KR20230129828A - Composition for preventing or treating of eye posterior segment diseases - Google Patents

Abstract

The present invention discloses a composition for preventing or treating posterior ocular diseases or damage, comprising extracellular vesicles derived from stem cells or isolated from their culture medium as an active ingredient.

Description

Composition for preventing or treating posterior eye segment diseases}

The present invention relates to compositions for preventing or treating posterior ocular diseases or injuries.

There are two types of macular degeneration: dry and wet macular degeneration. Dry macular degeneration is caused by an increase in aging deposits and drusen on the retina and degeneration/degeneration of retinal pigment epithelial cells and photoreceptor cells. About 90% of macular degeneration patients are dry. Dry macular degeneration can develop into a habitual form called neovascularization of the choroid. Currently, in the case of wet macular degeneration, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is a specific treatment method, but as there is currently no specific treatment for dry macular degeneration, new treatments and treatments are being developed. This is urgent.

The causes of macular and retinal degeneration are related to body aging, cardiovascular disease, smoking, body inflammation, environmental pollution, and exposure to sunlight, and are also caused by side effects from therapeutic compositions, including anticancer drugs. Accordingly, prevention and development of treatments for posterior ocular diseases, including macular and retinal degeneration due to senile, traumatic, and iatrogenic causes, are urgently needed.

Patent Documents 1 and 2 propose an eye treatment using stem cells. Stem cells are cells with multipotent potential, and are known to have regenerative medicine therapeutic efficacy and anti-inflammatory immunomodulatory abilities. However, the composition of the cells themselves is limited by inflow and proliferation into other organs, problems with distribution in the body, tumorigenicity, and immunity. This causes toxicity problems. In addition, the cell culture medium itself also contains cellular waste products including ammonium ions, resulting in side effects and decreased pharmacological effects.

Therefore, the development of new treatments that can solve the above problems is required.

KR 10-2016-0033068 A (2016.03.25) KR 10-2015-0009541 A (2015.01.26)

Ratnesh Singh et al., Pluripotent Stem Cells for Retinal Tissue Engineering: Current Status and Future Prospects, Stem Cell Reviews and Reports, 19 April 2018. Akiko Maeda et al., Gene and Induced Pluripotent Stem Cell Therapy for Retinal Diseases, Annu. Rev. Genome. Hum. Genet. 24, April, 2019.

The inventors of the present invention studied to develop a treatment for posterior ocular diseases using extracellular vesicles derived from stem cell culture medium, and as a result, using the regenerative and anti-inflammatory antioxidant effects of extracellular vesicles derived from stem cells as described, Through intravitreal injection and systemic intravenous injection of mesenchymal stem cell extracellular vesicles, death of pigment epithelial cells and photoreceptor cells present in the rear of the eye is prevented, and subretinal migration of immune cells, microglia, and macrophages is prevented. It was found that it has an improvement effect on posterior ocular diseases by reducing the expression of inflammatory genes and suppressing the expression of oxidative genes, leading to the development of a treatment for macular degeneration and retinal degeneration.

Accordingly, the purpose of the present invention is to provide a composition for preventing or treating posterior ocular diseases or damage.

In order to achieve the above object, the present invention provides a composition for preventing or treating posterior ocular diseases or damage, comprising extracellular vesicles derived from stem cells or isolated from their culture medium as an active ingredient.

At this time, the stem cells are mesenchymal stem cells.

The mesenchymal stem cells may be induced pluripotent stem cells (iPSC) derived from human or animal tissue, autologous or allogeneic mesenchymal stem cells, or cell lines derived from mesenchymal stem cells.

Additionally, the human or animal tissue is isolated from the group consisting of bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood fluid, and amniotic fluid.

In particular, the exosomes or extracellular vesicles of the present invention have an average particle diameter of 50 nm to 1000 nm and have CD9, CD63, and CD81 membrane protein markers.

At this time, the ratio of CD63/CD9 among the membrane protein markers is characterized as satisfying 2.5 or more.

The number of extracellular vesicles isolated per 1 L of culture medium is 1x10 ⁹ to 1x10 ¹² and the protein amount is 0.1 mg to 20 mg.

According to one embodiment, the extracellular vesicles are prepared by culturing stem cells; Obtaining the extracellular vesicles from cultured stem cells; and is produced through a step of separating extracellular vesicles.

The separation process includes aqueous biphasic separation, ultracentrifugation, antibody affinity separation, polymer precipitation, filtration, or tangential flow filtration.

According to another embodiment, the composition contains 0.0001 to 95% by weight of extracellular vesicles within 100% by weight of the total.

The posterior ocular diseases include degenerative macular degeneration, retinal degeneration, retinitis pigmentosa, Levett congenital amaurosis, Stargardt disease, Usher syndrome, Rod-Cone dystrophy, Cone-Rod dystrophy, ciliopathies, mitochondrial disorders, or retinal diseases. Atrophy, etc.

The damage to the back of the eye is macular and retinal damage that occurs during anticancer treatment, and the damage to the back of the eye may be traumatic or iatrogenic retinal damage caused by exposure to strong light or ultraviolet rays.

The composition can be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, intravitreal, or intradermal routes. .

At this time, the composition of the present invention may be any one formulation selected from the group consisting of eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops, and solutions.

The composition according to the present invention can be preferably applied for the prevention and treatment of posterior ocular diseases, especially macular degeneration and retinal degeneration.

The composition contains exosomes and vesicles derived from stem cells, which do not contain cellular waste and prevent inflow and proliferation into other organs, problems with body distribution, tumorigenicity, and immunotoxicity that occur in conventional treatments using stem cells. The problem does not occur.

Figure 1 is a graph showing the particle size distribution of extracellular vesicles isolated in Test Example 1.
Figure 2(a) is a planar photograph of the retinal pigment epithelial cell layer within the mouse eye, Figure 2(b) is a photograph of the cell nuclei of the photoreceptor cell layer within the mouse eye, and Figure 2(c) is a change in gene expression in macular degeneration eyes. This is an RT-qPCR (reverse transcription polymerase chain reaction) graph showing.
Figure 3 is a diagram showing the intravitreal administration schedule in macular degeneration-induced mice.
Figure 4 is a photograph showing eye retention after intraocular vitreous administration of fluorescently labeled extracellular vesicles to normal mice.
Figure 5(a) is a photograph of the retinal pigment epithelial cell layer, and Figure 5(b) is a description of the location of the retinal region in the confocal photograph and a method of measuring the number of cells per unit area (315 μm This is an explanation, and Figure 5(c) is a graph of the average and variance values of the number of retinal pigment epithelial cells measured per unit area (315 μm x 315 μm) of the R2 region.
Figure 6(a) is a photograph of the photoreceptor cell layer at the rear of the eye of a dry macular degeneration animal model, and Figure 6(b) is a graph showing the degree of photoreceptor damage.
Figure 7 is an RT-qPCR graph for inflammation-related genes after administration of extracellular vesicles into the eye vitreous body in an animal model of dry macular degeneration.
Figure 8 is an RT-qPCR graph showing changes in transcript expression of oxidative stress-related genes NOX1, NOX4, DOUX1, and DOUX2 after administration of extracellular vesicles into the eye vitreous body in an animal model of dry macular degeneration.
Figure 9 is an electrophysiological response electroretinogram (ERG) waveform image of the eye showing recovery after administration of extracellular vesicles in the vitreous body of the eye in an animal model of dry macular degeneration.
Figure 10 is a- electroretinogram (ERG) electrophysiological graph measuring the physiological response of the eye to light stimulation (-4 dB, -2 dB) after administration of extracellular vesicles in the vitreous body of the eye in an animal model of dry macular degeneration. This is a graph of wave potential difference and b-wave potential difference values.
Figures 11(a) to (d) are for experiments on establishing an animal model for retinal degeneration. Figure 11(a) is an image showing that degeneration is induced in the retinal photoreceptor layer by cisplatin administration, and Figure 11(b) is an image showing eye damage. This image does not affect retinal pigment epithelial cells, but shows the movement of subretinal immune cells. Figures 11(c) and 11(d) are graphs showing the degree of overexpression of inflammatory genes.
Figure 12 is a diagram showing the intravenous injection administration schedule for mice with induced retinal degeneration.
Figure 13(a) shows a cross-sectional photograph of the retina showing the therapeutic effect of extracellular vesicle administration, and Figure 13(b) is a graph showing the therapeutic effect of extracellular vesicle administration by measuring the number of subretinal locations of non-specific cells. .
Figure 14 is an image showing that administration according to changes in the concentration of extracellular vesicles suppresses the increased number of subretinal immune cells (macrophages and microglia) in a retinal degeneration model.

Term Definition

The present invention provides a composition for preventing or treating posterior ocular diseases, comprising extracellular vesicles derived from stem cells or isolated from their culture medium as an active ingredient.

As used herein, the term 'culture medium' refers to a culture medium obtained by culturing stem cells in a culture medium, or a dried, filtered and/or concentrated product of the culture medium. The culture may or may not contain stem cells.

In the present invention, extracellular vesicles refer to biological nanoparticles present in stem cells or secreted into their culture.

In this specification, the term 'extracellular vesicles (EVs)' refers to vesicles with a membrane structure composed of a lipid-bilayer that cells secrete to the outside of the cell or exist within the cell, and are found in the body fluids of almost all eukaryotic organisms. exist. The extracellular vesicles are classified into exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, and nanovesicles based on their origin, secretion mechanism, size, etc. It is used interchangeably with terms such as nanovesicles and outer membrane vesicles, and is an encompassing concept. Unless specifically stated herein, extracellular vesicles may be exosomes. The extracellular vesicles have a diameter of 50-1000 nm and are released from the cell when multivesicular bodies fuse with the cell membrane, or are released directly from the cell membrane. It is well known that the extracellular vesicles play a role in transporting proteins, bioactive lipids, and RNA, which are biomolecules within cells, in order to perform functional roles in mediating coagulation, cell-cell communication, and cellular immunity. Known marker proteins of the extracellular vesicles include CD9, CD63, and CD81, and others include receptors on the cell surface such as EGFR, molecules related to signal transduction, cell adhesion-related proteins, mesenchymal stem cell (MSC)-related antigens, Proteins such as heat shock protein and Alix related to vesicle formation are known.

As used herein, the term 'containing as an active ingredient' means containing a sufficient amount to achieve the improvement, prevention or treatment activity of posterior ocular diseases of stem cells or their culture medium or extracellular vesicles isolated therefrom.

As used herein, the term 'posterior eye disease or damage' refers to an abnormal condition or symptom that occurs in the eye.

The term “prevention” used in the present invention refers to any action that suppresses or delays the progression of posterior ocular disease or damage by administering the composition of the present invention.

As used herein, the term “treatment” refers to (a) inhibiting the development of posterior ocular disease or damage; (b) alleviation of posterior ocular disease or damage; and (c) the elimination of posterior ocular disease or damage.

Stem Cells

The stem cells of the present invention are mesenchymal stem cells, where the mesenchymal stem cells are derived from induced pluripotent stem cells (iPSC) derived from human or animal tissue, autologous and allogeneic mesenchymal stem cells, or mesenchymal stem cells. It may be a derived cell line.

As used herein, the term 'mesenchymal stem cell' refers to a stem cell with multipotency that can differentiate into cells such as fat, cartilage, bone, muscle, skin, and nerve. The mesenchymal stem cells can be differentiated from induced pluripotent stem cells or isolated from bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood, synovial membrane, amniotic fluid, etc.

‘Induced pluripotent stem cell (iPSC)’ refers to cells that return to their initial undifferentiated state by inducing dedifferentiation in already differentiated cells, such as somatic cells, and have pluripotency. it means. The dedifferentiation can be induced by introducing and expressing a specific gene (e.g., Sox2, c-Myc, Klf4, Oct-4, etc.) or by injecting a dedifferentiation-inducing protein produced in cells into which the specific gene has been introduced. .

The pluripotency refers to the ability to differentiate into tissues or organs originating from the three germ layers that make up the living body, that is, endoderm, mesoderm, and ectoderm.

The induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all mammals such as humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, and rabbits, but preferably induced pluripotent stem cells derived from humans. It's a cell.

Method for producing extracellular vesicles

The extracellular vesicles of the present invention can be produced through culturing stem cells in medium.

The term "medium" used in the present invention refers to the growth of cells such as stem cells in vitro, which contains essential elements such as sugar, amino acids, various nutrients, serum, growth factors, and minerals. It refers to a mixture for growth and proliferation. In particular, the medium of the present invention is a medium for culturing mesenchymal stem cells. The above-mentioned “mesenchymal stem cells” are cells isolated from stem cells derived from mammals, including humans, and have the ability to proliferate indefinitely and form various cell types (e.g., adipocytes, cartilage cells, muscle cells, These are cells that can differentiate into bone cells, etc.). The above “culture” is meant to include the growth and proliferation of mesenchymal stem cells.

The “basic medium” of the present invention is a mixture containing essential sugars, amino acids, water, etc. necessary for cells to survive, excluding serum, nutrients, and various mesenchymal stem cell growth factors. The basic medium of the present invention can be artificially synthesized and used, or a commercially produced medium can be used. Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, and α-Minimal Medium (α-MEM). Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium, but are not limited to these.

Serum is a supernatant obtained by centrifugation from animal or human blood. In addition to the essential nutrients required for cell growth, the serum contains trace elements including various factors such as various inorganic salts, polypeptide growth factors, and polypeptide hormones that are essential for growth but have not been clearly identified.

The “serum” of the present invention can be used as fetal bovine serum, commercial human serum, commercial human fetal serum, and autologous serum.

The concentration of serum used in the medium during cell culture of the present invention is within 30%, preferably within 25%, and more preferably within 20% of the total medium composition.

The culture medium of the present invention may additionally include a nutrient mixture. The nutrient mixture is a mixture containing various amino acids, vitamins, mineral salts, etc. commonly used in cell culture. It can be prepared by mixing the amino acids, vitamins, mineral salts, etc., or a commercially prepared nutrient mixture can be used. Commercially manufactured nutrient mixtures include, for example, F-12, M199, MCDB110, MCDB202, MCDB302, etc. Preferably, F-12 Nutrient Mixture, M199, MCDB culture medium, etc. are used. You can. The nutrient mixture can be used by diluting 1 to 10:1 in the basic medium, preferably 1 to 5:1. In addition, Transferrin, Selenium, Glutamine, etc. can also be used in combination.

In a specific embodiment, the cell culture medium of the present invention may additionally or instead of serum contain mesenchymal stem cell growth factors that affect the growth of mesenchymal stem cells. Growth factors for mesenchymal stem cells include, for example, insulin, hydrocortisone, EGF (Epidermal Growth Factor), LIF (Leukemia Inhibitory Factor), GM-CSF (Granulocyte-macrophage colony stimulating factor), These include EPO (Erythropoietin), FGF (Fibroblast Growth Factor), IGF (Insulin-like growth factor), PDGF (Platelet-derived growth factor), SCF (Stem cell factor), and TGF (Transforming growth factor). A distinction is made between the general culture medium for cells and the isolation culture medium for the separation of extracellular vesicles in the final step. The extracellular vesicle isolation culture medium contains serum from which extracellular vesicles have been removed, or preferably does not contain serum. However, it may contain nutrient mixtures and growth factors.

The production of extracellular vesicles from mesenchymal stem cells or their cultures follows the following steps.

First, mesenchymal stem cells are cultured in cell culture medium. As an example, mesenchymal stem cells are cultured in a cell culture dish/flask containing the general culture medium of the present invention at a concentration of 1 to 10,000 cells/cm ² , preferably 50 to 6000 cells/cm ² do. It is generally carried out at a temperature of 30°C to 37°C, 2% to 7% CO ₂ environment, and 5% to 21% CO ₂ environment. Generally, it does not exceed passage number 8, and preferably does not exceed passage number 6, but the passage and passage maintenance dates of stem cells and stem cell lines are not particularly limited as long as they are appropriate for each cell. In the cell culture, the passage interval should not exceed 150 hours at any passage.

Next, replace the normal culture medium with extracellular vesicle isolation culture medium. The replacement time is not limited, but preferably does not exceed passage number 6, and the extracellular vesicle isolation culture medium is collected within 1 to 4 days after replacing the culture medium.

As a culture medium for the isolation of extracellular vesicles, fetal serum from which vesicles have been removed is used, or serum-free medium from which fetal serum has been removed step by step is used. Influence mixtures and growth factors may be included.

The serum-free medium is a medium that does not contain animal serum as an additive and is not particularly limited. Compositions containing other additives excluding animal serum in a known basic medium can be accepted and used. The composition of the basic medium can be appropriately selected depending on the type of cells to be cultured.

In the embodiment of the present invention, at the last passage, the extracellular vesicle isolation culture medium was replaced, and bone marrow mesenchymal stem cell culture, passage number 6 or higher, was not used. However, it is possible to culture with other combinations such as specific stem cells or stem cell lines, Therefore, in the present invention, there is no limitation to a specific passage number. Additionally, the final incubation period does not exceed 100 hours. The collected culture medium for isolation of extracellular vesicles is centrifuged and the supernatant is stored frozen for isolation of extracellular vesicles. Centrifugation is a step to remove dead cells and cell debris, and in the present invention, 500Хg for 10 minutes was used, followed by 2,000Хg for 10 minutes or 10,000Хg for 10 minutes, but the combination of centrifugation and time is suitable for application. possible.

The separation process may be performed using aqueous biphasic separation, ultracentrifugation, antibody affinity separation, polymer precipitation, filtration, or tangential flow filtration, preferably KR 10-2016-0116802 A, KR 10-2016-0114336 A, and a method using an aqueous biphasic phase separation composition mentioned in KR 10-2020-0058631 A. The aqueous solution biphasic separation method is a separation method that utilizes the phenomenon that different particles are separated differently by two different phases, and is useful for removing impurities and polymers outside the extracellular endoplasmic reticulum.

Specifically, the aqueous biphasic phase separation composition according to the present invention includes a first aqueous solution phase forming a dispersed phase; and a second aqueous liquid phase that is phase-separated from the dispersed phase and forms a continuous phase, wherein the tension (γ) at the interface where the first aqueous liquid phase and the second aqueous liquid phase are phase-separated satisfies Equation 1 below. .

[Equation 1]

^{2 _ _ _}

The first aqueous phase containing extracellular vesicles has a bulk form and exists in a flowing state within the second aqueous phase due to gravitational force or buoyancy. Extracellular vesicles are trapped at the interface where the first aqueous solution phase and the second aqueous solution phase contact, and only proteins with small particle sizes escape the interface and move to the second aqueous solution phase, thereby causing cells present in the first aqueous solution phase. External endoplasmic reticulum can be recovered with high purity. In this separation method, the critical particle size can be controlled by controlling the interfacial tension, allowing separation of impurities and extracellular vesicles up to a size difference of about 10 nm. For example, the first aqueous solution contains dextran, and the second aqueous solution contains polyethylene glycol.

The separation process using an aqueous biphasic system is performed to sufficiently recover extracellular vesicles, and is performed within 2 hours, preferably within 5 minutes to 1 hour, more preferably within 5 minutes to 30 minutes, and most preferably within 15 minutes. do. This time has the advantage of significantly reducing the time compared to the conventional ultracentrifugation process, which takes at least 2 hours.

In the case of the existing method using ultracentrifugation, the size of extracellular vesicles is small (hundreds of nm) and there is not much difference in density from the protein, so there is a disadvantage in the efficiency of separating extracellular vesicles from plasma with high purity and efficiency. When using, there is a problem that the gravitational acceleration received by actual cells reaches up to 100,000 Хg, so there is a high possibility of damage to the endoplasmic reticulum. This means that the separation process using the aqueous solution biphasic system can solve the problems of existing separation methods.

The separated extracellular vesicles are analyzed for exosome size, number, and distribution of membrane protein markers using a nanoparticle tracking analysis device and TIRF (total internal fluorescence) analysis.

At this time, the quality of extracellular vesicles is confirmed by the number of extracellular vesicles isolated per 1 L of culture medium, the amount of protein in extracellular vesicles, and the number of extracellular vesicles per unit protein. The number of exosomes, which are the final extracellular vesicles of bone marrow stem cells in the example, is 1x10 ⁹ to 1x10 ¹² per 1 L of culture medium, and the amount of protein is 0.1 mg to 20 mg. The number and protein amount of isolated extracellular vesicles may vary depending on the type of stem cell, culture passage number, and type of culture medium, and the final isolated extracellular vesicles have the same or similar final size distribution, purity, and same or similar physiological activity. If it has, the present invention does not limit the specific separation method.

Preferably, the extracellular vesicles have an average particle diameter of 50 nm to 1000 nm and have a membrane protein marker (i.e., an extracellular vesicle-specific marker, more specifically an exosome-specific marker).

At this time, the extracellular vesicles present in the composition are 90% by weight or more, preferably 95% by weight or more, of the total, with a particle diameter distribution of 10 nm or more and 300 nm or less. If the particle diameter is less than 10 nm or more than 10% by weight of extracellular vesicles of 300 nm or more, it is undesirable because there is a problem of mixing with high molecular weight proteins or apoptotic bodies.

The suitable/appropriate cell culture method used in this example and separation using an aqueous biphasic system minimizes impurities in polymer proteins and also minimizes impurities in apoptotic bodies. In addition, in order to minimize and remove dead body contaminants, in an embodiment of the present invention, filtering is performed to a desired size (250 μm or 450 μm), and nanoparticles in the desired size range are produced.

In addition, when the number of extracellular vesicles present in the composition is within a certain number range, the effect of posterior ocular diseases can be further increased. Protein is quantitatively analyzed using methods such as Bradford or Bicinchoninic acid (BCA). Referring to Test Example 2 of the present invention, the efficacy of the extracellular vesicles with a purity of 5x10 ⁷ or more and 5x10 ⁸ or less per 1 μg of protein based on Bradford quantification was confirmed to be effective in animal experiments for retro-ocular diseases.

The term “membrane protein marker” of the present invention refers to a protein abundantly present in the membrane of extracellular endoplasmic reticulum. Membrane protein markers of the extracellular vesicles, especially exosomes, may be CD9, CD63, CD81, etc., and the isolated exosomes contain 25% or less of the CD9 membrane protein marker compared to the number of exosomes expressing CD9 or CD63. , preferably expressed at 20% or less, most preferably at 15% or less. In addition, the isolated exosomes express at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 70% of the CD63 membrane protein marker compared to the number of CD9 or CD63 expressing exosomes. .

According to one embodiment of the present invention, compared to total CD9, CD63, and CD81, CD9 ranges from 2 to 25%, CD63 ranges from 13 to 70%, and CD81 ranges from 10 to 60%. In particular, there is a difference in efficacy depending on the ratio of CD63 and CD9, and when the ratio of CD63/CD9 is 2.5 or more, preferably 3.5 or more, and more preferably 5 or more, the effect for posterior ocular diseases can be further increased.

Disease or injury to the back of the eye

As described above, extracellular vesicles isolated from stem cells or their culture medium can be used in compositions for prevention or treatment related to posterior ocular diseases or damage.

The posterior ocular disease refers to a disease that occurs in the eye, and particularly includes diseases related to the posterior ocular.

The back of the eye refers to the retina, choroid, and the subretinal area between them. Degenerative and inflammatory diseases of the back of the eye include not only tissue degeneration and degeneration, but also abnormal expression of genes such as inflammatory cytokines and chemokines, and oxidative stress-related genes. It includes diseases caused by abnormal expression, increase and activation of immune cells, and subretinal migration and activation of macrophages and microglia.

Specifically, eye diseases related to the rear of the eye include degenerative macular degeneration, retinal degeneration, retinitis pigmentosa, Levett congenital amaurosis, Stargardt disease, Usher syndrome, Rod-Cone dystrophy, Con-Rod dystrophy, ciliopathies, It may be a mitochondrial disorder, retinal atrophy, etc. At this time, macular degeneration refers to degeneration of the rear center of the eye (macula), including the retina, choroid, and subretinal area.

In addition, expected damage to the posterior eye includes traumatic and iatrogenic retinal damage, including macular and retinal damage, accompanied by posterior eye inflammation due to intense light, exposure to ultraviolet rays, or therapeutic compounds or compositions thereof, including anti-cancer agents.

Extracellular vesicles isolated from stem cells or their culture medium presented in the present invention are used to prevent or treat posterior ocular diseases, wherein they suppress damage and diseases related to ocular diseases or ocular damage and diseases expected upon administration of a therapeutic agent. Or, it can be used for relief and recovery.

Specifically, it has anti-apoptotic, anti-inflammatory and antioxidant effects, inhibits apoptosis/degeneration/degeneration of the pigment epithelial cell layer, inhibits apoptosis/degeneration/degeneration of the photoreceptor cell layer, and inhibits subretinal microglial cells. and prevents the increase of macrophages. In addition, it suppresses the expression of a number of inflammatory genes (TNFa, IL-1b, ICAM1, CCL2, CCL3, CCL4, CCL7, CCL8, CXCL1, CXCL9) in the posterior ocular area and a number of oxidative stress-inducing genes (NOX1, NOX4, DOUX1, DOUX2 ) prevents its appearance in the rear of the eye. In addition, it suppresses the expression of vascular endothelial growth factor (VEGF), a gene that stimulates the creation of new blood vessels, in the rear of the eye.

In particular, according to an embodiment of the present invention, after intracellular administration of the extracellular vesicles of the present invention into the eye vitreous body of mice, ocular retention and retinal distribution are maintained for several weeks or more compared to small molecule compounds or antibody drugs. Expect a long stir. In the case of small molecule compounds, when administered to the human eye, retention in the vitreous body drops to less than half within a day, and in the case of antibodies, about half disappears within 4 days, making it difficult for the distribution within the eye vitreous and retina to be maintained for more than a month. In the case of mice, metabolism is fast, so it is impossible to analyze the activity of small molecules and antibodies remaining after administration. Currently, in the case of fluorescently attached extracellular vesicles, when administered into the vitreous body at the same concentration that showed ocular efficacy, it has been observed that the residual period is more than 2 weeks in mice. When converted to humans, it is interpreted that it will remain in the vitreous body for more than 6 months.

The extracellular vesicle of the present invention functions as a nano-drug carrier, and in particular, functions as a drug nano-carrier for retention and distribution within the vitreous body of the eye, and is expected to maintain the efficacy of the drug for a long period of time. , the single-dose concentration and number of administrations of the loaded drug can be reduced, thereby reducing complications that may occur during administration, and the effect of reducing patient hospital visits and medical expenses is also expected.

pharmaceutical use

The preventive and therapeutic composition containing extracellular vesicles according to the present invention contains 0.0001 to 95% by weight of extracellular vesicles within 100% by weight of the total composition, and is formulated into a pharmaceutical preparation and administered or combined with other drugs. It may be administered simultaneously or at an appropriate time before or after administration of other medications.

As used herein, the term “administration” means providing a substance to a subject by any suitable method. The composition of the present invention can be administered orally or parenterally through any general route as long as it can reach the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells or organs.

The term "subject" herein is not particularly limited, but includes, for example, a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or It includes guinea pigs, preferably mammals and more preferably humans.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous systemic administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transdermal administration, ocular administration, or local ocular administration. there is. Ocular topical administration, for example, can be administered directly intraocularly, periocularly, retrobulbally, subretinal, central retinal, extrafovea, subconjunctival, or intravitreous. ), intracameral, or suprachoroidal administration. Compositions of the present invention may also be administered via an ocular insertion device.

The preventive or therapeutic composition of the present invention can be prepared into an appropriate preparation or dosage form by commercial methods. The dosage form of the preparation may be a solid preparation such as powder or granule, but from the viewpoint of obtaining excellent preventive treatment effects, it may be used as eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops and liquids. It is desirable. In particular, when used as an eye injection or eye drop, a solution is preferable. Suitable examples of the liquid preparation method include, for example, mixing pre-prepared stem cell-derived microparticles or stem cell culture fluid (e.g., culture supernatant) with a solvent, or additionally mixing a suspending agent or emulsifier. there is.

As described above, in the formulation of the pharmaceutical composition of the present invention, appropriate pharmaceutically acceptable carriers such as excipients, binders, solvents, solubilizers, suspending agents, emulsifiers, isotonic agents, buffers, and stabilizers are used depending on the needs of the formulation. , analgesics, preservatives, antioxidants, colorants, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, diluents, etc. can be mixed. Additionally, when the composition of the present invention contains cells, components acceptable for cell preparations may be added.

The dosage of the preventive or therapeutic composition of the present invention may vary depending on the type of disease, the degree of its symptoms, the formulation, the weight of the subject to be administered, etc., but the amount of mesenchymal stem cell-derived microparticles is, for example, 1 per day. The range from pg/kg to 100 mg/kg can be suitably exemplified, and the range from 100 pg/kg to 10 mg/kg can be more suitably exemplified.

Administration of the preventive or therapeutic composition of the present invention can be administered as a single dose or multiple doses in the case of intraocular vitreous administration, and can be administered at least once every 12 months, especially once every 6 months, and can be administered twice or more. Among these, it is preferable to administer once or twice or more every three months. In particular, the preventive or therapeutic composition of the present invention can achieve dramatic effects with only one administration, as shown in Test Example 8.

Additionally, in the case of eye drops, the administration of the preventive or therapeutic composition of the present invention can be performed one to multiple times per day. Eye drops can also be administered single or multiple times. In the case of multiple administration, for example, they can be administered continuously 2 or more times at a frequency of 1 time or more every 7 days, and especially, 2 or more times at a frequency of 1 time or more every 3 days. It is preferable to administer it, and especially, it is preferable to administer it 3 or more times at a frequency of 1 time or more every 2 days, and it is more preferable to continuously administer it 2 or more times at a frequency of 1 time or more a day.

If the preparation of the composition for prevention or treatment of the present invention is for intravitreal administration or eye drops, it can be prepared using techniques commonly used in intravitreal administration and eye drops, using pharmaceutically acceptable additives as needed. there is.

For example, isotonic agents such as sodium chloride and glycerin; pH adjusters such as hydrochloric acid and sodium hydroxide; Buffering agents such as sodium phosphate and sodium acetate; Surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, and polyoxyethylene hydrogenated castor oil; Stabilizers such as sodium citrate and sodium edetate; It can be prepared by selecting from preservatives such as benzalkonium chloride and paraben as needed. When administered into the ocular vitreous body, the pH of the sterilized preparation is usually 7.3, and the pH of the eye drops should be within the range acceptable for ophthalmic preparations, but a range of pH 4 to 8 is usually preferable.

If the preventive or therapeutic composition of the present invention is an injectable solution, intravenous administration (I.V.) is possible.

For intravenous administration, a pharmaceutically acceptable carrier may be used, such as phosphate buffered saline, saline solution, or other suitable substances that can be used for intravenous systemic administration of the drug.

The compositions of the present invention can be dissolved in any of a variety of buffers. Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer and sodium phosphate (eg 150 mM sodium phosphate). Insoluble polynucleotides can be dissolved in a weak acid or base and then diluted to a desired volume using a buffer solution. The pH of the buffer solution can be adjusted appropriately. Additionally, appropriate osmotic properties can be provided using pharmaceutically acceptable additives. Such additives are within the purview of those skilled in the art. When the composition is used in vivo, sterilized, pyrogen-free water can be used. Such formulations may contain an effective amount of the polynucleotide together with a suitable amount of aqueous solution to prepare a composition suitable for administration to humans. In particular, as a buffer solution for the ocular vitreous body and eye drops, a commercial or similar ocular Balanced Salt Solution (BSS) can be used. It is an isotonic solution with a pH of 6.8-7.4, an osmotic pressure of about 300 mOsm/kg, and a solution containing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate, sodium citrate, and sodium hydroxide.

Animals to which the preventive or therapeutic composition of the present invention is administered are not particularly limited, but humans, monkeys, mice, rats, hamsters, ferrets, cows, horses, rabbits, sheep, goats, cats, dogs, etc. are preferable. Humans are more desirable. In addition, when the prophylactic or therapeutic composition of the present invention contains cells and/or their culture medium, the one that matches the type of animal to which the preparation of the prophylactic or therapeutic composition of the present invention is administered is more stable against the disease. This is desirable from the viewpoint of obtaining excellent preventive and/or therapeutic effects.

food use

Meanwhile, the composition for preventing or treating posterior ocular diseases containing exosomes of the present invention can be applied as a functional food composition containing this and a functional food acceptable carrier.

As used herein, the term 'functional food composition' includes food products, foodstuffs, dietary supplements, nutritional supplements, or supplementary compositions for food products or foodstuffs.

The animal feed comprising the pet food composition may include treats (e.g. dog biscuits) or other food supplements, as well as foodstuffs that advantageously supply the necessary dietary requirements, such as supplements, for example gravies, beverages, yoghurts. , powder, suspension, chew, treat (e.g. biscuit) or any other delivery form.

Dietary supplements of the present invention may be delivered in any suitable format. In a preferred embodiment, it may be a liquid, gel, gel cap, capsule, powder, solid tablet (coated or uncoated), tea, etc.

[Example]

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. At this time, in the following test examples, extracellular vesicles are exosomes unless otherwise indicated. In addition, MSC-Ev, indicated in the drawing, is an abbreviation for mesenchymal stem cell-derived extracellular vesicles.

Test Example 1: Isolation of extracellular vesicles from stem cells

(1) Stem cell culture

11 ㎖ of bone marrow donated from a normal healthy person or from the patient himself was diluted with DMEM medium at a ratio of 1:2 and then placed on a previously prepared 10 ㎖ Histopaque (Sigma, density 1.077 g/㎖) layer. It was dropped and centrifuged at 400Хg for 30 minutes at room temperature. The mononuclear cell layer was separated, DMEM medium was added, and centrifuged at 400Хg for 5 minutes at room temperature. The obtained mononuclear cells were washed twice with DMEM medium and then cultured in MSCGM medium at 37°C and 5% C0 ₂ . . After 48 hours of culture, cells that did not stick to the bottom of the flask were removed by replacing them with new medium, and cells that stuck to the bottom of the flask were cultured with the medium changed once every 3 to 4 days. When the cultured cells grow to about 80%, they are subcultured to a new flask. Some of the cultured cells are continued to be subcultured, and some are placed in a cryopreservation solution containing MSCGM™, 10% fetal bovine serum (FBS), and 5% DMSO. Alternatively, it was turbid in a Stem Cell Banker and stored in liquid nitrogen at -176°C.

Specifically, mesenchymal stem cells (Cell Therapy Business Group, Catholic Seoul St. Mary's Hospital) are purchased (distributed), stored in a nitrogen tank at the headquarters, and used within one year of purchase. Cells of passage number 2 were purchased and cultured in DMEM culture medium containing 20% FBS (fetal bovine serum) and penicillin/streptomycin/amphotegicin B. After culturing using general culture medium until passage number 4, it was replaced with an isolation culture medium before extraction of exosomes and vesicles, and after 48 hours of culture, the cell culture supernatant was recovered.

The recovered cell culture supernatant was collected after 10 minutes at 500Хg, centrifuged again at 2,000Хg for 10 minutes, and the supernatant was aliquoted and stored frozen. Specifically, the frozen cell culture supernatant is a culture medium from which cells and cell debris have been removed, and contains exosomes secreted by 1x10 to 1x10 ³ cells per ml of culture medium, and has a protein concentration of 100 μg to 5000 μg per ml of culture medium. The one that corresponds to the category was used.

(2) Separation of extracellular vesicles using an aqueous biphasic phase separation composition

To create an aqueous biphasic system, polyethylene glycol and dextran were dissolved in phosphate buffered saline (PBS) and prepared at concentrations of 10.5 wt% and 45 wt%, respectively. In the aqueous solution ideal system of this composition, the interfacial tension between the dextran aqueous solution and the polyethylene glycol aqueous solution was 5.36X10 ^-6 J/m ² .

5 ml of polyethylene glycol aqueous solution was added to a horizontal*vertical test tube. Next, 500 ㎕ of the culture medium was mixed with 0.5 ml of dextran aqueous solution and dissolved for 1 hour, and the resulting solution was added to the test tube to induce phase separation.

Extracellular vesicles from the dextran layer were extracted with a pipette.

Next, the number and protein concentration of recovered extracellular vesicles were measured. In order to increase the purity of the number of extracellular vesicles per protein, that is, to completely remove residual proteins other than extracellular vesicles in the culture medium, this process was repeated more than twice.

Test Example 2: Confirmation of characteristics of isolated extracellular vesicles

The characteristics of the extracellular vesicles isolated in Test Example 1 were analyzed, and the results are shown in the drawing and table.

Figure 1 is a graph showing the particle size distribution of extracellular vesicles isolated in Test Example 1. At this time, the extracellular vesicles are exosomes, and the particle diameter distribution of the separated exosomes is 1000 nm or less, but it can be seen that more than 96% by weight is densely distributed between 50 nm and 300 nm.

Six replicate experiments were performed to analyze the characteristics of the isolated exosomes (number of exosomes extracted from 1 L media, content of exosomes in protein per 1 L, and number of exosomes per 1 ug of protein). Their average values are shown. The number of exosomes extracted from 1 L media was 3.66x10 ¹⁰ , the content of exosomes in protein per 1 L was 15 mg, and the number of exosomes per 1 ug of protein was measured to be 1.96x10 ⁸ .

At this time, as a result of measuring the distribution and content of membrane protein markers in the isolated exosomes, CD9, CD63, and CD81 were mainly present, accounting for 14.3%, 75.79%, and 2.5%, respectively, and these distributions accounted for 90% of the total exosomes. It can be seen that more than % of these protein markers are expressed.

Among the membrane protein markers, the ratio of CD63 and CD9 was 5.3.

Establishment of dry macular degeneration mouse model

The following is the establishment of a dry macular degeneration model. A dry mouse macular degeneration model was established using mouse BLAB/C. A small molecule chemical agent was used to treat macular degeneration in mice, and degeneration was induced in the retinal pigment epithelium and photoreceptor cell layer by systemic administration of sodium iodate.

Increased gene expression

Figure 2 is an experiment to establish a dry macular degeneration model, showing degeneration of retinal pigment epithelial cells in the center of the eye caused by sodium iodate, rosette formation due to loss of photoreceptor cells, increased expression of inflammatory genes, increased expression of genes related to oxidative stress, and apoptosis genes. This is a description of the animal model of increased expression.

Figure 2(a) is a planar photograph of the retinal pigment epithelial cell layer within the mouse eye, Figure 2(b) is a photograph of the cell nuclei of the photoreceptor cell layer within the mouse eye, and Figure 2(c) is a change in gene expression in macular degeneration eyes. This is a Polymerase Chain Reaction (PCR) graph showing.

Figure 2(a) is a planar photograph of the retinal pigment epithelial cell layer within the mouse eye, showing normal and sodium iodate-degenerated and degenerated retinal pigment epithelial cells stained with phallodin. In normal cases, phallodin staining shows a uniformly distributed retinal pigment epithelial cell layer of hexagonal to octagonal shape, but in the experimental group with induced macular degeneration, the absence of a uniformly distributed retinal pigment epithelial cell layer was observed.

Figure 2(b) is a photograph of the cell nuclei of the photoreceptor cell layer in the mouse eye, showing that photoreceptor cells, which are uniformly distributed in the normal eye, form rosettes in the eye with induced macular degeneration.

Figure 2(c) shows the results of an experiment obtained through RT-qPCR to show changes in expression of genes in eyes with induced macular degeneration, including an increase in the expression of genes related to inflammation, an increase in the expression of genes related to oxidation, and genes related to apoptosis, according to the present invention. The suitability of the macular degeneration model used can be seen.

Experimental design of macular degeneration animal model with extracellular vesicle administration

Animal models of macular degeneration cause damage to posterior ocular cells, retinal pigment epithelial cells, and photoreceptor cells. In the macular degeneration model, vitreous administration of extracellular vesicles was used, and drug efficacy was observed through electroretinogram (ERG), ocular staining, and RT-qPCR.

The ocular administration composition containing the mesenchymal stem cell extracellular vesicles (MSC-Ev) isolated through Test Example 1 was administered once with a solution of pH 7.3 sterile phosphate-buffered saline prepared in a manner suitable for protein concentration and number of extracellular vesicles. Each dose was divided and supplied. Specifically, an ocular administration composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) was prepared with 50 mg of isolated extracellular vesicles, 1 ml of PBS, and pH of 7.3. At this time, the test was performed based on the intravitreal administration schedule for macular degeneration-induced mice in Figure 3.

In the case of macular degeneration-induced mice, after induction, a single dose of 50 ng of extracellular vesicle protein was administered to the vitreous body. After 3 days, changes in genes related to inflammation and oxidation were observed using RT-qPCR, and on the 7th day, retinal non-invasive eye examination was performed. Retinal function was examined through electrophoresis, and the retina of the eye extracted through an invasive method was stained and observed to measure the efficacy of the administered extracellular vesicles on macular degeneration.

[result]

Figure 4 is a photograph showing retention within the eye after administration of fluorescent extracellular vesicles into the vitreous body of a normal mouse.

Referring to Figure 4, after administering fluorescent (Cy5.5) attached extracellular vesicles into the eye vitreous body, through a tracking experiment with the IVIS in vivo imaging system, the fluorescently attached extracellular vesicles were observed within the eye for more than 2 weeks. It was confirmed that it was maintained. The administered amount was ~ ^1X108 . Maintenance for 2 weeks in mice means retention for about 6 months in humans, which is interpreted to be similar to or higher than that of nanocarriers currently under development or in ocular clinical trials.

Test Example 3: Inhibitory effect of extracellular vesicle administration on retinal cell epithelial cell death

[method]

An ocular administration composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) was administered into the ocular vitreous body in the same manner as described in (FIG. 3) using an animal model of dry macular degeneration induced by sodium iodate (FIG. 2). Afterwards, recovery of the degenerated/degenerated retinal pigment epithelial cell layer by administration of extracellular vesicles was confirmed in a dry macular degeneration animal model (FIG. 5).

To obtain the measurement value, the number of retinal pigment epithelial cells present per unit area was measured and each experimental group was compared. As a result, the completely destroyed pigment epithelial cells in the R2 retina region of the dry macular degeneration animal model were recovered by administration of extracellular vesicles. It is visible at 5.

[result]

Specifically, Figure 5(a) is a morphological photograph of retinal pigment epithelial cells through phalloidin staining. The epithelial cell layer consisting of hexagonal or octagonal retinal pigment epithelial cells in normal eyes was destroyed in all eye regions by sodium iodate, and administration of extracellular vesicles prevented or restored damage to the completely destroyed retinal pigment epithelial cells.

Figure 5(b) is a description of the location of the retinal region in the confocal image and a method for measuring the number of cells per unit area (315 μm Figure 5(c) is a graph of the average and variance values of the number of retinal pigment epithelial cells measured per unit area (315 μm x 315 μm) of the R2 region.

Looking at the diagram in Figure 5, in the macular degeneration animal model, degeneration of retinal pigment epithelial cells by sodium iodate, intravitreal injection of mesenchymal stem cell extracellular vesicles (MSC-Ev) causes retinal pigment epithelial cell death in the rear of the eye. It appears to suppress.

Test Example 4: Inhibition of degeneration of the photoreceptor cell layer by administration of extracellular vesicles

The animal model of dry macular degeneration caused by sodium iodate causes damage not only to posterior eye and retinal pigment epithelial cells, but also to photoreceptor cells, and was performed as follows to confirm the drug effect of extracellular vesicles on this.

[method]

The ocular administration composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) isolated through Test Example 1 was administered in the same manner as described in FIG. 3, cell nuclei were stained with Hoechst33342 to observe rosettes, and the entire The eyes were scanned with a confocal microscope to quantify the rosette area, and the results are shown below.

[result]

Figure 6(a) is a photograph of the photoreceptor cell layer at the rear of the eye of a sodium iodate dry macular degeneration animal model, and Figure 6(b) is a graph showing the degree of photoreceptor damage.

Specifically, Figure 6(a) shows that the number of rosettes was reduced through administration of an ocular composition containing extracellular vesicles. Figure 6(b) is a graph showing the degree of photoreceptor damage, and rosette formation was reduced upon administration of extracellular vesicles.

Test Example 5: Reduction of inflammatory cytokine and chemokine expression by administering extracellular vesicles to a dry macular degeneration model

[method]

After intraocular administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) 4 hours after macular degeneration induction, the eye was removed on the third day, and posterior ocular tissues including the retina and choroid were removed. RNA (ribonucleic acid) was extracted and RT-qPCR (reverse transcription polymerase chain reaction) was performed to measure the expression of inflammatory cytokines and chemokines.

[result]

Figure 7 is an RT-qPCR graph for each gene after administration of extracellular vesicles in the eye vitreous body.

Looking at Figure 7, It was shown that the expression of inflammatory cytokine genes (TNFa, IL-1b, ICAM1), which were increased in this animal model, was statistically significantly reduced by the administration of mesenchymal stem cell extracellular vesicles (MSC-Ev). In addition, the expression of inflammatory chemokine genes (CCL2, CCL3, CCL4, CCL7, CCL8, CXCL1, CXCL9), which was increased in this animal model, was decreased by administration of mesenchymal stem cell extracellular vesicles (MSC-Ev).

Test Example 6: Confirmation of decreased expression of oxidative stress-inducing genes by administration of extracellular vesicles in a dry macular degeneration model

[method]

After intraocular administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) 4 hours after macular degeneration induction, the eye was removed on the third day, and posterior ocular tissues including the retina and choroid were removed. RNA (ribonucleic acid) was extracted and RT-qPCR was performed to measure the transcript expression of genes (NOX1, NOX4, DOUX1, DOUX2) that are located in mitochondria and increase oxidation in the animal model.

[result]

Figure 8 is an RT-qPCR graph for each gene after administration of extracellular vesicles into the eye vitreous body.

Referring to Figure 8, in this animal model, mesenchymal stem cell extracellular vesicles (MSC-Ev) increase the expression of transcripts of NOX1, NOX4, DOUX1, and DOUX2, which increase oxidative stress-inducing genes, through increased mitochondrial function. It was confirmed that administration of external endoplasmic reticulum resulted in statistically significant inhibition.

Test Example 7: Improvement of ocular function following extracellular vesicle administration in dry macular degeneration

[method]

Four hours after inducing macular degeneration, an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) was administered intravitreally, and at one week, an electroretinogram (ERG), a non-invasive ophthalmic device, was used. The physiological response of the eye was measured by stimulating the eye with white light in a range that stimulates rod photoreceptors. Mice were dark-adapted in a dark room for 24 hours before measurement, and measurements were also performed in a dark room. During the procedure, the eye was dilated with a pupil dilator, and then an electrode was placed on the eye on a pad maintained at a warm temperature under anesthesia, and the electrophysiological response of the eye to light was measured.

[result]

Figure 9 is a graph of the electrophysiological response of the eye showing recovery after administration of extracellular vesicles into the vitreous body of the eye.

In Figure 9, the regular physiological potential change of the eye obtained by stimulation of -4 dB and -2 dB white light in the dark-adapted mouse eye is expressed as time ms (mili seconds) on the X axis and the change value as uV (micro) on the Y axis. volts).

9, systemic administration of sodium iodate (40 mg/kg) completely paralyzes the electrophysiological functions of ocular rod photoreceptor cells and retinal bipolar cells, but mesenchymal stem cell extracellular vesicles (MSC-) according to the present invention It can be confirmed that intravitreal administration of Ev) significantly improves its function compared to normal.

Test Example 8: Improvement of rod photoreceptor cell and retinal bipolar cell function following extracellular vesicle administration in dry macular degeneration

Rod photoreceptors are cells in the retina that first perceive light and transmit signals. Recognition of light by rod photoreceptor cells and transmission to retinal bipolar cells by administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) in an animal model of macular degeneration damaged by sodium iodate This is the measurement value for At this time, the closer the measured value is to normal, it means that the electrophysiological functions of damaged rod photoreceptors and retinal bipolar cells are restored.

[method]

Four hours after inducing macular degeneration, an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) was administered intravitreally, and at one week, an electroretinogram (ERG), a non-invasive ophthalmic device, was used. The physiological response of the eye was measured by stimulating the eye with a very faint light that can stimulate rod photoreceptors. The above electrophysiological graph waveform is expressed numerically. a-wave is located on the left side of the graph waveform, and b-wave is the absolute value (uV) moving in the negative direction on the Y-axis, and b-wave is the absolute value (uV) of the bottom and top vertices of the waveform. This is the measured difference (uV).

a-wave is the electrophysiological response caused by light in rod photoreceptor cells, and b-wave represents the response in bipolar cells transmitted from rod photoreceptor cells. In other words, the a-wave response is the response value of the rod photoreceptor cell after the first recognition of light in the retina, and the b-wave is the signal transmitted from the first cell, the rod photoreceptor cell, to the next transmitting cell, the retinal bipolar cell. -Indicates the synpse response.

[result]

Figure 10 shows changes in the potential difference of the a-wave and b-wave of the electrophysiological graph waveform measuring the physiological response of the eye to light stimulation (-4 dB, -2 dB) after administration of extracellular vesicles in the vitreous body of the eye. At this time, the analysis of a-wave and b-wave waveforms can be said to be the first signal recognition (a-wave) and transmission (b-wave) of the retina by physical light.

According to Figure 10, sodium iodate reduces the response of rod photoreceptors to light stimulation (-4 dB, 2 dB), but administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) It can be seen that the electrophysiological function of the damaged rod photoreceptor cells is improved. This value is not equivalent to normal, which is a complete recovery, and is different from recovery to the normal state. However, it is a statistically significant value and significantly increases the light perception function of the retina, which is offset in the macular degeneration model. This improvement is achieved with a single dose. It's a dramatic recovery.

Establishment of mouse reticular degeneration model

The following describes the establishment of a retinal degeneration model using mouse BLAB/C, which, in contrast to the dry macular degeneration model, induces only degeneration of the photoreceptor cell layer without causing damage to retinal pigment epithelial cells. .

Experiments to establish an animal model for retinal degeneration

Retinal degeneration was induced in the retinal photoreceptor layer by systemic intraperitoneal administration of cisplatin (20 mg/kg) to BALB/C mice [see FIG. 11]. Cisplatin is an anti-cancer treatment, and eye side effects such as permanent retinal damage/degeneration in patients have been reported, and animal testing has confirmed that the same or similar results are obtained.

Figures 11(a) to (d) are for experiments on establishing an animal model for retinal degeneration. Figure 11(a) is a photograph of the eye retina of a vertical section stained with hematoxylin-eosin (H & E), showing retinal damage by cisplatin administration. It shows that degeneration is induced in the photoreceptor layer. 11(b) is a plan view of the retinal pigment epithelial cell layer of the eye, showing that eye damage caused by cisplatin administration does not affect the retinal pigment epithelial cells. Additionally, Figure 11(b) shows that cisplatin administration induces subretinal migration of immune cells (macrophages and microglia). That is, Figure 11(a, b) shows that administration of cisplatin specifically induces degeneration of photoreceptor cells and increases subretinal immune cells without damaging the retinal pigment epithelial cell layer.

Figure 11(c) shows that administration of cisplatin significantly increases the expression of the CXCL10 chemokine, and Figure 11(d) shows that administration of cisplatin significantly increases the expression of various inflammatory cytokines and chemokine genes (ICAM1, CCL2, CCL3, CCL5, CCL7) in the eye. ) is a graph showing that it induces the expression of

Experimental administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) in a retinal degeneration animal model

Animal models of retinal degeneration specifically cause damage to photoreceptor cells, increase intraocular inflammation-related cytokine and chemokine gene expression, and lead to an increase in immune cells in the subretinal region. Therefore, one day after inducing retinal degeneration in mice, the efficacy of extracellular vesicles as a drug was studied by observing the retina on the 4th day after systemic administration of extracellular vesicles by intravenous injection.

The intravenous composition containing the mesenchymal stem cell extracellular vesicles isolated through Test Example 1 was prepared with 100 ug of isolated extracellular vesicles, 1 ml of PBS, and pH 7.3. At this time, the test was performed by intravenous systemic administration (200 ul) to mice with induced retinal degeneration according to the schedule in FIG. 12. Upon administration, the appropriate concentration was diluted with PBS and administered intravenously.

In the case of mice with induced retinal degeneration, 0.01 to 1 ug of extracellular vesicles were administered intravenously on the first day, and the degree of retinal degeneration was observed on the fourth day through vertical sectioning and planar staining.

At this time, the test was performed based on the intravenous administration schedule for retinal degeneration-induced mice in Figure 12.

Test Example 9: Confirmation of treatment of photoreceptor cell degeneration by administration of extracellular vesicles in a mouse model of eye retinal disease

[method]

The composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) prepared above was administered intravenously systemically to an animal model of retinal degeneration to confirm its therapeutic effect on eye retina disease and photoreceptor cell degeneration.

[result]

Figure 13(a) is a normal image of a vertical section of the retina stained with hematoxylin-eosin to show the therapeutic effect of extracellular vesicle administration, and Figure 13(b) is a graph measuring and quantifying the number of subretinal locations of non-specific cells. see.

13(a), in a retinal degeneration model, distribution of non-specific cells in the retinal photoreceptor cell layer (ONL) through systemic intravenous administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) It can be seen that the distribution of non-specific cells in the subretinal area is restored. This can be quantified by measuring the number of subretinal locations of non-specific cells in Figure 13(b). Systemic administration of 0.1 to 1 ug of extracellular vesicles was effective in recovering retinal degeneration, and systemic administration of at least 0.1 ug was effective in recovering retinal degeneration. Additional systemic intravenous administration of 0.01 ug had no significant effect on retinal efficacy.

Test Example 10: Efficacy on inhibition of subretinal immune cells increased by administration of extracellular vesicles in an ocular retinopathy mouse model

[method]

The ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) prepared above was administered intravenously systemically to an animal model of retinal degeneration to confirm its therapeutic effect on ocular retinal disease and inhibition of increased immune cells. .

[result]

Figure 14 is an image showing that the increase in the number of subretinal immune cells (macrophages and microglia) induced by cisplatin drug treatment is suppressed when administered according to changes in the concentration of extracellular vesicles. At this time, extracellular vesicles were administered at concentrations of 1x10 ⁷ (0.01 ug), 1x10 ⁸ (0.1 ug), 5x10 ⁸ (0.5ug), and 1x10 ⁹ (1 ug).

14, in a retinal degeneration model, systemic intravenous administration of an ocular composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) inhibits the increase in subretinal macrophages and microglia by cisplatin. . Systemic administration of at least 0.1 ug slightly inhibited the increase in immune cells, but 1 ug inhibited it more.

In addition, additional systemic intravenous administration of 0.01 ug failed to suppress not only the degeneration of photoreceptor cells but also the increase in immune cells. Stem cell extracellular vesicle drugs showed efficacy in recovering retinal degeneration in the eye when administered systemically at 0.1 ug to 1 ug. This is ~20 times the amount administered intravitreally in the eye.

These results show that systemic intravenous administration of the composition containing mesenchymal stem cell extracellular vesicles (MSC-Ev) according to the present invention has a therapeutic effect on eye retina diseases and photoreceptor cell degeneration. .

Claims (17)

A composition for preventing or treating posterior ocular diseases or damage, comprising as an active ingredient extracellular vesicles derived from stem cells or isolated from their culture medium. According to paragraph 1,
A composition for preventing or treating posterior ocular disease or damage, wherein the stem cells are mesenchymal stem cells. According to paragraph 2,
The mesenchymal stem cells are induced pluripotent stem cells (iPSC) derived from human or animal tissue, autologous and allogeneic mesenchymal stem cells, or cell lines derived from mesenchymal stem cells, and prevent diseases or damage in the posterior ocular region. or therapeutic composition. According to paragraph 3,
A composition for preventing or treating posterior ocular disease or damage, wherein the human or animal tissue is selected and isolated from any one or more of bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood fluid, and amniotic fluid. According to paragraph 1,
A composition for preventing or treating posterior ocular disease or damage, wherein the extracellular vesicles have an average particle diameter of within 50 nm to 1000 nm. According to paragraph 1,
The extracellular vesicles include exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. A composition for preventing or treating posterior ocular disease or damage, comprising any one or more of vesicles. According to paragraph 1,
The extracellular vesicles are exosomes, wherein the exosomes have CD9, CD63, and CD81 membrane protein markers. A composition for preventing or treating posterior ocular disease or damage. In clause 7,
A composition for preventing or treating posterior ocular disease or damage, wherein the ratio of CD63/CD9 among the membrane protein markers satisfies 2.5 or more. In clause 7,
The exosome is a composition for preventing or treating posterior ocular disease or damage, wherein the number of exosomes isolated per 1 L of culture medium is 1x10 ⁹ to 1x10 ¹² and the protein amount is 0.1 mg to 20 mg. According to paragraph 1,
The extracellular vesicles include culturing stem cells to produce extracellular vesicles; Culturing the extracellular vesicles; and a composition for preventing or treating posterior ocular disease or damage, which is produced through a step of isolating extracellular vesicles. According to clause 10,
The separation process is an aqueous solution biphasic separation method, ultracentrifugation method, antibody affinity separation method, polymer precipitation method, filtration method, or tangential flow filtration method. A composition for preventing or treating posterior ocular disease or damage. According to paragraph 1,
The composition is a composition for preventing or treating posterior ocular disease or damage, containing 0.0001 to 95% by weight of extracellular vesicles within 100% by weight of the total composition. According to paragraph 1,
The posterior ocular diseases include degenerative macular degeneration, retinal degeneration, retinitis pigmentosa, Levett's congenital amaurosis, Stargardt disease, Usher syndrome, Rod-Cone dystrophy, Cone-Rod dystrophy, ciliopathies, mitochondrial disorders, or retinal diseases. A composition for preventing or treating atrophy, posterior ocular disease or damage. According to paragraph 1,
A composition for preventing or treating posterior ocular disease or damage, wherein the posterior ocular damage is macular and retinal damage occurring during anticancer treatment. According to paragraph 1,
A composition for preventing or treating posterior ocular disease or damage, wherein the posterior ocular damage is traumatic and iatrogenic retinal damage caused by exposure to strong light or ultraviolet rays. According to paragraph 1,
The composition may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, intravitreal, or intradermal routes. Composition for preventing or treating posterior disease or injury. In article 11,
The composition is for the prevention or treatment of posterior ocular diseases or damage, which is any one dosage form selected from the group consisting of eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, drops and solutions. Composition.