Crystal of difloxacin and salt-based compound of difloxacin and preparation method of crystal

Abstract

The invention belongs to the field of medicines, and discloses a crystal of difloxacin and a salt-based compound thereof, the grain size of the crystal is 200nm-10mu m, and the crystal is of a sheet structure. The crystal provided by the invention has the particle size of 200nm-10mu m, is of a sheet structure, can stably exist in water in a suspension form, has the advantages of long acting time and small irritation, does not block a needle head, and can be prepared into preparations with various concentrations. Meanwhile, the invention also discloses a preparation method of the crystal.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a crystal of difloxacin and a salt-based compound thereof 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] In the prior art, there are two preparations of difloxacin hydrochloride, solution and injection, both of which have a specification of 2.5%. Since difloxacin hydrochloride is only slightly soluble in water, the existing injection dosage form can only be made into a low-content specification, and needs to be made into a relatively strong alkaline (pH ≥ 10) to exist in the form of a solution.

[0006] The disadvantage of the prior art is that the relatively strong alkaline injection is highly irritating and has a low concentration, making it difficult to prepare a long-acting injection preparation. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a crystal of difloxacin and its salt-based compound; the crystal provided by the present invention has a particle size of 200nm to 10μm and a flaky structure, the crystal can stably exist in the form of a suspension in water, has a long action time, low irritation, does not clog needles, and can be prepared into preparations of various concentrations.

[0008] Meanwhile, the invention also discloses a method for preparing the crystal.

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

[0010] The invention provides a crystal of difloxacin and a salt-based compound thereof, which has a particle size of 200nm-10μm and a flaky structure.

[0011] In some embodiments of the present invention, the particle size of the crystals may be 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm;

[0012] Preferably, the crystal is difloxacin or a difloxacin salt or a mixture of the two.

[0013] Preferably, the difloxacin salt is difloxacin hydrochloride or difloxacin organic acid salt.

[0014] Preferably, 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.

[0015] Preferably, the weight ratio of difloxacin to difloxacin salt is 10-90:90-10. More preferably, the weight ratio of difloxacin to difloxacin salt is 20-60:40-80.

[0016] Any of the above-mentioned crystals is obtained by the following preparation method, comprising the following steps:

[0017] Step 1: preparing a solution in which difloxacin salt is dissolved;

[0018] Step 2: Rapidly cool the solution containing difloxacin salt to 2-10° C. to precipitate the difloxacin crystals.

[0019] The flaky difloxacin crystals can be prepared by rapid cooling and crystallization.

[0020] In the above-mentioned method for preparing crystals, step 2 further includes step 3: filtering the solution of step 2, adding alkali to the filtrate, adjusting the pH value of the filtrate to 2-7, so that the 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 the crystals continue to precipitate.

[0021] 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;

[0022] 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;

[0023] In step 3, the temperature of step 2 is maintained. If the temperature exceeds 10°C, the crystals will be redissolved.

[0024] In industrial production, the preferred operation is to filter first, then combine the filtrates and crystallize.

[0025] 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.

[0026] In the above-mentioned method for preparing crystals, the concentration of difloxacin salt in the solution of step 1 is 10 to 100 g / L;

[0027] In step 1, the temperature of the solution is raised to 80-90° C. to promote the dissolution of difloxacin salt. Preferably, a surfactant is also added in step 1 to improve the stability of the solution.

[0028] In the above-mentioned method for preparing crystals, the cooling rate in 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.

[0029] 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 pipeline reactors.

[0030] In the above-mentioned method for preparing crystals, in step 2, seed crystals are added to the solution containing difloxacin salt in advance, or seed crystals are made to appear in the solution containing difloxacin salt by adding a base; the seed crystals are difloxacin salt; while or after the seed crystals are present in the solution containing difloxacin salt, the temperature is lowered to promote the precipitation of crystals.

[0031] In the above-mentioned method for preparing crystals, in step 2, the crystal precipitation time is 5 to 15 minutes; in step 3, the crystal precipitation time is 5 to 15 minutes;

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

[0033] (1) The difloxacin crystals provided by the present invention have a particle size of 200 nm to 10 μm and a flaky structure. The crystals can stably exist in the form of a suspension in water, have a long action time, low irritation, and can be prepared into preparations of various concentrations.

[0034] In order to prepare difloxacin crystals with the above characteristics, the solution containing difloxacin salt is rapidly cooled to make difloxacin and its salt in a saturated or supersaturated state, and flake crystals are formed and precipitated. The particle size is small and the flake structure does not clog the needle, and it can be used to prepare difloxacin suspension injections of different concentrations; it can also be prepared into dry suspension injections, and water for injection is added and mixed before use.

[0035] (2) Continue to neutralize the solution at low temperature to allow the remaining difloxacin and its salt in the solution to crystallize synchronously, and the proportion of difloxacin monomer in the crystal increases. Compared with difloxacin salt crystals, difloxacin monomer crystals have a slower release rate in vivo; 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 purpose of instantaneous high release in the initial stage and long-term sustained release in the system;

[0036] The flaky crystals of the present invention can be used to prepare an integrated suspension without the need for an alkaline system, thereby solving the problem of high irritation of difloxacin alkaline solution injection in the prior art.

[0037] Since the difloxacin crystals of the present invention can be partially dissolved and maintain a relatively stable suspension state in the drinking water system after being added to the animal drinking water system, they can have both rapid absorption and long-term effects after entering the animal intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a microscope image of Example A1;

[0039] Figure 2 is a scanning electron microscope image of the plate-like crystals in step 2 of Example A1;

[0040] Figure 3 is a scanning electron microscope image of the plate-like crystals in step 3 of Example A1;

[0041] Figure 4 is a microscope image of Example A2;

[0042] Figure 5 is a scanning electron microscope image of Example A2;

[0043] Figure 6 is a microscope image of Example A3;

[0044] Figure 7 is a scanning electron microscope image of Example A3;

[0045] Figure 8 is a scanning electron microscope image of the difloxacin lactate ultrafine crystals obtained in Example A4;

[0046] Fig. 9 is a scanning electron microscope image of the difloxacin ultrafine crystals obtained in Example A4;

[0047] Fig.10 is a microscope image of Example A5;

[0048] Fig.11 is a scanning electron microscope image of Example A5;

[0049] Fig.12 This is a microscope image of Comparative Example A1. DETAILED DESCRIPTION

[0050] 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.

[0051] Example 1

[0052] 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℃.

[0053] 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;

[0054] 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.

[0055] Example 2

[0056] 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℃.

[0057] 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.

[0058] 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.

[0059] Example 3

[0060] 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℃.

[0061] 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.

[0062] 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.

[0063] Example 4

[0064] 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℃.

[0065] 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.

[0066] 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.

[0067] The wet crystals were dried by freeze drying to obtain difloxacin lactate ( Figure 8 ) and difloxacin ultrafine crystals ( Fig. 9 ).

[0068] Example 5

[0069] 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.

[0070] Comparative Example 1

[0071] 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 .

[0072] Performance Test 1

[0073] 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.

[0074] 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:

[0075] Table 1 Crystal morphology and proportion test results

[0076]

[0077] Result analysis: The difloxacin crystal product prepared in Example 1 has a relatively slow cooling rate and the alkali solution is added earlier, so the crystal shape is slightly larger than that of the difloxacin crystal product. The crystals are mainly difloxacin monomers, and there are a small amount of needle-shaped crystals.

[0078] In Example 2, the temperature is rapidly lowered and then stirred at high speed. A large amount of crystals appear first and then an alkali solution is used to adjust the solution to near neutrality. 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.

[0079] Example 3 After adding liquid nitrogen to assist in rapid cooling, the crystal crystallization temperature is lower, the crystal shape is relatively small, and it is relatively uniform, in the form of a lamellar polyhedron. The difloxacin lactate crystals are first separated, and then the difloxacin salt in the solution is neutralized with an alkali to form difloxacin monomer crystals. After being dried separately, difloxacin lactate crystals and difloxacin monomer crystals are obtained respectively.

[0080] Performance Test 2

[0081] Needle blocking test of difloxacin crystals:

[0082] 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.

[0083] Table 2 Needle blocking test of difloxacin crystals

[0084]

[0085] Note: If the needle is blocked once during the withdrawal and injection process, it will be considered as needle blockage.

[0086] Performance Test 3

[0087] Pharmacokinetics of difloxacin microcrystals in yellow chickens

[0088] Animal experiment: The experimental animals were yellow chickens.

[0089] 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).

[0090] 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.

[0091] The test results of Example 2 and Example 3 are as follows:

[0092] Table 3 Blood drug concentration of difloxacin crystals prepared by different processes in yellow chickens

[0093]

[0094]

[0095] 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.

[0096] 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 crystal of difloxacin and its salt-based compound, characterized in that: The crystal has a particle size of 200 nm to 10 μm and is in a flaky structure.

2. The crystal of difloxacin and its salt-based compound according to claim 1, characterized in that: The crystal is difloxacin or difloxacin salt or a mixture of the two.

3. The crystal of difloxacin and its salt-based compound according to claim 2, characterized in that: The difloxacin salt is difloxacin hydrochloride or difloxacin organic acid salt.

4. The crystal of difloxacin and its salt-based compound according to claim 3, 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.

5. The crystal of difloxacin and its salt-based compound according to claim 2, characterized in that: The weight ratio of difloxacin to difloxacin salt is 10-90:90-10.

6. The method for preparing a crystal according to any one of claims 1 to 5, characterized in that: The steps include: Step 1: preparing a solution in which difloxacin salt is dissolved; Step 2: Cooling the solution containing difloxacin salt to 2-10° C. at a rate of 5-50° C. / min to precipitate the difloxacin crystals.

7. The method for preparing a crystal according to claim 6, characterized in that: The method further comprises step 3 after step 2: filtering the solution of step 2, adding alkali to the filtrate, adjusting the pH value of the filtrate to 2-7, so that crystals are continuously precipitated; or directly adding alkali to the solution of step 2, adjusting the pH value of the solution to 2-7, so that crystals are continuously precipitated.

8. The method for preparing a crystal according to claim 6, characterized in that: The concentration of difloxacin salt in the solution of step 1 is 10-100 g / L; In step 1, the solution temperature is raised to 80-90° C. to promote the dissolution of the difloxacin salt.

9. The method for preparing a crystal according to claim 8, characterized in that: 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 the step 2, during the cooling process, alkali is continuously added to raise the pH of the system to 2-7, thereby increasing the proportion of difloxacin monomer.

10. The method for preparing a crystal according to claim 7, characterized in that: In step 2, the crystal precipitation time is 5 to 15 minutes; in step 3, the crystal precipitation time is 5 to 15 minutes; A surfactant is also added in step 1.

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