Dihydroxynaphthoate drug sustained release microsphere and preparation method thereof

Abstract

The invention relates to pamoate drug sustained release microspheres and a preparation method thereof. According to the sustained-release microsphere preparation for injection prepared by the preparation method disclosed by the invention, the particle size distribution of the microspheres is relatively narrow, and the release time of active pharmaceutical ingredients can last for about three months to four months. Besides, the microsphere preparation prepared by the invention can maintain stable blood concentration in an animal body, has no large fluctuation of the blood concentration within 3-4 months, and better avoids the occurrence of toxic and side effects.

Description

Technical Field

[0001] This invention relates to the technical field of sustained-release formulations, and more specifically to a sustained-release microsphere for a dihydroxynaphthyl acid salt and its preparation method. Background Technology

[0002] Leuprorelin, triptorelin, and goserelin are all synthetic gonadotropin drugs, currently used clinically primarily to treat hormone-responsive cancers such as prostate and breast cancer, as well as estrogen-dependent diseases such as endometriosis and uterine fibroids, and to treat precocious puberty and prevent premature ovulation after in-vitro fertilization. These drugs belong to the class of pituitary gonadotropin-releasing hormone (GnRH) receptor agonists, which reduce the secretion of testosterone and estradiol by lowering gonadotropin levels. GnRH is synthesized and released by GnRH neurons in the hypothalamus, stimulating the anterior pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH), forming the basic steps of the hypothalamic-pituitary-gonadal axis. Leuprorelin effectively inhibits the function of the pituitary-gonadal system. It exhibits stronger resistance to proteolytic enzymes and greater affinity for pituitary GnRH receptors than GnRH, and its activity in promoting luteinizing hormone (LH) release is approximately 20 times that of GnRH. Its inhibitory effect on pituitary-gonadal function is also stronger than that of GnRH. Initially, FSH, LH, estrogen, or androgen levels may show a transient 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 injectable products of leuprorelin and triptorelin available in China are all microsphere formulations, with onset times of 1 month, 3 months, and 6 months. Among them, the long-acting microsphere injectable products of triptorelin, marketed under the brand names Triptorelin and Dapijia, are available in strengths of 15mg (3 months) and 22.5mg (6 months), respectively. The active pharmaceutical ingredient in both formulations uses triptorelin dihydroxynaphthyl salt, resulting in a longer duration of therapeutic effect.

[0004] Double emulsion (W / O / W) is currently the most common preparation method for microsphere formulations. In W / O / W, the active pharmaceutical ingredient is first dissolved in an aqueous phase and then emulsified with an oil phase containing biodegradable materials to form a proemulsion. The microspheres are then solidified and shaped in the form of a double emulsion. W / O / W is one of the most common methods for industrial-scale microsphere production. However, because an aqueous phase is introduced during the W / O / W process, it is difficult to avoid the presence of pores of varying sizes in the microspheres, leading to burst release in vivo. Burst release results in excessively high blood drug concentrations at the beginning of administration, making the drug more susceptible to toxic side effects and significantly reducing the duration of sustained drug release. Summary of the Invention

[0005] Due to its poor water solubility, the active pharmaceutical ingredient, dihydroxynaphthyl salt, cannot be used to prepare sustained-release formulations that maintain drug release for 3 to 4 months using conventional double emulsion methods. Furthermore, it is impossible to maintain stable blood drug concentrations and good encapsulation efficiency, resulting in microspheres with irregular shapes and uniformity. The inventors discovered that a modified emulsification method can overcome these problems by preparing microsphere formulations containing both the active pharmaceutical ingredient and biocompatible polymers. This method requires control of the following steps:

[0006] 1. Controlling the particle size of the active pharmaceutical ingredient (API) bis(hydroxynaphthyl) salt. The API particles are pulverized, ground, or solvent-precipitated to ensure uniform dispersion in an organic solvent, forming a suspension emulsion. This improves the encapsulation efficiency of the microspheres and ensures the API is evenly distributed within them, thus promoting a sustained-release effect.

[0007] 2. Control the mass percentage of the active pharmaceutical ingredient to the biocompatible polymer. During the microsphere preparation process, control the mass percentage of the active pharmaceutical ingredient to the biocompatible polymer to ensure that the microspheres contain sufficient active pharmaceutical ingredient to achieve a sustained-release effect of 3-4 months, while not affecting the appearance of the microspheres and avoiding the occurrence of broken or irregular spherical shapes.

[0008] 3. Control the mass percentage of biocompatible polymeric material to the oil phase. In the microsphere preparation process, too low a content of biocompatible polymeric material in the oil phase can lead to subsequent droplet demulsification and stratification, while too high a content results in excessively high viscosity of the oil phase, causing insufficient encapsulation, poor microsphere formation, and irregular sphere shapes. Therefore, it is necessary to control the mass percentage of biocompatible polymeric material to the oil phase.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

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

[0011] B) Dissolve the biocompatible polymer material in an organic solvent to form an organic solution, add the above-mentioned active pharmaceutical ingredient microparticles, and disperse the active pharmaceutical ingredient in the organic solution to obtain an oil phase;

[0012] C) Dissolve the surfactant in water to prepare an external aqueous phase;

[0013] D) The obtained oil phase is added to the above-mentioned external aqueous phase and mixed and emulsified to obtain a mixed emulsion;

[0014] E) The mixed emulsion is cured and dried to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

[0015] Preferably, the active pharmaceutical ingredient is a small molecule drug, which may be any one or a mixture of the following: leuprorelin dihydroxynaphthyl acid, triptorelin dihydroxynaphthyl acid, or goserelin dihydroxynaphthyl acid.

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

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

[0018] Preferably, the mass percentage of the biocompatible polymeric 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 several of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), and polyacetin lactone (PCL).

[0023] Preferably, the surfactant is one or a mixture of several of the following: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pranic F88, Pranic 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 then dried.

[0025] Preferably, the preparation method further includes the following steps: 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 before drying to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

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

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

[0028] As another aspect of the present invention, an injectable sustained-release drug microsphere prepared according to any of the above methods is also provided.

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

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

[0031] According to the present invention, in step D) of the method for preparing sustained-release microspheres for injection, the emulsion preparation method can be a high-shear emulsifier, an ultrasonic instrument, a high-pressure homogenizer, or a combination thereof. The emulsion needs to be maintained between 0-15°C; the high-shear emulsifier can operate at a speed of 3000 rpm-20000 rpm, with a shearing time of 0.5 min-10 min; the high-pressure homogenizer can operate at a pressure of 100 bar-800 bar, and the number of continuous homogenization cycles can be 2-6.

[0032] In this 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 of the present invention, the mass percentage concentration of PLGA in the organic solution is preferably 9-28 wt%.

[0034] In a preferred embodiment of the present invention, the biocompatible polymer material is polylactic-glycolic acid copolymer (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 is 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 10000-90000 Daltons), PLGA (LA:GA is 55:45; Mw 15000-90000 Daltons), PLGA (LA:GA is 65:35; Mw 15000-90000 Daltons), PLGA (LA:GA is 75:25; Mw 10000-90000 Daltons), or PLGA (LA:GA is 85:15; Mw 55000-90000 Daltons).

[0035] In another preferred embodiment of the invention, the biocompatible polymer material is polylactic acid (PLA) with a molecular weight of 10-55 kilodaltons and a viscosity of 0.1-0.75 dL / g, for example, 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 may be selected from one or a mixture of several of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pranic F88, Pranic 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 this invention, the mass percentage concentration of the surfactant in the external aqueous phase is calculated as follows: mass percentage concentration of surfactant in the external aqueous phase = surfactant mass / (surfactant mass + water mass) * 100%.

[0038] In embodiments of 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 mass ratio of the organic solvent in the oil phase to the volume of the external aqueous phase is preferably 1:50-1:300, 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 can be 1500-5000 rpm and the stirring time can 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 can be 5°C-45°C and the curing time can be 2h-24h.

[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 microspheres for injection. This protective agent is an excipient added to the wet microspheres to increase the flowability and dispersibility of the obtained microsphere dry powder, and to adjust the osmotic pressure of the microspheres and protect the stability and activity of the small molecule drug. The protective agent may be selected from one or a mixture of several 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 sustained-release microspheres for injection prepared according to the above method, wherein the sustained-release microspheres for injection comprise active ingredients such as leuprorelin dihydroxynaphthyl acid, triptorelin dihydroxynaphthyl acid, and goserelin dihydroxynaphthyl acid, a biocompatible polymeric carrier material, and other pharmaceutically acceptable excipients.

[0044] Other pharmaceutically acceptable excipients include surfactants and excipients. Additionally, other pharmaceutically acceptable excipients may include protective agents.

[0045] The sustained-release microsphere formulation for injection has a duration of approximately 3-4 months, or approximately twelve to sixteen weeks. The difference between "approximately" and "about" is "(±1-6 days)".

[0046] According to the present invention, the injectable sustained-release microspheres prepared by the above method have a microsphere particle size (D50) of 5-200 μm, exhibiting not only a narrow microsphere size distribution (SPAN value (SPAN = (D90-D10) / D50) less than 2.8), but also significantly improved encapsulation efficiency, reduced burst release rate, and prolonged release time. Furthermore, compared to existing leuprorelin, triptorelin, and goserelin products, the injectable sustained-release microsphere formulation containing the active pharmaceutical ingredient according to the present invention provides an optimized preparation method, achieving a longer sustained release time than currently marketed products of the same active pharmaceutical ingredient. Based on a 3-month marketed product, a sustained release time of 4 months can be achieved using the preparation method of the present invention.

[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 and can be used for industrial operation, and has universality; at the same time, the preparation method produces an injectable sustained-release microsphere formulation with a narrow microsphere size distribution, and its encapsulation efficiency is significantly improved and the release time is prolonged.

[0049] 1) This invention provides a novel method for preparing microspheres of leuprorelin, triptorelin and goserelin dihydroxynaphthyl salts. The active pharmaceutical ingredient can be further pulverized and added to the oil phase to obtain better dispersion effect, so that the oil phase forms a suspension containing solid drug particles, which not only improves the encapsulation rate, but also makes the drug release stable and continuous.

[0050] 2) The injectable sustained-release microsphere formulation prepared according to the method of the present invention has a narrow particle size distribution and the release time of the active pharmaceutical ingredient can be sustained for approximately three to four months. Furthermore, the microsphere formulation prepared by the present invention can maintain a stable blood drug concentration in animals, without significant fluctuations in blood drug concentration over three to four months, thus better avoiding the occurrence of toxic side effects.

[0051] 3) The injectable sustained-release microsphere formulation prepared by the method of the present invention, in pharmacokinetic experiments in rats, showed that the inhibitory level of testosterone in rat serum remained effective for three to four months, indicating that the active pharmaceutical ingredient could be maintained at an effective therapeutic concentration for three to four months (see Examples 1-5). Even if the molar mass of the biocompatible polymer material is less than 25,000 Daltons, the microspheres prepared by the method of the present invention can maintain relatively excellent performance (see Examples 6-7). Attached Figure Description

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

[0053] Figure 2 Here is an electron microscope image of the microspheres prepared in Example 3;

[0054] Figure 3 Here is an electron microscope image of the microspheres prepared in Example 5;

[0055] Figure 4 These are the in vitro cumulative release curves for Examples 1-7;

[0056] Figure 5 These are in vivo blood drug concentration-time graphs in rats for Examples 1-5 and Comparative Formulations 1-3;

[0057] Figure 6 These are in vivo testosterone concentration-time graphs in rats for Examples 1-5 and Comparative Preparations 1-3;

[0058] Figure 7This is a blood drug concentration-time graph in rats from Examples 6-7. Detailed Implementation

[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 embodiments, injectable microsphere formulations will be prepared by way of the method for preparing injectable microspheres according to the present invention, wherein the molecular formula of leuprolide dihydroxynaphthyl acid used is C 59 H 84 N 16 O 12 ·C 23 H 16 O6, with a molar mass of 1597.8 g / mL; the molecular formula of the used triptorelin dihydroxynaphthyl acid is C 87 H 98 N 18 O 19 The molar mass is 1699.8 g / mL (CAS: 124508-66-3); the molecular formula of the dihydroxynaphthyl goserelin used is C 59 H 84 N 18 O 14 ·C 23 H 16 O6 has a molar mass of 1657.8 g / mL.

[0061] The active pharmaceutical ingredients were pulverized using a micro-differentiation airflow mill. Triptorelin dihydroxynaphthyl acid raw material was pulverized to obtain micro-powders with D50 values ​​of 1.3 μm, 2.8 μm, and 3.4 μm, used in the example experiments; a 4.6 μm micro-powder was used in the comparative experiments. Leuprorelin dihydroxynaphthyl acid raw material was pulverized to obtain a micro-powder with a D50 of 3.1 μm, used in the example experiments; a 6.2 μm micro-powder was used in the comparative experiments. Goserelin dihydroxynaphthyl acid raw material was pulverized to obtain a micro-powder with a D50 of 3.4 μm, used in the example experiments; a 6.7 μm micro-powder was used in the comparative experiments. Unless otherwise specified, the methods used in the following examples are methods commonly used by those skilled in the art.

[0062] Example 1

[0063] 69.89 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 526 g of dichloromethane, and then 2.46 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 1.3 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

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

[0065] Example 2

[0066] 21.3 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 105 g of dichloromethane, and then 1.57 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 2.8 μm) was added and uniformly dispersed to prepare the oil phase. 20000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes, until a secondary emulsion was formed. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

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

[0068] Example 3

[0069] 26.3 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 80.8 g of dichloromethane, and then 1.57 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 2.8 μm) was added and uniformly dispersed to prepare the oil phase. 34000 mL of a 1.0% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

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

[0071] Example 4

[0072] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of leuprolide dihydroxynaphthyl acid micro powder (D50 of 2.8 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the leuprolide dihydroxynaphthyl acid microsphere formulation.

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

[0074] Example 5

[0075] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of goserelin dihydroxynaphthyl acid micro powder (D50 of 2.8 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes, until a secondary emulsion was formed. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the dihydroxynaphthyl goserelin microsphere formulation.

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

[0077] Example 6

[0078] 28.3 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 10000-25000 Daltons) was dissolved in 107 g of dichloromethane, and then 1.57 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 1.8 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

[0079] The average particle size (D50) of the microspheres in this formulation is 29 μm, and the SPAN value (SPAN = (D90 - D10) / D50) is 1.18. The prepared 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, Mw = 10000-25000 Daltons) was dissolved in 78 g of dichloromethane, and then 1.62 g of triptorelin dihydroxynaphthyl acid micropowder (D50 = 1.8 μm) was added and uniformly dispersed to prepare the oil phase. 20000 mL of a 0.5% (w / w) polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion was mixed with the external aqueous phase at 0 °C using a high-shear emulsifier at a shear rate of 4000 rpm for 10 minutes until homogeneous mixing was achieved, forming a secondary emulsion. The secondary emulsion was stirred at 300 rpm and 25 °C to remove organic solvents, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered through a filter to collect the microspheres, washed repeatedly with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

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

[0083] Example 1: Detection of micronized particle size of active pharmaceutical ingredients

[0084] Take approximately 10 mg of the active pharmaceutical ingredient powder, add approximately 1 ml of olive oil, vortex and mix thoroughly, then ultrasonically disperse until no obvious agglomeration of particles is visible to the naked eye. Add the dispersion to a laser particle size analyzer (Chinese Pharmacopoeia 2020 Edition, General Chapter 0982, Method III) for measurement (Malvern Mastersizer 3000 laser particle size analyzer or an equivalent instrument). Use olive oil as the dispersion medium, a stirring speed of 2000 rpm, an opacity of 5%-15%, a background / sample measurement time of 10 s, a dispersion medium refractive index of 1.47, a particle refractive index of 1.65, an absorptivity of 0.1, and a particle density of 1 g / cm³. Perform three consecutive measurements, and use the average of the three results as the reported value to obtain the D50 test result.

[0085] Test Example 2: In Vitro Release Rate Test

[0086] In this test example 2, the sustained-release microsphere formulations of leuprolide, triptorelin, and goserelin prepared in Examples 1-7 above were used as examples to conduct in vitro release rate tests. The specific method is as follows:

[0087] Accurately weigh 20 mg of each of the sustained-release microsphere formulations prepared in Examples 1-7 above, add them to a 15 mL centrifuge tube, add 15 mL of preheated release medium (0.05 M pH 7.4 phosphate buffer), and then place the tube in a 37°C incubator. At the corresponding time points, take 1 mL of sample and add 1 mL of the corresponding release medium. See Table 1 for the 24-hour burst release rate and total cumulative release rate of Examples 1-7 obtained from the tests.

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

[0089]

[0090] For the in vitro cumulative release curves of Examples 1-7 obtained from the test, please refer to... Figure 4 .

[0091] Comparative Example 1: Comparison of Large-Particle-Size Drug Active Ingredient Micropowders

[0092] In Comparative Example 1, based on Examples 1-7 of the invention described above, Comparative Formulations 1-3 were prepared using microspheres with a D50 particle size of 4.6 μm for triptorelin dihydroxynaphthyl acid, 6.2 μm for leuprorelin dihydroxynaphthyl acid, and 6.7 μm for goserelin dihydroxynaphthyl acid. The method for preparing Comparative Formulations 1-3 also employed an emulsification method to prepare the microspheres; the main difference was that the particle size of the active pharmaceutical ingredient micropowder was greater than 3.5 μm.

[0093] Preparation of comparative formulation 1

[0094] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 4.6 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

[0095] The average particle size (D50) of the microspheres in this formulation is 44 μm, and the SPAN value (SPAN = (D90 - D10) / D50) is 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.

[0096] Preparation of comparative formulation 2

[0097] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of leuprolide dihydroxynaphthyl acid micro powder (D50 of 4.6 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the leuprolide dihydroxynaphthyl acid microsphere formulation.

[0098] The average particle size (D50) of the microspheres in this formulation is 38 μm, and the SPAN value (SPAN = (D90 - D10) / D50) is 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.

[0099] Preparation of comparative formulation 3

[0100] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of goserelin dihydroxynaphthyl acid micro powder (D50 of 4.6 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the dihydroxynaphthyl goserelin microsphere formulation.

[0101] The average particle size (D50) of the microspheres in this formulation is 46 μm, and the SPAN value (SPAN = (D90 - D10) / D50) is 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.

[0102] According to the in vitro release test method described in Example 2 of the present invention, the in vitro release duration of comparative formulations 1-3 is less than 120 days.

[0103] Comparative Example 2: Comparison of Active Ingredients in Unpulverized Drugs

[0104] In Comparative Example 2, based on Examples 1-7 of the invention described above, Comparative Formulation 4 compared the preparation of microspheres using triptorelin dihydroxynaphthyl acid with a D50 particle size of 17.3 μm that had not undergone micronization treatment. The method for preparing Comparative Formulation 4 also employed an emulsification method to prepare microspheres, the main difference being that the particle size of the active pharmaceutical ingredient microparticles was greater than 3.5 μm.

[0105] Preparation of comparative formulation 4

[0106] 27.2 g of PLGA (with a molar ratio of glycolide to lactide of 75:25, Mw = 25000-55000 Daltons) was dissolved in 149 g of dichloromethane, and then 1.83 g of triptorelin dihydroxynaphthyl acid micro powder (D50 of 17.3 μm) was added and uniformly dispersed to prepare the oil phase. 28000 mL of a 0.5% polyvinyl alcohol (PVA) injection aqueous solution was prepared as the external aqueous phase. The primary emulsion and the external aqueous phase were mixed at 0 °C using a high-shear emulsifier with a shear rate of 4000 rpm and an emulsification time of 10 minutes until uniform mixing was achieved to form a secondary emulsion. The organic solvent was removed by stirring at 300 rpm and at 25°C, and the microspheres were volatilized and solidified for 6 hours. After solidification, the emulsion was filtered to collect the microspheres, washed multiple times with distilled water, collected, and then freeze-dried after adding a protective agent to obtain the triptorelin dihydroxynaphthyl acid microsphere formulation.

[0107] Scanning electron microscopy revealed numerous broken microspheres with uneven morphology and abundant particulate matter on their surface, failing to meet the requirements for microsphere formulations. Quantitative analysis of the prepared sustained-release microspheres using HPLC showed an encapsulation rate of 45.12%. Due to the excessively large particle size, the droplets could not be properly encapsulated, resulting in significant drug loss.

[0108] Example 3: Pharmacokinetic and pharmacodynamic experiments of a single dose in rats.

[0109] In Example 3 of this test, the sustained-release microsphere formulations of dihydroxynaphthyl leuprorelin, dihydroxynaphthyl triptorelin, and dihydroxynaphthyl goserelin for injection, prepared in Examples 1-7 and Comparative Formulations 1-3 respectively, were used as examples to conduct pharmacokinetic and pharmacodynamic experiments in rats. The specific methods are as follows:

[0110] Male adult SD rats weighing 300-400g were selected as research subjects and administered the drugs via intramuscular injection. The sustained-release microsphere formulations of leuprorelin dihydroxynaphthyl acid, triptorelin dihydroxynaphthyl acid, and goserelin dihydroxynaphthyl acid prepared according to this invention were administered at a dose of 1.6 mg / kg (Examples 1-7, Comparative Formulations 1-3). At specific time points after administration, 0.3 mL of blood was collected from the jugular vein and transferred to centrifuge tubes containing 33.33 mg / mL of EDTA-K2 anticoagulant. The centrifuge tubes were inverted 5-10 times to thoroughly mix the anticoagulant with the blood, and temporarily stored at room temperature on moist ice. Plasma samples were then rapidly centrifuged at 4000 rpm for 15 min at 4°C and stored at -80°C. The drug concentrations and testosterone concentrations (ng / mL) in the plasma samples at each time point were determined using LC / MS-MS. The in vivo drug concentration-time curves are shown below. Figure 5 and Figure 7 The in vivo testosterone concentration-time curve is shown in the figure. Figure 6 .

[0111] from Figure 5 It can be seen that the sustained-release microsphere formulation for injection prepared according to the method of the present invention can maintain drug release in rats for up to 120 days, and the drug concentration can be maintained within a stable range for 4 months. In contrast, the drug duration of the comparative formulation is 95 days. Figure 5 It can be seen that the sustained-release microsphere formulation for injection prepared according to the method of the present invention can effectively inhibit the concentration level of testosterone in rat serum under effective conditions (1.0 ng / mL), and the effect can be stably maintained for up to 120 days.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing sustained-release microspheres of a bis(hydroxynaphthyl) sodium naphthalate drug, characterized in that, The preparation method includes the following steps: A) Process the active pharmaceutical ingredient to obtain active pharmaceutical ingredient microparticles; B) Dissolve the biocompatible polymer material in an organic solvent to form an organic solution, add the active pharmaceutical ingredient microparticles, and disperse the active pharmaceutical ingredient in the organic solution to obtain an oil phase; C) Dissolve the surfactant in water to prepare an external aqueous phase; D) The obtained oil phase is added to the external aqueous phase and mixed and emulsified to obtain a mixed emulsion; E) The mixed emulsion is cured and dried to obtain sustained-release microspheres containing the active pharmaceutical ingredient.

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

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 active pharmaceutical ingredient microparticles to the biocompatible polymer material is 3-7.5 wt%.

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

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

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 several of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), and polyacetin lactone (PCL).

10. The preparation method according to claim 1 or 2, characterized in that, The surfactant is one or a mixture of several of the following: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pranic F88, Pranic 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, It also includes the following steps: After obtaining the mixed emulsion, the mixed emulsion is stirred, solidified, filtered, and washed with water to form wet microspheres. A protective agent is added to the wet microspheres. The protective agent is one or a mixture of several of polyethylene glycol (PEG), gelatin, glycerin, mannitol, sucrose, trehalose, lactose, glucose, propylene glycol, sorbitol, zinc chloride, zinc sulfate, zinc acetate, and human serum albumin. After adding the protective agent, the microspheres are dried to obtain dihydroxynaphthyl salt 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 excipients are surfactants and / or excipients.

14. A sustained-release microsphere of a bis(hydroxynaphthyl) salt prepared according to any of the preceding claims.

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