CN117530922A - A high-stability compound injection for veterinary use and its preparation method and application - Google Patents

Abstract

The present invention is a high-stability compound injection for veterinary use and its preparation method and application. Compound injection based on W/V: enrofloxacin 2.0~10.0%, polymyxin sulfate 1.0~5.0%, co-solvent 0.5%~2.0%, acidity regulator 0.5~3.0%, latent solvent 20.0%~45.0 %, complexing agent 0.2~0.5%, and the rest is water for injection; the compatibility of enrofloxacin and polymyxin sulfate can effectively expand the antibacterial spectrum and enhance the antibacterial efficacy; the compound preparation uses an acid-base neutralization method Combined with latent solution technology, a highly stable compound injection with a neutral pH value (pH=4.5~5.0) and no crystallization during long-term storage (180 d) at low temperature (4°C) and accelerated (30°C) was prepared. , which is conducive to widespread clinical application.

Description

A high-stability compound injection for veterinary use and its preparation method and application

Technical field

The invention belongs to the field of veterinary drug preparation preparation, and specifically relates to a high-stability compound injection for veterinary use and its preparation method and application.

Background technique

Enrofloxacin, as a new fluoroquinolone drug specifically for animals, has strong bacteriostatic effect against a variety of pathogenic bacteria, is widely distributed in the body, and has a long elimination half-life. It is widely used in veterinary clinics to treat Klebsiella pneumoniae, Escherichia coli, and parasol. Infections caused by Haemophilus, Salmonella, Mycoplasma, Chlamydia, Pasteurella multocida, Pasteurella hemolyticus, Staphylococcus aureus and other bacteria. However, reports indicate that bacterial resistance to enrofloxacin is increasingly serious. The barrier effect of the bacterial outer membrane and the efflux effect of the efflux pump greatly restrict the effective accumulation of enrofloxacin in bacterial cells, which is an important reason for affecting the efficacy of the drug.

Polymyxin sulfate is a polypeptide antibiotic that can destroy the outer membrane of bacteria and has excellent antibacterial activity against negative bacteria. Its combined use with drugs such as ciprofloxacin, rifampin, and vancomycin can significantly increase the accumulation of drugs in the bacterial cytoplasm (doi:10.1111/nyas.13598). At the same time, the outer membrane structure is damaged, which also limits the activity of the efflux pump. Therefore, combining with polymyxin is an effective means to improve the penetration of other antibacterial drugs through the outer membrane barrier and efficient accumulation in the bacteria. The present invention intends to prepare an enrofloxacin-polymyxin sulfate compound preparation to improve the antibacterial efficacy of enrofloxacin and its therapeutic effect on drug-resistant bacteria.

Although there have been relevant patent reports on the combination of enrofloxacin and polymyxin sulfate to form an oral solution (Authorization number: CN 111821258 B). However, as shown in Figure 2, according to the technical solution provided by the patent (Authorization No.: CN 111821258 B), the "stable, non-crystallization solution preparation" described by it cannot be produced. Moreover, it is well known in the art that enrofloxacin has an extremely bitter taste and is easily rejected by pigs when administered orally. It is extremely difficult for oral dosage forms to achieve the purpose of "good palatability" stated in the patent (Authorization No.: CN 111821258 B). In addition, the European Union issued a document in 2016 stating that oral administration of colistin sulfate will accelerate the development of intestinal flora resistance to it, and oral colistin sulfate products should be withdrawn from the market as soon as possible. From the perspective of practical application and public health safety, the compound of enrofloxacin and colistin sulfate should not be made into an oral product. Injection is a dosage form that is more in line with public health and usage requirements. Enrofloxacin and polymyxin sulfate compound injection has great application prospects in clinical breeding.

However, a large number of studies have shown that solution formulations of fluoroquinolones (such as enrofloxacin) are prone to crystallization during long-term storage (Xie Yong, 2015). According to reports in the "Notice of the General Office of the Ministry of Agriculture and Rural Affairs on the Third Issue of Veterinary Drug Quality Supervision and Sampling Inspections in 2021", some commercially available enrofloxacin injection products are facing the problem of crystallization. In addition to causing insufficient drug content and affecting drug efficacy, the crystallization of the drug may also cause local inflammation, capillary blockage, and granuloma during injection. In severe cases, it may affect the life and health of the target animal, or even endanger the life. According to reports, increasing the solubility of drugs in solution is an effective means to reduce drug crystallization and improve stability. Among them, the addition of solubilizers, co-solvents and salt formation are important means to increase the solubility of poorly soluble drugs. For example, the use of sodium hydroxide and enrofloxacin to react to form enrofloxacin sodium can improve the enrofloxacin solution dosage form. stability. However, injection solutions with high pH values are highly irritating and can easily cause damage to the injection site. The instructions for commercially available Enrofloxacin Injection (Baili) state that the product may cause temporary damage to local tissues (Figure 1). At the same time, polymyxin sulfate is a polypeptide compound, and high pH can easily hydrolyze the amide bond, affecting its activity. In summary, a more reasonable strategy must be adopted to prepare enrofloxacin-polymyxin sulfate compound injection with stable quality and suitable pH.

The present invention uses one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide as a co-solvent, and uses one or a combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid. The combination is an acidity regulator, using one or a combination of polyethylene glycol-400, n-butanol, ethanol as a latent solvent, and one or a combination of disodium edetate and calcium edetate as the complex. mixture. First, the acid-base neutralization method was used to prepare enrofloxacin sodium salt, which improved the solubility of enrofloxacin. It was also treated with an acidity regulator to ensure the stability of polymyxin sulfate and stabilize the pH in a low-irritation range. , combined with latent solution technology to further enhance the hydrogen bonding between enrofloxacin and latent solvent molecules, making the enrofloxacin molecules more stable in aqueous solutions, and solving the problem that enrofloxacin solution dosage forms are prone to crystallization during long-term storage. The invention cleverly combines polymyxin and enrofloxacin. The two have different antibacterial targets, allowing them to exert maximum antibacterial activity, delay the development of bacterial resistance, and destroy the intrinsic outer membrane of bacteria. Adaptive compensation increases, inhibits the production of bacterial biofilm subpopulations and retained bacterial subpopulations, and reduces the probability of clinical stubborn infections and repeated infections. The combined use can improve the antibacterial efficacy of the two while reducing the dosage, which is in line with the requirements of my country's "dose reduction, efficiency increase" policy. More importantly, the present invention takes into account the practicability of the dosage form, the safety of the formula, and the storage stability, solves the application and production limitations of strong irritation in clinical use and low product stability, and is beneficial to the formulation of enrofloxacin and polyviscosity sulfate. The wide application of bacteriocin compound products. In summary, the enrofloxacin-polymyxin sulfate compound injection provided by the present invention will undoubtedly contribute to the prevention and treatment of bacterial infections in livestock and poultry in clinical breeding and delay the development of bacterial resistance.

Contents of the invention

The object of the present invention is to provide a highly stable enrofloxacin-polymyxin sulfate compound injection, which can be used for intramuscular injection in order to achieve low irritation; and can also inhibit the development of bacterial tolerance subgroups. Produce a compound preparation that can reduce the frequency of drug-resistant mutations of pathogenic bacteria. The development of the invention will not only be beneficial to the treatment of drug-resistant bacterial infections in animals in the clinic, but also delay the development of drug resistance of pathogenic bacteria.

The present invention uses one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide as a co-solvent, and uses one or a combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid. The combination is an acidity regulator, using one or a combination of polyethylene glycol-400, n-butanol, ethanol as a latent solvent, and one or a combination of disodium edetate and calcium edetate as the complex. mixture. The innovative combination of acid-base neutralization method and latent solution technology solves the problems of enrofloxacin solution formulation being easy to crystallize, highly irritating, and difficult to be compatible with polymyxin sulfate. The present invention cleverly utilizes the characteristics of polymyxin targeting the intrinsic bacterial outer membrane. Its combination with enrofloxacin will help enrofloxacin cross the bacterial outer membrane and increase its concentration in the bacterial cytoplasm, thereby promoting Enrofloxacin is combined with DNA polymerase to achieve maximum antibacterial efficacy. At the same time, the characteristics of polymyxin sulfate targeting the outer membrane will also help to eliminate dormant bacterial subpopulations (highly resistant to enrofloxacin). The compound injection provided not only significantly improves the antibacterial activity of enrofloxacin and polymyxin sulfate against drug-resistant bacteria, but also solves the problem of easy crystallization of commercially available enrofloxacin solution dosage forms. The compound injection not only has a suitable pH, but can also be stored at low temperature and room temperature for a long time. Further development of the present invention will not only be beneficial to the treatment of clinical animal bacterial infections, especially drug-resistant bacterial infections, but can also delay the development of drug resistance of pathogenic bacteria. It is a practical practice of my country's "reduce dosage and increase efficiency" policy.

Specifically, the present invention provides the following technical solutions:

The first aspect of the present invention provides a veterinary enrofloxacin-polymyxin sulfate compound injection, which is characterized in that the ingredients of the injection form the following preparation composition before mixing according to mass/volume:

(1) Enrofloxacin 2.0~10.0%;

(2) Polymyxin sulfate 1.0~5.0%;

(3) Co-solvent 0.5%~2.0%;

(4) Acidity regulator 0.5~3.0%;

(5) Latent solvent 20.0%~45.0%;

(6) Complexing agent 0.2~0.5%;

(7) The rest is water for injection.

In a preferred embodiment, the components of the injection form the following preparation composition before mixing based on mass/volume:

(1) Enrofloxacin 2.0~5.0%;

(2) Polymyxin sulfate 1.0~2.5%;

(3) Co-solvent 0.5%~2.0%;

(4) Acidity regulator 0.5~3.0%;

(5) Latent solvent 20.0%~45.0%

(6) Complexing agent 0.2~0.5%

(7) The rest is water for injection.

In a preferred embodiment, the co-solvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine, sodium hydroxide; preferably, the co-solvent is selected from sodium hydroxide.

In another specific embodiment, the acidity regulator is one of n-butyric acid, lactic acid, malic acid, tartaric acid, citric acid or a combination thereof; preferably, the acidity regulator is malic acid and/or lactic acid; most preferred is lactic acid.

In another specific embodiment, the latent solvent is one or a combination of polyethylene glycol-400, n-butanol, ethanol; preferably, the latent solvent is 1,2-propanediol and /or polyethylene glycol-400.

In another specific embodiment, the complexing agent is one of disodium edetate, calcium edetate, or a combination thereof; disodium edetate is preferred.

The second aspect of the present invention is to provide a method for preparing the veterinary enrofloxacin-polymyxin sulfate compound injection described in the first aspect. Specifically, the method includes the following steps:

1) Weigh the co-solvent and complexing agent according to the formula amount, dissolve it with water for injection, add the formula amount of enrofloxacin, stir at room temperature until completely dissolved, filter and sterilize with a membrane, and set aside;

2) Weigh the acidity regulator and latent solvent according to the formula amount, dissolve it with water for injection, add the formula amount of colistin sulfate, stir at room temperature until completely dissolved, filter and sterilize with the filter membrane, and set aside;

3) Add the filtrate in 1) to the filtrate 2) and stir continuously until a clear mixed solution is obtained. After filtration and sterilization with a membrane under negative pressure vacuum, the compound injection is obtained.

In a preferred embodiment, the filtration in steps 1)-3) is sterilization using a 0.22 μm membrane filtration;

In another specific embodiment, the negative pressure vacuum in step 3) is -0.1~0.05 atmospheres.

The third aspect of the present invention is a method for improving the stability of enrofloxacin-containing compound injection. The method is: adding cosolvents, latent solvents, acidity regulators and complexes to the compound injection. mixture;

In a preferred embodiment, the co-solvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine, sodium hydroxide; preferably, the co-solvent is selected from sodium hydroxide.

In another specific embodiment, the acidity regulator is one of n-butyric acid, lactic acid, malic acid, tartaric acid, citric acid or a combination thereof; preferably, the acidity regulator is malic acid and/or lactic acid; most preferred is lactic acid.

In another specific embodiment, the latent solvent is one or a combination of polyethylene glycol-400, n-butanol, ethanol; preferably, the latent solvent is 1,2-propanediol and /or polyethylene glycol-400.

In another specific embodiment, the complexing agent is one of disodium edetate, calcium edetate, or a combination thereof; disodium edetate is preferred.

In another preferred embodiment, the enrofloxacin-containing compound injection is enrofloxacin-polymyxin compound injection.

The fourth aspect of the present invention is the application of the veterinary enrofloxacin-polymyxin sulfate compound injection described in the first aspect in the preparation of drugs for treating bacterial infections.

In a specific embodiment, the bacteria include, but are not limited to, Klebsiella pneumoniae, Escherichia coli, Actinobacillus thoracicidal pneumoniae, Salmonella, MRSA, and Pasteurella multocida.

The beneficial effects of the present invention are as follows:

The highly stable enrofloxacin-polymyxin sulfate compound injection for veterinary use provided by the present invention utilizes the characteristics of polymyxin to destroy the outer membrane of bacteria, which greatly enhances the entry of enrofloxacin into bacterial cells. The qualitative ability of enrofloxacin improves the antibacterial activity of enrofloxacin against pathogenic bacteria, especially drug-resistant bacteria; on the other hand, the characteristic of polymyxin sulfate targeting the outer membrane is also conducive to the development of dormant subpopulations that are resistant to enrofloxacin. Clear. While improving the effectiveness of enrofloxacin and polymyxin sulfate, it can also delay the development of drug resistance. At the same time, this compound injection not only improves the solubility and stability of enrofloxacin, but also ensures the use of injections in clinical applications, avoids the problems of poor palatability and high irritation of the original dosage form, and can be guaranteed to be used at low temperatures (4°C) and It does not crystallize during long-term storage under accelerated (30°C) conditions, which is beneficial to the clinical application of enrofloxacin and polymyxin sulfate. The acid-base neutralization crystallization method and latent solution method used in this suspension process have low requirements on equipment and personnel quality, and the auxiliary materials are cheap and easy to obtain, which is conducive to large-scale industrial production. In addition, the introduction of this compound injection will compete with similar foreign products for market share, reduce the impact of foreign products on my country's veterinary drug market, and will help improve the international competitiveness of my country's veterinary drug companies. The compound injection has broad market prospects and can be injected intramuscularly for the prevention and treatment of Pasteurella multocida, Klebsiella pneumoniae, Haemophilus parasuis, Actinobacter pleuropneumoniae, and pneumonia in pigs, cattle, sheep, chickens and other animals. Livestock and poultry infections caused by Streptococcus, Mycoplasma, etc., especially related drug-resistant bacteria.

Description of drawings

Figure 1 Instructions for commercially available enrofloxacin injection (Baili injection)

Figure 2 The crystallization situation of the compound oral solution prepared based on the technical solution in the reference invention patent (Authorization No.: CN 111821258 B): A The solution made of propylene glycol alginate and buckwheat proteoglycan according to the material-to-liquid mass ratio of 1:4 is actually a paste Like semi-solid material, B, there is a large amount of precipitation in the lower layer of the 100 mL system prepared according to the reference invention patent method;

Figure 3 Synergistic efficacy of enrofloxacin and polymyxin sulfate against animal pathogenic bacteria: A Klebsiella pneumoniae WNX-1, B Salmonella 80048, C Klebsiella pneumoniae 25-2, D Klebsiella pneumoniae Botrytis 29-1, E Klebsiella pneumoniae 37-1, F MRSA T144;

Figure 4 Antibacterial advantages of enrofloxacin combined with polymyxin sulfate against enrofloxacin-resistant Klebsiella pneumoniae;

Figure 5 Skin irritation and injection site irritation of the compound injection of the present invention: A, C, and E are the skin conditions of the injection site on the left hind leg (physiological saline) of the rabbit on the 7th day of continuous injection, and B, D, and F are The skin condition of the rabbit's right hind leg (compound injection) injection site on the 7th day of continuous injection;

Figure 6 The effect of the compound injection of the present invention on blood routine and blood biochemistry in rabbits;

Figure 7 The histopathological effects of the compound injection of the present invention on muscle damage at the injection site and liver and kidneys in rabbits;

Figure 8 The therapeutic effect of the compound injection of the present invention on the mouse Pasteurella multocida infection model, A the survival curves of mice in the PBS group and the compound injection treatment group, B the bacterial load in the lungs of surviving mice.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention. However, these modifications and substitutions are fall within the protection scope of the present invention.

Example 1 Determination of co-solvent types in compound injection

Prepare saturated sodium glutamate, meglumine, triethanolamine, and sodium hydroxide solutions. Dissolve 2.5g of enrofloxacin in the saturated solutions until they are completely dissolved. Record the volume of the saturated solution used. The results showed that the dissolving effect of cosolvents on enrofloxacin from strong to weak was sodium hydroxide, triethanolamine, sodium glutamate, and meglumine. Therefore, sodium hydroxide was selected as the cosolvent for the compound injection.

Example 2 Determination of the type of acidity regulator in compound injection

Use 37 mL of sodium hydroxide saturated solution to completely dissolve 2.5 g of enrofloxacin to obtain five portions of enrofloxacin saturated solution under alkaline conditions, to which n-butyric acid, lactic acid, malic acid, and tartaric acid were added dropwise. , citric acid. During this process, the solution quickly becomes turbid. Continue to add dropwise, and the solution gradually becomes clear until it is completely clear. Record the amount of acid solution used in this process, and let the clear solution stand at room temperature to observe the precipitation of the solution. The results are shown in Table 1. The volume used to adjust the acidity of lactic acid and malic acid is small and no precipitation occurs when left at room temperature for 48 hours. However, the raw material cost of malic acid is high, so lactic acid is selected as the acidity regulator of the compound injection.

Table 1 Determination of acidity regulator for compound injection

Example 3 Determination of latent solvent types in compound injection

The types and amounts of raw materials and auxiliary materials in the formula are shown in Table 2:

Table 2. Types and dosage of raw materials and excipients of a compound injection

According to the types and amounts of raw materials and auxiliary materials listed in Table 2, weigh the required amount and prepare according to the following preparation method:

1) Weigh the co-solvent and complexing agent according to Table 2, dissolve them in 30 mL of water for injection, add the formula amount of enrofloxacin, stir at room temperature until completely dissolved, filter and sterilize with a 0.22 µm filter, and set aside;

2) Weigh the acidity regulator according to Table 2, dissolve it in 20 mL of water for injection, then add one of ethanol/n-butanol/propylene glycol/polyethylene glycol-400 as a latent solvent according to the amount shown in Table 2, and add Stir the formula amount of polymyxin sulfate at room temperature until completely dissolved, filter and sterilize with a 0.22 µm filter, and set aside;

3) Add the filtrate in 1) to the filtrate 2) and stir continuously until a clear light yellow solution is obtained. After filtering and sterilizing with a 0.22 µm filter membrane at 0.08 atmospheric pressure, seal it in five 30 mL bottles of brown medium borosilicate. In the glass bottle, you can get the compound injection.

The above four kinds of compound injections containing different latent solvents were placed in a 30°C incubator for 15 days. During this period, the crystallization conditions were observed every day and recorded in Table 3. As shown in Table 3, compared with the group without latent solvent, the Tween-80, n-butanol, ethanol, 1,2-propanediol and polyethylene glycol-400 groups all had some effects on the crystallization of the compound injection. Improvement, among which no crystallization was observed in the 1,2-propanediol and polyethylene glycol-400 groups at 15 days, so 1,2-propanediol and/or polyethylene glycol-400 were planned as potential candidates in the subsequent preparation process. Solvent.

Table 3. Crystallization conditions of compound injections containing different latent solvents

Example 4 Determination of the dosage of latent solvent, pH and complexing agent in compound injection

Since the present invention prepares an aqueous injection of enrofloxacin-polymyxin sulfate, enrofloxacin can chelate with heavy metal ions such as Ca ²⁺ , Mg ²⁺ , Fe ^{3 +} or Al ³⁺ , so it is necessary to Consider adding a complexing agent to remove potential heavy metal ions in the preparation water and pipelines to maximize the stability of enrofloxacin in the compound injection. At the same time, pH value also affects the crystallization stability of enrofloxacin (Xie Yong & Dong Qiaoqiao, 2015), and the interactive effects of complexing agent, pH and cosolvent need to be comprehensively considered. Therefore, in the present invention, an orthogonal experimental design is adopted to comprehensively investigate the dosage of complexing agent, pH and latent solvent.

In this study, the commonly used disodium edetate was used as a chelating agent. According to the dosage range of disodium edetate for injection recommended by the FDA (0.01%~0.1%) (https://db.yaozh.com/), Set up three gradient levels of 0.02~0.03%, 0.05~0.06%, and 0.1~0.11% for testing; use 1,2-propanediol as a latent solvent and set three gradient levels of 30~35%, 35~40%, and 40~45% respectively. The test was conducted at different levels; the pH values were set at 3 levels of 3.5~4.0, 4.5~5.0, and 5.0~5.5 (Table 3). That is, an orthogonal design with 9 different combinations of 3 factors and 3 variables was used to examine the crystallization of the compound solution within 90 days at 30°C under different amounts of complexing agent, pH, and latent solvent to finally determine the above parameters.

Table 4. Orthogonal experimental design table

Table 5 Orthogonal test results

Example 5 Comparison of pH between compound injection provided by the present invention and commercially available enrofloxacin injection (Baili injection)

Use a syringe to draw 5 mL each of the compound injection of the present invention and the commercially available enrofloxacin injection (Bailley injection, batch number KV03XF8, Figure 1 shows the product instructions) into a 10 mL centrifuge tube, and use a calibrated pH meter to pH testing was performed with 3 replicates in each group. The results show that the pH value of commercially available enrofloxacin injection (Baili injection) is 10.95 ± 0.13, and the pH value of the present invention is 5.0 ± 0.11. It can be seen that the pH range of the enrofloxacin-polymyxin sulfate compound injection provided by the present invention is closer to the physiological pH of animals. Compared with the commercially available enrofloxacin injection (Baili injection), it is expected to have better effects on target animals. Better compliance.

Example 6 Repetition of the compound oral liquid provided in the invention patent (Authorization No.: CN 111821258 B)

The enrofloxacin-polymyxin sulfate compound oral solution was prepared according to the method described in the examples of the invention patent (Authorization No.: CN 111821258 B). There are two main issues:

(1) The auxiliary materials cannot be completely dissolved according to the description of the patent: the content described in the patent "Propylene glycol alginate and buckwheat proteoglycan are made into a solution according to the mass ratio of material to liquid 1:4" is actually a paste-like semi-solid substance rather than a patent (Authorization number: CN 111821258 B) solution (Fig. 2A). After trying, it turns out that propylene glycol alginate and buckwheat proteoglycan should be added to water for injection at a material-to-liquid ratio of 1:8, and they need to be heated in a 90°C water bath for 20 minutes before they can be dissolved into a solution;

(2) The prepared solution has obvious precipitation and should be a suspension, which is inconsistent with its description of "solution-type appearance and no crystallization".

As shown in Figure 2B, the 100mL system prepared according to the technical solution in the invention patent (Authorization No.: CN 111821258 B) has a large amount of precipitation in the lower layer, which is not a "uniform clear solution" as described in the patent. At the same time, after being protected from light at room temperature for 7 days, centrifuge the upper clarified liquid and use high performance liquid chromatography (HPLC) to detect the content of enrofloxacin. The results show that the content of enrofloxacin is only 2.8%, which is consistent with the patent (authorization number: CN 111821258 B) is seriously inconsistent with the theoretical value of 5.0%, indicating that the preparation form prepared according to the technical solution of the patent (Authorization No.: CN 111821258 B) is very unstable. Moreover, it is well known in the art that enrofloxacin tastes extremely bitter. When used as an oral liquid, it is not difficult to speculate that a large amount of free enrofloxacin in the supernatant will cause problems with palatability. Therefore, the technical method described in the invention patent (Authorization No.: CN 111821258 B) cannot produce the enrofloxacin-polymyxin sulfate compound oral solution that is “not easy to crystallize and has good palatability”. Based on this, in order to provide an efficient and high-quality enrofloxacin-polymyxin sulfate compound preparation product for my country's veterinary clinics and enhance the international competitiveness of my country's veterinary drug companies, the present invention has undergone careful formulation and compatibility research. A more reasonable technical solution was proposed.

Example 7 Investigation of the synergistic efficacy of enrofloxacin and polymyxin sulfate against swine pathogenic bacteria when used in combination

Klebsiella pneumoniae (WNX-1, 37-1, 29-1, 25-2), Escherichia coli (OF B2 D19, OF 25922D38), Actinobacillus pleuropneumoniae (Hang8), methicillin-resistant gold Staphylococcus aureus (T144), Salmonella (80048) and Pasteurella multocida (P32 D7) were recovered and passaged.

The checkerboard test was conducted according to the method specified by CLSI to verify the efficacy of enrofloxacin combined with polymyxin sulfate. The specific methods are as follows:

1) Use fresh broth culture medium to dilute the original bacterial solution to a concentration of ~10 ⁶ CFU/mL;

2) Place two rows of test tubes on the test tube rack, with 7 test tubes in each row. The first row is used to dilute drug A, and the second row is used to dilute drug B. In the first row of test tubes, use the broth culture medium for drug A as Dilute 2 times so that the concentrations of drug A in tubes 1 to 7 are 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/16 MIC. The dilution steps for drug B are the same as those for drug A;

3) Add the A and B bacterial solutions in order to each well in the horizontal and vertical rows of the 96-well cell culture plate; add the A solution in the first tube to the 1st row of 8 of the cell culture plate in sequence. In each well, 50 µL per well, the solution in the second tube was added to the 8 wells in the second row, 50 µL per well. Add liquid A from tubes 3 to 7 to each hole in rows 3 to 7 in the same way. Row 8 serves as a separate drug sensitivity control for drug B, without adding drug A; drug B follows the method of drug A;

4) Add vertical rows 1 to 7 wells in sequence; then add 50 µL MH broth to each of the horizontal rows and vertical row 8, and then add 100 µL of the above diluted bacterial solution to each well to achieve the final concentration of the bacterial solution. It is 10 ⁵ CFU/mL, and negative control holes are set at the same time. Place the 96-well plate in a 37 ^° C constant-temperature incubator for 20 hours and then take out the observation and recording tubes. Each experiment is repeated three times.

5) Result judgment: Calculate the synergy or additive index of enrofloxacin and polymyxin sulfate through the following formula. Among them, when FICI ≤ 0.5, it is a synergistic effect, when 0.5 < FICI ≤ 1, it is an additive effect, when 1 < FICI ≤ 4, it is an irrelevant effect, and when FICI > 4, it is an antagonism.

Table 6. Synergistic efficacy of enrofloxacin and polymyxin sulfate against swine pathogens

Note: MRSA: methicillin-resistant Staphylococcus aureus; ENR: enrofloxacin; Coli: polymyxin sulfate

The synergistic efficacy of enrofloxacin and polymyxin sulfate against common swine pathogenic bacteria is shown in Table 1. It can be seen from the table that Klebsiella pneumoniae (WNX-1), methicillin-resistant Staphylococcus aureus (T144) and Salmonella (80048) have higher resistance levels to enrofloxacin. When polymyxin sulfate is added At this time, the sensitivity of the above high-level drug-resistant strains to enrofloxacin increased significantly, and the maximum synergistic effect could reach 16 times. Although Actinobacillus pleuropneumoniae (Hang 8) is relatively sensitive to enrofloxacin, its MIC value still dropped 16-fold after adding polymyxin sulfate. And the sensitivity of methicillin-resistant Staphylococcus aureus (T144), a Gram-positive bacterium, to enrofloxacin was also significantly reduced. In addition, when the two are used together, the sensitivity of the above strains to polymyxin sulfate is also significantly reduced, up to 16 times. The above results show that the combination of enrofloxacin and polymyxin sulfate can significantly increase the antibacterial activity of both, especially the antibacterial activity against drug-resistant bacteria, proving the rationality of the compatibility of the compound provided by the present invention.

Example 8 Inhibitory effect of enrofloxacin combined with colistin sulfate on Klebsiella pneumoniae resident bacterial subpopulations

1.Materials and methods

Drug concentration settings: 10 times and 1 times the MIC of enrofloxacin and polymyxin sulfate, and 0.5 times the MIC for both drugs.

Bacterial liquid preparation: Pick a single colony of bacteria, inoculate it into 1ml of LB broth, adjust it to 0.5 McFarland turbidity using a McFarland turbidimeter, and add it to a culture medium containing different drug concentrations. The initial bacterial concentration is 10 ⁶ CFU/ml. .

Test operation: Add the bacterial liquid to the culture medium containing different concentrations of drugs. Take 100 µL of the bacterial liquid at 0, 2, 4, 6, and 12 hours, dilute it with PBS, and apply 100 µL of the liquid when the appropriate dilution factor is reached. Incubate on a BHA plate in a 37°C incubator overnight and count colonies.

2.Test results

The results of this test are shown in Figure 4 of the instruction manual. The WNX-1 strain is an enrofloxacin-resistant strain (MIC value is 256 µg/mL), and obvious retained bacteria appeared when it was treated with 2560 µg/mL enrofloxacin (10×MIC) for 4-6 hours. The characteristic is that after rapid sterilization in the first 4 hours, the curve shows a flatter trend, and after 6 hours, bacteria begin to gradually multiply and grow. Compared with enrofloxacin, 10×MIC polymyxin sulfate can completely kill bacteria in 4 hours, but under the action of 1 times MIC concentration alone, neither enrofloxacin nor polymyxin sulfate can kill bacteria. The bacteria were completely killed. After 4-6 hours of drug action, the retained bacteria also developed temporary tolerance to polymyxin sulfate and began to reproduce and grow. This suggested that the subpopulation of retained bacteria of Klebsiella pneumoniae was resistant to antibiotics, especially reproduction. The fungicide (enrofloxacin) has strong resistance. Regrowth in the context of a subpopulation of retained bacteria predicts the risk of recurrent Klebsiella pneumoniae infection and a high probability of resistance mutations in this subpopulation.

As shown in Figure 3A-Figure 3F, the combination group showed obvious advantages. When used in combination, both drugs showed superior bactericidal efficacy against drug-resistant Klebsiella pneumoniae at 0.5 times the MIC. The bactericidal efficacy for 12 hours was equivalent to that of polymyxin sulfate at 10×MIC, and there was no bactericidal effect during the sterilization process. The emergence of a plateau eliminates the risk of recurrent infection caused by the retained bacterial subpopulation. And when used in combination, the bactericidal speed of polymyxin sulfate has also been significantly improved; the specific results are as follows: Figure 3A for WNX-1 strain, Figure 3B for Salmonella 80048, Figure 3C for Klebsiella pneumoniae 25-2, Figure 3D for pneumonia Klebsiella 29-1, Figure 3E Klebsiella pneumoniae 37-1, Figure 3F MRSA T144. The above results indicate that the combined use of enrofloxacin and polymyxin sulfate is a good strategy to reduce dosage and delay the development of drug resistance.

Example 9 Accelerated stability and low temperature stability of compound injection

Prepare the compound solution for injection according to the preparation process described in this patent, and distribute it into brown medium borosilicate bottles, each bottle is about 20 mL.

Accelerated stability test: Place the packaged compound solution at 30°C, take out a small amount of liquid at 1, 2, 3, and 6 months to measure its pH, and use high-performance liquid chromatography (HPLC) to detect the content and content of active ingredients in the solution. Related substance (ciprofloxacin) content. At the same time, the stability of the compound preparation of the present invention can be judged by observing the visible foreign matter, crystallization situation, appearance color, etc.

Low-temperature stability test: Place the packaged compound solution at 4°C, and mainly examine the crystallization of the prepared compound solution in the first, second, third, and sixth months to evaluate the low-temperature stability of the compound solution.

test results

The following table shows the crystallization conditions of the 30°C accelerated stability test group. It can be seen that groups 1 and 6 have long-term stability, and no crystals have been precipitated for 6 months. In group 1, moderate crystal precipitation was seen in the 4°C accelerated stability test for 30 days. Overall, the stability of group 6 in the orthogonal test was the strongest.

Table 7 Crystallization conditions and pH changes in accelerated stability test at 30°C

Note: -: No crystal precipitation; +: A small amount of crystal precipitation; ++: Moderate crystal precipitation; +++: A large amount of crystal precipitation

Table 8 Relative content of enrofloxacin (%) in accelerated stability test at 30℃

Table 9 Crystallization conditions in accelerated stability test at 4℃

Example 10 Investigation of skin irritation and safety of compound injection

The compound injection solution was prepared according to the preparation process described in this patent, and the experiment was conducted on the compound injection preparation stored for 6 months according to the above-mentioned Example Group 6. Adult male rabbits were injected intramuscularly with 2 times the recommended dose for 7 consecutive days. From the 1st to the 7th day, the compound solution was injected into the muscle of the right hind leg of the rabbit every day, and the same volume of normal saline was injected into the muscle of the left hind leg. After a week of recovery, Sampling observations were conducted again on the 14th day. On days 0, 7, and 14, blood was collected from the marginal ear vein of the experimental rabbits. 0.5 mL of blood was added to the EDTA anticoagulant tube for routine blood testing, and another 1 mL of blood was taken and left to stand at room temperature for 20-30 min, and centrifuged at 2000-3000 rpm. After 10 minutes, the upper serum was separated for detection of blood biochemical indicators such as liver function (serum alanine aminotransferase ALT, serum aspartate aminotransferase AST, total protein TP, albumin ALB), renal function (urea UREA, creatinine CREA). On the 7th day, the skin irritation and injection site irritation of the injection site were examined. On the 14th day, the rabbit liver, kidney, and injection site muscle tissue were fixed in 4% paraformaldehyde, and HE stained to observe histological changes. .

As shown in Figure 5, A, C, and E are the injection sites of the rabbit's left hind leg (physiological saline), and B, D, and F are the injection sites of the rabbit's right hind leg (compound injection) on the skin on the 7th day of continuous injection. status, no abnormal changes were seen. As shown in Figure 6, liver function indicators such as serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), total protein (TP), albumin (ALB), and renal function indicators such as urea (UREA) and creatinine (CREA) No significant changes occurred after 7 days of continuous injection, and there was no significant impact on the number of red blood cells (RBC), white blood cells (WBC), and platelets (PLT). As shown in Figure 7, there were no obvious pathological changes in the muscle tissue of the treatment group and the blank control group. Therefore, the compound injection will not cause pathological damage to the injection site. In addition, no obvious inflammatory cell infiltration and hepatocyte necrosis were found in the liver tissue of the treatment group and the blank group, and no pathological changes such as inflammatory cell infiltration, interstitial edema, congestion, microthrombi, and renal tubular necrosis were found in the kidney tissue. Therefore, the compound injection of the present invention has no skin irritation or injection site irritation, and does not affect the basic physiological state, liver function and kidney function of rabbits when administered continuously for 7 days at 2 times the recommended dose, and has good safety.

Example 11 Therapeutic effect of compound injection on mouse Pasteurella multocida infection model

1. Establish a respiratory infection model of Pasteurella multocida

The compound injection solution was prepared according to the preparation process described in this patent, and the experiment was conducted on the compound injection preparation stored for 6 months according to the above-mentioned Example Group 6.

Select 20-25g female ICR mice for the experiment. Before the experiment begins, each mouse is anesthetized by intraperitoneal injection of 100 μL of 1% sodium pentobarbital. The mouse is suspended vertically, pinches the nose and pulls it out with tweezers. Use a pipette to pour 75 μL of bacterial solution into the trachea of the mouse (the MICs of the bacteria for enrofloxacin and polymyxin sulfate are: 16 μg/mL and 1 μg/mL respectively). The concentration of the solution is ~6×10 ¹⁰ CFU/mL, and it is suspended for 2-3 minutes. After all the bacterial solution enters the trachea of the mouse, place it horizontally in the cage. Infected mice were treated by intramuscular injection with the recommended dose of the compound 1 hour after the challenge, and again at 24 hours and 48 hours after infection, which was in line with the recommended dose and course of treatment for clinical use of the compound product.

Observe the status of the mice every 6 hours after infection. After the mice die, take the lung tissue and grind it. Use PBS to dilute the grinding solution 10 times in a gradient and spread it on a sheep blood plate containing 1/4 MIC enrofloxacin. , count the number of colonies on the plate after 24 hours in a 37°C incubator. All mice were sacrificed 120 h after infection, and the lung tissues were ground and plated for counting.

2.Test results

Within 120 hours of infection, the survival curves of mice in the PBS group and compound injection treatment group are shown in Figure 8A. The results prove that compound injection can effectively slow down the degree of infection in mice and effectively improve the survival rate. As shown in Figure 8B, the bacterial load in the lungs of surviving mice decreased significantly. In short, the compound injection provided by the present invention has superior therapeutic effects on multi-drug-resistant Pasteurella multocida infections, and will have significant therapeutic effects on common clinical bacterial and mycoplasma infections in livestock and poultry.

The above embodiments are the most preferred modes of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention. However, these modifications and substitutions All fall within the protection scope of the present invention.

Claims (10)

1. The veterinary enrofloxacin-polymyxin sulfate compound injection is characterized in that the injection comprises the following components in parts by mass/volume before being mixed:

(1) 2.0-10.0% of enrofloxacin;

(2) 1.0-5.0% of polymyxin sulfate;

(3) 0.5% -2.0% of cosolvent;

(4) 0.5-3.0% of acidity regulator;

(5) 20.0% -45.0% of latent solvent;

(6) 0.2-0.5% of complexing agent;

(7) The balance of water for injection.

2. The compound injection according to claim 1, wherein the components in the injection before being mixed according to mass/volume are composed of the following preparation components:

(1) 2.0-5.0% of enrofloxacin;

(2) 1.0-2.5% of polymyxin sulfate;

(3) 0.5% -2.0% of cosolvent;

(4) 0.5-3.0% of acidity regulator;

(5) 20.0% -45.0% of latent solvent;

(6) 0.2-0.5% of complexing agent;

(7) The balance of water for injection.

3. The veterinary enrofloxacin-polymyxin sulfate compound injection according to claim 1 or 2, wherein the cosolvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide.

4. The veterinary enrofloxacin-polymyxin sulfate compound injection according to claim 1 or 2, wherein the acidity regulator is one or a combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid.

5. The veterinary enrofloxacin-polymyxin sulfate compound injection according to claim 1 or 2, wherein the latent solvent is one or a combination of polyethylene glycol-400, n-butanol and ethanol.

6. The veterinary enrofloxacin-polymyxin sulfate compound injection according to claim 1 or 2, wherein the complexing agent is one or a combination of disodium edentate and calcium sodium edentate.

7. The method for preparing veterinary enrofloxacin-polymyxin sulfate compound injection according to any one of claims 1-6, which is characterized by comprising the following steps:

1) Weighing cosolvent and complexing agent according to the formula amount, dissolving with water for injection, adding enrofloxacin according to the formula amount, stirring at room temperature until completely dissolving, filtering with a filter membrane, and sterilizing for later use;

2) Weighing an acidity regulator and a latent solvent according to the formula amount, dissolving the acidity regulator and the latent solvent by using water for injection, adding the colistin sulfate according to the formula amount, stirring at room temperature until the colistin sulfate is completely dissolved, filtering with a filter membrane, and sterilizing for later use;

3) Adding the filtrate in the step 1) into the filtrate 2), continuously stirring until a clear mixed solution is obtained, and filtering and sterilizing the mixed solution by a filter membrane under negative pressure vacuum to obtain the compound injection.

8. A method for improving stability of a compound injection containing enrofloxacin is characterized by comprising the following steps: adding a cosolvent, a latent solvent, an acidity regulator and a complexing agent into the compound injection;

the cosolvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide;

the acidity regulator is one or the combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid;

the latent solvent is one or the combination of polyethylene glycol-400, n-butanol and ethanol;

the complexing agent is one or the combination of disodium edentate and calcium edentate.

9. The method of claim 8, wherein the enrofloxacin-containing compound injection is enrofloxacin-polymyxin sulfate compound injection.

10. Use of a veterinary enrofloxacin-polymyxin sulfate compound injection according to any one of claims 1-6 in the manufacture of a medicament for the treatment of a bacterial infection.