Difloxacin suspension injection and its preparation method

Abstract

This invention belongs to the field of veterinary drugs and discloses a method for preparing difloxacin suspension injection, comprising the following steps: Step 1: Difloxacin is added to an aqueous solution containing gluconolactone to prepare a difloxacin solution; Step 2: A crystallization inhibitor is added to the difloxacin solution in Step 1; Step 3: The solution is dispensed into glass vials for injection, sealed, and sterilized with high-temperature steam; Step 4: The sterilized injection solution is immediately subjected to cold water immersion or cooled to room temperature in air for leak testing and crystallization, simultaneously obtaining difloxacin injection solution. Based on previous research results, this invention further proposes that, in the further optimization process, gluconolactone is used as a co-solvent for difloxacin, dissolved by heating, dispensed into injection bottles, sterilized at high temperature, and then recrystallized after cooling to obtain a suspension with small crystal particle size. This process is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drugs, specifically relating to a difluorofloxacin suspension injection and its preparation method. Background Technology

[0002] Quinolones are a class of synthetic antibacterial drugs, broadly classified into four generations based on their invention order and structural differences. Their primary mechanism of action is the inhibition of bacterial DNA gyrase. Currently, the main quinolones approved for use in livestock and poultry farming in China are the third-generation quinolones enrofloxacin (ENR), difloxacin (DIF), danofloxacin (DAN), and sarafloxacin (SAR), and the second-generation flumequine (FLU), all specifically for animal use. Additionally, methimazole is approved for use in pets. Ciprofloxacin (CIP) is also approved for use in livestock and poultry farming; this drug can be used in both humans and animals. Norfloxacin, pefloxacin, ofloxacin, and lomefloxacin, which were previously used in animal husbandry, are now discontinued and no longer permitted for use in animal husbandry.

[0003] Difluorofloxacin is an animal-specific drug (CAS#: 98106-17-3). It is currently not approved for human medical use and is only used in animals. It exerts its bactericidal effect by acting on the DNA gyrase A subunit of bacteria, inhibiting bacterial DNA replication and transcription. It has strong antibacterial activity against most Gram-negative and Gram-positive bacilli and cocci, including Klebsiella spp., Staphylococcus spp., Escherichia coli, Enterobacter spp., Curvularia spp., Shigella spp., Proteus spp., and Pasteurella spp. It can be used to treat chronic respiratory diseases, tracheitis, enteritis, pneumonia, fowl cholera, streptococcal disease, typhoid fever, and other diseases in livestock and poultry. It is particularly effective against Escherichia coli infection in chickens and red, yellow, and white diarrhea in piglets.

[0004] Because difluorofloxacin hydrochloride is only slightly soluble in water, existing injectable formulations can only be made in low concentrations and must be formulated to be strongly alkaline (pH ≥ 10) to exist in solution form. Currently, in domestically registered and approved difluorofloxacin hydrochloride solutions and injections, the difluorofloxacin content in the solution is 2.5%, and in the injection, it is 2.0%.

[0005] The applicant has previously filed two invention patents: CN119405601A and CN119504580A. These two patent applications prepared mixed crystals by rapid cooling, then added an appropriate amount of water for injection, mixed them, and prepared a 7.5% suspension for injection, and conducted pharmacokinetic tests on difluorofloxacin microcrystals in yellow chickens.

[0006] In industrial applications, the raw materials are generally not provided to aquaculture enterprises for them to prepare suspensions themselves. Instead, sterilized suspensions are provided for direct use.

[0007] However, the problem that troubles us is that although the preparation of suspensions is a conventional technique, the only sterilization method for suspensions is radiation sterilization.

[0008] The main drawbacks of existing technologies are that strongly alkaline injections are highly irritating, have low concentrations, require large injection volumes, and are rapidly absorbed and eliminated after injection, failing to achieve a sustained-release, long-lasting effect. Furthermore, conventional suspension-type injections, due to their thermodynamic instability, are difficult to meet the requirements of high-temperature sterilization processes. When the temperature of the suspension reaches the sterilization temperature, some particles dissolve in the solvent due to the high temperature. After the injection returns to room temperature, the supersaturated solution easily forms large crystals, which can cause needle blockage during use.

[0009] This phenomenon has been observed by many researchers. For example, the patent application with publication number CN113855630A, entitled "Betamethasone Sodium Phosphate & Betamethasone Acetate Suspension Injection and Preparation Method", specifically states in its description that the final product is not subjected to high-temperature sterilization. The specific characteristics are: A. In step (1), micronized sterile betamethasone acetate raw material is obtained first; B. In step (2), the betamethasone sodium phosphate solution is filtered through a 0.2um filter membrane or filter element; C. The mixing and homogenization described in steps (3) and (4) are carried out under Class 100 sterile conditions; D. The final product is filled into a container under stirring; E. Neither step (2) nor (3) is subjected to high-temperature sterilization; F. The homogenization pressure described in step (4) is 5.0-7.0MPa, and the time is 5-20 minutes.

[0010] In some suspension preparation processes in this field, a few documents also record the use of high-temperature sterilization processes. For example, patent application CN113209014A, entitled "Long-acting cefquinome sulfate suspension injection and its preparation process," uses one or more of aluminum monostearate, hydrogenated castor oil, lecithin, and beeswax as suspending agents, and uses soybean oil, olive oil, or peanut oil for injection as solvents. It uses ultra-high temperature sterilization for sterilization, but does not describe how it crystallizes by controlling the temperature.

[0011] Generally speaking, we believe that crystallization through non-rapid cooling will result in a significant increase in crystal size.

[0012] In patent application CN115006412A, which discloses a compound tobramycin eye drops and its preparation method, the method employed is to integrate the grinding and high-temperature sterilization of dexamethasone into the same equipment. This avoids the loss of active ingredients caused by filtration sterilization and saves on raw material processing steps. In particular, by using a specific suspending agent in the high-temperature moist heat sterilization process, the particle size of dexamethasone can be effectively prevented from increasing during the moist heat sterilization process. Combined with subsequent grinding operations, this ensures better dexamethasone dispersion, improves the dispersion and redispersibility of drug particles, and thus ensures the stability and efficacy of the eye drops. A jacket can be designed around the cavity of the nano-grinding machine to heat the drug solution with steam for high-temperature sterilization. Afterward, cooling water can be circulated through the jacket of the nano-grinding machine cavity to cool it down before subsequent grinding operations are performed, so that the D90 value of the drug particle size (the particle size corresponding to a cumulative distribution of 90%) reaches a certain range.

[0013] Although the proposed method claims to prevent particle size growth during moist heat sterilization using a suspending agent (polyvinylpyrrolidone), our verification has shown that it is not applicable, at least not to difluoroquinolone. Summary of the Invention

[0014] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing difluorofloxacin injection.

[0015] This invention builds upon previous research findings (CN119405601A, CN119504580A). In previous studies, a small-sized, stable suspension was obtained through rapid cooling. Further optimization involved dissolving the crystals with glucono-delta-lactone by heating and then sterilizing them at high temperatures. The resulting recrystallized suspension exhibited small crystal size, making this process suitable for large-scale production.

[0016] To achieve the objective of this invention, the following technical solution is adopted:

[0017] A method for preparing a difluorofloxacin suspension injection includes the following steps:

[0018] Step 1: Add difloxacin to an aqueous solution containing gluconolactone, stir, keep the temperature not lower than 80℃, add pH adjuster, adjust the pH value to 3~5, and prepare difloxacin solution;

[0019] Step 2: Keep the temperature not lower than 80℃, add a crystallization inhibitor to the difluorofloxacin solution in step 1, filter to remove impurities, and add water to make up to the total volume;

[0020] Step 3: Keep the difluorofloxacin solution from Step 2 at a temperature not lower than 80°C, dispense it into glass vials containing the injection solution, seal them, and sterilize them with high-temperature steam.

[0021] Step 4: The injection solution after high-temperature sterilization is immediately cooled to room temperature by cold water immersion and air immersion, and leak detection and crystallization are carried out simultaneously to obtain difluorofloxacin injection solution.

[0022] The difluorofloxacin injection provided by this invention comprises difluorofloxacin dissolved in an aqueous solution and difluorofloxacin microcrystals existing in a solid suspension in the aqueous solution. During the production process, the difluorofloxacin is in a dissolved state and can be sterilized using high-temperature steam. Upon returning to room temperature, the injection transforms into a light silvery-yellow suspension that can be stably stored. After intramuscular injection, the dissolved difluorofloxacin is rapidly absorbed, forming a high concentration in the body and quickly exerting its bactericidal effect. The solid suspension crystal portion temporarily remains in the interstitial space, continuing to slowly dissolve and be absorbed under the influence of body fluids, providing a long-term stable drug concentration in the body and exerting a long-lasting antibacterial effect. The difluorofloxacin crystals in the suspension portion of the injection have a particle size of 400 nm to 5 μm, exhibiting a polymorphic microcrystalline structure, making remixing easy, preventing needle clogging, and facilitating operation. This injection has the characteristics of a long-acting formulation, reducing the frequency of animal administration and significantly reducing the workload of veterinarians and animal stress when using difluorofloxacin injection to treat animal diseases. In addition, the present invention also provides a method for preparing the injection solution, and can prepare difluorofloxacin injection solutions with various concentration specifications.

[0023] The difference between this applicant's earlier application and the previous application is that the applicant's earlier application required rapid cooling to prepare plate-like crystals with a particle size distribution of 200nm~10μm in order to achieve the effect of not clogging the syringe.

[0024] However, suspensions prepared using this method can only be produced through radiation sterilization after dispensing, and cannot be produced through steam sterilization. If radiation sterilization is used, it introduces an additional step: leak detection.

[0025] This invention utilizes a suitable co-solvent, gluconolactone, along with a crystallization inhibitor, to enable difluorofloxacin solution to crystallize into 400 nm to 5 μm crystals in room-temperature cold water, while simultaneously performing leak testing. This allows the high-temperature sterilization process to be adapted to suspension processing.

[0026] In the above preparation method, the total content of difluorofloxacin, the active ingredient, in every 100 ml of the injection solution is 5-15 g. The active ingredient in the injection solution includes difluorofloxacin in a dissolved state and difluorofloxacin in a solid microcrystalline state.

[0027] In the above preparation method, the content of gluconolactone in each 100ml of the injection solution is 5~15g; the pH adjuster is gluconic acid or sodium gluconate, and the amount used is adjusted according to the pH requirements of the injection solution; the crystallization inhibitor is dextran, and the content of dextran in each 100ml of the injection solution is 2~10g.

[0028] In the above preparation method, the injection solution also contains an antioxidant, which is sodium bisulfite or sodium metabisulfite, and the amount of antioxidant used in each 100ml of the injection solution is 0.01~0.2g.

[0029] In the above preparation method, the temperature of the cold water is 5~30℃, preferably, the temperature of the cold water is room temperature, which refers to 20~30℃.

[0030] In addition, the present invention also discloses a difluorofloxacin suspension injection, which is prepared by any of the methods described above.

[0031] In the above-mentioned difluorofloxacin suspension injection, the crystal particle size of the difluorofloxacin microcrystals in the difluorofloxacin suspension injection is 400 nm to 5 μm.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The difluorofloxacin injection of the present invention selects gluconolactone as a cosolvent, which can generate small-sized suspension particles of difluorofloxacin during the cooling process, and is not limited by the cooling rate.

[0034] (2) The production process of this invention also solves the problem that traditional processes for producing suspension-type liquid preparations cannot meet the requirements for high-temperature sterilization for injection sterilization. This sterilization process is currently the most economical and reliable sterilization method in the production of water-based injections. During high-temperature sterilization, some of the particles in the suspension dissolve as the temperature rises. When the suspension cools, supersaturated components in the liquid crystallize and can form large crystals. Therefore, traditionally, suspension-type injections can only be sterilized using radioactive irradiation. This invention solves the sterilization problem of suspension-type difluorofloxacin injections, enabling the entire production process of difluorofloxacin injections to be completed in the same workshop, eliminating the need to outsource sterilization to a professional radioactive irradiation sterilization company, thus reducing the production cost of the sterilization process for this product.

[0035] (3) The difluorofloxacin injection prepared by this invention contains both dissolved difluorofloxacin and difluorofloxacin in a suspended crystalline state. After the injection prepared by this invention is injected into the body, the dissolved difluorofloxacin can be rapidly absorbed by the body at the injection site, quickly forming a high concentration of drug in the body to exert a bactericidal effect; the suspended crystalline difluorofloxacin is temporarily stored in the interstitial space at the injection site, slowly dissolving under the action of body fluids and further absorbed by the body to exert a long-lasting effect. At the same time, during the preparation of the difluorofloxacin injection, the ratio of fast-absorbing and slow-absorbing difluorofloxacin components in the injection can be adjusted according to clinical needs; the production process of the difluorofloxacin injection of this invention can produce injections with different specifications of high content (e.g., 15%) and low content (e.g., 5%) of difluorofloxacin according to clinical needs (e.g., for large or small animals). This invention solves the formulation process problem of difluorofloxacin hydrochloride injection, which has poor solubility and requires pH adjustment to a strong alkaline state (pH≥10) for existing technology, and the difluorofloxacin content in the injection is only no higher than 2.5%. Attached Figure Description

[0036] Figure 1 Microscopic photograph of Example 1;

[0037] Figure 2 The particle size distribution results are for Example 1;

[0038] Figure 3 Microscopic photograph of Example 2;

[0039] Figure 4 The particle size distribution results are for Example 2;

[0040] Figure 5 Microscopic photograph of Example 3;

[0041] Figure 6 The particle size distribution results are for Example 3;

[0042] Figure 7 Microscopic photograph of Example 4;

[0043] Figure 8 The particle size distribution results are for Example 4;

[0044] Figure 9 The pharmacokinetic curves for Example 1 are shown below.

[0045] Figure 10 The pharmacokinetic curves for Example 2 are shown below.

[0046] Figure 11 These are photographs of samples I through III after cooling.

[0047] Figure 12 Microscopic photograph of sample I;

[0048] Figure 13 Microscopic photograph of sample No. II;

[0049] Figure 14 Photographs of specimens IV through VI;

[0050] Figure 15 Microscopic photograph of sample IV;

[0051] Figure 16 Microscopic photograph of sample V;

[0052] Figure 17 Microscopic photograph of sample VI;

[0053] Figure 18 Photographs of samples VII through IX;

[0054] Figure 19 Microscopic photograph of sample VII;

[0055] Figure 20 Microscopic photograph of sample VIII;

[0056] Figure 21 Microscopic photograph of sample IX;

[0057] Figure 22 Microscopic photograph of difluorofloxacin raw material;

[0058] Figure 23 This is a product photo of Example 1. Detailed Implementation

[0059] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0060] Example 1

[0061] Step 1: Pour 800 mL of hot water for injection into a round-bottom flask, place it in an electric heating mantle, add 55 g of gluconolactone, and stir until dissolved to prepare a hot gluconolactone solution.

[0062] Step 2: Add 51 g of difluorofloxacin raw material (purity 98.5%) to the solution in Step 1, stir until dissolved, turn on the electric heating mantle to raise the temperature to 80℃, add gluconic acid or sodium gluconate to adjust the pH to 3.5±0.3, add 0.5 g of sodium metabisulfite as an antioxidant, stir until dissolved to prepare difluorofloxacin salt solution, and maintain the temperature.

[0063] Step 3: Add 80 g of dextran-20 to the difluorofloxacin salt solution prepared in step 2, stir until dissolved, add water for injection to make up to 1.02 L, filter the solution into an insulated storage tank, and maintain the temperature.

[0064] Step 4: Fill the difluorofloxacin salt solution prepared in Step 3 into clean ampoules or injection vials, seal the vials (or melt-seal the ampoules), place them in a sterilizer, spray the injection vials with 80°C water for injection and drain the water from the container, and then inject high-temperature steam for sterilization.

[0065] Step 5: After sterilization in Step 4, spray the injection vial with 80°C water for injection and drain it from the container. Immediately, pour cooled water for injection (25°C) into the sterilizer. At this time, perform a leak test on the injection solution (check the seal of the vial). Under the action of cold water, the partially dissolved difluorofloxacin in the injection solution begins to crystallize, and under the action of a crystallization inhibitor, it disperses in each inner packaging vial to form microcrystals. This process achieves simultaneous leak detection and crystallization of the injection solution. After cooling to room temperature, difluorofloxacin injection solution is obtained, with a difluorofloxacin content of 5% (5 g of difluorofloxacin per 100 mL).

[0066] Take 20 μL of the difluorofloxacin injection solution prepared in Example 1, spot it onto a glass slide, cover it with a coverslip, and observe the morphology of the particles in the suspension using a high-power microscope. Figure 1 As shown, the microparticles in the injection solution exhibit a relatively uniform polymorphic crystalline structure. An appropriate amount of the difluorofloxacin injection solution prepared in Example 1 was injected into the sample cell of a laser particle size analyzer to analyze the main particle size of the crystals in the injection solution, such as... Figure 2 As shown, the crystal grain size is mainly distributed in the range of 800 nm to 1600 nm.

[0067] Product photos for reference Figure 23 .

[0068] Example 2

[0069] Step 1: Pour 800 mL of hot water for injection into a round-bottom flask, place it in an electric heating mantle, add 75 g of gluconolactone, and stir until dissolved to prepare a hot gluconolactone solution.

[0070] Step 2: Add 76.5 g of difluorofloxacin raw material (purity 98.5%) to the solution in Step 1, stir until dissolved, turn on the electric heating mantle power supply, raise the temperature to 85℃, add gluconic acid or sodium gluconate to adjust the pH to 3.3±0.3, add antioxidant and 0.5 g of sodium metabisulfite, stir until dissolved, and prepare difluorofloxacin salt solution, maintain the temperature.

[0071] Step 3: Add 80 g of dextran-20 to the difluorofloxacin salt solution prepared in step 2, stir until dissolved, add water for injection to make up to 1.02 L, filter the solution into an insulated storage tank, and maintain the temperature.

[0072] Step 4: Fill the difluorofloxacin salt solution prepared in Step 3 into clean ampoules or injection vials, seal the vials (or melt-seal the ampoules), place them in a sterilizer, spray the injection vials with 85°C water for injection and drain it out of the container, and then inject high-temperature steam for sterilization.

[0073] Step 5: After sterilization in Step 4, spray the injection vial with 85°C water for injection and drain it from the container. Immediately, pour cooled water for injection (25°C) into the sterilizer. At this time, perform a leak test on the injection solution (check the seal of the vial). Under the action of cold water, the partially dissolved difluorofloxacin in the injection solution begins to crystallize, and under the action of a crystallization inhibitor, it disperses in each inner packaging vial to form microcrystals. This process achieves simultaneous leak detection and crystallization of the injection solution. After cooling to room temperature, difluorofloxacin injection solution is obtained, with a difluorofloxacin content of 7.5% (7.5 g of difluorofloxacin per 100 mL).

[0074] Samples of the difluorofloxacin injection solution prepared in Example 2 were taken and the morphology of the particles in the suspension was observed using a high-power microscope, such as... Figure 3 As shown, the microparticles in the injection solution exhibit a relatively uniform polymorphic crystalline structure; samples were taken and analyzed using a laser particle size analyzer to determine the main particle size of the crystals in the injection solution, such as... Figure 4 As shown, the crystal grain size is mainly distributed in the range of 700 nm to 1300 nm.

[0075] Example 3

[0076] Step 1: Pour 800 mL of hot water for injection into a round-bottom flask, place it in an electric heating mantle, add 95 g of gluconolactone, and stir until dissolved to prepare a hot gluconolactone solution.

[0077] Step 2: Add 101.5 g of difluorofloxacin raw material (purity 98.5%) to the solution in Step 1, stir until dissolved, turn on the electric heating mantle power supply, raise the temperature to above 80℃, add gluconic acid or sodium gluconate to adjust the pH to 3.3±0.3, add 0.5 g of sodium metabisulfite as an antioxidant, stir until dissolved, and prepare difluorofloxacin salt solution, maintaining the temperature.

[0078] Step 3: Add 80 g of dextran-20 to the difluorofloxacin salt solution prepared in step 2, stir until dissolved, add water for injection to make up to 1.02 L, filter the solution into an insulated storage tank, and maintain the temperature.

[0079] Step 4: Fill the difluorofloxacin salt solution prepared in Step 3 into clean ampoules or injection vials, seal the vials (or melt-seal the ampoules), place them in a sterilizer, spray the injection vials with water for injection at 80°C or higher and drain the water from the container, then inject high-temperature steam for sterilization.

[0080] Step 5: After sterilization in Step 4, spray the injection vial with water for injection at a temperature above 80°C and drain it from the container. Immediately pour cooled water for injection (temperature 25°C) into the sterilizer. At this time, leak testing is performed on the injection solution (checking the seal of the vial). Under the action of cold water, the partially dissolved difluorofloxacin in the injection solution begins to crystallize, and under the action of a crystallization inhibitor, it disperses in each inner packaging vial to form microcrystals. This process achieves simultaneous leak testing and crystallization of the injection solution. After cooling to room temperature, difluorofloxacin injection solution is obtained, with a difluorofloxacin content of 10% (10 g of difluorofloxacin per 100 mL).

[0081] Samples of the difluorofloxacin injection solution prepared in Example 3 were taken and the morphology of the particles in the suspension was observed using a high-power microscope, such as... Figure 5 As shown, the microparticles in the injection solution exhibit a relatively uniform polymorphic crystalline structure; samples were taken and analyzed using a laser particle size analyzer to determine the main particle size of the crystals in the injection solution, such as... Figure 6 As shown, the crystal grain size is mainly distributed in the range of 700 nm to 3600 nm.

[0082] Example 4

[0083] To further verify the effect of different cooling rates on crystallization, the cooling rate was changed based on Example 1.

[0084] Specifically, the preparation method in this embodiment is largely the same as in embodiment 1, except for step 5;

[0085] Step 5 is as follows: After sterilization in step 4, spray the injection bottle with water for injection at 80°C and remove it from the container. Place the bottle in room temperature air to cool naturally. After cooling to room temperature, difluorofloxacin injection is obtained, in which the difluorofloxacin content is 5% (5g of difluorofloxacin per 100 mL).

[0086] Observe the morphology of particles in the suspension using a high-powered microscope, such as Figure 7 As shown, the microparticles in the injection solution exhibit a relatively uniform polymorphic crystalline structure; samples were taken and analyzed using a laser particle size analyzer to determine the main particle size of the crystals in the injection solution, such as... Figure 8 As shown, the crystal particle size is mainly distributed in the range of 500 nm to 3000 nm.

[0087] Based on the results of Examples 1 and 4, slow cooling increases the crystal size, but the increase is still insufficient to cause adverse consequences such as clogging of the syringe.

[0088] Performance testing:

[0089] 1. Crystal particle size test: The difluorofloxacin injection solution prepared in Examples 1 to 4 was analyzed and tested for crystal morphology and particle size using a high-power microscope and laser particle size analyzer.

[0090] 2. Needle clogging test: After mixing the difluorofloxacin injection solution prepared in Examples 1 to 4, use a 1 mL disposable sterile syringe (equipped with a No. 5 needle) to draw in and expel the injection solution, and observe the needle clogging situation during the process.

[0091] 3. Determination of effective content and analysis of the ratio of dissolved to crystalline states of difluorofloxacin injection: Take an appropriate amount of the difluorofloxacin injection prepared in Examples 1-4, mix well, and transfer it as sample A. Filter sample A through a 0.22 μm microporous membrane, and collect the filtrate as sample B. Accurately transfer 1 mL each of sample A and sample B into separate 50 mL volumetric flasks, add mobile phase to the mark, mix well, and accurately transfer 2 mL into a 50 mL volumetric flask, add mobile phase to the mark, mix well, and then perform high-performance liquid chromatography (HPLC) analysis. Take an appropriate amount of difluorofloxacin reference standard, dissolve and dilute it with the mobile phase to prepare a reference solution containing 40 mg of difluorofloxacin per 1 mL. Perform high-performance liquid chromatography (HPLC) analysis using the same method. Calculate the difluorofloxacin content in the test sample using the external standard method based on the peak area of ​​difluorofloxacin. Where test sample A represents the difluorofloxacin content in the injection solution, test sample B represents the dissolved difluorofloxacin content, and B / (AB) is the ratio of dissolved to crystalline difluorofloxacin. The results are shown in the performance test section below. Specific HPLC conditions are as follows:

[0092] High-performance liquid chromatography (HPLC) instrument equipped with a UV detector; mobile phase: 6.82 mL of phosphoric acid (analytical grade) was injected into a 2 L beaker, 1.4 L of water and 500 mL of acetonitrile were added, a pH meter electrode was placed, triethylamine was added dropwise and the pH was adjusted to 2.5 ± 0.1, transferred to a 2 L volumetric flask, and water was added to the mark to prepare a mobile phase of 0.05 mol / L triethylamine phosphate:acetonitrile (75:25); chromatographic column: C 18 Chromatographic column: 4.6 mm × 250 mm, particle size: 5 μm; injection volume: 10 μL; detection wavelength: 280 nm.

[0093] The test results for the samples in each embodiment are as follows:

[0094] Table 1. Results of Crystal Morphology and Proportion Testing

[0095]

[0096] Results Analysis: The difloxacin injection solutions prepared in Examples 1-4 all yielded uniformly suspended difloxacin injection solutions. The active ingredient, difloxacin, existed in two forms: a dissolved state and a solid crystalline state. Reducing the total difloxacin content and increasing the proportion of gluconolactone in the formulation increased the proportion of dissolved difloxacin in the injection solution. The crystals in the injection solution exhibited polymorphic crystalline morphology, with a main particle size distribution ranging from 500 nm to 3600 nm. Different concentrations of difloxacin injection solutions could be prepared by adjusting the formulation ratios. All concentration specifications of the injection solutions showed good needle-passing performance and were convenient to use.

[0097] Performance Test 2

[0098] Pharmacokinetic testing of difluorofloxacin injection in yellow chickens

[0099] Animal experiment: The experimental animal was a yellow chicken.

[0100] Test drug: Difluorofloxacin injection prepared in Examples 1 and 2.

[0101] Experimental Methods: Fifteen adult Sanhuang chickens, 55 days old, were purchased from a local poultry market. After acclimatization in the laboratory for 5 days with no abnormalities observed, they were randomly divided into three groups: two treatment groups (6 chickens per group) and one blank control group (3 chickens per group). The treatment groups were weighed and administered a single dose of difluorofloxacin injection solution prepared in Examples 1 and 2, at a dose of 0.2 mL per kilogram of body weight via intramuscular injection in the pectoral region. Blood samples were drawn from the wing vein at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after administration to measure the concentration of difluorofloxacin in the blood, thus testing the pharmacokinetic behavior of the difluorofloxacin injection solutions prepared in different examples in Sanhuang chickens. The specific pharmacokinetic patterns (blood drug concentration curves) for Examples 1 and 2 are shown below. Figure 9 and Figure 10 .

[0102] Comparative Example 1

[0103] Add 4 g of trimethoprim (an antibacterial synergist), 3 g of dextran-20, and 40 mL of water to three 50 mL beakers, designated as samples I, II, and III, respectively. Add 2 g, 2.7 g, and 4 g of gluconolactone to samples I, II, and III, respectively. Heat the samples to dissolve the trimethoprim. Clear solutions can be obtained at temperatures above 90 °C. Stop heating and allow the samples to cool to room temperature.

[0104] The results are as follows Figure 11As shown, after cooling, the solution of sample III became clear without any crystallization or precipitate. After cooling, a small amount of crystals appeared on the surface and bottom of the solution of sample II. After standing for 30 minutes, the crystallization and precipitate at the bottom of the solution became more obvious. After cooling, a large amount of crystallization and precipitate appeared on the surface and bottom of the solution of sample I.

[0105] refer to Figure 12 and Figure 13 Microscopic examination of the crystals of samples I and II revealed that sample I could be clearly observed using a low-power microscope. Figure 12 ) and Sample No. II ( Figure 13 The crystals are all rectangular or elongated rhomboid, with relatively coarse crystal particles. The particle size of the main crystal is between 30 and 200 μm, as measured by microscopy.

[0106] As can be seen from the above experiments, the cosolvent gluconolactone used in this invention is only effective in controlling the particle size of difluorofloxacin during cooling crystallization.

[0107] Comparative Example 2

[0108] Add 1 g of difluorofloxacin raw material and 50 mL of water to three 50 mL beakers, designated as samples IV, V, and VI, respectively. Add 0.8 g, 1 g, and 1.5 g of citric acid monohydrate to samples IV, V, and VI, respectively. Heat to a temperature above 90°C to dissolve the difluorofloxacin, then stop heating and allow to cool to room temperature.

[0109] The results are as follows Figure 14 As shown, all three sample solutions became turbid after cooling. After standing for 30 minutes, obvious crystal precipitates appeared at the bottom of all three sample solutions. Microscopic examination of the crystals from the three samples revealed that, under low magnification, the crystals were all thin, flat, and roughly square or rhomboid in shape. The crystals were relatively coarse; measurements using the micrometer scale on the eyepiece showed that the main crystal particle size ranged from 20 μm to 150 μm. The crystal morphology of the three samples was essentially similar. Figures 15-17 Low-power microscope images of the crystallization of samples IV, V and VI.

[0110] Comparative Example 3

[0111] 2.5 g, 5 g, and 10 g of polyvinylpyrrolidone were added to three 50 mL beakers, respectively. Hot water was added, and the mixture was stirred until dissolved and adjusted to a volume of 50 mL. These were designated as samples VII, VIII, and IX. 1 g of difluorofloxacin was added to samples VII, VIII, and IX, and the mixture was heated to above 90 °C with stirring, then cooled to room temperature. The results showed that all three samples remained turbid during heating. After heating was stopped and the mixture was allowed to cool to room temperature, the difluorofloxacin precipitated quickly to the bottom of the solution. Microscopic examination of the particles at the bottom of the three samples was performed and compared with difluorofloxacin powder. Under a low-power microscope, it was clearly observed that the crystals were all rod-shaped, resembling broken needles or elongated rhombuses. The crystals were relatively coarse; measurements using the micrometer scale on the eyepiece showed that the main crystals had a length between 100 μm and 500 μm and a width between 40 μm and 100 μm. The experimental results show that polyvinylpyrrolidone has no significant solubilizing effect on difluorofloxacin, nor can it dissolve the difluorofloxacin raw material and produce microcrystals. Figure 18 These are photographs of samples VII, VIII, and IX after they were heated to a temperature greater than 90°C and then cooled to room temperature. Figures 19-21 The images are low-powered microscopic images of the precipitated particles in samples VII, VIII, and IX, respectively. Figure 22 This is a low-power microscope image of difluorofloxacin raw material.

[0112] As shown in Comparative Examples 2 and 3 above, conventional solubilizers or suspending agents cannot effectively improve the crystallization properties of difluorofloxacin.

[0113] Based on the above experiments, the following conclusions can be drawn:

[0114] 1. The co-solvent gluconolactone is only effective in controlling the crystallization size of difluorofloxacin during cooling. The mechanism may be that it forms a complex with difluorofloxacin molecules, creating a gluconolactone-difluorofloxacin double salt, which increases the solubility of difluorofloxacin in water. During the cooling process, steric hindrance (the complexation with gluconolactone combined with the high molecular weight of dextran) inhibits the aggregation of the double salt crystals, resulting in microcrystals of the gluconolactone-difluorofloxacin double salt. Currently, gluconolactone has no effect on crystallization control for other drugs that we have already verified. However, the above research results are not without reference value for other drugs. In research on other drugs, suitable co-solvents should be sought to inhibit crystal aggregation.

[0115] 2. Citric acid, polyvinylpyrrolidone, and other conventional solubilizers or suspending agents have no significant effect on the particle size control of difluorofloxacin cooling crystallization.

[0116] 3. As can be seen from Examples 1 to 4 of the present invention, the present invention can achieve simultaneous high-temperature sterilization and leak testing by optimizing the cosolvent and the process, effectively solving the defects of existing suspensions that cannot be adapted to high-temperature sterilization processes and that sterilization and leak testing require two separate steps.

[0117] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a difluorofloxacin suspension injection, characterized in that, Includes the following steps: Step 1: Add difluorofloxacin to an aqueous solution containing gluconolactone, stir, keep the temperature not lower than 80℃, add pH adjuster, adjust the pH value to 3~5, and prepare difluorofloxacin solution; Step 2: Keep the temperature not lower than 80℃, add a crystallization inhibitor to the difluorofloxacin solution in step 1, filter to remove impurities, and add water to make up to the total volume; Step 3: Keep the difluorofloxacin solution from Step 2 at a temperature not lower than 80°C, dispense it into glass vials containing the injection solution, seal them, and sterilize them with high-temperature steam. Step 4: The injection solution after high-temperature sterilization is immediately soaked in cold water or cooled to room temperature in air for leak detection and crystallization, and difluorofloxacin injection solution is prepared simultaneously. The total content of difluorofloxacin, the active ingredient, in every 100ml of the injection solution is 5-15g, and the content of gluconolactone in every 100ml of the injection solution is 5-15g. The pH adjuster is gluconic acid or sodium gluconate, and its usage is adjusted according to the pH requirements of the injection solution; the crystallization inhibitor is dextran, and the content of dextran in each 100ml of the injection solution is 2~10g.

2. The preparation method according to claim 1, characterized in that, The active ingredients in the injection solution include difluorofloxacin in a dissolved state and difluorofloxacin in a solid microcrystalline state.

3. The preparation method according to claim 1, characterized in that, The injection solution also contains an antioxidant, which is sodium bisulfite or sodium metabisulfite, and the amount of antioxidant used per 100 ml of the injection solution is 0.01~0.2 g.

4. The preparation method according to claim 1, characterized in that, The temperature of the cold water is 5~30℃.

5. The preparation method according to claim 4, characterized in that, The temperature of the cold water is room temperature, which is 20~30℃.

6. A difluorofloxacin suspension for injection, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

7. The difluorofloxacin suspension injection according to claim 6, characterized in that, The difluorofloxacin microcrystals in the difluorofloxacin suspension injection have a crystal particle size of 400 nm to 5 μm.

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