Dexamethasone single-phase zero-order release sustained-release microsphere as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to dexamethasone single-phase zero-order release sustained-release microspheres as well as a preparation method and application thereof. The dexamethasone sustained-release microspheres are prepared by an emulsification-solvent evaporation method, the release platform phase of the microspheres is improved by adding a release accelerator, and finally the dexamethasone sustained-release microspheres with a single-phase zero-order release behavior are obtained. Experimental results show that various representations of the sustained-release microspheres meet the requirements of preparations, and in-vivo pharmacodynamic studies of rats also show that the sustained-release microspheres can protect articular cartilage and maintain a normal cartilage structure, and have the same curative effect as commercially available dexamethasone acetate injection. Therefore, the dexamethasone sustained-release microsphere which is used for articular cavity injection and has a good release behavior is successfully developed, the advantages of articular cavity injection drugs can be fully played, the drugs can be continuously and stably released, long-acting treatment of arthritis is achieved, and the dexamethasone sustained-release microsphere has important clinical application value and wide market prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a dexamethasone single-phase zero-order release sustained-release microsphere, its preparation method, and its uses. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative disease characterized by joint pain, swelling, stiffness, and limited mobility. Without timely intervention, it can lead to permanent disability, severely impacting patients' social activities and quality of life. Drug therapy is used throughout the entire OA treatment process to relieve pain symptoms and improve joint function. Intra-articular injection of dexamethasone can effectively relieve pain, improve joint function, and reduce systemic drug exposure, preventing adverse reactions. However, due to the unique structure of the joint cavity, the drug is rapidly cleared after injection, requiring frequent medication use and resulting in poor patient compliance.

[0003] Currently, the dexamethasone formulation available for intra-articular injection in clinical practice is dexamethasone acetate injection, a suspension administered intra-articularly every two weeks. Dexamethasone acetate is a poorly soluble drug; preparing it as a suspension prolongs the duration of sustained release and reduces sudden release. However, the duration of efficacy is still relatively short, with clinical therapeutic effects lasting only two weeks. This is mainly because small-molecule drugs can be rapidly cleared through lymphatic vessels and synovial capillaries, significantly shortening the drug's residence time in the joint cavity, preventing the drug from targeting cartilage tissue for an extended period and maximizing its efficacy. Additionally, there is dexamethasone palmitate injection, a chylous injection with excipients such as refined soybean oil and refined egg yolk lecithin; administered intravenously every two weeks. Originally developed and marketed by Tanabe Mitsubishi Pharmaceutical in Japan under the brand name Limetasone, it is a sustained-release formulation of dexamethasone. Dexamethasone palmitate is a prodrug of dexamethasone, inactive in itself, and requires enzymatic hydrolysis in the body to slowly release the active substance dexamethasone to exert its effect. This preparation can also be injected into the joint cavity, but because its particle size is small, it can be non-specifically phagocytosed and cannot stay for a long time. If necessary, it is necessary to give a booster injection every 2-4 weeks to consolidate the therapeutic effect, which brings inconvenience to patients.

[0004] Microspheres are tiny spherical entities with particle sizes ranging from a few micrometers to hundreds of micrometers, formed by dissolving or dispersing drugs in polymer materials. Microspheres encapsulate or adsorb various poorly soluble small molecule drugs and peptide biopharmaceuticals onto or within the polymer surface through physical means. As the polymer slowly degrades, the drug is slowly released from the microsphere into the body to exert its effect. Compared to traditional drug delivery methods, microspheres can significantly prolong the duration of drug action, reduce the frequency of dosing, improve patient compliance, and reduce systemic drug toxicity. As a long-acting sustained-release delivery system, microspheres can extend the duration of drug action, reduce the frequency of dosing, and improve patient compliance. However, their three-phase release behavior greatly limits their clinical application.

[0005] Therefore, there is a need to develop a dexamethasone sustained-release microsphere with good release behavior for intra-articular injection, so that it can not only give full play to the advantages of intra-articular drug injection, but also continuously and stably release the drug to achieve long-term treatment of OA. Summary of the Invention

[0006] To address the deficiencies and shortcomings of existing technologies, this invention provides dexamethasone single-phase zero-level release sustained-release microspheres, their preparation method, and applications.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a dexamethasone single-phase zero-order release sustained-release microsphere, wherein the raw materials for preparing the microsphere include: dexamethasone, a carrier material and a release promoter; the carrier material is a PLGA copolymer, and the PLGA copolymer is one or more PLGA copolymers with a molecular weight in the range of 10,000 Da to 50,000 Da.

[0008] Alternatively, in the above-mentioned sustained-release microspheres, the PLGA copolymer is one or more PLGA copolymers with a molecular weight in the range of 10,000 Da to 30,000 Da.

[0009] Preferably, the PLGA copolymer is a 50:50 acid-terminated PLGA copolymer.

[0010] Alternatively, in the above-mentioned sustained-release microspheres, those skilled in the art of pharmaceutical formulation can select excipients with pore-forming and release-promoting effects as release promoters used in this invention, according to the actual needs of the formulation.

[0011] For example, the release promoter includes one or more of the following: P188, Tween 80, PEG 400, PLGA-PEG-PLGA or 2000 Da PLGA.

[0012] Alternatively, in the above-mentioned sustained-release microspheres, the mass percentage of dexamethasone is 5% to 40%, and the mass ratio of the carrier material to the release promoter is 3:1 to 80:1.

[0013] Alternatively, in the above-mentioned sustained-release microspheres, the mass percentage of dexamethasone is 5% to 30%, and the mass ratio of the carrier material to the release promoter is 4:1 to 40:1.

[0014] Alternatively, in the above-mentioned sustained-release microspheres, the mass percentage of dexamethasone is 10% to 20%, and the mass ratio of the carrier material to the release promoter is 7:1 to 3:1.

[0015] More preferably, the mass percentage of dexamethasone is 12% to 18%, and the mass ratio of the carrier material to the release promoter is 6:1 to 4:1.

[0016] Alternatively, in the above-mentioned sustained-release microspheres, the carrier material is a mixture of 10,000 Da PLGA and 30,000 Da PLGA.

[0017] Preferably, the release promoter is selected from 2000 Da PLGA.

[0018] Alternatively, in the above-mentioned sustained-release microspheres, the carrier material is a mixture of 10,000 Da PLGA and 30,000 Da PLGA, wherein the mass ratio of the 10,000 Da PLGA to the 30,000 Da PLGA is 9:1 to 7:5, and the release promoter is selected from 2,000 Da PLGA.

[0019] Alternatively, in the above-mentioned sustained-release microspheres, the mass ratio of 10000 Da PLGA, 30000 Da PLGA and 2000 Da PLGA is 3:7:2, and the mass percentage of dexamethasone in the microspheres is 20% to 25%.

[0020] Alternatively, the sustained-release microspheres may contain 60 mg of dexamethasone, 60 mg of PLGA (50:50 acid-capped, Mw = 10000 Da), 140 mg of PLGA (50:50 acid-capped, Mw = 30000 Da), 40 mg of 2000 Da PLGA, 2 mL of dichloromethane, and 80 mL of 1% PVA aqueous solution.

[0021] In a second aspect, the present invention provides a method for preparing the sustained-release microspheres described in the first aspect, characterized by comprising the following steps: Accurately weigh the prescribed amount of carrier material and release promoter into a vial, dissolve them in an appropriate amount of organic solvent to form the oil phase, accurately weigh the prescribed amount of dexamethasone, add it to the oil phase, stir and sonicate to form a pre-emulsion, add the pre-emulsion to an appropriate amount of external aqueous solution containing 0.2%-5% PVA by mass, and sonicate on a high-speed shear press at 1500-15000 r·min -1 The emulsion was sheared and emulsified under conditions of 15-60 s, and then dispersed in an appropriate amount of deionized water containing 0.02%-2% PVA. The mixture was then stirred at low speed for 2-8 h to allow the organic solvent to evaporate naturally. After solidification, the drug-loaded microspheres were obtained by centrifugation, washing, and freeze-drying.

[0022] Preferably, in the above preparation method, the preparation method includes the following steps: Accurately weigh the prescribed amount of carrier material and release promoter into a vial, dissolve them in an appropriate amount of dichloromethane to form the oil phase, accurately weigh the prescribed amount of dexamethasone, add it to the oil phase, stir and sonicate to form a pre-emulsion, add the pre-emulsion to an appropriate amount of external aqueous solution containing 0.5%-2% PVA by mass, and shear at 3000-10000 r·min on a high-speed shear press. -1 The emulsion was sheared and emulsified under conditions of 15-60 s, and then dispersed in an appropriate amount of deionized water containing 0.5%-2% PVA. The mixture was then stirred at low speed for 4 h to allow the dichloromethane to evaporate naturally. After solidification, the drug-loaded microspheres were obtained by centrifugation, washing, and freeze-drying.

[0023] Alternatively, in the above preparation method, the concentration of the carrier material is 50-200 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:20-1:80, and the drug loading ratio is 1:2-1:8 by weight.

[0024] Alternatively, in the above preparation method, the concentration of the carrier material is 50-150 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:20-1:60, and the drug-excipient ratio is 0.2:1-0.4:1 by weight.

[0025] Alternatively, in the above preparation method, the concentration of the carrier material is 75-150 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:30-1:50, and the drug loading ratio is 1:2-1:4 by weight.

[0026] Alternatively, in the above preparation method, the concentration of the carrier material is 100 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:40, and the drug loading ratio is 0.3:1 by weight.

[0027] Alternatively, in the above preparation method, the carrier material is a mixture of 10,000 Da PLGA and 30,000 Da PLGA, and the release promoter is selected from 2,000 Da PLGA, with a mass ratio of 3:7:2 for 10,000 Da PLGA, 30,000 Da PLGA, and 2,000 Da PLGA.

[0028] Alternatively, in the above preparation method, the primary emulsion is added to an appropriate amount of external aqueous solution containing 0.5%-2% PVA, and the mixture is subjected to high-speed shearing at 4000-8000 r·min. -1 Shear emulsification was performed under conditions of 20-40 s, and the resulting emulsion was then dispersed in an appropriate amount of deionized water containing 0.5%-2% PVA, with low-speed stirring at 250-300 r·min. -1 .

[0029] Alternatively, in the above preparation method, the colostrum is added to an appropriate amount of external aqueous solution containing 1% PVA, and the mixture is subjected to high-speed shearing at 6000 r·min. -1 Shear emulsification was performed under conditions of 30 s, and the resulting emulsion was then dispersed in an appropriate amount of deionized water containing 1% PVA. The low-speed stirring was 250-300 r·min. -1 .

[0030] In a third aspect, the present invention provides the use of the sustained-release microspheres described in the first aspect above, or the sustained-release microspheres prepared by the preparation method described in the second aspect above, in the preparation of a medicament for treating arthritis.

[0031] Alternatively, in the above-described uses, the arthritis referred to is rheumatoid arthritis or osteoarthritis.

[0032] Preferably, the drug is an injectable sustained-release formulation for intra-articular injection.

[0033] Compared with the prior art, the beneficial effects of the present invention are: This invention prepares dexamethasone sustained-release microspheres via an emulsification-solvent evaporation method. By adding a release promoter to improve the release plateau phase of the microspheres, dexamethasone sustained-release microspheres with single-phase zero-order release behavior are obtained. Experimental results show that all characterizations of the sustained-release microspheres of this invention meet formulation requirements. In vivo pharmacodynamic studies in rats also demonstrate that the sustained-release microspheres of this invention can protect articular cartilage and maintain normal cartilage structure, exhibiting efficacy consistent with commercially available dexamethasone acetate injection. Therefore, this invention successfully develops a dexamethasone sustained-release microsphere with excellent release behavior for intra-articular injection. It not only fully leverages the advantages of intra-articular drug injection but also provides sustained and stable drug release, achieving long-term treatment of arthritis. This invention has significant clinical application value and broad market prospects. Attached Figure Description

[0034] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 In vitro release curves of the drug in PBS at pH 7.4 at 37 °C using Span 80, P188, Tween 80 and PEG 400 as release promoters, as measured by Dex-MS.

[0035] Figure 2 In vitro release curves of PLGA 3w: PLGA 1w: PEG 400 in PBS at pH 7.4 at 37 °C using different ratios.

[0036] Figure 3 In vitro release curves of PLGA 3w:PLGA 1w:PLGA-PEG-PLGA in PBS pH 7.4 at 37 °C.

[0037] Figure 4 In vitro release curves of PLGA 3w:PLGA 1w:PLGA 2k in PBS pH 7.4 at 37 °C using different ratios.

[0038] Figure 5 In vitro release curves of Dex-MS-1 and Dex-MS-2 in PBS pH 7.4 at 37 °C (n=3).

[0039] Figure 6 SEM images of Dex-MS-2.

[0040] Figure 7 SEM images of dexamethasone microspheres during degradation in PBS pH 7.4 at 37 °C.

[0041] Figure 8 Changes in knee circumference before and after treatment in the blank group, model group, dexamethasone acetate injection group, Dex-MS-1 group and Dex-MS-2 group (n=5).

[0042] Figure 9 H&E staining results of the knee joint for pathological analysis 4 weeks after treatment.

[0043] Figure 10 Results of Safranin O-Fix Green staining of the knee joint for pathological analysis 4 weeks after treatment.

[0044] Figure 11 The changes in body weight of SD rats in the blank group, model group, dexamethasone acetate injection group, Dex-MS-1 group and Dex-MS-2 group (n=5).

[0045] Figure 12 H&E and Safranin O-Fixed Green staining results of the knee joint on days 0, 7 and 28. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used are commercially available.

[0047] Example 1: Formulation screening and in vitro drug release behavior study of dexamethasone sustained-release microspheres 1. Experimental Methods 1.1 Preparation of dexamethasone sustained-release microspheres Accurately weigh the prescribed amount of PLGA and release promoter into a vial, dissolve them in 2.0 mL of dichloromethane to form the oil phase, wherein PLGA with a molecular weight of 30,000 Da and PLGA with a molecular weight of 10,000 Da are blended in a ratio of 7:3. Accurately weigh 60.00 mg of dexamethasone active pharmaceutical ingredient, add it to the oil phase, stir and sonicate to form a primary emulsion. Add the primary emulsion to 80 mL of external aqueous solution containing 1% PVA, and shear at 6000 r·min on a high-speed shear press. -1 Shear emulsification was performed under 30 s conditions, and the resulting emulsion was then dispersed in 200 mL of deionized water containing 0.1% PVA, followed by stirring at low speed (250 ~ 300 r·min). -1 The dichloromethane was allowed to evaporate naturally over 4 hours. After curing, the drug-loaded microspheres were obtained by centrifugation, washing, and freeze-drying.

[0048] 1.2 Effect of release promoters on the in vitro release behavior of microspheres (1) Effect of surfactant type on the in vitro release behavior of microspheres With relevant parameters fixed, different types of surfactants, namely Span 80, P188, Tween 80, and PEG400, were weighed and tested with PLGA 3w:PLGA 1w:surfactant = 7:3:0.25. Dexamethasone microspheres were prepared according to the experimental method in section "1.1". The freeze-dried microspheres were accurately weighed and their in vitro release behavior was investigated.

[0049] (2) Effect of PEG 400 dosage on the in vitro release behavior of microspheres With relevant parameters fixed, PEG 400 was weighed out in proportions of PLGA 3w:PLGA 1w:PEG 400 = 7:3:0.25, PLGA 3w:PLGA 1w:PEG 400 = 7:3:0.3, PLGA 3w:PLGA 1w:PEG 400 = 7:3:0.4, and PLGA 3w:PLGA 1w:PEG 400 = 7:3:0.5 for experiments. The microsphere preparation method and detection indicators were the same as above.

[0050] (3) Effect of PLGA-PEG-PLGA dosage on the in vitro release behavior of microspheres With relevant parameters fixed, PLGA-PEG-PLGA were weighed in specific proportions, and experiments were conducted with the following ratios: PLGA 3w:PLGA 1w:PLGA-PEG-PLGA = 7:3:0.5, PLGA 3w:PLGA 1w:PLGA-PEG-PLGA = 7:3:1, PLGA 3w:PLGA 1w:PLGA-PEG-PLGA = 7:3:1.5, and PLGA 3w:PLGA 1w:PLGA-PEG-PLGA = 7:3:2. The microsphere preparation method and detection indicators were the same as above.

[0051] (4) Effect of 2000 Da PLGA dosage on the in vitro release behavior of microspheres Fix the relevant parameters, and weigh 2000 Da PLGA proportionally. Conduct experiments with the ratios of PLGA 3w:PLGA 1w:PLGA 2k being 7:3:1.25, PLGA 3w:PLGA 1w:PLGA 2k being 7:3:1.5, PLGA 3w:PLGA 1w:PLGA 2k being 7:3:2, PLGA 3w:PLGA 1w:PLGA 2k being 7:3:2.5, and PLGA 3w:PLGA 1w:PLGA 2k being 7:3:3. The microsphere preparation method and detection indexes are the same as above.

[0052] 1.3 Release Kinetics Fitting The fourth part of the Chinese Pharmacopoeia (2025 edition), "General Principles for Sustained Release, Controlled Release and Delayed Release Preparations (0913)", has clear regulations on judging the release behavior of drugs: the release data of sustained release preparations can be fitted with the first-order equation and Higuchi equation; controlled release preparations show a constant release rate within the set release time, and their release data can be fitted with the zero-order equation. The Korsmeyer-pappas model fitting can help judge the drug release mechanism, and the model formula is: Q t =Kt n , where K is the drug release rate constant and n is the release exponent. The release exponent n is a characteristic parameter representing the release mechanism. When the drug shape is spherical, n ≤ 0.43, and the drug release mechanism is Fickian Diffusion; 0.43 < n < 0.85, the drug release mechanism is Anomalous Transport, that is, the combined action of drug diffusion and matrix erosion; when n ≥ 0.85, it indicates that the drug release mechanism is matrix erosion.

[0053] 2. Experimental Results 2.1 Effects of Different Types of Release Promoters on the in Vitro Release Behavior of Microspheres (1) Surfactants According to their different hydrophilic-lipophilic balance values (HLB values), surfactants can be divided into W / O emulsifiers and O / W emulsifiers. Among them, Span 80 belongs to the W / O emulsifier, and PEG 400, Tween 80, and P188 belong to the O / W emulsifiers. The effects on the microsphere release behavior are shown in Figure 1When using O / W emulsifiers, all three types of microspheres exhibited a three-phase release. These trends suggest that when the surfactant is more hydrophilic, more water can enter the polymer droplets, leading to a more porous structure as the microparticles solidify and the pore-forming agent dissolves. The W / O emulsifier Span 80 exhibited a two-phase release, with a cumulative release of 65.59% in the first 10 days, showing the fastest initial release rate and highest cumulative release among various surfactants. Subsequently, it entered a sustained release phase with a slower release rate.

[0054] (2) Effect of PEG 400 dosage on the in vitro release behavior of microspheres To further investigate the effect of surfactant dosage on the in vitro release behavior of microspheres, PEG 400 was selected as a representative. Figure 2 It is evident that with the increase of PEG 400 dosage, more drug is deposited on the pore surface, enabling direct contact with the release medium, resulting in a prolonged initial continuous drug release time and an increased cumulative release amount. The effect of surfactant dosage on drug release behavior is consistent with the above surfactant type screening results; both can increase drug distribution on the particle surface, but only alter the initial release amount. The release plateau phase still exists. When the surface pores increase to a certain extent, they actually hinder the autocatalytic degradation process of the polymer matrix, resulting in a two-phase release where the rapid release phase is difficult to observe.

[0055] (3) Effect of PLGA-PEG-PLGA dosage on the in vitro release behavior of microspheres Depend on Figure 3 It is known that the higher the proportion of PLGA-PEG-PLGA, the faster the drug release rate, the change in the release curve, the shortening or even disappearance of the plateau phase, and the shift from the initial three-phase release to two-phase or single-phase release, with significant differences in release kinetics.

[0056] (4) Effect of 2000 Da PLGA dosage on the in vitro release behavior of microspheres The release curves described above clearly show that the microparticles only enter the rapid release phase after a certain period of time, primarily due to the lack of drug release pathways and autocatalytic acceleration of release before the polymer matrix degrades. Specifically, the large molecular weight of the selected PLGA, the tightly wound polymer chains, and the dense structure of the microparticles make it difficult for the drug to dissolve and diffuse through the polymer barrier. Furthermore, PLGA degradation is an acid-catalyzed reaction; the polymer ester bonds break to generate acidic oligomers, which can in turn catalyze the degradation reaction and lead to overall degradation, accelerating drug release. Based on this, 2000 Da PLGA was chosen as a multifunctional excipient. Different molecular weight PLGAs exhibit better compatibility, and changing its dosage does not cause significant changes in the release curve, allowing for precise and effective regulation of the drug release rate. Increasing the dosage of 2000 Da PLGA gradually reduces the difference in release rates between the plateau and rapid release phases, leading to a convergence. When a certain proportion is exceeded, the initial drug release significantly increases, exhibiting a biphasic release. Therefore, the addition of 2000 Da PLGA improves the three-phase release behavior of the microparticles, and an ideal release curve can be obtained by adjusting its dosage ratio. The closest to linear release is achieved when PLGA 3w:PLGA 1w:PLGA 2k = 7:3:2 (see...). Figure 4 ).

[0057] 2.2 Determination of the optimal formulation and investigation of in vitro release behavior (1) Determination of the optimal formulation of dexamethasone sustained-release microspheres By investigating the effects of different types and amounts of release promoters on the in vitro release behavior of microspheres, the final release promoter selected was 2000 Da PLGA, with a specific dosage ratio of PLGA 3w:PLGA 1w:PLGA 2k = 7:3:2. Other components in the optimal formulation and specific preparation methods are detailed in section "1.1".

[0058] (2) Investigation of in vitro release behavior and fitting of release kinetics The in vitro release behavior of ordinary dexamethasone sustained-release microspheres (Dex-MS-1, which is identical to Dex-MS-2 in composition and preparation method except for the absence of a release enhancer) and dexamethasone sustained-release microspheres with single-phase zero-order release behavior prepared according to the optimal formulation (Dex-MS-2) was investigated. Release kinetics were fitted according to various drug release models, and the results are as follows: Figure 5 As shown.

[0059] The results showed that Dex-MS-2 exhibited good in vitro release behavior and achieved a long-lasting sustained-release effect. The release behavior of both microspheres was consistent for the first 3 days. Subsequently, Dex-MS-1 entered a release plateau, and the release rates of the two curves differed significantly. Dex-MS-2 released the drug at a relatively constant rate from 4 to 44 days, with almost no plateau, achieving a total release of 91.55%. This release behavior was significantly better than Dex-MS-1, approaching zero-order release, which met the expected requirements. The pharmacokinetic fitting results of the in vitro release curves also confirmed the above conclusions. The in vitro release behavior of Dex-MS-2 showed a higher degree of fit to the zero-order equation than Dex-MS-1 (Dex-MS-2: r = 0.9938, Dex-MS-1: r = 0.9550), indicating that 2000 Da PLGA, as a multifunctional release promoter, can significantly improve the in vitro release behavior of Dex-MS-1, resulting in dexamethasone sustained-release microspheres with single-phase zero-order release behavior.

[0060] Among them, the n value of the Korsmeyer-Pappas equation for Dex-MS-1 is 1.2007, which is greater than 0.85, indicating that the release mechanism is skeleton dissolution; while the n value of Dex-MS-2 is 0.7978, which is between 0.43 and 0.85, indicating that the release mechanism is the combined effect of drug diffusion and skeleton dissolution (Non-Fick diffusion). The above results collectively indicate that the drug release mechanism of Dex-MS-2 is the combined effect of drug diffusion and skeleton dissolution.

[0061] Example 2: Evaluation and characterization of dexamethasone sustained-release microspheres 1. Experimental Methods 1.1 Sample Preparation 1.1.1 Preparation of dexamethasone sustained-release microspheres Different molecular weight PLGAs (Dex-MS-1: 140 mg 30000 Da PLGA, 60 mg 10000 Da PLGA; Dex-MS-2: 140 mg 30000 Da PLGA, 60 mg 10000 Da PLGA, 40 mg 2000 Da PLGA) were weighed according to the prescription and the microsphere preparation method under "1.1" in Example 1 was followed to obtain Dex-MS-1 and Dex-MS-2.

[0062] 1.1.2 Preparation of Physical Mixtures Accurately weigh the prescribed amounts of Dex and PLGA, and mix them thoroughly to obtain the physical mixture (PM).

[0063] 1.2 Observation of microsphere surface morphology Take an appropriate amount of the prepared Dex-MS-2 and place it on the conductive adhesive. Coat it evenly and vacuum gold plating for 50 seconds. Then, observe and record the surface morphology of the microsphere particles using an S-3400N scanning electron microscope.

[0064] 1.3 Determination of drug loading, encapsulation efficiency and particle size distribution Weigh 80-90 mg of the prepared Dex-MS-2 and disperse it uniformly in 1 mL of 0.1% PVA (w / v) solution to prepare a microsphere suspension. Analyze the suspension using Sympatec Helos... TM A laser particle size analyzer was used to determine the particle size distribution of microspheres. The testing software automatically fitted given particle size parameters and calculated the particle size distribution coefficient (Span value). All measurements were performed in triplicate. 10 D 50 and D 90 These represent the particle sizes when the microspheres have accumulated to 10%, 50%, and 90% of their volume, respectively. A smaller Span value indicates a more uniform microsphere size distribution.

[0065] 1.4 Observation of microsphere surface morphology at different time points after in vitro release Take several portions of Dex-MS-1 and Dex-MS-2 for in vitro release. Collect the samples on days 0, 5, 10, 20, 30, and 40, discarding all release media outside the dialysis bag. Then collect the solution and preparation inside the dialysis bag using EP tubes and centrifuge (13000 r·min). -1 The microspheres were washed three times with deionized water (10 min), and then all liquid was absorbed. The remaining residue was freeze-dried, and the surface morphology of the microspheres at different release times was recorded.

[0066] 2. Experimental Results (1) Observation of microsphere surface morphology like Figure 6 As shown, Dex-MS-2 has a round shape, smooth and porous surface; the overall size is about 20 ~ 60 μm, which is not much different from the microsphere particle size measurement results, and there is no adhesion or other undesirable situation between the microspheres.

[0067] (2) Determination of drug loading, encapsulation efficiency and particle size distribution The drug loading of Dex-MS-2 was determined to be (15.89 ± 0.21)%, and the encapsulation efficiency was (79.45 ± 1.05)%. The particle size and particle size distribution were (38.76 ± 0.11) μm and 1.09 ± 0.01 μm, respectively. Specifically, 10% of the microspheres had a diameter below 17.88 µm, 50% had a diameter below 38.76 µm, and 90% had a diameter below 59.98 µm. Within this particle size distribution, the active pharmaceutical ingredient (Dex) can be completely encapsulated within the microspheres. Furthermore, due to their small overall size and relatively uniform particle size distribution, the microspheres exhibit good needle permeability and can be successfully administered via a 26 G needle without causing needle blockage.

[0068] (3) Observation of the surface morphology of microspheres at different time points after in vitro release Depend on Figure 7It was observed that, at day 0 of the initial release phase, both microspheres exhibited certain pores on their surfaces, and the number of pores did not show a significant difference, indicating that the addition of 2000 Da PLGA had no significant impact on the surface morphology of the prepared microspheres; simultaneously, the initial release behavior of the two microspheres was consistent. By day 5, the differences in surface morphology between the two formulations became increasingly significant. For Dex-MS-1 microspheres, only pores formed during preparation were observed, and these pores were located near the surface of the microspheres and did not penetrate deep into the formulation. At this point, the particle structure was dense, and the drug could not diffuse through the polymer barrier, resulting in an extremely low release rate and a plateau phase. Dex-MS-2, however, was different; in addition to the initial pores, small and dense pores visible to the naked eye appeared on the surface of the formulation, indicating significant drug release. By day 10, the number and size of pores on the surface of Dex-MS-1 increased, indicating that under the influence of moisture, a polymer erosion process occurred from the outside in on the particle surface, while the internal structure remained unchanged. The surface of Dex-MS-2 not only exhibits a greater number of pores but also more "ripples," indicating a significantly faster degradation rate than Dex-MS-1. This difference can be attributed to the occurrence of an autocatalytic process. The presence of acidic degradation products of 2000 Da PLGA continuously catalyzes the degradation of high molecular weight polymers within the microparticles, leading to the formation of "ripples" through this inside-out autocatalytic action. On day 20, "ripples" were observed on the surface of Dex-MS-1 microspheres, indicating that the polymers within the microparticles were degrading. As autocatalysis progressed within Dex-MS-2, the surface pores continuously merged and gradually expanded inwards. Based on the comparison of the surface morphology of the two, it can be determined that the degradation rate of Dex-MS-2 was faster, and its release rate was also significantly higher than that of Dex-MS-1. This further indicates that after optimization, Dex-MS-2 can eliminate the release plateau phase, ensuring continuous drug release and essentially exhibiting single-phase zero-order release behavior. On day 30, the ripples on the surfaces of both formulations gradually developed into inward depressions, and a small amount of microsphere degradation fragments were observed in Dex-MS-2. During this process, the intensifying autocatalytic effect leads to the degradation of most of the PLGA inside the microspheres into water-soluble degradation products that flow out through the channels. Therefore, after freeze-drying, numerous inward "collapses" appear on the surface. By day 40, the microsphere framework structure of both formulations had disappeared, existing entirely in fragment form. At this point, PLGA degradation was nearing its end, with a significant reduction in the degree of polyester chain entanglement, and most of the polyester chains existing as water-soluble oligomers or monomers, leaving only a small amount of short polyester chains, insufficient to support the intact microsphere structure. This indicates that the number and diameter of pores during microsphere degradation are key factors affecting drug release. It also demonstrates that Dex-MS-2 can eliminate the release plateau, primarily due to the rapid degradation of low molecular weight 2000 Da PLGA, generating drug diffusion channels and autocatalyzing the degradation of the bulk PLGA, thus promoting drug release and exerting its effect.

[0069] Example 3: Pharmacodynamic Study of Dexamethasone Sustained-Release Microspheres 1. Laboratory animals Healthy male SD rats were provided by Liaoning Changsheng Biotechnology Co., Ltd., with animal production license number SCXK (Liaoning) 2022-0001.

[0070] 2. Experimental Methods 2.1 Establishment of an osteoarthritis model Preparation of sodium iodoacetate solution: Accurately weigh 100.00 mg of sodium iodoacetate and dissolve it in 2.5 mL of sterile physiological saline to obtain a concentration of 40 mg / mL. -1 The sodium iodoacetate solution should be stored away from light for later use.

[0071] Establishment of an osteoarthritis model: After one week of acclimatization, SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. The rats were placed ventrally on a worktable, and leg hair was removed using an electric balm. The balms were then disinfected with 75% ethanol. Before injection, the knee joint was fixed and flexed at 90 degrees. The needle was inserted at the midpoint of the patellar ligament. A noticeable loss of resistance after the needle broke through the joint space indicated entry of the needle into the joint space. A micro-syringe was then gently pushed to inject 50 μL of sodium iodoacetate solution into the rat's joint cavity. After injection, the needle was slowly withdrawn, and a cotton swab was used to press and prevent leakage. The control group rats received an equal volume of saline injected into their knee joint cavity. Joint swelling and mobility were observed. Drug administration was initiated 7 days after model establishment.

[0072] 2.2 Preparation of drug delivery formulations Preparation of microsphere solvent: Accurately weigh 0.50 g CMC-Na and dissolve it in 80 mL of PBS solution. Heat and stir until completely dissolved. Cool to room temperature and bring the volume to 100 mL to prepare a 0.5% CMC-Na solution. Accurately weigh 0.10 g Tween 80 and dissolve it in the aforementioned 0.5% CMC-Na solution. Bring the volume to 100 mL to prepare a phosphate buffer containing 0.1% Tween 80 and 0.5% (w / v) sodium carboxymethyl cellulose as the microsphere solvent.

[0073] Dex-MS-1 microsphere injection solution: Accurately weigh a certain amount of prepared Dex-MS-1 microspheres and dissolve them in the microsphere solvent mentioned above to obtain the solution.

[0074] Dex-MS-2 microsphere injection solution: Accurately weigh a certain amount of the prepared Dex-MS-2 microspheres and dissolve them in the microsphere solvent mentioned above to obtain the solution.

[0075] 2.3 Grouping and Dosing Grouping: The rats that were successfully modeled were randomly divided into five groups, with 5 rats in each group: Blank control group, Model group, commercially available injection control group, Dex-MS-1 microsphere injection group, and Dex-MS-2 microsphere injection group.

[0076] Administration: On day 7 after modeling, rats in both the control and model groups were intra-articularly injected with 0.1 mL of the injection solvent. The commercially available injection control group rats were given dexamethasone acetate injection (dose: 0.63 mg / kg). -1 The rats were administered intra-articular injections twice a week for a total of two times. The Dex-MS-1 group rats were given Dex-MS-1 microsphere injection (equivalent to a dose of 1.14 mg / kg). -1 The rats in the Dex-MS-2 group were administered Dex-MS-2 microsphere injection (equivalent to a dose of 1.14 mg / kg) via intra-articular injection. -1 Dex), intra-articular injection, once in total.

[0077] 2.4 Evaluation Indicators 2.4.1 Measurement of knee joint dimensions To assess the changes in knee joint dimensions of rats throughout the experimental period, knee joint dimensions of rats in each group were measured every two days using vernier calipers. The changes in knee joint circumference of each rat were tracked and recorded. The circumference was calculated using the formula L=2(a+b), where a is the transverse diameter and b is the longitudinal diameter. A curve showing the changes was plotted with time on the x-axis and knee joint circumference on the y-axis.

[0078] 2.4.2 Histopathological examination 2.4.2.1 H&E staining To evaluate the inflammatory changes in the knee joint of rats after drug administration, rats in each group were anesthetized and sacrificed after treatment. The knee joints were dissected, the surrounding muscle tissue was removed, and the tissues were washed with 0.9% saline. After fixation with 4% paraformaldehyde for 24 h, the tissues were transferred to EDTA for decalcification. After decalcification, the bone tissues were placed in a dehydrator for dehydration, then embedded in paraffin, trimmed and sectioned using a semi-automatic microtome, and finally stained with H&E using a staining machine. The sections were then observed and photographed under an optical microscope.

[0079] 2.4.2.2 Safranin O-Fixed Green Staining To evaluate the cartilage damage in the knee joint of rats after drug administration, rats in each group were anesthetized and sacrificed after treatment. The knee joints were dissected, the surrounding muscle tissue was removed, and the tissues were washed with 0.9% physiological saline. After fixation with 4% paraformaldehyde for 24 h, the tissues were transferred to EDTA for decalcification. After decalcification, the bone tissues were placed in a dehydrator for dehydration, then embedded in paraffin, trimmed and sectioned using a semi-automatic microtome, and finally stained with safranin O-fast green. The sections were then observed and photographed under an optical microscope.

[0080] 2.4.3 Accompanying safety and biocompatibility evaluation 2.4.3.1 Behavioral observation and weight changes Throughout the experiment, the general condition of the rats was observed, including their activity level, physical characteristics, coat color, and mental state. Changes in the body weight of each group were recorded regularly to evaluate the in vivo safety of the formulation. A curve showing the change in rat body weight over time was plotted to compare the weight differences between the groups.

[0081] 2.4.3.2 Biocompatibility evaluation Histocompatibility experiments were conducted using healthy male SD rats. Prepared dexamethasone sustained-release microspheres were injected into the rat joint cavity. Rats were sacrificed on days 0, 7, and 28 after microsphere injection, and the left knee joint was removed. Excess muscle tissue was removed, and the joint was fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, sectioned, and examined for histomorphological characteristics to observe the presence of inflammatory reactions and cartilage damage within the cartilage tissue.

[0082] 3 Results 3.1 Measurement of knee joint dimensions Changes in knee joint dimensions in each group of rats are as follows: Figure 8 As shown in the figure. This experiment established a rat osteoarthritis model by intra-articular injection of 2 mg sodium iodoacetate. Significant joint swelling was observed within one week, indicating rapid modeling and significant efficacy. During the experiment, the knee joint circumference of rats in the control group remained constant, while the knee joint circumference of rats in the model group was significantly higher than that in the control group (P<0.001), indicating successful establishment of the osteoarthritis model. Compared with the model group, the knee joint circumference of the injection control group, Dex-MS-1 group, and Dex-MS-2 group was significantly reduced (P<0.001), with no significant difference compared to the control group (P>0.05). All three formulations effectively reduced the knee joint circumference and inhibited joint swelling in OA rats. The swelling reduction rate in the Dex-MS-2 group was consistent with that in the injection control group, with knee joint dimensions gradually returning to normal after 10 days of administration. In contrast, the swelling reduction rate in the Dex-MS-1 group was slower within 4-12 days after administration, and knee joint dimensions returned to normal after 20 days of administration, indicating a poorer overall treatment effect compared to the Dex-MS-2 group.

[0083] The above results indicate that the optimized dexamethasone microspheres with single-phase zero-order release behavior can continuously release the drug for at least 28 days after a single dose, with minimal fluctuations in drug concentration. They exhibit superior anti-inflammatory and joint swelling-inhibiting effects compared to Dex-MS-1, while maintaining comparable therapeutic efficacy to commercially available injectables. However, they require less dosing frequency and have improved patient compliance. These results demonstrate that the dexamethasone sustained-release microspheres with single-phase zero-order release behavior have excellent therapeutic effects.

[0084] 3.2 Histopathological Results 3.2.1 H&E staining H&E staining can be used to assess the degree of cartilage damage, and the results are as follows: Figure 9 As shown in the figure, the joint cavity structure in the blank group was clear, the cartilage surface was smooth and flat, and the chondrocytes were regularly shaped and neatly arranged. In the model group, the cartilage surface was rough, the cells were disordered and irregular in shape, the synovial tissue was severely hyperplastic, and there was a large amount of inflammatory cell infiltration. The hyperplastic synovial tissue eroded into the cartilage and bone tissue areas, and no normal chondrocyte distribution was observed in the field of view, presenting typical pathological features of osteoarthritis. Compared with the model group, the degree of cartilage damage in the drug-treated groups was significantly improved. Among them, the protective effect of the injection control group and the Dex-MS-2 group on articular cartilage was more significant. In both groups, the chondrocytes were neatly arranged and clearly layered, with occasional chondrocyte hypertrophy, and no hyperplasia of connective tissue around the cartilage or inflammatory cell infiltration was observed. In contrast, although the cartilage structure in the Dex-MS-1 group was clear and intact, the number of chondrocytes in the matrix was greatly reduced, especially in the superficial and intermediate layers. This was also due to the existence of the Dex-MS-1 release plateau phase, which prevented the drug from maintaining an effective concentration, leading to irreversible damage to the chondrocytes. Therefore, the number of chondrocytes was reduced, resulting in poor efficacy. The above results indicate that the optimized dexamethasone sustained-release microspheres with single-phase zero-order release behavior can stably release the drug in vivo, maintain the integrity of cartilage structure, protect chondrocytes from further damage, and have a superior therapeutic effect.

[0085] 3.2.2 Safranin O-Fixed Green Staining Safranin O-Fixed Green staining can be used to differentiate between cartilage and bone tissue regions, and to assess the loss of proteoglycans in cartilage. Proteoglycans in articular cartilage bind to basophilic dyes, appearing red, while those in bone bind to eosinophilic dyes, appearing green. Figure 10It was observed that in the control group, the boundary between cartilage and bone tissue was clear, and the cartilage tissue showed strong red staining, indicating abundant and uniformly distributed proteoglycans within the matrix. Compared to the control group, the model group showed disrupted joint structure, blurred boundaries between cartilage and bone tissue, missing cartilage tissue, and severe destaining, indicating that sodium iodoacetate induction could cause cartilage tissue damage and significant proteoglycan loss, thus successfully establishing the OA model. Compared to the model group, the degree of cartilage damage in all treatment groups was improved, with the injection control group and the Dex-MS-2 group showing more significant effects in improving articular cartilage damage, and the cartilage tissue showing relatively strong red staining, indicating that both formulations could reduce cartilage damage and proteoglycan loss. In contrast, although the boundary between cartilage and bone tissue was relatively clear in the Dex-MS-1 group, the cartilage tissue showed lighter red staining and some superficial and intermediate matrix destaining, indicating cartilage tissue damage and proteoglycan loss. In summary, this again demonstrates that dexamethasone sustained-release microspheres with optimized release and single-phase zero-order release behavior can achieve efficacy comparable to commercially available injectable formulations, but with reduced dosing frequency and improved drug compliance.

[0086] 3.3 Accompanying safety and biocompatibility evaluation 3.3.1 Behavioral observation and weight changes like Figure 11 As shown, behavioral indicators and weight changes in rats were observed and monitored during the experiment. Rats in the control group drank water and ate normally, were in good spirits, showed no abnormal reactions, and their weight consistently increased. Rats in the model group experienced persistent joint swelling, reduced activity, poor coat color and mental state, decreased food intake, and significant weight loss. After treatment, compared to the model group, the joint swelling subsided, food and water intake increased, and weight showed an upward trend, indicating that all three formulations could alleviate the weight loss caused by osteoarthritis (OA), and the rate of weight gain was similar to that of the control group. No abnormalities were observed in joint swelling, behavioral indicators, or weight changes in rats. Furthermore, no rats in any group exhibited symptoms such as diarrhea, infection, tumors, or bleeding during the entire experiment, and no rats died. Preliminary assessment suggests that the three formulations have good in vivo safety.

[0087] 3.3.2 Biocompatibility evaluation Following intra-articular injection of dexamethasone sustained-release microspheres into healthy rats, H&E staining and Safranin O-Fix Green staining were performed at the injection sites on days 0, 7, and 28 to assess the in vivo biocompatibility of the dexamethasone sustained-release microspheres. Results are as follows: Figure 12As shown, on day 0 after administration, the cartilage tissue structure was clear, uniformly stained, and no obvious inflammatory cell infiltration was observed. On day 7 after administration, fibrous tissue proliferation and inflammatory cell infiltration were observed in some areas, the cartilage tissue was eroded, and a large amount of proteoglycan in the matrix was lost (black arrow), indicating an acute inflammatory reaction. On day 28 after administration, the articular cartilage structure was clear and intact, the cartilage surface was smooth, the chondrocytes were neatly arranged and regularly shaped, no inflammatory cell infiltration was observed, and the proteoglycan in the matrix returned to normal levels. The above results indicate that intra-articular injection of dexamethasone sustained-release microspheres can cause a certain degree of acute inflammatory reaction, but the inflammatory reaction gradually disappears over time, and the cartilage structure returns to normal, indicating that the dexamethasone sustained-release microspheres have good biocompatibility and high safety in vivo.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A dexamethasone monophasic zero-order release sustained release microsphere characterized by: The raw materials for preparing the microspheres include: dexamethasone, a carrier material and a release promoter; the carrier material is a PLGA copolymer, and the PLGA copolymer is one or more PLGA copolymers with a molecular weight in the range of 10000 Da-50000 Da.

2. The sustained release microspheres according to claim 1, characterized by: The PLGA copolymer is one or more PLGA copolymers with a molecular weight in the range of 10000 Da-30000 Da.

3. The sustained release microspheres according to claim 1, wherein: The mass percentage of dexamethasone in the microspheres is 5%-40%, and the mass ratio of the carrier material to the release promoter is 3:1-80:

1.

4. The sustained release microspheres according to claim 3, characterized by: The carrier material is a mixture of 10000 Da PLGA and 30000 Da PLGA, and preferably, the release promoter includes one or more of the following: P188, Tween 80, PEG 400, PLGA-PEG-PLGA or 2000 Da PLGA.

5. The method of preparing sustained release microspheres according to any one of claims 1 to 4, characterized by: The method comprises the following steps: Precisely take the carrier material and release promoter in the prescription amount in the Schlenk bottle, add an appropriate amount of organic solvent to dissolve as the oil phase, precisely take the dexamethasone in the prescription amount, add it to the oil phase, stir and ultrasonic, form the primary emulsion, add the primary emulsion to an appropriate amount of external aqueous phase solution containing 0.2%-5% PVA in mass percentage, perform shearing emulsification on a high-speed shearing machine at 1500-15000 r·min -1 , 15-60 s, then disperse the formed emulsion in an appropriate amount of deionized water containing 0.02%-2% PVA, then low-speed stirring for 2-8 h to naturally volatilize the organic solvent, after solidification, centrifugation, washing and freeze-drying, the drug-loaded microspheres are obtained.

6. The method of claim 5, wherein: The concentration of the carrier material is 50-200 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:20-1:80, and the drug loading ratio is 1:2-1:8 by weight.

7. The method of claim 6, wherein: The concentration of the carrier material is 75-150 mg / mL, the volume ratio of the oil phase to the external aqueous phase solution is 1:30-1:50, and the drug loading ratio is 1:2-1:4 by weight.

8. The method of claim 7, wherein: In the preparation method, the colostrum is added into a proper amount of external aqueous phase solution containing 0.5%-2% PVA, and sheared emulsified on a high-speed shearing machine at 4000-8000 r·min -1 for 20-40 s, and then the formed emulsion is dispersed in a proper amount of deionized water containing 0.5%-2% PVA, and the low-speed stirring is 250 ~ 300 r·min -1 .

9. Use of the sustained-release microspheres of any one of claims 1-3 or the sustained-release microspheres prepared by the preparation method of any one of claims 4-8 in the preparation of a medicament for treating arthritis.

10. Use according to claim 9, characterized in that: The arthritis is rheumatoid arthritis or osteoarthritis, and preferably, the medicament is an injectable sustained-release preparation for intra-articular injection. The arthritis is rheumatoid arthritis or osteoarthritis, and preferably, the medicament is an injectable sustained-release preparation for intra-articular injection.

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