Triptorelin acetate sustained-release microspheres and preparation method thereof

Abstract

The present disclosure relates to a triptorelin acetate sustained-release microsphere and a preparation method thereof. The preparation method of the triptorelin acetate sustained-release microsphere of the present disclosure comprises the following steps: 1) dissolving triptorelin acetate in water to prepare an inner aqueous phase solution; dissolving PLGA in an organic solution to prepare an oil phase solution; dissolving PVA in water to prepare an outer aqueous phase solution; 2) mixing the inner aqueous phase solution and the oil phase solution, emulsifying to form colostrum; then mixing the colostrum with the outer aqueous phase solution, emulsifying to form a double emulsion; 3) solidifying the double emulsion, freeze-drying, and obtaining the triptorelin acetate sustained-release microsphere. The method of the present disclosure can be used to prepare a triptorelin acetate sustained-release microsphere with low burst release and high castration maintenance rate.

Description

Technical Field

[0001] The present disclosure relates to the field of medicine, and in particular, to a kind of triptorelin acetate sustained-release microspheres and a preparation method thereof. Background Art

[0002] Triptorelin is a synthetic gonadotropin-releasing hormone (GnRH) analog. Studies have shown that long-term use of triptorelin acetate can secrete gonadotropins, thereby inhibiting testicular or estradiol levels. Taking the treatment of prostate cancer patients as an example, its mechanism of action is: GnRH agonists can stimulate the anterior pituitary to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH further stimulates spermatogonia in the patient's testicles to produce testosterone. When high concentrations of GnRH agonists are continuously administered, they will stimulate a sharp increase in LH and testosterone levels, causing the GnRH receptors to enter a refractory period, thereby causing LH and testosterone levels to drop to castration levels. When the GnRH receptors are downregulated / desensitized, low doses of the drug concentration can maintain castration levels. Based on the mechanism of action of the hypothalamus-pituitary-gonadal axis, triptorelin acetate is widely used clinically to treat diseases that require steroid hormones to be reduced to low levels, such as prostate cancer, endometriosis, etc. In addition, triptorelin acetate can also be used for assisted reproduction.

[0003] At present, endocrine therapy for prostate cancer usually uses serum testosterone levels as a standard. The European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) believe that 50 ng / dL is the target for all castration drug treatments. The results of many studies and analyses have indeed pointed to a strong suggestion - maintaining lower testosterone levels during castration therapy will result in better androgen deprivation therapy (ADT) treatment effects (Klotz L, O'Callaghan C, Ding K, et al, J Clin Oncol 2015; 33: 1151-6). On the other hand, testosterone testing was previously mostly done by radioimmunoassay, and its accuracy was affected to a certain extent by this method. However, with the development of new analytical techniques, the European Association of Urology (EAU) believes that the threshold of 50 ng / dL is too high and strongly recommends that the castration level should be ≤20 ng / dL.

[0004] According to the characteristics of the clinical indications of triptorelin acetate, patients often need to take the drug for a long time and endure the pain of frequent injections. Therefore, in order to improve the compliance of patients with medication, Ipsen of France has developed a long-acting sustained-release formulation with a dosing cycle of 1 month - Daphline, with a specification of 3.75mg. After intramuscular injection of Daphline, there will be an obvious "burst effect" in a short period of time, that is, the blood concentration of triptorelin increases rapidly, and then maintains a low level of blood concentration for a long time. Since the GnRH agonist can cause an increase in gonadotropin in the initial administration, which in turn causes a surge in testosterone levels, it usually occurs 2 to 3 days after medication and lasts for about 2 weeks. Patients may experience "rebound phenomenon" (or "tumor flash") symptoms, which can cause aggravated bone pain, spinal cord compression, acute urinary tract obstruction and cardiovascular death caused by hypercoagulable state. Daphline (3.75mg) will have a more significant "burst effect" in the early stage after administration, which may more easily lead to "acute-chronic" phenomenon in the subsequent administration process, increasing the safety risk of medication.

[0005] Patent CN105169366B discloses a method for preparing triptorelin acetate sustained-release microspheres, which can obtain triptorelin acetate sustained-release microspheres with a small burst release effect and a gentle sustained-release curve by using dichloromethane / ethyl acetate to add benzyl alcohol to dissolve PLGA and saturating the external aqueous phase with an organic solvent. However, the one-day burst release of the sustained-release microspheres obtained by this method is greater than 8%, and the burst release rate still needs to be reduced.

[0006] Therefore, there is an urgent need to develop a long-acting preparation of triptorelin acetate that has a lower burst release and meets the high castration level maintenance rate. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a long-acting inhibitor of triptorelin acetate with low burst release and high maintenance rate of castration level and a preparation method thereof.

[0008] In order to achieve the above objectives, the present disclosure adopts the following specific technical solutions:

[0009] In one aspect, the present disclosure provides a method for preparing triptorelin acetate sustained-release microspheres, comprising the following steps:

[0010] 1) dissolving triptorelin acetate in water to prepare an inner aqueous phase solution; dissolving PLGA in an organic solvent to prepare an oil phase solution; and dissolving PVA in water to prepare an outer aqueous phase solution;

[0011] 2) mixing the inner aqueous phase solution and the oil phase solution, emulsifying to form colostrum; and then mixing the colostrum with the outer aqueous phase solution, emulsifying to form a double emulsion;

[0012] 3) solidifying the multiple emulsion and freeze-drying it to obtain triptorelin acetate sustained-release microspheres.

[0013] On the other hand, the present disclosure provides triptorelin acetate slow-release microspheres prepared by the above preparation method.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned triptorelin acetate sustained-release microspheres and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides a use of the above-mentioned triptorelin acetate sustained-release microspheres in the preparation of drugs for treating prostate cancer, endometriosis, female infertility, precocious puberty and / or uterine fibroids.

[0016] The beneficial effects achieved by the present disclosure are at least as follows:

[0017] 1) The present disclosure uses PLGA excipients with a molecular weight of 15,000 to 35,000 Daltons to obtain a triptorelin acetate microsphere preparation with a small burst release and a long-lasting effect of one month.

[0018] 2) The volume and concentration of PVA used in the present invention can fully dissolve the free drugs on the surface / superficial layer of the microspheres in the solidification liquid during the solidification stage while ensuring the spheroidization of the preparation process, thereby reducing the drugs on the surface / superficial layer of the microspheres that are prone to burst release, thereby obtaining a triptorelin acetate microsphere preparation with a smaller burst release of microspheres.

[0019] 3) The emulsion speed used in the present invention can make the skeleton thickness of the microsphere preparation of the present invention more appropriate, so that triptorelin acetate is sufficient to stimulate the down-regulation / desensitization of GnRH receptors in the early release stage, thereby reducing LH secretion and testosterone levels, achieving the purpose of biochemical castration, but not causing the safety hazard caused by the "rebound phenomenon" of GnRH agonists, and in the later release stage, the skeleton slowly and continuously disintegrates, and the drug is slowly and continuously released, which can better suppress the testosterone concentration level.

[0020] Compared with the shortcomings of high burst release and high incidence of side effects of the marketed preparation of Diphereline (3.75 mg), the low burst release feature of the present invention improves the safety of medication. In addition, the microspheres obtained by the present invention have lower testosterone levels after administration, which has obvious clinical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Scanning electron micrograph of the triptorelin acetate microspheres with adhesion phenomenon in Example 1.

[0022] Figure 2 : Scanning electron micrograph of the triptorelin acetate microspheres of Example 8.

[0023] Figure 3 : In vitro release curve of triptorelin acetate microspheres of Example 8.

[0024] Figure 4 : Scanning electron micrograph of the triptorelin acetate microspheres of Example 14.

[0025] Figure 5 : Scanning electron micrograph of the poorly spherical triptorelin acetate microspheres of Example 15.

[0026] Figure 6 : Pharmacodynamics (PD) diagram of the unqualified triptorelin acetate microspheres in rats of Example 18.

[0027] Figure 7 : Comparative graph of the pharmacokinetics (PK) of the triptorelin acetate microspheres of Example 8 and Comparative Example 1 in rats.

[0028] Figure 8 : Comparison of the in vivo pharmacodynamics (PD) of the triptorelin acetate microspheres of Example 8 and Comparative Example 1 in rats.

[0029] Fig. 9 : Comparison of the pharmacokinetics (PK) of Example 8, triptorelin acetate microspheres and daptomycin in rats.

[0030] Fig.10 : Comparison of the in vivo pharmacodynamics (PD) of triptorelin acetate microspheres and diphenylamine in rats of Example 8. DETAILED DESCRIPTION

[0031] The experimental methods in the following examples of the present disclosure that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

[0032] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meanings as those commonly understood by those skilled in the art to which the present disclosure belongs. The terms used in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0033] The terms "including" and "having" and any variations thereof in the present disclosure are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0034] The "plurality" mentioned in the present disclosure refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0035] The term "homogeneous emulsification" used in the present disclosure refers to the effect of uniformly dispersing one liquid in another immiscible liquid in extremely fine droplets.

[0036] The term "curing" used in the present disclosure refers to a process in which a substance is transformed from a low molecule to a high molecule.

[0037] The term "lyophilization" as used in the present disclosure refers to freeze drying, which is a drying method in which the substance to be dried is rapidly frozen and then the ice therein is removed by sublimating into water vapor under high vacuum conditions.

[0038] The term "medical castration" or "castration drug" as used in this disclosure refers to the use of gonadotropin-releasing hormone analogs or antagonists to suppress testosterone secretion in prostate cancer patients to castration levels. The European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) use 50 ng / dL as the castration level standard for all castration drug treatments.

[0039] The term "microsphere" used in this disclosure refers to a tiny spherical entity formed by dissolving or dispersing a drug in a carrier excipient. Usually, particles with a diameter between 1-250 μm are called microspheres, particles with a diameter between 0.1-1 μm are called submicrospheres, and particles with a diameter between 10-100 nm are called nanospheres.

[0040] The term "burst release" as used in the present disclosure refers to the phenomenon that a large amount of drug is released from an injectable microsphere sustained-release preparation within the first day after administration, and its extent is generally measured by the cumulative drug release (%) within 24 hours.

[0041] In one aspect, the present disclosure provides a method for preparing triptorelin acetate sustained-release microspheres, comprising the following steps:

[0042] 1) dissolving triptorelin acetate in water to prepare an inner aqueous phase solution; dissolving PLGA in an organic solvent to prepare an oil phase solution; and dissolving PVA in water to prepare an outer aqueous phase solution;

[0043] 2) mixing the inner aqueous phase solution and the oil phase solution, emulsifying to form colostrum; and then mixing the colostrum with the outer aqueous phase solution, emulsifying to form a double emulsion;

[0044] 3) solidifying the multiple emulsion and freeze-drying it to obtain triptorelin acetate sustained-release microspheres.

[0045] In some embodiments of the present disclosure, in step 1) of the method, the mass concentration of triptorelin acetate in the inner aqueous phase solution is about 10%-50%, and the mass concentration of the oil phase solution is about 10%-45%.

[0046] In some embodiments of the present disclosure, the mass concentration of the above-mentioned triptorelin acetate is about 18%-45%, preferably 35-45%; preferably about 18%, 35%, 40% or 45%; more preferably about 40% or 45%.

[0047] In some embodiments of the present disclosure, the mass concentration of the oil phase solution is about 11%-40%, preferably about 20-40%, preferably about 12%, 20%, 25%, 40%; more preferably about 25% or 40%.

[0048] In some embodiments of the present disclosure, the molecular weight of the PLGA is about 13,000 to 40,000 Daltons.

[0049] In some embodiments of the present disclosure, the molecular weight of the PLGA is about 14,900-36,000 Daltons, preferably about 25,000-35,100 Daltons.

[0050] In some embodiments of the present disclosure, in step 1) of the above method, the organic solution used in the above oil phase solution is selected from a mixed solution of dichloromethane and acetone or a mixed solution of dichloromethane and ethyl acetate or one of dichloromethane, and the mixing ratio of the mixed solution is about (3-7):1.

[0051] In some embodiments of the present disclosure, the mixing ratio of the mixed solution of dichloromethane and acetone or the mixed solution of dichloromethane and ethyl acetate is 5:1.

[0052] In some embodiments of the present disclosure, the concentration of the PVA aqueous solution is about 0.1 wt%-1 wt%.

[0053] In some embodiments of the present disclosure, the concentration of the PVA aqueous solution is about 0.20 wt%-0.5 wt%.

[0054] In some embodiments of the present disclosure, after PVA is dissolved in water, it is prepared into an external aqueous phase solution with or without saturation with an organic solvent; in the above-mentioned PVA solution saturated with an organic solvent, the above-mentioned organic solvent is one of dichloromethane, acetone or ethyl acetate.

[0055] In some embodiments of the present disclosure, in step 2), the mass volume ratio (g / ml) of the colostrum to the external aqueous phase solution is about 1:(10-100), preferably 1:(13-80); more preferably 1:(35-70), further 1:(45-70).

[0056] In some embodiments of the present disclosure, the mass volume ratio (g / ml) of the colostrum to the external aqueous phase solution is about 1:35, 1:37, 1:46, 1:60 or 1:70.

[0057] In some embodiments of the present disclosure, in step 2), colostrum is formed by homogenization emulsification.

[0058] In some embodiments of the present disclosure, in step 2), a double emulsion is formed by homogenizing and emulsifying.

[0059] In some embodiments of the present disclosure, the apparatus used to form colostrum and / or form multiple emulsion in step 2) is any one of the following: an emulsifier, a homogenizer, a tissue homogenizer, a tabletop disperser or a colloid mill.

[0060] In some embodiments of the present disclosure, the rotation speed used in the emulsification is about 3000-7000 rpm.

[0061] In another aspect, the present disclosure provides triptorelin acetate sustained-release microspheres prepared according to the above preparation method.

[0062] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned triptorelin acetate sustained-release microspheres and a pharmaceutically acceptable carrier.

[0063] In some embodiments of the present disclosure, the aforementioned pharmaceutically acceptable carrier is selected from a suspension, a pH adjuster, an isotonicity adjuster, a surfactant, water, and physiological saline.

[0064] In some embodiments of the present disclosure, the aforementioned suspending agent is selected from sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate, or glycerol.

[0065] In some embodiments of the present disclosure, the aforementioned isotonicity adjusting agent is selected from sodium chloride, glucose, mannitol, or sorbitol.

[0066] In some embodiments of the present disclosure, the aforementioned surfactant is a non-ionic surfactant selected from the polysorbate series and the poloxamer series.

[0067] On the other hand, the present disclosure provides a method for preparing the above-mentioned triptorelin acetate and / or use of the above-mentioned triptorelin acetate sustained-release microspheres in preparing drugs for prostate cancer, endometriosis, female infertility, precocious puberty and / or uterine fibroids.

[0068] The technical solution of the present disclosure is further described below by way of specific implementation. It should be clear to those skilled in the art that the embodiments are only used to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

[0069] Example

[0070] In the following examples, unless otherwise specified, the average molecular weight of poly(lactide-co-glycolide) (PLGA) is 26,000 Daltons.

[0071] Example 1 (polyvinyl alcohol (PVA) 0.10%, colostrum mass: PVA volume ≈ 1: 18.5 (g / ml))

[0072] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0073] 2) Weigh 2.57 g of a lactide-glycolide copolymer, add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0074] 3) Prepare 0.10 wt% PVA (saturated with dichloromethane) 0.2 L as the external aqueous phase.

[0075] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0076] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0077] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0078] The freeze-dried microspheres are difficult to disperse and have adhesion. See the scanning electron microscopy image. Figure 1 .

[0079] Example 2 (PVA 0.20%, colostrum mass: PVA volume ≈ 1: 13.9)

[0080] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0081] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0082] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.15 L as the external aqueous phase.

[0083] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0084] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0085] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0086] The freeze-dried microspheres are difficult to disperse and have adhesion.

[0087] Example 3 (PVA 0.50%, colostrum mass: PVA volume ≈ 1: 29.6)

[0088] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0089] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0090] 3) Prepare 0.50 wt% PVA (saturated with dichloromethane) 0.32 L as the external aqueous phase.

[0091] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0092] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0093] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0094] The freeze-dried microspheres are difficult to disperse and have adhesion.

[0095] Example 4 (PVA 0.10%, colostrum mass: PVA volume ≈ 1: 27.7)

[0096] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0097] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0098] 3) Prepare 0.10 wt% PVA (saturated with dichloromethane) 0.3 L as the external aqueous phase.

[0099] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0100] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0101] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0102] The freeze-dried microspheres are difficult to disperse and have adhesion.

[0103] Example 5 (PVA 0.20%, colostrum mass:PVA volume ≈1:36.97, triptorelin acetate concentration in the aqueous phase solution is about 40%, and lactide-co-glycolide copolymer concentration in the oil phase solution is about 25%)

[0104] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0105] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0106] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.4 L as the external aqueous phase.

[0107] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0108] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0109] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0110] The freeze-dried microspheres were round and easy to disperse. The drug loading of the microspheres was 9.4%, the burst release in 1 day (1d) was 7%, and the sustained release was 28d.

[0111] Example 6 (PVA 0.20%, colostrum mass: PVA volume ≈ 1: 60.1)

[0112] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0113] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0114] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.65 L as the external aqueous phase.

[0115] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0116] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0117] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0118] The freeze-dried microspheres were round and easy to disperse, with a drug loading of 9.7%, a burst release of 5% in 1 day, and a sustained release of 28 days.

[0119] Example 7 (PVA 0.10%, colostrum mass: PVA volume ≈ 1: 46.2)

[0120] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0121] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0122] 3) Prepare 0.10 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0123] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0124] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0125] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0126] The freeze-dried microspheres were round and easy to disperse, with a drug loading of 9.7%, a burst release of 4% in 1 day, and a sustained release of 28 days.

[0127] Example 8 (PVA 0.20%, colostrum mass: PVA volume ≈ 1: 46.2)

[0128] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0129] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0130] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0131] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0132] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0133] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0134] The freeze-dried microspheres are easy to disperse, as shown in the scanning electron microscopy image. Figure 2 As can be seen from the figure, the microspheres are round and dispersed, and the particle size is relatively uniform. The drug loading is 9.5%, and the in vitro release curve is shown in Figure 3 , burst release is 3% in 1 day, and sustained release is 28 days.

[0135] Example 9 (PVA 0.50%, colostrum mass: PVA volume ≈ 1: 46.2)

[0136] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0137] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0138] 3) Prepare 0.5 L of 0.50 wt% PVA (saturated with dichloromethane) as the external aqueous phase.

[0139] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0140] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0141] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0142] The freeze-dried microspheres were easy to disperse, with a drug loading of 9.2%, a burst release of 4% in 1 day, and a sustained release of 28 days.

[0143] Example 10 (PVA 1.0%, colostrum mass: PVA volume ≈ 1: 46.2)

[0144] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0145] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0146] 3) Prepare 0.5 L of 1.0 wt% PVA (saturated with dichloromethane) as the external aqueous phase.

[0147] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0148] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0149] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0150] The freeze-dried microspheres were easy to disperse, with a drug loading of 9.2%, a burst release of 4% in 1 day, and a sustained release of 28 days.

[0151] Example 11 (PLGA molecular weight: 13000 Daltons)

[0152] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0153] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer (molecular weight 13,000 Daltons) and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0154] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0155] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0156] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0157] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0158] The freeze-dried microspheres were easy to disperse, with a drug loading of 8.9%, a burst release of 17% in 1 day, and a sustained release of 21 days.

[0159] Example 12 (PLGA molecular weight: about 15000 Daltons)

[0160] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0161] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer (molecular weight 14980 Daltons) and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0162] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0163] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0164] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0165] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0166] The freeze-dried microspheres were easy to disperse, with a drug loading of 9.3%, a burst release of 6% in 1 day, and a sustained release of 28 days.

[0167] Example 13 (PLGA molecular weight: about 35,000 Daltons)

[0168] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0169] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer (molecular weight 35030 Daltons) and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0170] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0171] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0172] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0173] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0174] The freeze-dried microspheres were easy to disperse, with a drug loading of 10.1%, a burst release of 3% in 1 day, and a sustained release of 28 days.

[0175] Example 14 (PLGA molecular weight: 53200 Daltons)

[0176] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0177] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer (molecular weight 53200 Daltons) and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0178] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0179] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0180] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0181] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0182] The freeze-dried microspheres are not easy to disperse and have serious agglomeration. Some of the microspheres are elliptical. The scanning electron microscopy image shows Figure 4 The drug loading of the prepared microspheres was 9.9% and the burst release in 1 day was 1%.

[0183] Example 15 (Emulsion speed 2000 rpm)

[0184] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0185] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0186] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0187] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0188] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 2000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0189] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0190] The microspheres are irregular in shape, not round, as shown in the SEM image. Figure 5 , drug loading 9.1%, 1d burst release 2%.

[0191] Example 16 (Emulsion speed 3000 rpm)

[0192] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0193] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0194] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0195] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0196] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0197] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0198] The microspheres are rounded and spherical in shape, with a drug loading of 9.3%, a burst release of 4% in 1 day, and a sustained release of 28 days.

[0199] Example 17 (Emulsion speed 7000 rpm)

[0200] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0201] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0202] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0203] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0204] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 7000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0205] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0206] The microspheres are rounded and spherical, with a drug loading of 9.8%, a burst release of 6% in 1 day, and a sustained release of 28 days. The burst release is higher than that of 3000 rpm, but still meets the low burst release requirements.

[0207] Example 18 (Emulsion speed 8000 rpm)

[0208] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0209] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0210] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0211] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0212] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 8000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0213] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0214] The microspheres are rounded and spherical in shape, with a drug loading of 9.1% and a burst release of 27% per day. The in vivo drug efficacy (PD) in rats is unqualified. Figure 6 .

[0215] Example 19 (Emulsion speed 3000 rpm)

[0216] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0217] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and acetone (mixing ratio 5:1) to prepare an oil phase solution.

[0218] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0219] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0220] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0221] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0222] The microspheres are rounded and spherical in shape, with a drug loading of 9.6%, a burst release of 5% in 1 day, and a sustained release of 28 days in vitro.

[0223] Example 20 (Emulsion speed 7000 rpm)

[0224] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0225] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and acetone (mixing ratio 5:1) to prepare an oil phase solution.

[0226] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0227] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0228] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 7000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0229] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0230] The microspheres are rounded and spherical in shape, with a drug loading of 9.4%, a burst release of 6% in 1 day, and a sustained release of 28 days in vitro.

[0231] Example 21 (PVA 0.20%, colostrum mass:PVA volume ≈1:69.7, triptorelin acetate concentration in the aqueous phase solution is about 45%, and lactide-co-glycolide copolymer concentration in the oil phase solution is about 40%)

[0232] 1) Weigh 0.22 g of triptorelin acetate and add 0.27 g of water for injection to prepare an internal aqueous phase solution.

[0233] 2) Weigh 2.67 g of a lactide-co-glycolide copolymer and add 4.01 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0234] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0235] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0236] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0237] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0238] The freeze-dried microspheres were easy to disperse, with a drug loading of 10.0%, a burst release of 5% in 1 day, and a sustained release of 28 days.

[0239] Example 22 (emulsification speed 3000 rpm)

[0240] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0241] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio is 7:1) to prepare an oil phase solution.

[0242] 3) Prepare 0.20 wt% PVA (saturated with ethyl acetate) 0.5 L as the external aqueous phase.

[0243] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0244] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0245] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0246] The microspheres are rounded and spherical in shape, with a drug loading of 9.1%, a burst release of 5% in 1 day, and a sustained release of 28 days in vitro.

[0247] Example 23 (emulsification speed 3000 rpm)

[0248] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0249] 2) Weigh 2.57 g of a lactide-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio is 3:1) to prepare an oil phase solution.

[0250] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0251] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0252] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0253] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0254] The microspheres are rounded and spherical in shape, with a drug loading of 9.9%, a burst release of 6% in 1 day, and a sustained release of 28 days in vitro.

[0255] Example 24 (Emulsion speed 3000 rpm)

[0256] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0257] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0258] 3) Prepare 0.20 wt% PVA (saturated with ethyl acetate) 0.5 L as the external aqueous phase.

[0259] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0260] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0261] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0262] The microspheres are rounded and spherical in shape, with a drug loading of 9.7%, a burst release of 7% in 1 day, and a sustained release of 28 days in vitro.

[0263] Example 25 (emulsification speed 3000 rpm)

[0264] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0265] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and acetone (mixing ratio 5:1) to prepare an oil phase solution.

[0266] 3) Prepare 0.20 wt% PVA (saturated with acetone) 0.5 L as the external aqueous phase.

[0267] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0268] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 3000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0269] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0270] The microspheres are rounded and spherical in shape, with a drug loading of 9.2%, a burst release of 5% in 1 day, and a sustained release of 28 days in vitro.

[0271] Example 26 (PVA 0.20%, colostrum mass:PVA volume ≈1:35.4, triptorelin acetate concentration in the aqueous phase solution is about 18%, and lactide-glycolide copolymer concentration in the oil phase solution is about 11.7%)

[0272] 1) Weigh 0.22 g of triptorelin acetate and add 1.00 g of water for injection to prepare an internal aqueous phase solution.

[0273] 2) Weigh 2.0 g of a lactide-co-glycolide copolymer and add 15.13 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1) to prepare an oil phase solution.

[0274] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.65 L as the external aqueous phase.

[0275] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0276] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0277] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0278] The freeze-dried microspheres were easy to disperse, with a drug loading of 9.2%, a burst release of 7% within 1 day, and a sustained release of 28 days in vitro.

[0279] Example 27 (Emulsion speed 7000 rpm)

[0280] 1) Weigh 0.22 g of triptorelin acetate and add 0.33 g of water for injection to prepare an internal aqueous phase solution.

[0281] 2) Weigh 2.57 g of a lactide-co-glycolide copolymer and add 7.70 g of a mixture of dichloromethane and acetone (mixing ratio 3:1) to prepare an oil phase solution.

[0282] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0283] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0284] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 7000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0285] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0286] The microspheres are rounded and spherical in shape, with a drug loading of 9.7%, a burst release of 7% in 1 day, and a sustained release of 28 days in vitro.

[0287] Example 28 (PVA 0.20%, colostrum mass:PVA volume ≈1:69.7, triptorelin acetate concentration in the aqueous phase solution is about 45%, and lactide-co-glycolide copolymer concentration in the oil phase solution is about 40%)

[0288] 1) Weigh 0.22 g of triptorelin acetate and add 0.27 g of water for injection to prepare an internal aqueous phase solution.

[0289] 2) Weigh 2.67 g of lactide-co-glycolide copolymer and add 4.01 g of dichloromethane to prepare an oil phase solution.

[0290] 3) Prepare 0.20 wt% PVA (saturated with dichloromethane) 0.5 L as the external aqueous phase.

[0291] 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersion and emulsification for 1 to 6 minutes.

[0292] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0293] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0294] The freeze-dried microspheres were easy to disperse, with a drug loading of 9.7%, a burst release of 5% in 1 day, and a sustained release of 28 days.

[0295] Example 29 (PVA 0.20%, colostrum mass:PVA volume ≈1:35.4, triptorelin acetate concentration in the aqueous phase solution is about 18%, and lactide-co-glycolide copolymer concentration in the oil phase solution is about 11.7%)

[0296] 1) Weigh 0.22 g of triptorelin acetate and add 1.00 g of water for injection to prepare an internal aqueous phase solution.

[0297] 2) Weigh 2.0 g of lactide-glycolide copolymer and add 15.13 g of a mixture of dichloromethane and ethyl acetate (mixing ratio 5:1)

[0298] Prepared into oil phase solution.

[0299] 3) Prepare 0.20 wt% PVA (without saturation with organic solvent) 0.65 L as the external aqueous phase.

[0300] 0 4) Add the oil phase into the internal water phase and form W / O type colostrum by dispersing and emulsifying for 1 to 6 minutes.

[0301] 5) After the colostrum is completed, the colostrum liquid is added to the external water phase and dispersed at an emulsifier speed of 4000 rpm to further emulsify and disperse to form a W / O / W type multiple emulsion.

[0302] 6) After the emulsion is completed, solidify it and stir and solidify it for 3.5 to 4.5 hours. Collect the wet solid and freeze-dry it. After the freeze-drying is completed, the solid is crushed to obtain powdered microspheres.

[0303] 5 The freeze-dried microspheres were easy to disperse, with a drug loading of 9.1%, a burst release of 6% in 1 day, and a sustained release of 28 days.

[0304] Comparative Example 1

[0305] According to the process of Example 2 of patent CN105169366B, triptorelin acetate sustained-release microspheres were prepared as comparative example 1. The microspheres were rounded spherical in shape, with a drug loading of 10.5%, a burst release of 10%, and a sustained release of 28 days.

[0306] 0 Compared with the in vitro burst release, the burst release of Comparative Example 1 is 10% greater than the burst release of the microspheres of the embodiments of the present disclosure (except Examples 11 and 18), and is particularly significantly greater than the burst release of Example 8 of the present disclosure. In addition, the pharmacokinetics (PK) of the triptorelin acetate microspheres of Example 8 of the present disclosure and Comparative Example 1 in rats are compared, and the experimental data are shown in Figure 7 As can be seen from the figure, the early release (first 1d) of Comparative Example 1 is much greater than the early release of Example 8 of the present disclosure, and the early release is too fast.

[0307] It is easy to cause insufficient release in the later stage. From the PK curve, it can be seen that the concentration of triptorelin in comparative example 1 at the later stage of 7 to 28 days is lower than that of Example 8 of the present disclosure, and the content is low at the 28th day, which is almost undetectable, while the release of the microspheres of Example 8 of the present disclosure in rats is maintained for a longer time, and the corresponding triptorelin concentration can still be detected at the 32nd day. This pharmacokinetic release phenomenon also has an effect on the significant effect of the castration effect of triptorelin acetate microspheres on testosterone in vivo. The comparison diagram of the in vivo pharmacodynamics (PD) of the triptorelin acetate microspheres of Example 8 of the present disclosure and comparative example 1 in rats is shown in Figure 8 As can be seen from the figure, due to the implementation of the present disclosure

[0308] The low burst release advantage of Example 8 is that the testosterone castration concentration in the later 7 to 28 days is much lower than that in Comparative Example 1, and its testosterone concentration meets the strong recommendation of the European Association of Urology (EAU) that the castration level should be ≤20 ng / dL, which shows that the testosterone castration level maintenance effect of the present invention is

[0309] The result is stronger than that of Comparative Example 1.

[0310] In summary, the triptorelin acetate microspheres obtained in the present disclosure have a smaller burst release amount and a lower testosterone castration level in the later stage of in vivo administration, and the maintenance effect is good.

[0311] 5 Comparative Example 2

[0312] The triptorelin acetate microspheres and dicaprolactone (Ipsen Pharma Biotech, 3.75 mg / vial) in the present disclosure were administered to rats for comparison. The pharmacokinetics (PK) comparison of the triptorelin acetate microspheres and dicaprolactone in rats in Example 8 of the present disclosure is shown in FIG. Fig. 9 , the comparison of drug efficacy (PD) in rats is shown in Fig.10 From the PK curve, it can be seen that the early release of Dafilin (first 1 day) is much greater than the early release of Example 8 of the present disclosure, and the early release is too fast, resulting in insufficient release in the later stage. The concentration of Dafilin in 1-25 days is lower than that in Example 8 of the present disclosure, and the content is low and almost undetectable on the 25th day. The release of the microspheres of Example 8 of the present disclosure in rats is maintained for a longer time, and the corresponding triptorelin can still be detected on the 32nd day.

[0313] The comparison chart of the pharmacodynamics PD in rats shows that, compared with Diphereline, the testosterone castration level of the microspheres of Example 8 of the present disclosure was far lower than 50 ng / dL during the 3-32 days after administration, and was basically maintained at around 20 ng / dL, with a stable testosterone castration maintenance level.

[0314] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0315] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. A method for preparing triptorelin acetate sustained-release microspheres, comprising the following steps: 1) dissolving triptorelin acetate in water to prepare an inner aqueous phase solution; dissolving PLGA in an organic solvent to prepare an oil phase solution; and dissolving PVA in water to prepare an outer aqueous phase solution; 2) mixing the inner aqueous phase solution and the oil phase solution, emulsifying to form colostrum; then mixing the colostrum with the outer aqueous phase solution, emulsifying at a rotation speed of 3000-7000 rpm to form a double emulsion; 3) solidifying the emulsion and freeze-drying it to obtain triptorelin acetate sustained-release microspheres, Wherein, the mass concentration of triptorelin acetate is 18%-45%, the mass concentration of the oil phase solution is 11%-40%, and the molecular weight of PLGA is 14900-36000 Daltons; In step 1), the organic solution used in the oil phase solution is selected from a mixed solution of dichloromethane and acetone, a mixed solution of dichloromethane and ethyl acetate, or any one of dichloromethane, and the mixing ratio of the mixed solution is (3-7):1; in step 2), the mass volume ratio of the colostrum to the external aqueous phase solution is g / ml 1:(35-70).

2. The preparation method according to claim 1, wherein In step 1), the mass concentration of triptorelin acetate is 35-45%.

3. The preparation method according to claim 1, wherein In step 1), the mass concentration of triptorelin acetate is 18%, 35%, 40% or 45%.

4. The preparation method according to claim 3, wherein In step 1), the mass concentration of triptorelin acetate is 40% or 45%.

5. The preparation method according to claim 1, wherein In step 1), the mass concentration of the oil phase solution is 20-40%.

6. The preparation method according to claim 1, wherein In step 1), the mass concentration of the oil phase solution is 12%, 20%, 25%, and 40%.

7. The preparation method according to claim 6, wherein: In step 1), the mass concentration of the oil phase solution is 25% or 40%.

8. The preparation method according to claim 1, wherein The molecular weight of the PLGA in step 1) is 25000-35100 Daltons.

9. The preparation method according to claim 1, wherein In step 1), the mixing ratio of the mixed solution is 5:

1.

10. The preparation method according to claim 1, wherein: In step 1), the concentration of the external aqueous phase solution is 0.1wt%-1wt%.

11. The preparation method according to claim 10, wherein: In step 1), the concentration of the external aqueous phase solution is 0.20wt%-0.5wt%.

12. The preparation method according to claim 1, wherein: In step 1), after PVA is dissolved in water, it is saturated with an organic solvent to prepare an external aqueous phase solution; the organic solvent is one of dichloromethane, acetone or ethyl acetate.

13. The preparation method according to claim 1, wherein In step 2), the mass volume ratio of the colostrum to the external aqueous phase solution is 1:35, 1:37, 1:46, 1:60 or 1:70 in g / ml.

14. The preparation method according to claim 1, wherein: In step 2), colostrum is formed by homogenization and emulsification.

15. The preparation method according to claim 1, wherein: In step 2), a double emulsion is formed by homogenizing and emulsifying.

16. The preparation method according to claim 14 or 15, wherein: The apparatus used to form colostrum and / or multiple emulsion is any one of the following: an emulsifier, a homogenizer, a tissue homogenizer, a tabletop disperser or a colloid mill.

17. Triptorelin acetate sustained-release microspheres prepared by the preparation method according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising the triptorelin acetate sustained-release microspheres according to claim 17 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, wherein The pharmaceutically acceptable carrier is selected from a suspending agent, a pH adjusting agent, an isotonicity adjusting agent, a surfactant or water.

20. The pharmaceutical composition according to claim 18, wherein The pharmaceutically acceptable carrier is physiological saline.

21. The pharmaceutical composition according to claim 19, wherein The suspending agent is selected from sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate or glycerol.

22. The pharmaceutical composition according to claim 19, wherein The isotonicity adjusting agent is selected from sodium chloride, glucose, mannitol or sorbitol.

23. The pharmaceutical composition according to claim 19, wherein The surfactant is a non-ionic surfactant selected from the polysorbate series or the poloxamer series.

24. Use of the triptorelin acetate sustained-release microspheres according to claim 17 or the pharmaceutical composition according to any one of claims 18 to 23 in the preparation of drugs for treating prostate cancer, endometriosis, female infertility, precocious puberty and / or uterine fibroids.

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