CN117530922B - High-stability compound injection for livestock and preparation method and application thereof - Google Patents
High-stability compound injection for livestock and preparation method and application thereof Download PDFInfo
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- CN117530922B CN117530922B CN202410029664.4A CN202410029664A CN117530922B CN 117530922 B CN117530922 B CN 117530922B CN 202410029664 A CN202410029664 A CN 202410029664A CN 117530922 B CN117530922 B CN 117530922B
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- enrofloxacin
- injection
- compound injection
- cosolvent
- polymyxin sulfate
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- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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Abstract
The invention relates to a high-stability compound injection for livestock and a preparation method and application thereof. The compound injection is calculated according to the W/V: 2.0-10.0% of enrofloxacin, 1.0-5.0% of polymyxin sulfate, 0.5-2.0% of cosolvent, 0.5-3.0% of acidity regulator, 20.0-45.0% of cosolvent, 0.2-0.5% of complexing agent and the balance of water for injection; the enrofloxacin and the polymyxin sulfate are combined, so that the antibacterial spectrum can be effectively enlarged, and the antibacterial curative effect can be enhanced; the compound preparation combines an acid-base neutralization method and a latent solvent technology to prepare the high-stability compound injection which is neutral in pH value (pH=4.5-5.0) and is not devitrified after long-term storage (180 d) at low temperature (4 ℃) and acceleration (30 ℃) and is favorable for wide clinical application.
Description
Technical Field
The invention belongs to the field of veterinary drug preparation, and in particular relates to a high-stability compound injection for livestock, a preparation method and application thereof.
Background
Enrofloxacin is used as a novel fluoroquinolone drug special for animals, has strong antibacterial effect on various pathogenic bacteria, wide in-vivo distribution and long elimination half-life, and is widely used for treating infections caused by bacteria such as klebsiella pneumoniae, escherichia coli, haemophilus parasuis, salmonella, mycoplasma, chlamydia, pasteurella multocida, pasteurella haemolytica, staphylococcus aureus and the like in veterinary clinics. However, it has been reported that bacterial resistance to enrofloxacin is increasingly severe. The barrier effect of the outer membrane of the bacteria and the discharge effect of the discharge pump greatly restrict the effective accumulation of enrofloxacin in bacterial cells, and are important reasons for influencing the drug effect.
The polymyxin sulfate is polypeptide antibiotics, can destroy the outer membrane of bacteria, and has excellent antibacterial activity on negative bacteria. The combination of the medicine with ciprofloxacin, rifampin, vancomycin and other medicines can obviously increase the accumulation amount of the medicines in bacterial cytoplasm (doi: 10.1111/nyas.13598). Meanwhile, the outer membrane structure is broken, and the activity of the efflux pump is limited. Therefore, the combination with polymyxin is an effective means for enhancing the efficient accumulation of other antibacterial agents in the cell body through the outer membrane barrier. The invention is intended to prepare an enrofloxacin-polymyxin sulfate compound preparation so as to improve the antibacterial efficacy of enrofloxacin and the treatment effect of enrofloxacin on drug-resistant bacteria.
Although related patents have reported the use of enrofloxacin in combination with polymyxin sulfate to make oral solutions (grant number: CN 111821258B). However, as shown in FIG. 2, the solution-type preparation which is stable and does not crystallize cannot be obtained according to the technical scheme provided in the patent (No. CN 111821258B). And the enrofloxacin has extremely bitter taste, is easy to refusal to eat when orally administrated to pigs, and the purpose of 'good palatability' described in the patent (grant number: CN 111821258B) is extremely difficult to achieve by oral dosage forms. And the European Union called oral administration of polymyxin sulfate in 2016 will accelerate the development of drug resistance of intestinal flora, oral administration of colistin sulfate products should be withdrawn from the market as early as possible. From the practical application and public health safety, the enrofloxacin and colistin sulfate compound is not suitable to be prepared into oral products. The injection is a dosage form which meets the public health and the use requirement, and the enrofloxacin and polymyxin sulfate compound injection has a great application prospect in the clinic of cultivation.
However, numerous studies have shown that solution formulations of fluoroquinolones (e.g., enrofloxacin) are prone to crystallization out during long-term storage (Xie Yong, 2015). According to the report of the third-stage veterinary drug quality supervision and spot check condition of 2021 in office of agricultural rural area, part of the commercial enrofloxacin injection products face crystallization problems. In addition to the fact that the drug content is insufficient and the drug effect is affected, the crystallization of the drug can cause local inflammation, capillary blockage and granuloma during injection, and serious patients can affect the life health of target animals and even endanger life. Increasing the solubility of a drug in solution is reported to be an effective means of reducing drug crystallization and improving stability. Among them, the addition of solubilizer and cosolvent and salifying are important means for increasing the solubility of poorly soluble drugs, for example, the use of sodium hydroxide to react with enrofloxacin to form enrofloxacin sodium can improve the stability of enrofloxacin solution dosage forms. However, high pH injections are more irritating and can easily cause damage to the injection site. Commercial enrofloxacin injection (bayer benefit) the product may show temporary damage to local tissues (fig. 1). Meanwhile, the polymyxin sulfate is a polypeptide compound, and the high pH value easily hydrolyzes amide bonds to influence the activity of the polymyxin sulfate. In conclusion, more reasonable strategies are needed to prepare enrofloxacin-polymyxin sulfate compound injection with stable quality and proper pH.
The invention takes one or the combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide as a cosolvent, one or the combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid as an acidity regulator, one or the combination of polyethylene glycol-400, n-butanol and ethanol as a cosolvent, and one or the combination of edetate disodium and edetate calcium as a complexing agent. Firstly, an acid-base neutralization method is used for preparing enrofloxacin sodium salt, so that the solubility of enrofloxacin is improved, the stability of polymyxin sulfate is ensured by treating with an acidity regulator, the pH is stabilized in a low-irritation range, and then the hydrogen bonding effect of enrofloxacin and latent solvent molecules is further enhanced by combining a latent solvent technology, so that enrofloxacin molecules are more stable in aqueous solution, and the problem that enrofloxacin solution dosage forms are easy to crystallize after long-term storage is solved. According to the invention, polymyxin and enrofloxacin are ingeniously combined, and the antibacterial targets of the polymyxin and enrofloxacin are different, so that the polymyxin and enrofloxacin exert the maximum antibacterial activity, the development of bacterial drug resistance is delayed, the bacterial adaptability compensation is increased due to the damage to the inherent outer membrane, the generation of bacterial biomembrane subgroups and detention bacteria subgroups is inhibited, and the probability of clinical intractable infection and repeated infection is reduced. The combination reduces the dosage while improving the antibacterial efficacy of the two, and meets the requirements of the national 'decrement and synergy' policy. More importantly, the invention takes the practicability, the formula safety and the storage stability of the preparation into consideration, solves the problems of application and production limitation of strong clinical use irritation and low product stability, and is beneficial to the wide application of the enrofloxacin and polymyxin sulfate compound product. In conclusion, the enrofloxacin-polymyxin sulfate compound injection provided by the invention is certainly beneficial to preventing and treating bacterial infection of livestock and poultry in the breeding clinic and delaying the development of bacterial drug resistance.
Disclosure of Invention
The invention aims to provide a high-stability enrofloxacin-polymyxin sulfate compound injection, and the injection can be used for intramuscular injection administration in order to obtain small irritation; but also can inhibit the generation of bacterial tolerance subgroup and reduce the drug resistance mutation frequency of pathogenic bacteria. The development of the invention is not only beneficial to the treatment of animal drug-resistant bacteria infection in clinic, but also can delay the development of pathogenic bacteria drug resistance.
The invention takes one or the combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide as a cosolvent, one or the combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid as an acidity regulator, one or the combination of polyethylene glycol-400, n-butanol and ethanol as a cosolvent, and one or the combination of edetate disodium and edetate calcium as a complexing agent. Innovative combination of acid-base neutralization and latent solvent technology solves the problems of easy crystallization and precipitation of enrofloxacin solution formulation, large irritation and difficult compatibility with polymyxin sulfate. The invention skillfully utilizes the characteristic of inherent outer membrane of polymyxin targeted bacteria, and the combination of the polymyxin targeted bacteria and the enrofloxacin can be favorable for crossing the outer membrane of the bacteria and improving the concentration of the enrofloxacin in the cytoplasm of the bacteria, thereby promoting the combination of the enrofloxacin and DNA polymerase to exert the maximum antibacterial effect. Meanwhile, the characteristics of the polymyxin sulfate targeting outer membrane also contribute to the elimination of the resting state bacterial subpopulation (highly tolerant enrofloxacin). The compound injection provided not only remarkably improves the antibacterial activity of enrofloxacin and polymyxin sulfate on drug-resistant bacteria, but also solves the defect that the dosage form of the commercially available enrofloxacin solution is easy to crystallize. The compound injection not only has proper pH, but also can be stored for a long time at low temperature and room temperature. The further development of the invention is not only beneficial to the treatment of animal bacteria, especially drug-resistant bacteria infection in clinic, but also can delay the development of drug resistance of pathogenic bacteria, and is one-time practical practice of the policy of decrement and synergy in China.
Specifically, the invention provides the following technical scheme:
the first aspect of the invention provides a veterinary enrofloxacin-polymyxin sulfate compound injection, which is characterized in that the injection comprises the following components in percentage 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.
In a preferred embodiment, the components of the injection are formulated as follows before being mixed by mass/volume:
(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 to 45.0 percent of latent solvent
(6) Complexing agent 0.2-0.5%
(7) The balance of water for injection.
In a preferred embodiment, the cosolvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide; preferably, the co-solvent is selected from sodium hydroxide.
In another specific embodiment, the acidity regulator is one or a combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid; 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 and ethanol; preferably, the latent solvent is 1, 2-propanediol and/or polyethylene glycol-400.
In another specific embodiment, the complexing agent is one or a combination of disodium edentate and calcium edentate; disodium edentate is preferred.
The second aspect of the invention provides a preparation method of the veterinary enrofloxacin-polymyxin sulfate compound injection, which specifically comprises 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.
In a preferred embodiment, the filtration in steps 1) -3) is sterilization by membrane filtration with 0.22 μm;
in another specific embodiment, the negative pressure vacuum in the step 3) is-0.1 to 0.05 atm.
The third aspect of the present invention is a method for improving the stability of a compound injection containing enrofloxacin, said method comprising: adding a cosolvent, a latent solvent, an acidity regulator and a complexing agent into the compound injection;
In a preferred embodiment, the cosolvent is selected from one or a combination of sodium glutamate, meglumine, triethanolamine and sodium hydroxide; preferably, the co-solvent is selected from sodium hydroxide.
In another specific embodiment, the acidity regulator is one or a combination of n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid; 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 and ethanol; preferably, the latent solvent is 1, 2-propanediol and/or polyethylene glycol-400.
In another specific embodiment, the complexing agent is one or a combination of disodium edentate and calcium edentate; disodium edentate is preferred.
In another preferred embodiment, the enrofloxacin-containing compound injection is an enrofloxacin-polymyxin compound injection.
The fourth aspect of the invention is the application of the veterinary enrofloxacin-polymyxin sulfate compound injection in preparing medicines for treating bacterial infection.
In a specific embodiment, the bacteria include, but are not limited to, klebsiella pneumoniae, escherichia coli, actinomyces thoracomium, salmonella, MRSA, pasteurella multocida.
The beneficial effects of the invention are as follows:
The veterinary high-stability enrofloxacin-polymyxin sulfate compound injection provided by the invention utilizes the characteristic that polymyxin damages the outer membrane of bacteria on one hand, greatly enhances the capability of enrofloxacin entering the cytoplasm of bacteria, and improves the antibacterial activity of enrofloxacin on pathogenic bacteria, especially drug-resistant bacteria; on the other hand, the feature of the polymyxin sulfate targeting the outer membrane also contributes to the clearance of dormant subpopulations that are resistant to enrofloxacin. The drug resistance can be delayed while the using efficacy of enrofloxacin and polymyxin sulfate is improved. Meanwhile, the compound injection not only improves the solubility and stability of enrofloxacin, but also ensures the use of the injection in clinical application, avoids the problems of poor palatability and large irritation of the original formulation, can ensure that enrofloxacin and polymyxin sulfate are not crystallized after long-term storage under the conditions of low temperature (4 ℃) and acceleration (30 ℃), and is beneficial to the clinical application of enrofloxacin and polymyxin sulfate. The acid-base neutralization crystallization method and the latent solvent method adopted in the suspension process have lower requirements on instruments and equipment and personnel quality, and the auxiliary materials have low cost and are easy to obtain, thereby being beneficial to industrial mass production. In addition, the compound injection can compete with foreign similar products for market share, reduces the impact of foreign products on veterinary drug markets in China, and is beneficial to improving the international competitiveness of veterinary drug enterprises in China. The compound injection has wide market prospect, and can be used for preventing and treating livestock and poultry infection of animals such as pigs, cattle, sheep, chickens and the like caused by pasteurella multocida, klebsiella pneumoniae, haemophilus parasuis, actinobacillus pleuropneumoniae, streptococcus pneumoniae, mycoplasma and the like, in particular related drug-resistant bacteria.
Drawings
FIG. 1 Instructions for a commercially available enrofloxacin injection (Bayer beneficial injection)
Fig. 2 is a diagram based on the crystallization of a compound oral solution prepared by the technical scheme in the reference patent (issued: CN 111821258B): a, propylene glycol alginate and buckwheat protein polysaccharide are mixed according to a mass ratio of 1:4 preparing a solution into a pasty semisolid substance, wherein a large amount of sediment exists on the lower layer of a 100 mL system prepared by the method of the reference invention;
FIG. 3 synergistic efficacy of enrofloxacin in combination with polymyxin sulfate against pathogenic bacteria of animal origin: klebsiella pneumoniae WNX-1, salmonella B80048, klebsiella pneumoniae C25-2, klebsiella pneumoniae D29-1, klebsiella pneumoniae E37-1, MRSA T144;
FIG. 4 shows the antimicrobial advantage of enrofloxacin resistant Klebsiella pneumoniae when enrofloxacin is combined with polymyxin sulfate;
figure 5 skin irritation and injection site irritation of the compound injection of the present invention: A. c, E is skin state of injection site of left hind leg (normal saline) of rabbit when continuously injecting 7 th d, B, D, F is skin state of injection site of right hind leg (compound injection) of rabbit when continuously injecting 7 th d;
FIG. 6 is a graph showing the effect of the compound injection of the present invention on rabbit blood convention and blood biochemistry;
FIG. 7 shows the histopathological effect of the compound injection of the present invention on muscle injury and liver and kidney at the injection site of rabbits;
FIG. 8 shows the effect of the compound injection of the present invention on treatment of a mice infection model with Pasteurella multocida, the survival curves of mice in the PBS group and the compound injection treatment group, and the pulmonary bacterial load of the surviving mice.
Detailed Description
The present invention will be further described with reference to specific embodiments, and it will be understood by those skilled in the art that modifications and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions fall within the scope of the present invention.
Example 1 determination of the Co-solvent species in Compound injection
Saturated sodium glutamate, meglumine, triethanolamine, sodium hydroxide solutions were prepared and 2.5g enrofloxacin was dissolved separately to complete dissolution using saturated solutions and the volume of saturated solution used was recorded. The result shows that the cosolvent has the cosolvent effect on enrofloxacin, namely sodium hydroxide, triethanolamine, sodium glutamate and meglumine from strong to weak, so that the sodium hydroxide is selected as the cosolvent of the compound injection.
Example 2 determination of the type of acidity regulator in Compound injection
And (3) completely dissolving 2.5. 2.5 g enrofloxacin by using a sodium hydroxide saturated solution 37 mL to obtain five parts of enrofloxacin saturated solution under alkaline conditions, dropwise adding n-butyric acid, lactic acid, malic acid, tartaric acid and citric acid into the enrofloxacin saturated solution, wherein the solution is quickly turbid in the process, and continuously dropwise adding, and gradually clarifying until the solution is completely clarified. The amount of acid solution used in this process was recorded and the clear solution obtained was allowed to stand at room temperature to observe the precipitation of the solution. The results are shown in Table 1, the volume used for adjusting the acidity of the lactic acid and the malic acid is small, and no precipitate is separated out when the lactic acid and the malic acid are placed at room temperature 48 h, but the cost of the malic acid raw material is high, so that the 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 the type of latent solvent in Compound injection
The types and the amounts of the raw materials and the auxiliary materials in the formula are shown in the table 2:
TABLE 2 kinds and amounts of raw materials and auxiliary materials of a compound injection
The required dosage is weighed according to the types and the dosage of the raw materials listed in the table 2, and the preparation is carried out according to the following preparation method:
1) Weighing cosolvent and complexing agent according to Table 2, dissolving with 30 mL injectable water, adding enrofloxacin with formula amount, stirring at room temperature until completely dissolving, filtering with 0.22 μm filter membrane, and sterilizing;
2) Weighing acidity regulator according to table 2, dissolving with water for injection of 20 mL, adding one of ethanol/n-butanol/propylene glycol/polyethylene glycol-400 as latent solvent according to the amount shown in table 2, adding formula amount of polymyxin sulfate, stirring at room temperature until completely dissolving, filtering with 0.22 μm filter membrane, and sterilizing;
3) Adding the filtrate in the step 1) into the filtrate 2), continuously stirring until a clear light yellow solution is obtained, filtering and sterilizing the solution by using a 0.22 mu m filter membrane under the pressure of 0.08 atm, and packaging the solution in 5 brown medium borosilicate glass bottles of 30 mL to obtain the compound injection.
The above 4 compound injections containing different latent solvents were placed in a 30 ℃ incubator for 15 d hours, during which crystallization was observed daily and recorded in table 3. As shown in Table 3, tween-80, n-butanol, ethanol, 1, 2-propanediol and polyethylene glycol-400 groups all improved crystallization of the compound injection, wherein no crystallization was observed in the 1, 2-propanediol and polyethylene glycol-400 groups at 15 d, and thus 1, 2-propanediol and/or polyethylene glycol-400 were proposed as a latent solvent in the subsequent preparation.
TABLE 3 crystallization of compound injection containing different latent solvents
EXAMPLE 4 determination of the amount of cosolvent, pH, complexing agent in Compound injection
Because the water injection of enrofloxacin-polymyxin sulfate prepared by the invention can chelate enrofloxacin with heavy metal ions such as Ca 2+、Mg2+、Fe3 + or Al 3+, complexing agents are added to remove water for the preparation and potential heavy metal ions in a pipeline, so that the stability of enrofloxacin in the compound injection is ensured to the greatest extent. Meanwhile, the pH value has an influence on the crystallization stability of enrofloxacin (Xie Yong & Qiaoqiao, 2015), and the interaction influence of a complexing agent, pH and a latent solvent is required to be comprehensively considered. Therefore, the orthogonal test design is adopted in the invention to fully examine the dosage of the complexing agent, the pH and the latent solvent.
In the research, common disodium edentate is selected as a chelating agent, and tests are carried out by setting 0.02-0.03%, 0.05-0.06% and 0.1-0.11% of 3 gradient levels according to the dosage range (0.01% -0.1%) of the disodium edentate recommended by FDA (https:// db. Yaozh. Com /); taking 1, 2-propylene glycol as a latent solvent, and respectively setting 3 levels of 30-35%, 35-40% and 40-45% for testing; the pH values were set to 3.5 to 4.0, 4.5 to 5.0, 5.0 to 5.5 levels, respectively (Table 3). Namely, the crystallization condition of the compound solution in 90 d at 30 ℃ under the conditions of different complexing agents, pH and latent solvent dosage is examined by orthogonal design of 9 groups of different combinations of 3 factor and 3 variables, so that the parameters are finally determined.
TABLE 4 orthogonal test design table
TABLE 5 results of orthogonal experiments
Example 5 the present invention provides a pH comparison of Compound injection and commercially available enrofloxacin injection (Bayer beneficial injection)
The compound injection and the commercially available enrofloxacin injection (Bayer injection, batch KV03XF8, and the specification of the product shown in FIG. 1) of the invention are respectively sucked by a syringe into a centrifuge tube of 10 mL, and pH detection is carried out by a calibrated pH meter, wherein each group is repeated 3 times. The results show that the pH of the commercially available enrofloxacin injection (Bayer beneficial injection) is 10.95 + -0.13 and the pH of the present invention is 5.0 + -0.11. The invention provides a compound injection of enrofloxacin and polymyxin sulfate, which has a pH range closer to the physiological pH of animals, and is expected to have better compliance to target animals compared with the commercial enrofloxacin injection (Bayer beneficial injection).
Example 6 repetition of Compound oral liquid provided in the patent of the invention (grant number: CN 111821258B)
The preparation of enrofloxacin-polymyxin sulfate compound oral solution was carried out according to the method described in the examples of the invention patent (issued number: CN 111821258B). The main problems are two:
(1) According to patent description, the auxiliary materials cannot be completely dissolved: the patent content is that propylene glycol alginate and buckwheat protein polysaccharide are mixed according to the mass ratio of 1:4 to make a solution "the actual pasty semisolid mass is not the solution described in the patent (accession number: CN 111821258B) (fig. 2A). The actual practice is that propylene glycol alginate and buckwheat protein polysaccharide are mixed according to a feed liquid ratio of 1:8 adding water for injection, and heating in water bath at 90 ℃ for 20min to dissolve into solution;
(2) The prepared solution has obvious precipitation and should be suspension, which is not consistent with the description of the solution appearance and non-crystallization.
As shown in FIG. 2B, the lower layer of the 100 mL system prepared according to the technical scheme of the invention (patent No. CN 111821258B) has a large amount of sediment, and the lower layer is not a uniform clear solution. Meanwhile, after the transparent liquid at room temperature is protected from light by 7 d, the upper layer of clear liquid is centrifugally taken to detect the content of enrofloxacin by using High Performance Liquid Chromatography (HPLC), and the result shows that the content of enrofloxacin is only 2.8 percent and is seriously different from the theoretical value of 5.0 percent which is mentioned in the patent (the authority number: CN 111821258B), and the preparation form prepared according to the technical scheme of the patent (the authority number: CN 111821258B) is quite unstable. And as is well known in the art, enrofloxacin has extremely bitter taste, and when used as an oral liquid, the enrofloxacin can not be easily detected in a large amount of free enrofloxacin in the supernatant liquid, so that the problem of palatability is caused. Therefore, the technical method disclosed by the invention patent (the authority number: CN 111821258B) cannot be used for preparing the enrofloxacin-polymyxin sulfate compound oral solution which is difficult to crystallize and has good palatability. Based on the method, an efficient and qualified enrofloxacin-polymyxin sulfate compound preparation product is provided for veterinary clinics in China, the international competitiveness of veterinary medicine enterprises in China is improved, and a more reasonable technical scheme is provided through careful formula compatibility research.
EXAMPLE 7 examination of synergistic efficacy of enrofloxacin in combination with polymyxin sulfate against pathogenic bacteria of porcine origin
Klebsiella pneumoniae (WNX-1, 37-1, 29-1, 25-2), escherichia coli (OF B2D 19, OF 25922D 38), actinobacillus pleuropneumoniae (Hang8), methicillin-resistant Staphylococcus aureus (T144), salmonella (80048) and Pasteurella multocida (P32D 7) were resuscitated and passaged, respectively.
A checkerboard test was performed according to the method prescribed by CLSI to verify the efficacy of enrofloxacin in combination with polymyxin sulfate. The specific method comprises the following steps:
1) Diluting the original bacterial liquid to the concentration of 10 6 CFU/mL by using a fresh broth culture medium;
2) Placing two rows of test tubes on a test tube rack, wherein 7 test tubes are arranged in each row, the 1 st row is used for diluting the medicine A, and the 2 nd row is used for diluting the medicine B; in a row 1 test tube, diluting the A medicinal broth culture medium by 2 times to ensure that the A medicine concentration of 1-7 tubes is 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC in sequence, wherein the B medicine is consistent with the A medicine dilution step;
3) Sequentially adding the medicine liquid of the medicine bacteria A and the medicine liquid of the medicine bacteria B into each hole of the transverse row and the vertical row of the 96-hole cell culture plate respectively; the A liquid medicine of the 1 st tube is sequentially added into the 8 holes of the 1 st row of the transverse row of the cell culture plate, 50 mu L of each hole is formed, the liquid medicine of the 2 nd tube is sequentially added into the 8 holes of the 2 nd row, and 50 mu L of each hole is formed. And adding the liquid A of the 3 rd to 7 th pipes to the holes of the 3 rd to 7 th rows. Row 8 as a separate drug sensitive control for drug B without drug a; the medicine B is prepared according to the method of medicine A;
4) Sequentially adding 1-7 holes in the vertical row; subsequently, 50 mu L of MH broth is added to each of the horizontal row and the vertical row of column 8, and then 100 mu L of the diluted bacterial liquid is added to each well, so that the final concentration of the bacterial liquid is 10 5 CFU/mL, and meanwhile, a negative control well is arranged. The 96-well plate was placed in a 37 o C incubator for 20 h cycles, and the observation record was taken out and repeated three times for each test.
5) And (3) judging results: the synergy or addition index of enrofloxacin and polymyxin sulfate is calculated by the following formula. Wherein the synergy is when FICI is less than or equal to 0.5, the additive effect is when FICI is less than or equal to 0.5 and the independent effect is when FICI is less than or equal to 1 and the antagonism is when FICI is greater than or equal to 4.
TABLE 6 synergistic efficacy of enrofloxacin and polymyxin sulfate against swine pathogens when used in combination
Note that: MRSA: methicillin-resistant staphylococcus aureus; ENR: enrofloxacin; coli: polymyxin sulfate
The synergistic efficacy of enrofloxacin in combination with polymyxin sulfate against common swine pathogens is shown in table 1. From the table, the average resistance of klebsiella pneumoniae (WNX-1), methicillin-resistant staphylococcus aureus (T144) and salmonella (80048) to enrofloxacin is higher, and when polymyxin sulfate is added, the sensitivity of the high-level resistant strain to enrofloxacin is obviously increased, and the maximum synergistic effect can reach 16 times. Although actinobacillus pleuropneumoniae (Hang8) is more sensitive to enrofloxacin, the MIC value was still 16-fold reduced after the addition of polymyxin sulfate. And the sensitivity of methicillin-resistant staphylococcus aureus (T144) as gram-positive bacteria to enrofloxacin is also significantly reduced. In addition, when the two are combined, the sensibility of the strain to polymyxin sulfate is obviously reduced, and the highest sensibility is 16 times. The results show that the combination of enrofloxacin and polymyxin sulfate can obviously increase the antibacterial activity of both, especially the antibacterial activity on drug-resistant bacteria, and the rationality of the compound compatibility provided by the invention is proved.
EXAMPLE 8 inhibition of the resident subpopulation of Klebsiella pneumoniae by enrofloxacin in combination with colistin sulfate
1. Materials and methods
Drug concentration setting: 10-fold and 1-fold MIC concentrations of enrofloxacin and polymyxin sulfate, and a concentration of 0.5-fold MIC of both drugs.
Preparing bacterial liquid: a single colony of bacteria was picked, inoculated into 1ml of LB broth, adjusted to 0.5 Mirabilimeter turbidity by means of a Mirabilimeter, and added to a medium containing different drug concentrations, the initial bacteria concentration being 10 6 CFU/ml.
Test procedure: and adding the bacterial liquid into culture mediums containing medicines with different concentrations, taking 100 mu L of bacterial liquid when the bacterial liquid is 0, 2, 4, 6 and 12 h, diluting the bacterial liquid by PBS, taking 100 mu L of liquid when the bacterial liquid is at a proper dilution multiple, coating the bacterial liquid on a BHA flat plate, and placing the bacterial liquid in a 37 ℃ incubator for overnight culture, and counting bacterial colonies.
2. Test results
The results of this test are shown in FIG. 4 of the specification. The WNX-1 strain is an enrofloxacin drug-resistant strain (MIC value is 256 mug/mL), and has obvious bacterial retention characteristics when 2560 mug/mL enrofloxacin (10 XMIC) is treated by 4-6 h, namely the bacteria start to gradually multiply and grow after the front 4 h is rapidly sterilized and then the curve is more gentle. Compared with enrofloxacin, 10×MIC polymyxin sulfate can completely kill bacteria at 4 h, but under the action of 1-time MIC concentration alone, neither enrofloxacin nor polymyxin sulfate can completely kill bacteria, after the enrofloxacin is subjected to the drug action of 4-6 h, the resident bacteria are transiently tolerant to polymyxin sulfate and begin to reproduce and grow, which suggests that the resident bacteria subgroup of Klebsiella pneumoniae has extremely strong resistance to antibiotics, particularly a bactericide (enrofloxacin) in a reproduction period. Regrowth in the background based on the resident bacterial subpopulation predicts the risk of recurrent infection of klebsiella pneumoniae and the high drug resistance mutation probability of this subpopulation.
As shown in fig. 3A-3F, the combination group exhibited significant advantages. When combined, the two medicaments show excellent sterilization efficacy on drug-resistant klebsiella at the MIC of 0.5 times, the sterilization efficacy of 12h is equivalent to that of 10 XMIC of polymyxin sulfate, no plateau phase exists in the sterilization process, and the risk of recurrent infection caused by resident bacteria subgroup is eliminated. The sterilizing speed of the polymyxin sulfate is also obviously improved when the polymyxin sulfate is combined, and specific results are shown in FIG. 3A, FIG. 3B, FIG. 80048, FIG. 3C, FIG. 25-2, FIG. 3D, FIG. 29-1, FIG. 3E, FIG. 37-1 and FIG. 3F, MRSA T144 respectively. The results show that the combined use of enrofloxacin and polymyxin sulfate is a good strategy for reducing the dosage and delaying the development of drug resistance.
Example 9 accelerated stability and Low temperature stability of Compound injection
According to the preparation process, the compound solution for injection is prepared and is subpackaged into brown medium boron silicon bottles, wherein each bottle is about 20 mL.
Accelerated stability test: and (3) placing the split-packed compound solution at 30 ℃, taking out a small amount of liquid in 1 st, 2 nd, 3 rd and 6 th months, measuring the pH value of the solution, and detecting the content of active ingredients and related substances (ciprofloxacin) in the solution by using High Performance Liquid Chromatography (HPLC). And meanwhile, the stability of the compound preparation is judged by observing visible foreign matters, crystallization conditions, appearance colors and the like.
Low temperature stability test: the split-packed compound solution is placed at 4 ℃ and the crystallization condition of the prepared compound solution is mainly inspected in 1 st, 2nd, 3 rd and 6 th months so as to evaluate the low-temperature stability of the compound solution.
Test results
The following table shows the crystallization conditions of 30 ℃ accelerated stability test groups, and it is seen that groups 1 and 6 have long-term stability and no crystal precipitation is seen for 6 months. While group 1 showed medium crystal precipitation in the accelerated stability test at 4℃for 30 days, the orthogonal test group 6 showed the strongest stability in combination.
TABLE 7 crystallization conditions and pH variation in 30℃accelerated stability test
Note that: -: no crystal precipitation was observed; +: a small amount of crystals are separated out; ++: precipitation of moderate crystals; +++: a large amount of crystals are precipitated
TABLE 8 accelerated stability test of enrofloxacin relative content (%)
Crystallization condition of acceleration stability test at 9 4 DEG C
Example 10 investigation of skin irritation and safety of Compound injection
The compound injection solution prepared according to the preparation process of the patent is subjected to experiments in compound injection preparations stored for 6 months according to the example group 6. Adult male rabbits were intramuscular injected with the compound solution at 2 times of the recommended dose for 7 days, from 1d th to 7 th d th, the right hind legs of the rabbits were intramuscular injected with the same volume of physiological saline, and after one week of recovery, sampling and observation was again performed at 14 th d. Carrying out auricular vein blood sampling on experimental rabbits at the 0 th, 7 th and 14 d th, adding 0.5 mL th blood into an EDTA anticoagulation tube for carrying out blood routine detection, standing 1 mL th blood at room temperature for 20-30 min, centrifuging at 2000-3000 rpm for 10 min, separating upper serum, and carrying out blood biochemical index detection on liver functions (serum glutamic pyruvic transaminase ALT, serum glutamic oxaloacetic transaminase AST, total protein TP, albumin ALB), kidney functions (UREA UREA, creatinine CREA) and the like. And at 7d, skin irritation at injection site and irritation at injection site were examined, rabbit liver, kidney, muscle tissue at injection site were fixed in 4% paraformaldehyde at 14: 14 d, and HE staining was performed to observe histological changes.
As shown in fig. 5, A, C, E is a rabbit left hind leg (normal saline) injection site, B, D, F is a rabbit right hind leg (compound injection) injection site, and no abnormal change is seen in skin state when 7 th d is continuously injected. As shown in fig. 6, liver function indexes such as serum glutamic pyruvic transaminase (ALT), serum glutamic oxaloacetic transaminase (AST), total Protein (TP), albumin (ALB), and kidney function indexes such as UREA (ura) and Creatinine (CREA) were not significantly changed after continuous injection of 7 d, and the numbers of Red Blood Cells (RBC), white Blood Cells (WBC), and Platelets (PLT) were not significantly affected. As shown in fig. 7, the muscle tissues of the treatment group and the blank group did not have obvious pathological changes, so that the compound injection did not cause pathological damage to the injection site. In addition, the liver tissues of the treatment group and the blank group have no obvious inflammatory cell infiltration and liver cell necrosis, and the kidney tissues have no pathological changes such as inflammatory cell infiltration, interstitial edema, blood stasis, microthrombus, tubular necrosis and the like. Therefore, the compound injection provided by the invention has no skin irritation and injection site irritation, and the basic physiological state, liver function and kidney function of rabbits are not affected by continuously administering 7 d at 2 times of recommended doses, so that the compound injection has better safety.
Example 11 therapeutic Effect of Compound injection on mice infection model with Pasteurella multocida
1. Establishing a pasteurella multocida respiratory system infection model
The compound injection solution prepared according to the preparation process of the patent is subjected to experiments in compound injection preparations stored for 6 months according to the example group 6.
Before the experiment is started, 100 mu L of pentobarbital sodium with the concentration of 1% is injected into the abdominal cavity of each mouse to be anesthetized, the mouse is vertically hung, the nose is pinched, the tongue of the mouse is pulled out by forceps, 75 mu L of bacterial liquid (the MIC of the bacterial liquid for enrofloxacin and polymyxin sulfate is 16 mu g/mL and 1 mu g/mL respectively) is poured into the trachea of the mouse by using a pipette, the bacterial liquid concentration is 6 multiplied by 10 10 CFU/mL, 2-3 min is hung, and the bacterial liquid is horizontally placed in a cage after the bacterial liquid completely enters the trachea of the mouse. 1 h of the treated compound bacteria is used for treating infected mice by intramuscular injection at a recommended compound dose, and the mice are dosed again after 24h and 48h of the infected mice, so that the compound bacteria accords with the recommended clinical use dose and the medication course of the compound product.
Every 6 h mice were observed for status after infection, after mice died, lung tissue was ground, the ground fluid was diluted 10-fold with PBS and plated on sheep blood plates containing 1/4 of the MIC enrofloxacin, and the colony numbers on the plates were counted after 24 h in an incubator at 37 ℃. And all mice were sacrificed at 120 h post infection and lung tissue was ground and plated for enumeration.
2. Test results
In the infection 120 h, the survival curves of mice in the PBS group and the compound injection treatment group are shown in figure 8A, and the result proves that the compound injection can effectively slow down the infection degree of the mice and effectively improve the survival rate. As shown in fig. 8B, there was a significant decrease in lung load in surviving mice. In a word, the compound injection provided by the invention has a superior treatment effect on multi-drug-resistant pasteurellosis infection, and has a remarkable treatment effect on clinical common bacterial and mycoplasma infection diseases of livestock and poultry.
The above embodiments are the most preferred forms of the invention, and it should be understood by those skilled in the art that modifications and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions fall within the scope of the present invention.
Claims (4)
1. The veterinary enrofloxacin-polymyxin sulfate compound injection is characterized by comprising the following components in mass/volume:
(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) A latent solvent 40%;
(6) Complexing agent 0.075;
(7) The rest is water for injection;
wherein the cosolvent is sodium hydroxide;
the acidity regulator is lactic acid;
the latent solvent is 1, 2-propylene glycol;
The complexing agent is disodium edentate;
the pH value is 5+/-0.11.
2. The method for preparing the veterinary enrofloxacin-polymyxin sulfate compound injection as claimed in claim 1, 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 formula amount of polymyxin sulfate, stirring at room temperature until the polymyxin 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 in the step 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.
3. A method for improving the stability of a compound injection containing enrofloxacin, wherein the compound injection containing enrofloxacin is enrofloxacin-polymyxin sulfate compound injection,
The injection consists of the following components in mass/volume:
(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) A latent solvent 40%;
(6) Complexing agent 0.075;
(7) The rest is water for injection;
The method 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 sodium hydroxide;
the acidity regulator is lactic acid;
the latent solvent is 1, 2-propylene glycol;
The complexing agent is disodium edentate;
wherein the addition proportion of the latent solvent is 40%, and the addition proportion of the complexing agent is 0.075%; simultaneously, the pH of the injection is adjusted to 5+/-0.11.
4. The use of a veterinary enrofloxacin-polymyxin sulfate compound injection of claim 1 in the manufacture of a medicament for the treatment of a bacterial infection; the bacterial infection is specifically one or more of klebsiella pneumoniae, escherichia coli, actinobacillus pleuropneumoniae, methicillin-resistant staphylococcus aureus, salmonella and pasteurella multocida.
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