Nanometer preparation with antioxidant function and application in treating glaucoma

Abstract

The application provides a kind of nano preparation with antioxidant function and the use for treating glaucoma, the preparation is composed of oil phase and water phase.Oil phase is lecithin, medium-chain triglyceride, fat-soluble antioxidant, glaucoma treatment drug, water phase is water-soluble antioxidant or deionized water.The nano delivery system described in the application is by loading glaucoma treatment drug in the nano preparation containing antioxidant, can improve local drug concentration, prolong drug retention time, at the same time, through antioxidant scavenging excess active oxygen, reduce inflammatory response, reduce eye cell apoptosis, so as to achieve good therapeutic effect.The nano preparation described in the application can be nano emulsion, liposome, micelle or lipid nanoparticle and other drug delivery systems.The nano preparation can deliver therapeutic drugs while scavenging ROS, reducing eye inflammation and reducing cell apoptosis to achieve more efficient therapeutic effect, with the characteristics of double effect, easy to produce and prepare, wide application range, etc.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a nano-formulation with antioxidant function and its use in treating glaucoma. It is a type of nano-delivery system with antioxidant activity that can efficiently deliver drugs while clearing ROS from damaged cells, restoring intracellular redox balance, and reducing cell apoptosis. It also relates to the construction method of the nano-formulation and its application in ophthalmic diseases. Background Technology

[0002] Vision is fundamental to our perception of the world and is crucial at every stage of our lives. However, eye diseases are frequent and diverse, and if left untreated, they often affect the visual system. Among numerous eye diseases, glaucoma is the second leading cause of blindness after cataracts. According to a report by the World Health Organization, by 2040, the number of people with glaucoma worldwide will reach 118 million, accounting for nearly 4% of the total population (Blindness, VisionImpairment et al. 2021). It is important to note, however, that vision impairment can be avoided through prevention or timely treatment. Therefore, efficient, convenient, and multifunctional treatment options are crucial for eye diseases.

[0003] Recent studies have shown that the development and progression of glaucoma are accompanied by cellular oxidative stress. ROS (reactive oxygen species) are metabolic byproducts of mitochondrial respiration, containing free radicals and non-free radical oxygen derivatives such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Under normal circumstances, the produced ROS are cleared by the body's antioxidant system. Oxidative stress occurs when the cellular redox balance is disrupted. Excessive accumulation of ROS can attack polyunsaturated fatty acids on the cell membrane, forming covalent bonds between lipid peroxides and receptors, thereby disrupting cell membrane integrity. Oxidized phospholipids can also be recognized by scavenging receptors or Toll-like receptors, inducing programmed cell death. Furthermore, ROS can induce autophagy, apoptosis, or necrosis by altering protein modifications and causing damage to mitochondrial and chromosomal nucleic acids.

[0004] Oxidative stress in ocular tissues and cells is closely related to the occurrence and development of eye diseases. For fundus tissues, retinal pigment epithelial cells (RPE) and retinal ganglion cells (RGCs) require high levels of metabolism, resulting in high levels of reactive oxygen species (ROS) produced by their mitochondria, thus placing them under greater oxidative stress. Physical stress, ischemia, and hypoxia can all disrupt the redox homeostasis of the retina, triggering inflammation and autophagy, leading to optic nerve atrophy and visual impairment. Therefore, neutralizing ROS and enhancing sufficient antioxidant defense capacity is crucial for the homeostasis of ocular tissues and cells. Currently, many natural antioxidants are used for vision protection in glaucoma. Among these antioxidants, antioxidant vitamins are a commonly used class of natural compounds, including vitamin C, vitamin E, and beta-carotene. Data from a clinical study showed that 60 glaucoma patients who took α-tocopherol acetate 600 mg / day or 6 mg / day orally for 12 consecutive months experienced significant improvement in visual field deviation compared to the placebo group (Engin, Engin et al. 2007). However, the therapeutic effects of oral administration on the eyes remain limited. Another study confirmed that, in an 11-year study, oral administration of vitamin E, vitamin C, and beta-carotene did not prevent the development of AMD compared to placebo. However, when the antioxidant coenzyme Q10 and vitamin E were formulated into an ophthalmic solution as a supplementary treatment for glaucoma, patients experienced visual improvement after 24 months of treatment (Quaranta, Riva et al. 2019). Therefore, reducing cellular oxidative stress is crucial for preventing and mitigating visual field defects associated with glaucoma. Developing antioxidants into topical forms is also essential for adjunctive treatment of glaucoma.

[0005] For most eye diseases, topical medication is a commonly used and well-tolerated treatment method. Traditional ophthalmic preparations include eye drops, ointments, ophthalmic suspensions, and ophthalmic gels. Among these, eye drops account for over 95% of the ophthalmic drug market share. After topical administration, eye drops are easily cleared by blinking or drained through the nasolacrimal duct, resulting in low local drug concentrations and hindering the desired treatment efficacy for many diseases. Simultaneously, many ophthalmic medications have poor water solubility, making it difficult for traditional solutions to achieve effective therapeutic concentrations. Currently, many novel drug delivery systems, such as liposomes, emulsions, polymer nanoparticles, gels, and contact lenses, have demonstrated advantages in ophthalmic drug delivery. These delivery methods primarily improve the effectiveness of topical administration by altering system viscosity, increasing drug content, or even overcoming the ocular barrier. Compared to other systems, liposomes exhibit better biocompatibility and improve the solubility of lipid-soluble drugs, increasing local drug concentrations and thus enhancing drug utilization. Because the clinical translation process for lipid formulations is relatively mature, many ophthalmic lipid formulations such as Restasis (cyclosporine A emulsion) and Xelpros (latanoprost emulsion) have entered clinical use.

[0006] Xelpros is the first marketed ophthalmic emulsion for glaucoma treatment. According to Sun Pharmaceutical's patents, the main components of Xelpros, besides latanoprost, are castor oil and polyethylene glycol-15 hydroxystearate (Solutol HS15, or HS15 for short), which solubilize latanoprost. HS15 is a nonionic surfactant developed by BASF in Germany and is currently listed in the European Pharmacopoeia and the United States Pharmacopeia for solubilizing various injectable formulations. However, according to research reports, lipid nanoparticles using HS15 as a surfactant have shown toxicity in various cytotoxicity tests. This may pose a significant safety risk to the fragile retinal cells of glaucoma patients. Therefore, to increase the safety of the carrier while achieving efficient delivery of latanoprost, an ophthalmic lipid nanodelivery system without HS15 needs to be developed. Summary of the Invention

[0007] The purpose of this invention is to provide a nano-formulation with antioxidant function, which is composed of an aqueous phase and an oil phase, wherein the ratio of oil phase to aqueous phase is 1:1000-1:2, w / w.

[0008] The oil phase consists of 5-80% lecithin, w / w, 0-50% medium-chain triglycerides, 0.1-50% fat-soluble antioxidants (natural vitamin E and its derivatives), and 0.01%-20% glaucoma treatment drugs; the aqueous phase consists of 0.1-30% water-soluble antioxidants (L-ascorbic acid) or deionized water.

[0009] The nanoformulations of the present invention refer to drug-acceptable carriers, including but not limited to nanoemulsions, liposomes, micelles or lipid nanoparticles.

[0010] The nanoparticles are required to have a uniform and stable particle size, less than 200 nm.

[0011] The nano-formulation of the present invention is prepared by the following method: using lecithin, medium-chain triglycerides, lipid-soluble antioxidants, and glaucoma treatment drugs as the oil phase, and an aqueous solution containing water-soluble antioxidants as the aqueous phase, to prepare an oil-in-water (O / W) drug-loaded nano-formulation. The oil phase:water phase ratio is 1:1000-1:2, w / w.

[0012] In the oil phase mentioned above, apart from fat-soluble antioxidants and glaucoma treatment drugs, other oily components can be replaced by phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-diolenooxypropylammonium chloride (DOTMA), dimethyl dioctadecylammonium bromide (DDAB), soybean oil, olive oil, castor oil, various medium and long chain fatty acid esters, cholesterol, squalane, and squalene.

[0013] Lipid-soluble antioxidants account for 0.1%-50% of the oil phase weight. Besides natural vitamin E and its derivatives, such as α-tocopherol, vitamin E acetate, vitamin E succinate, and vitamin E nicotinate, lipid-soluble antioxidants can also be replaced by other lipid-soluble antioxidants, such as melatonin, polydopamine, glutathione, β-carotene, astaxanthin, butylated hydroxyanisole (BHA), curcumin, N-acetylcysteine ​​(NAC), and flavonoids (such as quercetin, kaempferol, catechin, and epigallocatechin gallate (EGCG)).

[0014] Water-soluble antioxidants account for 0.1%-30% of the weight of the aqueous phase. Besides L-ascorbic acid, other water-soluble antioxidants can also be substituted, such as tea polyphenols, superoxide dismutase, glutathione peroxidase, and glutathione reductase.

[0015] Glaucoma treatment drugs can be prostaglandin analogs, such as latanoprostine, betamipridanoprostine, travoprostine, and bimatoprostine; they can also be β-adrenergic receptor antagonists, such as timolol, carteolol, and betalolol; they can also be α-adrenergic agonists, such as brimonidine tartrate; or they can be carbonic anhydrase inhibitors, such as acetazolamide and brinzolamide. Glaucoma treatment drugs encapsulated in nanodelivery systems can be one or more of the above combinations. The glaucoma treatment drug can be presented as an oil phase, accounting for 0.1%–20% of the total lipid mass.

[0016] Another object of the present invention is to provide the application of the nanoformulation in the preparation of a glaucoma treatment drug. The nanoformulation can slow ocular surface clearance, thereby prolonging drug retention time and increasing local drug concentration. Simultaneously, it scavenges excess reactive oxygen species (ROS) through antioxidants, reducing inflammatory responses and decreasing ocular cell apoptosis, thereby achieving a good therapeutic effect.

[0017] The formulation of the drug of this invention is a liquid preparation, including topical liquid preparations or injectable liquid preparations. The nano-formulation of this invention can be administered via 1) local instillation, which prolongs retention on the ocular surface, increasing the accumulation of the drug and antioxidants in the anterior segment of the eye; some nano-formulations can also be absorbed through the conjunctival-scleral pathway to reach the fundus tissue, delivering antioxidants to the fundus non-invasively. Alternatively, the drug, along with a ROS scavenger, can be directly delivered to the fundus tissue via 2) intraocular injection.

[0018] This invention constructs a nano-formulation with antioxidant activity, achieving co-delivery of antioxidants and glaucoma treatment drugs. This nano-formulation uses commonly available, biocompatible, and low-cost lipid materials as its main components, with added water-soluble or lipid-soluble antioxidants and loaded ophthalmic drugs. While maintaining therapeutic effects, it achieves better therapeutic outcomes by maintaining the redox homeostasis of ocular cells. This nano-formulation is easy to produce, cost-controllable, and of stable quality. Furthermore, due to the good biocompatibility and high safety of the lipid materials used, it is more easily absorbed by ocular cells, enhancing drug bioavailability. It has promising industrialization and broad application prospects in ophthalmic formulations.

[0019] The nano-formulation prepared in this invention has the functions of scavenging ROS, reducing ocular inflammation, and decreasing cell apoptosis. Recent studies have shown a certain relationship between oxidative stress and cell apoptosis in glaucoma. In these disease processes, the cellular redox balance is disrupted, and the large amount of ROS produced can induce DNA damage, protein and lipid oxidation, leading to cell dysfunction and even apoptosis. The nano-formulation in this invention contains antioxidants such as natural vitamin E and its derivatives, which can effectively scavenge ROS in cells, inhibit or delay oxidation, thereby mitigating or eliminating the effects of oxidative stress through prevention, blocking, and repair.

[0020] The innovation of this invention lies in: 1) addressing the problems of low drug delivery content and low bioavailability in topical glaucoma medications by improving drug delivery efficiency through nano-formulations; and 2) addressing the widespread oxidative stress damage in glaucoma by reducing ROS accumulation in the fundus, alleviating inflammatory responses, protecting the retina, and reducing visual impairment. Attached Figure Description

[0021] Figure 1 It refers to the particle size of drug-loaded nanoemulsions.

[0022] Figure 2 This is a transmission electron microscope image of a drug-loaded nanoemulsion.

[0023] Figure 3 It is the particle size stability of drug-loaded nanoemulsions.

[0024] Figure 4 It refers to the drug release behavior of free drugs and drug-loaded nanoemulsions.

[0025] Figure 5 It is the 24-hour cytotoxicity of free drug and drug-loaded nanoemulsion.

[0026] Figure 6 This describes the 24-hour cellular uptake of free drug and drug-loaded nanoemulsions.

[0027] Figure 7 It is the ability of free drugs and drug-loaded nanoemulsions to remove DPPH in vitro.

[0028] Figure 8 It is the ROS scavenging ability of free drugs and drug-loaded nanoemulsions in ARPE-19 cells.

[0029] Figure 9 This refers to the retention of free drugs and drug-loaded nanoemulsions on the ocular surface after topical instillation.

[0030] Figure 10 This refers to the penetration of free drugs and drug-loaded nanoemulsions into the posterior part of the eye after being dropped into the eye.

[0031] Figure 11 This refers to the intraocular pressure-lowering function of drug-loaded nanoemulsions in a rat model of high intraocular pressure.

[0032] Figure 12 It is a marker of redox activity and expression of inflammatory factors in the retina of rats after treatment with drug-loaded nanoemulsions.

[0033] Figure 13 This describes the apoptosis of retinal cells in rats after treatment with drug-loaded nanoemulsions.

[0034] Figure 14 This describes the infiltration of inflammatory cells in the retina of rats after treatment with drug-loaded nanoemulsions.

[0035] Figure 15 It represents the density of optic nerve cells in the retina of mice after treatment with drug-loaded nanoemulsions. Detailed Implementation

[0036] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0037] Example 1: Preparation and Physicochemical Characterization of Nanoemulsions

[0038] Nanoemulsion prescription:

[0039] Egg yolk lecithin 20mg

[0040] Long-chain fatty acid glycerides 20mg

[0041] α-Tocopherol 0.05mg

[0042] Latanoprost 10mg

[0043] 1 mL of water.

[0044] Oil-in-water emulsions were prepared using a high-energy emulsification method according to the above formulation. Egg yolk lecithin, long-chain triglycerides, α-tocopherol, and latanoprost were first dissolved in a small amount of ethanol. Then, deionized water was added under high-speed vortexing to obtain the proemulsion. Nanoemulsions were then obtained by ultrasonication using an ice bath probe. Dynamic light scattering analysis showed that the particle size of the nanoemulsions prepared by this formulation was below 200 nm (see [link to product description]). Figure 1 The morphology of the nanoemulsions, observed by transmission electron microscopy (TEM), exhibits a uniform and smooth spherical structure (see...). Figure 2 This nanoemulsion can be stably stored for at least 7 days at 4°C (see...). Figure 3 This formulation achieves over 90% release of the encapsulated drug within 48 hours, significantly prolonging the duration of action compared to the free drug (see...). Figure 4 ).

[0045] Example 2: Cytotoxicity of Drug-Loaded Nanoemulsions

[0046] Nanoemulsion prescription:

[0047] Egg yolk lecithin 50mg

[0048] Olive oil 35mg

[0049] α-Tocopherol 0.1mg

[0050] Latanoprost 5mg

[0051] 1 mL of water.

[0052] Since topical ocular medications typically require frequent administration, to determine if the local accumulation of nanoemulsions poses any safety risks, the human retinal pigment epithelial cell line (ARPE-19) was used as a model to investigate the safety of the nanoemulsion prepared according to the above formulation. With drug concentration gradients ranging from 0 to 25 μg / mL, it was found that compared to free drug, this nanoemulsion exhibited almost no toxicity to ocular cells, even at higher concentrations (see...). Figure 5 This demonstrates that the nanoemulsion has good biosafety.

[0053] Example 3: Cellular uptake of drug-loaded nanoemulsions

[0054] Nanoemulsion prescription:

[0055] Soy lecithin 0.1mg

[0056] Castor oil 0.7mg

[0057] Vitamin E acetate 0.15mg

[0058] Latanoprost 0.05mg

[0059] 1 mL of water.

[0060] Nanoemulsions were prepared using the above formulation and labeled with the fluorescent probe DID. The uptake of the drug-loaded nanoemulsions over 24 hours was investigated using the ARPE-19 cell line as a model. Fluorescence inverted microscopy imaging revealed that the ARPE-19 cell line had a significantly higher uptake capacity for the drug-loaded nanoemulsions than for free drug, demonstrating that the nanoemulsions have the ability to improve drug bioavailability (see...). Figure 6 ).

[0061] Example 4: In vitro antioxidant capacity study of drug-loaded nanoemulsions

[0062] Taking the formulation of Example 3 as an example, the free radical scavenging ability was predicted using 2,2-diphenyl-1-picrylhydrazine (DPPH). After reacting with the nanoemulsion for 30 minutes, it could scavenge more than 50% of free radicals, and the free radical scavenging rate continued to increase with the increase of the nanoemulsion volume (see Example 3). Figure 7 Subsequently, H2O2 was added in vitro to simulate cellular oxidative stress. DCFH-DA was used as a ROS indicator probe, and flow cytometry was used to examine the ROS levels after treatment with free drug and drug-loaded nanoemulsions (see [link to relevant documentation]). Figure 8 Compared with free drugs, drug-loaded nanoemulsions can significantly reduce ROS levels and demonstrate good in vitro antioxidant capacity at the cellular level.

[0063] Example 5: Investigation of ocular surface retention after local administration of drug-loaded nanoemulsion

[0064] Nanoemulsion prescription:

[0065] Egg yolk lecithin 50mg

[0066] (2,3-Dioleoylpropyl)trimethylammonium chloride DOTAP 30mg

[0067] 10mg soybean oil

[0068] α-Tocopherol 5-10mg

[0069] Latanoprost 0.1mg

[0070] 1 mL of water.

[0071] A pressing issue with eye drops is rapid clearance; free drug solutions typically remain in the eye for only 1-5 minutes. To facilitate visualization of drug retention, nanoemulsions were labeled with the lipid-soluble fluorescent dye DIR. In vivo imaging results showed that nearly 80% of the free drug was cleared within 5 minutes of administration, while drug-loaded nanoemulsions maintained drug retention on the ocular surface for over 30 minutes (see...). Figure 9 This means that it can effectively prolong the duration of drug action.

[0072] Example 6: Fundus penetration after topical administration of drug-loaded nanoemulsion

[0073] Nanoemulsion prescription:

[0074] Egg yolk lecithin 130mg

[0075] Vitamin E succinate 150mg

[0076] Bemiprost 20mg

[0077] 1 mL of water.

[0078] Using rats as a model for fundus penetration, drug-loaded nanoemulsions (LA@VNE) were visualized using DIR. The nanoemulsions were administered via topical eye drops. Thirty minutes after administration, rats were sacrificed, and the treated eyeball was cut into 10 μm sections for DAPI staining. Compared to free drug, LA@VNE primarily accumulated in the retinal pigment epithelium (RPE) and photosensitive outer layer (POS), with a small portion penetrating the inner retinal layer (see [link to study]). Figure 10 Therefore, therapeutic drugs and antioxidants can be delivered to the fundus in a non-invasive manner.

[0079] Example 7: Therapeutic Effect of Antioxidant Drug-Loaded Nanoemulsions in a Chronic Glaucoma Model

[0080] Nanoemulsion prescription:

[0081] Soy lecithin 1.5mg

[0082] Phosphatidylethanolamine 0.75mg

[0083] Curcumin 1mg

[0084] Vitamin E niacinate 1.5mg

[0085] 0.25mg timarol maleate

[0086] 1 mL of water.

[0087] A rat glaucoma model was created using anterior chamber injection of microbeads. Glaucoma is a group of diseases characterized by elevated intraocular pressure, visual field defects, or decreased vision. Recent studies have shown a relationship between oxidative stress and optic nerve cell apoptosis in glaucoma patients. Data from some clinical studies also indicate significant differences in serum protein markers between glaucoma patients and normal individuals or patients with non-glaucoma-related visual impairment (such as cataracts). Serum superoxide dismutase (SOD) levels in glaucoma patients are significantly lower than in normal individuals, and malondialdehyde (MDA) levels are elevated. Latanoprost, a therapeutic drug in nanoemulsions, can reduce intraocular pressure by relaxing the ciliary muscle and increasing aqueous humor outflow through the uvea-sclera pathway (see [link to relevant documentation]). Figure 11 Antioxidants in nanoemulsions can reduce the production of lipid peroxides (MDA) and the inflammatory factor TNF-α in the retinal region, and maintain the activity of cellular superoxide dismutase (SOD) (see...). Figure 12 Nanoemulsions exert a protective effect on the optic nerve. Compared with free drug therapy, nanoemulsion treatment significantly reduced retinal cell apoptosis and inflammatory cell infiltration (see...). Figure 13-14 ).

[0088] Example 8: Therapeutic effect of antioxidant drug-loaded nanoemulsion in an acute glaucoma model

[0089] Nanoemulsion prescription:

[0090] 1mg of soybean lecithin

[0091] Dimethyl dioctadecyl ammonium bromide (DDAB) 0.5 mg

[0092] Castor oil 0.1mg

[0093] Vitamin E acetate 0.5mg

[0094] Timarol 0.05mg

[0095] Latanoprost 0.05mg

[0096] 1 mL of water.

[0097] In glaucoma cases, retinal ischemia-reperfusion injury (I / R injury) caused by high intraocular pressure is a common complication and one of the main causes of optic nerve cell death. Based on this, we used a saline anterior chamber perfusion method to create a mouse model of acute glaucoma. After 7 consecutive days of treatment with the nanoemulsion prepared according to this invention, the production of lipid peroxides (MDA) and the inflammatory factor TNF-α in the retina was significantly reduced; retinal tile plots showed that the survival rate of optic nerve cells treated with the nanoemulsion of this invention was significantly higher than that of free drug (see...). Figure 15 ).

[0098] Example 9: Direct intraocular injection of antioxidant drug-loaded nanoemulsion

[0099] Nanoemulsion prescription:

[0100] Egg yolk lecithin 20mg

[0101] Phosphatidylinositol 10mg

[0102] Medium chain triglycerides 10mg

[0103] Dexamethasone 9mg

[0104] Bimatoprost 1mg

[0105] 1 mL of water (containing 300 mg of L-ascorbic acid).

[0106] The prescribed amounts of egg yolk lecithin, phosphatidylinositol, and medium-chain triglycerides were heated and melted into a liquid state. Then, the prescribed amounts of dexamethasone and bimatoprost were added to prepare an oil phase. After thorough mixing, the mixture was slowly added to an aqueous phase containing 300 mg of L-ascorbic acid under high-speed stirring to form a colostrum. The colostrum was repeatedly homogenized and granulated using a high-pressure homogenizer to obtain drug-loaded nanoemulsions with uniform and stable particle size. For advanced glaucoma or during postoperative intervention, this invention encapsulates dexamethasone and other intraocular pressure-lowering drugs in an antioxidant nanoemulsion, achieving efficient drug delivery, prolonging drug release, and reducing side effects such as injection pain. Furthermore, the antioxidant α-tocopherol contained in the carrier can scavenge ROS, reduce the secretion of inflammatory factors, and synergistically exert an anti-inflammatory effect with dexamethasone.

[0107] Example 10: Preparation and application of ophthalmic drug-loaded liposomes with antioxidant function

[0108] Liposome prescription:

[0109] Dioleoylphosphatidylethanolamine 10mg

[0110] Phosphatidylinositol 4mg

[0111] cholesterol 2mg

[0112] Astaxanthin 1mg

[0113] bromonidin tartrate 3mg

[0114] 1 mL of water.

[0115] Antioxidant-functional drug-loaded liposomes were prepared using a thin-film dispersion method. In simple terms, the lipids in the above-mentioned formulation were completely dissolved in chloroform, and the solvent was removed by vacuum evaporation using a rotary evaporator to form a homogeneous and complete thin film. Ultrapure water was added to the obtained lipid film, and hydration was performed in a 45°C water bath. Next, the hydrated sample was subjected to ice-bath ultrasonication to obtain drug-loaded liposomes with uniform and stable particle size. In the liposomes prepared by this invention, astaxanthin can scavenge excess ROS; brimonidine tartrate, as an adrenergic receptor agonist, can lower intraocular pressure. The combined use of these two substances can reduce optic nerve damage while lowering intraocular pressure, thus maximizing the preservation of vision.

[0116] Example 11: Preparation and application of ophthalmic drug-loaded micelles with antioxidant function

[0117] micelle formulation:

[0118] Distearate phosphatidylethanolamine (DSPE-PEG) 50-200mg

[0119] Epicatechin-3-addol (EGCG) 2-10mg

[0120] Brinzodamine 10mg

[0121] 1 mL of water.

[0122] This invention prepares DSPE-PEG micelles via a thin-film hydration method. First, the prescribed amount of DSPE-PEG and brinzolamide are dissolved in 1 mL of chloroform solution and sonicated until completely dissolved. The organic phase is then removed by rotary evaporation to form a uniform drug-loaded lipid film. Pure water is then added and stirred, and hydrated at 40°C for 1 hour to obtain the drug-loaded micelles. EGCG can scavenge excess ROS, reduce ocular inflammation, and protect the optic nerve from damage. Brinzolamide loaded in the micelles is a carbonic anhydrase inhibitor with a strong affinity and inhibitory effect on carbonic anhydrase isoenzyme II (CAII) in the human ciliary body, selectively inhibiting CAII activity and effectively reducing intraocular pressure.

[0123] Example 12: Preparation of Ophthalmic Nanoemulsions with Antioxidant Functions for Nucleic Acid Delivery

[0124] Nanoemulsion prescription:

[0125] Egg yolk lecithin 8mg

[0126] (2,3-Dioleoyl-propyl)trimethylammonium chloride DOTAP 2mg

[0127] castor oil 2mg

[0128] α-Tocopherol 1mg

[0129] Nucleic acid encoding Bcl / xl: 0.05-0.3 mg

[0130] 1 mL of water.

[0131] This invention prepares nanoemulsions using a high-pressure homogenization method. The oil phase is completely dissolved in an appropriate amount of ethanol, and an aqueous phase is added under high-speed stirring to prepare a primary emulsion. The prepared primary emulsion is repeatedly granulated using a high-pressure homogenizer to obtain nanoemulsions with uniform particle size. Besides delivering small molecule compounds or biomacromolecules such as peptides, the nanoemulsions prepared by this invention can also be used to deliver glaucoma-related therapeutic genes. The anti-apoptotic protein Bcl / xl is a member of the Bcl-2 family of mitochondrial integrity mediators and can effectively reduce the death of retinal ganglion cells. This invention, by delivering nucleic acids encoding anti-apoptotic proteins to the fundus, can maximize the survival of retinal ganglion cells after transection while reducing the frequency of drug administration. The antioxidant components in the carrier can synergistically scavenge excess ROS, reduce ocular inflammation, and protect the optic nerve from damage.

Claims

1. The use of a nano-preparation with antioxidant function in the preparation of a drug for treating glaucoma, characterized in that, The nano-preparation is composed of an oil phase and an aqueous phase, wherein the oil phase:aqueous phase is 1:1000-1:2, w / w, the oil phase is lecithin 5-80%, w / w, medium-chain triglyceride 0-50%, w / w, fat-soluble antioxidant 0.1-50%, w / w, and glaucoma treatment drug 0.01%-20%, w / w; the aqueous phase is water-soluble antioxidant 0-30%, w / w or deionized water, wherein the fat-soluble antioxidant is selected from alpha-tocopherol, vitamin E acetate, vitamin E succinate, and vitamin E nicotinate, and the water-soluble antioxidant is selected from L-ascorbic acid; the nano-preparation is uniform and stable, and the particle size is less than 200 nm.

2. Use according to claim 1, characterized in that, The drug further comprises a pharmaceutically acceptable carrier, which is selected from nanoemulsion, liposome, micelle or lipid nanoparticle.

3. Use according to claim 1, characterized in that, The glaucoma treatment drug comprises one or more of prostaglandin analogues, b-adrenergic receptor antagonists, a-2 adrenergic agonists or carbonic anhydrase inhibitors, wherein the prostaglandin analogues are selected from one or more of latanoprost, bimatoprost, travoprost or unoprostone; the b-adrenergic receptor antagonists are selected from one or more of timolol, carteolol or betaxolol; the a-2 adrenergic agonists are selected from brimonidine tartrate; and the carbonic anhydrase inhibitors are selected from acetazolamide or brinzolamide.

4. Use according to claim 1, characterized in that, In addition to the fat-soluble antioxidant and the glaucoma treatment drug, other oily components in the oil phase are replaced by phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin, trimethyl-2, 3-dioleoyloxypropylammonium bromide, trimethyl-2, 3-dioleoyloxypropylammonium chloride, dimethyl dioctadecylammonium bromide, soybean oil, olive oil, castor oil, various medium and long chain fatty acid esters, cholesterol or squalane.

5. Use according to claim 1, characterized in that, The application is to deliver the drug while improving the local drug concentration, prolonging the drug retention time, removing excess reactive oxygen species through the antioxidant, reducing the inflammatory response, reducing the apoptosis of ocular cells, and slowing down or eliminating the influence of oxidative stress on the eye in a preventive, blocking and repairing manner.

6. Use according to claim 1, characterized in that, The preparation form of the drug is a liquid preparation.

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