Indomethacin PLGA microsphere vitreous injection with drug sustained release anti-inflammatory function and staining effect in vitreous cutting and application thereof

Abstract

The present application relates to the technical field of pharmaceutical preparations, in particular to an indomethacin PLGA microsphere glass body injection with drug sustained release anti-inflammatory function and staining effect in vitreous cutting operation and application thereof, the injection comprises indomethacin and PLGA as a drug carrier and a physical dyeing agent. The PLGA provides vitreous cavity staining and intraoperative imaging on the one hand, assisting the operator to clearly identify the vitreous, proliferative membrane and the edge of the hole; on the other hand, the sustained release of indomethacin maintains the effective treatment concentration in the eye. Tests show that the effective concentration of the microsphere is maintained for about 3 times longer than that of free drugs, the inflammatory reaction is limited to the vitreous cavity, there is no lens toxicity, the safety is better than that of free drugs, the effect of inhibiting IL-1beta in the acute stage in the RRD-PVR model is significantly better than that of free drugs, and the retinal function is effectively protected. The present application realizes the diagnosis and treatment integration function of "one ball two use", and has good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to an indomethacin PLGA microsphere intravitreal injection that combines sustained-release anti-inflammatory drug function with staining function during vitrectomy, as well as a method for preparing the injection and its application in the preparation of drugs for the prevention and / or treatment of rhegmatogenous retinal detachment and secondary proliferative vitreoretinopathy. Background Technology

[0002] Proliferative vitreoretinopathy (PVR) is a common complication of rhegmatogenous retinal detachment (RRD) and a major cause of failure after retinal detachment reattachment surgery. PVR occurs in approximately 5%–10% of RRD patients, rising to 25%–40% in high-risk individuals. PVR is essentially an abnormal wound repair process accompanied by an inflammatory response, and currently there are no approved drugs specifically for its prevention or treatment.

[0003] Non-steroidal anti-inflammatory drugs (NSAIDs) exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity and reducing prostaglandin synthesis. They do not have the typical side effects of glucocorticoids, such as increased intraocular pressure and accelerated cataract formation, making them theoretically an ideal choice for adjunctive treatment of perivitreal retinopathy (PVR). However, free NSAIDs have a short half-life after intravitreal injection (usually only 1-7 days), making long-term treatment difficult; repeated injections increase the risk of intraocular infection and damage.

[0004] Polylactic acid-glycolic acid copolymer (PLGA) is a biodegradable polymer material approved by the US FDA and has been widely used in intraocular sustained-release formulations (such as the dexamethasone intravitreal implant Ozurdex). Encapsulating NSAIDs in PLGA microspheres enables single-injection, sustained drug release, which is an effective strategy to overcome the short half-life of free drugs.

[0005] Chinese patent CN116036022A discloses a drug microsphere containing a carrier material, wherein the drug may be indomethacin and the carrier material may be PLGA, and discloses a scheme for the drug microsphere to be administered via injection for sustained release. However, this patent only describes the formulation and does not provide any in vivo pharmacokinetic, toxicological, or pharmacodynamic validation data, nor does it disclose any technical solutions or technical teachings for the use of the microsphere as an intraoperative staining agent.

[0006] In addition, staining agents (such as triamcinolone) are often used during vitrectomy to help identify the vitreous body, proliferative membrane, and tear margins, but there is currently no solution for staining agents that also have long-lasting, sustained-release anti-inflammatory functions. There is currently no formulation that can simultaneously achieve the dual functions of "intraoperative staining to assist surgery" and "postoperative long-lasting anti-inflammatory effects."

[0007] Therefore, developing an indomethacin PLGA microsphere intravitreal injection formulation that combines intraoperative staining and postoperative sustained-release anti-inflammatory functions, and providing complete preclinical pharmacokinetic, toxicological, and pharmacodynamic validation data, has significant clinical translational value. Summary of the Invention

[0008] In view of this, the purpose of this invention is to overcome the shortcomings of existing NSAIDs, such as short half-life after intravitreal injection and the need for repeated injections, as well as the problem of limited function of existing intravitreal staining agents, and to provide an indomethacin PLGA microsphere intravitreal injection agent that combines drug sustained-release anti-inflammatory function with intravitreal staining function, as well as its preparation method and application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides an indomethacin PLGA microsphere intravitreal injection formulation that combines sustained-release anti-inflammatory drug function with staining effect during vitrectomy, the injection formulation comprising: Indomethacin; and Biodegradable polylactic-co-glycolic acid copolymer (PLGA) as a drug carrier and physical staining agent. The injectable is in the form of a microsphere suspension. After a single intravitreal injection, the PLGA microspheres act as physical suspension particles to provide intravitreal staining and / or intraoperative imaging, while maintaining an effective therapeutic concentration in the eye through sustained release of indomethacin.

[0011] The technical principle is as follows: This invention utilizes the dual physical properties of PLGA microspheres—(1) as a drug carrier: Indomethacin is encapsulated in PLGA microspheres, and the drug is continuously released through the slow degradation of PLGA, thus prolonging the effective concentration maintenance time of the drug in the vitreous cavity; (2) as a physical staining agent: PLGA microspheres are white suspended particles. After being injected into the vitreous cavity, they can form a scattering or reflection effect, making the vitreous body, proliferative membrane, and tear edges easier to identify under a surgical microscope, assisting the surgeon in precise operation. The two functions are achieved synergistically in the same formulation, without the need to use two separate products.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned injection, comprising the following steps: (1) Dissolve PLGA and indomethacin in dichloromethane, add ethanol and ultrapure water, and emulsify by ultrasonication with a probe to form a primary emulsion; (2) Add the colostrum to the polyvinyl alcohol (PVA) solution and shear and stir to form a double emulsion; (3) Add the re-emulsion to the PVA solution and stir to solidify; (4) Remove organic solvent by rotary evaporation, centrifuge and wash to obtain drug-loaded PLGA microspheres; (5) Disperse the drug-loaded microspheres in an injectable solvent to prepare a microsphere suspension.

[0013] Thirdly, the present invention provides the use of the above-mentioned injectable in the preparation of a medicament for the prevention and / or treatment of proliferative vitreoretinopathy secondary to rhegmatogenous retinal detachment.

[0014] Specifically, the present invention provides the use of a composition in the preparation of formulations having staining effects in vitrectomy and / or sustained-release anti-inflammatory drug functions, said composition comprising indomethacin and PLGA microspheres.

[0015] In some specific embodiments of the present invention, the mass ratio of indomethacin and PLGA microspheres used above is 1:(2~10).

[0016] In some specific embodiments of the present invention, the mass ratio of indomethacin and PLGA microspheres used above is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0017] In some specific embodiments of the present invention, the indomethacin used above is the sole active pharmaceutical ingredient, and the PLGA microspheres are the sole carrier ingredient.

[0018] In some specific embodiments of the present invention, the composition described above is composed of indomethacin and PLGA microspheres; The composition is PLGA microspheres loaded with indomethacin.

[0019] The present invention also provides a formulation that combines drug sustained-release anti-inflammatory function with staining function in vitrectomy, comprising indomethacin and PLGA microspheres.

[0020] In some specific embodiments of the present invention, the PLGA microspheres of the above-mentioned formulation are white suspended particles that are suspended in a clump-like manner in the vitreous cavity. After being impacted by a whistle needle, they adhere to the surface of the residual vitreous body, thereby achieving the effect of staining and shaping the vitreous body.

[0021] In some specific embodiments of the present invention, the PLGA microspheres of the above-mentioned formulation are loaded with indomethacin, with a drug loading of 15% to 30% and a particle size of 20 to 35 μm.

[0022] In some specific embodiments of the present invention, the drug loading of the above-mentioned formulation is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

[0023] In some specific embodiments of the present invention, the particle size of the above-mentioned formulation is 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm or 35 μm.

[0024] The present invention also provides a method for preparing the above-mentioned formulation, comprising: S1: Mix PLGA and indomethacin with an organic solvent at a mass ratio of (2~10):1, then mix with ethanol and ultrapure water, and emulsify with ultrasound using a probe to obtain the primary emulsion; S2: Add the colostrum to the emulsifier, shear and stir to obtain the re-emulsion; S3: Add the re-emulsion to the emulsifier and stir to solidify; S4: Remove organic solvent, centrifuge and wash to obtain drug-loaded PLGA microspheres; S5: Disperse the drug-loaded microspheres in an injectable solvent to prepare a microsphere suspension.

[0025] In some specific embodiments of the present invention, the organic solvent in the above preparation method is dichloromethane.

[0026] In some specific embodiments of the present invention, the emulsifier in the preparation method S2 above is polyvinyl alcohol with a concentration of 0.5% to 2%, and the shearing and stirring time is 20 to 40 minutes.

[0027] In some specific embodiments of the present invention, the emulsifier in the preparation method S3 above is polyvinyl alcohol with a concentration of 0.05% to 0.5%, and the stirring and curing time is 2 to 5 hours.

[0028] The present invention also provides the use of the above-described formulation or the formulation prepared by the above-described method in the prevention and / or treatment of rhegmatogenous retinal detachment secondary to proliferative vitreoretinopathy.

[0029] The present invention also provides a method for preventing and / or treating proliferative vitreoretinopathy secondary to rhegmatogenous retinal detachment, comprising the step of injecting the above-described preparation into the vitreous cavity of a subject.

[0030] The present invention also provides a surgical method comprising the step of injecting the above-mentioned preparation into the vitreous cavity of a subject.

[0031] In some specific embodiments of the present invention, the above-mentioned preparation is an intraoperative staining / contrast agent with anti-inflammatory effects, and the preferred dosage form is an injection.

[0032] In some specific embodiments of the present invention, the staining in the vitrectomy of the above-mentioned application and preparation includes: after the PLGA microspheres are injected into the vitreous cavity, they form a clump of milky white suspension, which adheres to the surface of the residual vitreous body after being impacted by a whistle, thereby achieving the staining and shaping effect of the vitreous body and / or the internal limiting membrane.

[0033] This invention discloses an intravitreal injection of indomethacin-PLGA microspheres, which combines sustained-release anti-inflammatory drug function with intravitreal staining effects, along with its preparation method and applications. The injection comprises indomethacin and PLGA as both a drug carrier and a physical staining agent, existing in the form of a microsphere suspension. After a single intravitreal injection, the PLGA microspheres act as physical suspension particles, providing intravitreal staining and intraoperative visualization, assisting the surgeon in clearly identifying the vitreous body, proliferative membrane, and vitreous tear margins. Simultaneously, they maintain an effective therapeutic concentration within the eye through sustained-release indomethacin. Pharmacokinetic studies show that the effective concentration of these microspheres is maintained for approximately three times longer than that of the free drug. Toxicological studies indicate that the inflammatory response of these microspheres is confined to the vitreous cavity, with no lens toxicity, and a safety profile superior to the free drug. Pharmacodynamic studies show that in the RRD-PVR model, these microspheres significantly inhibit IL-1β more effectively in the acute phase than the free drug and effectively protect retinal function. This invention achieves a dual-purpose diagnostic and therapeutic function with promising clinical translation prospects. Specifically, compared with the prior art, the present invention has the following beneficial effects: (1) Synergistic innovation of dual functions. This invention integrates the two functions of PLGA microspheres as "drug carrier" and "intraoperative staining agent" into the same formulation for the first time, realizing "one ball for two uses" - intraoperative staining to assist surgical operation, and postoperative sustained-release anti-inflammatory to prevent PVR. The two functions are naturally connected in the time dimension (intraoperative immediate imaging + postoperative continuous anti-inflammatory), producing a synergistic technical effect of "1+1>2". Through the self-control experiment of Bama miniature pigs, it was confirmed that the distribution morphology and staining and shaping effect of indomethacin PLGA microspheres in the vitreous cavity are basically consistent with the clinically commonly used triamcinolone (TA): after injection, both are clump-shaped milky white suspensions with no tendency to dissolve or spread; after being impacted by a whistle, both can adhere to the surface of residual vitreous body, achieving a clear staining and shaping effect, which is conducive to the surgeon's identification and complete removal of residual vitreous body.

[0034] (2) Significantly prolonged pharmacokinetic characteristics. Following intravitreal injection in Bama miniature pigs, the effective concentration of the microspheres of this invention was maintained for approximately 21 days, which is 3 times longer than that of free indomethacin (approximately 7 days); the half-life (t...) 1 / 2Both the mean residence time (MRT) and peak concentration (Cmax) were significantly prolonged, resulting in a lower peak concentration and a smoother release curve, thus avoiding high concentration shocks from the free drug.

[0035] (3) Superior safety profile. Toxicological studies in cynomolgus monkeys showed that the inflammatory response in the microsphere group was limited to the vitreous cavity (incidence rate 50%, grade 1), with no lens degeneration or inflammation of surrounding tissues observed; while the free indomethacin group showed widespread inflammatory response (ciliary body, retina) and lens degeneration. ERG examination confirmed that neither formulation damaged the function of retinal photoreceptor cells and bipolar cells. No lesions were observed in the blank control group (puncture only, no drug administration), excluding interference from the surgical procedure itself.

[0036] (4) Excellent pharmacodynamic effects. Pharmacodynamic evaluation using the cynomolgus monkey RRD-PVR model showed that a single intravitreal injection of the microspheres of the present invention significantly inhibited intraocular IL-1β levels; the inhibitory effect on IL-1β during the acute phase 2 weeks after administration was significantly better than that of the free drug (P<0.05); ERG showed that retinal function was effectively protected in the drug treatment group. Based on a comprehensive evaluation of pharmacokinetics (stable release), toxicology (localized inflammation, no lens toxicity), and pharmacodynamics (superior anti-inflammatory effect in the acute phase), the microspheres of the present invention maintain comparable efficacy to the free drug while exhibiting superior safety and a more stable release curve.

[0037] (5) Complete preclinical data chain. This invention provides complete preclinical research data from “in vitro formulation preparation → in vivo pharmacokinetics → toxicology → pharmacodynamics”, which is the core advantage that distinguishes it from existing technologies (such as CN116036022A). Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0039] Figure 1 The morphology and solution appearance of PLGA microspheres are shown; Figure 2 The in vitro release curve of indomethacin PLGA microspheres is shown. Figure 3 The effects of different concentrations of PLGA microspheres on the cell viability of RAW264.7 (A) and HUVEC (B) are shown. Figure 4 The effects of different concentrations of indomethacin PLGA microspheres on the expression of IL-6 (A) and TNF-α (B) in macrophages under inflammatory conditions were shown. Figure 5 The effect of different concentrations of indomethacin PLGA microspheres on the levels of IL-1β (A) and COX-1 (B) in macrophages under inflammatory conditions was shown. Figure 6 This study demonstrates the effects of different concentrations of indomethacin PLGA microspheres on the levels of VEGF (A) and IL-1β (B) in vascular endothelial cells under inflammatory conditions. Figure 7 The image shows a comparison of the color and form of two drug formulations after extraction into the syringe. The drug in the empty syringe on the left is a TA suspension, and the drug in the syringe on the right is an indomethacin PLGA microsphere suspension. Figure 8 This shows the distribution pattern of TA after intravitreal injection without vitrectomy; Figure 9 The distribution morphology of indomethacin PLGA microspheres after intravitreal injection in the absence of vitreous resection is shown. Figure 10 The staining and shaping effect of TA on the surface of residual vitreous body after being impacted by a whistle needle is shown. In this diagram, A shows the staining and shaping effect after the whistle needle impact, and B shows the staining and shaping effect. Figure 11 The staining and shaping effect of indomethacin PLGA microspheres on the surface of residual vitreous body after being impacted by a whistle needle is shown. In this diagram, A shows the effect after the whistle needle impact, and B shows the staining and shaping effect. Detailed Implementation

[0040] This invention discloses an indomethacin PLGA microsphere intravitreal injection formulation and its application, which combines sustained-release anti-inflammatory drug function with staining effects during vitrectomy. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0041] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0042] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0043] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0044] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0045] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0046] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0047] The indomethacin PLGA microsphere intravitreal injection of the present invention, which combines drug sustained-release anti-inflammatory function with staining effect in vitrectomy, comprises: indomethacin and biodegradable polylactic-co-glycolic acid copolymer (PLGA) as drug carrier and physical staining agent.

[0048] In practice, the injectable agent exists in the form of a microsphere suspension. After a single intravitreal injection, the PLGA microspheres, acting as physical suspending particles, provide intravitreal staining and / or intraoperative imaging, while maintaining an effective therapeutic concentration within the eye through sustained-release indomethacin. The microspheres have a white opalescent or white suspension appearance and can create scattering or reflection effects within the vitreous cavity, aiding in the identification of the vitreous body, proliferative membrane, or vitreous tear margins during vitrectomy.

[0049] In one example, the mass ratio of indomethacin to PLGA is 1:2 to 1:10, preferably 1:4; the drug loading of the microspheres is 15% to 30%, preferably 22% to 23%; and the encapsulation efficiency is 85% to 95%, preferably 89% to 93%.

[0050] In one example, the microspheres have a particle size of 20-35 μm, preferably 26.38±4.03 μm.

[0051] In one example, the microspheres are prepared by an emulsification-solvent evaporation method, and their in vitro release characteristics are: they can be continuously released for no less than 5 days in PBS containing 5% Tween 20; and the effective concentration in vivo is maintained for no less than 14 days, preferably no less than 21 days.

[0052] In one example, the injection volume is 50-150 μL / eye, and the dosage is 1-3 mg / eye (calculated as indomethacin).

[0053] The specific method for preparing the injectable agent described in this invention includes the following steps: (1) Dissolve PLGA and indomethacin in dichloromethane at a mass ratio of (2~10):1, add ethanol and ultrapure water, and emulsify by ultrasonication with a probe to form a primary emulsion; (2) Add the colostrum to the polyvinyl alcohol solution, and shear and stir to form a double emulsion; (3) Add the re-emulsion to the polyvinyl alcohol solution and stir to solidify; (4) Remove organic solvent by rotary evaporation, centrifuge and wash to obtain drug-loaded PLGA microspheres; (5) Disperse the drug-loaded microspheres in an injectable solvent to prepare a microsphere suspension.

[0054] In one example, the mass ratio of PLGA to indomethacin in step (1) is 4:1.

[0055] In one example, the concentration of the polyvinyl alcohol solution in step (2) is 0.5%~2% (w / w), and the shearing and stirring time is 20~40 minutes; the concentration of the polyvinyl alcohol solution in step (3) is 0.05%~0.5% (w / w), and the stirring and curing time is 2~5 hours.

[0056] In practice, the above-mentioned injection or the injection prepared by the above method is used to prepare drugs for the prevention and / or treatment of proliferative vitreoretinopathy secondary to rhegmatogenous retinal detachment.

[0057] In some examples, the drug is administered via a single intravitreal injection for the prevention or inhibition of the occurrence or progression of PVR in one or more of the following situations: (a) Postoperative scleral buckling surgery for rhegmatogenous retinal detachment; (b) Postoperative vitrectomy and retinal detachment repair after RRD; (c) During the waiting period for observation of diabetic retinopathy or traumatic vitreous hemorrhage; (d) After diabetic retinopathy or traumatic vitreous hemorrhage vitrectomy.

[0058] In some examples, the drug is used for: (a) Inhibit the expression of the intraocular inflammatory cytokine interleukin-1β (IL-1β); and / or (b) Improve or protect retinal function as reflected by electroretinography.

[0059] The drug showed significantly better inhibition of IL-1β during the acute phase (days 0-14) after intravitreal injection than free indomethacin (P<0.05); and in terms of ocular toxicology, the inflammatory response was confined to the vitreous cavity, without causing lens degeneration or retinal structural damage, and its safety profile was superior to that of free indomethacin.

[0060] In practice, the amount of drug used for intraoperative staining is much smaller than the therapeutic amount, and the microspheres are continuously removed during vitrectomy, leaving very little residual drug and a weak anti-inflammatory effect. Therefore, in some embodiments, a therapeutic dose of drug microspheres is still injected after the procedure to prevent postoperative PVR or epiretinal membrane formation.

[0061] In practice, the above-mentioned indication for post-vitrectomy injection to prevent PVR or epiretinal membrane is only one of the indications for this drug. This invention is also applicable to non-vitrectomy post-operative medication scenarios, such as scleral buckling after RRD, vitreous hemorrhage caused by various reasons, etc.

[0062] The present invention will be further illustrated below with reference to the embodiments.

[0063] Example 1: Preparation of Indomethacin PLGA Microspheres

[0064] Materials: PLGA (50:50, Evonik, Germany), indomethacin (>99%, Dalian Meilun), polyvinyl alcohol 1788 (degree of alcoholysis 87.0%~89.0%, Aladdin), dichloromethane (analytical grade), anhydrous ethanol (analytical grade).

[0065] Preparation method (emulsification-solvent evaporation method): (1) Dissolve 200 mg PLGA and 50 mg indomethacin in 2 mL of dichloromethane. After complete dissolution, add 150 μL ethanol and 50 μL ultrapure water and emulsify using a probe (ultrasound power 330W, 50%). (2) Add the colostrum to 10 mL of PVA solution (1%, w / w) and stir for 30 minutes using a uniform shear stirrer; (3) Add the suspension to 80 mL of PVA solution (0.1%, w / w) and stir to solidify for 3 hours; (4) Use a rotary evaporator to remove residual organic solvents; (5) Centrifuge and wash 3 times to obtain drug-loaded PLGA microspheres; (6) Disperse the drug-loaded microspheres in an injectable solvent to prepare a microsphere suspension.

[0066] result: See Figure 1 The prepared microspheres are complete and uniform in shape, with a smooth surface and a hydrated particle size of 26.38±4.03 μm. The microsphere suspension is milky white and can form a scattering effect after intravitreal injection, achieving intraoperative staining / contrast function.

[0067] Example 2: Determination of encapsulation efficiency and drug loading of indomethacin PLGA microspheres

[0068] method: Methanol was used to disrupt the structure of drug-loaded PLGA microspheres, releasing indomethacin. The solution was filtered through a 0.22 μm polytetrafluoroethylene filter, and the drug content was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions for indomethacin were as follows: 0.1 M glacial acetic acid (pH=3.0) as the aqueous phase, acetonitrile as the organic phase, with a ratio of 20:80; a flow rate of 1 mL / min; a column temperature of 30℃; and a detection wavelength of λ=320 nm.

[0069] result: The drug loading was 22.3%, and the encapsulation efficiency was 89.1%.

[0070] Example 3: In vitro release behavior of indomethacin PLGA microspheres

[0071] Methods: The in vitro drug release behavior was investigated using dialysis. Drug-loaded PLGA microsphere suspensions were dispersed in PBS solution. 1 mL of the suspension was placed in a dialysis bag and then in PBS containing 5% Tween 20. Samples were taken at 0.5, 1, 2, 4, 9, 11, 25, 48, 72, and 120 hours. Signal intensity was detected using a microplate reader, and drug concentration and cumulative release were calculated to plot release curves.

[0072] result: Figure 2 The results showed that the free drug group released drugs rapidly, with almost all reaching peak levels within one day. The microsphere group of this invention exhibited a lower overall release rate, with sustained release lasting approximately 5 days, demonstrating excellent drug release performance. This release characteristic matches the in vivo pharmacokinetic data (effective concentration maintained for approximately 21 days)—in vitro release reflects the initial release behavior of the microspheres, while in vivo sustained release time is longer.

[0073] Example 4: Cell compatibility of indomethacin PLGA microspheres

[0074] method: RAW264.7 macrophages and HUVEC vascular endothelial cells were co-incubated with PLGA microspheres at different concentrations (0.25~2 mg / mL) for 12 hours, and cell viability was detected by MTT assay.

[0075] result: Figure 3The results showed that the survival rate of both cell types exceeded 90% within the concentration range of 0.25~2 mg / mL, indicating that the microspheres of the present invention have good cell compatibility.

[0076] Example 5: Cellular efficacy experiment of indomethacin PLGA microspheres

[0077] method: An inflammation model was established by stimulating RAW264.7 macrophages with LPS. The macrophages were then co-incubated with drug-loaded PLGA microspheres at different concentrations (25-400 μg / mL) for 12 hours. The levels of inflammatory factors IL-6, TNF-α, IL-1β and COX-1 were detected by ELISA.

[0078] An inflammation model was established by stimulating HUVEC cells with LPS. The cells were then co-incubated with drug-loaded PLGA microspheres at different concentrations (25-400 μg / mL) for 12 hours. The levels of inflammatory factors VEGF and IL-1β were detected by ELISA.

[0079] result: Figures 4-6 The results showed that indomethacin PLGA microspheres reduced the expression levels of IL-6, TNF-α, IL-1β, and COX-1 in a concentration-dependent manner. In vascular endothelial cells, the drug-loaded microspheres also reduced the secretion levels of VEGF and IL-1β in a concentration-dependent manner.

[0080] Example 6: Pharmacokinetic Study of Bama Miniature Pigs

[0081] Experimental animals: 38 ordinary Bama miniature pigs, aged 5-9 months and weighing 18-25 kg.

[0082] Dosage regimen: Single intravitreal injection in both eyes, dose 2.8 mg / eye (calculated as indomethacin), administration volume 150 μL. A free indomethacin control group was included.

[0083] Sampling and Detection: Vitreous samples were collected at 15 minutes, 6 hours, 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days after drug administration. Indomethacin concentration was determined by LC-MS / MS, with a limit of quantification of 1.0 ng / mL. Pharmacokinetic parameters were calculated using the WinNonlin non-compartmental model.

[0084] result: Table 1

[0085]

[0086] The half-life and mean residence time of the microsphere group of this invention are significantly longer than those of the free drug group, and the effective concentration maintenance time is extended by about 3 times. The peak concentration is lower (data from the continuous sampling group), the release curve is more stable, and the high concentration shock of the free drug is avoided.

[0087] Example 7: Toxicological Study of Crab-Eating Mammals

[0088] Experimental animals: 15 ordinary cynomolgus monkeys, male, 6-9 years old, weighing 8-15 kg.

[0089] Experimental design: A single intravitreal injection was administered to the left eye (the eye used for toxicity evaluation). The experiment included a free indomethacin group, a PLGA microsphere group of the present invention, and a blank control group (puncture only, no drug administration). The dose was 2 mg / eye, and the administration volume was 100 μL.

[0090] Observation indicators: slit-lamp examination, intraocular pressure measurement, fundus photography, OCT, ERG and histopathological examination.

[0091] result: Slit-lamp examination: Both the free group and the PLGA microsphere group showed varying degrees of anterior segment inflammation, with a higher incidence and severity in the PLGA microsphere group, which is considered to be related to the physical stimulation of PLGA particles; no abnormalities were observed in the blank control group, proving that the puncture trauma itself does not cause persistent lesions.

[0092] ERG examination: There was no significant difference between the two drug-treated groups and the pre-modeling group (P>0.05), confirming that the drug did not damage the function of retinal photoreceptor cells and bipolar cells.

[0093] Histopathology (key findings): Table 2

[0094]

[0095] In this invention, the inflammatory response in the PLGA microsphere group was limited to the vitreous cavity (incidence rate 50%, grade 1), with no lens degeneration or inflammation of surrounding tissues observed, demonstrating significantly better safety than free drugs. No lesions were observed in the blank control group, excluding interference from surgical procedures.

[0096] Example 8: Pharmacodynamic Study of the Crab-Eating Monkey RRD-PVR Model

[0097] Experimental animals: 15 male cynomolgus monkeys of common grade, aged 6-9 years and weighing 8-15 kg (as described in the toxicology study).

[0098] Model establishment: The RRD-PVR model was established using the previously developed novel method. Before modeling, animals were fully dilated with compound tropicamide eye drops, and anesthetized with 5 mg / kg of acetaminophen, 2 mg / kg of xylazine, and 4 mg / kg of ketamine hydrochloride. During anesthesia, the animals were intubated, given oxygen, and sustained with isoflurane. After anesthesia, the right eye was periodically instilled with oxybuprofen eye drops for topical anesthesia.

[0099] Pre-existing scleral tunnels were created using a 25G scleral tunneling needle at approximately 3 mm from the limbus, both superior nasal and superior temporal. The superior nasal tunnel served as the entry point for intraocular light, while the superior temporal tunnel served as the entry point for the injection needle, scissors, and vitrectomy tip. Using a 41G subretinal injection needle, balanced salt solution (BSS) was injected into the superior and superior temporal peripheries at 2-3 sites, with each site receiving 50-100 μL, for a total injection volume of approximately 150 μL. The puncture site was then enlarged using scissors to create a retinal tear (approximately 3 PD in size), fully exposing the pigment epithelium. Next, a vitrectomy tip was inserted through the tear and bluntly pulled downwards into the retina, causing extensive separation of the neuroepithelial and pigment epithelial layers in the superior and superior temporal retina, forming a rhegmatogenous retinal detachment model. The scleral tunneling needle was removed, and the tunnel puncture site was gently closed with a cotton swab. After the procedure, 1-2 drops of ofloxacin eye ointment were instilled to prevent infection. The model was observed for 8 weeks post-modeling.

[0100] Dosage regimen: One week after modeling, a single intravitreal injection of 2 mg / eye was administered to the right eye in a volume of 100 μL. The study included a free indomethacin group, a PLGA microsphere group of this invention, and a modeling-only group (no drug administration).

[0101] Efficacy evaluation: Vitreous samples were collected before modeling, before drug administration, and on days 9, 15, 30, 41, and 57 after drug administration. IL-1β levels were detected using the Luminex liquid chromatography-array method. ERG examinations were performed at each time point.

[0102] result: Dynamic changes in inflammatory factors (IL-1β): Table 3

[0103]

[0104] Phased analysis: In the acute phase (within 2 weeks after administration, i.e., days 9 and 15): the IL-1β level in the PLGA microsphere group of this invention (81.1±92.6 pg / mL on day 9; 104.9±104.8 pg / mL on day 15) was significantly lower than that in the free drug group (491.5±758.6 pg / mL on day 9; 583.0±720.7 pg / mL on day 15), and the difference was statistically significant (P<0.05). This indicates that the microspheres of this invention have a significantly better anti-inflammatory effect than the free drug in the acute phase.

[0105] Chronic phase (2-8 weeks, i.e., day 30 to day 57): There was no significant difference in IL-1β levels between the two groups (P>0.05), and the efficacy was comparable.

[0106] Retinal function protection (ERG): Compared with the model-only group at the same time point, the amplitudes of dark adaptation a and b waves in the right eye of the free indomethacin group and the PLGA microsphere group of this invention were significantly increased (P<0.05 or P<0.01), while there was no significant difference in ERG parameters between the two drug treatment groups at each time point (P>0.05), suggesting that the two drugs have comparable efficacy in protecting retinal function.

[0107] Example 9: Comparative Study of Indomethacin PLGA Microspheres and Triamcinolone Staining During Vitrectomy

[0108] Objective: To explore the feasibility of using indomethacin PLGA microspheres instead of TA as a staining excipient for vitreous and internal limiting membrane (UDM) during vitrectomy to facilitate complete removal of the vitreous and UDM. The experimental animals were Bama miniature pigs, as their eye size, vitreous volume, and retinal texture are similar to those of primates.

[0109] Experimental animals: 1 Bama miniature pig, with the right eye as the TA experimental eye and the left eye as the PLGA experimental eye (self-control to eliminate individual differences).

[0110] Experimental methods: (1) In vitro morphological comparison: TA suspension and indomethacin PLGA microsphere suspension were extracted respectively, and their color and morphological characteristics in the syringe were observed. (2) Distribution pattern in the vitreous cavity without vitrectomy: 50 μL of TA suspension was injected into the vitreous cavity of the right eye and 50 μL of indomethacin PLGA microsphere suspension was injected into the left eye. The distribution pattern of the two preparations in the vitreous cavity was observed. (3) Staining and shaping effect after partial vitreous resection: After the central vitreous body was removed, TA or PLGA microspheres were injected respectively, and the microspheres were diffused by the impact of a whistle needle. The staining and shaping effects of the two preparations on the residual vitreous body and the internal limiting membrane were observed.

[0111] Experimental results: 1. Comparison of in vitro traits See Figure 7 The left side of the image shows the extracted TA precipitate suspension, and the right side shows the extracted indomethacin PLGA microsphere suspension. Both are milky white and homogeneous suspensions.

[0112] 2. In cases where the vitrectomy has not been performed, two different preparations are injected into each eye.

[0113] Figure 8 The image shows a 50 μL intravitreal injection of TA in the right eye. The background consists of the vitreous body and retina. The vitreous body is a transparent medium, and branching vessels are visible in the retina. It is observed that after TA injection, a clump-like milky-white suspension forms within the vitreous cavity, showing no tendency to dissolve or spread.

[0114] Figure 9This image shows a 50 μL PLGA injection into the vitreous cavity of the left eye. The background consists of the vitreous body and retina; the vitreous body is a transparent medium, and branching vessels are visible in the retina. The upper right corner shows the intraocular illumination source. The suspended white clumps are the PLGA. The black shadow in the lower left corner is the projection of the PLGA clumps. It is evident that, similar to TA, PLGA, after injection, appears as a clump-like milky-white suspension within the vitreous cavity, showing no tendency to dissolve or spread.

[0115] 3. After partial vitrectomy, the preparation is injected.

[0116] Figure 10 After the ATA suspension is injected into the central vitreous body, the needle impact causes it to diffuse and adhere to the surface of the remaining vitreous body. Figure 10 The left side of B is the vitreous body that has been removed by the vitreous scalpel after staining, so it is transparent. The right side is mostly the remaining vitreous body that has been stained and shaped but has not yet been removed.

[0117] Figure 11 The PLGA suspension in A was injected into the central vitreous body and then diffused by the impact of the whistle needle, adhering to the surface of the remaining vitreous body. Figure 11 The left side of B is the vitreous body that has been removed by the vitreous scalpel after staining, so it is transparent. The right side is mostly the remaining vitreous body that has been stained and shaped but has not yet been removed.

[0118] Conclusion: The above comparison shows that PLGA microspheres can completely replace TA as a staining excipient in vitrectomy, facilitating the visualization and complete removal of the vitreous. Meanwhile, indomethacin-loaded PLGA microspheres also have a similar intraoperative local anti-inflammatory effect to TA.

[0119] Based on the above embodiments, the comparison between the indomethacin PLGA microspheres of the present invention and free indomethacin is summarized as follows.

[0120] Table 4

[0121]

[0122] Overall conclusion: The indomethacin PLGA microspheres of this invention maintain efficacy comparable to free drugs while exhibiting superior safety (localized inflammation, no lens toxicity), a more stable release curve, and intraoperative staining assistance, demonstrating clear advantages in clinical translation.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of the composition in the preparation of formulations having staining effects in vitrectomy and / or sustained-release anti-inflammatory drug functions, characterized in that, The composition comprises indomethacin and PLGA microspheres.

2. The application as described in claim 1, characterized in that, The mass ratio of indomethacin to PLGA microspheres is 1:(2~10).

3. The application as described in claim 2, characterized in that, The mass ratio of indomethacin to PLGA microspheres is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:

10.

4. The application as described in claim 1, characterized in that, Indomethacin is the sole active pharmaceutical ingredient, and PLGA microspheres are the sole carrier ingredient.

5. The application as described in claim 1, characterized in that, The composition consists of indomethacin and PLGA microspheres; The composition is PLGA microspheres loaded with indomethacin; The formulation is used for the prevention and / or treatment of rhegmatogenous retinal detachment followed by proliferative vitreoretinopathy.

6. A formulation that combines sustained-release anti-inflammatory drug function with staining effect during vitrectomy, characterized in that, Including indomethacin and PLGA microspheres.

7. The formulation according to claim 6, characterized in that, The PLGA microspheres are loaded with indomethacin, with a drug loading of 15% to 30% and a particle size of 20 to 35 μm.

8. The formulation as described in claim 7, characterized in that: The drug loading is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. The particle size is 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm or 35 μm.

9. The method for preparing the formulation according to claim 6, characterized in that, include: S1: Mix PLGA and indomethacin with an organic solvent at a mass ratio of (2~10):1, then mix with ethanol and ultrapure water, and emulsify with ultrasound using a probe to obtain the primary emulsion; S2: Add the colostrum to the emulsifier, shear and stir to obtain the re-emulsion; S3: Add the re-emulsion to the emulsifier and stir to solidify; S4: Remove organic solvent, centrifuge and wash to obtain drug-loaded PLGA microspheres; S5: Disperse the drug-loaded microspheres in an injectable solvent to prepare a microsphere suspension.

10. The preparation method according to claim 9, characterized in that: The organic solvent is dichloromethane; or The emulsifier described in S2 is polyvinyl alcohol with a concentration of 0.5% to 2%, and the shearing and stirring time is 20 to 40 minutes; or The emulsifier mentioned in S3 is polyvinyl alcohol with a concentration of 0.05% to 0.5%, and the stirring and curing time is 2 to 5 hours.

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