Methods and compositions for preventing or treating myopia

Abstract

To provide: a method for preventing or treating myopia; and a composition used in the method.SOLUTION: The present invention provides a method and composition for inhibiting, improving, or treating myopia by administering a substance that induces M2 macrophage polarization, a substance that regulates a function of choroid-resident immune cells, such as a mast cell stabilizer or a chemical mediator release inhibitor, or lactic acid bacteria. Regulating the function of choroid-resident immune cells, for example by inducing polarization of M2 macrophages in a choroid or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria, is effective in myopia prevention and treatment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for preventing or treating myopia, more particularly to compositions that act on immune cells in the choroid to inhibit the progression of myopia, and more particularly to methods and compositions that modulate the function of choroid-resident immune cells, such as by inducing polarization into M2 macrophages or by 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 focal spot is focused in front of the retina due to axial elongation of the eye; the longer the axial length, the more severe the myopia. As the prevalence of myopia increases, there has been growing interest in the factors involved in the development of myopia and methods for slowing and preventing myopia progression. Recent studies have suggested that the choroid is important in regulating ocular growth and the development of myopia, and choroidal thinning has been shown to be a structural characteristic of myopia. The negative correlation between choroidal thickness and axial length suggests that changes in choroidal thickness may be a predictive biomarker for axial elongation. However, the detailed mechanisms by which the choroid contributes to the development and progression of myopia remain unclear.

[0003] The choroid, a tissue rich in fine blood vessels that covers the outer retina, not only supplies oxygen and nutrients to retinal cells but also growth factors involved in tissue remodeling of the outer sclera and regulating eye growth. A decrease in choroidal thickness or blood flow contributes to scleral ischemia and hypoxia, which are thought to affect scleral structural changes that lead to axial elongation. 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 have been proposed for inhibiting the progression of myopia by maintaining or increasing choroidal thickness, such as crocetin intake (see Non-Patent Document 1) and violet light irradiation (Non-Patent Document 2).

[0005] Crocetin is known to have anti-inflammatory and immunomodulatory effects, and is known to exert its effects by adjusting the balance of Th1 / Th2 and Th17 / Treg in T cells and by suppressing the NF-β pathway in cells such as macrophages that produce inflammatory mediators (TNF-β, IL-6, IFN-β, etc.) (Non-patent documents 3-5).

[0006] The choroid contains 13 types of cells, four of which are immune cells. Tissue-resident immune cells are responsible for maintaining tissue homeostasis and structure in addition to immune responses. For example, it has been reported that the choroid thins in mice lacking macrophages or mice in which mast cell degranulation is induced (Non-Patent Documents 6-7). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Mori et al., Sci Rep. 2019. 22;9(1):295. [Non-patent document 2] Jiang et al. Proc Natl Acad Sci USA. 2021. 1;118(22):e2018840118. [Non-patent document 3] Oxid Med Cell Longev. 2021 Sep 10;2021:6631929. [Non-patent document 4] Eur J Pharmacol. 2012 Jan 15;674(2-3):391-6. [Non-Patent Document 5] Biofactors. 2023 Feb 6. doi: 10.1002 / biof.1942. [Non-patent document 6] Elife. 2020 Apr 1;9:e55564. [Non-Patent Document 7] FASEB J. 2020 Aug;34(8):10117-10131. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure aims to provide a method for preventing or treating myopia and a composition used therein, particularly a composition that targets choroid-resident immune cells, particularly macrophages and mast cells. [Means for solving the problem]

[0009] The present inventors have found that polarization of choroidal macrophages into M2 macrophages suppresses choroidal thinning and is effective in preventing and treating myopia.

[0010] Furthermore, mast cells are distributed in large numbers around small blood vessels and are also present in the choroid, a tissue composed of blood vessels, suggesting their involvement in maintaining the morphology of the choroid. The present inventors have found that the progression of myopia is suppressed by instilling a drug (mast cell stabilizer) that inhibits mast cell degranulation. Specifically, the present inventors have found that the progression of myopia is suppressed by instilling a mast cell stabilizer (sodium cromoglycate, pemirolast potassium), a type of antiallergic drug, into a mouse model of myopia induced by wearing minus lenses. However, the suppression of myopia progression was not observed when another antiallergic drug, an antihistamine (levocabastine), was instilled into mice undergoing myopia induction by wearing minus lenses.

[0011] Furthermore, it is known that administration of lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, induces and activates polarization into M2 macrophages. The present inventors have found that administration of lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus paracasei, suppresses the progression of myopia. Taking these findings into consideration, along with the findings of Non-Patent Documents 6 and 7 mentioned above, it is believed that maintaining the choroidal structure (thickness) through intervention targeting immune cells resident in the choroid would have an inhibitory effect on the progression of myopia. These findings suggest that a composition that targets immune cells present in the choroid, such as macrophages and mast cells, and induces their respective properties to an appropriate state would have an inhibitory effect on the progression of myopia. The present invention is based on these findings and encompasses the following aspects.

[0012] [Aspect 1] A method for suppressing, ameliorating, 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 in need of treatment. [Aspect 2] The method of embodiment 1, wherein the substance that modulates choroid-resident immune cell function is an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor. [Aspect 3] The method of embodiment 2, wherein the M2 macrophage polarization inducer is selected from the group consisting of a protein, a peptide, a nucleic acid, a small molecule compound, and a large molecule compound. [Aspect 4] The method of embodiment 2, wherein the M2 macrophage polarization inducer is selected from the group consisting of IL-4, IL-10, IL-13, TLR2, TLR4, TLR7, TLR9 and their ligands, bisantrene 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. [Aspect 5] The method of embodiment 2, wherein the mast cell stabilizer or chemical mediator release inhibitor is cromoglycate or pemirolast, or a salt thereof. [Aspect 6] The method of embodiment 1, wherein the lactic acid bacteria are of the genus Lactobacillus. [Aspect 7] The method of embodiment 1, wherein the lactic acid bacterium is Lactobacillus paracasei. [Aspect 8] The method according to embodiment 1, wherein the substance modulating the function of choroid-resident immune cells or lactic acid bacteria is contained in a pharmaceutical composition, a supplement, or a food product. [Aspect 9] The method of embodiment 1, wherein the loss of refraction, axial elongation, and / or choroidal thinning is inhibited. [Aspect 10] A composition for suppressing or treating myopia, comprising a therapeutically effective amount of a substance that regulates the function of choroidal resident immune cells or lactic acid bacteria. [Aspect 11] A supplement for suppressing or improving myopia, comprising an effective amount of a substance that regulates the function of choroidal resident immune cells or lactic acid bacteria. [Aspect 12] A food for suppressing or improving myopia, comprising an effective amount of a substance that regulates the function of choroidal resident immune cells or lactic acid bacteria. [Aspect 13] 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. [Aspect 14] Use of a substance that modulates the function of choroidal resident immune cells or lactic acid bacteria to suppress or treat myopia. [Effects of the Invention]

[0013] According to the present disclosure, a new method for preventing and treating myopia can be provided by adjusting the function of choroidal resident immune cells, more specifically, by inducing polarization into 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 drawings]

[0014] [Figure 1] (A) shows the experimental schedule. (B) shows graphs of changes in refractive index (left), axial length (center), and choroidal thickness (right). [Figure 2] 1 is a graph showing changes in the expression of M1 macrophage marker genes and oxidative stress-related genes due to LPS administration. [Figure 3] (A) Experimental schedule. (B) Graph showing changes in CD206 protein and phosphorylated STAT6 expression in the choroid following IL-4 administration. (C) Graph showing changes in CD163 and CD206 mRNA expression following IL-4 administration. [Figure 4] 1 is a graph showing changes in the expression of M2 macrophage marker genes and oxidative stress-related genes by IL-4 administration. [Figure 5]1 shows the results of an experiment to suppress myopia progression by IL-4 administration. (A) A diagram showing the experimental schedule. (B) A diagram showing changes in refraction (left), axial length (center), and choroidal thickness (right) due to IL-4 administration during the myopia induction period. [Figure 6] These figures show the results of myopia induction suppression and M2 macrophage polarization induction by IL-13 administration. (A) A diagram showing the experimental schedule. (B) Graphs showing changes in refraction (upper left), axial length (upper right), choroidal thickness (lower left), and blood flow (lower right) due to IL-13 administration during the myopia induction period. (C) Graphs showing changes in macrophage number and M2 macrophage ratio due to myopia induction and IL-13 administration. [Figure 7] These are results showing the myopia-suppressing effect of instilling mast cell stabilizer. (A) Graph showing the change in axial length. (B) Graph showing the change in refractive index. (C) Graph showing the change in choroidal thickness. The left graph shows the control group vs. the pemirolast potassium instillation group, and the right graph shows the control group vs. the cromoglycate instillation group. *p<0.05, **p<0.01, ***p<0.001, student's t-test [Figure 8] Graphs comparing the myopia suppression effects of instilling a mast cell stabilizer with those of a histamine receptor inhibitor. (A) Graph showing the change in axial length. (B) Graph showing the change in refractive index. (C) Graph showing the change in choroidal thickness. From the left, the graphs show the control group, the levocabastine instillation group (histamine receptor inhibitor), and the pemirolast potassium instillation group (mast cell stabilizer). *p<0.05, **p<0.01, ***p<0.001, student's t-test [Figure 9] These results show the effect of administering Lactobacillus paracasei in inhibiting the progression of myopia. (A) Graph showing the change in axial length. (B) Graph showing the change in refractive index. (C) Graph showing the change in choroidal thickness. The left graph shows the control group, and the right graph shows the Lactobacillus paracasei-administered group. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. The present invention is not limited to the following embodiments and examples, and includes various modifications and applications within the scope of the gist of the present invention.

[0016] [Methods for preventing or treating myopia] As described above, in recent years, it has become clear that choroidal thinning is involved in the onset of myopia. As a result of extensive research into factors that control this thinning, the present inventors have found that changes in the state (polarization) of macrophages in the choroid cause changes in choroidal thickness and control the progression of myopia. Macrophages are classified into M1 and M2 macrophages. M1 macrophages are primarily involved in the initiation of inflammation, whereas M2 macrophages play an antagonistic role in terminating and suppressing inflammation. The present inventors have first demonstrated that inducing M1 macrophage polarization in the choroid leads to choroidal thinning and progression of myopia, while inducing M2 macrophage polarization inhibits choroidal thinning and progression of myopia. Furthermore, mast cells are abundantly distributed around small blood vessels and are present in the choroid, a tissue composed of blood vessels, suggesting their involvement in maintaining choroidal morphology. The present inventors have found that instillation of a drug (mast cell stabilizer) that inhibits mast cell degranulation inhibits myopia progression. Furthermore, it is known that administration of lactic acid bacteria such as Lactobacillus paracasei induces and activates polarization into M2 macrophages, and the present inventors have found that administration of lactic acid bacteria such as Lactobacillus paracasei inhibits the progression of myopia. Thus, the present inventors have found that the progression of myopia can be inhibited by regulating the function of choroidal resident immune cells. Thus, one aspect of the present disclosure relates to methods for inhibiting, ameliorating, or treating myopia by modulating the function of choroidal resident immune cells, more specifically, by polarizing macrophages into M2 macrophages in the choroid, or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. One aspect of the present disclosure relates to a method for inhibiting, ameliorating, or treating myopia by modulating the function of choroidal resident immune cells, more specifically, by inducing polarization to M2 macrophages in the choroid using an M2 macrophage polarization inducer, or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. One aspect of the present disclosure relates to a method for suppressing refractive loss, axial elongation, and / or choroidal thinning in a subject in need of treatment, comprising modulating the function of choroidal resident immune cells, more specifically, delivering an M2 macrophage polarization inducer to the subject's choroid, or administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. Furthermore, one aspect of the present disclosure also relates to a screening method for searching for substances that regulate the function of choroidal resident immune cells, such as components that promote polarization into M2 macrophages, in order to control choroidal thinning, which is a mechanism for the development of myopia, and a screening method for searching for components that are effective in the prevention and treatment of myopia.

[0017] <Choroidal resident immune cell function regulator> The choroid resident immune cell function regulator is a substance that regulates the function of choroid resident immune cells. Substances that regulate the function of choroid resident immune cells preferably include substances that regulate inflammation suppression in the choroid and substances that regulate the choroid to an anti-inflammatory environment. Examples of choroid resident immune cells include macrophages, mast cells, and the like. Examples of choroid resident immune cell function regulators include M2 macrophage polarization inducers, mast cell stabilizers, chemical mediator release inhibitors, lactic acid bacteria, and the like.

[0018] <M2 macrophage polarization inducer> Macrophages are classified into inflammatory M1 macrophages and anti-inflammatory M2 macrophages with different functions, and they polarize in response to signals such as cytokines and stimulatory components to perform specific function expression. Therefore, the M2 macrophage polarization inducers in the present disclosure include various substances involved in signal transduction that induce polarization to M2 macrophages in the choroid, and proteins, peptides, nucleic acid drugs, low molecular weight compounds, high molecular weight compounds, etc. can be used without particular limitation. Examples thereof include cytokines such as IL-4 and IL-13 produced by Th2 cells, leucine zipper transcription factor c-Maf, carbohydrate-binding lectin galectin-3, and the like. It is also known that there are several populations of M2 macrophages. In addition to the aforementioned IL-4 and IL-13 that induce M2a macrophages, IL-10, glucocorticoid hormones (GC) involved in the induction of M2c macrophages, TLR2, TLR4, TLR7, TLR9 and their ligands (ligands for TLR) involved in the induction of M2d macrophages can also be mentioned as M2 macrophage polarization inducers in the present disclosure.

[0019] Furthermore, examples of M2 macrophage polarization inducers in the present disclosure include various low molecular weight compounds, such as bisantrene 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] Agonists of the above-mentioned substances can also be used as M2 macrophage polarization inducers. Those skilled in the art can evaluate whether a substance has the ability to induce M2 macrophage polarization by culturing cells, such as monocytic cell lines (RAW264 cells, J774 cells, U937 cells), mouse peritoneal macrophages, or bone marrow-derived macrophages, in a medium containing the substance to be evaluated, and using an in vitro evaluation system to evaluate the expression of M2 macrophage markers. In addition to the M2 macrophage polarization inducers specifically described herein, M2 macrophage polarization inducers suitable for use in the methods and compositions of the present disclosure can be obtained.

[0021] When the M2 macrophage polarization inducer is a protein or peptide, it can be administered to a subject in the form of DNA or RNA encoding it. The nucleic acid encoding the M2 macrophage polarization inducer can be administered to a subject using a plasmid or expression vector. The expression vector can be, for example, a viral vector, particularly an adenoviral vector, but is not limited to this. Other usable viral vectors include retrovirus, adeno-associated virus, poxvirus, baculovirus, vaccinia, herpes simplex, Epstein-Barr virus, geminivirus, and caulimovirus vectors. The nucleic acid encoding the M2 macrophage polarization inducer can contain regulatory elements for specifically expressing the protein in the choroid or RPE. That is, the nucleic acid encoding the M2 macrophage polarization inducer can be operably linked to regulatory elements such as a promoter or enhancer.

[0022] <Mast cell stabilizer> Mast cell stabilizers are also called mast cell stabilizers or mast cell stabilizing drugs. Mast cell stabilizers are, for example, drugs that suppress the release of allergens from mast cells, such as drugs that stabilize the cell membrane of mast cells to suppress the release of allergens from mast cells. Examples of mast cell stabilizers include ashitazanolast hydrate solution (Zeperin), amlexanox (Elix), pemirolast potassium (Alegisar), pemirolast potassium (Pemilaston), cromoglycate sodium (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonists, cromolyn sodium, cromoglycic acid, ketotifen, methylxanthines, omalizumab, pemirolast, and quercetin, and preferably cromoglycic acid or pemirolast, or a salt thereof.

[0023] <Chemical mediator release inhibitors> 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 stabilize the cell membrane of mast cells to prevent histamine and other substances from being released, and they inhibit the release of allergens (substances that cause allergic reactions), such as histamine, LTB4, LTC4, LTD4, PGD2, TXB2, and PAF, from mast cells. Examples of chemical mediator release inhibitors include ashitazanolast hydrate solution (Zeperin), amlexanox (Elix), pemirolast potassium (Alegisar), pemirolast potassium (Pemilaston), cromoglycate sodium (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonists, cromolyn sodium, cromoglycic acid, ketotifen, methylxanthines, omalizumab, pemirolast, and quercetin, and preferably cromoglycic acid or pemirolast, or a salt thereof.

[0024] <Lactic acid bacteria> Examples of lactic acid bacteria include homolactic acid bacteria and heterolactic acid bacteria. Examples of lactic acid bacteria include coccoid lactococci and rod-shaped lactobacilli. Examples of lactic acid bacteria include gram-positive, rod-shaped or coccoid bacteria, non-spore-forming, non-motile bacteria that produce 50% or more of lactic acid relative to the glucose consumed, and require niacin (B3). Examples of lactic acid bacteria include intestinal 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 Lactobacillales and lactic acid bacteria of the phylum Actinomycetes. Examples of lactic acid bacteria of the Lactobacillales 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, lactic acid bacteria of the genus Streptococcus, etc. Examples of lactic acid bacteria of the phylum Actinomycetes include lactic acid bacteria of the genus Bifidobacterium, etc. Examples of lactic acid bacteria of the genus Lactobacillus include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei Shirota, etc. Examples of lactic acid bacteria of the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium, etc.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. Administration of Lactobacillus paracasei has been shown to improve the symptoms of non-alcoholic steatohepatitis by shifting hepatic Kupffer cells (resident liver macrophages) to the M2 state (Sohn et al, Dig Dis Sci, 2015 Nov;60(11):3340-50). Administration of Lactobacillus paracasei has been shown to alleviate blue light-induced retinal degeneration by activating M2 macrophages (Morita et al, Nutrients, 2018 Dec15;10(12):1991). Administration of Lactobacillus plantarum has been shown to alleviate colitis by promoting polarization from M1 to M2 macrophages (Jang et al, Int Immunopharmacol, 2014 Jul;21(1):186-92). It is known that administration of Lactobacillus brevis alleviates colitis by promoting polarization from M1 macrophages to M2 macrophages (Jang et al., J Appl Microbiol, 2013 Sep;115(3):888-96). Thus, it is known that administration of lactic acid bacteria of the order Lactobacillales, such as the genus Lactobacillus, induces and activates polarization to M2 macrophages. Therefore, the lactic acid bacteria are preferably lactic acid bacteria of the order Lactobacillales, such as the genus Lactobacillus.

[0025] A DDS may be used to deliver substances that modulate the function of choroid-resident immune cells, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, to the subject's choroid. For example, when the choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, is a nucleic acid, DNA or RNA (messenger RNA) encoding the choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, may be administered to a subject using a DDS such as a liposome. Other available DDS include charged lipids, nucleic acid-protein complexes, and biopolymers. Furthermore, the choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, may be administered to a subject in the form of a protein. Administration can be performed, for example, by topical administration to the choroid.

[0026] Delivery of a choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor, to the subject's choroid may be achieved by transplanting cells that secrete a choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator, into the subject. Cells that secrete a choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator, may be, for example, retinal pigment epithelial cells (RPE). Alternatively, the cells may be genetically engineered to secrete a choroid-resident immune cell function modulator, such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator.

[0027] The subject in the method for inhibiting or treating myopia of the present disclosure can be a mammal, including a human, or a non-human mammal, including a dog, cat, cow, or horse, but is preferably a human.

[0028] [Composition for inhibiting or treating myopia] One aspect of the present disclosure relates to a composition for suppressing, ameliorating, or treating myopia using an M2 macrophage polarization inducer, a mast cell stabilizer, a choroid-resident immune cell function modulator such as a chemical mediator release inhibitor, or lactic acid bacteria. More specifically, one embodiment of the present disclosure relates to a composition for suppressing or treating myopia, comprising a therapeutically effective amount of an M2 macrophage polarization inducer, a mast cell stabilizer, a choroid-resident immune cell function modulator such as a chemical mediator release inhibitor, or lactic acid bacteria. Such a composition can be used to deliver an M2 macrophage polarization inducer, a mast cell stabilizer, a choroid-resident immune cell function modulator such as a chemical mediator release inhibitor, or lactic acid bacteria to the choroid of a subject. Such a composition can be a pharmaceutical composition, a food product, or a supplement.

[0029] The pharmaceutical composition according to the present disclosure is administered, for example, to the eye. Examples of the administration form of the composition include eye drops (including instillation of eye ointment and eyewash), subconjunctival administration, intraconjunctival administration, and sub-Tenon administration.

[0030] The dosage form of the composition is not particularly limited, but examples thereof include eye drops, eye ointments, injections, patches, gels, inserts, etc., and eye drops are preferred. These can be prepared using conventional techniques commonly used in the art.

[0031] Eye drops can be prepared using a selection of ingredients as needed from among isotonic agents such as sodium chloride, potassium chloride, and concentrated glycerin; buffers such as sodium phosphate, sodium acetate, and epsilon-aminocaproic acid; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, and polyoxyethylene hydrogenated castor oil; stabilizers such as sodium citrate and sodium edetate; and preservatives such as parabens. The pH should be within the range acceptable for ophthalmic preparations, but is usually preferably within the range of 4 to 8. Eye ointments can be prepared using commonly used bases such as white petrolatum and liquid paraffin.

[0032] Furthermore, the pharmaceutical composition according to the present disclosure is not limited to administration to the ocular topical site, but can also be administered by any administration route, such as enteral administration (oral, tube feeding, infusion, etc.), parenteral administration (intravenous, intraarterial, transdermal, intramuscular injection, etc.), etc. The dosage form of the composition used for these administration forms can be appropriately selected, and may be, for example, a solid preparation such as a tablet, granule, powder, capsule, or chewable agent, or a liquid preparation such as a liquid, syrup, injection, or drip infusion. The pharmaceutical composition of the present disclosure may contain other ingredients in appropriate amounts, provided that the effects of the present invention are not impaired. Examples of other ingredients include any carrier, buffer, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener. These ingredients may be formulated singly or in combination of two or more.

[0033] The content of the M2 macrophage polarization inducer, mast cell stabilizer, choroidal resident immune cell function regulator such as a chemical mediator release inhibitor, or lactic acid bacteria in 100% by weight of the pharmaceutical composition can be appropriately set, for example, within the range of 0.001 to 99.99% by weight.

[0034] The dosage of the pharmaceutical composition according to the present disclosure is not particularly limited and can be appropriately selected depending on the dosage form, the age and body weight of the recipient, the degree of desired effect, etc. The dosage of the choroid-resident immune cell function regulator, such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor, or lactic acid bacteria, can be, for example, 100 to 1,000,000 nmol, or preferably 150 to 100,000 nmol, per day, and the administration frequency can be, for example, 1 to 100 times per month.

[0035] In addition, the composition according to the present disclosure can be food or supplement.The form of food or supplement can be, for example, liquid, solid, tablet, granule, powder, capsule, paste, gel, etc., for example, the above-mentioned solid or liquid preparations, etc. can be arbitrarily selected.Specific examples of food include various general processed foods such as fruit juice drinks, vegetable juices, soft drinks, tea, soups, puddings, yogurt, cake premix products, confectioneries, cookies, candies, gummies, chewing gum, as well as special purpose foods, foods for specified health uses, nutrient-functional foods, functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally adjusted foods, etc., such as supplements, drinks, etc.

[0036] The food or supplement according to the present disclosure may contain any functional ingredients (vitamins, minerals, etc.), any excipient, any additive (flavoring agent, sweetener, acidulant, colorant, thickener, binder, strengthening agent, disintegrant, buffer, surfactant, solubilizer, resorption promoter, dispersant, stabilizer, gelling agent, emulsifier, antioxidant, surfactant, preservative, moisture-proofing agent, pH adjuster, colorant, soothing agent, isotonicity agent, etc.).

[0037] One aspect of the present disclosure relates to the use of an M2 macrophage polarization inducer, a mast cell stabilizer, a choroid-resident immune cell function modulator such as a chemical mediator release inhibitor, or lactic acid bacteria for suppressing or treating myopia. Furthermore, another aspect of the present disclosure relates to the use of an M2 macrophage polarization inducer, a mast cell stabilizer, a choroid-resident immune cell function modulator such as a chemical mediator release inhibitor, or lactic acid bacteria in the manufacture of a medicament for suppressing or treating myopia.

[0038] [Screening method] One aspect of the present disclosure relates to a screening method for searching for components that promote polarization of macrophages into M2 macrophages in order to control choroidal thinning, which is a mechanism of myopia onset, and a screening method for searching for components that are effective in preventing and treating myopia. In some embodiments, the screening method comprises (i) administering a candidate substance to a model animal, and (ii) measuring the axial length, choroidal thickness, and refractive index of the model animal. In some embodiments, the screening method comprises (i) administering a candidate substance to the model animal, and (ii) measuring the expression of a choroid-resident immune cell marker, such as an M2 macrophage marker, in a sample from the model animal. In some embodiments, the screening method further comprises (iii) measuring the expression of a choroid-resident immune cell marker, such as an M1 macrophage marker. In some embodiments, the present invention also includes an in vitro screening method in which cells such as monocytic cell lines such as RAW264 cells, J774 cells, and U937 cells, and choroid-resident immune cells such as mouse peritoneal macrophages and bone marrow-derived macrophages are cultured in a medium containing a candidate substance, and the expression of choroid-resident immune cell markers such as M2 macrophage markers is evaluated. In some embodiments, the model animal can be an animal undergoing a myopia-inducing procedure. In some embodiments, the choroid-resident immune cell marker, such as an M2 macrophage marker, may be CD163, CD206, arginase, or IL-10. Methods for measuring choroid-resident immune cell markers, such as macrophage markers, include quantitative PCR, flow cytometry, immunostaining, luciferase assay, and arginase activity staining.

[0039] Substances identified by such screening methods can be used to prevent or treat myopia.

[0040] While preferred embodiments of the present invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only, and that various modifications, changes, and substitutions may be made by those skilled in the art without departing from the scope of the present invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the present invention. This application also claims the benefit of priority from Japanese Patent Application No. 2022-72596, and the contents of that Japanese Patent Application and all publications, including patents and patent application documents referenced herein, are to be construed as being incorporated by reference as if expressly set forth herein. [Example]

[0041] The present invention will be explained in more detail below by way of experimental examples.

[0042] <Test Example 1: Choroidal thinning and myopia caused by LPS administration> To confirm whether polarization into M1 macrophages is involved in choroidal thinning and myopia, we measured the expression of polarization marker genes in the choroid after LPS administration, as well as choroidal thickness, axial length, and refractive index.

[0043] We measured choroidal thickness, axial length, and refractive index in mice treated with LPS for 2 weeks and in control mice treated with PBS, and calculated the changes (Fig. 1A, B).The eyes were then enucleated and the expression of polarization marker genes was measured by quantitative PCR (Fig. 2).

[0044] C57BL6J mice (n=4 per group) were used. To the LPS-administered group, LPS solution was intraperitoneally administered daily at a dose of 10 mg / kg BW. To the PBS-administered group, PBS was administered instead of the LPS solution.

[0045] (Measurement of axial length, choroidal thickness, and refractive index) The axial length, choroidal thickness, and refractive index of each group of mice were measured. Axial length and choroidal thickness were measured using a spectral-domain optical coherence tomography (Envisu R4310, Leica). Refractive index was measured using an infrared photorefractor for mice (made by Professor Schaeffel, University of Tübingen).

[0046] (Observation of the choroid using an electron microscope) Eyes from each group were harvested and fixed overnight in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) at 4°C. They were then rinsed for 1 hour in 0.1M sodium cacodylate buffer. They were then fixed for 2 hours in 1% OsO4 in 0.1M cacodylate buffer, followed by dehydration through graded ethanol solutions. The eyes were then infiltrated overnight in a 1:2 mixture of propylene oxide and Epon-Araldite and embedded in 100% resin. The blocks were sectioned and examined using a transmission electron microscope (JEM1400 plus; JEOL) at an accelerating voltage of 100 kV.

[0047] (Evaluation of gene marker expression) Choroidal, retinal, and scleral samples were collected from mice administered LPS for 2 weeks as described above, and the expression of M1 macrophage marker genes and oxidative stress-related genes was analyzed using quantitative PCR.

[0048] (result) Compared to the control group administered PBS, the LPS-administered group showed a decrease in refraction (Fig. 1B, left), axial elongation (Fig. 1B, center), and choroidal thinning (Fig. 1B, right) at both the 1-week and 2-week administration periods.

[0049] Furthermore, gene expression analysis revealed increased expression of M1 marker genes and oxidative stress-related genes, particularly in the choroid (Figure 2).

[0050] As shown in these results, it was confirmed that an increase in M1 macrophages in the choroid induces myopia.

[0051] <Test Example 2 Polarization into M2 Macrophages and Myopia Suppression Effect by Administration of IL-4 and IL-13> M1 macrophages are mainly involved in the induction of inflammation, while M2 macrophages have antagonistic effects in that they are involved in the termination and suppression of inflammation. Since M1 macrophages have an action of inducing myopia, it is considered that M2 macrophages act suppressively on myopia progression. Therefore, IL-4, a cytokine necessary for polarization into M2 macrophages, was administered to mice, and whether polarization into M2 occurred in the choroid was examined by analyzing the expression levels of CD163 and CD206, which are M2 macrophage markers (Figs. 3A and B).

[0052] <Administration of IL-4> C57BL6J mice (n = 4 per group) were intraperitoneally administered 0.1 μg / 100 μl of an IL-4 solution (10 μg / kg BW). Choroid, retina, and liver samples were collected at 0 hours (before administration) and 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 4 and 24 hours after administration, and the expression of oxidative stress-related genes was analyzed by real-time PCR (Fig. 4).

[0053] In addition, IL-4 was administered to mice during myopia induction, and the myopia suppression effect was evaluated (Fig. 4). C57BL6 mice (-30D lens, n = 4) during myopia induction were intraperitoneally administered 0.1 μg / 100 μl of an IL-4 solution (10 μg / kg BW) for 3 weeks. As a control group, C57BL6J mice (0D lens, -30D lens, n = 4 per group) during myopia induction were used. After 3 weeks, refraction, axial elongation of the eye, and choroidal thinning were measured in the same manner as in Test Example 1.

[0054] (Results) As a result, an increase in the expression levels of CD163 and CD206, which are M2 markers, was observed in the choroid following IL-4 administration (Figs. 3B and C), confirming polarization into M2 macrophages. Additionally, gene expression analysis revealed enhanced expression of M2 macrophage marker genes and suppressed expression of oxidative stress-related genes (Fig. 5). From these results, it was suggested that administration of IL-4 induces polarization into M2 macrophages, suppresses the expression of oxidative stress-related genes, and inhibits the progression of myopia (Fig. 5).

[0055] Moreover, suppression of refractive decline (left in Fig. 5B), suppression of axial length elongation (center in Fig. 5B), and suppression of choroidal thinning (right in Fig. 5B) were confirmed by administering IL-4 during the myopia induction period.

[0056] From the above results, it was confirmed that polarization of choroidal macrophages into M2 by IL-4 administration exhibits an anti-myopia effect.

[0057] <IL-13 Administration> By administering IL-13, which is known as a cytokine that polarizes macrophages into M2 similar to IL-4, during the myopia induction period, it was confirmed using C57BL6J mice that, similar to the results of IL-4, elongation of the axial length, myopic shift in refraction, and choroidal thinning caused by wearing a minus lens were suppressed, and polarization into M2 macrophages was induced (Fig. 6). Note that blood flow changes and macrophage counts were measured as follows. Blood flow changes were measured using a wavelength swept optical coherence tomography (OCT S-1, Canon). Macrophage counts were measured by staining with F4 / 80 antibody and CD11b antibody after digestion of the choroid and using a flow cytometer (CytoFLEX S, Beckman Coulter). Measurement of M2 macrophage counts was performed in the same manner as measurement of macrophage counts, except that the staining process with CD206 antibody was added.

[0058] As shown in the results of Test Examples 1 and 2, it was confirmed that myopia can be induced or suppressed by controlling the state of macrophages, particularly in the choroid. From the perspective of myopia progression suppression and treatment, this indicates that promoting polarization into M2 macrophages is particularly effective in achieving this.

[0059] <Test Example 3: Suppression of myopia by instilling mast cell stabilizer> To verify the inhibitory effect of instilling mast cell stabilizers on the progression of myopia, a lens-induced myopia model was used in which mice were induced to develop myopia by wearing minus lenses. Cromolyn solution (4% solution) or pemirolast potassium (0.1% solution) was administered as an instillation once daily during the myopia induction period.

[0060] According to the test method described above, axial length, refractive index, and choroidal thickness were measured in mice that underwent myopia induction for 3 weeks and instillation of cromoglycate solution or pemirolast potassium solution, as well as in control mice, and the amount of change was calculated (Figure 7A, B, C).

[0061] As a result, compared with the control eyes, the minus lens-wearing eyes in the PBS-administered control group showed axial elongation (Fig. 7A), myopia (Fig. 7B), and choroidal thinning (Fig. 7C). On the other hand, the groups administered with cromolyn or pemirolast potassium did not show the above-mentioned changes observed in the PBS-administered group.

[0062] These results confirm that instillation of a mast cell stabilizer inhibits the progression of myopia.

[0063] <Test Example 4: Comparison of the myopia suppression effects of mast cell stabilizer eye drops and histamine receptor inhibitor eye drops> Antiallergic drugs are broadly divided into two types: mast cell stabilizers, which inhibit mast cell degranulation, and histamine receptor inhibitors, which inhibit the action of histamine secreted by mast cell degranulation. To clarify whether the myopia-suppressing effect of the instilled mast cell stabilizer in Test Example 3 was due to the action of the antiallergic drug or to the inhibition of mast cell degranulation, mice with myopia induced by LIM were administered with pemirolast potassium (0.1% solution) as a mast cell stabilizer and levocabastine solution (0.025% solution) as a histamine receptor inhibitor, and their myopia-suppressing effects were compared. The results are shown in Figure 8.

[0064] As shown in Figure 8, the results showed that the pemirolast potassium administration group did not show the axial elongation, myopia in refractive index, and choroidal thinning seen in the control group, as in Test Example 3. On the other hand, the levocabastine administration group showed the axial elongation, myopia in refractive index, and choroidal thinning seen in the control group.

[0065] These results confirmed that, among anti-allergy drugs, mast cell stabilizers have the effect of suppressing myopia.

[0066] <Test Example 5: Inhibition of myopia progression by administration of Lactobacillus paracasei> Because induction of M2 macrophages can suppress the progression of myopia, Lactobacillus paracasei, a lactic acid bacterium contained in Yakult (Yakult Honsha Co., Ltd.), was cultured and grown and administered to myopic model mice. Similar to Test Examples 3 and 4, axial length, refractive index, and choroidal thickness were measured and the amount of change calculated (Figure 9). As a result, it was confirmed that administration of lactic acid bacteria suppressed the progression of myopia (Figure 9).

Claims

1. A composition for inhibiting or treating myopia, comprising a therapeutically effective amount of pemirolast or a salt thereof.

2. Use of pemirolast, or a salt thereof, in the manufacture of a composition for inhibiting or treating myopia.

3. A composition for inhibiting or treating myopia, comprising a therapeutically effective amount of pemirolast or a salt thereof, wherein the composition inhibits axial elongation or choroidal thinning.

4. Use of pemirolast or a salt thereof in the manufacture of a composition for inhibiting or treating myopia, the composition inhibiting axial elongation or choroidal thinning.

5. A composition for inhibiting or treating myopia in a subject who has been observed to have axial elongation or choroidal thinning, comprising a therapeutically effective amount of pemirolast or a salt thereof.

6. Use of pemirolast or a salt thereof in the manufacture of a composition for inhibiting or treating myopia in a subject with axial elongation or choroidal thinning.

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