Pamoate drug sustained-release microsphere and preparation method therefor

Abstract

The present invention relates to a pamoate drug sustained-release microsphere and a preparation method therefor. A sustained-release microsphere formulation for injection prepared according to the preparation method of the present invention has a relatively narrow microsphere particle size distribution, and the release time of active pharmaceutical ingredients of a drug can last for about three to four months. In addition, the microsphere formulation prepared by the present invention can maintain stable plasma concentration in an animal body, and has no relatively large plasma concentration fluctuation within three to four months, thereby better avoiding the occurrence of toxic and side effects.

Description

Pamoate drug sustained-release microspheres and preparation method thereof Technical Field

[0001] The present invention relates to the technical field of sustained-release preparations, and more particularly to pamoate drug sustained-release microspheres and a preparation method thereof. Background Art

[0002] Leuprorelin, triptorelin, and goserelin are all synthetic gonadotropin drugs, currently used clinically to treat hormone-responsive cancers such as prostate cancer and breast cancer, as well as estrogen-dependent diseases such as endometriosis and uterine fibroids, as well as precocious puberty and preventing premature ovulation during in vitro fertilization. These drugs are all pituitary gonadotropin-releasing hormone (GnRH) receptor agonists, which reduce the secretion of testosterone and estradiol by lowering gonadotropin levels. GnRH is a gonadotropin-releasing hormone synthesized and released by GnRH neurons in the hypothalamus, stimulating the anterior pituitary to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH), forming a fundamental step in the hypothalamic-pituitary-gonadal axis. Leuprorelin effectively inhibits the function of the pituitary-gonadal system. Its resistance to proteolytic enzymes and affinity for pituitary GnRH receptors are both stronger than GnRH. Its activity in promoting luteinizing hormone (LH) release is approximately 20 times that of GnRH, and its inhibitory effect on pituitary-gonadal function is also stronger than GnRH. Initially, FSH, LH, estrogen, or androgen levels may transiently increase. Subsequently, due to decreased pituitary responsiveness, the secretion of FSH, LH, and estrogen or androgen is suppressed, thus having a therapeutic effect on sex hormone-dependent diseases (such as prostate cancer and endometriosis).

[0003] Currently, the long-acting sustained-release injections of leuprorelin and triptorelin available in China are all microsphere formulations, with onset times of 1, 3, and 6 months. Among them, the long-acting microsphere injections of triptorelin, marketed under the trade names Dafilin and Dabija, are available in 15mg (3 months) and 22.5mg (6 months) strengths. Both use triptorelin pamoate as the active ingredient, achieving a longer duration of therapeutic effect.

[0004] The double emulsion method is currently the most commonly used method for preparing microsphere preparations. The double emulsion method (W / O / W) first dissolves the active pharmaceutical ingredient in an internal aqueous phase and emulsifies it with an oil phase containing biodegradable materials to form a colostrum. The microspheres are then solidified and formed in the form of a double emulsion. The double emulsion method for preparing microspheres is currently one of the most commonly used methods for industrial production. However, due to the introduction of an internal aqueous phase during the double emulsion preparation process, the microspheres inevitably develop pores of varying sizes, which causes a burst release of the microspheres in the body. The burst release phenomenon can lead to excessively high blood drug concentrations in the early stages of medication, making the drug's toxic side effects more likely to occur and greatly reducing the drug's sustained-release cycle time. Summary of the Invention

[0005] Due to its poor water solubility, the conventional double emulsion method cannot produce a sustained-release formulation that can maintain the drug for three or even four months. It also cannot maintain stable blood drug concentrations and good encapsulation efficiency, and the resulting microspheres are uneven in shape and regularity. The inventors discovered that a modified emulsification method can overcome these problems by preparing microsphere preparations containing the active pharmaceutical ingredient and a biocompatible polymer. This method requires controlling the following steps:

[0006] 1. Controlling the particle size of the active pharmaceutical ingredient, pamoate. The particles of the active pharmaceutical ingredient, pamoate, are crushed, ground, or subjected to solvent precipitation treatment so that the treated particles can be evenly dispersed in an organic solvent to form a suspension emulsion. This improves the encapsulation efficiency of the microspheres and allows the active pharmaceutical ingredient to be evenly distributed within the microspheres, facilitating a sustained-release effect.

[0007] 2. Control the mass percentage of the active pharmaceutical ingredient and the biocompatible polymer material. During the microsphere preparation process, the mass percentage of the active pharmaceutical ingredient and the biocompatible polymer material is controlled to ensure that the microspheres contain enough active pharmaceutical ingredients to achieve a sustained release effect for 3-4 months without affecting the appearance of the microspheres, causing spherical breakage and irregular spherical shapes.

[0008] 3. Control the mass percentage of the biocompatible polymer to the oil phase. During the microsphere preparation process, if the biocompatible polymer content in the oil phase is too low, subsequent emulsion droplets will easily break and delaminate. If it is too high, the viscosity of the oil phase will be too high, resulting in insufficient encapsulation efficiency, poor microsphere formation, and irregular spheres. Therefore, it is necessary to control the mass percentage of the biocompatible polymer to the oil phase.

[0009] In order to achieve the above objectives, the technical solutions of the present invention are as follows:

[0010] A) processing the pharmaceutical active ingredient to obtain pharmaceutical active ingredient microparticles;

[0011] B) dissolving a biocompatible polymer material in an organic solvent to form an organic solution, adding the above-mentioned active pharmaceutical ingredient microparticles, and dispersing the active pharmaceutical ingredient in the organic solution to prepare an oil phase;

[0012] C) dissolving a surfactant in water to prepare an external aqueous phase;

[0013] D) adding the prepared oil phase to the above-mentioned external aqueous phase and mixing and emulsifying to obtain a mixed emulsion;

[0014] E) solidifying and drying the mixed emulsion to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

[0015] Preferably, the active ingredient of the drug is a small molecule drug, which can be any one of the following or a mixture of several of them: leuprorelin pamoate, triptorelin pamoate, goserelin pamoate.

[0016] Preferably, the particle size D50 of the active pharmaceutical ingredient after pulverization, grinding or solvent precipitation is 1.3-3.5 μm.

[0017] Preferably, the mass percentage of the active pharmaceutical ingredient particles and the biocompatible polymer material is 3-7.5 wt %.

[0018] Preferably, the mass percentage of the biocompatible polymer material to the oil phase is 9-28 wt %.

[0019] Preferably, the mass percentage concentration of the surfactant in the external aqueous phase is 0.1-5.0 wt %.

[0020] Preferably, the ratio of the mass of the organic solvent to the volume of the external aqueous phase is 1:50-1:300 (g / mL).

[0021] Preferably, the organic solvent is dichloromethane.

[0022] Preferably, the biocompatible polymer material is one or a mixture of poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(glycolide) (PGA) and poly(vinyl lactone) (PCL).

[0023] Preferably, the surfactant is one or a mixture of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pluronic F88, Pluronic F127, gelatin, glycine, lysine, histidine, arginine, aspartic acid, glutamic acid, span and Tween.

[0024] Preferably, in step E), the mixed emulsion is solidified into wet microspheres, a protective agent is added, and the microspheres are dried.

[0025] Preferably, the preparation method further comprises the following steps: after obtaining the mixed emulsion, stirring, solidifying, filtering, and washing the mixed emulsion to form wet microspheres, and adding a protective agent to the wet microspheres and then drying them to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

[0026] Preferably, the protective agent is one or a mixture of polyethylene glycol (PEG), gelatin, glycerol, mannitol, sucrose, trehalose, lactose, glucose, propylene glycol, sorbitol, zinc chloride, zinc sulfate, zinc acetate and human serum albumin.

[0027] Preferably, other auxiliary materials are added at the same time as the protective agent, and the other auxiliary materials are surfactants and / or excipients.

[0028] As another aspect of the present invention, there is also provided a sustained-release microsphere for injection prepared according to any one of the above methods.

[0029] According to the present invention, in step B) of the method for preparing sustained-release injectable microspheres, the oil phase may be a suspension containing solid drug particles. The active pharmaceutical ingredient may be one or more of leuprorelin pamoate, triptorelin pamoate, and goserelin pamoate.

[0030] In an embodiment according to the present invention, the mass percentage of the active pharmaceutical ingredient particles to the biocompatible polymer material is 3-7.5 wt %, calculated as follows: mass percentage of the active pharmaceutical ingredient particles to the biocompatible polymer material = mass of the active pharmaceutical ingredient particles / mass of the biocompatible polymer material * 100%.

[0031] According to the present invention, in step D) of the method for preparing sustained-release injectable drug microspheres, the emulsion can be prepared using a high-shear emulsifier, an ultrasonicator, a high-pressure homogenizer, or a combination thereof. The emulsion needs to be maintained at a temperature between 0°C and 15°C. The speed of the high-shear emulsifier can be 3,000 rpm to 20,000 rpm, and the shear time can be 0.5 min to 10 min. The pressure of the high-pressure homogenizer can be 100 bar to 800 bar, and the number of continuous homogenizations can be 2 to 6 times.

[0032] In the present invention, the mass percentage concentration of the biocompatible polymer material in the organic solution is calculated as follows: mass percentage concentration of the biocompatible polymer material in the organic solution = mass of the biocompatible polymer material / (mass of the biocompatible polymer material + mass of the organic solvent) * 100%.

[0033] In an embodiment according to the present invention, the mass percentage concentration of PLGA in the organic solution is preferably 9-28 wt %.

[0034] In a preferred embodiment according to the present invention, the biocompatible polymer material is poly(lactic-co-glycolic acid) (PLGA), and the molar ratio of glycolide (LA) to lactide (GA) in the PLGA is 50:50-85:15, the molecular weight is 5-100 kilodaltons, and the viscosity characteristics are 0.1-0.6 dL / g; wherein, the type and molecular weight of PLGA include but are not limited to: PLGA (LA:GA is 50:50; Mw 10,000-90,000 Daltons), PLGA (LA:GA is 55:45; Mw 15,000-90,000 Daltons), PLGA (LA:GA is 65:35; Mw 15,000-90,000 Daltons), PLGA (LA:GA is 75:25; Mw 10,000-90,000 Daltons), or PLGA (LA:GA is 85:15; Mw 55,000-90,000 Daltons).

[0035] In another preferred embodiment according to the present invention, the biocompatible polymer material is polylactic acid (PLA) with a molecular weight of 10-55 kilodaltons and a viscosity characteristic of 0.1-0.75 dL / g, such as PLA (Mw 8000-55000 Daltons).

[0036] According to the present invention, in step C) of the method for preparing sustained-release microspheres for injection, the surfactant can be selected from one or a mixture of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pluronic F88, Pluronic F127, gelatin, glycine, lysine, histidine, arginine, aspartic acid, glutamic acid, span and Tween; preferably, the surfactant is PVA, PVP or a combination thereof, and particularly preferably, the surfactant is PVA.

[0037] In the present invention, the mass percentage concentration of the surfactant in the external water phase is calculated as follows: mass percentage concentration of the surfactant in the external water phase = mass of the surfactant / (mass of the surfactant+mass of water)*100%.

[0038] In an embodiment according to the present invention, the mass percentage concentration of the surfactant in the external aqueous phase is preferably 0.1-5.0 wt %, more preferably 0.1-2.5 wt %.

[0039] In a preferred embodiment of the present invention, in step D) of the method for preparing sustained-release microspheres for injection, the ratio of the mass of the organic solvent in the oil phase to the volume of the external aqueous phase is preferably 1:50-1:300, expressed in g / mL.

[0040] According to the present invention, in step E) of the method for preparing sustained-release microspheres for injection, if stirring is performed, the stirring speed may be 1500-5000 rpm, and the stirring time may be 15-120 min.

[0041] According to the present invention, in step E) of the method for preparing sustained-release microspheres for injection, the curing temperature may be 5° C.-45° C., and the curing time may be 2 h-24 h.

[0042] According to the present invention, a protective agent is added to the wet microspheres obtained by solidifying the mixed emulsion in step E) of the method for preparing sustained-release injectable drug microspheres. The protective agent is an excipient added to the wet microspheres to increase the fluidity and dispersibility of the resulting microsphere dry powder, regulate the osmotic pressure of the microspheres, and protect the stability and activity of the small molecule drug. The protective agent can be selected from one or a mixture of polyethylene glycol (PEG), gelatin, glycerol, mannitol, sucrose, trehalose, lactose, glucose, propylene glycol, sorbitol, zinc chloride, zinc sulfate, zinc acetate, and human serum albumin; preferably, the protective agent is mannitol.

[0043] The present invention also provides a sustained-release microsphere for injection, comprising the sustained-release microsphere for injection prepared according to the above method, wherein the sustained-release microsphere for injection comprises active ingredients of leuprorelin pamoate, triptorelin pamoate, and goserelin pamoate, a biocompatible polymer carrier material, and other pharmaceutically acceptable excipients.

[0044] The pharmaceutically acceptable other auxiliary materials include surfactants and excipients. In addition, the pharmaceutically acceptable other auxiliary materials may also include protective agents.

[0045] The sustained-release microsphere preparation for injection has a sustained-release time specification of about 3-4 months, i.e., about 12 to 16 weeks, wherein the difference between "about" and "approximately" is "(±1-6 days)".

[0046] According to the present invention, the sustained-release microspheres for injection prepared by the above method have a microsphere particle size (D50) of 5-200 μm. Not only does the microsphere particle size distribution have a narrow (SPAN value (SPAN = (D90-D10) / D50) less than 2.8), but the encapsulation efficiency is significantly improved, the burst rate is reduced, and the release time is prolonged. In addition, compared with existing leuprorelin, triptorelin, and goserelin products, the sustained-release microsphere preparation for injection containing the active pharmaceutical ingredient according to the present invention provides an optimized preparation method, achieving a more sustained release time than the currently marketed product of the active pharmaceutical ingredient. Based on a 3-month marketed product, the preparation method of the present invention can achieve a 4-month sustained release time.

[0047] The technical solution of the present invention has the following beneficial effects:

[0048] The preparation method of the present invention is relatively simple and easy to implement, can be used for industrial operation, and has universal applicability; at the same time, according to the preparation method, a sustained-release microsphere preparation for injection with a narrow microsphere particle size distribution is prepared, and its encapsulation rate is significantly improved and the release time is prolonged.

[0049] 1) The present invention provides a novel microsphere preparation method for leuprorelin, triptorelin, and goserelin pamoate. The active pharmaceutical ingredients are further crushed and added to the oil phase to achieve a better dispersion effect, so that the oil phase forms a suspension containing solid drug particles, which not only improves the encapsulation efficiency but also stabilizes and sustains the release of the drug.

[0050] 2) The sustained-release microsphere preparation for injection prepared according to the preparation method of the present invention has a narrow particle size distribution and can release the active pharmaceutical ingredient for approximately three to four months. Furthermore, the microsphere preparation prepared according to the present invention can maintain stable blood drug concentrations in animals, with no significant fluctuations in blood drug concentrations over three to four months, thereby effectively preventing the occurrence of toxic side effects.

[0051] 3) In vivo pharmacodynamic studies of sustained-release microspheres prepared by the present invention in rats revealed that the suppression of testosterone in rat serum remained effective for three to four months, indicating that the active ingredient remains within therapeutically effective concentrations for three to four months (see Examples 1-5). The microspheres prepared by the present invention maintain relatively excellent performance even when the molar mass of the biocompatible polymer is less than 25,000 daltons (see Examples 6-7). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is an electron microscope image of the microspheres prepared in Example 1;

[0053] FIG2 is an electron microscope image of the microspheres prepared in Example 3;

[0054] FIG3 is an electron microscope image of the microspheres prepared in Example 5;

[0055] FIG4 is an in vitro cumulative release curve of Examples 1-7;

[0056] FIG5 is a graph showing the blood drug concentration-time of Examples 1-5 and Comparative Formulations 1-3 in rats;

[0057] FIG6 is a graph showing testosterone concentration-time in rats of Examples 1-5 and Comparative Formulations 1-3;

[0058] FIG7 is a graph showing the blood drug concentration-time in rats of Example 6-7. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the present invention should not be limited to the specific embodiments disclosed below.

[0060] In the following examples, the preparation method of microspheres for injection according to the present invention is used to prepare microsphere preparations for injection, wherein the molecular formula of leuprorelin pamoate used is C 59 H 84 N 16 O 12 ·C 23 H 16 O6, molar mass 1597.8g / mL; the molecular formula of triptorelin pamoate used is C 87 H 98 N 18 O 19 , with a molar mass of 1699.8 g / mL (CAS: 124508-66-3); the molecular formula of goserelin pamoate used is C 59 H 84 N 18 O 14 ·C 23 H 16 O6, molar mass is 1657.8 g / mL.

[0061] The active pharmaceutical ingredients were pulverized using a micronized jet mill. The triptorelin pamoate API was pulverized to obtain powders with a D50 of 1.3 μm, 2.8 μm, and 3.4 μm for the example experiments, and a 4.6 μm powder for the comparative example experiments. The leuprorelin pamoate API was pulverized to obtain a D50 of 3.1 μm for the example experiments, and a 6.2 μm powder for the comparative example experiments. The goserelin pamoate API was pulverized to obtain a D50 of 3.4 μm for the example experiments, and a 6.7 μm powder for the comparative example experiments. The methods used in the following examples are commonly used by those skilled in the art unless otherwise specified.

[0062] Example 1

[0063] 69.89 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 526 g of dichloromethane, and 2.46 g of triptorelin pamoate micropowder (D50 of 1.3 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0064] The average particle size (D50) of the microspheres of the preparation was 31 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.22. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 97.37%.

[0065] Example 2

[0066] 21.3 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 105 g of dichloromethane, and 1.57 g of triptorelin pamoate micropowder (D50 of 2.8 μm) was added and uniformly dispersed to prepare an oil phase. 20,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0067] The average particle size (D50) of the microspheres of the preparation was 34 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.21. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 94.77%.

[0068] Example 3

[0069] 26.3 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 80.8 g of dichloromethane, and 1.57 g of triptorelin pamoate micropowder (D50 of 2.8 μm) was added and uniformly dispersed to prepare an oil phase. 34,000 mL of a 1.0% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0070] The average particle size (D50) of the microspheres of the preparation was 27 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.25. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 93.63%.

[0071] Example 4

[0072] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of leuprorelin pamoate micropowder (D50 of 2.8 μm) was added and uniformly dispersed to prepare an oil phase; 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase; and the colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a leuprorelin pamoate microsphere preparation.

[0073] The average particle size (D50) of the microspheres of the preparation was 38 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.19. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 89.12%.

[0074] Example 5

[0075] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of goserelin pamoate micropowder (D50 of 2.8 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent is removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres are volatilized and solidified for 6 hours. The emulsion after solidification is filtered and collected by a filter, and the microspheres are washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres are freeze-dried to obtain a goserelin pamoate microsphere preparation.

[0076] The average particle size (D50) of the microspheres of the preparation was 41 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.26. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 90.62%.

[0077] Example 6

[0078] 28.3 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 10,000-25,000 Daltons) was dissolved in 107 g of dichloromethane, and 1.57 g of triptorelin pamoate micropowder (D50 of 1.8 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0079] The average particle size (D50) of the microspheres of the preparation was 29 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.18. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 94.61%.

[0080] Example 7

[0081] 27.4 g of PLGA (with a molar ratio of glycolide to lactide of 75:25 and an Mw of 10,000-25,000 Daltons) was dissolved in 78 g of dichloromethane. 1.62 g of triptorelin pamoate micropowder (D50 of 1.8 μm) was added and uniformly dispersed to prepare the oil phase. 20,000 mL of a 0.5% (by weight) aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and external aqueous phase were mixed using a high-shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the double emulsion at a stirring speed of 300 rpm and a temperature of 25°C, and the microspheres were volatilized and solidified for 6 hours. The solidified emulsion was filtered and collected using a filter, washed with distilled water multiple times, and then collected. A protective agent was added and freeze-dried to obtain the triptorelin pamoate microsphere preparation.

[0082] The average particle size (D50) of the microspheres of the preparation was 27 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.22. The obtained sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 92.12%.

[0083] Test Example 1: Particle Size Detection of Pharmaceutical Active Ingredients

[0084] Take approximately 10 mg of the active ingredient powder, add approximately 1 ml of olive oil, vortex mix, and ultrasonically disperse until no particles are noticeably agglomerated. The dispersion is then measured using a laser particle size analyzer (Malvern Mastersizer 3000 or equivalent) (Chinese Pharmacopoeia 2020 General Chapter 0982, Method 3). Olive oil is used as the dispersion medium, stirring at 2000 rpm, an opacity range of 5%-15%, a background / sample measurement time of 10 seconds, a refractive index of the dispersion medium of 1.47, a refractive index of the particles of 1.65, an absorptivity of 0.1, and a particle density of 1 g / cm³. Three measurements are performed consecutively, and the average of the three results is reported as the D50 test result.

[0085] Test Example 2: In vitro release rate test

[0086] In this test example 2, the in vitro release rate test was conducted using the sustained-release microsphere preparations of leuprorelin pamoate, triptorelin pamoate, and goserelin pamoate prepared in Examples 1-7 above. The specific method is as follows:

[0087] 20 mg of each sustained-release microsphere preparation prepared in Examples 1-7 was accurately weighed and added to a 15 mL centrifuge tube. 15 mL of preheated release medium (0.05 M pH 7.4 phosphate buffer) was then added. The tube was then placed in a 37°C incubator. At the corresponding time points, 1 mL of sample was taken and 1 mL of the corresponding release medium was added. The 24-hour burst release rate and total cumulative release rate of Examples 1-7 obtained from the tests are shown in Table 1:

[0088] Table 1: 24-hour burst release rate and total cumulative release rate of Examples 1-7 obtained from the test:

[0089] See FIG4 for the in vitro cumulative release curves of Examples 1-7 obtained from the tests.

[0090] Comparative Example 1: Comparison of large particle size micropowders of active pharmaceutical ingredients

[0091] In Comparative Example 1, based on the above-described Examples 1-7, Comparative Formulations 1-3 compared microspheres prepared using triptorelin pamoate with a D50 particle size of 4.6 μm, leuprorelin pamoate with a D50 particle size of 6.2 μm, and goserelin pamoate with a D50 particle size of 6.7 μm. Comparative Formulations 1-3 also employed an emulsification method to prepare the microspheres, the primary difference being that the particle size of the active pharmaceutical ingredient micropowder was greater than 3.5 μm.

[0092] Preparation of Comparative Formulation 1

[0093] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of triptorelin pamoate micropowder (D50 of 4.6 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0094] The average particle size (D50) of the microspheres of the preparation was 44 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.34. The prepared sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 78.48%. The in vitro release duration was 98 days.

[0095] Preparation of Comparative Formulation 2

[0096] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of leuprorelin pamoate micropowder (D50 of 4.6 μm) was added and uniformly dispersed to prepare an oil phase; 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase; and the colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a leuprorelin pamoate microsphere preparation.

[0097] The average particle size (D50) of the microspheres of the preparation was 38 μm, and the SPAN value (SPAN=(D90-D10) / D50) was 1.31. The prepared sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 80.43%. The in vitro release duration was 96 days.

[0098] Preparation of Comparative Formulation 3

[0099] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of goserelin pamoate micropowder (D50 of 4.6 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent is removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres are volatilized and solidified for 6 hours. The emulsion after solidification is filtered and collected by a filter, and the microspheres are washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres are freeze-dried to obtain a goserelin pamoate microsphere preparation.

[0100] The average particle size (D50) of the microspheres of the preparation was 46 μm, and the SPAN value (SPAN = (D90-D10) / D50) was 1.29. The prepared sustained-release microspheres were quantitatively determined by HPLC, and the encapsulation efficiency was 77.65%. The in vitro release duration was 94 days.

[0101] According to the in vitro release test method described in Test Example 2 of the present invention, the in vitro release durations of comparative preparations 1-3 were all less than 120 days.

[0102] Comparative Example 2: Comparison of un-crushed active ingredients of medicines

[0103] In Comparative Example 2, based on the invention described in Examples 1-7, Comparative Formulation 4 was compared with microspheres prepared using unmicronized triptorelin pamoate with a D50 particle size of 17.3 μm. Comparative Formulation 4 also employed an emulsification method to prepare microspheres, differing primarily in that the particle size of the active pharmaceutical ingredient micropowder was greater than 3.5 μm.

[0104] Preparation of Comparative Formulation 4

[0105] 27.2 g of PLGA (wherein the molar ratio of glycolide to lactide is 75:25, Mw = 25,000-55,000 Daltons) was dissolved in 149 g of dichloromethane, and 1.83 g of triptorelin pamoate micropowder (D50 of 17.3 μm) was added and uniformly dispersed to prepare an oil phase. 28,000 mL of a 0.5% by weight aqueous solution of polyvinyl alcohol (PVA) for injection was prepared as the external aqueous phase. The colostrum and the external aqueous phase were mixed in a high shear emulsifier at 0°C at a shear rate of 4,000 rpm for 10 minutes to form a double emulsion. The organic solvent was removed from the emulsion at a stirring speed of 300 rpm and a temperature of 25° C., and the microspheres were volatilized and solidified for 6 hours. The emulsion after solidification was filtered and collected by a filter, and the microspheres were washed multiple times with distilled water, and then collected. After adding a protective agent, the microspheres were freeze-dried to obtain a triptorelin pamoate microsphere preparation.

[0106] Scanning electron microscopic observation of the microspheres in this preparation revealed numerous broken microspheres, uneven morphology, and numerous surface particles, indicating that their appearance did not meet the requirements for a microsphere preparation. Quantitative analysis of the prepared sustained-release microspheres by HPLC revealed an encapsulation efficiency of 45.12%. Due to the large size of the particles, the emulsions were unable to encapsulate the microspheres, resulting in the loss of most of the drug.

[0107] Test Example 3: Pharmacokinetic and pharmacodynamic experiments of a single dose in rats

[0108] In Test Example 3, pharmacokinetic and pharmacodynamic experiments were conducted in rats using the sustained-release microsphere preparations for injection of leuprorelin pamoate, triptorelin pamoate, and goserelin pamoate prepared in Examples 1-7 and Comparative Preparations 1-3, respectively. The specific methods are as follows:

[0109] Adult male SD rats weighing 300-400 g were used as research subjects and administered intramuscularly. Leuprorelin pamoate, triptorelin pamoate, and goserelin pamoate sustained-release microsphere formulations for injection, prepared according to the present invention, were administered at a dose of 1.6 mg / kg (Examples 1-7 and Comparative Formulations 1-3). At specific times after administration, 0.3 mL of blood was collected from the jugular vein and transferred to a centrifuge tube containing 33.33 mg / mL aprotinin in EDTA-K2 anticoagulant. The tube was inverted 5-10 times to thoroughly mix the anticoagulant and blood, and then temporarily stored at room temperature on wet ice. Plasma samples were then separated and stored frozen at -80°C using a high-speed refrigerated centrifuge at 4000 rpm for 15 minutes. LC / MS-MS was used to determine the drug concentration and testosterone concentration (ng / mL) in the plasma samples at each time point. The in vivo blood drug concentration-time curves are shown in Figures 5 and 7; the in vivo testosterone concentration-time curve is shown in Figure 6.

[0110] As can be seen from Figure 5, the sustained-release microsphere preparation for injection prepared according to the method of the present invention can release the drug in rats for up to 120 days, and the drug concentration can be maintained in a stable range within 4 months. In contrast, the drug duration of the comparative preparation is 95 days. As can be seen from Figure 5, the sustained-release microsphere preparation for injection prepared according to the method of the present invention can effectively suppress the testosterone concentration level in rat serum to an effective state (1.0 ng / mL), and the action time can be stably maintained for up to 120 days.

[0111] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing pamoate drug sustained-release microspheres, characterized in that: The preparation method comprises the following steps: A) processing the pharmaceutical active ingredient to obtain pharmaceutical active ingredient microparticles; B) dissolving a biocompatible polymer material in an organic solvent to form an organic solution, adding the pharmaceutically active ingredient microparticles, and dispersing the pharmaceutically active ingredient in the organic solution to prepare an oil phase; C) dissolving a surfactant in water to prepare an external aqueous phase; D) adding the prepared oil phase to the external aqueous phase and mixing to emulsify to obtain a mixed emulsion; E) solidifying and drying the mixed emulsion to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

2. The preparation method according to claim 1, characterized in that The active ingredient of the medicine is any one or a mixture of the following: leuprorelin pamoate, triptorelin pamoate and goserelin pamoate.

3. The preparation method according to claim 1 or 2, characterized in that The particle size D50 of the active pharmaceutical ingredient microparticles is 1.3-3.5 μm.

4. The preparation method according to claim 1 or 2, characterized in that The mass percentage of the pharmaceutical active ingredient particles and the biocompatible polymer material is 3-7.5 wt %.

5. The preparation method according to claim 1 or 2, characterized in that The mass percentage concentration of the biocompatible polymer material in the organic solution is 9-28 wt %.

6. The preparation method according to claim 1 or 2, characterized in that The mass percentage concentration of the surfactant in the external water phase is 0.1-5.0 wt %.

7. The preparation method according to claim 1 or 2, characterized in that The ratio of the mass of the organic solvent to the volume of the external aqueous phase is 1:50-1:300 g / mL.

8. The preparation method according to claim 1 or 2, characterized in that The organic solvent is dichloromethane.

9. The preparation method according to claim 1 or 2, characterized in that: The biocompatible polymer material is one or a mixture of polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolide (PGA) and polyacetic acid lactone (PCL).

10. The preparation method according to claim 1 or 2, characterized in that: The surfactant is one or a mixture of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pluronic F88, Pluronic F127, gelatin, glycine, lysine, histidine, arginine, aspartic acid, glutamic acid, span and Tween.

11. The preparation method according to claim 1 or 2, characterized in that: The following steps are also included: After obtaining the mixed emulsion, the mixed emulsion is stirred, solidified, filtered, and washed with water to form wet microspheres, and a protective agent is added to the wet microspheres. The protective agent is one or a mixture of polyethylene glycol (PEG), gelatin, glycerol, mannitol, sucrose, trehalose, lactose, glucose, propylene glycol, sorbitol, zinc chloride, zinc sulfate, zinc acetate, and human serum albumin. After adding the protective agent, the mixed emulsion is dried to obtain pamoate drug sustained-release microspheres. 12 . The preparation method according to claim 11 , wherein the protective agent and other pharmaceutically acceptable excipients are added to the wet microspheres.

13. The preparation method according to claim 12, characterized in that The other auxiliary materials are surfactants and / or excipients.

14. Pamoate sustained-release microspheres prepared according to any one of the preceding claims.

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