Preparation method of goserelin microspheres and product thereof

Abstract

The invention relates to a preparation method of goserelin microspheres and a product thereof, and the preparation method comprises the following steps: mixing a goserelin raw material solution and a degradable polymer solution to obtain an oil phase; mixing a surfactant with water to obtain a water phase; mixing the oil phase with the water phase to form an emulsion, stirring and curing to obtain goserelin microspheres; wherein solvents of the goserelin raw material solution and the degradable polymer solution are respectively and independently cosolvents, and the cosolvents are obtained by mixing a first solvent and a second solvent. Compared with a traditional multiple emulsion method, the preparation method is simpler to operate, meanwhile, the prepared goserelin microspheres have higher drug loading rate and smaller particle size, injection administration of a No.7 needle can be achieved, the surfaces of the microspheres are free of holes, burst release is low, the drugs can be evenly distributed in the microspheres, and acetic acid residues are basically avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparation production, and relates to a preparation method of serrelin microspheres and a product thereof. Background Art

[0002] Goserelin is a gonadotropin-releasing hormone analogue, which is mainly used clinically for the treatment of breast cancer, prostate cancer and endometriosis. Goserelin is a polypeptide molecule, which is easily degraded by gastrointestinal peptidases when taken orally, and has an extremely low bioavailability of only 0.1%. Although the bioavailability of nasal and vaginal administration has increased to about 20%, its nasal and vaginal mucosa is easily affected by physiological conditions (such as ovarian cycle stage or upper respiratory tract infection), which is not convenient for long-term use and requires two routes of administration, subcutaneous or intramuscular injection. And because its indications are hormone-dependent chronic diseases, the treatment cycle is long, and patient compliance and compliance are poor, it is necessary to develop long-acting injection preparations.

[0003] The long-acting injection of goserelin currently on the market is mainly in two dosage forms: implant and microsphere. Among them, the main preparation process of the implant (US5366734) is: dissolve goserelin and PLGA in glacial acetic acid, remove the glacial acetic acid after freeze drying, and then extrude it into a rod by hot melt, and finally sterilize it by radiation to make an implant. This method requires a high freeze-drying vacuum and time to remove acetic acid, and the acetic acid residue is high. Celex Pharmaceuticals (CN102755627) announced a way to reduce acetic acid residues, that is, based on the original Zoladex implant preparation, through solvent screening, ethanol-glacial acetic acid is selected instead of pure glacial acetic acid to dissolve drugs and polymers, and the obtained goserelin implant has the advantages of low solvent residue and uniform drug dispersion. Shengzhao Pharmaceutical (CN115778909A) has announced a method of using a turntable device to pre-treat goserelin acetate, a sustained-release polymer, and a glacial acetic acid-dichloromethane mixed solution to form microparticles, and then feed the microparticles into a hot-melt extruder for implant preparation. This method does not require freeze-drying, has low energy consumption, and can significantly reduce related substances. Although the goserelin implant has good sustained-release performance, its injection needle is equivalent to a No. 16 puncture needle, with a length of about 30 mm, causing high pain during injection and causing more subcutaneous bleeding after injection. In addition, since the drug is pre-filled in a disposable syringe, a special production syringe is required, and the production and use costs are high.

[0004] In comparison, microsphere preparations can use small diameter needles, greatly reduce the pain and bleeding of patients, and do not require specially produced syringes. The microsphere preparation process also has the advantages of not requiring high temperature, aseptic operation without radiation sterilization, and thus less impurities. For example, Luye Pharmaceuticals has announced a goserelin acetate microsphere composition and preparation method (CN104107434A), which uses water-in-oil-in-water particles (S / O / W) to prepare microspheres. The goserelin acetate and poloxamer mixture needs to be pre-treated into a micropowder with a particle size of less than 2 microns before it can be mixed with an organic solvent and a sustained-release polymer for emulsification and solidification into spheres. Due to the limitation of the particle size of the raw material particles, this method has high requirements for the emulsification equipment. For equipment such as membrane emulsification with a pore size of less than 2 microns, there is a risk of membrane pore clogging and low processing capacity. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing goserelin microspheres and a product thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing goserelin microspheres, the preparation method comprising the following steps:

[0008] (1) mixing a goserelin raw material solution and a degradable polymer solution to obtain an oil phase; mixing a surfactant and water to obtain an aqueous phase;

[0009] (2) mixing the oil phase with the water phase to form an emulsion, stirring and solidifying to obtain goserelin microspheres;

[0010] Wherein, the solvents of the goserelin raw material solution and the degradable polymer solution in step (1) are both co-solvents obtained by mixing the first solvent and the second solvent.

[0011] The preparation method of goserelin microspheres involved in the present invention creatively adopts a co-solvent to prepare goserelin microspheres by a single emulsion method. Compared with the traditional double emulsion method, the preparation method is simpler, does not require special equipment, does not need to reduce the particle size of the goserelin API, and does not need to homogenize and emulsify to reduce the particle size of the API colostrum, and can directly achieve full mixing of the polymer and the API, and also solves the problems of colostrum rupture and particle clogging of membrane pores in the double emulsion method. At the same time, the goserelin microspheres prepared by the present invention have a higher drug loading rate and a smaller particle size, can achieve injection administration with a No. 7 needle, the microsphere surface is non-porous, the burst release is low, the drug can be evenly distributed in the microsphere, and there is basically no acetic acid residue.

[0012] The present invention also creatively adopts a specific oil phase preparation method, firstly mixing the first solvent and the second solvent to prepare a co-solvent system, and then independently dissolving the goserelin raw material and the degradable polymer in the co-solvent system to obtain the goserelin raw material solution and the degradable polymer solution, and then mixing them. Compared with other oil phase preparation methods, this method can further increase the solubility of the goserelin raw material and the degradable polymer, thereby improving the drug loading and encapsulation rate of the goserelin microspheres.

[0013] Preferably, the first solvent is selected from anhydrous acetic acid.

[0014] The second solvent is independently selected from any one of dichloromethane, chloroform, ethyl acetate or N-methylpyrrolidone, or a combination of at least two thereof.

[0015] Preferably, the volume ratio of the first solvent to the second solvent is independently (0.01-0.7):1, for example 0.01:1, 0.02:1, 0.03:1, 0.05:1, 0.07:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0016] Preferably, the volume ratio of the first solvent to the second solvent is independently (0.2-0.5):1, for example 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.25:1, 0.27:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0017] The present invention creatively finds that using a first solvent and a second solvent in a specific ratio as a co-solvent system can further increase the drug loading and encapsulation efficiency of goserelin microspheres and reduce the size of the microspheres.

[0018] Preferably, the goserelin raw material includes goserelin or a pharmaceutically acceptable salt thereof.

[0019] Preferably, the pharmaceutically acceptable salt of goserelin includes goserelin acetate or goserelin pamoate.

[0020] Preferably, the concentration of goserelin raw material in the goserelin raw material solution in step (1) is 10-50wt%, for example, 10wt%, 12wt%, 14wt%, 16wt%, 18wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0021] Preferably, the concentration of the degradable polymer in the degradable polymer solution is 10-40wt%, for example 10wt%, 12wt%, 14wt%, 16wt%, 18wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0022] Preferably, the mass ratio of the goserelin raw material to the degradable polymer is 1:(5-15), for example 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0023] Preferably, the degradable polymer in step (1) comprises lactide-glycolide copolymer and / or polylactide.

[0024] Preferably, the weight average molecular weight of the degradable polymer is 5000-50000Da, for example 5000Da, 6000Da, 7000Da, 8000Da, 9000Da, 10000Da, 15000Da, 20000Da, 30000Da, 40000Da, 50000Da, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0025] Preferably, the weight average molecular weight of the degradable polymer is 8000-30000Da, for example 8000Da, 9000Da, 10000Da, 11000Da, 13000Da, 15000Da, 18000Da, 20000Da, 22000Da, 25000Da, 30000Da, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0026] Preferably, the first solvent in step (1) further comprises a release regulator.

[0027] Preferably, the release modifier comprises any one of arginine, stearic acid or xinafoic acid, or a combination of at least two thereof.

[0028] Further adding a release modifier to the first solvent can effectively increase the drug loading and encapsulation efficiency of goserelin microspheres, reduce the size of microspheres, and reduce the burst release of drugs, thereby improving the sustained release and controlled release effects of goserelin microspheres. Arginine, stearic acid or xinafoic acid can all play the above effects. Compared with stearic acid or xinafoic acid, arginine is more effective in increasing drug loading and encapsulation efficiency, reducing the size of microspheres, and reducing the burst release of drugs.

[0029] Preferably, the mass ratio of the release modifier to the goserelin raw material is (0.006-0.6):1, for example 0.006:1, 0.008:1, 0.01:1, 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0030] Preferably, the mass ratio of the release modifier to the goserelin raw material is (0.01-0.5):1, for example 0.01:1, 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0031] The present invention also creatively finds that the use of a specific added amount of a release regulator can further increase the drug loading and encapsulation rate of the microspheres and reduce the size of the microspheres.

[0032] Preferably, the surfactant in step (1) comprises any one of polyvinyl alcohol, carboxymethyl cellulose, gelatin, poloxamer or Tween, or a combination of at least two thereof.

[0033] Preferably, the amount of the surfactant is 0.01-10wt% of the aqueous phase, for example 0.01wt%, 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%, 0.1wt%, 0.5wt%, 1wt%, 2wt%, 4wt%, 6wt%, 8wt%, 10wt%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0034] Preferably, the aqueous phase in step (1) also includes a osmotic agent.

[0035] Preferably, the penetrant includes any one of phosphate, carbonate, chloride, sulfate or citrate, or a combination of at least two of them.

[0036] Further addition of osmotic agent to the aqueous phase can effectively increase the drug loading and encapsulation rate of goserelin microspheres and reduce the size of microspheres. It also affects the surface and internal morphology of microspheres. The osmotic pressure of the aqueous phase increases, making it difficult for the drug in the oil phase to diffuse into the aqueous phase. The surface and interior of the microspheres are smooth and dense, with few cavities, thereby reducing the release of drugs from the pores and further improving the sustained and controlled release effects of goserelin microspheres. Phosphates, carbonates, chlorides, sulfates or citrates can all play the above effects, among which chlorides are more effective in increasing drug loading and encapsulation rate, reducing the size of microspheres, and reducing drug burst release.

[0037] Preferably, the amount of the penetrant is 0.1-3wt% of the aqueous phase, for example 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt%, 1wt%, 2wt%, 3wt%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0038] The present invention also creatively discovered that by using a specific amount of added osmotic agent, the drug loading and encapsulation rate of the microspheres, the size of the microspheres, and the sustained release effect of the drug can be further improved.

[0039] Preferably, the aqueous phase in step (1) also includes a pH regulator.

[0040] Preferably, the pH adjuster includes any one of sodium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate or disodium hydrogen phosphate, or a combination of at least two thereof.

[0041] Preferably, the pH adjuster is a combination of sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0042] Preferably, the pH value of the pH adjuster added to the aqueous phase is 7-9, such as 7, 7.5, 8, 8.5, 9, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0043] The present invention creatively discovered that when a specific pH adjuster is selected and the pH value of the aqueous phase is adjusted to 7-9, the acetic acid residue in the goserelin microspheres can be effectively reduced without affecting the stability of the goserelin polypeptide.

[0044] Preferably, the mixing in step (2) includes homogenous emulsification, shear emulsification, membrane emulsification or microchannel emulsification.

[0045] Preferably, the volume ratio of the oil phase to the water phase is 1:(50-300), for example 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0046] Preferably, the volume ratio of the oil phase to the water phase is 1:(70-200), for example 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:120, 1:140, 1:160, 1:180, 1:200, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0047] In a second aspect, the present invention provides goserelin microspheres prepared according to the method for preparing goserelin microspheres according to the first aspect.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The preparation method of goserelin microspheres involved in the present invention creatively adopts a co-solvent to prepare goserelin microspheres by a single emulsion method. On the one hand, compared with the traditional double emulsion method, the preparation method is simpler, does not require special equipment, does not need to reduce the particle size of the goserelin API, and does not need homogenization and emulsification to reduce the particle size of the API colostrum, and can directly achieve full mixing of the polymer and the API, and also solves the problems of colostrum rupture and particle clogging of membrane pores in the double emulsion method. On the other hand, the goserelin microspheres prepared by the present invention have a higher drug loading rate and a smaller particle size, can achieve injection administration with a No. 7 needle, the microsphere surface is non-porous, the burst release is low, the drug can be evenly distributed in the microsphere, and there is basically no acetic acid residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is an electron microscope photograph of goserelin microspheres prepared in Example 1;

[0051] Figure 2 This is an in vitro release curve of goserelin microspheres prepared in Example 1 in accelerated stability testing. DETAILED DESCRIPTION

[0052] 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.

[0053] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0054] The polyvinyl alcohol involved in the following examples was purchased from Merck Chemicals with a product model of MXP 3-82.

[0055] Example 1

[0056] This embodiment provides a method for preparing goserelin microspheres:

[0057] (1) adding arginine to glacial acetic acid, and then mixing with dichloromethane in a volume ratio of 0.4:1 to prepare a co-solvent system, wherein the content of arginine in the co-solvent system is 0.85 wt %;

[0058] (2) Weighing 0.4 g of goserelin acetate and dissolving it in 0.5 g of the co-solvent system to obtain an API solution; weighing 2.8 g of PLGA (weight average molecular weight of 24,000 Da) and dissolving it in 11.25 g of the co-solvent system to obtain a PLGA solution; adding the above PLGA solution to the above API solution to obtain a PLGA-API solution;

[0059] (3) Using a 50 mL syringe to draw PLGA-API solution as the oil phase, place it on the syringe pump and set the speed to 80 mL / min. Separately, take a 0.25 wt% polyvinyl alcohol solution containing 1.5 wt% NaCl, add sodium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 1:1, adjust the pH to 8, and use it as the external aqueous phase. Set the aqueous phase flow rate to 9.6 L / min, and the volume ratio of the oil phase to the aqueous phase to be 1:120. Adjust the speed of the online homogenizer to 4000 rpm, turn on the syringe pump to inject the PLGA-API solution to obtain a microsphere suspension, and then stir the microsphere suspension at a speed of 200 rpm for solidification. The solidified microspheres are repeatedly washed with pure water for multiple times to remove residual polyvinyl alcohol, and then freeze-dried to obtain goserelin microspheres.

[0060] Example 2

[0061] This embodiment provides a method for preparing goserelin microspheres:

[0062] (1) adding arginine to glacial acetic acid, and then mixing with dichloromethane in a volume ratio of 0.5:1 to prepare a co-solvent system, wherein the content of arginine in the co-solvent system is 1.5 wt %;

[0063] (2) Weighing 0.4 g of goserelin and dissolving it in 0.9 g of the co-solvent system to obtain an API solution; weighing 4 g of PLGA (weight average molecular weight of 8000 Da) and dissolving it in 11.25 g of the co-solvent system to obtain a PLGA solution; adding the above PLGA solution to the above API solution to obtain a PLGA-API solution;

[0064] (3) Using a 50 mL syringe to draw PLGA-API solution as the oil phase, place it on the syringe pump and set the speed to 80 mL / min. Separately, take a 0.5 wt % polyvinyl alcohol solution containing 0.75 wt % NaCl, add sodium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 2:1, adjust the pH to 7, and use it as the external aqueous phase. Set the aqueous phase flow rate to 5.6 L / min, and the volume ratio of the oil phase to the aqueous phase to be 1:70. Adjust the speed of the online homogenizer to 4000 rpm, turn on the syringe pump to inject the PLGA-API solution to obtain a microsphere suspension, and then stir the microsphere suspension at a speed of 200 rpm for solidification. The solidified microspheres are repeatedly washed with pure water for multiple times to remove residual polyvinyl alcohol, and then freeze-dried to obtain goserelin microspheres.

[0065] Example 3

[0066] This embodiment provides a method for preparing goserelin microspheres:

[0067] (1) adding arginine to glacial acetic acid, and then mixing with dichloromethane in a volume ratio of 0.3:1 to prepare a co-solvent system, wherein the content of arginine in the co-solvent system is 0.56 wt %;

[0068] (2) Weighing 0.4 g of goserelin acetate and dissolving it in 1.6 g of the co-solvent system to obtain an API solution; weighing 2.4 g of PLGA (weight average molecular weight of 30,000 Da) and dissolving it in 5.6 g of the co-solvent system to obtain a PLGA solution; adding the above PLGA solution to the above API solution to obtain a PLGA-API solution;

[0069] (3) Using a 50 mL syringe to draw PLGA-API solution as the oil phase, place it on the syringe pump and set the speed to 80 mL / min. Separately, take a 0.1 wt% polyvinyl alcohol solution containing 2.25 wt% NaCl, add sodium dihydrogen phosphate and sodium dihydrogen phosphate in a mass ratio of 1:2, adjust the pH to 9, use it as the external aqueous phase, set the aqueous phase flow rate to 12 L / min, and the volume ratio of the oil phase to the aqueous phase is 1:150. Adjust the speed of the online homogenizer to 4000 rpm, turn on the syringe pump to inject the PLGA-API solution to obtain a microsphere suspension, and then stir the microsphere suspension at a speed of 200 rpm for solidification. The solidified microspheres are repeatedly washed with pure water for multiple times to remove residual polyvinyl alcohol, and then freeze-dried to obtain goserelin microspheres.

[0070] Example 4

[0071] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that in step (1), arginine is added to glacial acetic acid, and then mixed with dichloromethane in a volume ratio of 1:1 to prepare a co-solvent system, and other conditions remain unchanged.

[0072] Example 5

[0073] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that in step (1), arginine is added to glacial acetic acid, and then mixed with dichloromethane at a volume ratio of 0.01:1 to prepare a co-solvent system, and other conditions remain unchanged.

[0074] Example 6

[0075] This embodiment provides a method for preparing goserelin microspheres, which is different from Example 1 only in that the content of arginine in the co-solvent system in step (1) is 2.8 wt %, and other conditions remain unchanged.

[0076] Example 7

[0077] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that arginine is not added in step (1), and other conditions remain unchanged.

[0078] Example 8

[0079] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that in step (1), arginine is replaced by stearic acid, and other conditions remain unchanged.

[0080] Example 9

[0081] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that in step (1), arginine is replaced by xinafoic acid, and other conditions remain unchanged.

[0082] Example 10

[0083] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that the external aqueous phase in step (3) is a 0.25 wt % polyvinyl alcohol solution without NaCl, and other conditions remain unchanged.

[0084] Embodiment 11

[0085] This example provides a method for preparing goserelin microspheres, which differs from Example 1 only in that in step (3), sodium dihydrogen phosphate and sodium hydrogen phosphate are not added to adjust the pH. At this time, the pH of the external aqueous phase is measured to be 6.5, and other conditions remain unchanged.

[0086] Example 12

[0087] This embodiment provides a method for preparing goserelin microspheres, which differs from Embodiment 1 only in that sodium dihydrogen phosphate and disodium hydrogen phosphate in step (3) are replaced with sodium hydroxide, and the pH of the external aqueous phase is adjusted to 8, while other conditions remain unchanged.

[0088] Comparative Example 1

[0089] This comparative example provides a method for preparing goserelin microspheres, which differs from Example 1 only in that step (1) is not performed, and step (2) is replaced by: weighing 0.4 g of goserelin acetate and dissolving it in 0.5 g of glacial acetic acid (containing 0.1 g of arginine) to obtain an API solution; weighing 2.8 g of PLGA (weight average molecular weight of 24000 Da) and dissolving it in 11.25 g of dichloromethane to obtain a PLGA solution; adding the above PLGA solution to the above API solution to obtain a PLGA-API solution. Other conditions remain unchanged.

[0090] Comparative Example 2

[0091] This comparative example provides a method for preparing goserelin microspheres, which differs from Example 1 only in that step (2) is replaced by: weighing 0.4 g of goserelin acetate and 2.8 g of PLGA (weight average molecular weight of 24000 Da) and dissolving them in 11.75 g of a co-solvent system to obtain a PLGA-API solution. Other conditions remain unchanged.

[0092] Comparative Example 3

[0093] This comparative example provides a method for preparing goserelin microspheres:

[0094] Weigh 0.4g of goserelin acetate and dissolve it in 0.5g of pure water to obtain an inner water phase, and then weigh 2.8g of PLGA (Mw≈24k) and dissolve it in 11.25g of dichloromethane to obtain a middle oil phase. Add the above oil phase to the above inner water phase to form an O / W emulsion, and homogenize it with a high-speed homogenizer at 6000rpm for 3-5min to obtain colostrum. Use a 50mL syringe to absorb the colostrum and place it on a syringe pump, set the speed to 80mL / min, take another 0.25wt% polyvinyl alcohol solution of 1.5wt% NaCl, add sodium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 1:1, adjust the pH to 8, as the outer water phase, set the water phase flow rate to 9.6L / min, adjust the speed of the online homogenizer to 4000rpm, turn on the syringe pump to inject colostrum, obtain a microsphere suspension, and then stir the microsphere suspension at 200rpm for solidification. The solidified microspheres were repeatedly washed with pure water to remove residual polyvinyl alcohol, and then freeze-dried to obtain goserelin microspheres.

[0095] Test Example 1

[0096] This test example measured the drug loading of goserelin microspheres prepared in Examples 1-12 and Comparative Examples 1-3:

[0097] Accurately weigh 5 mg of goserelin acetate or goserelin, add 0.1% acetic acid solution to dissolve and quantitatively dilute to a solution containing 0.1 mg of goserelin per 1 mL as a reference solution; accurately weigh 5 mg of the microspheres prepared in Examples 1-12 and Comparative Examples 1-3, place in a 50 mL volumetric flask, add 15 mL of dichloromethane, ultrasonicate for 15 min, add 20 mL of 0.1% acetic acid solution, ultrasonicate for 15 min, dilute to the scale with 0.1% acetic acid solution, shake well, take an appropriate amount, centrifuge at 10000 rpm / min for 15 min, and take the supernatant for high performance liquid chromatography detection.

[0098] Chromatographic conditions: octadecyl bonded silica gel was used as the filler (4.6 mm × 250 mm, 5 μm); triethylamine solution (15.2 g of triethylamine was taken, 800 mL of water was added to dissolve it, the pH was adjusted to 3.0 with phosphoric acid, and then diluted to 1000 mL with water and shaken well)-[acetonitrile-n-propanol (3:2)] (17:3) was used as the mobile phase; the detection wavelength was 220 nm; the column temperature was 35 °C; the flow rate was 1.0 mL / min; the injection volume was 20 μL.

[0099] The retention time and peak area of ​​the chromatogram were recorded, and the amount of goserelin in the microspheres was calculated by the external standard method. The drug loading and encapsulation efficiency were calculated according to the following formula. The specific test results are shown in Table 1.

[0100] Microsphere drug loading (%) = (amount of goserelin in microspheres / total weight of microspheres) × 100%

[0101] Microsphere encapsulation efficiency (%) = (actual drug loading / theoretical drug loading) × 100%

[0102] Table 1

[0103]

[0104]

[0105] It can be seen from the data in Table 1 that the preparation method of goserelin microspheres involved in the present invention can effectively improve the drug loading and encapsulation efficiency of goserelin microspheres.

[0106] Comparing the results of Example 1 with those of Example 4-5, it can be seen that when the ratio of the first solvent to the second solvent in the co-solvent is within a reasonable range, the drug loading and encapsulation efficiency of the goserelin microspheres can be significantly improved; comparing the results of Example 1 with those of Example 6, it can be seen that the amount of release modifier added will also have a significant effect on the drug loading and encapsulation efficiency; comparing the results of Example 1 with those of Example 7-9, it can be seen that adding a release modifier to the first solvent can enable the prepared goserelin microspheres to obtain a higher drug loading and encapsulation efficiency, and when the release modifier selects arginine, the drug loading and encapsulation efficiency can be further improved; comparing the results of Example 1 with those of Example 10, it can be seen that adding an osmotic agent to the aqueous phase can enable the prepared goserelin microspheres to obtain a higher drug loading and encapsulation efficiency. Comparing the results of Example 1 with those of Example 11, it can be seen that adding a pH modifier to the aqueous phase is beneficial to improving the drug loading and encapsulation efficiency of the microspheres. Comparing the results of Example 1 with those of Example 12, it can be seen that using sodium dihydrogen phosphate aqueous solution and disodium hydrogen phosphate as pH modifiers can further improve the drug loading and encapsulation efficiency of the microspheres.

[0107] By comparing the results of Example 1 with Comparative Examples 1-2, it can be seen that the oil phase preparation method involved in the present invention can further improve the drug loading and encapsulation efficiency of goserelin microspheres compared with other methods. In addition, the present invention has found in experiments that the method of dissolving the goserelin raw material in the first solvent and the polymer in the second solvent in Comparative Example 1 has a significantly poor dissolution effect and may even cause precipitation.

[0108] By comparing the results of Example 1 with those of Comparative Example 3, it can be seen that the goserelin microspheres prepared by the co-solvent method can obtain higher drug loading and encapsulation efficiency than those prepared by the W / O / W emulsion method.

[0109] Test Example 2

[0110] This test example measured the acetic acid content of the goserelin microspheres prepared in Examples 1-3 and 11-12:

[0111] Add 0.7 mL of phosphoric acid to 1000 mL of water, adjust the pH value to 3.0 with 0.42% sodium hydroxide solution, and use it as a diluent (mobile phase A). Accurately weigh an appropriate amount of acetic acid, dilute it with the diluent to make 1 mL of a solution containing 100 μg of acetic acid, and use it as a reference solution; accurately weigh 0.1 g of goserelin microspheres prepared in Examples 1-14 and Comparative Examples 1-3, place them in a 50 mL stoppered centrifuge tube, add 10 mL of dichloromethane and shake to disintegrate, add 10 mL of diluent, shake for 1 min, let stand to separate, take the supernatant, and use it as the test solution for high performance liquid chromatography detection. The specific acetic acid content is shown in Table 2.

[0112] Chromatographic conditions: Sepax-GP C18 column (4.6 mm × 250 mm, 5 μm) was used; phosphoric acid solution was used as mobile phase A, methanol was used as mobile phase B, and the detection wavelength was 210 nm; column temperature was 35°C; flow rate was 1.2 mL / min; injection volume was 20 μL.

[0113] Table 2

[0114] Group Acetic acid content (%) Example 1 0.0035 Example 2 0.0058 Example 3 0.0043 Embodiment 11 0.38 Example 12 0.0069

[0115] It can be seen from the data in Table 2 that the goserelin microspheres prepared by the preparation method of the goserelin microspheres involved in the present invention have low acetic acid residue. Comparing Example 1 with Example 11, it can be seen that the present invention effectively reduces the acetic acid residue of the goserelin microspheres by adding an alkaline buffer solution to the aqueous phase.

[0116] Test Example 3

[0117] This test example analyzes the particle size of goserelin microspheres prepared in Examples 1-12 and Comparative Examples 1-3:

[0118] Weigh an appropriate amount of goserelin acetate microspheres, mix the microspheres with the attached injection solvent, place them in 1000 mL of water, stir at 2000 rpm / min, and ultrasonicate for 2 minutes at the same time, and measure the particle size and particle size distribution according to the particle size and particle size distribution determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0982, Method 3), using a laser particle size analyzer Mastersizer 3000 for determination (the detector shading rate meets the requirements), and the specific test results are shown in Table 3.

[0119] Table 3

[0120]

[0121]

[0122] From the data in Table 3, it can be seen that the microspheres prepared by the preparation method involved in this application have a volume average particle size of less than 32.4 μm, which can meet the requirements of injection preparations. Factors such as the ratio of the first solvent to the second solvent, the selection of the release modifier and its addition amount, the addition of the permeabilizer, and the preparation method of the oil phase will affect the particle size of the prepared goserelin microspheres.

[0123] Test Example 4

[0124] This test example conducted an in vitro dissolution test on the goserelin microspheres prepared in Examples 1-12 and Comparative Examples 1-3:

[0125] 50 mg of goserelin microspheres prepared in Examples 1-12 and Comparative Examples 1-3 (3 replicates) were accurately weighed and placed in a 50 mL glass container. 50 mL of release medium (PBS solution with a pH of 7.4, containing 0.5% Tween 80) preheated to 37°C ± 0.5°C was accurately added. The container was sealed with a stopper and an aluminum cap. The suspension was immediately shaken. Each container was fixed in an inverted state and immersed in a water bath at 37°C ± 0.5°C. The water bath temperature was adjusted to 0.5°C. / h, the temperature was raised from 37°C to 61°C, the horizontal oscillation amplitude was about 4 cm, and the oscillation speed was 125 times / min. After 1h, 24h, 30h, 48h, and 72h of oscillation, the mixture was taken out from the shaker, 1mL of the suspension was accurately extracted with a syringe, and 1mL of the release medium (preheated to the corresponding temperature) was accurately added, the suspension was filtered (polyethersulfone was tentatively used as the filter membrane material, and the filter membrane pore size was 0.45μm), and the filtrate was taken as the test solution.

[0126] Determination method: HPLC detection was performed with the release medium as blank solution and a series of concentrations of goserelin acetate solution as reference solution. The mobile phase was 2.72 g of potassium dihydrogen phosphate dissolved in 450 mL of water, then 550 mL of methanol was added and mixed, and the pH was adjusted to 3.0 with phosphoric acid. The cumulative release was calculated, and the specific release results are shown in Table 4.

[0127] Table 4

[0128]

[0129] It can be seen from the data in Table 4 that the microspheres prepared by the preparation method of the present invention have low 1h cumulative release, significant effect of suppressing burst release, and slow overall release, close to the zero-order release curve, which can achieve a stable and slow release effect.

[0130] Comparing the results of Example 1 with those of Examples 4-5, it can be seen that when the ratio of the first solvent to the second solvent in the co-solvent is within a reasonable range, the sustained release effect of the microspheres can be effectively improved; comparing the results of Example 1 with those of Example 7, it can be seen that the addition of a release regulator can significantly improve the sustained release effect of the microspheres; comparing the results of Example 1 with those of Examples 8-9, it can be seen that when arginine is selected as the release regulator, the drug loading and encapsulation efficiency can be further improved; comparing the results of Example 1 with those of Example 10, it can be seen that the addition of an osmotic agent can also improve the sustained release effect of the microspheres, and the amount of osmotic agent added can further affect the sustained release effect of the microspheres. Comparing the results of Example 1 with those of Example 11, it can be seen that adding an alkaline buffer solution to the aqueous phase can effectively inhibit the burst release of the microspheres while reducing the acetic acid content.

[0131] By comparing the results of Example 1 with those of Comparative Example 3, it can be seen that the goserelin microspheres prepared by the co-solvent method have a better sustained-release effect than those prepared by the W / O / W emulsion method.

[0132] Test Example 5

[0133] In this test example, the goserelin microspheres prepared in Example 1 were scanned by electron microscope to observe the surface morphology of the microspheres:

[0134] An appropriate amount of the goserelin microsphere sample prepared in Example 1 was placed on the conductive adhesive, and after gold spraying treatment under vacuum conditions, the microsphere surface morphology was observed under a scanning electron microscope. The results were as follows: Figure 1 As shown in the figure, it can be seen that the surface of the microspheres prepared in Example 1 is smoother and has fewer holes.

[0135] Test Example 6

[0136] This test example tests the stability of the microspheres prepared in Example 1:

[0137] The microspheres prepared in Example 1 were packaged, sealed with a roll cap, and placed in a stability box (40°C 75% RH) for accelerated stability testing. The drug loading, acetic acid content, and in vitro release of the microspheres after 1 month, 2 months, 3 months, and 6 months of acceleration were tested. The stability data are shown in Tables 5 and Figure 2 shown.

[0138] Table 5

[0139]

[0140] The data in Table 5 show that when the accelerated stability test was conducted at 40°C and 75% RT, there was no significant difference in the drug loading, acetic acid content and in vitro release of the microspheres of Example 1 after 1, 2, 3 and 6 months of acceleration, indicating that the addition of alkaline buffer solution to the external aqueous phase would not affect the stability of the raw material.

[0141] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of 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.

[0142] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0143] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing goserelin microspheres, characterized in that: The preparation method comprises the following steps: (1) mixing a goserelin raw material solution and a degradable polymer solution to obtain an oil phase; mixing a surfactant and water to obtain an aqueous phase; (2) mixing the oil phase with the water phase to form an emulsion, stirring and solidifying to obtain goserelin microspheres; Wherein, the solvents of the goserelin raw material solution and the degradable polymer solution in step (1) are both co-solvents obtained by mixing the first solvent and the second solvent.

2. The method for preparing goserelin microspheres according to claim 1, characterized in that: The first solvent is selected from anhydrous acetic acid; The second solvent is independently selected from any one or a combination of at least two of dichloromethane, chloroform, ethyl acetate or N-methylpyrrolidone; Preferably, the volume ratio of the first solvent to the second solvent is independently (0.01-0.7):1, and more preferably, independently (0.2-0.5):

1.

3. The method for preparing goserelin microspheres according to claim 1 or 2, characterized in that: The goserelin raw material in step (1) includes goserelin or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutically acceptable salt of goserelin includes goserelin acetate or goserelin pamoate; Preferably, the concentration of goserelin raw material in the goserelin raw material solution is 10-50wt%; Preferably, the concentration of the degradable polymer in the degradable polymer solution is 10-40 wt %; Preferably, the mass ratio of the goserelin raw material to the degradable polymer is 1:(5-15).

4. The method for preparing goserelin microspheres according to any one of claims 1 to 3, characterized in that: The degradable polymer in step (1) comprises lactide-glycolide copolymer and / or polylactide; Preferably, the weight average molecular weight of the degradable polymer is 5000-50000 Da, more preferably 8000-30000 Da.

5. The method for preparing goserelin microspheres according to any one of claims 1 to 4, characterized in that: The oil phase in step (1) also includes a release regulator; Preferably, the release modifier comprises any one or a combination of at least two of arginine, stearic acid or xinafoic acid; Preferably, the mass ratio of the release modifier to the goserelin raw material is (0.006-0.6):1, and more preferably (0.01-0.5):

1.

6. The method for preparing goserelin microspheres according to any one of claims 1 to 5, characterized in that: The surfactant in step (1) comprises any one of polyvinyl alcohol, carboxymethyl cellulose, gelatin, poloxamer or Tween, or a combination of at least two thereof; Preferably, the surfactant is used in an amount of 0.01-10 wt % of the aqueous phase.

7. The method for preparing goserelin microspheres according to any one of claims 1 to 6, characterized in that: The aqueous phase in step (1) further includes a penetrant; Preferably, the penetrant includes any one or a combination of at least two of phosphate, carbonate, chloride, sulfate or citrate; Preferably, the amount of the penetrant is 0.1-3 wt % of the aqueous phase; Preferably, the aqueous phase in step (1) further comprises a pH regulator; Preferably, the pH adjuster includes any one of sodium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate or disodium hydrogen phosphate, or a combination of at least two thereof; Preferably, the pH value of the pH adjuster added to the aqueous phase is 7-9.

8. The method for preparing goserelin microspheres according to any one of claims 1 to 7, characterized in that: The mixing in step (2) includes homogenous emulsification, shear emulsification, membrane emulsification or microchannel emulsification.

9. The method for preparing goserelin microspheres according to any one of claims 1 to 8, characterized in that: The volume ratio of the oil phase to the water phase is 1:(50-300), and more preferably 1:(70-200).

10. Goserelin microspheres prepared according to the method for preparing goserelin microspheres according to any one of claims 1 to 9.

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