Alarelin acetate microspheres for injection and preparation method and use thereof

Abstract

The present invention provides aprolin acetate microspheres for injection, which contain aprolin acetate and a polymer; wherein the polymer includes a polylactic-co-glycolic acid copolymer; preferably, the average particle size of the aprolin acetate microspheres for injection is ≤45 μm. The present invention also provides aprolin acetate microsphere composition for injection, which contains the aprolin acetate microspheres for injection of the present invention, and a water-soluble polysaccharide or amino acid. The present invention also provides a method for preparing the aprolin acetate microspheres for injection of the present invention. The present invention also provides a method for preparing the aprolin acetate microsphere composition for injection of the present invention. The present invention also provides the use of the aprolin acetate microspheres for injection of the present invention or the aprolin acetate microsphere composition for injection of the present invention. The aprolin acetate microspheres for injection of the present invention have excellent drug loading and release behavior, no burst release phenomenon, and can achieve good in vivo drug efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to alanine acetate microspheres for injection, and a preparation method and use thereof. Background Art

[0002] Alarelin, also known as alarelin, is a synthetic nonapeptide analog of gonadotropin-releasing hormone (GnRH). It stimulates the pituitary gland to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH), causing a transient increase in ovarian steroid hormones. Repeated use can inhibit the pituitary gland from releasing LH and FSH, causing a decrease in the level of estradiol in the blood, thereby exerting its efficacy. Alarelin can be used clinically to treat hormone-dependent diseases such as prostate cancer, endometriosis, uterine fibroids, estrogen receptor-positive premenopausal breast cancer, and central precocious puberty, and is widely used clinically.

[0003] Currently, the dosage form of alanine injection on the domestic market is lyophilized powder injection. Since the half-life of alanine in the human body is very short, this dosage form requires long-term and frequent injections, and patient compliance is poor, which not only increases the patient's pain, but also increases the cost of treatment.

[0004] Studies have proposed preparing alanine into microspheres to achieve sustained-release administration, in order to overcome the defect that marketed products require long-term and frequent injections.

[0005] In "Optimization of preparation process and in vitro release study of alanine microspheres" by Zhang Min et al., Pharmaceutical Services and Research, 2012, 012(005): 358-361, a method for preparing alanine microspheres is disclosed. However, data show that the alanine acetate sustained-release microspheres prepared by the method have large particle size, low drug loading rate, too fast initial rate, reach the peak testosterone concentration 1h after injection, effectively suppress testosterone level 5 days after injection, but maintain the testosterone concentration to return to normal level after 21 days, and cannot meet the requirements of a one-month sustained-release preparation. In addition, the microspheres prepared by the method contain gelatin, although gelatin can help reduce the burst release of microspheres, not using gelatin under the same conditions will lead to an increase in burst release. However, the addition of gelatin is prone to cause allergies in users, and there is a safety risk.

[0006] In summary, most of the current alanine microspheres require the addition of gelatin, have low drug loading, and generally have a too-fast initial release, which cannot meet the requirements of a one-month sustained-release preparation.

[0007] Therefore, it is very necessary to research and develop a new alanine acetate microsphere to improve the drug loading and release performance of the microspheres and bring convenience to clinical use. Summary of the invention

[0008] The present invention overcomes the deficiencies in the prior art and provides an alarelin acetate microsphere, which has excellent drug loading and release performance and can provide good release in vivo.

[0009] In a first aspect, the present invention provides aprorelin acetate microspheres for injection, which comprise aprorelin acetate and a high molecular polymer;

[0010] Wherein, the high molecular polymer includes polylactic acid-co-glycolic acid (PLGA);

[0011] Preferably, the average particle size of the aloperin acetate microspheres for injection is ≤45 μm, preferably ≤35 μm, and more preferably ≤30 μm.

[0012] Preferably, in the alorellin acetate microspheres for injection of the present invention, the high molecular weight polymer is polylactic acid-glycolic acid copolymer, or a mixture of polylactic acid-glycolic acid copolymer and polylactide.

[0013] Preferably, in the alorelin acetate microspheres for injection described in the present invention, the molecular weight of the polylactic acid-glycolic acid copolymer is 7622-16616, more preferably 7622-16600, further preferably 11000-16600, further preferably 11800-16600, further preferably 12000-15000, and most preferably 12000-13500.

[0014] Preferably, in the alorelin acetate microspheres for injection of the present invention, the molar ratio of lactide (LA) to glycolide (GA) in the polylactic acid-glycolic acid copolymer is 50:50 to 85:15, more preferably 75:25 to 85:15.

[0015] More preferably, the weight percentage of the polylactide in the high molecular weight polymer is 5-40%, further preferably 10-40%, further preferably 15-35%, further preferably 25-35%.

[0016] Preferably, in the alorelin acetate microspheres for injection of the present invention, the molecular weight of the polylactide is 10000-30000, more preferably 12283-23184, further preferably 14000-22000, further preferably 14000-20000, and most preferably 16000-19000.

[0017] Preferably, in the alorelin acetate microspheres for injection of the present invention, the mass ratio of alorelin acetate to high molecular weight polymer is 1:5 to 1:15, more preferably 1:5 to 1:10, and further preferably 1:7 to 1:8.

[0018] In an embodiment of the present invention, the aloperin acetate microspheres for injection of the present invention do not contain gelatin.

[0019] Preferably, in the alorellin acetate microspheres for injection described in the present invention, the alorellin acetate microspheres for injection are prepared by a double emulsion solvent evaporation method.

[0020] Preferably, in the alorellin acetate microspheres for injection described in the present invention, the external aqueous phase used in the double emulsion solvent evaporation method is a polyvinyl alcohol solution.

[0021] Preferably, in the alorellin acetate microspheres for injection of the present invention, the external aqueous phase further comprises an inorganic salt, and more preferably, the inorganic salt is sodium chloride.

[0022] Preferably, in the alorelin acetate microspheres for injection of the present invention, the mass ratio of alorelin acetate to polyvinyl alcohol is 1:0.1 to 1:10, more preferably 1:0.1 to 1:5, further preferably 1:0.5 to 1:5, and further preferably 1:1 to 1:3.

[0023] Preferably, in the alorelin acetate microspheres for injection of the present invention, the mass ratio of alorelin acetate to inorganic salt is 1:0.5 to 1:100, more preferably 1:1 to 1:50, further preferably 1:1 to 1:25, and most preferably 1:1 to 1:20.

[0024] In a second aspect, the present invention further provides an alarelin acetate microsphere composition for injection, which comprises the alarelin acetate microsphere for injection of the present invention, and a water-soluble polysaccharide or amino acid.

[0025] Preferably, in the alanine acetate microsphere composition for injection of the present invention, the water-soluble polysaccharide is selected from one or more of mannitol, lactose and glucose, and the amino acid is selected from one or more of glycine and leucine.

[0026] Preferably, in the alorelin acetate microsphere composition for injection of the present invention, the mass ratio of alorelin acetate to water-soluble polysaccharide or amino acid is 1:0.1 to 1:10, more preferably 1:0.5 to 1:5, and further preferably 1:1 to 1:2.

[0027] In a third aspect, the present invention provides a method for preparing the alanine acetate microspheres for injection of the present invention, comprising the following steps:

[0028] (1) adding alorelin acetate to water to form an inner water phase;

[0029] (2) dissolving the high molecular weight polymer in an organic solvent to form an oil phase;

[0030] (3) mixing and emulsifying the internal water phase prepared in step (1) and the oil phase prepared in step (2) to obtain colostrum;

[0031] (4) mixing the colostrum prepared in step (3) with external water to form a double emulsion;

[0032] (5) removing the organic solvent in the emulsion prepared in step (4) to solidify the microspheres, and collecting the solid portion thereof.

[0033] Preferably, in step (1) of the method of the present invention, the mass ratio of alorelin acetate to water for injection is 1:0.8 to 1:2.4, more preferably 1:1 to 1:2.4, most preferably 1:1.2. Preferably, the water is water for injection.

[0034] Preferably, in step (2) of the method of the present invention, the organic solvent is selected from one or more of halogenated hydrocarbons, ethers, alcohols, aromatic hydrocarbons, tetrahydrofuran or acetonitrile; more preferably, the organic solvent is selected from one or more of dichloromethane, chloroform, dichloroethane, carbon tetrachloride, ether, n-propanol, methanol, toluene or xylene; most preferably, the organic solvent is dichloromethane.

[0035] Preferably, in step (2) of the method of the present invention, the total concentration of the high molecular weight polymer is 20-40 wt %, more preferably 25-40 wt %, most preferably 30-35 wt %, based on the total weight of the oil phase.

[0036] Preferably, in step (3) of the method of the present invention, the emulsification is carried out by high-speed stirring or ultrasonic crushing; more preferably, the rotation speed of the high-speed stirring is 3000-10000 rpm, and further preferably 7000-10000 rpm; more preferably, the intensity of the ultrasonic crushing is 20-80%, and further preferably 30-50%.

[0037] Preferably, in step (3) of the method of the present invention, the emulsification is carried out at 15-40°C, more preferably at 22-40°C, further preferably at 22-36°C, and most preferably at 25-35°C.

[0038] Preferably, in the method of the present invention, the volume ratio of the colostrum prepared in step (3) to the external aqueous phase in step (4) is 1:200 to 1:50, more preferably 1:150 to 1:95.

[0039] Preferably, in the method of the present invention, the colostrum prepared in step (3) is added to the external aqueous phase by a high-speed dispersion method.

[0040] Preferably, in the method of the present invention, the external aqueous phase is a polyvinyl alcohol aqueous solution.

[0041] Preferably, in the method of the present invention, the concentration of polyvinyl alcohol in the external aqueous phase is 0.01-0.50% (w / v, g / ml), more preferably 0.1-0.50% (w / v, g / ml).

[0042] Preferably, the external aqueous phase further comprises inorganic salts; preferably, in the method described in the present invention, the concentration of the inorganic salts in the external aqueous phase is 0.01-10% (w / v, g / ml), more preferably 0.05-5% (w / v, g / ml), further preferably 0.1-5% (w / v, g / ml), further preferably 0.1-1% (w / v, g / ml), and most preferably 0.1-0.25% (w / v, g / ml).

[0043] Preferably, in step (5) of the method of the present invention, the organic solvent in the emulsion prepared in step (4) is removed by stirring and evaporating under reduced pressure at 10-30°C.

[0044] In a fourth aspect, the present invention also provides a method for preparing an alorelin acetate microsphere composition for injection, comprising the following steps: adding a water-soluble polysaccharide solution or an amino acid solution to the alorelin acetate microsphere for injection, followed by freeze-drying.

[0045] Preferably, in the method for preparing alanine acetate microsphere composition for injection described in the present invention, the water-soluble polysaccharide solution is a mannitol aqueous solution; more preferably, the mass concentration of the mannitol aqueous solution is 0.5-20%, further preferably 5-20%, and most preferably 9-15%.

[0046] On the other hand, the present invention provides use of the alorellin acetate microspheres for injection according to the present invention, the alorellin acetate microsphere composition for injection according to the present invention, the alorellin acetate microspheres for injection prepared by the method of the present invention, or the alorellin acetate microsphere composition for injection prepared by the method of the present invention in the preparation of drugs for treating hormone-dependent diseases such as prostate cancer, endometriosis, uterine fibroids, estrogen receptor-positive premenopausal breast cancer and central precocious puberty.

[0047] The present invention has the following advantages and positive effects:

[0048] 1. The microspheres of the present invention have small and uniform particle sizes and greatly reduce burst release, ensuring long-term and stable release of active ingredients both in vivo and in vitro.

[0049] 2. Further, the inventors of the present application unexpectedly found that the alorelin acetate microspheres for injection of the present invention, based on the characteristics of alorelin acetate, can be combined with PLGA and PLA to make the alorelin acetate microspheres of the present invention have excellent drug loading and release performance, and can provide good release in vivo. The present invention prepared alorelin microspheres that showed excellent sustained release effects both in vitro and in vivo.

[0050] 3. The microspheres of the present invention do not need to be specially controlled for the temperature of colostrum during preparation, which reduces the operational risk and shortens the production time. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0052] Figure 1 The rat serum testosterone concentration-time curves of microspheres with different LA:GA ratios in Example 8 are shown;

[0053] Figure 2 The effect of different PLA addition amounts on the release of microspheres in Example 9 is shown;

[0054] Figure 3 The experimental results of the release of microspheres with different PLA addition amounts in Example 9 in rats are shown;

[0055] Figure 4 The rat serum testosterone concentration-time curves of microspheres with different PLA addition amounts in Example 9 are shown;

[0056] Figure 5 The testosterone content-time curve of rat plasma samples measured by the microspheres of the comparative example and the example of the present invention using the LC-MS / MS quantitative analysis method is shown. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0058] Example 1

[0059] Weigh 1g of alanine acetate, add 1.2g of water for injection to dissolve as the inner water phase, weigh 7.4g of PLGA (lactide: glycolide = 75:25, weight average molecular weight of 12539), add 17g of dichloromethane to dissolve as the oil phase, stir and emulsify the above oil-water phases at 8000rpm (about 25°C) to form a primary emulsion (about 20ml). The primary emulsion is quickly added to 2L of 0.25% NaCl-0.1% (w / v, g / ml) PVA aqueous solution within 3min through high-speed dispersion (7000rpm), the temperature of the PVA aqueous solution is controlled at 10°C to 30°C, and the organic solvent is evaporated for 3h under reduced pressure to solidify the microspheres, and the solid part is collected by centrifugation and filtration, and 16.0g of a 9% (g / g) mannitol aqueous solution is added and evenly dispersed, freeze-dried, and the freeze-dried powder is passed through a 120-mesh sieve to obtain.

[0060] Example 2

[0061] Weigh 1g of alanine acetate, add 1.5g of water for injection to dissolve as the inner water phase, weigh 6.3g of PLGA (lactide: glycolide = 75:25, weight average molecular weight of 11854), add 23.3g of dichloromethane to dissolve as the oil phase, and emulsify the above oil-water phases (at an intensity of 40%) by ultrasonic crushing (about 25°C) to form a primary emulsion (about 24ml). The primary emulsion is added to 3L of 0.25% NaCl-0.25% (w / v, g / ml) PVA aqueous solution within 3min by high-speed dispersion (5000rpm), the temperature of the PVA aqueous solution is 10°C, and the organic solvent is evaporated under reduced pressure and stirred for 3h to solidify the microspheres, and the solid part is collected by centrifugation and filtration, and 9.6g of a mannitol aqueous solution with a concentration of 15% (g / g) is added and evenly dispersed, and freeze-dried, and the freeze-dried powder is passed through a 120-mesh sieve to obtain.

[0062] Example 3

[0063] Weigh 7 g of alorelin acetate, add 8.5 g of water for injection to dissolve as the inner aqueous phase, weigh 51.8 g of PLGA (lactide: glycolide = 75:25, weight average molecular weight of 12539), add 119 g of dichloromethane to dissolve as the oil phase, and emulsify the above oil and water phases (about 25°C) under high-speed stirring (stirring speed 8000-10000 rpm) to form a primary emulsion (about 141 ml). The primary emulsion was rapidly added into 14 L of 0.1% NaCl-0.1% (w / v, g / ml) PVA aqueous solution within 4 min by high-speed dispersion (6000 rpm). The temperature of the PVA aqueous solution was controlled at 10°C to 20°C, and the organic solvent was evaporated by continuous stirring for 3 h under reduced pressure to solidify the microspheres. The solid part was collected by centrifugation and filtration, and 12.1 g of 12% (g / g) mannitol aqueous solution was added and dispersed evenly, and freeze-dried. The freeze-dried powder was passed through a 120-mesh sieve to obtain the microspheres.

[0064] Example 4 Study on PLGA Molecular Weight

[0065] According to the conditions of Example 1, the following PLGA with different molecular weights were selected to prepare alanine acetate microspheres, and the drug loading, 48h accelerated release and 35-day release of the obtained microspheres are shown in Tables 1 and 2, respectively.

[0066] Table 1

[0067]

[0068] Table 2

[0069] PLGA molecular weight 7622 11854 12539 14197 16616 Microsphere drug loading% 7.0 8.1 8.4 8.5 7.7 35 days release 4h (%) 30.35 21.42 18.52 14.64 9.69 35 days release 1d (%) 42.76 29.32 26.80 22.66 18.27 35 days release 2d (%) 51.55 38.97 34.20 27.69 21.95 35 days release 3d(%) 56.35 48.02 38.89 33.12 26.50 35 days release 7d (%) 66.77 60.05 50.02 45.46 35.63 35 days release 14d(%) 78.29 73.63 65.00 57.63 43.16 35 days release 21d (%) 84.91 80.18 74.69 69.89 52.32 35 days release 28d (%) 94.58 90.08 88.20 86.03 72.07 35 days release 35d(%) 98.67 97.52 96.28 94.83 83.79

[0070] The results show that the drug loading of alanine microspheres prepared by PLGA with a lower molecular weight of 7622 is low; as the molecular weight gradually increases, the 9h release of the prepared microspheres gradually slows down, and the release of microspheres with a molecular weight between 11800 and 16600 meets the demand. The microspheres prepared by PLGA with a molecular weight of 7622 have a faster initial release, and 85% of the release has been achieved at 21 days, and the overall release rate is faster. The 35-day release of microspheres prepared by PLGA with a molecular weight of 16616 did not reach 85%, and the overall release was slow. Preferably, the molecular weight range of PLGA is 12000-15000.

[0071] Example 5 Study on PLGA concentration

[0072] According to the conditions of Example 1, the drug loading, 48h accelerated release and 35-day release of microspheres prepared at PLGA concentrations of 20%-40% (w / w, PLGA weight remains unchanged, solvent volume is reduced) were studied. The results are shown in Tables 3-4 below.

[0073] Table 3

[0074]

[0075] Table 4

[0076] PLGA concentration% 20 25 30 35 40 Microsphere drug loading% 3.0 7.0 8.4 8.2 8.8 35 days release 4h (%) 3.83 10.29 15.48 19.41 22.11 35 days release 1d (%) 5.45 16.41 22.55 28.20 32.25 35 days release 2d (%) 8.33 19.50 29.37 37.21 40.00 35 days release 3d(%) 10.30 23.58 37.78 41.05 43.52 35 days release 7d (%) 28.62 42.18 52.72 58.68 59.72 35 days release 14d(%) 43.11 55.54 66.09 72.78 73.52 35 days release 21d (%) 51.70 63.58 79.45 83.35 83.80 35 days release 28d (%) 57.21 67.60 88.14 90.65 91.83 35 days release 35d(%) 58.56 72.93 94.75 95.49 95.92

[0077] The results showed that the drug content of microspheres prepared with a lower concentration (20%) of PLGA was low and the release was slow, which was related to the poor ability of low concentration PLGA to encapsulate alanine; the drug content of microspheres prepared with a higher concentration of PLGA (25% to 40%) was slightly higher and the release was normal. Preferably, the PLGA concentration range is 25 to 40%, and more preferably, the PLGA range is 30 to 35%.

[0078] Study on the concentration of alanine in the aqueous phase in Example 6

[0079] According to the conditions of Example 1, different amounts (g) of water were used to dissolve alorelin acetate at room temperature. The drug loading and 48h accelerated release of the prepared microspheres are shown in the following table.

[0080] Table 5

[0081]

[0082] The results showed that when the concentration of the internal water phase was in the range of 0.8-2.4 g / g Ala, the particle size of the prepared microspheres was basically unaffected. However, when the concentration of the internal water phase was low (0.8 g / g Ala), Ala was difficult to dissolve at room temperature and had a high viscosity after dissolution, resulting in a low drug content in the prepared microspheres and a slow release. When the concentration of the internal water phase was in the range of 1.0-2.4 g / g Ala, the release of the microspheres was relatively stable.

[0083] Example 7 Study on Mannitol Concentration

[0084] The difference between the preparation of microspheres and Example 1 is that no sodium chloride is added, and the concentrations of mannitol are 12%, 9%, 15%, 5%, and 1% respectively (the amount of solvent is changed, and the amount of mannitol remains unchanged). The drug loading and 48h accelerated release of the prepared microspheres are shown in the following table.

[0085] Table 6

[0086]

[0087] Experiments have shown that different concentrations of mannitol have no significant difference in release performance. Increasing the mannitol concentration is to reduce the freeze-drying volume and facilitate later scale-up.

[0088] Example 8 Study on the molar composition ratio of LA and GA in PLGA

[0089] Weigh 1.0 g of alorelin acetate, add 1.2 g of water for injection to dissolve as the inner aqueous phase, weigh 7.4 g of PLGA (the ratio of LA to GA is in the range of 50:50 to 85:15, namely 50:50, 75:25, 85:15, and 100:0, and the molecular weight is 12539), add 17 g of dichloromethane to dissolve as the oil phase, and emulsify the above oil-water phases by stirring at 8000 rpm to form a primary emulsion (about 20 ml). The primary emulsion was quickly added to 2L 0.25% NaCl-0.1% (w / v, g / ml) PVA aqueous solution within 3min by high-speed dispersion (7000rpm), the temperature of the PVA aqueous solution was controlled at 10°C to 30°C, and the organic solvent was evaporated by continuous stirring for 3h under reduced pressure to solidify the microspheres. The solid part was collected by centrifugation and filtration, 12.1g of 12% (g / g) mannitol aqueous solution was added and dispersed evenly, and freeze-dried. The freeze-dried powder was passed through a 120-mesh sieve to obtain the drug content and release rate of different microspheres. The results are shown in the following table.

[0090] Table 7

[0091]

[0092] Table 8

[0093] LA:GA 50:50 75:25 85:15 100:0 Microsphere drug loading% 7.63 8.25 8.14 8.32 Volume average particle size μm 15.68 18.73 19.25 18.63

[0094] 35d (35 days) release:

[0095] Table 9

[0096] LA:GA 50:50 75:25 85:15 100:0 35 days release 4h (%) 30.99 20.85 9.78 1.71 35 days release 1d (%) 36.77 28.11 20.59 11.69 35 days release 2d (%) 44.76 34.39 29.11 16.77 35 days release 3d(%) 53.41 40.06 37.44 23.37 35 days release 7d (%) 69.79 57.98 52.2 36.63 35 days release 14d(%) 83.29 70.43 66.74 53.68 35 days release 21d (%) 89.53 79.45 75.49 63.88 35 days release 28d (%) 96.35 86.34 85.47 73.57 35 days release 35d(%) 99.12 91.64 88.23 80.15

[0097] The results show that different LA to GA ratios have the same significant effect on the burst release and mid-term release of microspheres. The greater the proportion of GA, the stronger the hydrophilicity of PLGA, and the relatively enhanced burst release of microspheres. The 50:50 PLGA microspheres have a faster burst release, and the release in the 4th hour exceeds 30% of the total release. The 75:25 PLGA release meets the current standards, but the release in 4 hours is still more than 20%. The 85:15 release burst is significantly reduced, but due to the slow release in the early stage, the inhibition time of testosterone in the animal body is delayed, and the ignition effect is delayed. In the 100:0 PLA ​​group, the burst release is very low, but the overall release is slower. It can be seen that 75:25 and 85:15 are better overall.

[0098] The serum testosterone concentration-time curve of rats with microspheres of different LA:GA ratios

[0099] Thirty-two male SD rats were divided into four groups, with eight rats in each group. Alarelin acetate microspheres for injection with a dose of 0.375 mg / kg (calculated as alarelin acetate) and a ratio of LA to GA of 50:50, 75:25, 85:15, and 100:0 were injected subcutaneously once. Blood was collected from the jugular vein by puncture using a disposable syringe before administration and at 0.25 h, 1 h, 3 h, 6 h, 24 h (D1) and D2, D3, D5, D7, D11, D14, D21, D28, D35, and D42 after administration. About 0.35 ml of blood was collected from the blood collection points before administration and within 24 h after administration, and about 0.5 ml of blood was collected from the subsequent blood collection points. The blood was then transferred to a 5 μl centrifuge tube containing 100 TIU / ml aprotinin and 15% EDTA-K2 solution, and the centrifuge tube was immediately placed in an ice bath after gently mixing. After whole blood was collected, it was centrifuged at 4°C and 2400×g for 5 minutes, and the supernatant was separated to obtain plasma. Except during the analysis period, the plasma samples were stored in an ultra-low temperature refrigerator (-60°C to -90°C). The testosterone content in rat plasma samples was determined by LC-MS / MS testosterone quantitative analysis method.

[0100] Table 10

[0101] Blood collection time 50:50 75:25 85:15 100:0 0.25h 6.62±2.33 5.34±3.67 2.4±0.41 1.15±0.5 1h 26.44±4.85 22.99±8.08 15.7±2.26 15.31±2.13 3h 21.92±3.31 26.67±5.59 24.03±1.46 24.36±2.18 6h 14.6±7.59 16.77±2.82 19.73±3.03 22.46±3.93 1d 7.79±1.35 10.3±0.32 12.27±3.49 14.57±3.64 2d 3.65±0.72 6.99±2.18 5.37±1.99 7.91±0.68 3d 2.64±0.4 4.07±0.76 1.6±0.93 1.52±1.25 5d 1.43±0.74 1.25±1.06 1.14±0.83 0.66±0.64 7d 1.69±0.33 1.18±0.83 0.63±0.25 0.54±0.36 11d 1.47±1.19 0.78±1.06 0.72±0.43 0.72±0.95 14d 1.19±0.24 0.97±0.26 0.6±0.3 0.56±0.63 21d 1.21±0.56 1.05±0.07 0.89±0.08 0.45±0.37 28d 3.14±1.26 0.88±0.26 0.56±0.68 0.51±0.66 35d 6.04±1.92 1.7±0.95 0.88±0.49 0.56±0.44 42d 6.27±3.08 2.02±1.12 0.98±0.38 0.41±0.32

[0102] For ease of viewing, Table 10 has been plotted as Figure 1 . The results show that the four PLGA microspheres with different LA:GA ratios show different testosterone concentration curves. After injection, testosterone has an acute-chronic effect (ignition effect). Due to the fastest release in the early stage, the testosterone concentration of PLGA with LA:GA of 50:50 reaches a peak 1h after injection and then falls back. It maintains a low level of testosterone from 5 to 21 days. After 21 days, the testosterone inhibition effect weakens and the testosterone concentration gradually returns to normal levels. The testosterone concentration of 75:25 PLGA microspheres reaches a peak level 3h after injection, maintains a low level from 5 to 28 days after injection, and slowly recovers after 28 days. The testosterone concentration of 85:15 PLGA microspheres reaches a peak 3h after injection and maintains a low level within 5 to 42 days. The testosterone peak time of 100:0 PLGA microspheres after injection is relatively late, and the testosterone inhibition level is continuously maintained from 5 to 42 days after injection.

[0103] Since the 85:15 release burst was significantly reduced and the ignition effect was delayed, the testosterone inhibition time in the animal body was delayed. In the 100:0 PLA ​​group, the burst release was very low and the overall release was slower. 85:15 generally met the requirements.

[0104] Example 9: Addition of PLA to PLGA

[0105] Weigh 1.0g of alanine acetate, add 1.2g of injection water to dissolve, weigh 7.4g of PLGA (75:25, 12539), add different amounts of PLA (accounting for 0% to 40% of the weight percentage of the polymer, molecular weight 18080), dissolve in 17g of dichloromethane as the oil phase, stir and emulsify the two phases at 8000rpm (about 25°C), add 2.0L of 0.25% NaCl-0.1% (w / v, g / ml) 10-30°C PVA aqueous solution in a high-speed disperser at 7000rpm within 3min. Stir continuously for 3h under reduced pressure, evaporate the organic solvent to solidify the microspheres. Collect the precipitate by centrifugation, add 12.1g of 12% mannitol solution, disperse, and freeze-dry. After freeze-drying, pass through a 120-mesh sieve. The drug content and release of microspheres prepared with different PLA addition amounts are shown in the following table.

[0106] Table 11

[0107]

[0108] Table 12

[0109] PLA addition % 0 10 20 30 40 Microsphere drug loading% 8.34 8.55 8.43 8.51 8.60

[0110] 35d Release:

[0111] Table 13

[0112] PLA addition amount 0％ 10％ 20％ 30％ 40％ 35 days release 4h (%) 11.29 10.23 8.72 6.70 1.96 35 days release 1d (%) 26.14 23.76 20.62 18.60 10.98 35 days release 2d (%) 37.67 34.45 30.94 26.89 16.99 35 days release 3d(%) 43.50 41.22 37.69 34.13 24.59 35 days release 7d (%) 61.00 58.00 54.39 49.52 36.97 35 days release 14d(%) 73.03 69.77 65.84 60.66 49.52 35 days release 21d (%) 82.23 77.89 73.61 68.26 59.78 35 days release 28d (%) 88.95 87.42 83.65 80.29 71.45 35 days release 35d(%) 94.97 92.94 91.26 86.31 76.06

[0113] For ease of viewing, Table 13 has been plotted as Figure 2 Conclusion: Different addition amounts of PLA have significant effects on the drug loading and release of microspheres.

[0114] Comparative release experiment of microspheres with different PLA addition amounts in rats

[0115] Each of the above samples was divided into 24 bottles, each containing 0.9 mg of alanine acetate, and 0.5 ml of the corresponding suspension solvent was added and mixed thoroughly. The above suspension was injected into the subcutaneous part of the back of male SD rats of about 6 weeks old that were randomly grouped and numbered using a 2.5 ml disposable syringe and a No. 8 needle. Three experimental animals were used in parallel for each data point to detect the amount of injection loss. The rats were fed and rested normally, and the residual microspheres in the tissues were removed for content detection at 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d, and 56d after injection. The test results are shown in the table below.

[0116] Table 14

[0117]

[0118] For ease of viewing, Table 14 has been plotted as Figure 3 . Conclusion: The addition of different amounts of PLA to PLGA has a significant effect on the release behavior of the prepared microspheres in rats. When the amount of PLA added is 10%, the microspheres can be continuously released in rats for 28 days; when added at 20%, the microspheres can be continuously released for 35 days; when added at 30%, the microspheres can be continuously released for 42 days; when added at 40%, the microspheres can be continuously released for 56 days. However, the slower the early release, the slower the drug effect, and the longer the clearance period in the body in the later stage, so it is necessary to further explore the more preferred amount of PLA added.

[0119] The concentration-time curve of testosterone in rat serum with different PLA microspheres

[0120] The above five groups of samples were taken, and eight six-week-old male SD rats were selected in each group. A single subcutaneous injection of 0.375 mg / kg (in terms of aprolin acetate) aprolin acetate microspheres for injection was given. Blood was collected from the jugular vein puncture using a disposable syringe before administration and 0.25h, 1h, 3h, 6h, 24h (D1) after administration, and D2, D3, D5, D7, D11, D14, D21, D28, D35 and D42. About 0.35 ml of blood was collected from the blood collection point before administration and within 24h after administration, and about 0.5 ml of blood was collected from the subsequent blood collection points, and then transferred to a 5 μl centrifuge tube containing 100 TIU / ml aprotinin and 15% EDTA-K2 solution. After gently mixing, the centrifuge tube was immediately placed in an ice bath. After collecting whole blood, centrifuge at 4°C and 2400×g for 5 minutes to separate the supernatant to obtain plasma. Except during the analysis period, the plasma samples were stored in an ultra-low temperature freezer (-60°C to -90°C). The testosterone content in rat plasma samples was determined by LC-MS / MS testosterone quantitative analysis method.

[0121] Table 15

[0122] Blood collection time 0％ 10％ 20％ 30％ 40％ 0.25h 7.93±5.38 5.72±1.59 5.28±3.3 4.16±2.76 5.27±4.01 1h 25.93±4.68 22.54±5.03 22±3.26 20.06±4.28 17.58±1.69 3h 23.44±6.33 25.79±3.52 24.43±10.78 25.91±3.00 26.55±3.79 6h 15.65±1.39 17.94±5.11 20.45±10.43 19.47±2.02 21.32±1.72 1d 10.75±4.46 12.66±3.43 14±7.36 15.07±2.69 15.71±3.34 2d 3.47±2.25 4.47±2.07 6.8±6.68 10.2±1.84 11.14±5 3d 2.37±2.47 2.83±1.41 4.54±1.07 3.82±1.63 4.19±2.34 5d 0.79±0.93 2.12±1.62 2.53±2.52 0.78±0.36 1.11±0.36 7d 0.96±0.94 0.92±1.04 0.82±1.22 0.97±0.18 1.03±0.85 11d 0.74±0.56 0.68±0.65 0.42±0.54 0.83±0.38 0.9±0.21 14d 0.89±0.73 0.91±1.67 0.72±1.28 0.58±0.44 0.9±0.63 21d 1.02±1.3 0.93±1.59 0.55±0.87 0.43±0.24 0.38±0.17 28d 0.84±0.43 0.61±0.39 0.66±1.34 0.60±0.41 0.68±0.3 35d 1.84±0.66 1.57±1.37 1.02±0.56 0.54±0.19 0.48±0.32 42d 2.5±0.63 1.82±0.39 1.19±1.01 1.09±0.65 0.39±0.33

[0123] For ease of viewing, the data in Table 15 are plotted as Figure 4 . From the results, the microspheres prepared by adding different proportions of PLA to PLGA showed similar testosterone suppression levels in the early and middle stages after injection. The testosterone concentration of the microspheres without PLA began to return to normal levels 28 days after injection. After adding 10% PLA, it was seen that the recovery rate of testosterone concentration slowed down after 28 days. The addition of 20-30% and 40% PLA can maintain the testosterone suppression effect to 35 and 42 days. Therefore, taking all factors into consideration, a good testosterone suppression effect can be achieved when 20-30% PLA is added.

[0124] Embodiment 10 Research on sodium chloride dosage

[0125] The difference between the microsphere preparation and Example 8 (the molar ratio of LA to GA is 75:25) is the different amount of sodium chloride used.

[0126] Table 16

[0127]

[0128] As can be seen from Table 16, the addition of sodium chloride can effectively reduce the burst release and control the early release, which is manifested in the accelerated release model as the 1h release rate is reduced to less than 10%.

[0129] Example 11 Study on the influence of colostrum temperature

[0130] Weigh 1.0g of alanine acetate, add 1.2g of injection water to dissolve, weigh 7.4g of PLGA (75:25, 12539), add 30% of PLA (molecular weight 18080), dissolve in 17g of dichloromethane as the oil phase, stir and emulsify the two phases at 8000rpm, and control the colostrum temperature to 15℃, 20℃, 25℃, 30℃, 35℃ and 37℃ respectively, and add 2.0L of 0.25% NaCl-0.1% 10-30℃ PVA aqueous solution within 3min at 7000rpm by a high-speed disperser. Stir continuously for 3h under reduced pressure, evaporate the organic solvent to solidify the microspheres. Collect the precipitate by centrifugation, add 12.1g of 12% mannitol solution, disperse, and freeze-dry. After freeze-drying, pass through a 120-mesh sieve. The content and release of microspheres prepared at different colostrum temperatures are shown in the following table.

[0131] Table 17

[0132]

[0133] Generally speaking, for example, in the preparation process of leuprorelin, the colostrum temperature is controlled at a low level, which is conducive to increasing the drug loading. However, in the preparation process of alanine microspheres, it was unexpectedly found that when the colostrum temperature was 20°C or below, the drug loading rate would be significantly reduced, and the initial burst release was very large, and the microspheres prepared above 35°C (37°C) had a low release rate of 9h. The results show that there is no significant difference in the drug loading and release of the microspheres prepared by the present invention at a colostrum temperature of 25 to 35°C, with a high drug loading and meeting the one-month sustained release requirement. The temperature of the colostrum at the end of room temperature is generally between 25 and 35°C. Therefore, compared with other microsphere preparation methods, the method of the present invention has no special requirements for colostrum temperature and viscosity, which greatly reduces the risk of controlling the colostrum temperature in production and shortens the operation time.

[0134] Example 12 Investigation of adding different molecular weight PLA to PLGA

[0135] Weigh 1.0g of alanine acetate, add 1.2g of injection water to dissolve, weigh 7.4g of PLGA (75:25, 12539), add 30% of PLA (molecular weights are 12283, 15960, 18080, 21669 and 23184 respectively), dissolve in 17g of dichloromethane as the oil phase, stir and emulsify the two phases at 8000rpm, and add 2.0L of 0.25% NaCl-0.1% 10-30℃ PVA aqueous solution in 3min at 7000rpm by a high-speed disperser. Stir continuously for 3h under reduced pressure, evaporate the organic solvent to solidify the microspheres. Collect the precipitate by centrifugation, add 12.1g of 12% mannitol solution, disperse, and freeze-dry. After freeze-drying, pass through a 120-mesh sieve. The content and release of microspheres prepared by adding different molecular weight PLA are shown in the following table.

[0136] Table 18

[0137]

[0138] The microspheres prepared by adding 30% of PLA with a molecular weight of 12283 had a large burst release, the PLA with a molecular weight of 23184 had a slow early release, and the release of microspheres prepared by PLA with molecular weights of 15960, 18080, and 21669 met the expected target. The preferred PLA molecular weight range is 15960-21669.

[0139] Comparative Example 1

[0140] This comparative example is a method for preparing leuprorelin microspheres with gelatin added, which is prepared according to the colostrum preparation method in the document "Zhang Min et al., Optimization of preparation process and in vitro release study of alanine microspheres, Pharmaceutical Services and Research, 2012, 012(005): 358-361" (PLGA model is 75 / 25, PLGA molecular weight is 14000):

[0141] See Table 19 for the prescription.

[0142] Table 19

[0143]

[0144] After testing, the drug loading of the microspheres was 7.3%, the 1h accelerated release was 18%, and the particle size was 54 μm. It can be seen that the drug loading of Comparative Example 1 was low, the burst release was large, and the particle size was large.

[0145] Comparative Example 2

[0146] This comparative example is a method for preparing leuprorelin microspheres with gelatin added, and the recipe is as follows: Dissolve the peptide and gelatin in deionized water at 60°C. Dissolve PLGA in dichloromethane and mix and stir. Cool to 15-18°C and pour into the external aqueous phase. Stir for 3 hours and remove the solvent to obtain microparticles. Sieve, wash with water, centrifuge, disperse in mannitol solution, and freeze-dry.

[0147] Table 20

[0148]

[0149] The 48h accelerated release test results of the prepared microspheres are as follows.

[0150] Table 21

[0151]

[0152] The microspheres prepared in Comparative Example 1 and Comparative Example 2 were tested for content and 35-day long-term release, and the test results are as follows:

[0153] Table 22

[0154] Comparative Example 1 Comparative Example 2 Microsphere drug loading% 7.3％ 6.8％

[0155] 35d Release:

[0156] Table 23

[0157] Comparative Example 1 Comparative Example 2 35 days release 4h (%) 9.58 8.17 35 days release 1d (%) 23.70 19.19 35 days release 2d (%) 41.49 32.74 35 days release 3d(%) 55.33 44.04 35 days release 7d (%) 70.87 60.53 35 days release 14d(%) 80.49 72.02 35 days release 21d (%) 87.00 81.07 35 days release 28d (%) 93.80 88.72 35 days release 35d(%) 98.90 96.21

[0158] It can be seen that the microspheres prepared in Comparative Example 1 have the defects of large particle size and poor sustained release effect. Compared with Comparative Example 1, the microspheres of the embodiment of the present invention have smaller particle size and better sustained release effect, which has obvious advantages. The drug loading of Comparative Example 2 is low.

[0159] PD in rats:

[0160] Take the PLGA microspheres added with 30% PLA in Example 9 and the microspheres of Comparative Examples 1 and 2. Eight 6-week-old male SD rats were selected in each group, and a single subcutaneous injection of 0.375 mg / kg (in terms of aprolin acetate) of aprolin acetate microspheres for injection was given, and blood was collected from the jugular vein puncture using a disposable syringe before and 0.25h, 1h, 3h, 6h, 24h (D1) after administration and D2, D3, D5, D7, D11, D14, D21, D28, D35 and D42. About 0.35 ml of blood was collected from the blood collection point before and within 24h after administration, and about 0.5 ml of blood was collected from the subsequent blood collection points, and then transferred to a 5 μl centrifuge tube containing 100 TIU / ml aprotinin and 15% EDTA-K2 solution, and the centrifuge tube was immediately placed in an ice bath after gently mixing. After whole blood was collected, it was centrifuged at 4°C and 2400×g for 5 minutes, and the supernatant was separated to obtain plasma. Except during the analysis period, the plasma samples were stored in an ultra-low temperature refrigerator (-60°C to -90°C). The testosterone content in rat plasma samples was determined by LC-MS / MS quantitative analysis method.

[0161] Table 24

[0162] Blood collection time Comparative Example 1 Comparative Example 2 Example 9 (30%) 0.25h 9.99±2.62 7.42±2.96 4.16±2.76 1h 26.74±4.27 25.33±2.23 20.06±4.28 3h 23.07±4.84 22.76±7.42 25.91±3.00 6h 18.16±7.91 21.25±1.98 19.47±2.02 1d 10.01±2.75 10.24±3.27 15.07±2.69 2d 3.91±0.80 4.12±0.84 10.2±1.84 3d 1.90±0.21 2.31±2.4 3.82±1.63 5d 0.89±0.27 1.07±0.97 0.78±0.36 7d 0.59±0.08 0.65±0.81 0.97±0.18 11d 0.55±0.36 0.83±0.45 0.83±0.38 14d 0.68±0.45 0.96±0.78 0.58±0.44 21d 0.68±0.65 0.48±0.53 0.43±0.24 28d 2.24±0.9 0.76±0.87 0.60±0.41 35d 4.10±0.32 1.66±0.21 0.54±0.19 42d 3.96±0.55 3.23±0.56 1.09±0.65

[0163] Conclusion: If Figure 5 As shown, the initial release rate of the microspheres in Comparative Example 1 was faster, reaching the peak testosterone concentration 1 hour after injection, and effectively inhibiting the testosterone level 5 days after injection, but the testosterone concentration began to return to normal levels after 21 days. The testosterone inhibition curve of the microspheres in Comparative Example 2 was similar to that of Comparative Example 1, but began to return to normal after 28 days and failed to maintain 35 days. The PLGA microspheres added with 30% PLA in Example 9 reached the peak testosterone concentration 3 hours after injection, and continued to maintain the testosterone inhibition effect to 35 days after the 5th day of injection. After 35 days, the testosterone level slowly recovered, showing a more ideal in vivo inhibition effect.

[0164] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A kind of aprorelin acetate microspheres for injection, comprising aprorelin acetate and a high molecular polymer; in, The high molecular polymer includes polylactic acid-glycolic acid copolymer; Preferably, the average particle size of the aloperin acetate microspheres for injection is ≤45 μm.

2. The alorellin acetate microspheres for injection according to claim 1, wherein: The high molecular polymer is polylactic acid-glycolic acid copolymer, or a mixture of polylactic acid-glycolic acid copolymer and polylactide; Preferably, the molecular weight of the polylactic acid-co-glycolic acid is 7622-16616, more preferably 11000-16600, most preferably 12000-13500; Preferably, the molar ratio of lactide to glycolide in the polylactic acid-co-glycolic acid is 50:50 to 85:15, more preferably 75:25 to 85:15; More preferably, the weight percentage of the polylactide in the high molecular weight polymer is 5-40%, further preferably 10-40%, further preferably 15-35%, further preferably 25-35%; Preferably, the molecular weight of the polylactide is 10000-30000, more preferably 14000-22000, further preferably 14000-20000, most preferably 16000-19000; Preferably, the mass ratio of aloperin acetate to the high molecular weight polymer is 1:5 to 1:15, more preferably 1:5 to 1:

10.

3. The alorellin acetate microspheres for injection according to claim 1, wherein: The alanine acetate microspheres for injection are prepared by a double emulsion solvent evaporation method; Preferably, the external aqueous phase used in the double emulsion solvent volatilization method is a polyvinyl alcohol aqueous solution; Preferably, the external aqueous phase further comprises an inorganic salt, more preferably, the inorganic salt is sodium chloride; Preferably, the mass ratio of alorelin acetate to polyvinyl alcohol is 1:0.1 to 1:10, more preferably 1:0.1 to 1:5; Preferably, the mass ratio of aloperin acetate to the inorganic salt is 1:0.5 to 1:100, more preferably 1:1 to 1:

20.

4. An alorelin acetate microsphere composition for injection, comprising the alorelin acetate microsphere for injection according to any one of claims 1 to 3, and a water-soluble polysaccharide or amino acid; Preferably, the water-soluble polysaccharide is selected from one or more of mannitol, lactose and glucose, and the amino acid is selected from one or more of glycine and leucine; Preferably, the mass ratio of alorellin acetate to the water-soluble polysaccharide or amino acid is 1:0.1 to 1:10, more preferably 1:0.5 to 1:

5.

5. A method for preparing the alanine acetate microspheres for injection according to any one of claims 1 to 3, comprising the following steps: (1) adding alorelin acetate to water to form an inner water phase; (2) dissolving the high molecular weight polymer in an organic solvent to form an oil phase; (3) mixing and emulsifying the internal water phase prepared in step (1) and the oil phase prepared in step (2) to obtain colostrum; (4) mixing the colostrum prepared in step (3) with external water to form a double emulsion; (5) removing the organic solvent in the emulsion prepared in step (4) to solidify the microspheres, and collecting the solid portion thereof.

6. The method according to claim 5, wherein: In step (1), the mass ratio of alorelin acetate to water is 1:0.8 to 1:2.4; more preferably 1:1.2; preferably, the water is water for injection; Preferably, in step (2), the organic solvent is selected from one or more of halogenated hydrocarbons, ethers, alcohols, aromatic hydrocarbons, tetrahydrofuran or acetonitrile; more preferably, the organic solvent is selected from one or more of dichloromethane, chloroform, dichloroethane, carbon tetrachloride, ether, n-propanol, methanol, toluene or xylene; most preferably, the organic solvent is dichloromethane.

7. The method according to claim 5, wherein: In step (2), based on the total weight of the oil phase, the total concentration of the high molecular weight polymer is 25-40 wt %; more preferably 30-35 wt %; Preferably, in step (3), the emulsification is performed by high-speed stirring or ultrasonic crushing; more preferably, the speed of the high-speed stirring is 3000-10000 rpm; more preferably, the intensity of the ultrasonic crushing is 20-80%; Preferably, in step (3), the emulsification is carried out at 15-40°C, more preferably at 22-40°C, further preferably at 22-36°C, most preferably at 25-35°C.

8. The method according to claim 5, wherein: The volume ratio of the colostrum prepared in step (3) to the external aqueous phase in step (4) is 1:200 to 1:50, more preferably 1:150 to 1:95; Preferably, the colostrum prepared in step (3) is added to the external aqueous phase by a high-speed dispersion method; Preferably, the external aqueous phase is a polyvinyl alcohol aqueous solution; Preferably, the concentration of polyvinyl alcohol in the external aqueous phase is 0.01-0.50% (w / v, g / ml); Preferably, the external aqueous phase further comprises an inorganic salt; preferably, the concentration of the inorganic salt in the external aqueous phase is 0.01-10% (w / v, g / ml), more preferably 0.1-5% (w / v, g / ml), further preferably 0.1-1% (w / v, g / ml), and most preferably 0.1-0.25% (w / v, g / ml); Preferably, in step (5), the organic solvent in the emulsion prepared in step (4) is removed by stirring and evaporating under reduced pressure at 10-30°C.

9. A method for preparing the alanine acetate microsphere composition for injection according to claim 4, comprising the following steps: adding a water-soluble polysaccharide solution or an amino acid solution to alanine acetate microspheres for injection, followed by freeze-drying; Preferably, the water-soluble polysaccharide solution is a mannitol aqueous solution; preferably, the concentration of the mannitol aqueous solution is 0.5-20% (g / g), more preferably 5-20% (g / g), most preferably 9-15% (g / g).

10. Use of the alorellin acetate microspheres for injection according to any one of claims 1 to 3, the alorellin acetate microsphere composition for injection according to claim 4, the alorellin acetate microspheres for injection prepared according to the method of any one of claims 5 to 8, or the alorellin acetate microsphere composition for injection prepared according to the method of claim 9 in the preparation of drugs for treating hormone-dependent diseases such as prostate cancer, endometriosis, uterine fibroids, estrogen receptor-positive premenopausal breast cancer and central precocious puberty.

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