Extracellular vesicles loaded with drug to be delivered to retina through transscleral pathway, and composition for preventing or treating optic neurodegenerative diseases, comprising same

Abstract

The present invention relates to extracellular vesicles loaded with a drug, and a use thereof. The extracellular vesicles according to one aspect allow the drug to reach the retina through a transscleral pathway so as to be efficient and have fewer side effects compared to other administration methods, and thus can be effectively used for preventing or treating optic neurodegenerative diseases.

Description

Title of Invention: EXTRACELLULAR VESICLES LOADED WITH DRUG TO BE DELIVERED TO RETINA THROUGH TRANSSCLERAL PATHWAY, AND COMPOSITION FOR PREVENTING OR TREATING OPTIC NEURODEGENERATIVE DISEASES, COMPRISING SAME Technical Field [1] The present disclosure relates to extracellular vesicles loaded with a drug and uses thereof. Background Art [2] Current treatment strategies for optic nerve degenerative diseases such as glaucoma focus on lowering intraocular pressure. Glaucoma is a disease in which the optic nerve is compressed or blood supply is impaired due to elevated intraocular pressure, thereby causing dysfunction of the optic nerve, and the progressive and irreversible degeneration of retinal ganglion cells (RGCs) and axons is a representative pathophysiological phenomenon of glaucoma. Lowering intraocular pressure to reduce the blockade of neurotransmitter transport at the optic nerve head, the point at which the axons of the optic nerve bend, can suppress optic nerve degeneration. However, it has been reported that in at least 20 % of patients, glaucoma progresses even when intraocular pressure is well controlled. If the energy that has become deficient in the process of the already-reduced neurotransmission is not replenished, optic nerve degeneration progresses. That is, to improve the protection of the optic nerve, nonintraocular pressure-related factors must also be addressed. [3] The neuroprotective effects of various drugs against glaucomatous retinal ganglion cell damage have been reported in many in vivo studies and randomized clinical trials. For example, it has been confirmed in ex vivo and in vivo models that an excellent neuroprotective effect is exhibited in case that nicotinamide (NAM), which is a precursor of nicotinamide adenine dinucleotide (NAD) serving as a basic component of ATP, is supplied to neuronal cells (Gustavsson ST et al., 2023, Investigative Ophthalmology & Visual Science 64:34-34; Tribble JR et al., 2021, Redox Biol 43:101988; Williams PA et al., 2017, Journal of Glaucoma 26:1161). However, to deliver NAM to human optic nerve cells via oral administration through the systemic absorption system, an amount of about 2 to 3 g or more per day is required for a person weighing 60 kg. This means that 6 tablets must be taken per day to provide effective neuroprotection against glaucoma progression. As a method of overcoming such an inefficient drug delivery system and delivering a high dose of nicotinamide to the retina, direct intraocular delivery, for example, intravitreal injection, subconjunctival injection, sub-Tenon injection, topical (eye drop) administration, and the like, which correspond to eye drop treatment or intraocular injection procedures, may also be selected. However, intravitreal injection causes severe pain and has a high risk of intraocular infection, and has the drawback that, considering the short half-life of nicotinamide, daily injections are required to maintain retinal nicotinamide levels. Eye drop administration may be an attractive alternative in terms of convenience and safety. However, because the efficacy of eye drops is affected by patient compliance, corneal penetration kinetics, aqueous humor dilution, the vitreous, and the like, there are cooperation issues as well as side effects upon instillation. [4] On the other hand, in case that compliance issues are overcome, delivery via the subconjunctival route is safer than intravitreal injection and more efficient than eye drops. This also enables the delivery of depot formulations capable of delivering a substance over a long period (from several weeks to several months). In case that a drug is placed 2 in the subconjunctival space through a small-gauge needle, the sclera, which is the next structure encountered by the carrier, can serve to control delivery. The extracellular matrix (ECM) structure and biomechanical properties of the sclera are known to vary depending on tissue water content associated with permeability to exogenous compounds. [5] In view of this, the inventors of the present disclosure have discovered a drug delivery system (DDS) using a subconjunctival route for the treatment of optic nerve degenerative diseases. In particular, to overcome the disruptive effects of subconjunctival fibrosis, which is an obstacle to multiple injection approaches, and the like, extracellular vesicles (EVs), which are biodegradable and biocompatible materials and bio-derived carriers, were proposed as a solution. The present disclosure has been completed by confirming that exogenous processed EVs loaded with a proven drug such as nicotinamide deliver a high dose of the therapeutic drug to the inside of the retina and retinal ganglion cells via a trans-scleral route in vivo. Disclosure of Invention Technical Problem [6] An aspect is to provide a pharmaceutical composition for preventing or treating an optic nerve degenerative disease, the pharmaceutical composition including extracellular vesicles loaded with a drug, wherein the drug includes one or more selected from the group consisting of an optic nerve protective drug, an intraocular pressure-lowering agent, and a neurotrophic factor. [7] Another aspect is to provide a method of preventing, ameliorating or treating an optic nerve degenerative disease, the method including administering an effective amount of extracellular vesicles loaded with a drug to a subject in need thereof. [8] Another aspect is to provide a method of delivering a drug to the retina, the method including administering an effective amount of extracellular vesicles loaded with a drug to a periocular region of a subject in need thereof to deliver the drug to the retina trans-sclerally. [9] Another aspect is to provide the use of extracellular vesicles loaded with a drug for preventing, ameliorating or treating an optic nerve degenerative disease.

[10] Another aspect is to provide the use of extracellular vesicles loaded with a drug for the preparation of a formulation for preventing, ameliorating or treating an optic nerve degenerative disease. Solution to Problem

[11] An aspect provides a pharmaceutical composition for preventing or treating an optic nerve degenerative disease, the pharmaceutical composition including extracellular vesicles loaded with a drug, wherein the drug includes one or more selected from the group consisting of an optic nerve protective drug, an intraocular pressure-lowering agent, and a neurotrophic factor.

[12] As used herein, "extracellular vesicles (EVs)" refers to nano-sized membrane structures, and means all types of vesicles that are surrounded by a membrane generated in an endosomal compartment of most eukaryotic cells, and generated extracellularly. Extracellular vesicles deliver various active molecules from producer cells to recipient cells. Extracellular vesicles are particles that can reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on the cell from which they are secreted, and play an important role in intercellular interactions while transferring genetic material and proteins between cells. Various molecules, including proteins, microRNA (miRNA), and lipids, may be involved in regulating the behavior of recipient cells through extracellular vesicles. That is, extracellular vesicles may be used as a drug delivery platform that contributes to intercellular communication as an important carrier of biologically active molecules in numerous physiological and pathological processes. Compared to synthetic materials, extracellular vesicles elicit a weaker immune response due to their biological origin, and have the advantage of being relatively safe because they are non-mutagenic, non-replicative, and free of tumorigenic changes, as confirmed through numerous in vivo studies and clinical trials.

[13] "Extracellular vesicles," "extracellular vesicular bodies," "vesicles or sacs released to the outside of cells," and the like are all used interchangeably, and extracellular vesicles may include a plurality of different species such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles.

[14] In an embodiment, the extracellular vesicles may be derived from mesenchymal stem cells (MSCs). Specifically, the extracellular vesicles may be derived from adipose-derived mesenchymal stem cells (AD-MSCs).

[15] The drug according to an aspect may be delivered trans-sclerally to the retina.

[16] For the extracellular vesicles loaded with a drug of the present disclosure, after the drug is administered periocularly, the sclera plays a role in controlling the delivery of the drug. For example, in case that the drug is placed in a subconjunctival space or the like through a small-gauge needle, the next structure that the carrier encounters is the sclera, and the drug can reach retinal optic nerve cells or the like, which are recipient cells, through a trans-scleral route.

[17] As used herein, "sclera" refers to a highly elastic and structurally complex connective tissue that makes up at least three-quarters of the outer tunic of the eyeball, and that performs various functions important for vision. The sclera mainly provides a stable and rigid structure for the retina, thereby preventing off-axis light emission from the 5 outside. The scleral stroma consists of collagen and elastin fibers, and the posterior scleral fibers are less densely packed than the anterior sclera, which is advantageous for trans-scleral delivery to the posterior retina. The arrangement of these fibers and the presence of negatively charged proteoglycans in the scleral stroma affect drug diffusion. Due to a large and accessible surface area and a high level of hydration resulting from hypocellularity, the sclera may be beneficial to water-soluble substances, be less likely to have drugs degraded by proteolytic enzymes, and deliver substances for a long period of several weeks to several months.

[18] Therefore, the trans-scleral route may be used as an alternative that can overcome potential obstacles of other routes. For example, an oral administration method may be hindered by blood retinal and blood aqueous barriers and generally requires a high concentration of a drug, and thus is inefficient and has a potential risk of systemic side effects. Intravitreal injection is efficient but invasive, and thus pain associated with the procedure is severe, and because the drug residence time is short, periodic injections are required, and thus there are disadvantages in terms of patient compliance. In topical administration, delivery to the retina is difficult for various reasons, such as lacrimal glands, relatively low corneal permeability, reverse intraocular convection, and, most importantly, diffusion distance.

[19] In an embodiment, the composition of the present disclosure is for preventing or treating an optic nerve degenerative disease. That is, "drug" as used herein refers to a drug that may be used for preventing, ameliorating or treating an optic nerve degenerative disease, and should be construed to include, without limitation, any type of drug as long as it corresponds to use for preventing, ameliorating or treating an optic nerve degenerative disease. For example, the drug may be an optic nerve protective drug, an 6 intraocular pressure-lowering agent, and / or a neurotrophic factor.

[20] In an embodiment, the optic nerve protective drug may include one or more selected from the group consisting of a precursor of nicotinamide adenine dinucleotide (NAD), memantine, citicholine, a Ginko biloba extract (GBE), calcium channel blockers (CCBs), coenzyme Q10, and a statin.

[21] The precursor of NAD may include one selected from the group consisting of nicotinamide, nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid mononucleotide (NaMN), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid, and combinations thereof.

[22] NAD metabolism is closely associated with ATP production, NAD / NADPH homeostasis, and maintenance of DNA stability. Because mitochondrial dysfunction and ATP depletion contribute to glaucoma pathophysiology, supplements of NAM, which is a precursor of NAD, may have a positive effect on neuroprotection in patients with optic nerve degenerative diseases, particularly glaucoma patients. NAD itself is an allosteric inhibitor of SARM1, and thus may support neuronal survival by preventing Wallerian degeneration.

[23] In an embodiment, the optic nerve protective drug may be a neurotrophic factor, and the neurotrophic factor may be a drug involved in a tyrosine kinase signaling pathway. Specifically, the neurotrophic factor may include one or more selected from the group consisting of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin growth factor (IGF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurturin.

[24] In an embodiment, the intraocular pressure-lowering agent may include one or more selected from the group consisting of Rho-kinase inhibitors, carbonic anhydrase 7 inhibitors, brimonidine, and prostaglandin analogues.

[25] In an embodiment, the intraocular pressure-lowering agent may include one or more selected from the group consisting of betaxolol, carteolol, timolol, and levobunolol.

[26] In an embodiment, the intraocular pressure-lowering agent may be a prostaglandin analogue, and the prostaglandin analogue may include one or more selected from the group consisting of latanoprost, bimatoprost, tafluprost, travoprost, and unoprostone.

[27] The term "prevention" as used herein refers to any act of inhibiting or delaying a disease via administration of the composition of the present disclosure to a subject. For preventive benefit, the composition may be administered to a subject at risk of developing a specific disease, disorder, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even though the disease, disorder, or symptom has not yet appeared.

[28] The term "treat" as used herein refers to any act of ameliorating or benefiting the symptoms of a disease via administration of the composition of the present disclosure to a subject. "Treat," "alleviate," and "ameliorate" as used herein may be used interchangeably. Therapeutic benefit refers to any therapeutically significant amelioration in, or effect on, one or more diseases, disorders, or symptoms under treatment.

[29] In an embodiment, the optic nerve degenerative disease may be one selected from the group consisting of hereditary optic neuropathy, traumatic optic neuropathy, ischemic optic neuropathy, compressive optic neuropathy, glaucoma, papilledema, toxic optic neuropathy, optic neuritis, neuromyelitis optica, multiple sclerosis, optic nerve transection, optic nerve atrophy, Leber hereditary optic neuropathy (LHON), and amblyopia.

[30] In an embodiment, the composition may reduce dendritic atrophy.

[31] In an embodiment, the composition may inhibit death or promote proliferation of retinal ganglion cells (RGCs).

[32] The expression that the composition "includes" extracellular vesicles loaded with a drug means that the extracellular vesicles loaded with a drug of the present specification are added to a degree capable of exhibiting the effects mentioned above, and also encompasses being formulated into various forms by adding various components as auxiliary components for drug delivery, stabilization, and the like.

[33] The extracellular vesicles loaded with a drug may include a compound or a salt thereof in an amount of, with respect to the total weight of the composition, 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%.

[34] The composition of the present disclosure may be formulated according to conventional methods and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, topical preparations, suppositories, sterile injectable solutions, and implantable preparations. The pharmaceutical composition may further include, in addition to the active ingredient, a pharmaceutically acceptable excipient usable for formulation.

[35] The excipient may include one or more selected from the group consisting of: a carrier, a vehicle, a diluent, a solvent, for example, a monohydric alcohol, for example, ethanol, isopropanol, and a polyhydric alcohol, for example, glycerol, and an edible oil, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, an oily ester, 9 for example, ethyl oleate, isopropyl myristate; and a binder, an adjuvant, a solubilizer, a thickener, a stabilizer, a disintegrant, a glidant, a lubricant, a buffer, an emulsifier, a wetting agent, a suspending agent, a sweetening agent, a coloring agent, a flavoring agent, a coating agent, a preservative, an antioxidant, a processing agent, a drug delivery modifier and enhancer, for example, calcium phosphate, magnesium stearate, talc, a monosaccharide, a disaccharide, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-p-cyclodextrin, polyvinylpyrrolidone, a low-melting-point wax, an ion exchange resin, and the like, but the present disclosure is not limited thereto.

[36] The carrier is commonly used in formulation, and may include, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present disclosure may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[37] The composition of the present disclosure may be in any form suitable for the intended method of administration. In the composition of the present disclosure, "administration" means introducing a predetermined substance into a patient by any suitable method, and the composition of the present disclosure may be in the form of a parenteral formulation, that is, a formulation for periocular administration.

[38] The administration may include administering the composition according to an embodiment in an amount of 0.00001 mg to 1,000 mg, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg, per subject per day.

[39] However, the dosage may be variously prescribed depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, diet, time of administration, route of administration, excretion rate, and sensitivity to response, and those of ordinary skill in the art may appropriately adjust the dosage in consideration of these factors. The frequency of administration may be once a day or two or more times within a range of clinically acceptable side effects, and the administration may be performed at one site or two or more sites, and the total number of administration days may be from 1 day to 30 days per treatment, administered daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period of time. Even for animals other than humans, the dose may be the same as that for humans per kg, or, for example, an amount obtained by converting the above dose based on a volume ratio (for example, an average value) of an organ (such as the heart) between a target animal and a human, or the like, may be administered.

[40] The composition may be an injectable formulation. The periocular administration refers to administration by an administration method such as subconjunctival injection, intraconjunctival injection, sub-Tenon injection, or retrobulbar injection.

[41] To formulate the pharmaceutical composition into the parenteral formulation, the active ingredient may be mixed with a stabilizer or a buffer in water to prepare a solution 11 or suspension, and the solution or suspension may be prepared into a unit dosage form of an ampoule or a vial. In addition, the composition may be sterilized, or may further include auxiliary agents such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts for regulating osmotic pressure, and / or buffers, and may further include other therapeutically useful substances.

[42] The content of the extracellular vesicles in the composition of the present disclosure may be appropriately adjusted according to the intended use of the composition, the form of the formulation, or the like, and may be, for example, 0.001 to 99 wt%, 0.001 to 90 wt%, 0.001 to 50 wt%, 0.01 to 50 wt%, 0.1 to 50 wt%, or 1 to 50 wt% with respect to the total weight of the composition.

[43] The composition of the present disclosure may be a pharmaceutical composition and may be administered in a pharmaceutically effective amount. "Pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including a patient's disease type, severity of the disease, type of active ingredient to be administered, type of formulation, patient's age, gender, body weight, health condition, diet, sensitivity, administration time and administration method of a drug, combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[44] The composition of the present disclosure may prevent or treat a disease in a subject by a method including administering the composition to the subject in an amount effective to prevent or treat the disease.

[45] The dosage of the pharmaceutical composition for preventing or treating a disease according to the present disclosure may range from 0.01 pg / kg to 10 g / kg, specifically 12 0.01 mg / kg to 1 g / kg, per day, depending on the patient's condition, body weight, gender, age, severity of the patient's condition, and route of administration. The administration may be performed once a day or divided into several doses. This dosage should not be construed as limiting the scope of the present disclosure in any respect.

[46] The subject may be a mammal. The mammal may be a human, a dog, a cat, a cow, a goat, or a pig.

[47]

[48] Another aspect provides a method of preventing, ameliorating or treating an optic nerve degenerative disease, the method including administering an effective amount of extracellular vesicles loaded with a drug to a subject in need thereof.

[49] Another aspect provides a method of delivering a drug to the retina, the method including administering an effective amount of extracellular vesicles loaded with a drug to a periocular region of a subject in need thereof to deliver the drug to the retina trans-sclerally.

[50] Another aspect provides the use of extracellular vesicles loaded with a drug for preventing, ameliorating or treating an optic nerve degenerative disease.

[51] Another aspect provides the use of extracellular vesicles loaded with a drug for the preparation of a formulation for preventing, ameliorating or treating an optic nerve degenerative disease.

[52]

[53] As the present disclosure allows for various changes and numerous examples, particular examples will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present disclosure to particular modes of practice, and it should be construed as including all changes, equivalents, and substitutes within the spirit and scope of the present disclosure. In describing the present disclosure, detailed descriptions of related art are omitted when it is deemed that they may obscure the gist of the present disclosure. Advantageous Effects of Invention

[54] Vesicles loaded with a drug, according to an aspect, are drug carriers that can be administered periocularly and delivered to the retina trans-sclerally, and delivering the drug to the retina via a trans-scleral route allows for treatment even at a lower drug concentration compared to other administration methods, thereby offering high efficiency and a reduced potential risk of systemic side effects. Periocular administration of the vesicles of the present disclosure and delivery thereof through a trans-scleral route can effectively reduce dendritic atrophy in an explant model of optic nerve degeneration. Brief Description of Drawings

[55] FIG. 1 is a schematic view illustrating a method of preparing drug-loaded extracellular vesicles (EVs) according to an embodiment.

[56] FIG. 2 illustrates graphs showing the size distribution of EVs prepared according to an embodiment.

[57] FIG. 3 is a graph showing the pH of EVs prepared according to an embodiment.

[58] FIG. 4 illustrates transmission electron microscope (TEM) images showing the structure of EVs prepared according to an embodiment.

[59] FIG. 5 is an image showing the results of Western blotting for markers of EVs prepared according to an embodiment.

[60] FIG. 6 is a graph showing the drug loading efficiency of EVs prepared according to an embodiment.

[61] FIG. 7 is a schematic view illustrating a method of inducing optic nerve degeneration in a mouse model according to an embodiment.

[62] FIG. 8 is a graph showing cumulative intraocular pressure (IOP) for RNA-binding protein with multiple splicing (RBPMS) and DiOlistics groups.

[63] FIG. 9 illustrates images and graphs showing the levels of retinal ganglion cell (RGC) loss in low ocular hypertension (OHT) and high OHT groups.

[64] FIG. 10 illustrates an image and a graph showing dendritic degeneration in low OHT and high OHT groups.

[65] FIG. 11 is a schematic view illustrating a method of explanting and culturing a mouse retina.

[66] FIG. 12 illustrates images showing the results of comparatively observing dendritic degeneration of drug-loaded EVs according to an embodiment.

[67] FIG. 13 is a graph showing the results of comparative analysis of dendritic degeneration of drug-loaded EVs according to an embodiment.

[68] FIG. 14 is a schematic view illustrating the purification of DiO-labeled drug-loaded EVs according to an embodiment.

[69] FIG. 15 is a graph showing the results of measuring the DiO intensity of each fraction by size exclusion chromatography.

[70] FIG. 16 is a graph showing the results of comparatively measuring the DiO intensities of concentrated DiO-labeled drug-loaded EVs according to an embodiment and a PBS solution.

[71] FIG. 17 is a graph showing the size distribution of DiO-labeled drug-loaded EVs according to an embodiment.

[72] FIG. 18 illustrates images showing the scleral penetration of DiO-labeled drug-loaded EVs according to an embodiment.

[73] FIG. 19 illustrates retinal tissue images taken 2 hours after injection of DiO-labeled drug-loaded EVs according to an embodiment.

[74] FIG. 20 is a graph showing the release profile of nicotinamide released from drug-loaded EVs according to an embodiment. Mode for the Invention

[75] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, these examples are intended to illustratively describe one or more embodiments, and the scope of the present disclosure is not limited to these examples.

[76]

[77] Example 1. Preparation of extracellular vesicles (EVs)

[78] Extracellular vesicles loaded with a drug used in the composition according to an example were prepared as follows.

[79]

[80]   1.1 Isolation of extracellular vesicles

[81] Human adipose-derived mesenchymal stem cells (ADMSCs; Lonza, Basel, Switzerland) were cultured using CellCor™ CD MSC medium (CDM; Xcell Therapeutics, Seoul, Korea) supplemented with a 1 % antibiotic-antifungal solution. The ADMSCs were seeded at a density of 5x105 cells / plate in 150 pi culture plates and cultured at 37 °C in a humidified atmosphere containing 5 % CO2. To isolate EVs, conditioning media were collected every 24 hours for a total of 120 hours. The collected CDM was centrifuged at 1,300 rpm for 3 minutes, followed by filtration through a 0.22 mm vacuum filter / storage bottle system to remove non-exosomal large particles such as cells, cell debris, microvesicles, and apoptotic bodies. To isolate EVs, tangential flow filtration (TFF; Repligen, Waltham, MA, USA) was performed using a 500 kDa molecular weight cut-off (MWCO) filter, and the diafiltration volume was adjusted to 7. The isolated EVs were concentrated using an Amicon Ultra-15 centrifugal filter unit (Merck, MA, USA).

[82]

[83]   1.2 Drug loading into extracellular vesicles

[84] The EVs isolated in Example 1.1 were mixed with nicotinamide (NAM; 500 mM) and sequentially extruded 11 times through 400 nm, 200 nm, and 100 nm polycarbonate membrane filters using a mini-extruder (Avanti Polar Lipids, AL, USA). The extruded solution was purified three times using an Amicon Ultra-15 centrifugal filter unit, and a schematic view is shown in FIG. 1.

[85]

[86] Example 2. Verification of extracellular vesicles

[87] To verify the EVs isolated according to the EV engineering of Example 1, the following experiments were conducted.

[88] The amount and size of the isolated EVs were measured using MONO ZetaView® (Particle Metrix, Meerbusch, Germany) in 488 nm scattering mode. The EV samples were diluted with filtered PBS solution (HyClone laboratories, UT, USA) to 107-108 particles / ml for use, and the detailed parameters for accurate analysis of all samples were adjusted to a sensitivity of 75, a shutter of 100, a minimum trace length of 15, and a cell temperature of 25 °C. The results thereof are shown in FIG. 2.

[89] The pH of the EVs was measured with a pH meter using a microelectrode (InLab Ultra-Micro-ISM, Mettler Toledo, OH, USA) on a small amount of sample, and the results thereof are shown in FIG. 3.

[90] The morphology of the EVs was examined using a transmission electron 17 microscope (TEM; Hitachi, H-7600, 80 kV, Japan). After drying the EV solution on a 150-mesh carbon-coated Formvar / copper grid (FCF150-CU, Electron Microscopy Sciences, USA), the EVs were stained with a 7 % uranyl acetate or gadolinium acetate solution and dried on the copper grid for negative staining. After drying, the Formvar / copper grid was placed on a grid box for TEM examination, and the imaging results are shown in FIG. 4.

[91] Western blot analysis was performed to analyze the content of the EVs. An equal number of EVs (1*109 particles) was used, and after 10 % SDS-PAGE, the EVs were placed on a nitrocellulose (NC) membrane, and then the NC membrane was blocked using a TBST solution diluted in 5 % skim milk. The NC membrane onto which the EV proteins had been transferred was incubated with CD63 (Abcam, MA, USA), TSG101, and Apo-A1 primary antibodies (Santa Cruz Biotechnology, CA, USA), followed by the addition of an HRP-linked secondary antibody (Cell Signaling Technology, MA, USA) for incubation. The blots were pretreated with an enhanced chemiluminescent solution (GE Healthcare, WI, USA) and visualized with a ChemiDoc™ XRS+ and ImageLab software (Bio-Rad, CA, USA), and the results thereof are shown in FIG. 5.

[92] Additionally, to confirm whether the drug (nicotinamide) was well loaded into the EVs according to an embodiment, the total amount of NAM contained in the Waste that passed through an Amicon Ultra-15 centrifugal filter unit and in the NAM-EV was quantified by HPLC. The loading efficiency was determined according to the formula NAM EV / (NAM EV+Waste) x 100, and the results thereof are shown in FIG. 6.

[93]

[94] As shown in FIG. 2, the average sizes of ASC EV and NAM EV were observed to be similar between the two groups (ASC EV, 161 nm; NAM-EV, 155 nm).

[95] As shown in FIG. 3, the pH values of ASC EV and NAM EV were neutral, at about 7, which corresponds to a pH suitable for ocular application.

[96] As shown in FIG. 4, a typical cup-shaped morphology was observed in ASC EV and NAM EV.

[97] As shown in FIG. 5, the surface marker (CD63), internal marker (TSG101), and negative marker (ApoA1) of the EVs were confirmed by Western blot, and it was demonstrated that ASC EV and NAM-EV are EVs.

[98] As shown in FIG. 6, the NAM loading efficiency was approximately 48 %, and 5.38 pg of NAM was loaded per particle.

[99]

[100] Through the above results, the presence of EVs was confirmed, which indicates that using an extruder in the drug loading process according to Example 1 helps reform the particles into a structure similar to that of EVs.

[101]

[102] Example 3. Preparation of mouse model and retinal explant tissue

[103] 3.1 Preparation of mouse model

[104] All experimental procedures were performed in accordance with the UK Animals Scientific Procedures Act 1986 and the guidelines of the Association for Research for Vision and Ophthalmology regarding the use of animals. The individual research protocols complied with the ethical guidelines of Cardiff University, UK, and CHA University, Republic of Korea. The animals were raised on a 12-hour light / 12-hour dark cycle, and food and water were provided without limit. Adult male C57BL / 6J (B6) mice weighing 20-25 g were bred, unilateral ocular hypertension was induced at 8-12 weeks of age, and the mice were used in an ex vivo retinal explant model for retinal flat mounts.

[105]

[106] 3.2 Preparation of mouse model with induced optic nerve degeneration

[107] To induce ocular hypertension, polyurethane microbeads with a diameter of 17 pm (SUNPU-170, Sunjin Chemical, Gyeonggi-do, Republic of Korea) were injected into the anterior chamber, and after sterilization with ultraviolet light for 30 minutes, the beads were diluted in phosphate-buffered saline (PBS) to a concentration of 250 mg / mL. Before injecting the microbeads into the mice, the mice were anesthetized by intraperitoneal injection of ketamine (37.5 mg / kg) and medetomidine hydrochloride (1.25 mg / kg), and the prepared microbeads were then injected only into the left eye of each mouse. Specifically, a 1 mL syringe filled with at least 0.3 mL of the microbead solution was fitted with a 30-gauge needle and inserted subconjunctivally 2 mm from the limbus, with the tip advanced to the limbus within the subconjunctival space and injected into the anterior chamber through the iridocorneal angle. While maintaining a constant flow into the anterior chamber to minimize leakage of the beads, 5 pL of the microbead solution was injected.

[108] FIG. 7 is a schematic view illustrating a method of inducing an increase in intraocular pressure (IOP) by intraocular injection of polyurethane microbeads into the mouse eye.

[109]

[110] 3.3 Preparation of ex vivo retinal explant model for retinal flat mounts

[111] For retinal explant culture from the adult mice of Examples 3.1 and 3.2, up to 16 retinal explant tissues were generated from 8 mice. Retinal flat mounts were placed, with the ganglion cell side facing upward, on cell culture inserts (Millicell®, pore size 0.4 pm) positioned in a 6-well plate. The cells were cultured at the air / medium interface and were not immersed in the culture medium. Thereafter, 4 retinas were assigned to each group: (i) control, (ii) EV, (iii) NAM, and (iv) NAM-EV. For the groups other than the control, EV, 20 NAM, and NAM-EV were dissolved in the culture medium at a concentration of 50 mM. The retinal explant tissue cultures were maintained in a humidified incubator containing 5 % CO2 at 37 °C. Half of the retinal explant tissue culture medium was replaced at 1 day ex vivo (DEV), and the retinas were removed from the culture medium after 3 DEV.

[112]

[113] Example 4. Correlation between elevated intraocular pressure and retinal ganglion cells (RGCs)

[114] The following experiments were performed to evaluate the relationship between the level of intraocular pressure elevation and changes in two different RGC structures (number of RGC somas and dendritic arborization), in order to determine whether the elevation of IOP is a determining factor for changes in RGCs.

[115]

[116] 4.1 Confirmation of elevated intraocular pressure

[117] First, to calculate a baseline, the intraocular pressure of both eyes of the mice was measured before microbead injection. Following microbead injection into the mice according to Example 3, the intraocular pressure was measured on day 1, day 3, and thereafter at weekly intervals for up to 4 weeks, using a rebound tonometer (iCare TONOLAB tonometer; Tonovet, Vantaa, Finland) specifically calibrated for use on mouse eyes. All measurements were performed in conscious animals under gentle restraint following topical anesthesia using 0.5 % proparacaine hydrochloride ophthalmic solution (Alcon Laboratories, Fort Worth, Texas). Mice were placed in an open-apex cloth cone to expose the animal's head for easy positioning. Intraocular pressure was measured first in the right eye and then immediately in the left eye (treated eye), within 5 seconds for each eye while maintaining the same posture. Six single measurements were recorded, 21 the first and last measurements were discarded, and then the intermediate four measurements were averaged to obtain an average intraocular pressure estimate. The same process was repeated three times, and then the final IOP value was defined as the average of the three measurements. The contralateral eye (right eye) was used as an untreated control. In addition, to minimize the effect of circadian rhythms on IOP, IOP recordings were always performed between 9:00 AM and 10:00 AM.

[118] One week after microbead injection, using an IOP of 17 mmHg as a discriminative cutoff (proven to be a value below the threshold for causing RGC depletion), mice were divided into a low IOP increase group (6 mice) and a high IOP increase group (7 mice) according to the level of IOP increase, and it was identified which ocular hypertension (OHT) mouse model could detect RGC changes earlier. Retinal flat mounts were obtained on day 28, and the cumulative IOP of each group is shown in FIG. 8.

[119] As shown in FIG. 8, there was a statistically significant increase in IOP in the RBPMS_High and DiOlistics_High groups compared to the RBPMS_Low and DiOlistics_Low groups, and it was confirmed that the IOP continuously and gradually increased after a single injection of microbeads.

[120]

[121] 4.2 Observation of changes in number of RGC somas and dendritic arborization according to elevated intraocular pressure

[122] RGC soma loss was quantified after immunohistochemical labeling of wholemount retinas with RNA-binding protein with multiple splicing (RBPMS). Globes were incised and fixed in 4 % PFA for 2 days, and then retinas were dissected in a small dish containing HBSS. Permeabilization was performed with 0.1 % Triton-X 100 in PBS at room temperature for 30 minutes, and then antigen retrieval was performed in 300 pL of sodium citrate buffer and a water bath at 90 °C for 30 minutes. Thereafter, protein blocking was performed in 2.5 % NHS (normal horse serum) for 1 hour. Then, the retinas were placed in a solution containing a primary antibody of RBPMS Novus (1:1000), incubated overnight at 4 °C, and then washed twice with PBS. The washed retinas were placed in a solution containing an Alexa fluor Plus-conjugated secondary antibody (1:1000) and left for 3 hours at room temperature in the dark. Thereafter, the retinas were washed twice more with PBS and then stained with Hoechst (1:1000) in PBS for 30 minutes. After the final wash, the stained retinas were mounted with FluorSave (Merck) and coverslipped. Images of individual RBPMS+ cells were captured using an Olympus IX71 microscope (Olympus), and four images per retina (20X magnification, 0.25 pm / pixel) were captured at equidistant positions at 0, 3, 6, and 9 o'clock, centered on a superior-inferior line passing through the optic nerve head (~1000 pm eccentricity). The images were cropped to 400 pm2, and RBPMS+ cells were counted using the Cell Counter plugin for Fiji. Only round nuclei were counted to exclude vascular endothelium, and the counts were averaged across the four images.

[123] Additionally, dendritic pruning was evaluated in retinal flat mounts on day 28 after induction of ocular hypertension, through DiOlistics labeling of individual cells using a custom-made gene gun. The bullets were prepared by dissolving the dye in 400 pl of methylene chloride, followed by application onto tungsten and natural drying, wherein 2 mg of DiI, 4 mg of DiO, and 80 mg of tungsten (diameter of 1.7 pm) were mixed in a ratio in 30.5 cm of Tezfel tubing (Biorad). The coated tungsten was collected, transferred to a tube, and then vortexed to disperse it along the length of the tube. For labeling, the retinas were mounted flat on a glass slide, and all liquid was removed. A culture insert (Falcon®, pore size 3.0 pm) was inverted onto the retina to serve as a filter for large particle clumps, and the contents of a single bullet were discharged at a gene gun pressure of 100-120 psi. The tissue was transferred to a culture dish containing Neurobasal-A medium and maintained at 37 °C and 5 % CO2 for 30 minutes. Then, the retinas were fixed in 4 % PFA for 1 hour, washed with PBS, and mounted with FluorSave reagent (Merck). After drying, the coverslips were sealed. Images of individual RGC dendritic arbors were acquired using a Zeiss LSM880 confocal microscope (20X magnification, 0.45 pm / pixel, 1 pm z-thickness), and whole dendritic arbors were reconstructed using Imaris (version 9.3.1, Bitplane). RGC dendrites were automatically tracked using the Imaris filaments tool, and Sholl analysis was performed using Imaris software to provide a quantitative measure of dendritic integrity (dendritic intersections per bin distance from the soma center, 10 pm steps).

[124] The level of RGC loss was determined by assigning 3 retinal explants from the low OHT group and 3 retinal explants from the high OHT group to RNA-binding protein with multiple splicing (RBPMS), and dendritic degeneration was determined by subjecting 3 retinal explants from the low OHT group and 4 retinal explants from the high OHT group to DiOlistics labeling, and the results thereof are shown in FIGS. 9 and 10.

[125] As shown in FIG. 9, the high OHT group showed a significantly lower number of RBPMS+ cells compared to the control (p=0.050), whereas the low OHT group showed no significant difference compared to the control (p=0.500).

[126] As shown in FIG. 10, in contrast, Sholl analysis showed a statistically significant difference in both the low OHT group and the high OHT group compared to the control.

[127]

[128] As described above, the observation that dendritic arborization decreased when 24 intraocular pressure increased relatively gradually at 17 mmHg or lower indicates that evaluating dendritic pruning has higher sensitivity than evaluating RGC soma loss for detecting RGC damage caused by increased intraocular pressure.

[129]

[130] Example 5. Evaluation of therapeutic effect of drug-loaded EVs

[131] According to the mouse glaucoma model of Example 3, media containing additives (EV, NAM, and NAM-EV) were compared with a control (additive-free medium). All of the additives were allowed to pass through a cell culture insert (Millicell®, pore size 0.4 pm) to reach the whole-mount retinas of mice, and a schematic view thereof is shown in FIG. 11.

[132] The control was not administered any additive, and the EV group was administered EVs, the NAM group was administered NAM, and the NAM-EV group was administered NAM-loaded EVs during 3 days of culture. Images and results of the evaluation of dendritic pruning in each group are shown in FIGS. 12 and 13.

[133] As shown in FIGS. 12 and 13, significant preservation of dendritic branches was observed in the NAM and NAM-EV groups compared to the NT group (control). The area under the Sholl curve was significantly increased in the NAM and NAM-EV groups compared to the control and the EV group, and there was no significant difference in Sholl AUC between the control and the EV group.

[134]

[135] Example 6. Confirmation of scleral penetration of drug-loaded EVs via subconjunctival injection

[136] To explore the possibility of utilizing drug-loaded EVs via a trans-scleral route in eyes larger than those of rodents, and for further research toward full-scale EV production, the following experiments were performed.

[137]

[138] 6.1 Preparation of rabbit model

[139] All experimental procedures were performed in accordance with the UK Animals Scientific Procedures Act 1986 and the guidelines of the Association for Research for Vision and Ophthalmology regarding the use of animals. The individual research protocols complied with the ethical guidelines of Cardiff University, UK, and CHA University, Republic of Korea. To evaluate scleral penetration of DiO-labeled EVs via subconjunctival injection, male New Zealand White (NZW) rabbits (n=3) weighing 1.9-2.2 kg were used.

[140]

[141] 6.2 Preparation of cryosections of rabbit eyes

[142] A solution containing a concentrated DiO-labeled EV preparation was subconjunctivally injected into the eyes (n=6) of NZW rabbits. To evaluate the depth of penetration of the EVs into the eye over time, the animals were sacrificed immediately, 1 hour, or 2 hours after injection. For cryosectioning, the eyes were enucleated, washed with PBS, and immediately embedded in OCT compound (Sakura) to prepare blocks. The prepared blocks were stored at -80 °C until use. Cryosections of 14 pm (from the anterior side to the dorsal side) were cut using a cryotome (CM3050S, Leica), followed by DAPI staining. Images were digitized using an Axio Scan.Z1 slide scanner (Carl Zeiss).

[143]

[144] 6.3 Confirmation of EV distribution

[145] To confirm the distribution of EVs after subconjunctival injection, the EVs were labeled with DiO, a green fluorescent lipophilic carbocyanine dye. The EVs isolated in 26 Example 1 were incubated with Fast DiO green fluorescent membrane dye (D3898, Invitrogen, CA, USA) at a final concentration of 2 pg / mL at room temperature for 1 hour, and then, to completely remove unlabeled free DiO, the incubated solution was purified using a size exclusion chromatography column packed with Sepharose CL-2B (Sigma-Aldrich, MO, USA). The reacted solution was loaded onto the column, and eleven 1 ml fractions were collected. The DiO intensity of all fractions was measured using a microplate reader (Molecular Devices, CA, USA) (excitation at 484 nm, emission at 540 nm, and cut-off at 530 nm), and the collected fractions 4 to 8 were concentrated using an Amicon Ultra-15 centrifugal filter unit. The results thereof are shown in FIGS. 15 and 16. FIG. 14 is a schematic view illustrating the purification of DiO-labeled EVs.

[146] As shown in FIG. 15, fraction 10 exhibited a considerably higher intensity than the other fractions, indicating the presence of free DiO in this fraction. In addition, fraction 9 was discarded together with fraction 10 because fraction 9 was also likely to contain some free DiO. Fractions 4 to 8 were considered to contain pure DiO-labeled EVs and were subsequently subjected to concentration, and the concentrated DiO-labeled EVs (DiO-EV) appeared colorless and transparent to the naked eye. FIG. 16 illustrates the results of measuring the DiO intensity thereof.

[147] As shown in FIG. 16, when the DiO intensity of the solution containing DiO-EV was compared with that of a PBS solution, the former exhibited a much higher intensity, indicating the presence of DiO-EV in the solution.

[148]

[149] In addition, the size distribution of the EVs was evaluated using MONO ZetaView®, and the results thereof are shown in FIG. 17, and the results of imaging at each time point are shown in FIGS. 18 and 19. FIG. 18 illustrates images of scleral penetration of EVs observed at each time point after injection. FIG. 19 illustrates high-magnification images of the retina taken 2 hours after injection.

[150]

[151] As shown in FIG. 17, the measured values had a mean of 149 nm, which was observed to be consistent with the typical size range of EVs.

[152] As shown in FIGS. 18 and 19, EVs were not detected in the retina immediately after injection, but were observed to be detected in the retina 1 hour after injection.

[153]

[154] Example 7. Drug detection profile of drug-loaded EVs

[155] The nicotinamide release profile of NAM-EV was measured. A dialysis system was used to estimate the release kinetics of NAM from NAM-loaded EVs. The system was configured using a membrane with a cutoff size of 3.5 kDa such that the EVs were prevented from escaping from the inside of the membrane while NAM was released into the external environment.

[156] Specifically, a balanced salt solution (BSS; Alcon, TX, USA) was used as the release medium for the examination. Then, 1 milliliter of NAM-loaded EVs (NAM-EV) was packed into a 3.5 kDa dialysis membrane bag, and the sealed dialysis bag was placed in 50 mL of release medium (100 rpm). At regular intervals, 10 mL of the release medium was collected and replaced with fresh medium. The concentration of nicotinamide was quantified at 40 °C using high-performance liquid chromatography (HPLC; Vanquish VC-P20-A, Thermo Fisher Scientific, OH, USA) and an Agilent Eclipse column (5 micron, 4.6 x 150 mm C-18). The mobile phase consisted of water and acetonitrile (50:50) and was flowed at a flow rate of 1.0 mL^min-1, which was observed at a wavelength of 210 nm, and the results thereof are shown in FIG. 20.

[157] As shown in FIG. 20, the amount of drug released at each time point was converted into a percentage, and the data were observed to follow a first-order release kinetics model with a regression coefficient (R2) of 0.9713. The release profile of nicotinamide released from NAM-EV showed an initial burst for up to 24 hours, followed by a sustained release pattern up to the 96-hour time point.

[158]

[159] Taken together, the drug-loaded EVs according to an aspect have the ability to pass through a physical barrier such as the sclera, and can reach the outer retina to treat optic nerve degenerative diseases, such as by inhibiting dendritic pruning. That is, because the drug can be delivered to the inner retina via a trans-scleral route, the drug-loaded EVs have great potential as a new DDS platform.

[160]

[161] The foregoing description of the present disclosure is provided for illustrative purposes only, and it will be understood by those of ordinary skill in the art to which the present disclosure pertains that the present disclosure may be easily modified in other particular forms without changing the technical spirit or essential characteristics of the present disclosure. Thus, the above-described examples should be construed as being provided for illustrative purposes only and not for purposes of limitation.

[162]

[163] Abbreviations

[164] ASC: Adipose-Derived Stem Cell

[165] ATP: Adenosine Triphosphate

[166] DDS: Drug Delivery System

[167] EFNA3: Ephrin-A3

[168] eNAMPT: Extracellular Nicotinamide Phosphoribosyltransferase

[169] EV: Extracellular Vesicle

[170] IOP: Intraocular Pressure

[171] miR: MicroRNA

[172] NAD: Nicotinamide Adenine Dinucleotide

[173] NAD+: Nicotinamide Adenine Dinucleotide (oxidised form)

[174] NADH: Nicotinamide Adenine Dinucleotide (reduced form)

[175] NADP+: Nicotinamide Adenine Dinucleotide Phosphate (oxidised form)

[176] NADPH: Nicotinamide Adenine Dinucleotide Phosphate (reduced form)

[177] NAM: Nicotinamide

[178] NMN: Nicotinamide Mononucleotide

[179] RGC: Retinal Ganglion Cell

[180] TEM: Transmission Electron Microscopy

[181] TSG101: Tumor Susceptibility Gene 101

[182] ApoA1: Apolipoprotein A1

Claims

1. A pharmaceutical composition for preventing or treating an optic nerve degenerative disease, the pharmaceutical composition comprising extracellular vesicles loaded with a drug, wherein the drug comprises one or more selected from the group consisting of an optic nerve protective drug, an intraocular pressure-lowering agent, and a neurotrophic factor.

2. The pharmaceutical composition of claim 1, wherein the optic nerve protective drug comprises one or more selected from the group consisting of a precursor of nicotinamide adenine dinucleotide (NAD), memantine, citicholine, Ginko biloba extract (GBE), calcium channel blockers (CCBs), coenzyme Q10, a statin, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin growth factor (IGF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurturin.

3. The pharmaceutical composition of claim 1, wherein the intraocular pressure-lowering agent comprises one or more selected from the group consisting of Rho-kinase inhibitors, carbonic anhydrase inhibitors, brimonidine, betaxolol, carteolol, timolol, levobunolol, latanoprost, bimatoprost, tafluprost, travoprost, and unoprostone.

4. The pharmaceutical composition of claim 2, wherein the precursor of NAD comprises one selected from the group consisting of nicotinamide, nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid mononucleotide (NaMN), nicotinic acid adenine dinucleotide (NaAD),nicotinic acid, and combinations thereof.

5. The pharmaceutical composition of claim 1, wherein the optic nervedegenerative disease is one selected from the group consisting ofhereditary optic neuropathy, traumatic optic neuropathy, ischemic optic neuropathy, compressive optic neuropathy, glaucoma, papilledema, toxic optic neuropathy, optic neuritis, neuromyelitis optica, multiple sclerosis, optic nerve transection, optic nerve atrophy, Leber hereditary opticneuropathy (LHON), and amblyopia.

6. The pharmaceutical composition of claim 1, wherein the compositionreduces dendritic atrophy.

7. The pharmaceutical composition of claim 1, wherein the compositioninhibits death or promotes proliferation of retinal ganglion cells (RGCs).

8. The pharmaceutical composition of claim 1, wherein the extracellularvesicles are derived from adipose-derived mesenchymal stem cells.

9. The pharmaceutical composition of claim 1, wherein the composition is forperiocular administration, and the drug is delivered trans-sclerally to theretina.

10. The pharmaceutical composition of claim 9, wherein the periocularadministration is subconjunctival injection, intraconjunctival injection, sub-Tenon injection, or retrobulbar injection.

11. The pharmaceutical composition of claim 1, wherein the composition is aninjectable formulation.

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