Low-burst release leuprolide acetate PLGA microspheres, preparation method and application thereof

Abstract

This invention belongs to the field of sustained-release drug formulation technology, specifically relating to a low-burst-release leuprolide acetate PLGA microsphere and its preparation method. The microsphere comprises leuprolide acetate, polylactic acid-hydroxyacetic acid copolymer, and a ternary interfacial barrier system. The ternary interfacial barrier system includes Tween-80, polyoxyethylene 15-hydroxystearate, and cholesterol acetate; cholesterol acetate is a steroidal hydrophobic barrier agent. The hydrophobic chain structures of Tween-80 and HS15 in this invention are different, and they can form a mixed interfacial layer at the oil / water interface, which helps reduce the accumulation defects of a single surfactant adsorption layer. Furthermore, CA has a rigid steroidal hydrophobic structure, which can accumulate on the surface or near-surface region of the microsphere during droplet solidification, and together with the mixed interfacial layer formed by Tween-80 and HS15, constitutes a surface-enriched dense barrier layer, which can significantly reduce the distribution of leuprolide acetate on the surface or near-surface region of the microsphere, reduce the proportion of rapidly elutable drugs on the surface, and thus reduce the initial burst release at 24 h.

Description

Technical Field

[0001] This invention belongs to the field of sustained-release and controlled-release drug formulation technology, specifically relating to a low-burst-release leuprolide acetate PLGA microsphere, its preparation method, and its application. Background Technology

[0002] Leuprorelin acetate is a gonadotropin-releasing hormone analogue, belonging to the polypeptide class of drugs. Because polypeptide drugs are easily enzymatically broken down in the body and have a short half-life, conventional injectable formulations often fail to maintain long-term effective blood drug concentrations. Therefore, preparing leuprorelin acetate into long-acting sustained-release microspheres is a common technical approach to improve administration convenience, extend dosing intervals, and enhance patient compliance.

[0003] Polylactic acid-glycolic acid copolymer (PLGA) possesses excellent biocompatibility and biodegradability, making it a commonly used polymer material for preparing long-lasting injectable microspheres. Leuprolide acetate PLGA microspheres are typically prepared using a double emulsion-solvent evaporation method. This involves first dissolving leuprolide acetate in an inner aqueous phase, then emulsifying it with a PLGA-containing organic phase to form a W / O primary emulsion. The primary emulsion is then added to an outer aqueous phase containing a stabilizer to form a W / O / W double emulsion. After solvent evaporation or extraction, the mixture solidifies to form microspheres.

[0004] However, leuprolide acetate is a hydrophilic peptide that readily migrates to the oil / water interface during double emulsion preparation and droplet solidification, accumulating on or near the surface of the microspheres. This portion of the drug tends to rapidly enter the release medium during the initial release phase, causing an initial burst release. Excessive initial burst release can lead to problems such as excessive initial drug exposure, insufficient subsequent release, and unstable release profiles, posing a significant challenge in the development of leuprolide acetate PLGA microspheres.

[0005] Existing techniques for reducing the burst release of PLGA microspheres mainly include adjusting the molecular weight of PLGA, changing the lactic acid / glycolic acid ratio, changing the end-group type, increasing the microsphere size, altering the composition of the internal and external aqueous phases, adding salts or stabilizers, changing the evaporation rate of organic solvents, and adjusting the emulsifier concentration. While these methods can influence the release behavior to some extent, they typically achieve this primarily by changing particle size, porosity, encapsulation efficiency, or polymer degradation rate. They are difficult to directly control the radial distribution of leuprolide acetate in the microspheres, and particularly difficult to reduce drug accumulation on the surface or near-surface region of the microspheres.

[0006] In addition, conventional external aqueous phase stabilizers such as polyvinyl alcohol are mainly used to stabilize multi-emulsion droplets, while Tween-type or other surfactants are typically used to improve emulsification, particle size distribution, or encapsulation efficiency. The adsorption layer of a single surfactant at the oil / water interface may have molecular stacking defects or uneven coverage, potentially allowing hydrophilic drugs to migrate to the interfacial region during droplet solidification. Conventional binary surfactant formulations are also mostly used to adjust HLB values ​​or emulsion stability, rather than to form a dense barrier structure retained on the microsphere surface after solidification.

[0007] Therefore, there is an urgent need to develop a new leuprolide acetate PLGA microsphere and its preparation method, which can reduce the distribution of leuprolide acetate on the surface or near the surface of the microsphere while maintaining high drug loading, encapsulation efficiency and long-term stable drug release performance, thereby reducing the initial burst release. Summary of the Invention

[0008] In view of this, the present invention provides a novel leuprolide acetate PLGA microsphere and its preparation method, in order to solve the technical problems mentioned in the background art.

[0009] The terminology used in this invention is explained below: Unless otherwise stated, PLGA in this invention refers to polylactic acid-glycolic acid copolymer.

[0010] The polyoxyethylene 15 hydroxy stearate described in this invention can be simply referred to as HS15.

[0011] The cholesterol acetate described in this invention can be abbreviated as CA.

[0012] In this invention, the initial burst release refers to the cumulative percentage of drug released by the microspheres after 24 hours in the release medium.

[0013] The low burst release mentioned in this invention refers to a reduction in the initial burst release at 24 h compared to control microspheres without CA; in some embodiments, the reduction is not less than 20%.

[0014] In this invention, the surface or near-surface region refers to the outer surface of the microsphere and the shallow region near the outer surface. The drug in this region can be characterized by a rapid surface elution experiment.

[0015] In this invention, the total amount of Tween-80 and HS15 added refers to the total mass of Tween-80 and HS15 introduced into the external aqueous phase or emulsion system during the microsphere preparation process. This total amount is calculated based on the mass of polylactic acid-glycolic acid copolymer added, and does not imply that all the added material will remain in the microspheres.

[0016] The amount of CA mentioned in this invention refers to the mass of CA added to the organic phase during the microsphere preparation process, calculated based on the mass of polylactic acid-glycolic acid copolymer added.

[0017] To achieve the above objectives, the present invention adopts the following technical solution: A low-burst-release leuprolide acetate PLGA microsphere, the microsphere comprising leuprolide acetate, polylactic acid-glycolic acid copolymer and a ternary interfacial barrier system; The ternary interface barrier system includes Tween-80, polyoxyethylene 15-hydroxystearate, and cholesterol acetate. The cholesterol acetate is a steroidal hydrophobic barrier agent.

[0018] In this invention, Tween-80 has an oleate hydrophobic chain, and polyoxyethylene 15-hydroxystearate has a saturated hydroxystearate hydrophobic structure. The difference in their hydrophobic chain structures may lead to the formation of a mixed interface layer at the oil / water interface of the PLGA emulsion droplets. Cholesterol acetate has a rigid steroidal hydrophobic structure and limited compatibility with the PLGA matrix. During the emulsion droplet curing process, it can accumulate on or near the surface of the microspheres, and together with the mixed interface layer formed by Tween-80 and polyoxyethylene 15-hydroxystearate, constitute a surface-enriched dense barrier layer.

[0019] In a preferred embodiment, the weight ratio of leuprolide acetate to polylactic acid-glycolic acid copolymer is 1:5 to 1:20, preferably 1:8 to 1:15, and more preferably 1:10.

[0020] In a preferred embodiment, the amount of cholesterol acetate is 0.25% to 5% of the mass of the polylactic acid-glycolic acid copolymer, more preferably 2% to 5%.

[0021] In a preferred embodiment, the mass ratio of Tween-80 to polyoxyethylene 15-hydroxystearate is 8:1 to 1:4, more preferably 4:1 to 1:1.

[0022] In a preferred embodiment, the total amount of Tween-80 and polyoxyethylene 15-hydroxystearate is 0.5% to 10% of the mass of polylactic acid-hydroxyacetic acid copolymer, more preferably 2% to 5%.

[0023] In a preferred embodiment, the ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50 to 85:15, preferably 75:25.

[0024] The organic solvent for dissolving the polylactic acid-glycolic acid copolymer is a mixture of dichloromethane and ethyl acetate, with a volume ratio of 9:1 to 5:5, preferably 5:5.

[0025] In a preferred embodiment, Tween-80 and polyoxyethylene 15-hydroxystearate are present in the external aqueous phase during the microsphere preparation process, and cholesterol acetate is present in the organic phase containing polylactic acid-hydroxyacetic acid copolymer; the cholesterol acetate is enriched on the surface or near-surface region of the microsphere during the droplet solidification process, and reduces the enrichment of leuprolide acetate on the surface of the microsphere.

[0026] The present invention also provides a method for preparing the above-mentioned low-burst release leuprolide acetate PLGA microspheres, comprising the following steps: (1) Dissolve leuprolide acetate in the aqueous phase to obtain the inner aqueous phase; (2) Dissolve polylactic acid-glycolic acid copolymer and cholesterol acetate in an organic solvent to obtain an organic phase; (3) The aqueous phase is added to the organic phase and emulsified to obtain the primary emulsion; (4) The primary emulsion is added to an aqueous phase containing polyvinyl alcohol, Tween-80 and polyoxyethylene 15-hydroxystearate, and emulsified to obtain a secondary emulsion; (5) Remove the organic solvent to solidify the emulsion droplets into microspheres; (6) Collect, wash and dry to obtain low-burst release leuprolide acetate PLGA microspheres.

[0027] The present invention also provides the use of the above-mentioned microspheres in the preparation of medicaments for treating hormone-dependent diseases.

[0028] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a ternary interfacial barrier system composed of Tween-80, HS15, and CA. Instead of simply adjusting the molecular weight, particle size, or PVA concentration of PLGA to reduce burst release, it reduces initial burst release by regulating the droplet interfacial structure and radial drug distribution.

[0029] 2. Tween-80 and HS15 have different hydrophobic chain structures. They can form a mixed interface layer at the oil / water interface, which helps to reduce the accumulation defects of a single surfactant adsorption layer.

[0030] 3. CA has a rigid steroidal hydrophobic structure, which can be enriched on the surface or near-surface region of microspheres during the emulsion solidification process, and together with the mixed interface layer formed by Tween-80 and HS15, it constitutes a surface-enriched dense barrier layer.

[0031] 4. This invention can significantly reduce the distribution of leuprolide acetate on or near the surface of microspheres, reduce the proportion of rapidly elutable drugs on the surface, and thus reduce the initial burst release at 24 h.

[0032] 5. The method of this invention is compatible with the conventional re-emulsion-solvent evaporation method, does not require a complex post-coating process, and has good process feasibility and scale-up potential.

[0033] 6. After entering the body, CA can be hydrolyzed into cholesterol and acetic acid by endogenous esterases. Both of these are endogenous substances in the human body and have high biological safety. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 The particle size distribution of leuprolide acetate PLGA microspheres prepared in Example 1 is shown.

[0036] Figure 2 This is a scanning electron microscope image of the leuprolide acetate PLGA microspheres prepared in Example 1.

[0037] Figure 3 In vitro release curves of leuprolide acetate PLGA microspheres with different formulations.

[0038] Figure 4 The effect of different CA / PLGA mass ratios on the surface rapid elution ratio and initial burst release at 24 h of leuprolide acetate PLGA microspheres was investigated, where A represents the surface rapid elution ratio and B represents the initial burst release at 24 h.

[0039] Figure 5 The effect of different Tween-80 to HS15 mass ratios on the surface rapid elution ratio and initial burst release at 24 h of leuprolide acetate PLGA microspheres was investigated, where A represents the surface rapid elution ratio and B represents the initial burst release at 24 h.

[0040] Figure 6 The effect of different total amounts of Tween-80 and HS15 on the surface rapid elution ratio and initial burst release at 24 h of leuprolide acetate PLGA microspheres was investigated, where A represents the surface rapid elution ratio and B represents the initial burst release at 24 h.

[0041] Figure 7 The effect of different combinations of interfacial components on the surface rapid elution ratio and initial burst release at 24 h of leuprolide acetate PLGA microspheres was investigated, where A represents the surface rapid elution ratio and B represents the initial burst release at 24 h.

[0042] Figure 8 This shows the distribution of CA in the cured PLGA.

[0043] Figure 9The distribution results of leuprolide acetate in CA-containing cured PLGA are shown.

[0044] Figure 10 The distribution results of leuprolide acetate in CA-free cured PLGA are shown. Detailed Implementation

[0045] The present invention will be further described below with reference to the embodiments. It should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Without departing from the spirit and substance of the present invention, those skilled in the art can make conventional adjustments to the types of raw materials, the feeding ratio, the emulsification method, the solvent system, the curing method, and the drying method, and all such adjustments should fall within the scope of protection of the present invention.

[0046] Unless otherwise stated, the percentages in the following examples are mass-volume percentages or mass percentages.

[0047] Example 1: Preparation of Leuprolide Acetate PLGA Microspheres using a Tween-80 / HS15 / CA Ternary System 1. Prescription Composition Internal aqueous phase W1: 50 mg of leuprolide acetate dissolved in 0.25 mL of water for injection.

[0048] Organic phase O: 500 mg PLGA (75:25) and 5 mg CA were dissolved in 3 mL of a dichloromethane / ethyl acetate mixed solvent, wherein the volume ratio of dichloromethane to ethyl acetate was 7:3.

[0049] External aqueous phase W2: 40 mg Tween-80, 10 each HS15, dissolved in 100 mL of water for injection (containing 0.5% polyvinyl alcohol, w / v).

[0050] In this embodiment, the amount of CA used is 1.0% of the mass of PLGA; the mass ratio of Tween-80 to HS15 is 4:1.

[0051] 2. Preparation method (1) Weigh 50 mg of leuprolide acetate and add it to 0.25 mL of water for injection. Gently vortex until completely dissolved to obtain the internal aqueous phase W1.

[0052] (2) Weigh 500 mg PLGA and 5 mg CA, add them to 3.0 mL of dichloromethane / ethyl acetate mixed solvent, and gently shake or magnetically stir at room temperature until completely dissolved to obtain organic phase O.

[0053] (3) The aqueous phase W1 was slowly added dropwise to the organic phase O, and ultrasonic emulsification was performed using a probe under ice bath conditions. The ultrasonic power was 30 W to 60 W, with a working time of 2 s and an intermittent time of 2 s, and the total emulsification time was 30 s to 60 s, to obtain the W / O promulgated emulsion.

[0054] (4) The obtained W / O colostrum was immediately added to 100 mL of pre-cooled external aqueous phase W2 and emulsified using a high-speed shear emulsifier. The shearing speed was 3000 rpm to 6000 rpm and the emulsification time was 1 min to 3 min to obtain W / O / W double emulsion.

[0055] (5) Transfer the emulsion to a beaker and stir at 300 to 600 rpm for 4 h at room temperature to allow the organic solvent to evaporate and the microspheres to solidify.

[0056] (6) Centrifuge the solidified microsphere suspension at 3000 rpm to 5000 rpm for 5 min to 10 min, discard the supernatant, and wash 3 times with water for injection.

[0057] (7) The washed microspheres were resuspended in an appropriate amount of aqueous solution containing 2% mannitol, pre-frozen, and then freeze-dried for 24 to 48 hours to obtain leuprolide acetate PLGA microspheres.

[0058] 3. Testing The particle size, morphology, drug loading, encapsulation efficiency, surface rapid drug elution ratio, in vitro release curve, and 24-hour burst release were determined using the following detection methods.

[0059] Particle size determination: Take an appropriate amount of dried microspheres and disperse them in purified water containing 0.02% Tween-80. Gently shake to ensure uniform dispersion of the microspheres, and determine D10, D50, and D90 using a laser particle size analyzer. Each batch of samples was measured in triplicate.

[0060] Morphological observation: A small amount of dried microspheres were fixed on conductive adhesive, sputtered with gold, and then the surface morphology of the microspheres was observed using a scanning electron microscope, including sphericity, surface pores, wrinkles, cracks, crystal precipitation, and aggregation.

[0061] Drug loading and encapsulation efficiency determination: Accurately weigh approximately 10 mg of microspheres and add an appropriate amount of acetonitrile, dimethyl sulfoxide, or other solvent capable of dissolving PLGA to ensure complete dissolution. Then, add an appropriate amount of water or buffer solution to allow leuprolide acetate to enter the aqueous phase. After centrifugation or filtration, collect the supernatant and determine the leuprolide acetate content using HPLC.

[0062] Drug loading capacity is calculated using the following formula: Drug loading (%) = Actual drug mass in microspheres / Total mass of microspheres × 100%.

[0063] Encapsulation efficiency (%) = Actual drug mass in microspheres / Drug mass added × 100%.

[0064] In vitro release assay: Accurately weigh approximately 20 mg of microspheres and place them in a centrifuge tube or release bottle, adding the release medium. The release medium can be pH 7.4 phosphate buffer; if necessary, 0.02% sodium azide can be added as a preservative. Release the sample under constant temperature and shaking conditions at 37°C. Release sampling times were set at 1 h, 6 h, 24 h, 3 d, 7 d, 14 d, 21 d, and 28 d. After each sampling, centrifuge the sample, collect the supernatant to determine the leuprolide acetate content, and add an equal volume of fresh release medium. The cumulative release percentage over 24 h is defined as the initial burst release.

[0065] Surface-rapid drug elution assay: Accurately weigh approximately 20 mg of dried microspheres, add 5 mL of pH 7.4 phosphate buffer, and gently shake for 5 min at 25°C. After centrifugation, collect the supernatant and determine the leuprolide acetate content. This fraction of drug release is defined as rapidly elutable drug on or near the surface.

[0066] Surface rapid elution rate (%) = (mass of drug eluted in 5 min / total drug mass in microspheres) × 100%.

[0067] 4. Experimental Results Figure 1 The particle size distribution of the microspheres prepared in Example 1 is shown in Table 1. The D10, D50 and D90 values ​​are 3.2 μm, 9.0 μm and 15.7 μm, respectively.

[0068] Its scanning electron microscopy results ( Figure 2 The results showed that the leuprolide acetate PLGA microspheres prepared in Example 1 were spherical and no obvious drug crystals were observed on the surface of the microspheres.

[0069] The leuprorelin acetate PLGA microspheres prepared in Example 1 had an encapsulation efficiency of 75.1% ± 2.6% and a drug loading of 6.3% ± 0.2%.

[0070] Figure 3 The results of the accelerated release test of the leuprolide acetate PLGA microspheres prepared in Example 1 show that the cumulative release amount at 24 h was 8.0 ± 1.5%, with no obvious burst release effect. The cumulative release amount at 28 days was 100%, indicating complete release.

[0071] Table 1. Basic properties and release results of the microspheres in Example 1

[0072] Example 2: Effect of CA dosage on microsphere burst release Microspheres were prepared according to the method in Example 1. Except for the amount of CA, the formulation and process were the same as in Example 1. The concentration of Tween-80 in the aqueous phase was 0.04% (w / v), and the concentration of HS15 was 0.01% (w / v). The amount of CA in the organic phase is shown in Table 2.

[0073] Table 2. Screening prescriptions for CA dosage

[0074] The particle size, drug loading, encapsulation efficiency, proportion of drug rapidly eluted from the surface, and in vitro release of each group of microspheres were determined. The results (Table 3) showed that when the CA / PLGA mass ratio was 20%, the system exhibited a flocculated state and could not form microsphere structures. Therefore, subsequent encapsulation efficiency, drug loading, and drug release tests did not examine the group with a CA / PLGA mass ratio of 20%.

[0075] The results of the drug loading and encapsulation efficiency experiments showed that when the CA / PLGA mass ratio was 0.25%-10%, the drug loading of each group of microspheres exceeded 5% and the encapsulation efficiency exceeded 50%.

[0076] Table 3 Effect of CA dosage on microsphere properties and release

[0077] Compared to the control group (C0 group) without CA, the addition of CA (content 0.25%-10%) reduced the proportion of rapidly eluted drug from the surface from 35.87% to below 25%. CA content between 0.5%-5% of PLGA mass reduced the proportion of rapidly eluted drug to below 20%, and the 24-hour burst release rate was reduced to below 25%. Therefore, the amount of CA used should be 0.25% to 5% of the mass of the polylactic-co-glycolic acid copolymer. Furthermore, when the amount of CA is 2% to 5% of the mass of the polylactic-co-glycolic acid copolymer, the 24-hour burst release rate can be reduced to below 20%. Figure 4 (See Table 3). Therefore, the amount of CA was further optimized to 2% to 5% of the mass of the polylactic acid-glycolic acid copolymer.

[0078] Example 3: Effect of Tween-80 to HS15 ratio on microsphere burst release Microspheres were prepared according to the method in Example 1. Except for the different ratio of Tween-80 to HS15 in the aqueous phase, the formulations and processes were the same as in Example 1. The total concentration of Tween-80 and HS15 was fixed at 0.05% (w / v), and the ratio of Tween-80 to HS15 was adjusted according to the scheme in Table 4. The amount of CA was fixed at 1.0% of the mass of PLGA.

[0079] Table 4 Screening prescriptions with Tween-80 to HS15 ratio

[0080] The particle size, drug loading, encapsulation efficiency, surface drug elution ratio, and in vitro release curves of each group of microspheres were determined. Experimental results (Table 5 and...) Figure 5 The results showed that when the Tween-80:HS15 mass ratio was between 8:1 and 1:4, the leuprorelin acetate loading exceeded 4%. Compared with the control group containing only Tween-80, the burst release rate at 24 h was significantly lower in the control group with a Tween-80:HS15 mass ratio between 8:1 and 1:4, all below 30%. However, when the Tween-80:HS15 mass ratio was between 4:1 and 1:1, the burst release rate at 24 h could be reduced to below 20%. The cumulative release rate at 28 days for all the above groups exceeded 95%. Therefore, the optimal mass ratio of Tween-80 to HS15 is 8:1 to 1:4, and more preferably 4:1 to 1:1.

[0081] Table 5. Effect of Tween-80 to HS15 ratio on microsphere properties and burst release.

[0082] The preferred formulation is one that, under conditions of similar particle size, drug loading, and encapsulation efficiency, has a lower surface rapid elution ratio and a lower 24-hour burst release.

[0083] Example 4: Effect of total dosage of Tween-80 and HS15 on microsphere burst release Each group of microspheres was prepared according to the method of Example 1. The ratio of Tween-80 to HS15 in the external aqueous phase was set to 4:1, and its total amount accounted for 0.25% to 30% of the mass of polylactic acid-glycolic acid copolymer (as shown in Table 6). The remaining formulations and processes were the same as in Example 1.

[0084] Table 6 Screening prescriptions for total dosage of Tween-80 and HS15

[0085] The particle size, drug loading, encapsulation efficiency, proportion of drug rapidly eluted from the surface, and in vitro release of each group of microspheres were determined. Results (Table 7 and...) Figure 6 The results showed that when the addition of Wen-80 and HS15 was 0, complete microspheres could not be formed. Therefore, data such as drug loading and encapsulation efficiency were not measured for this group of samples.

[0086] The drug loading and encapsulation efficiency results showed that when the total mass of Tween-80 and HS15 was 0.25% of the PLGA mass, a large amount of drug could not be effectively encapsulated. This may be because the low amount of surfactant used could not fully emulsify the primary and secondary emulsions. Therefore, the total mass of Tween-80 and HS15 should exceed 0.25% of the PLGA amount. When the total mass of Tween-80 and HS15 was 0.5%-20% of the PLGA amount, the 24-hour burst release was less than or equal to 20%, and when its amount was 1.0%-10% of the PLGA amount, the 24-hour burst release could be reduced to below 15%. However, when the total mass of Tween-80 and HS15 was 30% of the PLGA amount, the 24-hour burst release increased to 26.83%. This may be because the excessive amount of surfactant reduced the microsphere size and increased its specific surface area, thus allowing more drug to be distributed on the microsphere surface. The increased surface rapid elution ratio in this group (C8) verified the above hypothesis. The total amount of Tween-80 and HS15 fed had no significant effect on the cumulative drug release percentage at 28 days.

[0087] Based on the above experimental results, the total amount of Tween-80 and HS15 fed is 0.5% to 20% of the mass of polylactic acid-glycolic acid copolymer, and more preferably 1% to 10%.

[0088] Table 7. Effect of total mass of Tween-80 and HS15 on microsphere properties and release.

[0089] Example 5: Effect of PLGA lactic acid / glycolic acid ratio on microsphere burst release Each group of microspheres was prepared according to the method in Example 1. Except for the lactic acid / glycolic acid ratio in PLGA, which was adjusted according to Table 8, all other formulation process parameters remained unchanged.

[0090] Table 8 Screening Formulations for PLGA Lactic Acid / Glycolic Acid Ratio

[0091] The in vitro release of microspheres from each group was measured. The results (Table 9) showed that when the lactate / glycolic acid ratio in PLGA was 75:25, the PLGA microspheres exhibited the lowest leuprolide acetate burst release at 24 h. When the lactate / glycolic acid ratio was between 50:50 and 85:15, the 24-hour burst release of the PLGA microspheres was controlled below 15%, and the cumulative release over 28 days was consistently above 95%. These structural findings indicate that the lactate / glycolic acid ratio in PLGA should be between 50:50 and 85:15, with 75:25 being the preferred ratio.

[0092] Table 9. Effect of PLGA lactate / glycolic acid ratio on drug release rate from microspheres

[0093] Example 6: Effect of organic solvent system on microsphere burst release Each group of microspheres was prepared according to the method of Example 1. Except for adjusting the type and proportion of organic solvents in the oil phase according to Table 10, the other formulations and process conditions remained unchanged.

[0094] Table 10 Organic Solvent Screening Formulations

[0095] Particle size, morphology, drug loading, encapsulation efficiency, surface rapid elution ratio, and 24-hour burst release were determined. As shown in Table 11, the microspheres prepared with pure dichloromethane or pure ethyl acetate had larger particle sizes than those prepared with a dichloromethane / ethyl acetate mixed solvent. Microspheres prepared with dichloromethane / ethyl acetate ratios between 9:1 and 5:5 exhibited higher encapsulation efficiency and drug loading, but lower surface rapid elution ratios and 24-hour burst release rates. The microspheres prepared with a dichloromethane / ethyl acetate ratio of 5:5 showed the lowest surface rapid elution ratio and 24-hour burst release rate. Therefore, dichloromethane / ethyl acetate was chosen as the organic phase, with a ratio between 9:1 and 5:5, preferably 5:5.

[0096] Table 11 Effects of the type and proportion of organic solvents in the organic phase on the properties and release of microspheres

[0097] Comparative Example 1 Leuprolide acetate microspheres were prepared according to the method in Example 1. The types and methods of surfactant addition were as shown in Table 12, while other formulations and processes remained unchanged.

[0098] Table 12 Screening formulations for surfactant combinations

[0099] Particle size, morphology, drug loading, encapsulation efficiency, surface rapid elution ratio, and 24-hour burst release were determined. See Table 13 and... Figure 7As shown, when no surfactant (S1) is used or when only CA (S2) is used alone, regular microspheres cannot be formed. This may be because CA is mainly distributed in the organic phase of dissolved PLGA and cannot form an effective emulsification effect on the organic phase. When Tween-80 (S3) or HS15 (S4) is used alone, microspheres can be formed, but when the two are used together (S5), the particle size of the formed microspheres is significantly smaller than when the two are used alone. The combination of Tween-80 and CA (S6) and the simultaneous use of the three surfactants (S8) can also obtain microspheres with smaller particle sizes. However, when HS15 and CA are used together (S7), the particle size of the generated microspheres increases, which may be due to the small amount of HS15 used in this system.

[0100] The drug loading and encapsulation efficiency results showed that, compared with the three binary surfactant systems, the microspheres formed by the ternary surfactant system had higher drug loading and encapsulation efficiency.

[0101] The surface rapid elution ratio results showed that, within the same single surfactant group, binary surfactant groups with added CA (e.g., S6 group compared to S3 group, S7 group compared to S4 group) all had lower surface rapid elution ratios, while the ternary surfactant system had an even lower surface rapid elution ratio. The 24-hour burst release percentage results were consistent with the surface rapid elution ratio results. This suggests that, compared to single or no CA binary surfactant systems, the addition of CA can effectively reduce the distribution of leuprolide acetate on the microsphere surface.

[0102] Table 13 Effects of surfactant combinations on microsphere properties and release

[0103] Example 7: Validation of CA Surface Enrichment 500 mg PLGA (75:25) and 5 mg CA were dissolved in 3 mL of a dichloromethane / ethyl acetate mixture (7:3 volume ratio). 0.25 mL of water for injection containing 50 mg of leuprolide acetate was added. After ultrasonic emulsification, the mixture was transferred to a 50 mL round-bottom flask and rotary evaporated to obtain a PLGA film. The film was refrigerated at -20°C overnight, and then 0.5 mL of dichloromethane at -20°C was added. The mixture was gently vortexed for 2 seconds and immediately aspirated. This washing process with dichloromethane was repeated ten times. 2.5 mL of methanol was added to each of the ten samples to precipitate PLGA. The samples were centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. The CA content was determined by gas chromatography. The leuprolide acetate content was determined by liquid chromatography. Simultaneously, the above experiment was repeated under the same conditions except without the addition of CA to investigate the effect of CA addition on the distribution area of ​​leuprolide acetate in the PLGA film.

[0104] The gas chromatography conditions were as follows: a DB-5 capillary column (30 m × 0.25 mm × 0.25 μm) was used, with high-purity nitrogen or helium as the carrier gas. The injection port temperature was set to 290°C and the FID detector temperature to 310°C. The column temperature program was set to an initial temperature of 200°C held for 1 min, followed by a ramp-up to 290°C at a rate of 15°C / min and a hold for 10 min. The injection volume was 1 μL (split ratio 10:1).

[0105] The liquid chromatography conditions were as follows: a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a mobile phase of trifluoroacetic acid aqueous solution (0.1%): acetonitrile (30:70, V / V) at a flow rate of 1 mL / min. The column temperature was 35°C, and the detection wavelength was 220 nm.

[0106] result( Figure 8 The results showed that the CA content was highest in the initial two dichloromethane washes (eluting the surface layer of the PLGA film). As the number of washes increased, the eluted layer gradually shifted to the deeper layers of the PLGA film, and the CA concentration gradually decreased. This result demonstrates that CA is more distributed on the surface layer of the PLGA during the curing process.

[0107] Figure 9 The results showed that the leuprolide acetate content was lowest on the PLGA film surface (i.e., in the initial washing solution), and began to increase with each subsequent washing. In the group without CA (…), Figure 10 Leuprolide acetate is more distributed on the surface of PLGA films.

[0108] The above results demonstrate that the addition of CA leads to a greater distribution of leuprolide acetate in the deeper layers of the cured PLGA, possibly because CA occupies more of the surface space of the PLGA film. This suggests that CA may alter the interfacial composition through surface enrichment and reduce the distribution of leuprolide acetate in the surface region.

[0109] Although the invention has been illustrated and described with reference to specific embodiments, it will be apparent to those skilled in the art that many other variations and modifications can be made without departing from the spirit and scope of the invention. Therefore, all such variations and modifications falling within the scope of the invention are intentionally included in the appended claims.

Claims

1. A low-burst-release leuprolide acetate PLGA microsphere, characterized in that, The microspheres comprise leuprolide acetate, polylactic acid-glycolic acid copolymer, and a ternary interfacial barrier system. The ternary interface barrier system includes Tween-80, polyoxyethylene 15-hydroxystearate, and cholesterol acetate. The cholesterol acetate is a steroidal hydrophobic barrier agent.

2. The low-burst-release leuprolide acetate PLGA microspheres according to claim 1, characterized in that, The weight ratio of leuprolide acetate to polylactic acid-glycolic acid copolymer is 1:5 to 1:

20.

3. The low-burst-release leuprolide acetate PLGA microspheres according to claim 1, characterized in that, The amount of cholesterol acetate used is 0.25% to 5% of the mass of the polylactic acid-glycolic acid copolymer.

4. The low-burst-release leuprolide acetate PLGA microspheres according to claim 1, characterized in that, The mass ratio of Tween-80 to polyoxyethylene 15-hydroxystearate is 8:1 to 1:

4.

5. The low-burst-release leuprolide acetate PLGA microspheres according to claim 1, characterized in that, The total amount of Tween-80 and polyoxyethylene 15-hydroxystearate fed into the reactor is 0.5% to 20% of the mass of the polylactic acid-hydroxyacetic acid copolymer.

6. The low-burst-release leuprolide acetate PLGA microspheres according to any one of claims 1 to 5, characterized in that, The ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is from 50:50 to 85:

15.

7. The low-burst-release leuprolide acetate PLGA microspheres according to any one of claims 1 to 5, characterized in that, The organic solvent for dissolving the polylactic acid-glycolic acid copolymer is a mixture of dichloromethane and ethyl acetate in a volume ratio of 9:1 to 5:

5.

8. The low-burst-release leuprolide acetate PLGA microspheres according to claim 1, characterized in that, The Tween-80 and polyoxyethylene 15-hydroxystearate are present in the external aqueous phase during the microsphere preparation process, and the cholesterol acetate is present in the organic phase containing polylactic acid-hydroxyacetic acid copolymer; the cholesterol acetate is enriched on the surface or near-surface region of the microsphere during the droplet solidification process and reduces the enrichment of leuprolide acetate on the surface of the microsphere.

9. A method for preparing low-burst-release leuprolide acetate PLGA microspheres according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Dissolve leuprolide acetate in the aqueous phase to obtain the inner aqueous phase; (2) Dissolve polylactic acid-glycolic acid copolymer and cholesterol acetate in an organic solvent to obtain an organic phase; (3) The aqueous phase is added to the organic phase and emulsified to obtain the primary emulsion; (4) The primary emulsion is added to an aqueous phase containing polyvinyl alcohol, Tween-80 and polyoxyethylene 15-hydroxystearate, and emulsified to obtain a secondary emulsion; (5) Remove the organic solvent to solidify the emulsion droplets into microspheres; (6) Collect, wash and dry to obtain low-burst release leuprolide acetate PLGA microspheres.

10. The use of low-burst-release leuprolide acetate PLGA microspheres according to any one of claims 1 to 8 in the preparation of a medicament for treating hormone-dependent diseases.

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