described in further detail below in conjunction with specific embodiments.

Detailed ways

The present invention is a part of the national "Eleventh Five-Year" science and technology support project (project name: development and application of new veterinary compound preparation; project number: 2006BAD31B08).

In order to provide a compound injection that can be generally used for mixed infectious diseases of various animals, the present invention takes a variety of common pathogenic bacteria isolated from clinical cases of different animals such as pigs, cattle, and chickens as research objects. A compound combination with good synergistic effect and strong effect on Gram-positive bacteria and Gram-negative bacteria was screened out from various commonly used drugs such as floxacin, amoxicillin, tylosin, and colistin sulfate ( Amoxicillin and colistin sulfate), combined with the anti-inflammatory effect of prednisolone, successfully developed amoxicillin, colistin sulfate, prednisolone which can be widely used in the prevention and treatment of mixed infectious diseases of various animals Compound injection.

The present invention has the advantage of developing three kinds of medicines, amoxicillin, colistin sulfate and prednisolone, into a compound preparation, which overcomes the stress response caused by simultaneous administration of multiple medicines to animals and reduces the workload of veterinary personnel. It is especially important that this compound preparation not only overcomes the defects of the narrow antibacterial spectrum of the single preparation and the small scope of application of the existing compound preparation, but also enhances the antibacterial efficacy of amoxicillin and colistin sulfate, and delays the drug resistance of these two drugs. sexual production.

The invention provides a compound preparation containing amoxicillin, colistin sulfate and prednisolone. In the process of compound formulation, special consideration should be given to the synergy between drugs, and appropriate pharmaceutical excipients should be screened to form a preparation with definite curative effect and good stability.

The medicine used in the present invention, optimal use has time-dependent antibiotic amoxicillin and concentration-dependent antibiotic colistin sulfate, two kinds of antibiotics are used in combination, can synergize antibacterial advantage, expand antibacterial spectrum, strengthen antibacterial effect (referring to follow-up experiment one and Experiment 2); combined with the use of prednisolone, which has anti-inflammatory and anti-inflammatory advantages, the combined use of the three has anti-infection and anti-inflammatory effects (see follow-up experiments 3 and 4—clinical effect tests). Wherein the content of each drug component in the injection: amoxicillin 5-30% (g/100ml), preferably 10-20% (g/100ml); colistin sulfate 0.25-3% (g/100ml), preferably 0.5-2.5% (g/100ml); prednisolone 0.01-0.1% (g/100ml), preferably 0.02-0.05% (g/100ml).

In the present invention, compound medicine is combined with suitable pharmaceutical auxiliary materials to prepare veterinary suspension, including the use of pharmaceutical auxiliary materials such as dispersing medium, suspending agent, wetting agent, antioxidant and preservative.

Dispersion medium: It can be selected from soybean oil for injection, ethyl oleate, glyceryl triacetate, isopropyl myristate and castor oil for injection, preferably ethyl oleate and injection Composite dispersion medium for soybean oil. In the present invention, an oily solvent is specially selected as the dispersion medium of the preparation, which solves the problem that amoxicillin is easily degraded in an aqueous solution, and in the oily suspension, colistin sulfate is slowly released in the body, and the blood drug concentration is stable, reducing Toxic and side effects of colistin sulfate.

Suspending agent: can be selected from aluminum stearate, polyvinylpyrrolidone, lecithin, cholesterol, petrolatum, sodium carboxymethylcellulose and hydrogenated castor oil, the type and amount of suspending agent Adjust according to the amount of drug and the type of dispersing medium, so as to form a stable injection. The suspending agent content in the injection is 0.1-2% (g/100ml).

Wetting agent: can be selected from one of Tween-80, Span-60, Span-80, and epoxyethylene-40-hydrogenated castor oil, and a single component should be used as much as possible in the pharmaceutical process, but it does not exclude Use multiple combinations. The wetting agent content in the injection is 0.2-2% (ml/100ml).

Antioxidant: one of vitamin E, thiourea, tert-butyl hydroxyanisole, sodium metabisulfite and propyl gallate can be selected, and a single component should be used as much as possible in the pharmaceutical process, but it does not exclude the use of multiple compounds The way. The antioxidant content in the injection is 0.01-1% (g/100ml).

Preservatives: can be selected from one of benzyl alcohol, methylparaben and propylparaben, and use a single component as much as possible in the pharmaceutical process, but it does not exclude the use of multiple compound methods. The content of preservative in the injection is 0.005-1% (g/100ml).

The content of each component in the veterinary suspension formed according to the present invention can be: amoxicillin 5-30% (g/100ml), preferably 10-20% (g/100ml); colistin sulfate 0.25-3 % (g/100ml), preferably 0.5-2.5% (g/100ml); prednisolone 0.01-0.1% (g/100ml), preferably 0.02-0.05% (g/100ml), suspending agent 0.1- 2% (g/100ml), 0.2-2% (g/100ml) wetting agent, 0.01-1% (g/100ml) antioxidant, 0.005-1% (g/100ml) preservative, and the rest is dispersing medium (ml).

The present invention is further illustrated below with specific examples. The following examples 1-10 adopt the best experimental scheme of the present invention, and those of ordinary skill in the art can obviously think of some similarities and alternatives according to the disclosure of the present invention, which should all belong to the disclosure of the present invention. The methods used in the examples are conventional methods unless otherwise specified.

Example 1

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 20kg,

Colistin Sulfate 1.5kg,

Prednisolone 0.04kg,

Aluminum stearate 1kg,

Span-80 1L,

Propyl gallate 0.02kg,

Benzyl alcohol 0.5kg

Add soybean oil for injection to 100L.

Prepare the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone with the present invention, and concrete method comprises the following steps:

1) Adding the formulated amount of aluminum stearate and propyl gallate into 2L of soybean oil for injection, heating and stirring to dissolve them completely, adding the formulated amount of benzyl alcohol to obtain liquid A;

2) Take 60L of soybean oil for injection and pour it into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until it is completely added, add the formula amount of Span-80 while stirring;

3) After all the excipients are added completely, sequentially add amoxicillin, colistin sulfate and prednisolone in the prescribed amount, and then grind in an alternate manner of cyclic grinding and non-circulating grinding;

4) Check the particle fineness, the result meets the requirements (the standard is that the number of particles with a particle size greater than 15 μm should be less than 10%), stop grinding, add soybean oil for injection to 100L, mix, fill, seal, sterilize, and obtain The veterinary suspension containing amoxicillin, colistin sulfate and prednisolone of the present invention.

Example 2

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 20kg,

Colistin Sulfate 1kg,

Prednisolone 0.03kg,

Hydrogenated castor oil 1kg,

Span-60 0.5L,

Propyl gallate 0.02kg,

Methylparaben 0.05kg,

Add isopropyl myristate to 100L.

Prepare the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone with the method of the present invention, concrete method comprises the following steps:

1) Add hydrogenated castor oil, propyl gallate and methylparaben in 2L of isopropyl myristate into 2L of isopropyl myristate, heat and stir to completely dissolve to obtain liquid A;

2) Take 40L of isopropyl myristate and pour it into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until it is completely added, add the formula amount of Span-60 while stirring;

3) After all the excipients are added completely, sequentially add amoxicillin, colistin sulfate and prednisolone in the prescribed amount, and then grind in an alternate manner of cyclic grinding and non-circulating grinding;

4) check particle fineness, the result meets the requirements, stop grinding, add isopropyl myristate to 100L, mix, fill, seal, sterilize, obtain the present invention containing amoxicillin, colistin sulfate and splash Veterinary suspension of Nisolone.

Example 3

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 10kg,

Colistin Sulfate 1kg,

Prednisolone 0.05kg,

Hydrogenated castor oil 0.5kg,

Vaseline 0.25kg,

Span-80 0.2L,

Thiourea 0.2kg,

Methylparaben 0.05kg,

Ethyl oleate 30L,

Add soybean oil for injection to 100L.

Prepare the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone with the method of the present invention, concrete method comprises the following steps:

1) Add hydrogenated castor oil, vaseline, thiourea and methylparaben in 2L of soybean oil for injection into 2L of soybean oil, heat and stir to dissolve completely, and obtain liquid A;

2) Pour the prescribed amount of ethyl oleate and 30L soybean oil for injection into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until all the ingredients are added, add the prescribed amount of Span-80 while stirring ;

3) After all the excipients are added completely, sequentially add amoxicillin, colistin sulfate and prednisolone in the prescribed amount, and then grind in an alternate manner of cyclic grinding and non-circulating grinding;

4) Check particle fineness, the result meets the requirements, stop grinding, add soybean oil for injection to 100L, mix, fill, seal, sterilize, obtain the present invention containing amoxicillin, colistin sulfate and prednisone Dragon's Veterinary Suspension.

Example 4

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 15kg,

Colistin Sulfate 2.5kg,

Prednisolone 0.04kg,

Lecithin 0.8kg,

Span-80 1L,

Vitamin E 0.05kg,

Propylparaben 0.05kg,

Add castor oil for injection to 100L.

Prepare the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone with the method of the present invention, concrete method comprises the following steps:

(1) Get lecithin, vitamin E and propylparaben of formula quantity and add in 2L castor oil for injection, heat and stir to make it dissolve completely, obtain A liquid;

(2) Take 80L of castor oil for injection and pour it into the colloid mill, start the colloid mill, then slowly add the above-mentioned liquid A, wait until it is completely added, add the formula amount of Span-80 while stirring;

(3) After all the auxiliary materials are added completely, sequentially add amoxicillin, colistin sulfate and prednisolone in the prescribed amount, and then grind in an alternate mode of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add castor oil for injection to 100L, mix, fill, seal, sterilize, and obtain the present invention containing amoxicillin, colistin sulfate and prednisone Dragon's Veterinary Suspension.

Example 5

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 15kg,

Colistin Sulfate 2kg,

Prednisolone 0.05kg,

Lecithin 0.2kg,

Hydrogenated castor oil 0.5kg,

Span-60 0.8L,

tert-butylhydroxyanisole 0.05kg,

Methylparaben 0.05kg,

Add ethyl oleate to 100L.

Prepare the veterinary suspension containing amoxicillin, colistin sulfate and prednisolone with the method of the present invention, concrete method comprises the following steps:

1) Add hydrogenated castor oil, lecithin, tert-butyl hydroxyanisole and methyl paraben into 2L ethyl oleate, heat and stir to dissolve completely, and obtain liquid A;

2) Take 60L of ethyl oleate and pour it into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until it is completely added, add the formula amount of Span-60 while stirring;

3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in the prescribed amount in sequence, start grinding, and grind in an alternate manner of cyclic grinding and non-circulating grinding;

4) Check the particle fineness, the result meets the requirements, stop grinding, add ethyl oleate to 100L, mix, fill, seal, sterilize, and obtain the present invention containing amoxicillin, colistin sulfate and prednisolone veterinary suspension.

Example 6

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 20kg

Colistin Sulfate 2kg

Prednisolone 0.04kg

Hydrogenated castor oil 0.3kg

Lecithin 0.5kg

Span-80 0.6L

Vitamin E 0.05kg

Methylparaben 0.05kg

Add ethyl oleate to 100L

The preparation method of the veterinary suspension of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) Dissolve hydrogenated castor oil, lecithin and methylparaben in 2L of hot ethyl oleate in the prescribed amount to be liquid A;

(2) Take 60L of ethyl oleate and pour it into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until all the addition is complete, add the formula amount of Span-80 while stirring;

(3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in the prescribed amount in turn, start grinding, and grind in an alternate manner of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add ethyl acetate to 100 L, mix evenly, fill, seal, and sterilize to obtain the suspension.

Example 7

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 20kg

Colistin Sulfate 2kg

Prednisolone 0.04kg

Hydrogenated castor oil 0.3kg

Lecithin 0.3kg

Span-80 1.5L

Vitamin E 0.075kg

Methylparaben 0.05kg

Ethyl Oleate 10L

Add soybean oil for injection to 100L

The preparation method of the veterinary suspension of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) Get the hydrogenated castor oil, lecithin and methylparaben of formula quantity and be dissolved in 2L hot soybean oil for injection, be A liquid;

(2) Take 50L of soybean oil for injection and ethyl oleate in the prescribed amount and pour it into the colloid mill, start the colloid mill, then slowly add the above-mentioned liquid A, wait until it is completely added, add the prescribed amount of Span- 80;

(3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in the prescribed amount in turn, start grinding, and grind in an alternate manner of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add soybean oil for injection to 100 L, mix evenly, fill, seal, and sterilize to obtain the suspension.

Example 8

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 15kg

Colistin Sulfate 2.5kg

Prednisolone 0.05kg

Lecithin 1.5kg

Span-60 0.5L

Vitamin E 0.075kg

Methylparaben 0.05kg

Isopropyl myristate Add to 100L

The preparation method of the veterinary suspension of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) Dissolve lecithin, vitamin E and methylparaben in 2L hot isopropyl myristate in the prescribed amount to form A liquid;

(2) Take 60L of isopropyl myristate and pour it into the colloid mill, start the colloid mill, then slowly add the above liquid A, wait until it is completely added, add the formula amount of Span-60 while stirring;

(3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in the prescribed amount in turn, start grinding, and grind in an alternate manner of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add isopropyl myristate for injection to 100 L, mix evenly, fill, seal, and sterilize to obtain the suspension.

Example 9

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 30kg

Colistin Sulfate 0.5kg

Prednisolone 0.02kg

Cholesterol 0.1kg

Ethylene oxide-40-hydrogenated castor oil 1.0L

tert-butylhydroxyanisole 0.01kg

Methylparaben 0.005kg

Triacetin Add to 100L

The preparation method of the veterinary suspension of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) Dissolve the cholesterol, methylparaben and tert-butyl hydroxyanisole in 2L triacetin in the prescribed amount to form A liquid;

(2) Get 60L of glyceryl triacetate and pour it into the colloid mill, start the colloid mill, then slowly add the above-mentioned liquid A, wait until all the addition is complete, add the ethylene oxide-40-hydrogenated castor oil of the formula while stirring;

(3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in the prescribed amount in turn, start grinding, and grind in an alternate manner of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add triacetin for injection to 100 L, mix well, fill, seal, and sterilize to obtain the suspension.

Example 10

The present invention contains the formula of the veterinary suspension of amoxicillin, colistin sulfate and prednisolone as follows:

Amoxicillin 10kg

Colistin Sulfate 2kg

Prednisolone 0.1kg

Lecithin 0.5kg

Cholesterol 0.5kg

Methylparaben 0.5kg

Ethyl oleate 50L

Add soybean oil for injection to 100L

The preparation method of the veterinary suspension of described amoxicillin, colistin sulfate and prednisolone comprises the steps:

(1) Dissolve lecithin, cholesterol and methylparaben in 2L of ethyl oleate according to the measured amount to form A solution;

(2) Take the remaining amount of ethyl oleate and pour it into the colloid mill, start the colloid mill, then slowly add the above-mentioned liquid A, wait until all the addition is complete, add ethylene oxide-40-hydrogenated castor oil according to the amount while stirring;

(3) After all the excipients are added completely, add amoxicillin, colistin sulfate and prednisolone in sequence, start grinding, and grind in an alternating mode of cyclic grinding and non-circulating grinding;

(4) After checking that the particle fineness meets the requirements, stop grinding, add soybean oil for injection to 100 L, mix evenly, fill, seal, and sterilize to obtain the suspension.

Experiment 1. The combined antibacterial effect of amoxicillin and colistin sulfate

In order to explore whether the combined application of amoxicillin and colistin sulfate can enhance the antibacterial effect of E. , Streptococcus mammatus and other common pathogenic bacteria infection, the present invention has carried out the joint antibacterial test of two medicines in vitro.

1 Materials and Instruments

1.1 Test drugs

Amoxicillin (AMO), content is 99.5%, batch number 20080312, Zhuhai United Laboratories Co., Ltd.;

Colistin sulfate (COS), content 23694IU/mg, batch number N0710003J, Livzon Group Fuzhou Fuxing Pharmaceutical Co., Ltd.;

1.2 Tested strains

Isolated strains: 17 strains of porcine Escherichia coli; 6 strains of Pasteurella suis; 8 strains of Salmonella porcine and chicken; 10 strains of Staphylococcus aureus mastitis in dairy cows; .

Quality control strains: ATCC25922, ATCC29213, ATCC49619, all purchased from China Veterinary Drug Control Institute.

1.3 Medium

MH broth medium, batch number 20071102, MH agar medium, batch number 20071103, Beijing Aoboxing Biotechnology Co., Ltd. The above-mentioned various media were prepared according to conventional methods, and after passing the sterility inspection, they were stored in a refrigerator at 4°C for cultivating Escherichia coli, Salmonella choleraesuis and Staphylococcus aureus. For the culture medium of Pasteurella multocida and Streptococcus suis, 5% (V/V) premium fetal bovine serum is added in the above preparation method. The medium used to cultivate Actinobacillus pleuropneumoniae is PPLO medium.

1.4 Main Instruments

(1) Spectrophotometer: Model 721, Shandong Gaomi Analytical Instrument Factory.

(2) Micro checkerboard dilution method combined with drug susceptibility test plate: Tianjin Jinzhang Technology Development Co., Ltd.

(3) Continuous micro-sampler, eight-channel micro-sampler: American Eppendorf Company.

2 test method

2.1 Preparation and storage of drug stock solution

Amoxicillin stock solution: use 0.1mol/L phosphate buffer (pH6.0) to make amoxicillin into a drug stock solution with a concentration of 1280 μg/mL, filter it with an autoclaved 0.2 μm pore size filter head, and dispense to In a 2mL centrifuge tube, store in a -20°C refrigerator until use.

Colistin sulfate stock solution: use distilled water to make colistin sulfate into a drug stock solution with a concentration of 2560 μg/mL, filter it with an autoclaved 0.2 μm pore size filter, dispense it into a sterilized 2mL plastic centrifuge tube, seal it, Store in -20°C refrigerator until use.

2.2 Preparation of bacteria solution

2.2.1 Preparation of Escherichia coli, Salmonella and Staphylococcus aureus

Dip a few colonies from the slant of the culture medium where the strains were preserved, inoculate them in the MH broth culture solution, cultivate them at 37°C for 18 hours, and dilute the original culture solution by 10 ^-1 times, respectively as follows: 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 ; the concentrations of 10 ^-6 , 10 ^-7 , and 10 ^-8 were added to each bacterial solution in a small amount. Take 100 μL of the sampler into the pre-prepared MH agar plate, and sterilize the L-shaped glass rod by burning. In a water-proof constant temperature incubator, observe after 24 hours of cultivation, and calculate the concentration of the bacterial solution. Determine the concentration of the original bacterial stock solution, then dilute it to 10 ⁶ CFU/ml with sterilized physiological saline, store it in a 4°C refrigerator for later use, and store it for no more than 6 hours.

2.2.2 Preparation of Pasteurella multocida and Streptococcus bacteria liquid

Dip a little bacterial lawn from the slant where the strains were preserved, inoculate it into the blood MH broth culture solution, incubate at 37°C for 18 hours, and dilute the original bacterial solution by 10 ^-1 times as follows: 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 ; take 10 ^-6 , 10 ^-7 , 10 ^-8 concentrations of each bacterial solution and take 100 μL with a micro-sampler Put the pre-prepared blood MH agar plate, burn the L-shaped glass rod on the flame to sterilize, after cooling, spread the bacterial solution evenly on the surface of the medium (sterile test is qualified); then put the plate medium into In a water-proof constant temperature incubator, observe after 24 hours of cultivation, and calculate the concentration of the bacterial solution. Determine the concentration of the bacterial stock solution, then dilute it with sterilized physiological saline to 10 ⁶ CFU/ml, store it in a 4°C refrigerator for later use, and store it for no more than 6 hours.

2.2.3 Preparation of Actinobacillus pleuropneumoniae bacteria liquid

Dip a little bacterial lawn from the slant where the strains were preserved, inoculate it in PPLO medium, incubate at 37°C for 18 hours, and dilute the original bacterial solution by 10 ^-1 times as follows: 10 ^-1 , 10 ^-2 , 10 ^{- 3} , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 ; take 10 ^-6 , 10 ^-7 , and 10 ^-8 concentrations of each bacterial solution respectively, and take 100 μL to the pre- In the prepared PPLO medium, burn the L-shaped glass rod on the flame to sterilize it. After cooling, spread the bacterial solution evenly on the surface of the medium (the sterility test is qualified); then put the plate medium into the water-proof type In a constant temperature incubator, observe after 24 hours of cultivation, and calculate the concentration of the bacterial solution. Determine the concentration of the bacterial stock solution, then dilute it with sterilized physiological saline to 10 ⁶ CFU/ml, store it in a 4°C refrigerator for later use, and store it for no more than 6 hours.

2.3 Determination of MIC

2.3.1 Determination of MIC of Escherichia coli, Salmonella and Staphylococcus aureus

Broth micro-dilution method: Dilute amoxicillin stock solution and colistin sulfate stock solution to 11 concentrations in sterilized MH broth (0.125-512 μg/mL for amoxicillin, 0.0625-128 μg/mL for colistin sulfate mL), 100 μL was added to the microwells of the 96-well plate, and then a certain concentration of bacterial suspension was added to make the final bacterial concentration in each well 5×10 ⁵ CFU/mL. Bacterial growth control and blank control were set up and cultured at 37°C. Cultivate in the box for 24 hours, and use the lowest antibacterial single-drug concentration without bacterial growth as the MIC. Escherichia coli ATCC25922 was used as the quality control bacteria for MIC determination.

2.3.2 Determination of MIC of Pasteurella and Streptococcus

Broth microdilution method: dilute amoxicillin stock solution and colistin sulfate stock solution to 11 concentrations in serum broth (from 0.125-128 μg/mL for amoxicillin and 0.031-64 μg/mL for colistin sulfate) , respectively take 100 μL into the micro wells of 96-well plate, and then add a certain concentration of bacterial suspension, so that the final bacterial concentration in each well is 5×10 ⁵ CFU/mL, and set up bacterial growth control and blank control, and culture in a 37°C constant temperature incubator 24h, the lowest concentration of antibacterial single drug without bacterial growth is the MIC.

2.3.3 Determination of MIC of Actinobacillus pleuropneumoniae

Dilute amoxicillin stock solution and colistin sulfate stock solution to 11 concentrations in PPLO medium (0.125-128 μg/mL for amoxicillin, 0.031-64 μg/mL for colistin sulfate), and add 100 μL to 96 wells Then add a certain concentration of bacterial suspension to make the final bacterial concentration of each well 5×10 ⁵ CFU/mL, and set up a bacterial growth control and a blank control, and culture it in a constant temperature incubator at 37°C for 24 hours. The minimum antibacterial single drug concentration is MIC.

2.4 Combined drug susceptibility test

According to the results of the MIC of the single drug, the amoxicillin stock solution and the colistin sulfate stock solution were respectively diluted to 6 different working concentrations with broth, and the two drugs were added to the 96-well plate successively according to the checkerboard design. In each well, take 50 μl of each antibacterial drug in each well, so that the final concentration of each drug in each well is 1/8MIC, 1/4MIC, 1/2MIC, 1MIC, 2MIC and 4MIC of its single drug, and The single-drug control, bacterial growth control and blank control of the two drugs were respectively set up. Add 100 μL of 10 ⁶ CFU/mL bacterial solution to each well to make the final concentration of bacteria in each well 5×10 ⁵ CFU/mL, incubate at 37°C for 24 hours, observe and record the results. The checkerboard test was repeated 3 times for each strain. When selecting the best combination effect, the respective MIC _{A drug combination} and MIC _{B drug combination are used to} calculate the FIC index.

FIC index calculation and interpretation standards:

FIC index ≤0.5, synergistic effect; >0.5-1, additive effect; >1-2, irrelevant effect; >2, antagonistic effect.

3 test results

3.1 The in vitro susceptibility test results of amoxicillin and colistin sulfate against Escherichia coli and Staphylococcus aureus when used alone or in combination are shown in Table 1-1 to Table 1-6.

Table 1-1 The in vitro susceptibility test results of AMO and COS alone and in combination against porcine Escherichia coli (MIC unit is ug/mL)

Table 1-2 In vitro susceptibility test results of AMO and COS alone and in combination against Staphylococcus aureus (MIC unit is ug/mL)

Table 1-3 The in vitro drug susceptibility test results of AMO and COS alone and in combination against Streptococcus (MIC unit is ug/ml)

Table 1-4 The in vitro drug susceptibility test results of AMO and COS alone and in combination against Salmonella (MIC unit is ug/ml)

Table 1-5 The in vitro drug susceptibility test results of AMO and COS alone and in combination to Pasteurella (MIC unit is ug/ml)

Table 1-6 The in vitro drug susceptibility test results of AMO and COS alone and in combination against Actinobacillus pleuropneumoniae (MIC unit is ug/ml)

According to the combined drug susceptibility test, the MIC of amoxicillin after combined use is 1/16-1 of that of single use, and the MIC value of some strains decreased significantly; especially for some amoxicillin-resistant Escherichia coli strains and For Staphylococcus aureus, the MIC value of AMO dropped below the drug-resistant breakpoint (breakpoint) after combined use, making drug-resistant strains become sensitive strains; the MIC after colistin sulfate combined is 1/8-1 of the MIC when used alone MIC values also decreased to varying degrees.

3.2 The FIC index distribution of the joint application of AMO and COS (see Table 1-7), the joint application of AMO and COS, the FIC index distribution is concentrated in 0.5~1, indicating that the combination of the two has an additive effect on most bacteria (70%~ 100%), 30% synergistic effect on Staphylococcus aureus, 33.33% irrelevant effect on Pasteurella, but no antagonistic effect.

Table 1-7 Effect of AMO combined with COS on bacterial MIC

4 Conclusion

The above experimental results show that the use of amoxicillin and colistin sulfate as a compound preparation has synergistic or additive effects on Streptococcus, Staphylococcus aureus, Pasteurella, Escherichia coli, Actinobacillus pleuropneumoniae, and Salmonella , Broaden the antibacterial spectrum and enhance the antibacterial efficacy.

Experiment 2: Study on the Antimutagenic Concentration of Escherichia coli Combined with Amoxicillin and Colistin Sulfate

In order to explore whether the combination of amoxicillin and colistin sulfate can delay the generation of drug resistance, the present invention has measured the minimum inhibitory concentration (MIC) of amoxicillin and colistin sulfate to escherichia coli with standard broth dilution method. ), and the antimutagenic concentration (MPC) of amoxicillin to Escherichia coli was determined by agar dilution method, and the changes of the antimutagenic concentration and drug resistance frequency of amoxicillin to Escherichia coli after combined use with colistin sulfate were investigated.

1 Materials and Instruments

1.1 Test drugs

Amoxicillin (AMO), content is 99.5%, batch number 20080312, Zhuhai United Laboratories Co., Ltd.;

Colistin sulfate (COS), content 23694IU/mg, batch number N0710003J, Livzon Group Fuzhou Fuxing Pharmaceutical Co., Ltd.;

1.2 Tested strains

Nine strains were tested, among which the standard strain of Escherichia coli (ATCC25922) was purchased from China Veterinary Drug Control Institute; the other eight strains of Escherichia coli were clinical isolates.

1.3 Medium

MH broth medium, batch number 20071102, MH agar medium, batch number 20071103, Beijing Aoboxing Biotechnology Co., Ltd. The above-mentioned various culture media were prepared according to conventional methods, and after passing the sterility test, they were stored in a refrigerator at 4°C.

1.4 Main Instruments

(1) Spectrophotometer: Model 721, Shandong Gaomi Analytical Instrument Factory.

(2) Micro checkerboard dilution method combined with drug susceptibility test plate: Tianjin Jinzhang Technology Development Co., Ltd.

(3) Continuous micro-sampler, eight-channel micro-sampler: American Eppendorf Company.

2 test method

2.1 Preparation and storage of drug stock solution

Amoxicillin stock solution: use 0.1mol/L phosphate buffer (pH6.0) to make amoxicillin into a drug stock solution with a concentration of 1280 μg/mL, filter it with an autoclaved 0.2 μm pore size filter head, and dispense to In a 2mL centrifuge tube, store in a -20°C refrigerator until use.

Colistin sulfate stock solution: use distilled water to make colistin sulfate into a drug stock solution with a concentration of 2560 μg/mL, filter it with an autoclaved 0.2 μm pore size filter, dispense it into a sterilized 2mL plastic centrifuge tube, seal it, Store in -20°C refrigerator until use.

2.2 Preparation of bacteria solution

Dip a few colonies from the slant of the culture medium where the strains were preserved, inoculate them in the MH broth culture solution, cultivate them at 37°C for 18 hours, and dilute the original culture solution by 10 ^-1 times, respectively as follows: 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 ; the concentrations of 10 ^-6 , 10 ^-7 , and 10 ^-8 were added to each bacterial solution in a small amount. Take 100 μL of the sampler into the pre-prepared MH agar plate, and sterilize the L-shaped glass rod by burning. In a water-proof constant temperature incubator, observe after 24 hours of cultivation, and calculate the concentration of the bacterial solution. Determine the concentration of the original bacterial stock solution, then dilute it to 10 ⁶ CFU/ml with sterilized physiological saline, store it in a 4°C refrigerator for later use, and store it for no more than 6 hours.

2.3 Determination of MIC

Broth micro-dilution method: Dilute amoxicillin stock solution and colistin sulfate stock solution to 11 concentrations in sterilized MH broth (0.5-512 μg/mL for amoxicillin, 0.031-32 μg/mL for colistin sulfate mL), 100 μL was added to each microwell of the 96-well plate successively, and then a certain concentration of bacterial suspension was added to each well so that the final bacterial concentration in each well was 5×10 ⁵ CFU/mL, and the bacterial growth The control and the blank control were cultured in a constant temperature incubator at 37°C for 24 hours, and the MIC value was determined according to the standards of the American Clinical Laboratory Standards Institute (CLSI). Escherichia coli ATCC25922 was used as the quality control bacteria for MIC determination.

2.4 Determination of mutation prevention concentration (MPC):

The anti-mutation concentration (MPC) refers to the minimum concentration of antibacterial drug required to prevent the selective enrichment and amplification of drug-resistant mutant strains. The MPC theory provides new ideas and references for effectively suppressing bacterial resistance and formulating antimicrobial drug application strategies.

Single-drug MPC assay: inoculate a single colony in 20mL MH broth, shake culture overnight at 37°C, centrifuge enrichment at 3000r/min at 4°C, resuspend the bacteria in 200mL MH broth (10 times the original solution), and shake culture for 6h When the concentration of the bacterial solution reaches 6×10 ⁹ ～9×10 ⁹ CFU/mL, collect all the bacterial solution, centrifuge at 3000 r/min at 4°C for 30 minutes to enrich, and adjust the concentration of the bacterial solution to 3×10 ¹⁰ CFU with fresh broth /mL. Take 100 μL of bacterial solution and put them on MH plates with different concentrations of AMO and COS with a diameter of 90 mm, spread the bacteria evenly with L sticks, incubate at 35°C for 72-96 hours, and count the colonies according to the literature (Zhao X, Drlica K. Restricting the Selection of antibiotic resistant mutant bacteria: measurement and potential use of the mutant selection window [J]. J Infect Dis, 2002, 185: 561-565.) to calculate the MPC of a single drug. All experiments were repeated 3 times, and the mean value was taken.

MPC determination of combined drugs: Take 100 μL of the above bacterial solution and place them in the wells of AMO containing 1 ×, 2 ×, 4 ×, 8 ×, 16 ×, 32 × MIC and COS 2 mg/L respectively, and use an L stick to Spread the bacteria evenly, incubate at 35°C for 72-96 hours, and count the colonies. The frequency of drug resistance was calculated according to the literature (Zhanel G G, Mayer M, Laing N, et al. aeruginosa[J].Antimicrob, Agents Chemother.2006.50(5):2228-2230.): Divide the number of colonies on different concentrations of amoxicillin and colistin sulfate plates by the number of inoculated colonies. All experiments were repeated 3 times, and the mean value was taken.

2.5 Data analysis SPSS 10.0 statistical software was used to perform rank sum test on each group of data.

3 results

3.1 The MIC value of amoxicillin and colistin sulfate single drug against porcine E. coli is shown in Table 2-1.

Table 2-1AMO and COS to the MIC value of porcine Escherichia coli (mg/L)

3.2 The MPC and MPC/MIC values of amoxicillin alone and in combination with colistin sulfate against porcine Escherichia coli are shown in Table 2-2.

Table 2-2 The MPC and MPC/MIC values (mg/L) of AMO used alone and in combination with COS on porcine Escherichia coli

Note: Compared with the MPC/MIC used alone with AMO and COS, the difference is significant (P<0.05)

3.3 The drug resistance frequency of amoxicillin combined with colistin sulfate against porcine Escherichia coli is shown in Table 2-3.

Table 2-3 The drug resistance frequency of Escherichia coli in pigs when combined with AMO and COS

Note: There is a significant difference in the frequency of drug resistance when AMO and COS are used together (P<0.05)

4 Conclusion

The above experimental results show that the MPC/MIC of amoxicillin combined with colistin sulfate is 2-16 times lower than that of amoxicillin alone. Significantly decreased (P<0.05). Since the antibacterial mechanisms of AMO and COS are different and have different targets, bacteria need to be resistant to both drugs to survive. After the combination of the two drugs, the selection window for drug resistance is narrowed, the threshold of bacterial mutation is increased, and the threshold for bacterial mutation is reduced. MPC/MIC ratio, so that MPC/MIC and drug resistance frequency are greatly reduced. Therefore, after being used in combination with colistin sulfate, amoxicillin's anti-drug resistance ability is enhanced, and the generation of drug-resistant strains is reduced, so the generation of drug resistance can be delayed.

Experiment 3. Drug efficacy experiment of veterinary suspension of amoxicillin, colistin sulfate and prednisolone

1. Test materials and methods

1.1 Test drugs

1. The veterinary suspension containing amoxicillin, colistin sulfate and prednisolone of the present invention is prepared according to Example 3 and is called A-C-P.

2. Amoxicillin Sodium for Injection, the trade name is Mosa, Shandong Lukang Pharmaceutical Co., Ltd.; I

③ Colistin Sulfate Water-Soluble Injection, the trade name is Xieliuting, Xiangtan Veterinary Medicine Factory of Beijing Zhongnong University Animal Health Products Group;

4. Ampicillin-colistin sulfate-dexamethasone suspension, the trade name is Multibio d, France Vick company pharmaceutical factory;

⑤ Amoxicillin and colistin sulfate suspensions are prepared according to Example 3 and do not contain prednisolone, which are called A-C.

1.2 Bacterial inoculation

Actinobacillus pleuropneumoniae was purchased from the China Institute of Veterinary Drug Control, serotype I, number 259. After being cultured on PPLO agar for 24 hours, it was counted and diluted to 1.0×10 ⁶ CFU/ml for use. The pig inoculation procedure is briefly described as follows. Each pig is tied on a special support in a dorsal position, and the inoculation site is coated with 1% procaine for local anesthesia, and a 35mm, 18-gauge needle is passed between the two cartilage rings of the trachea Puncture, inoculate Actinobacillus pleuropneumoniae at a dose of 0.05ml/kg (1.0×10 ⁶ CFU/ml) body weight, and inject sterile normal saline at the same dose in the negative control group. Dosing was done 4 hours after inoculation.

1.3 Experimental animals and handling

Eighty-six Duroc Landrace binary hybrid pigs weighing 20-25 kg were fed conventional feed without antibiotic additives, and the pigs had free access to water. After a one-week rest period, they were randomly divided into 9 groups. Except for the blank control group, each pig in the other groups was inoculated with Actinobacillus pleuropneumoniae at a dose of 0.05ml/kg (1.0×10 ⁶ CFU/ml) body weight, and the negative control group was injected with sterile saline at the same dose. After 4 hours of inoculation, administer according to the experimental design method in Table 3-1. All pigs were collected nasal secretions for bacteria isolation and culture before the experiment. As a result, Actinobacillus pleuropneumoniae could not be detected in the nasal secretions. Animals in each group were fed under the same conditions, with exactly the same diet composition and nutrient level, and the test period was 2 weeks.

Table 3-1 Experimental design

group Number of animals (head) drugs 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, once a day for 5 consecutive days. Middle dose group 10 A-C-P Intramuscular injection, AMO 10mg/kg, COS 25,000IU/kg and PRE 0.05mg/kg body weight, once a day for 5 consecutive days. low dose group 10 A-C-P Intramuscular injection, AMO 5mg/kg, COS 12,500IU/kg and PRE 0.025mg/kg body weight, once a day for 5 consecutive days. drug control group I 10 Mosa Intramuscular injection, AMO 10mg/kg body weight, 2 times a day, for 5 consecutive days. drug control group II 10 Diarrhea Intramuscular injection, COS 25,000IU/kg body weight, 2 times a day for 5 consecutive days. Drug control group III 10 A-C Intramuscular injection, AMO 10mg/kg and COS 25,000IU/kg body weight, once a day for 5 consecutive days. drug control group IV 10 Multibio d Intramuscular injection, ampicillin 10mg/kg, COS 25,000IU/kg and dexamethasone 0.025mg/kg body weight, once a day for 5 consecutive days. positive control group 10 No drug for infection - negative control group 6 no infection no medication -

Note: AMO in the table stands for amoxicillin, COS stands for colistin sulfate, and PRE stands for prednisolone.

1.4 Feeding management and observation

Each test group weighed the body weight of each pig at the beginning of the test and at the end of the test. Clinical manifestations such as breathing, appetite, and mental state of each pig were closely observed every day after artificial infection, and rectal body temperature was measured. The incidence and mortality of pigs in each group were recorded. Necropsy was carried out on the dead pigs, and the changes of lung, liver and spleen, heart, mesentery, etc. were observed. Slaughter all the dead pigs and some pigs after the test, take the liver and spleen, apply tryptone soybean agar plate medium, and incubate at 37°C for 20-48°C to check whether there is any bacterial growth, and conduct a series of laboratory tests on the detected bacteria To check whether it is Actinobacillus pleuropneumoniae.

1.5 Efficacy Judgment Criteria

Cure: During the test period, the clinical symptoms of the pigs treated with the drug disappeared, the spirit, appetite, body temperature, breathing, etc. returned to normal, and the weight gain was close to or exceeded that of the blank control group; old lesions were found in the lungs after necropsy, and no Actinobacillus pleuropneumoniae was isolated from the bacteria . The cure rate was calculated according to the ratio of the number of cured heads to the number of experimental heads in this group.

Significantly effective: During the test period, the symptoms were significantly reduced or disappeared, and the body weight had no obvious change or a slight increase before and after the test. After autopsy, certain lesions were seen in the lungs, and Actinobacillus pleuropneumoniae was found to be carried after the bacteria were isolated. The significant rate was calculated according to the ratio of the number of markedly effective to the number of test heads in this group.

Effective: During the test period, the mental state improved significantly, the appetite increased, and there was mild abdominal breathing or coughing. The effective rate is calculated according to the ratio of effective heads to the number of test heads in this group. When calculating the effective rate, the number of effective heads includes the number of markedly effective heads and the number of cured heads.

Invalid: During the test period, the test pigs' spirit, appetite, breathing, body temperature and other symptoms still did not improve or even worsened and died. The inefficiency was calculated according to the ratio of the number of invalid heads to the number of experimental heads in this group.

1.6 weight gain

The body weight of each pig was weighed at the beginning and end of the experiment, and the average weight gain was calculated.

1.7 Statistical analysis of data

Chi-square test (SPSS10.0 statistical analysis software) was used for statistical analysis. P<0.05 means significant difference, P<0.01 means extremely significant difference, P>0.05 means no significant difference.

2 Test results and summary

2.1 During the test period, the pigs in the blank control group were in good spirits, had a normal desire to eat, and had a body temperature of 38°C to 40°C, which was within the normal range.

2.2 Positively infected pigs in the control group began to experience transient vomiting, shortness of breath, and 0.5-1°C rise in body temperature 2-3 hours after inoculation. After 4 to 6 hours, shortness of breath and obvious abdominal respiration, and body temperature increased by 1.5-2.5°C. Finally, dyspnea occurred, in dog sitting position, and died soon. The most acute type of death occurred in 4-24 hours. Seven pigs in the positive infection control group died within 24 hours, and another 3 pigs died within 24-48 hours of infection. .

2.3 After the death of the sick pig, a large amount of bloody secretions flowed out of the mouth and nostrils. The autopsy showed that there was light red bloody fluid in the pleural cavity, the lungs were dark red, congested and swollen, and a large amount of bloody fluid flowed out. The trachea and bronchi are full of bloody mucus foam-like exudates. In pigs with a longer course of disease, white fibrinous exudates can be found on the surface of the lungs, and even adhere to the chest cavity. In pigs with a longer course of disease, the spleen and liver are dark black.

2.4 After tracheal inoculation of Actinobacillus pleuropneumoniae bacteria solution, all inoculated pigs had typical clinical symptoms of pleuropneumonia. Compared with the positive control group, the clinical symptoms of each group of drugs were improved to varying degrees after drug treatment, that is, the symptoms of cough, dyspnea, and depression of the diseased pigs were significantly alleviated, and the appetite gradually recovered; the number of pigs in each drug group survived All were significantly higher than the positive control group (P<0.05), indicating that each drug had a certain protective effect on swine pleuropneumonia. The cure rate, effective rate and average weight gain rate of the A-C-P high and medium dose groups were significantly higher than those of the A-C-P low dose group, Mosa group and Xieliting group (P<0.05), indicating that the A-C-P high and medium dose groups had superior curative effect. In the A-C-P low-dose group, Mosa group and Xieliating group; the cure rate, effective rate, and average weight gain rate between the A-C-P high-dose group and the middle-dose group had no significant difference (P>0.05), indicating that it is effective in the clinical treatment of porcine infection. During the course of acute pleuropneumonia infection, it is reasonable to use the medium dose of A-C-P as the clinically recommended dose; compared with the commercially available drug Multibio d group, there was no significant difference in the cure rate, marked rate and effective rate between the A-C-P medium dose group (P> 0.05), but the cure rate and effective rate of the A-C-P middle dose group were all higher by 10%, and the effective rate was higher by 20%, and the commercially available drug Multibiod had 20% inefficiency, showing that the overall curative effect of A-C-P was better than that of The marketed drug Multibio d. See Table 3-2 for specific test data.

Table 3-2 Treatment test results of each group (X±SD)

*: Compared with A-C-P high-dose and middle-dose groups and drug control group IV, there is a significant difference (p<0.05);

★: Significantly different from the healthy control group (p<0.05)

2.5 During the observation of respiration, appetite and mental state, it was found that 7, 6 and 5 pigs in the A-C-P high-dose and middle-dose groups and the commercially available drug Multibio d group were able to recover to a healthy state 7 days after infection, However, there were only 2, 1, and 0 pigs in groups A-C, Mosa group, and Xieliting group, respectively. The recovery time of A-C group, Mosa group and Xieliting group to a healthy state is mostly concentrated on the 9th to 14th day after infection, and the recovery time is much longer than that of the A-C-P high, medium-dose group and the commercially available drug Multibio d group. The reason is that the A-C-P The drug formulas of the group and the commercially available drug Multibio d group all contain glucocorticoid anti-inflammatory ingredients (A-C-P and Multibio d formulas contain prednisolone and dexamethasone respectively), and glucocorticoid drugs have anti-inflammatory, anti- Therefore, it plays the role of adjuvant therapy in the anti-infection treatment, which is beneficial to the recovery of the healthy physique of sick animals. It can also be seen from the results of the 15-day weight gain that the treatment group containing glucocorticoids has no significant difference in weight gain from the healthy control group at an appropriate dosage, which is consistent with the recovery of the body after treatment. There is a certain correlation between the length of time to fitness level.

2.6 This test chose Multibio d as one of the control drugs, mainly considering that the main components of the drug are similar to the components of the suspension of the present invention. The drug is produced by the French Vic company and is used for dairy cows in Europe and the United States. Treatment of inflammation (currently the product has not been registered in China). Result of the test shows that suspension product of the present invention is all superior to Multibiod aspect curative effect judging indicators such as cure rate, effective rate and the time that body returns to normal state, shows that on the medicine selection of prevention and treatment porcine contagious pleuropneumonia, the present invention's The product is the drug of choice. There was a significant difference (p<0.05) in time to return to normal and weight gain compared to the control A-C product (without prednisolone), suggesting that the anti-inflammatory component played an important role in the treatment process.

Experiment 4: Curative Effects of Veterinary Suspension of Amoxicillin, Colistin Sulfate and Prednisolone on Mixed Infection of Porcine Pleuropneumonia and Colibacillosis

Experimental purpose: To investigate the curative effect of the suspension product of the present invention and the commercially available product on the mixed infection of porcine infectious pleuropneumonia and colibacillosis.

1. Test materials and methods

1.1 Test drugs

1. The veterinary suspension containing amoxicillin, colistin sulfate and prednisolone of the present invention is prepared according to Example 3 and is called A-C-P.

② Ampicillin-colistin sulfate-dexamethasone suspension, the trade name is Multibio d, France Vic Pharmaceuticals (unregistered in China);

③ Amoxicillin and colistin sulfate suspensions, prepared according to Example 3, do not contain prednisolone, and are called A-C.

1.2 Experimental animals: Piglets aged 2-3 months from pig farms in Guangxi, Hunan, Beijing, Shandong, etc. were selected, and the breeds were Duroc×Landrace×Dark York Sanyuanza, Duroc×Landrace Biyuanza, etc. Natural infection with Escherichia coli and infectious pleuropneumoniae.

1.3 Diagnosis basis

1.3.1 Clinical symptoms: Sudden onset of pigs, depression, loss of appetite or food waste, high fever 40.5-42 ℃, visible mucous membrane flushing; coughing, sneezing, rapid breathing, panting, and the dog sitting in a sitting position and breathing openly; Some sick pigs coughed continuously, and in severe cases, it was extremely difficult to breathe, with serous or mucous nasal fluid, walking swaying, ataxia, or lying on the ground, driving the limbs into a duck-swimming shape; the head, neck, jaw, The skin of the abdomen and extremities appeared purplish red, and the sick pig finally collapsed and died of suffocation.

1.3.2 Pathological changes: the whole body of the dead pig was pale, and the mouth and nose flowed out of light red or red foam-like liquid. The trachea and bronchi are filled with frothy fluid, the pleural cavity has light yellow effusion, the pleural cavity serosa and the visceral capsule have fibrous films, which are often adhered to the pleural cavity; the lungs, heart, and liver are enlarged, congested, and hemorrhage; the fundus of the stomach Stomach wall thickening, jelly-like liquid, strip-shaped bleeding spots and bleeding points, intestinal mucosal edema, spot-shaped and strip-shaped bleeding, and some intestinal mucosa have hemorrhagic inflammation.

1.3.3 Pathogen isolation and identification

1.3.3.1 Inoculate common agar and MacConkey agar with the collected contents of the front part of the small intestine, liver, spleen, heart, and lymph node tissue of the lesion. Observation after culture: ①Medium-sized, round, translucent, off-white, dewdrops grow on the common agar plate Resembling colonies; MacConkey agar plates grow red colonies. ②Staining microscopic examination: Gram staining microscopic examination was negative for Brevibacterium. ③Biochemical characteristics: The bacteria can ferment maltose, lactose, glucose, mannitol, produce indigo matrix, M-R test is positive, no hydrogen sulfide is produced, and urea is not decomposed. The bacterium was determined to be Escherichia coli.

1.3.3.2 The collected lung, heart, spleen, kidney and other tissues were inoculated on PPLO agar plate, and observed after culture: ①The colony was the size of a needle tip, round, with neat edges, smooth surface, and off-white dewdrop shape; ②Staining microscopic examination: Gram Staining microscopic examination was negative for P. rubrum; ③Biochemical characteristics: The bacteria can ferment fructose and lactose, but not mannitol, sorbitol, rhamnose and mannose. Indigo, methyl red, and V-P tests were negative. Hydrolyzable urease. It was determined to be Actinobacillus pleuropneumoniae.

1.4 Experimental animals and handling

112 piglets from 6 different farms were selected as cases in this experiment by using multiple centers, multiple farms, and random control grouping. Pigs were fed conventional feed without antibiotic additives and had free access to water. All pigs were randomly divided into 4 groups. Dosing according to the experimental design method in Table 4-1.

Table 4-1 Experimental design

drugs 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, once a day for 4 consecutive days. Multibio d 32 Intramuscular injection, AMP 10mg/kg, COS 25,000IU/kg and DEX 0.025mg/kg body weight, once a day for 4 consecutive days. A-C 30 Intramuscular injection, AMO 10mg/kg and COS 25,000IU/kg body weight, once a day for 4 consecutive days. Sick no drug group 15 -

Note: In the table, AMO stands for amoxicillin, AMP stands for ampicillin, COS stands for colistin sulfate, DEX stands for dexamethasone, and PRE stands for prednisolone.

1.5 Observation of drug effect: From the beginning of administration to the end of the observation, a total of 12 days, the clinical symptoms such as breathing, appetite, and mental state of the test pigs in each group were observed, and detailed records were made.

1.6 Efficacy Judgment Criteria

Cure: After 4 days of administration, the test pig's mental state, appetite, respiration, and body temperature all returned to normal, and clinical symptoms such as lameness and diarrhea no longer appeared, and there was no recurrence after drug withdrawal. The cure rate was counted at the 3rd, 6th and the end of the test respectively after drug withdrawal. The cure rate was calculated according to the ratio of cured heads to the number of heads in this group.

Effective: After 4 days of administration, the mental state of the test pigs improved significantly, their appetite increased, they had mild abdominal respiration or cough, lameness and diarrhea. The sum of the number of effective heads and the number of cured heads is the total number of effective heads, and the total effective rate is calculated according to the ratio of the total number of effective heads to the number of test heads in this group.

Ineffective: After 4 days of administration, the test pigs' spirit, appetite, respiratory temperature, lameness, diarrhea and other symptoms still did not improve or even worsened and died. The inefficiency was calculated according to the ratio of the number of invalid heads to the number of experimental heads in this group.

1.6 Statistical analysis of data

Chi-square test (SPSS10.0 statistical analysis software) was used for statistical analysis. P<0.05 means significant difference, P<0.01 means extremely significant difference, P>0.05 means no significant difference.

2 Test results and summary

2.1 The pigs in the disease control group without medication were observed, and the symptoms did not change significantly or aggravated or even died within 5 days, and treatment was taken to reduce losses.

2.2 The cure rates of mixed infection of porcine infectious pleuropneumonia and colibacillosis in A-C-P group, Multibio d group and A-C group were 82.9%, 75.0% and 73.3% respectively, and the total effective rates were 91.4%, 81.3% and 80.0% respectively; See Table 4-2 for specific test data.

Table 4-2 Treatment test results of each group (X±SD)

*: Significant difference compared with A-C-P group (p<0.05); ★: Indicates self-healing rate.

2.3 Statistical analysis shows: A-C-P group, Multibio d group and A-C group are compared with sick non-administration group, and its cure rate, total effective rate difference are extremely significant (P＜0.01), show that three kinds of medicines are effective to porcine contagious pleuropneumonia. Mixed infection with colibacillosis has good curative effect. Compared with the A-C group and the commercially available drug Multibiod group, the total effective rate of the A-C-P group was not significantly different (P>0.05), but the total effective rate of the A-C-P group was 11.4% and 11.4% higher than the A-C group and the commercially available drug Multibiod group respectively. 10.1%, indicating that the overall curative effect of A-C-P is better than that of A-C and the marketed drug Multibio d.

2.4 In this test, the main indicator of cure rate was investigated in three stages. The test results showed that the three drugs all had high cure rates on the third day after drug withdrawal, but the effects were different. 3 days, the cure rate of A-C group is compared with A-C-P group, difference is significant (P＜0.05), the cure rate of A-C group is compared with commercially available drug Multibiotic group and has no significant difference (P＞0.05), but its cure rate is higher than market. Multibio d was 15.6% lower than that of the sold drug, and there was no significant difference (P>0.05) compared with other stages (the 6th day after stopping the drug and the end of the test). It shows that A-C-P group and Multibiod group can make sick pigs return to normal state shorter than A-C group. The reason is that the formulas of A-C-P group and Multibiod group contain anti-inflammatory ingredients, which are helpful for the recovery of sick animals.

2.5 From the comprehensive consideration of the total effective rate and the recovery time of the body, the overall curative effect of the suspension product of the present invention is better than A-C and the commercially available drug Multibio d, indicating that the suspension product of the present invention is more suitable for porcine infectious pleura Treatment of mixed infection of pneumonia and colibacillosis.

Claims (9)

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:

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% (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;

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 is sterilized, and obtains containing the animal use suspensoid liquid of amoxicillin, colistin sulfate and prednisolone.