Method and composition for preventing or treating myopia

Abstract

The present invention aims to provide a method for preventing and treating myopia and a composition used therein. By the present invention, a method and a composition for suppressing, improving, or treating myopia are provided by administering substances that regulate the function of choroid-resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria. Inducing polarization of M2 macrophages in the choroid, or regulating the function of choroid-resident immune cells, such as administering mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria, is effective for preventing and treating myopia.

Description

Technology Field

[0001] The present invention relates to a method and composition for preventing or treating myopia, and more specifically, to a composition that acts on immune cells in the choroid to inhibit the progression of myopia, and more specifically, to a method and composition that modulates the function of choroid-resident immune cells, such as by inducing polarization of M2 macrophages or administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. Background Technology

[0002] Myopia refers to a condition in which the focus is formed in front of the retina due to elongation of the eyeball's axial length; the longer the axial length, the more severe the myopia. With the increasing prevalence of myopia, interest is growing in the factors involved in its development as well as in inhibiting its progression and preventing it. Recent studies suggest that the choroid plays a crucial role in regulating eyeball growth and the development of myopia, and that choroidal thinning is a structural characteristic of myopia. Since there is a negative correlation between choroidal thickness and axial length, it is suggested that changes in choroidal thickness may serve as a predictive biomarker for axial elongation. However, the detailed mechanisms by which the choroid is involved in the development or progression of myopia remain unclear.

[0003] The choroid is a tissue that covers the outer surface of the retina and is rich in fine blood vessels. In addition to supplying oxygen and nutrients to retinal cells, it also supplies growth factors involved in the tissue remodeling of the sclera on the outer surface of the eye and the regulation of eye growth. It is believed that a decrease in choroidal thickness or blood flow contributes to scleral ischemia and hypoxia, thereby influencing changes in the scleral structure that cause the elongation of the eye's axial length. Therefore, maintaining or increasing choroidal thickness and blood flow is attracting attention as a new target for the prevention and treatment of myopia.

[0004] Methods to inhibit the progression of myopia by maintaining or increasing the thickness of the choroid have been proposed, for example, crocetin intake (see Non-patent Literature 1) and irradiation with violet light (Non-patent Literature 2).

[0005] Crocetin is known to have anti-inflammatory and immunomodulatory effects, and it is known to exert these effects by regulating the balance of Th1 / Th2 and Th17 / Treg in T cells or by inhibiting the NF-κB pathway in cells that produce inflammatory mediators (TNF-α, IL-6, IFN-γ, etc.), such as macrophages (Non-patent Literature 3-5).

[0006] Thirteen types of cells exist in the choroid, four of which are immune cells. Tissue-resident immune cells are responsible for tissue homeostasis and the maintenance of structure in addition to immune responses; for example, it has been reported that the choroid becomes thin in mice deficient in macrophages or in mice in which mast cell degranulation is induced (Non-patent Literature 6-7). Prior art literature

[0007] Mori et al., Sci Rep. 2019. 22;9(1):295.Jiang et al. Proc Natl Acad Sci USA. 2021. 1;118(22):e2018840118.Oxid Med Cell Longev. 2021 Sep 10;2021:6631929.Eur J Pharmacol. 2012 Jan 15;674(2-3):391-6.Biofactors. 2023 Feb 6. doi: 10.1002 / biof.1942.Elife. 2020 Apr 1;9:e55564.FASEB J. 2020 Aug;34(8):10117-10131. The problem to be solved

[0008] The present disclosure aims to provide a method for preventing or treating myopia and a composition used therein, in particular a composition targeting choroidal resident immune cells, in particular macrophages or mast cells. means of solving the problem

[0009] The inventors have discovered that the polarization of macrophages in the choroid to M2 macrophages inhibits the thinning of the choroid, making it effective for the prevention and treatment of myopia.

[0010] Furthermore, mast cells are widely distributed around small blood vessels, and their presence has been confirmed in the choroid, a tissue composed of blood vessels, suggesting their involvement in maintaining the choroid's shape. Accordingly, the inventors discovered that the progression of myopia is inhibited by the eye administration of a drug (mast cell stabilizer) that inhibits the degranulation of mast cells. Specifically, the inventors found that the progression of myopia is inhibited by the eye administration of a mast cell stabilizer (sodium chromoglate, potassium pemirorast), a type of anti-allergic drug, to mice with a myopia model induced by wearing concave lenses. Additionally, when another anti-allergic drug, an antihistamine (levocabastine), was eye administration to mice undergoing myopia induction by wearing concave lenses, inhibition of myopia progression was not confirmed.

[0011] Furthermore, it is known that lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, induce and activate polarization of M2 macrophages upon administration. Accordingly, the inventors have discovered that the administration of lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, inhibits the progression of myopia. Considering these points together with the findings of the aforementioned non-patent literatures 6 and 7, it is believed that if the choroidal structure (thickness) can be maintained through intervention targeting immune cells residing in the choroid, it will act inhibitorily against the progression of myopia. Based on these findings, it is believed that a composition that targets immune cells present in the choroid, such as macrophages or mast cells, and induces their respective properties to an appropriate state, possesses an effect that inhibits the progression of myopia. The present invention is based on such findings and includes the following aspects.

[0012] [Sun 1]

[0013] A method for suppressing, improving, or treating myopia, comprising administering a therapeutically effective amount of a substance that modulates the function of choroidal resident immune cells or lactic acid bacteria to a subject requiring treatment.

[0014] [Sun 2]

[0015] A method described in Sun 1 in which a substance regulating the function of choroidal resident immune cells is an M2 macrophage polarization-inducing substance, a mast cell stabilizer, or a chemical mediator release inhibitor.

[0016] [Sun 3]

[0017] A method described in Sun 2 in which the M2 macrophage polarization-inducing substance is selected from the group consisting of proteins, peptides, nucleic acids, low-molecular-weight compounds and high-molecular-weight compounds.

[0018] [Sun 4]

[0019] The method described in Example 2, wherein the M2 macrophage polarization-inducing substance is selected from the group consisting of IL-4, IL-10, IL-13, TLR 2, 4, 7, 9 and their ligands, arsentren dihydrochloride, triptolide, lovastatin, QS11, regorafenib, sorafenib, ixazomib, GW-843682X, KW 2449, axitinib, JTE 013, purmorphamine, arcyriaflavin A, dasatinib, NVP-LDE225, 1-naphthyl PP1, MGCD-265, and bosutinib.

[0020] [Sun 5]

[0021] A method described in Sun 2 in which a mast cell stabilizer, or a chemical mediator glass inhibitor, is chromoglic acid, or pemirorast, or a salt thereof.

[0022] [Sun 6]

[0023] A method described in Sun 1 in which the lactic acid bacteria is of the genus Lactobacillus.

[0024] [Sun 7]

[0025] A method described in Sun 1 in which the lactic acid bacteria is Lactobacillus paracasei.

[0026] [Sun 8]

[0027] A method described in Sun 1 in which a substance that regulates the function of choroidal resident immune cells, or lactic acid bacteria, is included in a pharmaceutical composition, supplement, or food.

[0028] [Sun 9]

[0029] A method described in Sun 1 in which the reduction of refraction, elongation of the ocular axis and / or non-thinning of the choroid is inhibited.

[0030] [Sun 10]

[0031] A composition for suppressing or treating myopia containing a therapeutically effective amount of a substance that modulates the function of choroidal resident immune cells, or lactic acid bacteria.

[0032] [Sun 11]

[0033] A supplement for suppressing or improving myopia containing an effective amount of a substance that regulates the function of choroidal resident immune cells, or lactic acid bacteria.

[0034] [Sun 12]

[0035] A food for suppressing or improving myopia containing an effective amount of a substance that regulates the function of choroidal resident immune cells, or lactic acid bacteria.

[0036] [Sun 13]

[0037] Use of a substance that regulates the function of choroidal resident immune cells, or lactic acid bacteria, in the manufacture of a medicine for suppressing or treating myopia.

[0038] [Sun 14]

[0039] Use of substances that regulate the function of choroidal resident immune cells, or lactic acid bacteria, to suppress or treat myopia. Effects of the invention

[0040] According to the present disclosure, a new method for the prevention and treatment of myopia can be provided by adjusting the function of choroidal resident immune cells, more specifically by inducing polarization of M2 macrophages in the choroid, or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. Brief explanation of the drawing

[0041] Figure 1 shows the results of myopia induction by LPS administration. (A) is a diagram showing the experimental schedule. (B) is a graph showing changes in refractive value (left), ocular axial length (center), and choroidal thickness (right). Figure 2 is a graph showing the changes in expression of M1 macrophage marker genes and oxidative stress-related genes due to LPS administration. Figure 3 shows the results of M2 macrophage polarization induction by IL-4 administration. (A) is a diagram showing the experimental schedule. (B) is a graph showing the changes in expression of CD206 protein and phosphorylated STAT6 in the choroid by IL-4 administration. (C) is a graph showing the changes in mRNA expression of CD163 and CD206 by IL-4 administration. Figure 4 is a graph showing the changes in expression of M2 macrophage marker genes and oxidative stress-related genes due to IL-4 administration. Figure 5 shows the results of an experiment on inhibiting myopia by IL-4 administration. (A) is a diagram showing the experimental schedule. (B) is a diagram showing the changes in refraction (left), axial length of the eyeball (center), and choroidal thickness (right) by IL-4 administration during the myopia induction period. Figure 6 shows the results of inhibiting myopia and inducing M2 macrophage polarization by IL-13 administration. (A) is a diagram showing the experimental schedule. (B) is a graph showing changes in refraction (top left), ocular axial length (top right), choroidal thickness (bottom left), and blood flow (bottom right) by IL-13 administration during the myopia induction period. (C) is a graph showing changes in the number of macrophages and the ratio of M2 macrophages by myopia induction and IL-13 administration. Figure 7 shows the results regarding the myopia-inhibiting effect of Mast Cell Stabilizer eye drops. (A) is a graph showing the change in ocular axial length. (B) is a graph showing the change in refractive value. (C) is a graph showing the change in choroidal thickness. In each case, the left side represents the control group vs. the pemirorast potassium eye drop group, and the right side represents the control group vs. the chromoglic acid eye drop group. *p<0.05, **p<0.01, ***p<0.001, Student's t-test Figure 8 is a graph comparing the myopia-inhibiting effects of Mast Cell Stabilizer eye drops and histamine receptor inhibitors. (A) is a graph showing the change in ocular axial length. (B) is a graph showing the change in refractive value. (C) is a graph showing the change in choroidal thickness. From left to right, they represent the control group, the levocavastin eye drop group (histamine receptor inhibitor), and the pemirorast potassium eye drop group (mast cell stabilizer). *p<0.05, **p<0.01, ***p<0.001, Student's t-test Figure 9 shows the results of the inhibitory effect on myopia progression by the administration of Lactobacillus paracasei. (A) is a graph showing the change in ocular axial length. (B) is a graph showing the change in refractive value. (C) is a graph showing the change in choroidal thickness. Each, the left side represents the control group, and the right side represents the group administered Lactobacillus paracasei. Specific details for implementing the invention

[0042] Details according to the present invention will be described. The present invention is not limited to the contents of the following embodiments and examples, and includes various modifications and applications within the scope that includes the gist of the present invention.

[0043] [Methods to suppress or treat myopia]

[0044] As mentioned above, it has recently become evident that a change known as non-thinning of the choroid is involved in the development of myopia. As a result of conducting thorough research on factors controlling this non-thinning, the inventors discovered that changes in the state (polarization) of macrophages in the choroid cause a change in the thickness of the choroid, thereby controlling the progression of myopia.

[0045] Macrophages are classified into M1 and M2 macrophages; while M1 macrophages are primarily involved in inducing inflammation, M2 macrophages exert an antagonistic effect involved in the termination and inhibition of inflammation. The inventors have discovered for the first time that inducing polarization to M1 macrophages in the choroid leads to choroidal thinning and progression of myopia, whereas inducing polarization to M2 macrophages inhibits choroidal thinning and progression of myopia. Furthermore, since mast cells are widely distributed around small blood vessels and their presence has been confirmed in the choroid—a tissue composed of blood vessels—suggesting their involvement in maintaining the choroid's shape, the inventors have discovered that the progression of myopia is inhibited by the instillation of an eye drop agent (mast cell stabilizer) that inhibits the degranulation of mast cells. In addition, it is known that lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, induce and activate polarization of M2 macrophages upon administration. The inventors of the present invention have discovered that the administration of lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, inhibits the progression of myopia. Thus, the inventors have discovered that the progression of myopia can be inhibited by adjusting the function of choroidal resident immune cells.

[0046] Accordingly, one aspect of the present disclosure relates to a method for inhibiting, improving, or treating myopia by adjusting the function of choroidal resident immune cells, more specifically by polarizing macrophages in the choroid to M2 macrophages, or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria.

[0047] One aspect of the present disclosure relates to a method for suppressing, improving, or treating myopia by modulating the function of choroidal resident immune cells, more specifically by using an M2 macrophage polarization-inducing substance to induce polarization of M2 macrophages in the choroid, or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria.

[0048] One aspect of the present disclosure relates to a method for inhibiting refractive loss, elongation of the ocular axis, and / or non-thinning of the choroid in a subject requiring treatment, comprising modulating the function of choroidal resident immune cells, more specifically delivering an M2 macrophage polarization-inducing substance to the subject choroid, or administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria.

[0049] In addition, one aspect of the present disclosure relates to a screening method for searching for substances that regulate the function of choroidal resident immune cells, such as components that induce polarization of M2 macrophages to control the non-thinning of the choroid, which is a mechanism of myopia development, and a screening method for searching for components effective in the prevention and treatment of myopia.

[0050] <Substances Regulating the Function of Choroidal Resident Immune Cells>

[0051] Substances that regulate the function of choroidal resident immune cells are substances that regulate the function of choroidal resident immune cells. Preferably, substances that regulate the function of choroidal resident immune cells include substances that regulate inflammation in the choroid and substances that regulate the choroid to become an anti-inflammatory environment. Examples of choroidal resident immune cells include macrophages and mast cells. Examples of substances that regulate the function of choroidal resident immune cells include M2 ​​macrophage polarization-inducing substances, mast cell stabilizers, chemical mediator release inhibitors, and lactic acid bacteria.

[0052] <M2 Macrophage Polarization Inducing Substance>

[0053] Macrophages are classified into inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which differ in function, and they polarize in response to signals from cytokines or stimulating components to express specific functions.

[0054] Accordingly, the M2 macrophage polarization-inducing substances in the present disclosure include various substances involved in signal transmission that induces polarization of M2 macrophages in the choroid, and proteins, peptides, nucleic acid drugs, small molecule compounds, large molecule compounds, etc., can be used without particular limitation.

[0055] Examples include cytokines such as IL-4 and IL-13 produced by Th2 cells, as well as the leucine zipper transcription factor c-Maf and the carbohydrate-binding lectin galectin-3. In addition, it is known that there are several forms of M2 macrophages, and in addition to the aforementioned IL-4 and IL-13 that induce M2a macrophages, IL-10 and glucocorticoid hormone (GC) involved in the induction of M2c macrophages, and TLR2, TLR4, TLR7, TLR9 and their ligands (ligands for TLRs) involved in the induction of M2d macrophages can also be cited as M2 macrophage polarization-inducing substances in the present disclosure.

[0056] In addition, examples of M2 macrophage polarization-inducing substances in the present disclosure include various small molecule compounds, such as arsentren dihydrochloride, triptolide, lovastatin, QS11, regorafenib, sorafenib, ixazomib, GW-843682X, KW 2449, axitinib, JTE 013, purmorphamine, arcyriaflavin A, dasatinib, NVP-LDE225, 1-naphthyl PP1, MGCD-265, bosutinib, etc.

[0057] As an M2 macrophage polarization-inducing substance, an agonist of the above substance may also be used. Furthermore, those skilled in the art may obtain M2 macrophage polarization-inducing substances suitable for use in the methods and compositions disclosed herein, in addition to the M2 macrophage polarization-inducing substances specifically described in this specification, by culturing cells such as monocyte cell lines including RAW264 cells, J774 cells, and U937 cells, mouse intraperitoneal macrophages, and bone marrow-derived macrophages in a culture medium containing the substance to be evaluated, and by using an in vitro evaluation system that evaluates the expression of M2 macrophage markers, thereby evaluating whether the substance possesses the ability to induce M2 macrophage polarization.

[0058] If the M2 macrophage polarization-inducing substance is a protein or peptide, it may be administered to the target in the form of DNA or RNA encoding them. The nucleic acid encoding the M2 macrophage polarization-inducing substance may be administered to the target using a plasmid or an expression vector. The expression vector may be, for example, a viral vector, particularly an adenovirus vector, but is not limited thereto. Other usable viral vectors include, for example, retroviruses, adeno-associated viruses, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, Geminivirus, and carrimovirus vectors. The nucleic acid encoding the M2 macrophage polarization-inducing substance may contain a regulatory element for specifically expressing the protein in the choroid or RPE. That is, the nucleic acid encoding the M2 macrophage polarization-inducing substance may be operably linked to a regulatory element such as a promoter or enhancer.

[0059] <Masked Cell Stabilizer>

[0060] A mast cell stabilizer is also referred to as a mast cell stabilizer or a mast cell stabilizing drug. A mast cell stabilizer is, for example, a drug that inhibits the release of allergens from mast cells; specifically, it is a drug that inhibits the release of allergens from mast cells by stabilizing the cell membrane of the mast cells. Examples of mast cell stabilizers include asitazanolast hydrate (Zeferin), amlexanox (Erix), femirolast potassium (Allegisal), femirolast potassium (Femirastone), sodium cromoglobin (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonist, cromoglobin sodium, cromoglobin, ketotifen, methylxanthine, omalizumab, femirolast, quercetin, etc., and preferably, cromoglobin or femirolast or its salts, etc.

[0061] <Chemical Mediator Glass Inhibitor>

[0062] Chemical mediator release inhibitors are drugs that suppress allergic reactions by inhibiting the release of chemical mediators from immune cells, such as mast cells, for example; they are drugs that stabilize the cell membrane of mast cells to prevent histamine, etc. from being released to the outside, for example; and they are drugs that inhibit the release of allergens (substances that cause allergic reactions), such as histamine, LTB4, LTC4, LTD4, PGD2, TXB2, PAF, etc., from mast cells. Examples of chemical mediator release inhibitors include asitazanolast hydrate (Zeferin), amlexanox (Erix), femirolast potassium (Allegisal), femirolast potassium (Pemirastone), sodium cromoglobin (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonists, cromoglobin sodium, cromoglobin, ketotifen, methylxanthine, omalizumab, femirolast, quercetin, etc., and preferably, cromoglobin or femirolast or its salts, etc.

[0063] <Lactic Acid Bacteria>

[0064] Examples of lactic acid bacteria include homolactic acid bacteria and heterolactic acid bacteria. Additionally, examples of lactic acid bacteria include spherical lactic acid cocci and rod-shaped lactic acid bacilli. Examples of lactic acid bacteria include Gram-positive bacteria that are bacilli or cocci, do not have spores, are non-motile, produce more than 50% lactic acid relative to the glucose consumed, and require niacin (B3). Examples of lactic acid bacteria include enteric lactic acid bacteria, animal lactic acid bacteria, plant lactic acid bacteria, and marine lactic acid bacteria. Examples of lactic acid bacteria include lactic acid bacteria of the order Lactobacillus and lactic acid bacteria of the phylum Actinomycetes. Examples of lactic acid bacteria of the Lactobacillus order include lactic acid bacteria of the genus Lactobacillus, lactic acid bacteria of the genus Enterococcus, lactic acid bacteria of the genus Lactococcus, lactic acid bacteria of the genus Pediococcus, lactic acid bacteria of the genus Leuconostoc, and lactic acid bacteria of the genus Streptococcus. Examples of lactic acid bacteria of the Actinomycetes phylum include lactic acid bacteria of the genus Bifidobacterium.Lactic acid bacteria of the genus Lactobacillus include, for example, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus fructivorans, and Lactobacillus Examples include Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei Shirota. Examples of lactic acid bacteria of the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium. Examples of lactic acid bacteria of the genus Lactococcus include Lactococcus lactis and Lactococcus cremoris. Examples of lactic acid bacteria of the genus Pediococcus include Pediococcus damnosus.Examples of lactic acid bacteria of the genus Leuconostoc include Leuconostoc mesenteroides. Examples of lactic acid bacteria of the genus Streptococcus include Streptococcus thermophiles and Streptococcus mutans. Examples of lactic acid bacteria of the genus Bifidobacterium include Bifidobacterium bifidum and Bifidobacterium adolescentis.

[0065] It is known that the administration of Lactobacillus paracasei improves the symptoms of non-alcoholic fatty liver disease by shifting hepatic Kupffer cells (hepatic resident macrophages) to M2 (Sohn et al, Dig Dis Sci, 2015 Nov;60(11):3340-50). It is also known that the administration of Lactobacillus paracasei alleviates blue light-induced retinal degeneration by activating M2 macrophages (Morita et al, Nutrients, 2018 Dec15;10(12):1991). It is known that the administration of Lactobacillus plantarum alleviates colitis by inducing polarization from M1 macrophages to M2 macrophages (Jang et al, Int Immunopharmacol, 2014 Jul;21(1):186-92). It is also known that the administration of Lactobacillus brevis alleviates colitis by inducing polarization from M1 macrophages to M2 macrophages (Jang et al, J Appl Microbiol, 2013 Sep;115(3):888-96). As such, it is known that lactic acid bacteria of the Lactobacillus order, such as Lactobacillus, induce and activate polarization to M2 macrophages upon administration. Therefore, the lactic acid bacteria are preferably lactic acid bacteria of the Lactobacillus order, such as the genus Lactobacillus.

[0066] DDS may be used for the delivery of substances that regulate the function of choroidal resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediator release inhibitors, to the target choroid.

[0067] For example, if the choroidal resident immune cell function modulators, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, are nucleic acids, the DNA or RNA (messenger RNA) encoding these choroidal resident immune cell function modulators may be administered to the target using a DDS such as liposomes. Other available DDSs include charged lipids, nucleic acid-protein complexes, and biopolymers. Additionally, choroidal resident immune cell function modulators, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, may be administered to the target in the form of proteins. Administration may be performed, for example, by local administration to the choroid.

[0068] The delivery of substances that regulate the function of choroidal resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediator release inhibitors, to the target choroid may be performed by transplanting cells that secrete substances that regulate the function of choroidal resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediators, into the target. Cells that secrete substances that regulate the function of choroidal resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediators, may be, for example, retinal pigment epithelial cells (RPE). Alternatively, they may be cells that have been genetically modified to secrete substances that regulate the function of choroidal resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediators.

[0069] In the method for suppressing or treating myopia of the present disclosure, the subject may be a mammal including humans, a dog, a cat, a cow, or a horse, but preferably a human.

[0070] [Composition for inhibiting or treating myopia]

[0071] One aspect of the present disclosure relates to a composition for suppressing, improving, or treating myopia using a substance that modulates the function of choroidal resident immune cells, such as an M2 macrophage polarization-inducing substance, a mast cell stabilizer, or a chemical mediator release inhibitor, or lactic acid bacteria. More specifically, one aspect of the present disclosure relates to a composition for suppressing or treating myopia containing a therapeutically effective amount of a substance that modulates the function of choroidal resident immune cells, such as an M2 macrophage polarization-inducing substance, a mast cell stabilizer, or a chemical mediator release inhibitor, or lactic acid bacteria. Such a composition may be used to deliver a substance that modulates the function of choroidal resident immune cells, such as an M2 macrophage polarization-inducing substance, a mast cell stabilizer, or a chemical mediator release inhibitor, or lactic acid bacteria to a target choroid. Such a composition may be a pharmaceutical composition, a food, or a supplement.

[0072] The pharmaceutical composition according to the present disclosure is administered locally to the eye, for example. Examples of forms of administration of the composition include ophthalmic administration (including ophthalmic ointment and eye wash), subconjunctival administration, intraconjunctival administration, and subtenon's administration.

[0073] The formulation of the present composition is not particularly limited, but examples include eye drops, eye ointments, injections, adhesives, gels, inserts, etc., and for example, eye drops are preferred. In addition, these can be prepared using conventional techniques commonly used in the field.

[0074] Eye drops can be prepared by selecting and using, as necessary, isotonic agents such as sodium chloride, potassium chloride, and concentrated glycerin; buffering agents such as sodium phosphate, sodium acetate, and ypsilone-aminocaproic acid; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl stearate 40, and polyoxyethylene hardened castor oil; stabilizers such as sodium citrate and sodium edetate; and preservatives such as parabens. The pH may be within the acceptable range for ophthalmic preparations, but is typically preferably within the range of 4 to 8. Eye ointments can be prepared using common bases such as white petroleum jelly and liquid paraffin.

[0075] In addition, the pharmaceutical composition according to the present disclosure is not limited to administration to the local eye and may be administered via any route of administration, such as enteral administration (oral, tube, cervical, etc.) and parenteral administration (jugular vein, carotid artery, dermal, intramuscular injection, etc.). The dosage form of the composition used for these forms of administration can be appropriately selected, but may be, for example, a solid dosage form such as a tablet, granule, powder, capsule, or chewing tablet, or a liquid dosage form such as a liquid, syrup, injection, or drop.

[0076] In the pharmaceutical composition according to the present disclosure, other components may be incorporated in appropriate amounts to the extent that the effects of the present invention are not impaired. Examples of other components include any carrier, buffer, diluent, excipient, suspending agent, lubricant, adjuvant, medium, delivery system, emulsifier, tablet degrading agent, absorbent, preservative, surfactant, coloring agent, flavoring agent, or sweetener. These components may be incorporated as a single type or in an appropriate combination of two or more types.

[0077] The content ratio of choroidal resident immune cell function modulating substances, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria in 100% by weight of the pharmaceutical composition can be appropriately set in the range of, for example, 0.001 to 99.99% by weight.

[0078] There are no particular limitations on the dosage of the pharmaceutical composition according to the present disclosure, and it can be appropriately selected depending on the form of administration, the age and body weight of the subject, the degree of desired effect, etc. The dosage of a substance that regulates the function of choroidal resident immune cells, such as an M2 macrophage polarization-inducing substance, a mast cell stabilizer, or a chemical mediator release inhibitor, or a lactic acid bacterium, may be, for example, 100 to 1,000,000 nmol per day, preferably 150 to 1,000,000 nmol, and the frequency of administration may be, for example, 1 to 100 times per month.

[0079] In addition, the composition according to the present disclosure may be a food or a supplement. As for the form of the food or supplement, examples include liquid, solid, tablet, granular, powder, capsule, paste, gel, etc., and the above-mentioned solid or liquid formulations may be arbitrarily selected. Specific examples of the food include, for example, beverages such as fruit juice, vegetable juice, soft drinks, and tea; various general processed foods such as soup, pudding, yogurt, cake premix products, confectionery, cookies, candy, jelly, and gum; as well as special purpose foods, specific health foods, nutritional functional foods, functional foods, nutritional supplements, health supplements, nutritional fortified foods, nutritional adjustment foods, etc., such as supplements and drinks.

[0080] The food or supplement according to the present disclosure may include any functional material (vitamins, minerals, etc.), any excipient, and any additive (flavoring agent, sweetener, acidulant, coloring agent, thickener, binder, reinforcing agent, disintegrant, buffering agent, surfactant, solubility aid, reabsorption promoter, dispersant, stabilizer, gelling agent, emulsifier, antioxidant, surfactant, preservative, moisture-proof agent, pH adjuster, coloring agent, analgesic agent, isotonic agent, etc.).

[0081] One aspect of the present disclosure relates to the use of choroidal resident immune cell function modulating substances, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria, for suppressing or treating myopia. Additionally, another aspect of the present disclosure relates to the use of choroidal resident immune cell function modulating substances, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria, in the manufacture of medicines used for suppressing or treating myopia.

[0082] [Screening Method]

[0083] One aspect of the present disclosure relates to a screening method for searching for a component that induces polarization of M2 macrophages to control the non-thinning of the choroid, which is a mechanism of myopia development, and a screening method for searching for a component effective for the prevention and treatment of myopia.

[0084] In some embodiments, the screening method comprises (i) a process of administering a candidate substance to a model animal, and (ii) a process of measuring the ocular axial length, choroidal thickness, and refractive index in the model animal. In some embodiments, the screening method comprises (i) a process of administering a candidate substance to a model animal, and (ii) a process of measuring the expression of choroidal resident immune cell markers, such as M2 macrophage markers, in a sample derived from the model animal. In some embodiments, the screening method further comprises (iii) a process of measuring the expression of choroidal resident immune cell markers, such as M1 macrophage markers.

[0085] In addition, in some embodiments, the method also includes an in vitro screening method for evaluating the expression of choroidal resident immune cell markers, such as M2 macrophage markers, by culturing cells, such as monocyte cell lines including RAW264 cells, J774 cells, and U937 cells, and choroidal resident immune cells such as mouse intraperitoneal macrophages and bone marrow-derived macrophages, in a medium containing a candidate substance.

[0086] In some embodiments, the model animal may be an animal undergoing myopia-inducing treatment.

[0087] In some embodiments, choroidal resident immune cell markers such as M2 macrophage markers may be CD163, CD206, arginase, and IL-10. Methods for measuring choroidal resident immune cell markers such as macrophage markers may include quantitative PCR, flow cytometry, immunohistochemistry, luciferase assay, and arginase activity staining.

[0088] Substances identified by such screening methods can be used to suppress or treat myopia.

[0089] Although preferred embodiments of the present invention are described herein, it is obvious to those skilled in the art that such embodiments are provided merely for illustrative purposes, and that those skilled in the art will be able to make various modifications, changes, and substitutions without departing from the present invention. It should be understood that various alternative embodiments of the invention described herein may be used when practicing the present invention. Furthermore, since this application claims the benefit of priority of the Japanese patent application with application number 2022-72596, the contents of said Japanese patent application and the contents described in all publications, including patents and patent application documents referenced in this specification, should be interpreted as being introduced by reference in the same manner as the contents specified in this specification.

[0090] (Example)

[0091] The present invention will be explained in more detail below through experimental examples.

[0092] <Test Example 1: Choroidal Thinning and Myopia Caused by LPS Administration>

[0093] To determine whether polarization by M1 macrophages is involved in the non-thinning and myopia of the choroid, the expression of polarization marker genes in the choroid, as well as choroidal thickness, ocular axial length, and refractive values, were measured after LPS administration.

[0094] The choroidal thickness, ocular axial length, and refractive value were measured in mice administered LPS for 2 weeks and control mice administered PBS, and the changes were calculated (Fig. 1A, B). Afterward, the eyes were enucleated, and the expression of polarization marker genes was measured using quantitative PCR (Fig. 2).

[0095] C57BL6J mice were used (n=4 in each group).

[0096] In the LPS administration group, LPS solution was administered intraperitoneally daily at a dose of 10 mg / kg BW.

[0097] PBS was administered to the PBS administration group instead of the LPS solution.

[0098] (Measurement of eye axis length, choroid thickness, and refractive power)

[0099] The ocular axial length, choroidal thickness, and refractive index of mice in each group were measured. The ocular axial length and choroidal thickness were measured using spectral domain optical coherence tomography (Envisu R4310, Leica). The refractive index was measured using an infrared photorefractor for mice (infrared photorefractor for mice, manufactured by Professor Schaeffel of the University of Tubingen).

[0100] (Observation of the choroid by electron microscopy)

[0101] The eyes of mice from each group were collected, fixed overnight at 4°C in 2.5% glutaraldehyde in PBS (Phosphate Buffered Saline), and rinsed for 1 hour with 0.1M sodium cacodylate buffer. Next, they were fixed for 2 hours in 1% OsO4 in 0.1M cacodylate buffer, followed by dehydration with stepwise ethanol solutions. Additionally, the eyes were infiltrated overnight in a 1:2 mixture of propylene oxide and epon-araldite and embedded in 100% resin. The blocks were cut and observed using a transmission electron microscope (JEM1400 plus; JEOL) at an acceleration voltage of 100kV.

[0102] (Evaluation of gene marker expression)

[0103] As described above, choroid, retina, and sclera samples were collected from mice administered LPS for 2 weeks, and the expression of M1 macrophage marker genes and oxidative stress-related genes was analyzed using quantitative PCR.

[0104] (result)

[0105] For the control group administered PBS, a decrease in refraction (left side of Fig. 1B), elongation of the ocular axis (center of Fig. 1B), and thinning of the choroid (right side of Fig. 1B) were observed in the LPS administration group during either the 1-week or 2-week administration period.

[0106] In addition, as a result of gene expression analysis, upexpression of M1 marker genes and oxidative stress-related genes was confirmed, particularly in the choroid (Fig. 2).

[0107] As shown in these results, it was confirmed that an increase in M1 macrophages in the choroid induces myopia.

[0108] <Test Example 2 Polarization and Myopia Inhibitory Effects on M2 Macrophages by Administration of IL-4 and IL-13>

[0109] While M1 macrophages are primarily involved in inducing inflammation, M2 macrophages have an antagonistic effect involved in terminating or suppressing inflammation. Since M1 macrophages have a myopia-inducing effect, M2 macrophages are thought to act inhibitorily on myopia and its progression.

[0110] Therefore, IL-4, a cytokine required for polarization toward M2 macrophages, was administered to mice, and whether polarization toward M2 occurred in the choroid was determined by analyzing the expression levels of M2 macrophage markers CD163 and CD206 (Fig. 3A, B).

[0111] <IL-4 Administration>

[0112] 0.1 μg / 100 μl (10 μg / kg BW) of IL-4 solution was administered intraperitoneally to C57BL6J mice (n=4 in each group), and choroid, retina, and liver samples were collected at 0 hours (before administration) and at 4, 24, and 48 hours after administration, and the expression of M2 macrophage marker genes was analyzed by Western blot and real-time PCR (Fig. 3). In addition, choroid and retina samples were collected at 0 hours (before administration) and at 4 and 24 hours after administration, and the expression of oxidative stress-related genes was analyzed by real-time PCR (Fig. 4).

[0113] In addition, IL-4 was administered to mice undergoing myopia induction, and whether there was a myopia-inhibiting effect was evaluated (Fig. 4).

[0114] 0.1 μg / 100 μl (10 μg / kg BW) of IL-4 solution was administered intraperitoneally to C57BL6 mice (-30D lens, n=4) undergoing myopia induction for 3 weeks. As a control group, C57BL6J mice (0D lens, -30D lens, n=4 in each group) undergoing myopia induction were used. After 3 weeks, refraction, elongation of the ocular axis, and choroidal thinning were measured in the same manner as in Test Example 1.

[0115] (result)

[0116] As a result, an increase in the expression levels of M2 markers CD163 and CD206 in the choroid was confirmed by the administration of IL-4 (Figs. 3B, C), confirming polarization toward M2 macrophages. In addition, gene expression analysis confirmed the upexpression of M2 macrophage marker genes and the suppression of oxidative stress-related genes (Fig. 5). From these results, it was suggested that the administration of IL-4 induces polarization toward M2 macrophages and suppresses the expression of oxidative stress-related genes, thereby inhibiting the progression of myopia (Fig. 5).

[0117] In addition, by administering IL-4 during the myopia induction period, inhibition of the decrease in refractive power (left side of Fig. 5B), inhibition of the elongation of the ocular axis (center of Fig. 5B), and inhibition of the non-thinning of the choroid (right side of Fig. 5B) were confirmed.

[0118] From the above results, it was confirmed that the polarization of choroidal macrophages to M2 by IL-4 administration exhibits a myopia-inhibiting effect.

[0119] <IL-13 Administration>

[0120] It was confirmed using C57BL6J mice that, similar to the results of IL-4, the elongation of the ocular axis, myopic refraction, and non-thinning of the choroid caused by wearing a concave lens were inhibited and polarization to M2 macrophages were induced by administering IL-13, a cytokine known to polarize macrophages to M2, similar to IL-4 (Fig. 6).

[0121] In addition, changes in blood flow and the measurement of the number of macrophages were performed as follows.

[0122] Changes in blood flow were measured using a wavelength sweep optical coherence tomograph (OCT S-1, Canon).

[0123] After digestion of the choroid, the macrophage count was measured using a flow spectrometer (CytoFLEX S, Beckman Coulter) after staining with F4 / 80 antibody and CD11b antibody. The M2 macrophage count was measured in the same manner as the macrophage count, except for the addition of the staining process with CD206 antibody.

[0124] As shown in the results of Test Examples 1 and 2, it was confirmed that myopia can be induced or inhibited by controlling the state of macrophages, particularly in the choroid. From the perspective of inhibiting and treating myopia progression, it was shown that inducing polarization of M2 macrophages is effective in achieving this.

[0125] <Test Example 3: Inhibition of Myopia by Instilling Mast Cell Stabilizer Eye Drops>

[0126] To verify the inhibitory effect on myopia progression by instilling Mast Cell Stabilizer eye drops, a lens-induced myopia model was used in which myopia was induced in mice by wearing concave lenses, and cromoglobin solution (4% solution) or femirolast potassium (0.1% solution) was administered eye drops once a day during the myopia induction period.

[0127] According to the above test method, the ocular axial length, refractive value, and choroidal thickness were measured in mice and control mice that underwent 3 weeks of myopia induction and ophthalmic administration of cromoglobin solution or pemirorast potassium solution, and the changes were calculated (Fig. 7A, B, C).

[0128] As a result, compared to the control eye with PBS, the eye wearing a concave lens showed elongation of the ocular axis (Fig. 7A), myopia of the refractive index (Fig. 7B), and thinning of the choroid (Fig. 7C). Meanwhile, in the group administered ophthalmic solution of cromoglobin or femirorast potassium solution, the aforementioned changes observed in the PBS group were not observed.

[0129] As shown in these results, it was confirmed that ophthalmic administration of Mast Cell Stabilizer inhibits the progression of myopia.

[0130] <Test Example 4: Comparison of Myopia Inhibitory Effects of Mast Cell Stabilizer Eye Drops and Histamine Receptor Inhibitor Eye Drops>

[0131] Anti-allergic drugs are broadly classified into two types: mast cell stabilizers that inhibit mast cell degranulation, and histamine receptor inhibitors that inhibit the action of histamine secreted by mast cell degranulation. In order to clarify whether the myopia-inhibiting effect of the mast cell stabilizer eye drops of Test Example 3 is due to the action of the anti-allergic drug or due to the inhibition of mast cell degranulation, femirolast potassium (0.1% solution) as a mast cell stabilizer and levocavastin solution (0.025% solution) as a histamine receptor inhibitor were eye drops administered to mice that had undergone myopia induction by LIM, and the myopia-inhibiting effects were compared. The results are shown in Fig. 8.

[0132] As shown in Figure 8, as a result, similar to Test Example 3, elongation of the ocular axis, myopia of refractive error, and non-thinning of the choroid were not observed in the group administered pemirorast potassium, which is observed in the control group. Meanwhile, in the group administered levocavastin, elongation of the ocular axis, myopia of refractive error, and non-thinning of the choroid were observed, similar to the control group.

[0133] From the above results, the myopia-inhibiting effect of the mast cell stabilizer was confirmed among anti-allergic drugs.

[0134] <Test Example 5 Inhibition of Myopia Progression by Administration of Lactobacillus paracasei>

[0135] Since the induction of M2 macrophages can inhibit the progression of myopia, Lactobacillus paracasei, which is included in Yakult (Yakult Co., Ltd.), was cultured and proliferated as a lactic acid bacterium and administered to myopia model mice. As in Test Examples 3 and 4, the ocular axial length, refractive value, and choroidal thickness were measured, and the changes were calculated (Fig. 9). As a result, it was confirmed that the administration of the lactic acid bacterium inhibits the progression of myopia (Fig. 9).

Claims

Claim 1 A composition for inhibiting or treating myopia, containing a therapeutically effective amount of pemirorast or its salt. Claim 2 A composition for inhibiting or treating myopia, containing a therapeutically effective amount of pemirorast or a salt thereof, for inhibiting elongation of the ocular axis or non-thinning of the choroid. Claim 3 A composition for inhibiting or treating myopia for subjects in whom elongation of the ocular axis or non-thinning of the choroid is confirmed, containing a therapeutically effective amount of pemirorast or its salt. Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete

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