Long-acting sustained-release pharmaceutical preparation and preparation method therefor

Abstract

A long-acting sustained-release pharmaceutical preparation and a preparation method therefor. The long-acting sustained-release pharmaceutical preparation is a microsphere preparation, which comprises brexpiprazole and PLGA, wherein the content of brexpiprazole is 45%-75% of the total weight of the microsphere, the content of PLGA is 55%-25% of the total weight of the microsphere, and PLGA has a viscosity of 0.15-0.25 dl / g and a molecular weight of 8000-32000. The microsphere has a relatively high drug loading capacity, can effectively reduce the administration dosage and reduce the pain in patients during subcutaneous or intramuscular administration; and the prepared sustained-release microsphere has no burst release phenomenon, and has a stable change in blood drug concentration within an effective period of release in rats.

Description

A long-acting sustained-release pharmaceutical preparation and its preparation method Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a long-acting sustained-release pharmaceutical preparation and a preparation method thereof, and more particularly to brepizole microspheres and a preparation method thereof. Background Art

[0002] Brepiprazole is a next-generation antipsychotic drug candidate that acts on multiple receptors. It is a dopamine D2 receptor partial agonist (improving positive and negative symptoms, cognitive impairment, and depressive symptoms), a 5-HT2A receptor antagonist (improving negative symptoms, cognitive impairment, depressive symptoms, and insomnia), an α1 adrenergic receptor antagonist (improving positive symptoms of schizophrenia), and a serotonin uptake / reuptake inhibitor (improving depressive symptoms). It is also a 5-HT1A partial agonist (with anxiolytic and antidepressant activity) and a 5-HT7 antagonist (regulating body temperature, circadian rhythms, learning and memory, and sleep). Brepiprazole oral tablets (Rexulti), jointly developed by Lundbeck of Denmark and Otsuka Pharmaceutical Co., Ltd. of Japan, were first approved by the FDA in July 2015 for the treatment of schizophrenia and as an adjunct therapy to antidepressants for major depressive disorder. They were approved for marketing in Japan and Europe in April and July 2018, respectively. However, due to the particularity of patients, the phenomenon of refusal or missed medication is particularly prominent. Long-acting sustained-release preparations can reduce the number of medications, reduce the relapse rate, improve patient compliance, and reduce the burden on family members and hospitals. Therefore, the development of long-acting sustained-release preparations has high clinical value.

[0003] Bangqing Wu et al. (Engineered PLGA microspheres for extended release of brexpiprazole: in vitro and in vivo studies) disclosed brexpiprazole microspheres with a maximum drug loading of only 23.79%. Furthermore, during in vivo release, there was a severe burst release due to the rapid release of brexpiprazole adsorbed on the microsphere surface. Blood drug concentrations fluctuated widely during the main release phase, posing a risk of increased side effects due to excessively high drug concentrations. Patent JP2013139441A discloses brexpiprazole microspheres and their preparation methods. These microspheres had a drug loading of 21.3%, a relatively low drug loading for sustained-release microspheres. This poses a risk of high dosages for clinical microsphere preparations. Since sustained-release microspheres are typically administered subcutaneously or intramuscularly, excessively high dosages can significantly increase patient pain.

[0004] However, when the drug loading in sustained-release microspheres increases, some of the drug will remain on the surface of the microspheres, causing a burst release phenomenon in the microsphere controlled-release system, leading to increased side effects. Therefore, the development of a brepizole sustained-release microsphere formulation with a high drug loading, no burst release, and smooth release is of great clinical significance.

[0005] Summary of the Invention

[0006] Researchers have found that the drug loading in brepirazole microspheres in the prior art is generally low. When using the publicly available formulations in the prior art to prepare microspheres with higher drug loading, there are problems with severe burst release or incomplete release. To address the above problems, the inventors, through extensive research, prepared brepirazole sustained-release microspheres with a drug loading of up to 45-75% and no burst release. The blood drug concentration in rats changes steadily during the release process, and there is no release plateau phase. The release period can reach 1-3 months. By increasing the drug loading, the microspheres effectively reduce the dosage and reduce the pain of subcutaneous or intramuscular administration. The microspheres also have high sphericity and uniform drug distribution, making them suitable for large-scale production.

[0007] The technical solutions for implementing the present invention are as follows:

[0008] In one aspect, the present invention provides brepizole microspheres, comprising brepizole and PLGA, wherein the content of brepizole is 45%-75% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%) of the total weight of the microspheres, and the content of PLGA is 55%-25% (e.g., 55%, 50%, 45%, 40%, 35%, 30%, 25%) of the total weight of the microspheres; preferably, the content of brepizole is 45%-70% of the total weight of the microspheres, more preferably, the content of brepizole is 45%-60% of the total weight of the microspheres, and most preferably, the content of brepizole is 50%-60% of the total weight of the microspheres.

[0009] Furthermore, in some embodiments of the present invention, the intrinsic viscosity of the PLGA is 0.15-0.25 dl / g (such as 0.15 dl / g, 0.16 dl / g, 0.17 dl / g, 0.18 dl / g, 0.19 dl / g, 0.20 dl / g, 0.21 dl / g, 0.22 dl / g, 0.23 dl / g, 0.24 dl / g, 0.25 dl / g); preferably, the intrinsic viscosity of the PLGA is 0.17-0.23 dl / g; more preferably, the intrinsic viscosity of the PLGA is 0.18-0.23 dl / g.

[0010] Further, in some embodiments of the present invention, the molar ratio of PL to PG in the PLGA is 50:50-95:5 (such as 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5); preferably, the molar ratio of PL to PG in the PLGA is 50:50-85:15, more preferably, the molar ratio of PL to PG in the PLGA is 50:50-75:25.

[0011] Further, in some embodiments of the present invention, the molecular weight of the PLGA is 8000-32000 (such as 8000, 9000, 10000, 14000, 15000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 30000), preferably, the molecular weight of the PLGA is 10000-30000, further preferably, the molecular weight of the PLGA is 12000-25000, more preferably, the molecular weight of the PLGA is 14000-25000, and most preferably, the molecular weight of the PLGA is 17000-23000.

[0012] Further, in some embodiments of the present invention, the D50 of the microspheres is 20-100 μm (such as 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm), preferably, the D50 of the microspheres is 25-75 μm, more preferably, the D50 of the microspheres is 25-50 μm, and most preferably, the D50 of the microspheres is 25-45 μm.

[0013] Furthermore, in some embodiments of the present invention, the brepizole microspheres are of a core-shell structure, wherein the core comprises brepizole and the shell comprises PLGA; the core is fully or partially encapsulated by the shell.

[0014] Furthermore, in some embodiments of the present invention, the average thickness of the shell is 0.5-20 μm; preferably, the average thickness of the shell is 0.5-10 μm; more preferably, the average thickness of the shell is 0.5-6 μm.

[0015] Furthermore, in some embodiments of the present invention, the core comprises amorphous brexpiprazole solid.

[0016] The second aspect of the present invention provides a method for preparing the brepizole sustained-release microspheres according to the first aspect, the method comprising the following steps:

[0017] ① preparing a solution containing brepazole or its salt, PLGA and an organic solvent;

[0018] ② Under conditions that effectively inhibit evaporation of the organic solvent, mixing the solution obtained in step ① with the PVA solution to obtain an O / W emulsion; and

[0019] ③ Under conditions such that brepizole is precipitated in the form of spherical particles, the organic solvent in the O / W emulsion obtained in step ② is removed.

[0020] Furthermore, in some embodiments of the present invention, the organic solvent described in step ① is not limited, as long as it can dissolve brepazole and PLGA. Examples of such organic solvents include chloroform, dichloroethane, trichloroethane, dichloromethane, carbon tetrachloride, ether, isopropyl ether, ethyl acetate, butyl acetate, benzene, toluene, xylene, acetonitrile, and mixtures thereof; preferably, the organic solvent is dichloromethane.

[0021] Further, in some embodiments of the present invention, the intrinsic viscosity of the PLGA in step ① is 0.15-0.25 dl / g (such as 0.15 dl / g, 0.16 dl / g, 0.17 dl / g, 0.18 dl / g, 0.19 dl / g, 0.20 dl / g, 0.21 dl / g, 0.22 dl / g, 0.23 dl / g, 0.24 dl / g, 0.25 dl / g); preferably, the intrinsic viscosity of the PLGA is 0.17-0.23 dl / g; more preferably, the intrinsic viscosity of the PLGA is 0.18-0.23 dl / g.

[0022] Further, in some embodiments of the present invention, the molar ratio of PL to PG in the PLGA is 50:50-95:5 (such as 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5); preferably, the molar ratio of PL to PG in the PLGA is 50:50-85:15, more preferably, the molar ratio of PL to PG in the PLGA is 50:50-75:25.

[0023] Further, in some embodiments of the present invention, the molecular weight of the PLGA is 8000-32000 (such as 8000, 9000, 10000, 14000, 15000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 30000, 31000, 32000), preferably, the molecular weight of the PLGA is 10000-30000, further preferably, the molecular weight of the PLGA is 12000-25000, more preferably, the molecular weight of the PLGA is 14000-25000, and most preferably, the molecular weight of the PLGA is 17000-23000. Further, the D50 of the microspheres is 20-100 μm (such as 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm), preferably, the D50 of the microspheres is 25-75 μm, more preferably, the D50 of the microspheres is 25-50 μm, and most preferably, the D50 of the microspheres is 25-45 μm.

[0024] Furthermore, in some embodiments of the present invention, the brepizole microspheres are of a core-shell structure, wherein the core comprises brepizole and the shell comprises PLGA; the core is fully or partially encapsulated by the shell.

[0025] Furthermore, in some embodiments of the present invention, the average thickness of the shell is 0.5-20 μm; preferably, the average thickness of the shell is 0.5-10 μm; more preferably, the average thickness of the shell is 0.5-6 μm.

[0026] Furthermore, in some embodiments of the present invention, the core comprises amorphous brexpiprazole.

[0027] Further, in some embodiments of the present invention, the volume weight ratio (mL / g) of the organic solvent to brepizole or its salt in step ① is 7:1-30:1 (such as 7:1, 8:1, 9:1, 10:1, 12:1, 15:1, 20:1, 23:1, 25:1, 30:1), preferably, the volume weight ratio (mL / g) of the organic solvent to brepizole or its salt is 10:1-25:1.

[0028] Furthermore, in some embodiments of the present invention, the weight ratio of PLGA to brepizole or its salt in step ① is 1:1-1:3 (such as 1:1, 1:1.2, 1:1.5, 1:1.9, 1:2.3, 1:3); preferably, the weight ratio of PLGA to brepizole or its salt is 1:1-1:2.3; more preferably, the weight ratio of PLGA to brepizole or its salt is 1:1-1:1.5.

[0029] Furthermore, in some embodiments of the present invention, the solubility of the PVA solution in step ② is 0.1%-3% (w / v) (such as 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%).

[0030] Furthermore, in some embodiments of the present invention, the volume ratio of the PVA solution to the organic solvent in step ② is 100:1-300:1 (such as 100:1, 133:1, 150:1, 200:1, 233:1, 250:1, 300:1), preferably, the volume ratio of the PVA solution to the organic solvent is 150:1-300:1.

[0031] Furthermore, in some embodiments of the present invention, step ② includes: under suitable temperature conditions, mixing the solution obtained in step ① with the PVA solution and stirring at a speed R1 to obtain an O / W emulsion; preferably, the R1 is 500-6000 rpm, more preferably, the R1 is 1000-6000 rpm, and most preferably, the R1 is 1500-3600 rpm.

[0032] Furthermore, in some embodiments of the present invention, step ③ includes stirring the O / W emulsion obtained in step ② at a certain rotation speed under suitable temperature conditions to remove the organic solvent.

[0033] Furthermore, in some embodiments of the present invention, step ③ also includes separation and freeze-drying steps.

[0034] Furthermore, in some embodiments of the present invention, no alkali is required to adjust the pH during the preparation of the microspheres. Preferably, no alkali is required to adjust the pH of the PVA solution during the preparation process. More preferably, the pH of the PVA solution is 6-7.5, and most preferably, the pH of the PVA solution is 6-7.

[0035] In another aspect, the present invention provides an injectable aqueous suspension, which protects the brepizole microspheres according to the first aspect of the present invention or the brepizole microspheres prepared according to the second aspect.

[0036] Furthermore, in some embodiments of the present invention, the suspension further includes a pharmaceutically acceptable carrier and water for injection. Preferably, the pharmaceutically acceptable carrier is selected from a suspending agent, a pH regulator, an isotonicity regulator, and a surfactant; the suspending agent is selected from sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate and glycerol; the isotonicity regulator is selected from sodium chloride, glucose, mannitol and sorbitol; the surfactant is a non-ionic surfactant selected from a polysorbate series and a poloxamer series; the pH regulator is selected from citric acid, sodium hydroxide and disodium hydrogen phosphate.

[0037] Furthermore, in some embodiments of the present invention, the amount of microspheres in the suspension is not limited, as long as the microspheres are dispersed therein.

[0038] Advantages of the present invention:

[0039] 1. Generally, under the premise that the particle size of the microspheres remains basically unchanged, the higher the drug loading of the microspheres, the more severe the burst release phenomenon and the faster the release rate of the microspheres. The present invention selects specific raw materials and ratios to make the drug loading of the brepirazole microspheres prepared by the present invention as high as 45-75%. Under the conditions of in vitro simulated release, the higher the drug loading, the slower the release rate of the prepared brepirazole microspheres. The prepared sustained-release microspheres have no burst release phenomenon in vitro, and the blood drug concentration changes steadily during the release process in rats. By increasing the drug loading, the dosage can be effectively reduced, reducing the pain of subcutaneous or intramuscular administration in patients.

[0040] 2. The high-drug-loaded brepizole sustained-release microspheres of the present invention have a core / shell structure. The drug is mainly encapsulated in the inner core polymer, and the outer layer serves as a release barrier, which can significantly reduce the burst release of the drug, and the blood drug concentration changes steadily during the release period.

[0041] 3. The release period of the brepizole sustained-release microspheres provided by the present invention can reach 1-3 months, which can effectively reduce the frequency of administration and improve medication compliance.

[0042] 4. The preparation method of breprazol sustained-release microspheres provided by the present invention has a simple process and a stable structure, and can be industrialized on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1. In vitro release curves of microspheres prepared from experimental groups 2-1 to 2-6;

[0044] Figure 2. Scanning electron microscopy images of microspheres prepared in experimental groups 2-1 and 2-6;

[0045] Figure 3. Scanning electron microscopy images of the cross-section of microspheres prepared in experimental group 2-1 before and after acid washing;

[0046] Figure 4. Scanning electron microscopy images of the cross-section of microspheres prepared in experimental group 2-2 before and after acid washing;

[0047] Figure 5. Scanning electron microscopy images of the cross-section of microspheres prepared in experimental groups 2-4 before and after acid washing;

[0048] Figure 6. Scanning electron microscopy images of the cross-section of microspheres prepared in experimental groups 2-5 before and after acid washing;

[0049] Figure 7. XRD patterns of brepizole raw materials and microspheres corresponding to experimental groups 2-3;

[0050] Figure 8. DSC graphs of brepizole API, PLGA, and microspheres corresponding to experimental groups 2-3;

[0051] Figure 9. Blood drug concentration-time curves of rats in experimental group 1-1, experimental group 1-4, and experimental group 2-2;

[0052] Figure 10. Blood drug concentration-time curves of rats in experimental groups 1-5;

[0053] Figure 11. Blood drug concentration-time curves of rats in experimental groups 1-2 and 1-3;

[0054] Figure 12 is a graph showing the cumulative release rate versus time for Comparative Example 1 and Comparative Example 2 in vitro;

[0055] Figure 13. Blood drug concentration-time curve of Comparative Example 2 and rats in Comparative Example 2. DETAILED DESCRIPTION

[0056] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or in accordance with the product instructions are used. Reagents or instruments used without indicating the manufacturer are conventional products that can be obtained commercially. The brepizole raw material selected for preparing microspheres herein can be crystalline or amorphous. In some embodiments of the present invention, the brepizole raw material used is crystalline.

[0057] As used herein, the term "PLGA" refers to poly(lactide-co-glycolide) or poly(lactic acid-co-glycolic acid), and "LA:GA molar ratio" or "PL to PG molar ratio" refers to the molar ratio of polylactide to glycolide in poly(lactide-co-glycolide).

[0058] As used herein, the term "molecular weight of PLGA" refers to the weight average molecular weight of PLGA.

[0059] As used herein, the term "intrinsic viscosity" refers to the concentrated viscosity of a polymer solution when its concentration approaches zero. This refers to the contribution of individual molecules to the viscosity of a solution. This viscosity reflects the properties of the polymer and is independent of concentration. Common methods used in the art can be used to measure the intrinsic viscosity of polymers, such as the capillary tube method, the rotating drum method, and the falling ball method.

[0060] As used herein, the term "PLA" refers to polylactide.

[0061] The term "burst release" used in this article refers to the rapid release of drugs adsorbed on the surface during an in vitro release test, which is called the burst release effect. Specifically, "burst release" means that in the determination of the in vitro release curve, the prepared microspheres release more than 20% of the drug cumulatively on the first day; the release medium used in the present invention is determined experimentally, and there is a certain correlation between the release curve measured in the release medium of the present invention using the test method of the present invention and the in vivo release curve of rats. When the in vitro release curve is determined using the release medium of the present invention, when the cumulative release of the microspheres exceeds 20% on the first day, the corresponding microspheres have large fluctuations in the blood drug concentration in rats in the early stage, resulting in unstable blood drug concentration in the body and increasing potential side effects.

[0062] The term "crystallization" as used herein refers to the preparation of brepirazole solid particles distributed between the microspheres or in the microsphere preparation solution in the brepirazole microspheres, and the brepirazole solid particles may be crystalline or amorphous.

[0063] The term "theoretical drug loading" as used herein refers to the weight percentage of brepizole raw material used in the total weight of PLGA and brepizole raw materials before microsphere preparation.

[0064] The term "drug loading" used herein refers to the actual drug loading, specifically the percentage of brepizole mass in the prepared microspheres to the total mass of the microspheres.

[0065] In the embodiments or comparative examples, the test methods are as follows:

[0066] Particle size of microspheres

[0067] The particle size determination methods include microscope method and laser particle size analyzer-wet method, and the detection instrument is Malvern 3000 particle size analyzer.

[0068] Microsphere morphology

[0069] 1. Polarizing microscope

[0070] After the microspheres are stirred and solidified for a certain period of time and then sieved and washed with water, a small amount of sample is taken and placed under a polarizing microscope to observe the morphology of the microspheres and whether crystallization occurs.

[0071] 2. Scanning Electron Microscopy

[0072] The freeze-dried sample was prepared and then sprayed with gold, and then observed under a scanning electron microscope (FSEM-2, Nova 450), photographed, and the morphology and internal structure of the microspheres were observed.

[0073] Observation of the internal structure of microspheres was divided into direct structural morphology observation and changes in the internal structure of microspheres after dissolution of brepazine. 1) Direct structural morphology observation. Microspheres were frozen with liquid nitrogen and then sectioned with a utility knife. 2) Changes in the internal structure of microspheres after dissolution of brepazine. A 10% to 20% citric acid solution cannot dissolve PLGA, but can dissolve a small amount of brepazine. Based on this, the microspheres were first sectioned with liquid nitrogen to produce cross-sectional samples. Then, citric acid solution was added to dissolve the brepazine in the microspheres. After the brepazine was completely dissolved, the acid solution was washed away, the microspheres were dried at room temperature, and observed under a scanning electron microscope. Compared to the unacidized microspheres, the missing portion of the cross-section represents brepazine.

[0074] 3. Study on the crystal form of breprazol in microspheres

[0075] XRD (Rigaku X-ray diffractometer Ultima IV) was used to study the crystal form and compatibility of brepizole and PLGA polymer in the microspheres.

[0076] DSC detection was performed using DSC3 (YQ20040540) equipment according to CP 2020 version, Part 4 - Method 0661 and Method 0981.

[0077] 4. Drug loading and encapsulation efficiency of brepizole microspheres

[0078] The drug loading capacity mentioned in the present invention refers to the amount of drug loaded per unit weight of microspheres, and the calculation formula is:

[0079] Drug loading = weight of brepizole in microspheres / (total weight of brepizole in microspheres + PLGA) × 100%.

[0080] The encapsulation efficiency of the present invention refers to the percentage of the weight of brepirazole encapsulated in the microspheres to the weight of brepirazole input during preparation, and is calculated as follows:

[0081] Encapsulation efficiency = weight of brepizole in microspheres / weight of brepizole added during preparation × 100%.

[0082] 5. Determination of in vitro release curve

[0083] Accurately weigh an appropriate amount of sample (containing approximately 2 mg of breprazol). Add 150 mL of release medium (2 mM potassium dihydrogen phosphate, 8 mM sodium dihydrogen phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, 0.05% benzalkonium chloride, 1% Tween 80) maintained at 37 ± 0.5°C. Place in a 37°C desiccator and allow to stand. Remove 5 mL of sample at 2 h, 4 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 9 days (sampling points adjusted based on actual sample release) and rehydrate with 5 mL of solution. Centrifuge the sample solution, and analyze the release amount of the supernatant using HPLC. Prepare a release curve.

[0084] Example 1

[0085] Experimental group 1-1

[0086] Mix 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 6mL of dichloromethane and dissolve in a water bath at 50-60°C (any temperature range). Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0087] The oil phase was added to 1200 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3600 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0088] Experimental groups 1-2

[0089] Mix 0.42g of breprazol and 0.28g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.22 dl / g, and molecular weight of 21,000). Add 6mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0090] The oil phase was added to 1200 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0091] Experimental groups 1-3

[0092] Mix 0.42g of breprazol and 0.28g of PLGA (LA:GA molar ratio of 90:10, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 6mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0093] The oil phase was added to 1200 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0094] Experimental groups 1-4

[0095] Mix 0.5g of breprazol and 0.5g of PLGA (LA:GA molar ratio of 90:10, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 3.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0096] The oil phase was added to 700 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 1000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0097] Experimental groups 1-5

[0098] Mix 0.35g of breprazol and 0.35g of PLGA (LA:GA molar ratio of 75:25, intrinsic viscosity of 0.21 dl / g, and molecular weight of 20,000). Add 6mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0099] The oil phase was added to 1400 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0100] Experimental groups 1-6

[0101] Mix 0.7g of breprazol and 0.3g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.22 dl / g, and molecular weight of 21,000). Add 9.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0102] The oil phase was added to 1900 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0103] Comparison group 1

[0104] Mix 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.12 dl / g, and molecular weight of 8000). Add 8.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0105] The oil phase was added to 1700 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0106] Comparison group 2

[0107] Mix 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.33 dl / g, and molecular weight of 39,000). Add 8.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0108] The oil phase was added to 1700 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0109] Comparison group 3

[0110] Mix 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.42 dl / g, and molecular weight of 56,000). Add 8.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0111] The oil phase was added to 1700 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0112] Comparison group 4

[0113] Mix 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 100:0, intrinsic viscosity of 0.22 dl / g, and molecular weight of 21,000). Add 8.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0114] The oil phase was added to 1700 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0115] The particle size, drug loading, encapsulation efficiency and in vitro release of the microspheres obtained in experimental groups 1-1 to 1-6 and comparison groups 1 to 4 were measured according to the methods described in the examples of the specification. The test results are shown in Table 1-3.

[0116] Table 1 Particle size, drug loading and encapsulation efficiency of microspheres obtained in experimental groups 1-1 to 1-6 and control group 1-4

[0117] Table 2 In vitro release results of microspheres obtained from experimental groups 1-1 to 1-6

[0118] Table 3 In vitro release results of microspheres obtained in comparison groups 1-4

[0119] Conclusion: As shown in Tables 1-3, when the theoretical drug loading of brepazine is 60%, different types of PLGA polymers can be used to prepare brepazine microspheres, with drug loadings of 53-60% and encapsulation efficiencies greater than 89.5%. However, the viscosity, molecular weight, and LA:GA molar ratio of the PLGA polymer greatly affect the release behavior of the brepazine microspheres. When the PLGA viscosity is 0.15-0.25 dl / g and the molecular weight is 12,000-25,000, the prepared microspheres have no burst release, the cumulative release in the initial 2 hours is less than 5%, and the release is relatively complete. The microspheres prepared in Comparative Groups 1 to 3 all have severe burst release, and the inventors found that when the PLGA of Comparative Groups 1-3 is used, the problem of severe burst release of microspheres with a drug loading of up to 60% cannot be solved by changing or optimizing the preparation process. Comparative Examples 1-3 are only some specific examples. Analysis shows that the possible reasons are that for control group 1, the LA:GA molar ratio of PLGA was 85:15, the viscosity was 0.12 dl / g, and the molecular weight was 8000. The polymer viscosity was too low to effectively encapsulate brepazole, resulting in a burst release in the prepared microspheres. For control groups 2 and 3, when the polymer viscosity was greater than 0.33 dl / g and the molecular weight was higher than 39000, due to the excessive polymer viscosity, during the microsphere formation process, PLGA competed to deposit in the microsphere core, resulting in some brepazole deposited on the shallow surface of the microspheres, resulting in a burst release in the prepared microspheres.

[0120] When brepazole microspheres were prepared using PLA with an LA:GA molar ratio of 100, a viscosity of 0.22 dl / g, and a molecular weight of 21,000, there was no burst release phenomenon. However, the release amount of the microspheres reached 7.0% at 0.08 days, and the cumulative release on the 5th day was 9.7%. The release rate was slow and it could not be completely released within a specific release period.

[0121] Example 2

[0122] Experimental group 2-1

[0123] Mix 0.24g of breprazol and 0.96g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 6.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0124] The oil phase was added to 1300 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2400 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0125] Experimental group 2-2

[0126] Mix 0.36g of breprazol and 0.94g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 7.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0127] The oil phase was added to 1500 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2600 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0128] Experimental groups 2-3

[0129] Mix 0.6g of breprazol and 0.6g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 9.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0130] The oil phase was added to 1900 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0131] Experimental groups 2-4

[0132] Mix 0.72g of breprazol and 0.48g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 10mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0133] The oil phase was added to 2000 mL of 0.5% PVA aqueous solution and homogenized and emulsified using a disperser emulsifier for 2 minutes at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0134] Experimental groups 2-5

[0135] Mix 0.7g of breprazol and 0.3g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 9.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0136] The oil phase was added to 2000 mL of 0.5% PVA aqueous solution and homogenized and emulsified using a disperser emulsifier for 2 minutes at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0137] Experimental groups 2-6

[0138] Mix 0.8g of breprazol and 0.2g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 10.5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0139] The oil phase was added to 2200 mL of 0.5% PVA aqueous solution and homogenized and emulsified using a disperser emulsifier for 2 minutes at a speed of 3000 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0140] The particle size, drug loading, encapsulation efficiency, in vitro release curve, and scanning electron microscopy images of the microspheres obtained in Example 2 were measured according to the methods described in the specification. The results of microsphere particle size, drug loading, and encapsulation efficiency are shown in Table 4, and the in vitro release curve of the microspheres is shown in Figure 1.

[0141] Table 4

[0142] Experimental conclusion: When the theoretical drug loading of the microspheres is 20%-70%, the actual drug loading of the prepared sustained-release microspheres is slightly less than the theoretical drug loading, the encapsulation efficiency is greater than 90%, and they are uniformly spherical. Specifically, it can be clearly seen from Figure 2 that the microspheres prepared in the implementation group 2-1 and the experimental group 2-6 are uniformly spherical; the prepared microspheres have a particle size D50 of 36-42 μm, and the particle size of the microspheres with different drug loadings is close, with no significant difference. When the theoretical drug loading is 80%, the prepared sustained-release microspheres have more crystallization, which shows that for the polymer used in Example 2, the theoretical drug loading of brepizole microspheres is preferably less than 80%.

[0143] Those skilled in the art generally believe that, assuming the microsphere particle size remains essentially unchanged, higher microsphere drug loading leads to more severe burst release and a faster release rate. However, after systematic research, the inventors unexpectedly discovered that, according to the formulation of the present invention, when the microsphere drug loading is less than 70%, the release rate of the microspheres decreases as the microsphere drug loading increases. Specifically, see the release curve shown in Figure 1.

[0144] In order to further explore the reason why the higher the drug loading of brepazine microspheres, the slower the release, the inventors further analyzed the internal structure of brepazine microspheres. The freeze-dried samples were sprayed with gold, placed under a scanning electron microscope for observation, and photographed to observe the morphology and internal structure of the microspheres. The observation of the internal structure of the microspheres was divided into direct structural morphology observation and changes in the internal structure of the microspheres after dissolution of brepazine. The scanning electron micrographs of the samples prepared in experimental group 2-1 before and after acid washing are shown in Figure 3. The scanning electron micrographs of the samples prepared in experimental group 2-2 before and after acid washing are shown in Figure 4. The scanning electron micrographs of the samples prepared in experimental group 2-4 before and after acid washing are shown in Figure 5. The scanning electron micrographs of the samples prepared in experimental group 2-5 before and after acid washing are shown in Figure 6.

[0145] The prepared microspheres were subjected to XRD and DSC analysis. For example, the XRD patterns of the brepizole raw material and microspheres from the experimental group 2-3 are shown in FIG7 , and the DSC patterns of the brepizole raw material, PLGA raw material, and microspheres from the experimental group 2-3 are shown in FIG8 .

[0146] As can be seen from Figures 3-6, for breprazol microspheres with no significant difference in particle size (particle size difference less than 20%), when the microsphere drug loading is 19.4%, the cross-section of the microspheres after slicing has no core-shell structure, and the cross-section of the microspheres after acid washing has multiple small pores; when the microsphere drug loading is 26.9%, the cross-section of the microspheres after slicing has no core-shell structure, and the cross-section of the microspheres after acid washing has more small pores; when the microsphere drug loading is 56.9%, the cross-section of the microspheres after slicing has a clear core-shell structure, the shell thickness is about 5μm, and the core inside the microspheres is acid washed away after acid washing; when the microsphere drug loading is 67.6%, the cross-section of the microspheres after slicing has a clear core-shell structure, the shell thickness is about 4μm, and the core inside the microspheres is acid washed away after acid washing. From the above experimental phenomena, it can be seen that when the microsphere drug loading is less than 40%, the prepared microspheres are honeycomb-shaped, and when the drug loading is higher than 50%, the prepared microspheres are core-shell structures. That is, the microstructure of the microspheres varies greatly with different drug loadings. The higher the drug loading, the easier it is for the brexpiprazole microspheres to form a core-shell structure. For core-shell structure microspheres, the outer polymer needs to be dissolved to release the drug inside. Therefore, the sustained-release microspheres with high drug loading have no burst release in the early stage, and the release rate is slower than that of the sustained-release microspheres with low drug loading.

[0147] Similarly, the applicant selected a LA:GA molar ratio of 75:25; an intrinsic viscosity of 0.17 dl / g, and a molecular weight of 14,000. There was no significant difference in the prepared particle size. For brexpiprazole microspheres with different drug loadings, similar experimental phenomena were found. When the microsphere drug loading was low, the prepared microspheres were honeycomb-shaped. When the microsphere drug loading was higher than 50%, the prepared microspheres were core-shell structures. Similarly, the early release rate of microspheres with high drug loading was lower than that of microspheres with low drug loading.

[0148] As can be seen from Figure 7, the brepizole raw material is crystalline, and the prepared microspheres have an amorphous structure. Furthermore, XRD experiments revealed that the XRD patterns of the microspheres prepared from Experimental Groups 2-4, 2-5, and 2-6 all exhibited amorphous diffraction peaks, without the characteristic peaks of brepizole.

[0149] As can be seen from Figure 8, the peak corresponding to 60°C is the endothermic peak of the glass transition of brepizole microspheres, and the peak corresponding to 120°C is the exothermic peak of the transformation of amorphous brepizole in brepizole microspheres into crystalline brepizole.

[0150] Example 3

[0151] The brepirazole microspheres prepared in Experimental Groups 1-1, 1-4, and 2-2 of the present invention were prepared into a formulation. Specifically, a predetermined amount of microspheres were weighed and dispersed with a prepared suspension (CMC-Na 0.6 wt %, sodium chloride 0.75 wt %, anhydrous citric acid 0.11 wt %, sodium hydroxide 0.055 wt %, and disodium hydrogen phosphate 0.1 wt %) to prepare a suspension having a brepirazole solution concentration of 15 mg / mL.

[0152] The sample was injected into the posterior rectus muscle of SD rats at a dose of 15 mg / kg. Blood samples were collected before administration (pre-dose), 6h (0.25d), 24h (1d), 3d, 5d, 7d, 9d, 11d, 14d, 16d, 18d, 21d, 24d, 28d, 31d, 35d, and 42d after administration. The drug concentration in plasma was determined by the established LC-MS method, and the rat blood drug concentration-time curve was drawn to evaluate the blood drug concentration-time relationship.

[0153] Specific results are shown in Figure 9. Blood drug concentrations in experimental groups 1-1 and 1-4 fluctuated steadily, remaining within the effective range during dosing and maintaining efficacy for up to 42 days. The curve for experimental group 2-2 showed a rapid early rise, peaking on day 7, and then rapidly declining. This may be due to the fact that the microspheres prepared in experimental group 1-1 had a core-shell structure. For core-shell microspheres, the outer polymer layer must dissolve to release the drug within. Therefore, the initial release rate of the microspheres was relatively stable, resulting in stable blood drug concentrations in rats. In contrast, the microspheres prepared in experimental group 2-2 had a honeycomb structure with a drug loading of 26.9%. During the early release phase, as the surface layer of the microspheres dissolves, the shallow brepizole layer is rapidly released from the microspheres. This rapid early release led to greater fluctuations in blood drug concentrations.

[0154] The brepirazole microspheres prepared in Experimental Groups 1-2, 1-3, and 1-5 of the present invention were prepared into a formulation. Specifically, a predetermined amount of microspheres were weighed and dispersed with a prepared suspension (CMC-Na 0.6 wt %, sodium chloride 0.75 wt %, anhydrous citric acid 0.11 wt %, sodium hydroxide 0.055 wt %, and disodium hydrogen phosphate 0.1 wt %) to prepare a suspension having a brepirazole concentration of 15 mg / mL.

[0155] The sample was injected into the posterior rectus muscle of SD rats. The dose of experimental groups 1-2 and 1-3 was 22.5 mg / kg, and the dose of experimental group 1-5 was 30 mg / kg. Blood samples were collected before administration (pre-dose), 6h (0.25d), 24h (1d), 3d, 5d, 7d, 10d, 11d, 14d, 17d, 19d, 21d, 24d, 28d, 31d, 35d, 42d, 49d, 56d, 63d, 70d, 77d, and 84d after administration (the sampling points were adjusted according to the actual changes in blood drug concentration). The drug concentration in plasma was determined by the established LC-MS method, the rat blood drug concentration and time curve was drawn, and the blood drug concentration-time relationship was evaluated.

[0156] The specific results are shown in Figures 10-11.

[0157] As can be seen from Figures 10-11, the brexpiprazole microsphere samples prepared in experimental groups 1-2, 1-3 and 1-5 showed no burst release in vitro, and the blood drug concentration in rats changed steadily during the release period, with no obvious peak-valley phenomenon throughout the release period. The release period could reach 40 days and more than 90 days, respectively.

[0158] Example 4

[0159] 1. Effect of different amounts of dichloromethane solution on the test

[0160] Weigh 0.42g of breprazol and 0.28g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add a certain amount of dichloromethane to each, dissolve in a 50-60°C water bath, and stir to obtain an oil phase. Prepare a 0.5% PVA solution.

[0161] The oil phase was added to 1400 mL of a 0.5% aqueous PVA solution and homogenized using a disperser emulsifier for 2 minutes at 2500 rpm. After homogenization, the mixture was mechanically stirred at room temperature, maintained at approximately 25°C, and cured for 5 hours. A 5 mL sample was taken and the microsphere morphology was observed under a microscope. The microspheres were collected by filtration and lyophilized. The particle size, drug loading, and encapsulation efficiency of the microspheres obtained in Experimental Groups 4-1 and 4-4 were determined according to the methods described in the examples in the manual. The experimental results are shown in Table 5.

[0162] Table 5

[0163] Conclusion: In order to explore the appropriate dichloromethane volume and brepazole weight ratio, in addition to the above-mentioned optimal experimental groups 4-1 to experimental groups 4-4, a series of studies were conducted. The results showed that when the dichloromethane volume and brepazole weight ratio was 4.3:1, dichloromethane could not completely dissolve brepazole; when the dichloromethane volume and brepazole weight ratio was 5.7:1, dichloromethane could completely dissolve brepazole, but the dissolution time would be longer. When the appropriate dichloromethane and brepazole dosage ratio was 7-25:1, under these experimental conditions, the experiment was carried out smoothly and efficiently, and the obtained microspheres had good morphology, uniform size, and encapsulation efficiency greater than 90%.

[0164] 2. Effect of different PVA concentrations on the test

[0165] Weigh 0.42g of breprazol and 0.28g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 6mL of dichloromethane to each, dissolve in a 50-60°C water bath, and stir to obtain an oil phase. Prepare PVA solutions of varying concentrations.

[0166] The oil phase was added to 1400 mL of PVA aqueous solution of varying concentrations and homogenized for 2 minutes using a disperser emulsifier at 2000 rpm. After homogenization, the mixture was mechanically stirred at room temperature, maintained at approximately 25°C, and cured for 5 hours. A 5 mL sample was taken and the microsphere morphology was observed under a microscope. The microspheres were collected by filtration and freeze-dried. The particle size, drug loading, encapsulation efficiency, and polarizing microscopy of the microspheres obtained in Experimental Groups 4-5 to 4-8 were determined according to the methods described in the examples in the manual.

[0167] Table 6

[0168] Conclusion: The test results are shown in Table 6. When the PVA concentration is 0.1-3%, spherical brexpiprazole microspheres can be prepared, and the prepared microspheres have no crystallization and good sphericity. Good sphericity specifically means that the prepared microspheres are uniformly spherical, with a smooth surface and no damage or breakage when observed under a polarizing microscope or a scanning electron microscope.

[0169] 3. Effect of different microsphere particle sizes on test results

[0170] Weigh 0.6g of breprazol and 0.4g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.19 dl / g, and molecular weight of 17,000). Add 8.5mL of dichloromethane to each, dissolve in a 50-60°C water bath, and stir to obtain an oil phase. Prepare a 0.5% PVA solution.

[0171] The oil phase was added to 1700 mL of PVA aqueous solution, and homogenized and emulsified for 2 minutes using a disperser emulsifier at different speeds. After the homogenization and emulsification, the room temperature was controlled at around 25°C, and mechanical stirring was turned on for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope. The microspheres were collected by filtration and freeze-dried.

[0172] The particle size, drug loading, encapsulation efficiency and polarizing microscope of the microspheres obtained in experimental groups 4-9 to 4-12 were measured according to the methods described in the examples of the specification.

[0173] Table 7

[0174] Conclusion: Microspheres with different particle sizes can be prepared by changing the rotation speed, and the microspheres have good spherical properties.

[0175] 4. Effect of different pH on test results

[0176] Experimental groups 4-13

[0177] Weigh 0.864g of breprazol and 0.576g of PLGA (LA:GA molar ratio of 75:25, intrinsic viscosity of 0.17 dl / g, molecular weight of 14,000). Add 12mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Prepare a 0.5% polyvinyl alcohol (PVA) solution. Without adjusting the pH of the PVA solution, the pH was between 6 and 7.

[0178] The oil phase was added to 2800 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2800 rpm. After the homogenization and emulsification, the room temperature was controlled at around 25°C, and mechanical stirring was started at the process design speed for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope. The microspheres were collected by filtration and freeze-dried.

[0179] Experimental groups 4-14

[0180] Weigh 0.864g of breprazol and 0.576g of PLGA (LA:GA molar ratio of 75:25, intrinsic viscosity of 0.17 dl / g, molecular weight of 14,000). Add 12mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Prepare a 0.5% PVA solution and adjust the pH to 11 with sodium hydroxide solution.

[0181] The oil phase was added to 2800 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2800 rpm. After the homogenization and emulsification, the room temperature was controlled at around 25°C, and mechanical stirring was started at the process design speed for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope. The microspheres were collected by filtration and freeze-dried.

[0182] Experimental groups 4-15

[0183] Weigh 0.864g of breprazol and 0.576g of PLGA (LA:GA molar ratio of 75:25, intrinsic viscosity of 0.17 dl / g, molecular weight of 14,000). Add 12mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Prepare a 0.5% PVA solution and adjust the pH to 12 with sodium hydroxide solution.

[0184] The oil phase was added to 2800 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 2800 rpm. After the homogenization and emulsification, the room temperature was controlled at around 25°C, and mechanical stirring was started at the process design speed for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope. The microspheres were collected by filtration and freeze-dried.

[0185] The particle size, drug loading, encapsulation efficiency, polarizing microscope, and in vitro release curves of the microspheres obtained in Experimental Groups 4-13 to 4-15 were measured according to the methods described in the examples in the specification. The test results are shown in Tables 8 and 9.

[0186] Table 8

[0187] Table 9

[0188] From the above experimental results, it can be seen that during the preparation process of experimental group 4-13, no sodium hydroxide was used to adjust the pH. After measurement, the pH of the system was 6-7, the prepared brepirazole microspheres had no crystallization, no burst release during the in vitro release process, and the cumulative release amount on the 9th day reached 64.4%, and the release was relatively complete; during the preparation process of experimental group 4-14, sodium hydroxide solution was used to adjust the pH of the PVA solution to 11, and the prepared brepirazole microspheres had a small amount of crystallization. During the in vitro release process, the early release was faster, with a cumulative release of 21.0% on the first day, and the later release was very slow, with a cumulative release of 5.7% from the 2nd to the 5th day, and there was an incomplete release problem; during the preparation process of experimental group 4-15, sodium hydroxide solution was used to adjust the pH of the PVA solution to 12, and the prepared brepirazole microspheres had a small amount of crystallization, a slow release rate, only 29.1% released on the 9th day, and almost no release in the later period, and there was also an incomplete release problem.

[0189] Patent JP2013139441A discloses that the preparation process of brepirazole microspheres requires the use of alkali to adjust the PVA external aqueous phase to alkaline, which makes the prepared microspheres susceptible to base catalysis and reduced stability. Alkaline adjustment also increases the complexity of the preparation process, which is not conducive to large-scale industrial production. The microspheres prepared in the present application do not require the use of sodium hydroxide solution to adjust the pH of the PVA solution. The prepared microspheres are free of crystallization, uniformly formed, and have no burst release, ensuring complete release.

[0190] Comparative Example

[0191] Comparative Example 1

[0192] Mix 0.3g of breprazol and 0.7g of PLGA (LA:GA molar ratio of 85:15, intrinsic viscosity of 0.33 dl / g, and molecular weight of 39,000). Add 5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0193] The oil phase was added to 1000 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3400 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0194] Comparative Example 2

[0195] Mix 0.3g of breprazol and 0.7g of PLGA (LA:GA molar ratio of 75:25, intrinsic viscosity of 0.32 dl / g, and molecular weight of 37,000). Add 5mL of dichloromethane and dissolve in a 50-60°C water bath. Stir thoroughly to obtain an oil phase. Simultaneously, prepare a 0.5% PVA solution.

[0196] The oil phase was added to 1000 mL of 0.5% PVA aqueous solution and homogenized and emulsified for 2 minutes using a disperser emulsifier at a speed of 3400 rpm. After the homogenization and emulsification, mechanical stirring was started at room temperature for room temperature curing for 5 hours. A 5 mL sample was taken to observe the microsphere morphology under a microscope, the microspheres were collected by filtration, and freeze-dried.

[0197] The particle size, drug loading, encapsulation efficiency and in vitro release curve of the microspheres obtained in Comparative Examples 1 and 2 were measured according to the methods described in the examples of the specification. The test results are shown in Table 10 and Figure 12.

[0198] Table 10

[0199] The brepirazole microspheres prepared in Comparative Examples 1 and 2 of the present invention were prepared into a formulation. Specifically, a certain amount of microspheres were weighed and dispersed with a prepared suspension (CMC-Na 0.6 wt %, sodium chloride 0.75 wt %, anhydrous citric acid 0.11 wt %, sodium hydroxide 0.055 wt %, and disodium hydrogen phosphate 0.1 wt %) to prepare a suspension having a brepirazole solution concentration of 15 mg / mL.

[0200] The sample was injected into the posterior rectus muscle of SD rats at a dose of 15 mg / kg. Blood samples were collected before administration (pre-dose), 6h (0.25d), 24h (1d), 3d, 5d, 7d, 9d, 11d, 14d, 16d, 18d, 21d, 24d, 28d, 31d, 35d, 42d, 49d, and 56d after administration. The drug concentration in plasma was determined by the established LC-MS method, and the blood drug concentration-time curve of rats was drawn to evaluate the blood drug concentration-time relationship.

[0201] The specific results are shown in Figure 13.

[0202] As shown in Figures 12-13, the cumulative release of the microspheres prepared in Comparative Examples 1 and 2 over the first five days of in vitro release was less than 10%, indicating a slow early release rate. This corresponds to a blood drug concentration greater than 1.0 ng / ml on the first day in rats, but subsequent blood drug concentrations from days 1 to 16 were less than 1 ng / ml, indicating a release plateau and unsuitable for clinical application.

[0203] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0204] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A breprazol microsphere, characterized in that: The microspheres include brepizole and PLGA, wherein the content of brepizole is 45%-75% of the total weight of the microspheres, and the content of PLGA is 55%-25% of the total weight of the microspheres; preferably, the content of brepizole is 45%-70% of the total weight of the microspheres; more preferably, the content of brepizole is 45%-60% of the total weight of the microspheres; Preferably, the intrinsic viscosity of the PLGA is 0.15-0.25 dl / g, and more preferably, the intrinsic viscosity of the PLGA is 0.17-0.23 dl / g.

2. The breprazol microspheres according to claim 1, characterized in that: The molar ratio of PL to PG in the PLGA is 50:50-95:5, preferably, the molar ratio of PL to PG in the PLGA is 50:50-85:15; Preferably, the molecular weight of the PLGA is 8000-32000, more preferably, the molecular weight of the PLGA is 12000-25000, most preferably, the molecular weight of the PLGA is 14000-25000.

3. The brepazine microspheres according to claim 1, characterized in that: The D50 of the microspheres is 20-100 μm, preferably, the D50 of the microspheres is 25-75 μm, more preferably, the D50 of the microspheres is 25-50 μm, and most preferably, the D50 of the microspheres is 25-45 μm.

4. The breprazol microspheres according to claim 1, characterized in that: The brepazine microspheres are of a core-shell structure, wherein the core comprises brepazine and the shell comprises PLGA; Preferably, the shell has an average thickness of 0.5-20 μm.

5. A method for preparing the breprazol microspheres according to claims 1-4, characterized in that: The method comprises the steps of: ① preparing a solution containing brepazole or its salt, PLGA and an organic solvent; ② Under the condition of effectively inhibiting the evaporation of the organic solvent, mixing the solution obtained in step ① with the PVA solution to obtain an O / W emulsion; as well as ③ Under the condition that brepiraazole is precipitated in the form of spherical particles, the organic solvent in the O / W emulsion obtained in step ② is removed.

6. The method for preparing brepazine microspheres according to claim 5, characterized in that: In step ①, the organic solvent is selected from chloroform, dichloroethane, trichloroethane, dichloromethane, carbon tetrachloride, ether, isopropyl ether, ethyl acetate, butyl acetate, benzene, toluene, xylene, acetonitrile and mixtures thereof; preferably, the organic solvent is dichloromethane.

7. The method for preparing breprazol microspheres according to claim 5, characterized in that: In step ①, the volume weight ratio (mL / g) of the organic solvent to brepazine or its salt is 7:1-25:1; preferably, the volume weight ratio (mL / g) of the organic solvent to brepazine or its salt is 10:1-20:1; Preferably, in step ①, the weight ratio of PLGA to brepizole or its salt is 1:1-1:3; more preferably, the weight ratio of PLGA to brepizole or its salt is 1:1-1:2.

3.

8. The method for preparing breprazol microspheres according to claim 5, characterized in that: In step ②, the solubility of the PVA solution is 0.1%-3% (w / v).

9. The method for preparing breprazol microspheres according to claim 5, characterized in that: The step ③ also includes separation and freeze-drying steps.

10. An injectable aqueous suspension, characterized in that: The suspension comprises the microspheres according to any one of claims 1 to 4 or the microspheres prepared by the method according to any one of claims 5 to 9, as well as a pharmaceutically acceptable carrier and water for injection; Preferably, the pharmaceutically acceptable carrier is selected from a suspending agent, a pH adjusting agent, an isotonicity adjusting agent, and a surfactant; Preferably, the suspending agent is selected from sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate and glycerol; Preferably, the isotonicity adjusting agent is selected from sodium chloride, glucose, mannitol and sorbitol; Preferably, the pH adjuster is selected from citric acid, sodium hydroxide and disodium hydrogen phosphate; Preferably, the surfactant is a nonionic surfactant selected from the polysorbate series and the poloxamer series.

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