Improved leuprorelin sustained release microsphere and preparation method thereof

Abstract

The invention belongs to the technical field of preparation of leuprorelin, and particularly relates to an improved leuprorelin sustained release microsphere and a preparation method of the improved leuprorelin sustained release microsphere. Comprising the following components: 10-15 parts of leuprorelin, 60-80 parts of a polylactic acid-glycolic acid copolymer, 3-6 parts of an amino-containing hydrophilic modifier, 1-3 parts of a water-soluble stabilizer, 2-4 parts of a buffer agent and 0.6-1.2 parts of an emulsifier. Wherein the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (1-3): 1, and the viscosity average molecular weight of the polylactic acid-glycolic acid copolymer is 30000-80000Da. The amino-containing hydrophilic modifier and the polylactic acid-glycolic acid copolymer form a synergistic effect, so that the hydrophobicity of the sustained-release microsphere is remarkably reduced, a core-shell structure is formed, and the release period of leuprorelin is further prolonged.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of leuprolide preparation, and particularly relates to an improved leuprolide sustained-release microsphere and a preparation method thereof. BACKGROUND

[0002] Leuprolide is an artificial synthetic gonadotropin-releasing hormone agonist, which can reduce the synthesis of sex hormones by continuously inhibiting the secretion of pituitary gonadotropin, and is widely used in the treatment of diseases such as prostate cancer, endometriosis and uterine fibroids.

[0003] Since leuprolide has a short half-life, conventional injections need to be administered daily, which not only brings inconvenience to patients, but also easily affects the treatment effect due to fluctuations in blood drug concentration. Therefore, at present, the drug is usually wrapped into microspheres by using biodegradable carrier materials, and the drug is slowly released in the body after injection, so as to maintain a stable blood drug concentration and achieve a treatment effect that can be maintained for several weeks or even months with one administration. Poly (lactic-co-glycolic acid) is the most commonly used carrier material for preparing leuprolide sustained-release microspheres.

[0004] However, poly (lactic-co-glycolic acid) is a hydrophobic material itself, and the surface of the prepared microspheres has poor hydrophilicity, which is easily recognized and removed by the body's immune system after injection, thereby reducing the bioavailability of the drug and possibly causing local inflammatory reactions. In addition, the hydrophobic carrier has poor compatibility with the water-soluble leuprolide, which easily leads to the aggregation of the drug in the microspheres, thereby affecting the uniformity of release. SUMMARY

[0005] In order to solve the problems existing in the prior art, the application provides an improved leuprolide sustained-release microsphere and a preparation method thereof. The amino-containing hydrophilic modifier and poly (lactic-co-glycolic acid) form a synergistic effect, which significantly reduces the hydrophobicity of the sustained-release microspheres, and forms a core-shell structure, thereby further prolonging the release period of leuprolide.

[0006] The specific technical scheme adopted by the application is as follows: An improved leuprolide sustained-release microsphere, which comprises the following components in parts by mass: leuprolide 10-15 parts, poly (lactic-co-glycolic acid) 60-80 parts, amino-containing hydrophilic modifier 3-6 parts, water-soluble stabilizer 1-3 parts, buffer 2-4 parts, and emulsifier 0.6-1.2 parts. The molar ratio of lactic acid to glycolic acid in the poly (lactic-co-glycolic acid) is 1-3:1, and the viscosity-average molecular weight of the poly (lactic-co-glycolic acid) is 30000-80000 Da.

[0007] Further, the amino-containing hydrophilic modifier is amino-modified polyvidone with a number average molecular weight of 3000-6000 Da or amino-terminated polyethylene glycol monomethyl ether with a molecular weight of 1500-3000 Da, wherein the amino-modified polyvidone has an amino substitution degree of 5-8%.

[0008] Further, the water-soluble stabilizer includes any one or both of ascorbic acid and EDTA-2Na.

[0009] Further, the buffer is a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate with a mass ratio of 1:2-4.

[0010] Further, the emulsifier is a mixture of Span 80 and Tween 80 with a molar ratio of 2.8-3.2:1.

[0011] A preparation method of the improved leuprolide sustained-release microspheres, the preparation method comprising the following steps: S1, oil phase preparation, poly (lactic-co-glycolic acid), a hydrophilic modifier and an emulsifier are added to an organic solvent, stirred uniformly and an oil phase is obtained for standby; S2, water phase preparation, leuprolide, a water-soluble stabilizer and a buffer are added to water, the pH value is adjusted to 6.5-7.5, stirred uniformly and a water phase is obtained for standby; S3, primary emulsion preparation, the water phase is added to the oil phase, and the emulsion is sheared at a speed of 12000-15000 r / min for 5-15 min to obtain a primary emulsion; S4, re-emulsion preparation, the primary emulsion is added to a polyvinyl alcohol aqueous solution, and the emulsion is stirred at a speed of 3000-5000 r / min for 15-30 min to obtain a re-emulsion; S5, microsphere collection, the re-emulsion is stirred at 15-25℃ for 4-6h, then stirred at 1500-3000 Pa and 25-30℃ for 2-3h, and then the precipitate is collected by centrifugation, and the precipitate is washed to obtain the sustained-release microspheres; S6, surface modification post-processing, the wet microspheres are added to an aqueous solution containing 0.1-0.15 wt% non-ionic surfactant, dispersed at a speed of 6000-8000 r / min for 8-12 min to obtain a microsphere suspension, and then the microsphere suspension is dispersed in a crosslinking agent aqueous solution with a pH value of 5.0-6.0, stirred at 20-22℃ for 3-6h, centrifuged at a speed of 10000-12000 r / min for 10-15 min after the reaction is completed, and the microspheres are collected, washed and dried to obtain the improved leuprolide sustained-release microspheres.

[0012] Further, the organic solvent is a mixture of dichloromethane and ethyl acetate with a volume ratio of 3-5:1, and the mass ratio of the organic solvent to poly (lactic-co-glycolic acid) is 5-8:1.

[0013] Further, the mass concentration of leuprolide in the aqueous phase is 10-15 mg / mL.

[0014] Further, the polyvinyl alcohol aqueous solution is water containing 1-3 wt% polyvinyl alcohol, and the volume ratio of colostrum to the polyvinyl alcohol aqueous solution is 1:5-10.

[0015] Further, the non-ionic surfactant is poloxamer 188.

[0016] Further, the crosslinking agent aqueous solution contains 0.3-0.8 wt% crosslinking agent, 0.05-0.1 wt% EDC and 0.03-0.06 wt% NHS, the crosslinking agent is genipin or protocatechuic aldehyde, and the solid-liquid ratio of the sustained-release microspheres to the crosslinking agent aqueous solution is 1:20-25 g / L.

[0017] wherein EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide.

[0018] The beneficial effects of the present application are: 1. The present application selects a specific number average molecular weight of hydrophilic modifier to form a synergistic effect with polylactic acid-glycolic acid copolymer, which not only exposes the hydrophilic segment to the surface of the microspheres, significantly reduces the hydrophobicity of the polylactic acid-glycolic acid copolymer carrier, avoids the recognition of the microspheres as a foreign body by the body's immune system and rapid clearance, but also does not cause the agglomeration of the modifier or affect the molding of the microspheres due to the too large molecular weight, thereby providing a guarantee for the sustained release of the microsphere drug.

[0019] In addition, the improvement of the hydrophilicity of the microsphere surface reduces the hydrophobic interaction with the tissue cells of the injection site, reduces the release of inflammatory factors caused by foreign body reaction, and can significantly reduce the incidence of redness and pain at the injection site in clinical application.

[0020] 2. The present application improves the interaction between the drug and the carrier interface through the molecular bridging effect of the hydrophilic modifier, avoids the aggregation of the drug to form crystals or local high concentration areas in the microspheres, makes the drug in the microspheres in a uniform dispersed state, and has good drug dispersion uniformity. At the same time, the synergistic effect of the water-soluble stabilizer and the buffer is utilized to inhibit the oxidative degradation and hydrolysis of leuprolide during preparation and in vivo release, ensure the drug activity, avoid the problem of excessively high blood drug concentration peak or insufficient release in the later stage caused by the burst effect of the existing microspheres, and improve the stability of the treatment effect.

[0021] 3、The crosslinking agent is used for condensation reaction with the groups on the surface of the microspheres to form a dense crosslinked film, and the improved leuprolide sustained-release microspheres finally obtained are of a core-shell structure, the crosslinked film fills the natural pores on the surface of the traditional microspheres, blocks the rapid leakage channel of the drug, the diffusion resistance of the crosslinked film delays the release of leuprolide, and thus the release period of leuprolide is prolonged. DETAILED DESCRIPTION

[0022] The application will be further described below in combination with specific examples: Example 1 S1, oil phase preparation, 70 parts of polylactic acid-glycolic acid copolymer (lactic acid: glycolic acid = 2:1, viscosity average molecular weight 50000 Da), 4.5 parts of aminated povidone (number average molecular weight 5000 Da, amino substitution degree 6%) and 0.9 parts of emulsifier (molar ratio of Span 80: Tween 80 is 3:1) are added into 420 parts of organic solvent (volume ratio of dichloromethane and ethyl acetate is 4:1), stirred uniformly and the oil phase is obtained for standby; S2, water phase preparation, 12 parts of leuprolide, 2 parts of ascorbic acid and 3 parts of buffer (mass ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is 1:2-4) are added into water, the pH value is adjusted to 7.0, and stirring is carried out until complete dissolution, to obtain the water phase for standby, and the mass concentration of leuprolide in the water phase is 12 mg / mL; S3, primary emulsion preparation, the water phase is added to the oil phase at a rate of 1 mL / min, the volume ratio of the water phase to the oil phase is 1:3, and shearing emulsification is carried out at a speed of 13000 r / min for 10 min to obtain the primary emulsion; S4, re-emulsion preparation, the primary emulsion is added into 2wt% polyvinyl alcohol aqueous solution, the volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution is 1:8, and stirring emulsification is carried out at a speed of 4000 r / min for 22 min to obtain the re-emulsion; S5, microsphere collection, the re-emulsion is stirred at 20℃ for 5h, then continues to be stirred at 2500Pa and 28℃ for 2.5h, and then 10000 r / min centrifugation is carried out for 15 min to collect the precipitate, and the precipitate is washed with deionized water for 3 times to obtain the sustained-release microspheres; S6, after surface modification treatment, the wet microspheres were added to an aqueous solution containing 0.12wt% poloxamer 188, and dispersed at a speed of 7000r / min for 10min to obtain a microsphere suspension, then the microsphere suspension was added dropwise to an aqueous solution of crosslinking agent (containing 0.6wt% genipin, 0.08wt% EDC and 0.045wt% NHS, the pH value of the aqueous solution of crosslinking agent was 5.5) at a rate of 0.8mL / min (the solid-liquid ratio of the sustained-release microspheres to the aqueous solution of crosslinking agent was 1:22g / L), then stirred at 21℃ for 5h, after the reaction was completed, the microspheres were collected by centrifugation at a speed of 11000r / min for 12min, washed with a phosphate buffer solution with a pH value of 7.0 for 3 times, and then freeze-dried at-50℃ to obtain the modified leuprolide sustained-release microspheres.

[0023] Example 2 S1, oil phase preparation, 65 parts of polylactic acid-glycolic acid copolymer (lactic acid: glycolic acid = 1:1, viscosity average molecular weight 60000Da), 5 parts of amino-terminated polyethylene glycol monomethyl ether (number average molecular weight 2500Da) and 1.1 parts of emulsifier (molar ratio of Span 80: Tween 80 is 3.2:1) were added to 455 parts of organic solvent (volume ratio of dichloromethane to ethyl acetate is 3:1), stirred uniformly and obtained for standby oil phase; S2, water phase preparation, 15 parts of leuprolide, 1.5 parts of EDTA-2Na and 3.5 parts of buffer (mass ratio of sodium dihydrogen phosphate to disodium hydrogen phosphate is 1:2.5) were added to water, the pH value was adjusted to 7.5, and stirred until completely dissolved to obtain the water phase for standby, the mass concentration of leuprolide in the water phase was 15mg / mL; S3, primary emulsion preparation, the water phase was added to the oil phase at a rate of 1.1mL / min, the volume ratio of the water phase to the oil phase was 1:3.5, and the primary emulsion was obtained by shearing emulsification at a speed of 15000r / min for 8min; S4, re-emulsion preparation, the primary emulsion was added to a 3wt% polyvinyl alcohol aqueous solution, the volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution was 1:5, and the re-emulsion was obtained by stirring emulsification at a speed of 3500r / min for 30min; S5, microsphere collection, the re-emulsion was stirred at 25℃ for 4h, then stirred at 3000Pa and 30℃ for 2h, then the precipitate was collected by centrifugation at 12000r / min for 10min, and the sustained-release microspheres were obtained after washing the precipitate with deionized water for 3 times; S6, after surface modification treatment, the wet microspheres were added to an aqueous solution containing 0.14wt% poloxamer 188, and dispersed at a speed of 8000r / min for 12min to obtain a microsphere suspension, then the microsphere suspension was added dropwise to a crosslinking agent aqueous solution (containing 0.5wt% protocatechualdehyde, 0.07wt% EDC and 0.04wt% NHS, the solid-liquid ratio of the slow-release microspheres to the crosslinking agent aqueous solution was 1:25g / L) with a pH value of 6.0 at a rate of 1.0mL / min, and then stirred at 22℃ for 4h, after the reaction was completed, the microspheres were collected by centrifugation at a speed of 12000r / min for 10min, washed with a phosphate buffer with a pH value of 7.0 for 3 times, and then freeze-dried at-50℃ to obtain the modified leuprolide slow-release microspheres.

[0024] Example 3 S1, oil phase preparation, 60 parts of polylactic acid-glycolic acid copolymer (lactic acid: glycolic acid = 1.5:1, viscosity average molecular weight 30000Da), 3 parts of aminated povidone (number average molecular weight 3000Da, amino substitution degree 5%) and 0.6 parts of emulsifier (molar ratio of Span 80: Tween 80 is 2.8:1) were added to 300 parts of organic solvent (volume ratio of dichloromethane to ethyl acetate is 3.5:1), stirred uniformly and obtained for standby oil phase; S2, water phase preparation, 10 parts of leuprolide, 1 part of ascorbic acid and 2 parts of buffer (mass ratio of sodium dihydrogen phosphate to disodium hydrogen phosphate is 1:2) were added to water, the pH value was adjusted to 6.5, and stirred until completely dissolved to obtain the water phase for standby, the mass concentration of leuprolide in the water phase was 10mg / mL; S3, primary emulsion preparation, the water phase was added to the oil phase at a rate of 0.9mL / min, the volume ratio of the water phase to the oil phase was 1:4, and the primary emulsion was obtained by shearing emulsification at a speed of 12000r / min for 15min; S4, re-emulsion preparation, the primary emulsion was added to a 1wt% polyvinyl alcohol aqueous solution, the volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution was 1:9, and the re-emulsion was obtained by stirring emulsification at a speed of 3000r / min for 15min; S5, microsphere collection, the re-emulsion was stirred at 15℃ for 6h, then stirred at 25℃ for 3h under a pressure of 1500Pa, and then the precipitate was collected by centrifugation at a speed of 10000r / min for 15min, and the slow-release microspheres were obtained after washing the precipitate with deionized water for 3 times; S6, after surface modification treatment, the wet microspheres were added into the aqueous solution containing 0.1 wt% poloxamer 188, and dispersed at a speed of 6000 r / min for 8 min to obtain a microsphere suspension, and then the microsphere suspension was added dropwise into the crosslinking agent aqueous solution (containing 0.3 wt% genipin, 0.05 wt% EDC and 0.03 wt% NHS, and the solid-liquid ratio of the sustained-release microspheres to the crosslinking agent aqueous solution was 1:20 g / L) with a pH value of 5.0 at a rate of 0.5 mL / min, and then stirred at 20°C for 6 h. After the reaction, the microspheres were collected by centrifugation at a speed of 10000 r / min for 15 min, washed with a phosphate buffer with a pH value of 7.0 for 3 times, and then freeze-dried at -50°C to obtain the modified leuprolide sustained-release microspheres.

[0025] Example 4 S1, oil phase preparation, 80 parts of polylactic acid-glycolic acid copolymer (lactic acid: glycolic acid = 2.5:1, viscosity average molecular weight 80000 Da), 6 parts of aminated povidone (number average molecular weight 6000 Da, amino substitution degree 8%) and 1.2 parts of emulsifier (molar ratio of Span 80: Tween 80 is 3.1:1) were added into 640 parts of organic solvent (volume ratio of dichloromethane and ethyl acetate is 5:1), stirred uniformly and obtained for standby; S2, water phase preparation, 14 parts of leuprolide, 3 parts of ascorbic acid and EDTA-2Na complex (mass ratio 1:1) and 4 parts of buffer (mass ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is 1:3.5) were added into water, the pH value was adjusted to 7.2, and stirred until completely dissolved to obtain the water phase for standby, and the mass concentration of leuprolide in the water phase was 14 mg / mL; S3, primary emulsion preparation, the water phase was added to the oil phase at a rate of 1.0 mL / min, and the volume ratio of the water phase to the oil phase was 1:3, and the primary emulsion was obtained by shearing emulsification at a speed of 14000 r / min for 12 min; S4, re-emulsion preparation, the primary emulsion was added into a 2.5 wt% polyvinyl alcohol aqueous solution, and the volume ratio of the primary emulsion to the polyvinyl alcohol aqueous solution was 1:10, and the re-emulsion was obtained by stirring emulsification at a speed of 5000 r / min for 28 min; S5, microsphere collection, the re-emulsion was stirred at 22°C for 5.5 h, then stirred at 2800 Pa and 29°C for 2.8 h, and then the precipitate was collected by centrifugation at 11000 r / min for 13 min, and the sustained-release microspheres were obtained after washing the precipitate with deionized water for 3 times; S6. Post-surface modification treatment: Wet microspheres were added to an aqueous solution containing 0.15 wt% poloxamer 188 and dispersed at 7800 r / min for 11 min to obtain a microsphere suspension. The microsphere suspension was then added dropwise at a rate of 0.9 mL / min to a crosslinking agent aqueous solution with a pH of 5.8 (the crosslinking agent aqueous solution contained 0.8 wt% protocatechuic aldehyde, 0.1 wt% EDC, and 0.06 wt% NHS, and the solid-liquid ratio of the sustained-release microspheres to the crosslinking agent aqueous solution was 1:24 g / L). The reaction was then stirred at 21 °C for 5.5 h. After the reaction was completed, the microspheres were collected by centrifugation at 11500 r / min for 12 min. The microspheres were washed three times with phosphate buffer solution with a pH of 7.0 and then freeze-dried at -50 °C to obtain modified leuprorelin sustained-release microspheres.

[0026] Comparative Example 1 Compared with Example 1, Comparative Example 1 does not contain amino-modified povidone and does not undergo surface modification post-treatment. The specific steps are as follows: S1. Preparation of the oil phase: 70 parts of polylactic acid-glycolic acid copolymer (lactic acid:glycolic acid = 2:1, viscosity-average molecular weight 50000 Da) and 0.9 parts of emulsifier (molar ratio of Span 80 to Tween 80 is 3:1) were added to 420 parts of organic solvent (a mixture of dichloromethane and ethyl acetate in a volume ratio of 4:1), stirred evenly, and the oil phase was prepared for use. S2. Preparation of aqueous phase: 12 parts of leuprorelin, 2 parts of ascorbic acid and 3 parts of buffer (sodium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 1:2-4) were added to water, the pH was adjusted to 7.0, and the mixture was stirred until completely dissolved to obtain an aqueous phase for later use. The mass concentration of leuprorelin in the aqueous phase was 12 mg / mL. S3. Preparation of the primary emulsion: The aqueous phase is added dropwise to the oil phase at a rate of 1 mL / min, with the volume ratio of the aqueous phase to the oil phase being 1:3. The mixture is sheared and emulsified at a speed of 13000 r / min for 10 min to obtain the primary emulsion. S4. Preparation of double emulsion: The primary emulsion is added to a 2wt% polyvinyl alcohol aqueous solution, with a volume ratio of primary emulsion to polyvinyl alcohol aqueous solution of 1:8. The mixture is stirred and emulsified at 4000 r / min for 22 min to obtain the double emulsion. S5. Microsphere collection: The re-emulsion was stirred at 20℃ for 5h, and then stirred at 2500Pa and 28℃ for 2.5h. The precipitate was then collected by centrifugation at 10000r / min for 15min. The precipitate was washed three times with deionized water and freeze-dried to obtain modified leuprolide sustained-release microspheres.

[0027] Comparative Example 2 Compared with Example 1, Comparative Example 2 did not undergo surface modification post-treatment. The specific steps are as follows: S1. Preparation of the oil phase: 70 parts of polylactic acid-glycolic acid copolymer (lactic acid:glycolic acid = 2:1, viscosity-average molecular weight 50000 Da), 4.5 parts of aminated povidone, and 0.9 parts of emulsifier (Span 80: Tween 80 molar ratio of 3:1) were added to 420 parts of organic solvent (a mixture of dichloromethane and ethyl acetate in a volume ratio of 4:1), stirred evenly, and the oil phase was prepared for use. S2. Preparation of aqueous phase: 12 parts of leuprorelin, 2 parts of ascorbic acid and 3 parts of buffer (sodium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 1:2-4) were added to water, the pH was adjusted to 7.0, and the mixture was stirred until completely dissolved to obtain an aqueous phase for later use. The mass concentration of leuprorelin in the aqueous phase was 12 mg / mL. S3. Preparation of the primary emulsion: The aqueous phase is added dropwise to the oil phase at a rate of 1 mL / min, with the volume ratio of the aqueous phase to the oil phase being 1:3. The mixture is sheared and emulsified at a speed of 13000 r / min for 10 min to obtain the primary emulsion. S4. Preparation of double emulsion: The primary emulsion is added to a 2wt% polyvinyl alcohol aqueous solution, with a volume ratio of primary emulsion to polyvinyl alcohol aqueous solution of 1:8. The mixture is stirred and emulsified at 4000 r / min for 22 min to obtain the double emulsion. S5. Microsphere collection: The re-emulsion was stirred at 20℃ for 5h, and then stirred at 2500Pa and 28℃ for 2.5h. The precipitate was then collected by centrifugation at 10000r / min for 15min. The precipitate was washed three times with deionized water and freeze-dried to obtain modified leuprolide sustained-release microspheres.

[0028] II. Performance Testing 1. Detection of hydrophilicity of microsphere surface Test method: Contact angle measurement: The microspheres were evenly spread on a glass slide using the seat drop method, and 5 μL of deionized water was added. The static contact angle was recorded by the contact angle meter after 30 seconds. Ten parallel samples were measured for each group, and the average value was taken.

[0029] Zeta potential measurement: Microspheres were ultrasonically dispersed in physiological saline, and the potential value was measured by a Zeta potential meter. Surface free energy calculation: The surface free energy was calculated using the Owens-Wendt method based on the contact angles of the two probe solutions, water and ethylene glycol.

[0030] The test results are shown in Table 1.

[0031] Table 1 As can be seen from Table 1, the static contact angles of Examples 1-4 are 57.3°-63.8°, all of which are in the low contact angle range, indicating that the surface of the microspheres in this invention is hydrophilic. Comparative Example 2 only added a hydrophilic modifier and did not perform surface modification. Therefore, its hydrophilicity is still weaker than that of the Example. This is because the surface modification in the Example further optimized the surface topology of the microspheres, reduced the exposure of hydrophobic regions, and synergistically improved the hydrophilicity.

[0032] The absolute value of the Zeta potential in the examples is smaller than that in the comparative example. This is because the amino group of the hydrophilic modifier undergoes a condensation reaction with the crosslinking agent, neutralizing part of the negative charge on the surface of the microspheres, reducing the surface charge density, reducing the aggregation between microspheres and the probability of the body's immune system recognizing foreign objects, thereby improving biocompatibility.

[0033] The surface free energy of the other embodiment is higher than that of the comparative example. The higher the surface free energy, the better the compatibility of the microspheres with the aqueous phase. After injection, the hydrophobic interaction with tissue cells can be reduced, thus reducing the risk of local inflammatory response.

[0034] 2. Blood drug concentration stability test Subjects: Male SD rats (200±20g), 10 rats per group, and the average value of the test results is taken; Detection method: A single subcutaneous injection of microsphere suspension (dose: leuprorelin 0.3 mg / kg) was administered. Serum samples were collected from the fundus venous plexus of rats at 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 48 h, and 72 h after administration. The concentration of leuprorelin in the blood samples was determined by liquid chromatography-tandem mass spectrometry (ng / mL) after solid-phase extraction and organic solvent treatment.

[0035] The test results are shown in Table 2.

[0036] Table 2 As shown in Table 2, the blood drug concentrations 1 hour after administration in Examples 1-4 were 2.63-3.12 ng / mL, while the blood drug concentrations 1 hour after administration in Comparative Examples 1-2 were 8.65 ng / mL and 5.32 ng / mL, respectively. This is because the molecular bridging effect of the hydrophilic modifier improves the interfacial compatibility between water-soluble leuprolide and the hydrophobic polylactic-co-glycolic acid copolymer, avoiding drug aggregation to form local high-concentration areas and reducing burst release.

[0037] Examples 1-4 showed that the blood drug concentration remained at 1.82-2.13 ng / mL 72 hours after administration, and the coefficient of variation (CV) for the entire release cycle was below 20%, indicating a significant advantage in the stability of release of the present invention. This is because the surface cross-linked film fills the natural pores on the surface of the microspheres, blocking the rapid leakage channels of the drug and ensuring sufficient release in the later stages.

[0038] 3. Duration of drug efficacy testing Subjects: Male SD rats (200±20g), 10 rats per group, and the average value of the test results is taken; Detection method: A single subcutaneous injection of microsphere suspension (dose: leuprorelin 0.3 mg / kg) was administered. Serum samples were collected from the fundus venous plexus of rats before administration (week 0) and at 1, 2, 4, 6, 8, 10, and 12 weeks after administration. The concentration of leuprorelin in the blood samples was determined by liquid chromatography-tandem mass spectrometry (ng / mL) after solid-phase extraction and organic solvent treatment.

[0039] The test results are shown in Table 3.

[0040] Table 3 The lower limit of clinically effective blood concentration of leuprorelin in treating diseases is approximately 0.1 ng / mL. As shown in Table 3, the blood concentrations of Examples 1-4 remained at 0.121-0.129 ng / mL at 12 weeks, consistently within the effective therapeutic window; while the concentration of Comparative Example 1 had decreased to 0.090 ng / mL at 4 weeks and only 0.010 ng / mL at 12 weeks; the concentration of Comparative Example 2 had decreased to 0.085 ng / mL at 8 weeks and only 0.020 ng / mL at 12 weeks, also failing to maintain long-term therapeutic effect.

[0041] This is because in Comparative Example 1, the lack of hydrophilic modification and surface modification resulted in the strong hydrophobicity of the polylactic acid-glycolic acid copolymer carrier, leading to rapid depletion of the drug after a large initial release. In Comparative Example 2, although a hydrophilic modifier was added, the lack of cross-linking membrane protection resulted in a relatively fast degradation rate of the polylactic acid-glycolic acid copolymer.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified leuprolide sustained-release microspheres, characterized by, The preparation method comprises the following steps: The preparation method comprises the following steps:

2. The improved leuprolide sustained-release microspheres according to claim 1, wherein The preparation method comprises the following steps:

3. The improved leuprolide sustained-release microspheres according to claim 1, wherein the leuprolide is present in an amount of 5-15% by weight. The preparation method comprises the following steps:

4. The improved leuprolide sustained-release microspheres according to claim 1, wherein the leuprolide is present in an amount of 5-15% by weight. The preparation method comprises the following steps:

5. The improved leuprolide sustained-release microspheres according to claim 1, wherein The preparation method comprises the following steps:

6. A method for preparing a modified leuprolide sustained-release microspheres for preparing a modified leuprolide sustained-release microspheres as claimed in claim 1, characterized by, The preparation method comprises the following steps: The preparation method comprises the following steps: The preparation method comprises the following steps: The preparation method comprises the following steps: The preparation method comprises the following steps: The preparation method comprises the following steps: The preparation method comprises the following steps:

7. The method of claim 6, wherein the modified leuprolide sustained-release microspheres are prepared by the steps of: The preparation method comprises the following steps:

8. The method of claim 6, wherein the modified leuprolide sustained-release microspheres are prepared by the steps of: The preparation method comprises the following steps:

9. 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10. The method for preparing a modified leuprolide sustained-release microsphere according to claim 6, characterized in that, The crosslinking agent aqueous solution contains 0.3-0.8wt% crosslinking agent, 0.05-0.1wt% EDC and 0.03-0.06wt% NHS, the crosslinking agent is genipin or protocatechuic aldehyde, and the solid-liquid ratio of the sustained-release microspheres and the crosslinking agent aqueous solution is 1:20-25g / L.

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