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

Extracellular vesicles containing drugs that are delivered to the retina through the transscleral route and a composition for preventing or treating optic nerve degenerative diseases comprising the same

[0001] The present invention relates to drug-loaded extracellular vesicles and their uses.

[0002] Current treatment strategies for optic nerve degenerative diseases such as glaucoma focus on reducing intraocular pressure (IOP). Glaucoma is a disease in which elevated intraocular pressure compresses the optic nerve or impairs its blood supply, leading to dysfunction of the optic nerve. Progressive and irreversible degeneration of retinal ganglion cells (RGCs) and their axons is a hallmark pathophysiological feature of glaucoma. Lowering IOP to reduce the blockage of neurotransmitter transport at the optic nerve head, where optic nerve axons bend, can slow optic nerve degeneration. However, it has been reported that more than 20% of patients progress to glaucoma even with well-controlled IOP. Optic nerve degeneration progresses if the energy deficit in already reduced neurotransmission is not replenished. Therefore, improving optic nerve protection requires addressing non-IOP-related factors as well.

[0003] The neuroprotective effects of various drugs on 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 nicotinamide (NAM), a precursor of nicotinamide adenine dinucleotide (NAD), a basic component of ATP, has an excellent neuroprotective effect when supplied to neurons (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, in the case of nicotinamide, when administered orally to supply optic nerve cells in humans, an amount of approximately 2-3 g or more per day is required for a 60 kg person to be delivered through the systemic absorption system. This means that six tablets must be taken daily to provide effective neuroprotection against glaucoma progression. Direct intraocular delivery, such as intravitreal injection, subconjunctival injection, subtenon injection, or topical (eyedrop) injection, can be used to overcome these inefficient drug delivery systems and deliver high doses of nicotinamide to the retina. However, intravitreal injections are painful and carry a high risk of intraocular infection. In addition, given the short half-life of nicotinamide, daily injections are necessary to maintain retinal nicotinamide levels. While eyedrop administration may be an attractive alternative in terms of convenience and safety, compliance is problematic because the efficacy of eyedrops is affected by factors such as patient compliance, corneal penetration kinetics, aqueous dilution, and the vitreous, and side effects are also a concern.

[0004] On the other hand, subconjunctival delivery is safer than intravitreal injection and more efficient than eye drops if compliance issues can be overcome. It also allows for the delivery of depot formulations capable of delivering substances over long periods (weeks to months). When a drug is placed into the subconjunctival space via a small-gauge needle, the sclera, the next structure encountered by the carrier, can play a role in controlling delivery. The structure and biomechanical properties of the sclera's extracellular matrix (ECM) are known to vary depending on tissue water content, which is related to its permeability to exogenous compounds.

[0005] Focusing on this point, the present researchers developed a drug delivery system (DDS) utilizing the subconjunctival route for the treatment of optic nerve degenerative diseases. Specifically, to overcome the confounding effects of subconjunctival fibrosis, a barrier to multiple injections, they proposed extracellular vesicles (EVs), bio-derived carriers made of biodegradable and biocompatible materials, as a solution. They demonstrated that exogenously engineered EVs loaded with proven drugs, such as nicotinamide, delivered high doses of therapeutic drugs to the retina and retinal ganglion cells via the transscleral route in vivo, thereby completing the present invention.

[0006] One aspect is to provide a pharmaceutical composition for the prevention or treatment of optic nerve degenerative disease, which comprises a drug-loaded extracellular vesicle, wherein the drug comprises at least one selected from the group consisting of an optic nerve protective drug, an intraocular pressure lowering agent, and a neurotrophic factor.

[0007] Another aspect provides a method for preventing, ameliorating or treating an optic nerve degenerative disease comprising administering to a subject in need thereof an extracellular vesicle containing an effective amount of a drug.

[0008] Another aspect provides a method of delivering a drug to the retina, comprising administering an effective amount of drug-loaded extracellular vesicles into the periphery of a subject in need thereof, thereby delivering the drug to the retina through the sclera.

[0009] Another aspect provides the use of drug-loaded extracellular vesicles for the prevention, improvement or treatment of optic nerve degenerative diseases.

[0010] Another aspect provides the use of drug-loaded extracellular vesicles for the manufacture of a formulation for preventing, ameliorating or treating optic nerve degenerative diseases.

[0011] One aspect provides a pharmaceutical composition for the prevention or treatment of optic nerve degenerative disease, comprising a drug-loaded extracellular vesicle, wherein the drug comprises at least one selected from the group consisting of an optic nerve protective drug, an intraocular pressure lowering agent, and a neurotrophic factor.

[0012] As used herein, "extracellular vesicles (EVs)" are nano-sized membrane-bound structures produced in the endosomal compartments of most eukaryotic cells, encompassing all types of vesicles produced outside the cell. Extracellular vesicles transport various active molecules from producer cells to recipient cells. EVs can reflect the state of the secreting source cell (donor cell), exhibit diverse biological activities depending on the cell from which they are secreted, and play a crucial role in cell-to-cell interactions by transporting genetic material and proteins between cells. Various molecules, including proteins, microRNAs (miRNAs), and lipids, can be involved in regulating the behavior of recipient cells via EVs. Thus, EVs can serve as important carriers of biologically active molecules in numerous physiological and pathological processes and can serve as drug delivery platforms that contribute to intercellular communication. Compared to synthetic substances, they have the advantage of being relatively safe because they are of biological origin and are less likely to cause immune system reactions, and are non-mutagenic, non-replicative, and non-tumorous, as confirmed by numerous in vivo studies and clinical trials.

[0013] The terms "extracellular vesicle," "extracellular vesicle," and "vesicle or vesicle released outside of a cell" are all used interchangeably, and extracellular vesicles can include many different types, including exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles.

[0014] In one specific example, the extracellular vesicles may be derived from mesenchymal stem cells (MSCs). Specifically, they may be derived from adipose-derived mesenchymal stem cells (AD-MSCs).

[0015] Depending on the nature of the drug, it may be delivered to the retina through the transscleral membrane.

[0016] The drug-loaded extracellular vesicles of the present invention act as a sclera-controlled drug delivery system after the drug is administered to the periorbital area. For example, if the drug is placed into the subconjunctival space or other space via a small-gauge needle, the next structure encountered by the carrier is the sclera, allowing the drug to reach recipient cells, such as retinal optic nerve cells, via the sclera-transit pathway.

[0017] As used herein, the term "sclera" refers to a highly elastic and structurally complex connective tissue that occupies more than three-quarters of the outer tunic of the eyeball and performs several functions crucial to vision. The sclera primarily provides a stable and rigid structure to the retina, preventing off-axis light emission from the outside. The scleral stroma is composed of collagen and elastin fibers, with the posterior scleral fibers being less densely packed than the anterior sclera, facilitating transscleral transmission to the posterior retina. The arrangement of these fibers and the presence of negatively charged proteoglycans in the scleral stroma influence drug diffusion. The sclera's large accessible surface area and high water content due to its hypocellularity make it conducive to water-soluble substances, making it less susceptible to proteolytic enzyme degradation and enabling drug delivery over extended periods of weeks to months.

[0018] Therefore, the transscleral route can be used as an alternative to overcome the potential obstacles of other routes. For example, oral administration is susceptible to interference with the blood-retinal and blood-aqueous barriers, typically requiring high drug concentrations, making it ineffective and potentially risking systemic side effects. Intravitreal injections, while effective, are invasive, resulting in significant procedure-related pain, and the short drug retention time necessitates regular injections, which can compromise patient compliance. Topical administration presents challenges in delivering the drug to the retina for several reasons, including the lacrimal gland, relatively low corneal permeability, adverse intraocular convection, and, most importantly, the diffusion distance.

[0019] In one specific embodiment, the composition of the present invention is for the prevention or treatment of optic nerve degenerative diseases. That is, the term "drug" as used herein refers to a drug that can be used for the prevention, improvement, or treatment of optic nerve degenerative diseases. It should be interpreted to include any type of drug without limitation, as long as it is used for the prevention, improvement, or treatment of optic nerve degenerative diseases. For example, the drug may be an optic nerve protective drug, an intraocular pressure lowering agent, and / or a neurotrophic factor.

[0020] In one specific example, the optic nerve protective drug may include at least one selected from the group consisting of a precursor of nicotinamide adenine dinucleotide (NAD), memantine, citicoline, Ginkgo biloba extract (GBE), calcium channel blockers (CCB), coenzyme Q10, and a statin.

[0021] The precursor of the above NAD may include any 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.

[0022] NAD metabolism is closely linked to ATP production, NAD / NADPH homeostasis, and DNA stability. Because mitochondrial dysfunction and ATP depletion contribute to the pathophysiology of glaucoma, supplementation with NAM, a precursor of NAD, may have a positive neuroprotective effect on patients with optic nerve degenerative diseases, particularly glaucoma. Because NAD itself is an allosteric inhibitor of SARM1, it may support neuronal survival by preventing Wallerian degeneration.

[0023] In one specific example, the optic nerve protective drug may be a neurotrophic factor, and the neurotrophic factor may be a drug involved in tyrosine kinase signaling. Specifically, the neurotrophic factor may include at least one 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.

[0024] In one specific example, the intraocular pressure lowering agent may include at least one selected from the group consisting of Rho-kinase inhibitors, carbonic anhydrase inhibitors, brimonidine, and prostaglandin analogues.

[0025] In one specific example, the intraocular pressure lowering agent may include at least one selected from the group consisting of betaxolol, carteolol, timolol, and levobunolol.

[0026] In one specific example, the intraocular pressure lowering agent may be a prostaglandin analogue, and the prostaglandin analogue may include at least one selected from the group consisting of latanoprost, bimatoprost, tafluprost, travoprost, and unoprostone.

[0027] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0028] As used herein, the term "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0029] In one specific example, the optic nerve degenerative disease may be any 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 atrophy, Leber hereditary optic neuropathy (LHON), and amblyopia.

[0030] In one specific example, the composition may reduce dendritic atrophy.

[0031] In one specific example, the composition may inhibit death or promote proliferation of retinal ganglion cells (RGCs).

[0032] The above composition “comprising” drug-loaded extracellular vesicles means that the drug-loaded extracellular vesicles of the present specification are added to an extent that can exhibit the above-mentioned effects, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

[0033] The drug-loaded extracellular vesicles are present in an amount of 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 %, 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 % of the compound or salt thereof.

[0034] The composition of the present invention may be formulated and used in oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, formulated according to conventional methods, or in parenteral formulations such as suspensions, emulsions, lyophilized preparations, topical preparations, suppositories, sterile injection solutions, and implantable preparations. The pharmaceutical composition may further comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient that can be used in formulation.

[0035] The above excipients include carriers, vehicles, diluents, solvents, for example, monohydric alcohols, for example, ethanol, isopropanol, and polyhydric alcohols, for example, glycerol, and edible oils, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters, for example, ethyl oleate, isopropyl myristate; It may include at least one selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0036] The carriers mentioned above are those commonly used in formulations, and include, but are 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 ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0037] The composition of the present invention may be in any form suitable for the intended method of administration. "Administration" in the context of the composition of the present invention means introducing a given substance into a patient by any suitable method, and the composition of the present invention may be in the form of a parenteral dosage form, i.e., a dosage form for periocular administration.

[0038] The above administration is 0.00001 mg to 1,000 mg of the composition according to one specific example per subject per day, 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, Alternatively, it may be administered in doses of 10 mg to 25 mg.

[0039] However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

[0040] The above composition may be in the form of an injection. The above periocular administration means administration by a method such as subconjunctival injection, intraconjunctival injection, sub-Tenon injection, or retrobulbar injection.

[0041] To formulate the above-described parenteral dosage form, the pharmaceutical composition may be prepared as a solution or suspension by mixing the active ingredient with a stabilizer or buffer in water, and such solution or suspension may be prepared as a unit dosage form in an ampoule or vial. In addition, the composition may be sterilized, or may further include auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control, and / or buffers, and may further include other therapeutically useful substances.

[0042] The content of extracellular vesicles in the composition of the present invention can be appropriately adjusted depending on the purpose of use of the composition, the form of the formulation, etc., 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% based on the total weight of the composition.

[0043] The composition of the present invention may be a pharmaceutical composition and may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means 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 the type of the patient's disease, the severity of the disease, the type of active ingredient administered, the type of formulation, the patient's age, sex, weight, health condition, diet, sensitivity, the time and method of drug administration, the combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[0044] The composition of the present invention can prevent or treat a disease in a subject, including a step of administering to the subject an amount effective to prevent or treat the disease.

[0045] The dosage of the pharmaceutical composition for the prevention or treatment of diseases according to the present invention may range from 0.01 ug / kg to 10 g / kg per day, specifically from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0046] The above entity may be a mammal. The mammal may be a human, a dog, a cat, a cow, a goat, or a pig.

[0047]

[0048] Another aspect provides a method of preventing, ameliorating or treating an optic nerve degenerative disease comprising administering to a subject in need thereof an effective amount of an extracellular vesicle loaded with a drug.

[0049] Another aspect provides a method of delivering a drug to the retina, comprising administering an effective amount of drug-loaded extracellular vesicles into the periphery of a subject in need thereof, thereby delivering the drug to the retina through the sclera.

[0050] Another aspect provides the use of drug-loaded extracellular vesicles for the prevention, amelioration or treatment of optic nerve degenerative diseases.

[0051] Another aspect provides the use of drug-loaded extracellular vesicles for the manufacture of a formulation for preventing, ameliorating or treating optic nerve degenerative diseases.

[0052]

[0053] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0054] The drug-loaded vesicles according to the present invention are drug carriers that can be administered periocularly and delivered to the retina through the scleral membrane. The transscleral route, which delivers the drug to the retina, is more efficient than other administration methods, allowing treatment with lower drug concentrations, and thus reduces the potential risk of systemic side effects. When the vesicles of the present invention are administered periocularly and delivered through the scleral route, they can effectively reduce dendritic atrophy in an ex vivo transplantation model of optic nerve degeneration.

[0055] Figure 1 is a schematic diagram of a method for manufacturing a drug-loaded EV according to one specific example.

[0056] Figure 2 is a graph confirming the size distribution of EVs manufactured according to one specific example.

[0057] Figure 3 is a graph confirming the pH of an EV manufactured according to one specific example.

[0058] Figure 4 is a TEM image confirming the structure of an EV manufactured according to one specific example.

[0059] Figure 5 is an image showing the results of confirming the marker of EV produced according to one specific example using Western blot.

[0060] Figure 6 is a graph showing the drug loading efficiency of an EV manufactured according to one specific example.

[0061] Figure 7 is a schematic diagram of a method for inducing optic nerve degeneration according to one specific example in a mouse model.

[0062] Figure 8 is a cumulative IOP graph by RBPMS and DiOlistics groups.

[0063] Figure 9 is an image and graph observing the level of RGC loss in the low OHT group and the high OHT group.

[0064] Figure 10 is an image and graph showing dendrite degeneration observed in the low OHT group and the high OHT group.

[0065] Figure 11 is a schematic diagram of a mouse retina transplantation and culture method.

[0066] Figure 12 is an image comparing and observing the dendrite degeneration of drug-loaded EVs according to one specific example.

[0067] Figure 13 is a graph comparing and analyzing the dendrite degeneration of drug-loaded EVs according to one specific example.

[0068] Figure 14 is a schematic diagram showing the purification of a DiO-labeled drug-loaded EV according to one specific example.

[0069] Figure 15 is a graph measuring the DiO intensity of size exclusion chromatography fractions by fraction.

[0070] Figure 16 is a graph comparing the DiO intensity of a concentrated DiO-labeled drug-loaded EV and a PBS solution according to one specific example.

[0071] Figure 17 is a graph showing the size distribution of EVs loaded with DiO-labeled drugs according to one specific example.

[0072] Figure 18 is an image showing the observation of the corneal penetration of a DiO-labeled drug-loaded EV according to one specific example.

[0073] Figure 19 is an image of retinal tissue taken 2 hours after injection of EV loaded with DiO labeled drug according to one specific example.

[0074] Figure 20 is a graph showing the release profile of nicotinamide released from a drug-loaded EV according to one specific example.

[0075] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0076]

[0077] Example 1. Preparation of extracellular vesicles (EVs)

[0078] Drug-loaded extracellular vesicles used in a composition according to one embodiment were prepared as follows.

[0079]

[0080] 1.1 Isolation of extracellular vesicles

[0081] Human adipose-derived mesenchymal stem cells (ADMSC; Lonza, Basel, Switzerland) were cultured in CellCor with 1% antibiotic-antimycotic solution. TM ADMSCs were cultured using CD MSC medium (CDM; Xcell Therapeutics, Seoul, Korea). ADMSCs were seeded at 5 × 10 in 150 pi plates. 5 Cells / plates were seeded and cultured in a humidified environment at 37°C containing 5% CO2. Conditioned media was collected every 24 h for a total of 120 h to isolate EVs. The collected CDM was centrifuged at 1,300 rpm for 3 min, and then a 0.22 mm vacuum filter / storage bottle system was used to remove large particles other than exosomes, such as cells, cell debris, microvesicles, and apoptotic bodies. To isolate EVs, a 500 kDa molecular weight cutoff filter was used for tangential flow filtration (TFF; Repligen, Waltham, MA, USA), and the diafiltration rate was adjusted to 7. An Amicon Ultra-15 centrifugal filter device (Merck, MA, USA) was used to concentrate the isolated EVs.

[0082]

[0083] 1.2 Drug loading into extracellular vesicles

[0084] The EV isolated in Example 1.1 was 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 device, and a schematic diagram is shown in Fig. 1.

[0085]

[0086] Example 2. Verification of extracellular vesicles

[0087] To verify the EV separated according to the EV engineering of Example 1, the following experiments were performed.

[0088] The amount and size of isolated EVs were measured using MONO ZetaView® Particle Metrix (Meerbusch, Germany) in 488 nm scattering mode. EV samples were diluted with filtered PBS solution (HyClone laboratories, UT, USA) for 10 7 -10 8 It was diluted to particles / ml and used, and the detailed parameters for accurate analysis for all samples were adjusted to sensitivity 75, shutter 100, minimum trace length 15, and cell temperature 25°C. The results are shown in Fig. 2.

[0089] The pH of EV was measured with a pH meter using a microelectrode (InLab Ultra-Micro-ISM, Mettler Toledo, OH, USA) for a small amount of sample, and the results are shown in Fig. 3.

[0090] The morphology of EVs was examined using a transmission electron microscope (TEM; Hitachi, H-7600, 80 kV, Japan). After drying the EV solution on 150-mesh carbon-coated Formvar / copper grids (FCF150-CU, Electron Microscopy Sciences, USA), the EVs were stained with a 7% uranyl acetate or gadolinium acetate solution and dried on the copper grids for negative staining. After drying, the Formvar / copper grids were placed in a grid box for TEM examination, and the results of the images are shown in Figure 4.

[0091] Western blot analysis was performed to analyze the content of EVs. The same number of EVs (1 × 10 9After EVs were subjected to 10% SDS-PAGE and placed on a nitrocellulose (NC) membrane, the NC membrane was blocked using a TBST solution diluted in 5% skim milk. The NC membrane onto which EV proteins were transferred was incubated with CD63 (Abcam, MA, USA), TSG101, and Apo-A1 primary antibodies (Santa Cruz Biotechnology, CA, USA), followed by the addition of HRP-linked secondary antibodies (Cell Signaling Technology, MA, USA). The blots were pretreated with an enhanced chemiluminescence solution (GE Healthcare, WI, USA) and analyzed by ChemiDoc TM Visualization was performed using XRS+ and ImageLab software (Bio-Rad, CA, USA) and is shown in Fig. 5.

[0092] Additionally, to confirm whether the drug (nicotinamide) was properly loaded into the EV according to one specific example, the total amount of NAM contained in the Waste and NAM-EV that passed through the Amicon Ultra-15 centrifugal Filter Unit was quantified by HPLC. The loading efficiency was determined according to the formula NAM EV / (NAM EV+Waste) x 100, and the results are shown in Fig. 6.

[0093]

[0094] As shown in Fig. 2, the average sizes of ASC EV and NAM EV were observed to be similar in both groups (ASC EV 161 nm; NAM-EV 155 nm).

[0095] As shown in Fig. 3, the pH values ​​of ASC EV and NAM EV are neutral at approximately 7, which corresponds to a pH suitable for ocular application.

[0096] As shown in Fig. 4, a typical cup-shaped morphology was observed in ASC EVs and NAM EVs.

[0097] As shown in Fig. 5, the surface marker (CD63), internal marker (TSG101), and negative marker (ApoA1) of EVs were confirmed by Western blot, and it was proven that ASC EVs and NAM-EVs were EVs.

[0098] As shown in Fig. 6, the NAM loading efficiency was approximately 48%, and 5.38 pg of NAM was loaded per particle.

[0099]

[0100] The above results confirmed the existence of EV, and imply that the use of an extruder in the drug loading process according to Example 1 helps to reform particles into a structure similar to EV.

[0101]

[0102] Example 3. Preparation of mouse model and retinal explant tissue

[0103] 3.1 Preparation of the mouse model

[0104] All experimental procedures were performed in accordance with the UK Animals Scientific Procedures Act 1986 and the Association for Research in Vision and Ophthalmology guidelines on the use of animals. Individual study protocols adhered to the ethical guidelines of Cardiff University, UK, and CHA University, South Korea. Animals were housed on a 12-h light / 12-h dark cycle, and food and water were provided ad libitum. Adult male C57BL / 6J (B6) mice weighing 20-25 g were bred to induce unilateral ocular hypertension at 8-12 weeks of age and used for ex vivo retinal explantation models for retinal flat mounts.

[0105]

[0106] 3.2 Preparation of a mouse model with induced optic nerve degeneration

[0107] To induce ocular hypertension, polyurethane microbeads (SUNPU-170, Sunjin Chemical, Gyeonggi-do, South Korea) with a diameter of 17 μm were injected into the anterior chamber, sterilized with ultraviolet light for 30 minutes, and 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). The prepared microbeads were then injected only into the left eye of the mice. Specifically, a 30-gauge needle was attached to a 1 mL syringe filled with at least 0.3 mL of the microbead solution and inserted 2 mm subconjunctivally from the limbus. The tip was advanced to the limbus within the subconjunctival space and injected into the anterior chamber through the iridocornea. To minimize bead leakage, 5 μL of microbead solution was injected while maintaining a constant flow in the forward direction.

[0108] Figure 7 is a schematic diagram showing a method for inducing IOP elevation by intraocularly injecting polyurethane microbeads into a mouse eye.

[0109]

[0110] 3.3 Preparation of an ex vivo retinal explant model for retinal flat mount

[0111] For retinal explant culture from adult mice of Examples 3.1 and 3.2, up to 16 retinal explants were generated from 8 mice. Retinal flat mounts were placed on cell culture inserts (Millicell®, pore size 0.4 μm) placed in 6-well plates, with the ganglion cells facing upward. Cells were cultured at the air / medium interface and were not immersed in the culture medium. Four retinas were then assigned to each group: (i) control; (ii) EV; (iii) NAM; and (iv) NAM-EV. Except for the control group, EV, NAM, and NAM-EV were dissolved in the culture medium at a concentration of 50 mM. Retinal explant 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.

[0112]

[0113] Example 4. Correlation between increased intraocular pressure and retinal ganglion cells (RGCs)

[0114] To determine whether increased intraocular pressure is a determinant of RGC changes, the following experiments were performed to evaluate the relationship between the level of increased intraocular pressure and changes in two different RGC structures (RGC cell body number and dendritic branching).

[0115]

[0116] 4.1 Confirmation of increased intraocular pressure

[0117] First, to calculate the baseline, the intraocular pressure (IOP) of both eyes of mice was measured before microbead injection. IOP was measured using a rebound tonometer (iCare TONOLAB tonometer; Tonovet, Vantaa, Finland), specifically calibrated for use in mouse eyes, on days 1 and 3, and then at weekly intervals for up to 4 weeks after microbead injection, as described in Example 3. All measurements were performed in awake animals under gentle restraint after topical anesthesia with 0.5% proparacaine hydrochloride eye drops (Alcon Laboratories, Fort Worth, Texas). Mice were placed in an open-topped cloth cone to expose the head for easy positioning. IOP was measured first in the right eye, followed by the left eye (the treatment eye), and measurements were taken within 5 seconds for each eye while maintaining the same posture. Six single measurements were recorded, the first and last measurements were discarded, and the middle four measurements were averaged to obtain an estimate of the mean IOP. This process was repeated three times, and the final IOP value was defined as the average of the three measurements. The contralateral eye (right eye) served as an untreated control. Furthermore, to minimize the influence of circadian rhythms on IOP, IOP measurements were always taken between 9:00 and 10:00 AM.

[0118] One week after microbead injection, the mice were divided into a hypotony group (6 mice) and an ocular hypertension group (7 mice) based on the level of IOP increase, with an IOP of 17 mmHg as the cutoff (a value determined to be below the threshold for inducing RGC depletion). This was done to identify which OHT (ocular hypertension) mouse model could detect RGC changes earlier. Retinal flat mounts were obtained on the 28th day, and the cumulative IOP of each group is shown in Fig. 8.

[0119] As shown in Fig. 8, there was a statistically significant increase in intraocular pressure in the RBPMS_High and DiOlistics_High groups compared to the RBPMS_Low and DiOlistics_Low groups, and it was confirmed that intraocular pressure increased continuously and gradually after a single injection of microbeads.

[0120]

[0121] 4.2 Observation of changes in the number of RGC cell bodies and dendritic branches according to increased intraocular pressure

[0122] RGC cell body loss was quantified after immunohistochemical labeling of whole-mount retinas with RNA-binding protein with multiple splicing (RBPMS). Globes were dissected and fixed in 4% PFA for 2 days, and retinas were dissected in small dishes containing HBSS. Permeabilization was performed with 0.1% Triton-X 100 in PBS for 30 minutes at room temperature, followed by antigen retrieval in 300 μL of sodium citrate buffer in a 90°C water bath for 30 minutes. Protein blocking was then performed in 2.5% NHS (normal horse serum) for 1 hour. Retinas were then placed in a solution containing primary antibodies (1:1000) from RBPMS Novus, incubated overnight at 4°C, and washed twice with PBS. Washed retinas were placed in a solution containing Alexa fluor Plus-tagged secondary antibodies (1:1000) and left in the dark at room temperature for 3 h. The retinas were then washed twice more with phosphate-buffered saline (PBS) and stained with Hoechst (1:1000) in PBS for 30 min. After a final wash, the stained retinas were mounted with FluorSave (Merck) and coverslipped. Individual RBPMS+ cells were imaged using an Olympus IX71 microscope (Olympus). Four images (20X magnification, 0.25 μm / pixel) per retina were captured equidistantly at 0, 3, 6, and 9 o'clock, centered on a line passing through the optic nerve head (~1000 μm eccentricity). Images were taken at a 400 μm magnification. 2 , and RBPMS+ cells were counted using the Cell Counter plugin for Fiji. Only round nuclei were counted, excluding vascular endothelium, and the number was averaged across four images.

[0123] Additionally, dendritic pruning was assessed in retinal flat mounts 28 days after induction of ocular hypertension via DiOlistics labeling of individual cells using a custom-made gene gun. Bullets were prepared by dissolving the dye in 400 μl of methylene chloride, coating the tungsten with the dye, and air-drying. 2 mg DiI, 4 mg DiO, and 80 mg tungsten (diameter 1.7 μm) were mixed in a 30.5 cm Tezfel tubing (Biorad). The coated tungsten was collected, transferred to the tube, and vortexed to disperse it along the length of the tube. For labeling, retinas were flat-mounted on glass slides, and all liquid was removed. A culture insert (Falcon®, pore size 3.0 μm) was inverted over the retina to act as a filter for large particle clumps, and the contents of a single bullet were ejected at 100–120 psi of gene gun pressure. The tissue was transferred to a culture dish containing Neurobasal-A medium and maintained at 37°C, 5% CO2 for 30 min. The retina was then fixed in 4% PFA for 1 h, washed in PBS, and mounted with FluorSave reagent (Merck). After drying, the coverslip was sealed. Images of individual RGC dendritic arbours were acquired with a Zeiss LSM880 confocal microscope (20x magnification, 0.45 μm / pixel, 1 μm z-thickness), and the entire dendritic arbour was reconstructed using Imaris (version 9.3.1, Bitplane).RGC dendrites were automatically traced using the Imaris filaments tool, and Sholl analysis was performed using Imaris software to provide a quantitative index of dendritic integrity (dendritic intersections per bin distance from the cell body center, 10 μm steps).

[0124] The level of RGC loss was determined by assigning three retinal explants from the low OHT group and three retinal explants from the high OHT group to RBPMS (RNA-binding protein with multiple splicing), and dendritic degeneration was determined by applying DiOlistics labeling to three retinal explants from the low OHT group and four retinal explants from the high OHT group, and the results are shown in Figures 9 and 10.

[0125] As shown in Figure 9, the high-pressure group had a significantly lower number of RBPMS+ cells compared to the control group (p=0.050), whereas the low-pressure group showed no significant difference compared to the control group (p=0.500).

[0126] As shown in Figure 10, on the other hand, Sholl analysis showed statistically significant differences in both the low-pressure and high-pressure groups compared to the control group.

[0127]

[0128] The observation that a decrease in dendritic arborization occurred when the intraocular pressure increased relatively gradually below 17 mmHg suggests that assessing dendritic pruning has higher sensitivity than assessing RGC cell body loss for detecting RGC damage due to elevated intraocular pressure.

[0129]

[0130] Example 5. Evaluation of the therapeutic effect of drug-loaded EVs

[0131] In accordance with the mouse glaucoma model of Example 3, media containing additives (EV, NAM, and NAM-EV) were compared with the control (medium without additives). All additives were allowed to pass through the cell culture insert (Millicell® pore size 0.4 μm) and reach the whole-mount retina of the mouse, and a schematic diagram is shown in Fig. 11.

[0132] The control group was not administered any additives, the EV group was administered EV, the NAM group was administered NAM, and the NAM-EV group was administered EV loaded with NAM during 3 days of culture. Images and results evaluating dendritic pruning in each group are shown in Figures 12 and 13.

[0133] As shown in Figures 12 and 13, significant preservation of dendritic branches was observed in the NAM and NAM-EV groups compared to the NT group. The area under the Sholl curve was significantly increased in the NAM and NAM-EV groups compared to the control and EV groups, and there was no significant difference in the Sholl AUC between the control and EV groups.

[0134]

[0135] Example 6. Confirmation of scleral penetration of drug-loaded EVs through subconjunctival injection.

[0136] To explore the possibility of utilizing drug-loaded EVs via a transscleral route in the eye, which is larger than that of rodents, and for further research toward full-scale EV production, the following experiments were conducted.

[0137]

[0138] 6.1 Preparation of the rabbit model

[0139] All experimental procedures were performed in accordance with the UK Animals Scientific Procedures Act 1986 and the Association for Research in Vision and Ophthalmology guidelines on the use of animals. Individual study protocols adhered to the ethical guidelines of Cardiff University, UK, and CHA University, South Korea. Male New Zealand White (NZW) rabbits (n=3), weighing 1.9–2.2 kg, were used to assess scleral penetration following subconjunctival injection of DiO-labeled EVs.

[0140]

[0141] 6.2 Preparation of frozen sections of rabbit eyes

[0142] A solution containing concentrated DiO-labeled EV preparations was injected subconjunctivally into the eyes of NZW rabbits (n=6). To assess the depth of EV penetration into the eye over time, animals were sacrificed immediately, 1 hour, or 2 hours after injection. For cryosectioning, the eyes were enucleated, washed with PBS, and blocks were immediately prepared using OCT compound (Sakura). The blocks were stored at -80°C until use. Frozen sections were cut into 14-μm sections (from the anterior to the dorsal surface) using a cryotome (CM3050S, Leica) and stained with DAPI. Images were digitized using a slide scanner Axio Scan.Z1 (Carl Zeiss).

[0143]

[0144] 6.3 Check the distribution of EV

[0145] To confirm the distribution of EVs after subconjunctival injection, EVs were labeled with DiO, a lipophilic carbocyanine dye with green fluorescent properties. The EVs isolated in Example 1 were incubated with Fast DiO green fluorescent membrane dye (D3898, Invitrogen, CA, USA) at a final concentration of 2 μg / mL at room temperature for 1 h. The incubated solution was purified using a size-exclusion chromatography column packed with Sepharose CL-2B (Sigma-Aldrich, MO, USA) to completely remove unlabeled free DiO. The reacted solution was loaded onto the column, and 11 fractions of 1 mL each were collected. DiO intensity was measured in all fractions (excitation at 484 nm, emission at 540 nm, cut-off at 530 nm) using a microplate reader (Molecular Devices, CA, USA), and the collected fractions 4 to 8 were concentrated using an Amicon Ultra-15 centrifugal filter device, and the results are shown in Figures 15 and 16. Figure 14 is a schematic diagram showing the purification of DiO-labeled EVs.

[0146] As shown in Figure 15, fraction 10 showed a significantly higher intensity than the other fractions, indicating the presence of free DiO in this fraction. In addition, fraction 9 was also discarded along with fraction 10 because it was likely to contain some free DiO. Fractions 4 to 8 were considered to contain pure DiO-labeled EVs and were subsequently concentrated. The concentrated DiO-labeled EVs (DiO-EVs) appeared colorless and transparent to the naked eye. The results of measuring the DiO intensity are shown in Figure 16.

[0147] As shown in Fig. 16, when the DiO intensity of the solution containing DiO-EV is compared with that of the PBS solution, the former shows a much higher intensity, indicating that DiO-EV is present in the solution.

[0148]

[0149] In addition, the size distribution of EVs was evaluated using MONO ZetaView®, and the results are shown in Fig. 17, and the results of image capture by time period are shown in Figs. 18 and 19. Fig. 18 is an image observing the corneal penetration of EVs by time period after injection, and Fig. 19 is a high-magnification image of the retina taken 2 hours after injection.

[0150]

[0151] As shown in Fig. 17, the measured value was observed to be consistent with the typical EV size range with an average of 149 nm.

[0152] As shown in Figures 18 and 19, EVs were not detected in the retina immediately after injection, but it was observed that EVs were detected in the retina 1 hour after injection.

[0153]

[0154] Example 7. Drug detection profile of drug-loaded EVs

[0155] The release profile of nicotinamide from NAM-EVs was measured. A dialysis system was used to estimate the release kinetics of NAM from NAM-loaded EVs. The system was configured to prevent EV leakage from the membrane interior using a membrane with a cutoff size of 3.5 kDa while simultaneously allowing NAM to be released into the external environment.

[0156] Specifically, balanced salt solution (BSS; Alcon, TX, USA) was used as the release medium and investigated. Then, 1 milliliter of NAM-loaded EV (NAM-EV) was packaged in 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 release medium was collected and replenished with fresh medium. The concentration of nicotinamide was quantified using high-performance liquid chromatography (HPLC; Vanquish VC-P20-A, Thermo Fisher Scientific, OH, USA) at 40°C with an Agilent Eclipse column (5 micron, 4.6 × 150 mm C-18). The mobile phase consisted of water and acetonitrile (50:50) and flowed at a flow rate of 1.0 mL / min-1. This was observed at a wavelength of 210 nm, and the results are shown in Fig. 20.

[0157] As shown in Figure 20, the amount of drug released at each time point was converted to 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-EVs showed an initial burst up to 24 hours, followed by a sustained release pattern up to 96 hours.

[0158]

[0159] In summary, drug-loaded EVs, depending on the modality, have the ability to cross physical barriers such as the sclera, reach the outer retina, and treat optic nerve degenerative diseases by interfering with dendritic pruning. This means that they can deliver drugs to the inner retina via a transscleral route, demonstrating great potential as a novel DDS platform.

[0160]

[0161] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0162]

[0163] abbreviation

[0164] ASC: Adipose-Derived Stem Cell

[0165] ATP: Adenosine Triphosphate

[0166] DDS: Drug Delivery System

[0167] EFNA3: Ephrin-A3

[0168] eNAMPT: Extracellular Nicotinamide Phosphoribosyltransferase

[0169] EV: Extracellular Vesicle

[0170] IOP: Intraocular Pressure

[0171] miR: MicroRNA

[0172] NAD: Nicotinamide Adenine Dinucleotide

[0173] NAD+: Nicotinamide Adenine Dinucleotide (oxidised form)

[0174] NADH: Nicotinamide Adenine Dinucleotide (reduced form)

[0175] NADP+: Nicotinamide Adenine Dinucleotide Phosphate (oxidised form)

[0176] NADPH: Nicotinamide Adenine Dinucleotide Phosphate (reduced form)

[0177] NAM: Nicotinamide

[0178] NMN: Nicotinamide Mononucleotide

[0179] RGC: Retinal Ganglion Cell

[0180] TEM: Transmission Electron Microscopy

[0181] TSG101: Tumor Susceptibility Gene 101

[0182] ApoA1: Apolipoprotein A1

Claims

1. A pharmaceutical composition for the prevention or treatment of optic nerve degenerative disease, comprising an extracellular vesicle containing a drug, A pharmaceutical composition comprising at least one drug selected from the group consisting of an optic nerve protective drug, an intraocular pressure lowering agent, and a neurotrophic factor.

2. A pharmaceutical composition according to claim 1, wherein the optic nerve protective drug comprises at least one selected from the group consisting of a precursor of nicotinamide adenine dinucleotide (NAD), memantine, citicoline, Ginkgo biloba extract (GBE), calcium channel blockers (CCB), coenzyme Q10, 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. A pharmaceutical composition according to claim 1, wherein the intraocular pressure lowering agent comprises at least one selected from the group consisting of Rho-kinase inhibitors, carbonic anhydrase inhibitors, brimonidine, betaxolol, carteolol, timolol, levobunolol, latanoprost, bimatoprost, tafluprost, travoprost, and unoprostone.

4. A pharmaceutical composition according to claim 2, wherein the NAD precursor comprises any 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. A pharmaceutical composition according to claim 1, wherein the optic nerve degenerative disease is any 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 atrophy, Leber hereditary optic neuropathy (LHON), and amblyopia.

6. A pharmaceutical composition according to claim 1, wherein the composition reduces dendritic atrophy.

7. A pharmaceutical composition according to claim 1, wherein the composition inhibits death or promotes proliferation of retinal ganglion cells (RGC).

8. A pharmaceutical composition according to claim 1, wherein the extracellular vesicles are derived from adipose-derived mesenchymal stem cells.

9. A pharmaceutical composition according to claim 1, wherein the composition is for intraocular administration, and the drug is delivered to the retina through the trans-scleral membrane.

10. A pharmaceutical composition according to claim 9, wherein the periocular administration is subconjunctival injection, intraconjunctival injection, sub-Tenon injection, or retrobulbar injection.

11. A pharmaceutical composition according to claim 1, wherein the composition is in the form of an injection.

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