Difloxacin microcrystal suspension injection and preparation method thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a difloxacin microcrystal suspension injection which comprises water and a difloxacin crystal existing in the water in a suspension state. The particle size of the difloxacin crystal is 200 nm to 10 [mu] m, and the difloxacin crystal is of a sheet structure. The particle size of the difloxacin crystal in the difloxacin microcrystalline suspension injection provided by the invention is 200nm-10mu m, the difloxacin crystal is of a sheet structure, the crystal can stably exist in a liquid carrier in a suspension form, and after being prepared into the injection, the difloxacin microcrystalline suspension injection has the advantages of long acting time, small irritation and no blockage of a needle tube, and can be prepared into preparations with various concentrations. Meanwhile, the invention also provides a preparation method of the suspension injection.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a difloxacin microcrystalline suspension injection and a preparation method thereof. Background Art

[0002] Quinolones are a class of synthetic antibacterial drugs, which are roughly divided into four generations according to the order of invention and different structures. Their main mechanism of action is to inhibit bacterial DNA helicase. At present, there are mainly three-generation quinolones such as Ciprofloxacin (CIP), Enrofloxacin (ENR), Difloxacin (DIF), Danofloxacin (DAN), Sarafloxacin (SAR), and the second-generation Flumequine (FLU) approved for use in livestock and poultry farming in China.

[0003] Difloxacin is a drug for animals. The raw material is difloxacin hydrochloride, CAS: 91296-86-5. The 2017 edition of "Quality Standards for Veterinary Drugs" includes difloxacin hydrochloride (raw material), difloxacin hydrochloride injection, difloxacin hydrochloride solution, difloxacin hydrochloride powder and difloxacin hydrochloride tablets.

[0004] Difloxacin produces a bactericidal effect by acting on the A subunit of bacterial DNA gyrase, inhibiting bacterial DNA replication and transcription. It has strong antibacterial activity against most Gram-negative bacteria and Gram-positive bacilli and cocci, including Klebsiella, Staphylococcus, Escherichia coli, Enterobacter, Campylobacter, Shigella, Proteus and Pasteurella. It can be used to treat chronic respiratory diseases, tracheitis, enteritis, pneumonia, fowl cholera, streptococcal disease, typhoid and other diseases in livestock and poultry, especially Escherichia coli disease in chickens and red, yellow and white diarrhea in piglets.

[0005] Since difloxacin hydrochloride is only slightly soluble in water, the existing injection dosage form can only be made into low-content specifications, and it needs to be made into a relatively strong alkaline (pH ≥ 10) to exist in the form of a solution. In the existing technology, in the two preparations of difloxacin hydrochloride solution and injection registered by domestic manufacturers and obtained the "veterinary drug product approval number", the content specifications of difloxacin in the solution are both 2.5%, and the content specifications of difloxacin in the injection are both 2.0%.

[0006] The disadvantages of the prior art are mainly that the relatively strong alkaline injection is highly irritating, has a low concentration, a large injection volume, and the drug is absorbed and eliminated quickly after injection, and cannot achieve a sustained-release and long-acting effect. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a difloxacin microcrystalline suspension injection. The difloxacin crystals in the difloxacin microcrystalline suspension injection provided by the present invention have a particle size of 200nm to 10μm and a flaky structure. The crystals can stably exist in a suspended form in a liquid carrier. After being made into an injection, it has a long action time, low irritation, does not block the needle tube, and can be prepared into preparations of various concentrations.

[0008] At the same time, the invention also provides a method for preparing the suspension injection.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] A difloxacin microcrystal suspension injection comprises water and difloxacin crystals in a suspended state in the water; the particle size of the difloxacin crystals is 200nm-10μm and they are in a flaky structure.

[0011] The difloxacin crystals in the suspension injection of the present invention have a small particle size and a flake structure, and are less likely to clog a needle than difloxacin with a needle structure; at the same time, the difloxacin crystals contain difloxacin salt and difloxacin monomer, and instantaneous release is achieved through the difloxacin salt, and sustained release is achieved through the difloxacin monomer, thereby achieving the purpose of initial high concentration and long-term release.

[0012] The suspension of the invention is weakly acidic, thus overcoming the problem that the traditional difloxacin injection must be adjusted to be alkaline.

[0013] In the above-mentioned difloxacin microcrystalline suspension injection, the content of difloxacin, an effective ingredient, in the injection is 2wt% to 15wt%, and the difloxacin crystals are difloxacin or difloxacin salt or a mixture of the two.

[0014] Preferably, the content of difloxacin as an active ingredient in the injection is 3wt% to 15wt%, more preferably, the content of difloxacin as an active ingredient in the injection is 4wt% to 15wt%, more preferably, the content of difloxacin as an active ingredient in the injection is 5wt% to 15wt%.

[0015] In the above-mentioned difloxacin microcrystalline suspension injection, the weight ratio of difloxacin to difloxacin salt is 10-90:90-10.

[0016] In the above-mentioned difloxacin microcrystalline suspension injection, the difloxacin salt is difloxacin hydrochloride or difloxacin organic acid salt.

[0017] In the above-mentioned difloxacin microcrystalline suspension injection, the organic acid involved in the difloxacin organic acid salt is one or more combinations of citric acid, tartaric acid, lactic acid and acetic acid.

[0018] In the above-mentioned difloxacin microcrystalline suspension injection, the pH of the suspension injection is 3-6.

[0019] In the above-mentioned difloxacin microcrystalline suspension injection, the suspension injection further contains one or more combinations of a pH regulator, an antioxidant, and a suspending agent.

[0020] Specifically, the pH adjuster can be selected from one or more combinations of NaOH, KOH, sodium gluconate, ethanolamine, diethanolamine or triethanolamine;

[0021] The antioxidant may be selected from one or more combinations of sodium bisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium pyrosulfite, and ascorbic acid;

[0022] The suspending agent can be selected from one or more combinations of sodium carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, and hydroxypropyl methyl cellulose.

[0023] Meanwhile, the invention also discloses a method for preparing the difloxacin microcrystal suspension injection, which comprises adding difloxacin crystals into water to prepare a suspension.

[0024] In the above-mentioned preparation method, the preparation method of the difloxacin crystal is:

[0025] Step 1: preparing a solution containing difloxacin salt;

[0026] Step 2: Rapidly cool the solution containing difloxacin salt to 2-10° C. to precipitate the difloxacin crystals.

[0027] The flaky difloxacin crystals can be prepared by rapid cooling and crystallization.

[0028] In the above preparation method, after step 2, step 3 is further included: filtering the solution of step 2, and adding alkali to the filtrate, adjusting the pH value of the filtrate to 2-7, so that crystals continue to precipitate; or, directly adding alkali to the solution of step 2, adjusting the pH value of the solution to 2-7, so that crystals continue to precipitate;

[0029] In the above step 1, after the difloxacin salt is dissolved in water, the system pH is less than 2; in step 2, after cooling and crystallization, most of the difloxacin salt is precipitated, and the system pH is slightly increased; in the step 2, during the cooling process, a base can be continuously added to increase the system pH to 2-7, thereby increasing the proportion of difloxacin monomer;

[0030] In step 3, adding a base can promote the precipitation of the difloxacin salt remaining in the solution in the form of difloxacin or difloxacin salt; generally speaking, the higher the pH, the more difloxacin monomers are in the product; conversely, the more difloxacin salt is in the product;

[0031] In step 3, the temperature of step 2 is maintained. If the temperature exceeds 10°C, the crystals will be redissolved.

[0032] In industrial production, the preferred operation is to filter first, then combine the filtrates and crystallize.

[0033] The advantage of step 3 of the present invention is that the solution is continuously neutralized at low temperature so that the remaining difloxacin and its salt in the solution are crystallized synchronously, the proportion of difloxacin monomer in the crystal is increased, and the release rate of difloxacin monomer crystal in the body is slow compared with difloxacin salt crystal; therefore, the proportion of difloxacin monomer in the crystal can be changed by adjusting the pH value, thereby enabling the crystal of the present invention to achieve the dual purposes of instantaneous high release in the initial stage and long-term sustained release in the system.

[0034] The concentration of difloxacin salt in the solution of step 1 is 10-100 g / L;

[0035] In step 1, the solution temperature is increased to 80-90° C. to promote the dissolution of difloxacin salt;

[0036] The cooling rate of step 2 is 5-50°C / min; generally speaking, the faster the cooling rate, the better. It is recommended that the cooling time be controlled within 10 minutes, preferably within 5 minutes, and more preferably within 2 minutes.

[0037] In the laboratory stage, the fastest cooling speed that can be achieved is 2 minutes to reach the target temperature; in industrial production, faster cooling can be achieved with the help of more effective cooling modules, such as pipe reactors or the addition of liquid nitrogen.

[0038] In the step 2, seed crystals are added to the solution containing difloxacin salt in advance, or seed crystals are generated in the solution containing difloxacin salt by adding a base; the seed crystals are difloxacin salt; and the solution containing difloxacin salt is cooled while or after the seed crystals are present to promote the precipitation of crystals;

[0039] In the step 2, during the cooling process, alkali is continuously added to raise the pH of the system to 2 to 7, thereby increasing the proportion of difloxacin monomer crystals;

[0040] In the step 2, the crystal precipitation time is 2 to 30 minutes; in the step 3, the crystal precipitation time is 2 to 30 minutes; in the step 2, the crystallization speed is positively correlated with the cooling speed; in the step 3, the crystallization speed is correlated with the stirring speed and the alkali solution addition speed;

[0041] A surfactant is also added in step 1.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The difloxacin crystals in the difloxacin microcrystal suspension injection of the present invention have a particle size of 200nm to 10μm and a flaky structure. The crystals can stably exist in a liquid carrier in a suspended form. After being made into an injection, the crystals have a long action time, low irritation, and no needle blocking, and can be prepared into preparations of various concentrations; at the same time, the difloxacin crystals contain difloxacin salts and difloxacin monomers, and instantaneous release is achieved through the difloxacin salts, and sustained release is achieved through the difloxacin monomers, achieving the purpose of initial high concentration and long-term release. The suspension of the present invention is weakly acidic, which overcomes the problem that traditional difloxacin injections must be adjusted to alkalinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a microscope image of Example A1;

[0045] Figure 2 is a scanning electron microscope image of the plate-like crystals in step 2 of Example A1;

[0046] Figure 3 is a scanning electron microscope image of the plate-like crystals in step 3 of Example A1;

[0047] Figure 4 is a microscope image of Example A2;

[0048] Figure 5 is a scanning electron microscope image of Example A2;

[0049] Figure 6 is a microscope image of Example A3;

[0050] Figure 7 is a scanning electron microscope image of Example A3;

[0051] Figure 8 is a scanning electron microscope image of the difloxacin lactate ultrafine crystals obtained in Example A4;

[0052] Fig. 9 is a scanning electron microscope image of the difloxacin ultrafine crystals obtained in Example A4;

[0053] Fig.10 is a microscope image of Example A5;

[0054] Fig.11 is a scanning electron microscope image of Example A5;

[0055] Fig.12 It is a microscope image of Comparative Example A1;

[0056] Fig.13 is a microscope image of Example B1;

[0057] Fig.14 is a microscope image of Example B2;

[0058] Fig.15 is a microscope image of Example B3;

[0059] Fig.16 is a microscope image of Example B4;

[0060] Fig.17 is a microscope image of Example B5;

[0061] Fig.18 is a microscope image of Example B6;

[0062] Fig.19 is the particle size distribution diagram of Example B1;

[0063] Fig. 20 is the particle size distribution diagram of Example B2;

[0064] Fig.21 is the particle size distribution diagram of Example B3;

[0065] Fig. 22 is the particle size distribution diagram of Example B4;

[0066] Fig.23 is the particle size distribution diagram of Example B5;

[0067] Fig.24 This is the particle size distribution diagram of Example B6. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] Part I Preparation, characterization and preliminary performance testing of difloxacin crystals

[0070] Example A1

[0071] Step 1: Add 2500mL of 90℃ hot water to a 316L mixing tank with a partition, then add 50g of difloxacin hydrochloride raw material, then add 1g of Tween-80, drop about 10mL of dilute hydrochloric acid (1mol / L) and stir to dissolve the difloxacin hydrochloride, filter into another identical mixing tank, the filtrate is liquid A, and keep the temperature not lower than 80℃.

[0072] Step 2: Take 4.5g of analytically pure sodium hydroxide and dissolve it in 200mL of water. Slowly drop 20mL of the above-mentioned alkali solution (concentration of 2.25g / 100ml) into solution A under rapid stirring at a speed of 600r / min to produce nanocrystalline seeds. Pass 0-4℃ ice water into the interlayer of the stirring tank, cool down while stirring, and quickly reduce the solution temperature to 5-10℃. The cooling time is 10min. During the cooling process, continue to stir while cooling and drop about 10mL of alkali solution. Keep 5-10℃ for 15 minutes, then filter with a 0.22μm microporous filter membrane, and then rinse the filter cake with 200mL of 0-4℃ ultrapure water to replace the salt water in the filter cake (the solubility of difloxacin hydrochloride crystals in 0-4℃ cold water is about 0.05%, so elution will not cause obvious redissolution of the crystals), drain, and transfer the filter cake to a drying dish for negative pressure drying to obtain difloxacin hydrochloride microcrystals. The crystals produced in step 2 were sampled and tested by scanning electron microscopy. Figure 2 , it can be seen that plate-like crystals have been produced;

[0073] Step 3: Take the filtrate from step 2 and adjust the pH to 6-7, keep it at 5-10℃ for 15 minutes, and then filter it with a 0.22μm microporous filter membrane. The filter cake is then rinsed with 50mL 0-4℃ ultrapure water to replace the brine in the filter cake, and then drained. The filter cake is transferred to a drying dish and dried under negative pressure to obtain difloxacin microcrystals. The microcrystals are tested by microscope and scanning electron microscope. The results show that Figure 1 and Figure 3 ;pass Figure 3 It can be seen that the final product is mainly in the form of lamellar crystals.

[0074] Example A2

[0075] Step 1: Add 2500mL of 90℃ hot water to a 316L mixing tank with a partition, then add 50g of difloxacin hydrochloride raw material, then add 1g of Tween-80, drop about 10mL of dilute hydrochloric acid (1mol / L) and stir to dissolve the difloxacin hydrochloride, filter into another identical mixing tank, the filtrate is liquid A, and keep the temperature not lower than 80℃.

[0076] Step 2: Take 4.5g of analytical pure sodium hydroxide and dissolve it in 200mL of water. Spray 20mL of alkali solution (concentration is 2.25g / 100ml) into solution A with rapid stirring at a speed of 1000r / min to produce nanocrystalline seeds. Pass 0-4℃ ice water into the interlayer of the stirring tank, and inject liquid nitrogen on the surface of the solution to quickly reduce the solution temperature to 2-6℃. The cooling time is 5min, and stir under heat preservation conditions.

[0077] Step 3: Slowly add alkali solution, adjust the pH to 3-4, maintain 2-6℃ for 15 minutes, and then filter with a 0.22μm microporous filter membrane. The filter cake is then rinsed with 200mL 0-4℃ ultrapure water to replace the brine in the filter cake, drained, and the filter cake is transferred to a drying dish for negative pressure drying to obtain mixed microcrystals of difloxacin and difloxacin hydrochloride. The microcrystals are tested by microscope and scanning electron microscope. The results show that Figure 4 and Figure 5 ;pass Figure 5 It can be seen that the final product is mainly in the form of lamellar crystals.

[0078] Example A3

[0079] Step 1: Take 50g of analytically pure lactic acid and add it to a 316L stirring tank with interlayer heating and 2500mL of 90℃ hot water, add 200g of difloxacin raw material, maintain the temperature, add 1g of Tween-80, stir to completely dissolve the difloxacin, filter the solution into another stirring tank of the same model as liquid A, and keep the temperature not lower than 80℃.

[0080] Step 2: Take another 9g of analytical pure sodium hydroxide and dissolve it in 400mL of water as liquid B. Spray 50mL of alkali solution (concentration of 2.25g / 100ml) into liquid A with rapid stirring at a speed of 1000r / min to produce nanocrystalline seeds. Pass ice water at about 0°C into the interlayer of the stirring tank to quickly reduce the solution temperature to 5-10°C within 5min. Maintain the temperature and continue stirring for 15min.

[0081] Step 3: Slowly add the remaining B solution and adjust the pH to 4-5, continue stirring, maintain 5-10℃ for 15 minutes, filter with a 0.22μm microporous filter membrane, rinse the filter cake with 400mL 0-4℃ ice water to remove salt, drain, transfer the filter cake to a drying dish and dry under negative pressure to obtain difloxacin and difloxacin lactate microcrystals. The microcrystals were tested by microscope and scanning electron microscope, and the results showed that Figure 6 and Figure 7 ;pass Figure 7 It can be seen that the final product is mainly in the form of lamellar crystals.

[0082] Example A4

[0083] Step 1: Take 50g of analytically pure lactic acid and add it to a 316L stirring tank with interlayer heating and 2500mL of 90℃ hot water, add 200g of difloxacin raw material, maintain the temperature, add 1g of Tween-80, stir to completely dissolve the difloxacin, filter the solution into another stirring tank of the same model as liquid A, and keep the temperature not lower than 80℃.

[0084] Step 2: Take another 9g of analytical pure sodium hydroxide and dissolve it in 400mL of water as liquid B. Spray 20mL of alkali solution (concentration of 2.25g / 100ml) into liquid A while rapidly stirring at a speed of 1000r / min to produce nanocrystalline seeds. Pass ice water at about 0°C into the interlayer of the stirring tank, and inject liquid nitrogen on the surface of the solution to quickly reduce the solution temperature to 2-5°C within 2 minutes. Maintain the temperature and continue stirring for 15 minutes.

[0085] Step 3: Filter the crystals with a 0.22 μm microporous membrane, and then rinse the filter cake with 200 mL of 0-4°C ultrapure water to remove excess lactic acid in the filter cake to obtain difloxacin lactate crystals. Slowly add the remaining B solution to the filtrate, adjust the pH to 6-7, continue stirring, maintain 2-5°C for 15 minutes, filter with a 0.22 μm microporous membrane, and then rinse the filter cake with 50 mL of 0-4°C ice water to remove salt, and drain to obtain difloxacin crystals.

[0086] The wet crystals were dried by freeze drying to obtain difloxacin lactate ( Figure 8 ) and difloxacin ultrafine crystals ( Fig. 9 ).

[0087] Example A5

[0088] The same as Example 1, except that the concentration of difloxacin hydrochloride in solution A in step 1 is 10 g / L. Fig.10 and Fig.11 ,pass Fig.11 It can be seen that the final product is mainly in the form of lamellar crystals.

[0089] Comparative Example A1

[0090] Take 10g of difloxacin hydrochloride raw material, add it to a beaker filled with 1000mL of 90℃ hot water, put in a magnetic stirrer, start stirring to dissolve difloxacin hydrochloride, and filter to remove impurities. Slowly add 2% NaOH solution to the filtrate, and adjust the pH to 6-7. Let the solution return to room temperature while stirring continuously, filter with a 0.22μm microporous filter membrane to separate the crystals, and then rinse the filter cake with 50mL of 0-4℃ ultrapure water to replace the desalted solution. The filter cake is transferred to a drying dish and dried under negative pressure. The obtained difloxacin crystals are mainly slender needle-shaped crystals, which can be seen. Fig.12 .

[0091] Performance Test 1

[0092] 1. Crystal particle size test: Disperse the crystal particles and use (microscope and scanning electron microscope) to analyze and test the crystal morphology and particle size.

[0093] 2. Difloxacin content test: Since the solubility of difloxacin salt-based crystals and difloxacin monomer crystals in 20°C pure water is very different, the mixed crystals can be separated by washing the difloxacin salt and difloxacin monomer crystals with 20°C pure water in small amounts for multiple times, so that the difloxacin salt-based crystals are dissolved in the solution, and then the concentration of difloxacin in the solution is detected by high performance liquid chromatography, and the ratio of difloxacin salt-based crystals to difloxacin monomer crystals in the mixed crystals can be calculated. The test results of the samples in each embodiment are as follows:

[0094] Table 1 Crystal morphology and proportion test results

[0095]

[0096]

[0097] Result analysis: The difloxacin crystal product prepared in Example A1 has a relatively gentle cooling rate and the alkali solution is added earlier, so the crystal shape is slightly larger. The crystals are mainly difloxacin monomers, and there are a small amount of needle-shaped crystals.

[0098] In Example A2, the temperature is rapidly lowered and then stirred at a high speed, and a large amount of crystals are first formed before the solution is adjusted to near neutrality with alkaline solution. The crystallization temperature is relatively low, so the crystals produced are mainly difloxacin hydrochloride crystals, which are relatively small and uniform, and are lamellar polyhedrons.

[0099] In Example A3, after adding liquid nitrogen to assist in rapid cooling, the crystallization temperature of the crystal is lower, the crystal shape is relatively small, and it is relatively uniform, and it is a lamellar polyhedron. After separating the difloxacin lactate crystals, the difloxacin salt in the solution is neutralized with alkali to form difloxacin monomer crystals. After being dried separately, difloxacin lactate crystals and difloxacin monomer crystals are obtained respectively.

[0100] Performance Test 2

[0101] Needle blocking test of difloxacin crystals:

[0102] Take difloxacin crystals, add water and mix well to prepare a 7.5% suspension. Use 1mL, 2.5mL and 5mL disposable sterile syringes connected to their own needles (No. 4.5, No. 5.5 and No. 7 needles, respectively) to pump the suspension 10 times to test the needle blockage of crystals prepared by different processes.

[0103] Table 2 Needle blocking test of difloxacin crystals

[0104]

[0105]

[0106] Note: If the needle is blocked once during the withdrawal and injection process, it will be considered as needle blockage.

[0107] Performance Test 3

[0108] Pharmacokinetics of difloxacin microcrystals in yellow chickens

[0109] Animal experiment: The experimental animals were yellow chickens.

[0110] Drug preparation: Take the crystals obtained in Example 2 and Example 3, add an appropriate amount of water for injection, mix well, and prepare a 7.5% suspension injection (75 mg per ml of difloxacin).

[0111] Experimental method: The experimental chickens were weighed and administered once by intramuscular injection of 0.2 mL per kg of body weight. Blood was drawn from the wing vein at 0.25 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 36 hours, and 48 hours after administration to detect the concentration of difloxacin in the blood, in order to test the release effect of difloxacin crystals obtained by different processes in the body.

[0112] The test results of Example 2 and Example 3 are as follows:

[0113] Table 3 Blood drug concentration of difloxacin crystals prepared by different processes in yellow chickens

[0114]

[0115]

[0116] As shown in the pharmacokinetic results of Table 3, after intramuscular injection of yellow chickens, the difloxacin crystals prepared in Examples 2 and 3 were slowly absorbed in the bodies of the test chickens. Within 1 to 48 hours after administration, the drug was always at a steady-state concentration, and at the 48-hour time point, the drug was still near the peak blood drug concentration, showing a good sustained-release effect, and it is expected that the purpose of only administering the drug once in a course of treatment can be achieved. Compared with Example 2, the difloxacin lactate and monomer mixed crystals of Example 3 showed better bioavailability and long-acting effect.

[0117] Part II Preparation, characterization and performance testing of difloxacin microcrystalline suspension

[0118] All the cases described below were performed in a sterile environment.

[0119] Example B1

[0120] Step 1: Add 2500mL of 90℃ hot water to a 316L mixing tank with a partition, then add 50g of difloxacin hydrochloride raw material, then add 1g of Tween-80, drop about 10mL of dilute hydrochloric acid (1mol / L) and stir to dissolve the difloxacin hydrochloride, filter into another identical mixing tank, the filtrate is liquid A, and keep the temperature not lower than 80℃.

[0121] Step 2: Take another 2g of analytical pure sodium hydroxide and dissolve it in 200mL of water. Spray 10mL of alkali solution (concentration is 1g / 100ml) into solution A under rapid stirring at a speed of 1000r / min to produce nanocrystalline seeds. Pass 0-4℃ ice water into the interlayer of the stirring tank, and inject liquid nitrogen on the surface of the solution to quickly reduce the solution temperature to 2-6℃. The cooling time is 2min, and stirring is continued under insulation conditions.

[0122] Step 3: slowly drop alkali solution, adjust the pH to 2.5, maintain at 2-6°C for 15 minutes, and then filter with a 0.22 μm microporous filter membrane. The filter residue is difloxacin hydrochloride microcrystals.

[0123] Step 4: Take an appropriate amount of sterile water, add the difloxacin hydrochloride microcrystals in step 3 according to the aseptic operation method and the final concentration requirement, adjust the pH to 3.0 with alkali solution under stirring, add sodium bisulfite solution (the final proportion of the preparation is 0.1%, w / v) adjusted to pH 3.0 as an antioxidant and hydroxypropyl cellulose (accounting for 0.5-1.5% of the final preparation, w / v) solution as a suspending agent, make up to volume, fill into a small-capacity sterile container, seal it by melting or covering it, and prepare difloxacin hydrochloride microcrystalline suspension injection.

[0124] Example B2

[0125] Step 1: Add 2500mL of 90℃ hot water to a 316L mixing tank with a partition, then add 50g of difloxacin hydrochloride raw material, and then add 1g of Tween-80, stir to dissolve the difloxacin hydrochloride, filter into another identical mixing tank, the filtrate is liquid A, and keep the temperature not lower than 80℃.

[0126] Step 2: Take another 2g of analytical pure sodium hydroxide and dissolve it in 200mL of water. Spray 10mL of alkali solution into solution A while stirring rapidly at a speed of 1000r / min to produce nanocrystalline seeds. Pass 0-4℃ ice water into the interlayer of the stirring tank to quickly reduce the solution temperature to 2-6℃. The cooling time is 5min, and continue stirring under insulation conditions.

[0127] Step 3: slowly drop alkali solution, adjust the pH to 3.0, maintain at 2-6°C for 15 minutes, and then filter with a 0.22 μm microporous filter membrane. The filter residue is difloxacin hydrochloride and difloxacin mixed microcrystals.

[0128] Step 4: Take an appropriate amount of sterile water, add the difloxacin hydrochloride microcrystals in step 3 according to the aseptic operation method and the final concentration requirement, adjust the pH to 3.4 with alkali solution under stirring, add sodium bisulfite solution (the final proportion of the preparation is 0.1%, w / v) adjusted to pH 3.4 as an antioxidant and hydroxypropyl cellulose (accounting for 0.5-1.5% of the final preparation, w / v) solution as a suspending agent, make up to volume, fill into a small-capacity sterile container, seal it by melting or covering it, and prepare difloxacin hydrochloride microcrystalline suspension injection.

[0129] Example B3

[0130] Step 1: Take 50g of analytically pure lactic acid and add it to a 316L stirring tank with interlayer heating and 2500mL of 90℃ hot water, add 200g of difloxacin raw material, maintain the temperature, add 1g of Tween-80, stir to completely dissolve the difloxacin, filter the solution into another stirring tank of the same model as liquid A, and keep the temperature not lower than 80℃.

[0131] Step 2: Take another 2g of analytical pure sodium hydroxide and dissolve it in 200mL of water as liquid B. Spray 20mL of alkali solution into liquid A while rapidly stirring at a speed of 1000r / min to produce nanocrystalline seeds. Pass ice water at about 0°C into the interlayer of the stirring tank, and inject liquid nitrogen on the surface of the solution to quickly reduce the solution temperature to 2-6°C within 2 minutes. Maintain the temperature and continue stirring for 15 minutes.

[0132] Step 3: slowly drop alkali solution, adjust the pH to 2.6, maintain 2-6°C for 15 minutes, filter and separate the crystals with a 0.22 μm microporous filter membrane, and the filter residue is difloxacin lactate crystals.

[0133] Step 4: Take sterile water, add the difloxacin lactate microcrystals of step 3 according to the aseptic operation method and the final concentration requirement, adjust the pH to 3.0 with alkali solution under stirring, add sodium bisulfite solution (the final proportion of the preparation is 0.1%, w / v) adjusted to pH 3.0 as an antioxidant and hydroxypropyl cellulose solution (accounting for 0.5-1.5% of the final preparation, w / v) as a suspending agent, make up to volume, fill into a small-capacity sterile container, seal it by melting or covering it, and prepare difloxacin lactate microcrystal suspension injection.

[0134] Example B4

[0135] Take the difloxacin hydrochloride and difloxacin lactate microcrystals obtained in step 3 of Example B1 and step 3 of Example B3, respectively, add sterile water according to the aseptic operation method and the final concentration requirement, adjust the pH to 3.0 with alkali solution under stirring, add sodium bisulfite solution (the final proportion of the preparation is 0.1%, w / v) adjusted to pH 3.0 as an antioxidant and hydroxypropyl cellulose (accounting for 0.5%, w / v of the final preparation) solution as a suspending agent, make up to volume, fill into a small capacity sterile container, seal it with a seal or cover it, and prepare a mixed microcrystalline injection of difloxacin hydrochloride and difloxacin lactate.

[0136] Example B5

[0137] The method is substantially the same as Example B3, except that the pH in step 3 is adjusted to 3.0, and the pH in step 4 is adjusted to 3.4.

[0138] Example B6

[0139] The method is substantially the same as Example B3, except that the pH in step 3 is adjusted to 3.4, and the pH in step 4 is adjusted to 4.2.

[0140] Performance Test 1

[0141] 1. Morphological analysis of suspended particles: A small amount (3-5 uL) of the difloxacin microcrystalline suspension injection (difloxacin content: 7.5 wt%) prepared in each example was taken and directly pressed with a slide and a cover glass. The crystal morphology of the particles in the suspension injection was observed under a high-power microscope. Fig.13 ~Attached Fig.18 shown.

[0142] 2. Particle size analysis of suspended particles: Take an appropriate amount of difloxacin microcrystalline suspension injection obtained in each example, and use a laser particle size analyzer to detect the particle size distribution of the crystals in the suspension injection, as shown in the attached Fig.19 ~Attached Fig.24 shown.

[0143] Result analysis: The microscopic analysis results of the suspended particles in the difloxacin microcrystalline suspension injection prepared in each example showed that the crystal morphology of the particles was basically small flakes and polygonal (quadrilateral, pentagon, hexagon, etc.) crystals with good uniformity and very few needle-shaped crystals.

[0144] The laser particle size analyzer test results of the suspended particles in the difloxacin microcrystalline suspension injection prepared in each embodiment showed that the hydrochloride particles prepared in Example B1 had a slightly larger particle size, ranging from 1 to 7 μm; the hydrochloride particles prepared in Example B2 had a slightly larger particle size, ranging from 2 to 7 μm; the hydrochloride and lactate composite particles prepared in Example B4 had two relatively concentrated particle size values, ranging from 1 to 3 μm and from 4 to 7 μm, respectively; the particle sizes of the lactate particles prepared in the remaining embodiments were mainly concentrated between 0.9 and 3 μm.

[0145] Performance Test 2

[0146] Needle blocking test of difloxacin crystals:

[0147] Take the difloxacin microcrystalline suspension injection (difloxacin content: 7.5%) prepared in each embodiment, use 1mL, 2.5mL and 5mL disposable sterile syringes connected with self-contained needles (4.5, 5.5 and 7 needles respectively), and pump the suspension 10 times to test the needle blocking of crystals prepared by different processes. The results show that the difloxacin microcrystalline suspension injection prepared in each embodiment can pass through 4.5, 5.5 and 7 needles smoothly, and the above-mentioned needles can be used for drug administration in actual use.

[0148] Table 4 Needle blocking test of difloxacin microcrystalline suspension injection

[0149]

[0150]

[0151] Note: If the needle is blocked once during the withdrawal and injection process, it will be considered as needle blockage.

[0152] Performance Test 3

[0153] Difloxacin microcrystalline suspension injection sedimentation volume ratio test:

[0154] Take the difloxacin microcrystalline suspension injection prepared in each embodiment (difloxacin content is: 5%, 7.5% and 10%, respectively), add them to the dry 50mL stoppered measuring cylinder to the 50mL mark, plug it tightly, shake it vigorously for 1min, record the initial volume H0 of the suspension, let it stand for 3 hours, and record the final volume H of the suspension, which is calculated as follows: Sedimentation volume ratio = H / H0. The test results show that the sedimentation volume ratio of the difloxacin microcrystalline suspension injection prepared in each embodiment is between 0.91 and 0.94, which can meet the requirements of the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China on the sedimentation volume ratio of suspension injection H / H0 ≥ 0.9.

[0155] Performance Test 4

[0156] Pharmacokinetics of difloxacin microcrystalline suspension injection in yellow chickens:

[0157] Animal experiment: The experimental animals were yellow chickens, 96 days old, weighing between 1.8 and 2.3 kg.

[0158] Test drug: difloxacin microcrystalline suspension injection (difloxacin content: 7.5%) prepared in Example B1 to Example B6.

[0159] Test method: The test chickens were weighed and administered once with a dose of 0.2 mL per kg of body weight injected into the chest muscle. Blood was drawn from the chicken wing vein at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, and 72 hours after administration. The concentration of difloxacin in the blood was detected by high performance liquid chromatography (with fluorescence detector) to test the release effect of each embodiment product in the chicken. The results are as follows:

[0160] Table 5 Blood drug concentration of difloxacin microcrystalline suspension injection prepared in each example in yellow chicken

[0161]

[0162]

[0163] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A difloxacin microcrystalline suspension injection, characterized in that: The suspension injection comprises water and difloxacin crystals in a suspended state in the water; the particle size of the difloxacin crystals is 200nm-10μm and the difloxacin crystals are in a flaky structure.

2. The difloxacin microcrystalline suspension injection according to claim 1, characterized in that: The content of the effective ingredient difloxacin in the injection is 2wt% to 15wt%, and the difloxacin crystal is difloxacin or difloxacin salt or a mixture of the two.

3. The difloxacin microcrystalline suspension injection according to claim 2, characterized in that: The weight ratio of difloxacin to difloxacin salt is 10-90:90-10.

4. The difloxacin microcrystalline suspension injection according to claim 2, characterized in that: The difloxacin salt is difloxacin hydrochloride or difloxacin organic acid salt.

5. The difloxacin salt crystal according to claim 4, characterized in that The organic acid involved in the difloxacin organic acid salt is one or more combinations of citric acid, tartaric acid, lactic acid and acetic acid.

6. The difloxacin microcrystalline suspension injection according to claim 1, characterized in that: The pH of the suspension injection is 3-6.

7. The difloxacin microcrystalline suspension injection according to claim 1, characterized in that: The suspension injection also contains one or more combinations of a pH regulator, an antioxidant, and a suspending agent.

8. The method for preparing the difloxacin microcrystalline suspension injection according to any one of claims 1 to 7, characterized in that: Difloxacin crystals are added to water to prepare a suspension.

9. The preparation method according to claim 8, characterized in that: The preparation method of the difloxacin crystal is: Step 1: preparing a solution containing difloxacin salt; Step 2: Rapidly cool the solution containing difloxacin salt to 2-10° C. to precipitate the difloxacin crystals.

10. The method for preparing difloxacin crystals according to claim 6, characterized in that: After step 2, the method further includes step 3: filtering the solution of step 2, and adding alkali to the filtrate to adjust the pH value of the filtrate to 2-7, so that crystals continue to precipitate; or, directly adding alkali to the solution of step 2 to adjust the pH value of the solution to 2-7, so that crystals continue to precipitate; The concentration of difloxacin salt in the solution of step 1 is 10-100 g / L; In step 1, the solution temperature is increased to 80-90° C. to promote the dissolution of difloxacin salt; The cooling rate of step 2 is 5 to 50°C / min; In the step 2, seed crystals are added to the solution containing difloxacin salt in advance, or seed crystals are generated in the solution containing difloxacin salt by adding a base; the seed crystals are difloxacin salt; and the solution containing difloxacin salt is cooled while or after the seed crystals are generated to promote the precipitation of crystals; In step 2, during the cooling process, alkali is continuously added to raise the pH of the system to 2 to 7, thereby increasing the proportion of difloxacin monomer; In the step 2, the crystal precipitation time is 2 to 30 minutes; in the step 3, the crystal precipitation time is 2 to 30 minutes; A surfactant is also added in step 1.

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