Methods and compositions for preventing or treating myopia

Abstract

The purpose of the present invention is to provide a method for preventing or treating myopia, and a composition used in said method. 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 the 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 the polarization of M2 macrophages in the choroid or by administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria, is effective in myopia prevention and treatment.

Description

Technical Field

[0001] The present invention relates to methods and compositions for preventing or treating myopia, and more particularly to compositions that act on immune cells in the choroid to suppress the progression of myopia, and more particularly to methods and compositions for inducing polarization into M2 macrophages or modulating the function of choroid resident immune cells such as administering mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria.

Background Art

[0002] Myopia refers to a state in which the focus is formed in front of the retina due to the elongation of the axial length of the eye, and it can be said that the longer the axial length, the stronger the myopia. With the increasing prevalence of myopia, there is growing interest in the factors involved in the onset of myopia and methods for suppressing and preventing myopia progression. Recent research has suggested that the choroid is important for the regulation of eye growth and the onset of myopia, and it has been shown that choroidal thinning is a structural feature of myopia. Since there is a negative correlation between choroidal thickness and axial length, it has been suggested that changes in choroidal thickness may be predictive biomarkers for axial length elongation. However, the detailed mechanisms by which the choroid is involved in the onset and progression of myopia are still unclear.

[0003] The choroid is a tissue that covers the outside of the retina rich in fine blood vessels, and in addition to the function of supplying oxygen and nutrients to retinal cells, it has the function of supplying growth factors involved in tissue remodeling of the sclera on the outside of the eyeball and regulation of eye growth. A decrease in choroidal thickness or a decrease in blood flow is thought to contribute to scleral ischemia and hypoxia, affecting the changes in scleral structure where the axial length increases. Therefore, maintaining and increasing choroidal thickness and blood flow have attracted attention as new targets in the prevention and treatment of myopia.

[0004] Methods for suppressing myopia progression by maintaining and increasing choroidal thickness have been proposed, for example, crocetin intake (see Non-Patent Document 1), violet light irradiation (Non-Patent Document 2), etc.

[0005] Crocin is known to have anti-inflammatory and immunomodulatory effects, and its effects are known to be expressed through the regulation of the balance between Th1 / Th2 and Th17 / Treg of T cells and the suppression of the NF-κB pathway of cells that produce inflammatory mediators such as macrophages (TNF-α, IL-6, IFN-γ, etc.) (Non-Patent Documents 3 to 5).

[0006] There are 13 types of cells in the choroid, 4 of which are immune cells. Tissue-resident immune cells are responsible for tissue homeostasis and structure maintenance in addition to immune responses. For example, it has been reported that the choroid becomes thinner in mice lacking macrophages or in mice in which mast cell degranulation is induced (Non-Patent Documents 6 to 7).

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present disclosure aims to provide a method for preventing or treating myopia, and a composition used therefor, particularly a composition targeting choroidal resident immune cells, particularly macrophages and mast cells.

Means for Solving the Problems

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

[0010] In addition, mast cells are abundantly distributed around small blood vessels and their presence is also recognized in the choroid, which is a tissue composed of blood vessels, and their involvement in maintaining the morphology of the choroid has been suggested. The present inventors have found that the progression of myopia is suppressed by instilling an agent that suppresses mast cell degranulation (mast cell stabilizer). That is, the present inventors have found that the progression of myopia is suppressed by instilling a mast cell stabilizer (sodium cromoglycate, pemirolast potassium), which is a type of anti-allergic drug, into myopia model mice wearing minus lenses. In addition, no suppression of the progression of myopia was observed when a histamine antagonist (levocabastine), which is another anti-allergic drug, was instilled into mice during myopia induction by wearing minus lenses.

[0011] Furthermore, it is known that lactic acid bacteria such as those belonging to the genus Lactobacillus, such as Lactobacillus paracasei, induce and activate polarization into M2 macrophages upon administration. The inventors of the present invention have found that administration of lactic acid bacteria such as those belonging to the genus Lactobacillus, such as Lactobacillus paracasei, suppresses the progression of myopia. Considering these facts in combination with the findings of Non-Patent Documents 6 to 7 described above, it is considered that if the choroidal structure (thickness) can be maintained by an intervention targeting immune cells resident in the choroid, it will 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 into an appropriate state is considered to have an effect of suppressing the progression of myopia. The present invention is based on such findings and includes the following aspects.

[0012] [Aspect 1] A method for suppressing, improving, or treating myopia, comprising administering a therapeutically effective amount of a choroid-resident immune cell function regulator or lactic acid bacteria to a subject in need of treatment. [Aspect 2] The method according to Aspect 1, wherein the choroid-resident immune cell function regulator is an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor. [Aspect 3] The method according to Aspect 2, wherein the M2 macrophage polarization inducer is selected from the group consisting of proteins, peptides, nucleic acids, low molecular weight compounds, and high molecular weight compounds. [Aspect 4] The method according to embodiment 2, wherein the M2 macrophage polarization inducer is selected from the group consisting of IL-4, IL-10, IL-13, TLR2, 4, 7, 9 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, bosutinib. [Embodiment 5] The method according to embodiment 2, wherein the mast cell stabilizer or the chemical mediator release inhibitor is cromolyn acid, or pemirolast, or a salt thereof. [Embodiment 6] The method according to embodiment 1, wherein the lactic acid bacterium belongs to the genus Lactobacillus. [Embodiment 7] The method according to embodiment 1, wherein the lactic acid bacterium is Lactobacillus paracasei. [Embodiment 8] The method according to embodiment 1, wherein the choroid resident immune cell function regulator or the lactic acid bacterium is contained in a pharmaceutical composition, supplement or food. [Embodiment 9] The method according to embodiment 1, wherein the reduction of refraction, the elongation of the eye axis, and / or the thinning of the choroid are suppressed. [Embodiment 10] A composition for suppressing or treating myopia, containing a therapeutically effective amount of a choroid resident immune cell function regulator or a lactic acid bacterium. [Embodiment 11] A supplement for suppressing or improving myopia, containing an effective amount of a choroid resident immune cell function regulator or a lactic acid bacterium. [Embodiment 12] A food for suppressing or improving myopia, containing an effective amount of a choroidal resident immune cell function regulator or lactic acid bacteria. [Aspect 13] Use of a choroidal resident immune cell function regulator or lactic acid bacteria in the manufacture of a medicament for suppressing or treating myopia. [Aspect 14] Use of a choroidal resident immune cell function regulator or lactic acid bacteria for suppressing or treating myopia.

Advantages of the Invention

[0013] According to the present disclosure, it is possible to provide a new method for preventing and treating myopia 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 Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] A detailed description of the present invention will be given. The present invention is not limited to the contents of the following embodiments and examples, and includes various modifications and application examples within the scope encompassing the gist of the present invention.

[0016] [Method for suppressing or treating myopia] As described above, in recent years, it has become clear that a change such as choroidal thinning is involved in the onset of myopia. As a result of intensive research by the present inventors on the factors that control this thinning, it has been found that the thickness of the choroid changes due to changes (polarization) in the state of macrophages in the choroid, and the progression of myopia is controlled. The state of macrophages includes M1 macrophages and M2 macrophages. M1 macrophages are mainly involved in the induction of inflammation, while M2 macrophages have an antagonistic function of being involved in the termination and suppression of inflammation. The present inventors have found that by inducing polarization to M1 macrophages in the choroid, choroidal thinning and the progression of myopia are observed, while by inducing polarization to M2 macrophages, choroidal thinning and the progression of myopia are suppressed for the first time. In addition, mast cells are abundantly distributed around small blood vessels, and their presence is also recognized in the choroid, which is a tissue composed of blood vessels, and their involvement in maintaining the morphology of the choroid is suggested. The present inventors have found that the progression of myopia is suppressed by instilling an agent (mast cell stabilizer) that suppresses mast cell degranulation. Furthermore, lactic acid bacteria such as Lactobacillus genus including Lactobacillus paracasei are known to induce and activate polarization to M2 macrophages by their administration. The present inventors have found that the administration of lactic acid bacteria such as Lactobacillus genus including Lactobacillus paracasei suppresses the progression of myopia. Thus, the present inventors have found that the progression of myopia can be suppressed by regulating the functions of choroid-resident immune cells. Accordingly, 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 polarizing macrophages in the choroid into M2 macrophages, 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, improving, or treating myopia by modulating the function of choroidal resident immune cells, more specifically, by inducing polarization of 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 a decrease in refraction, elongation of the eye axis, and / or thinning of the choroid, which includes modulating the function of choroidal resident immune cells in a subject in need of treatment, more specifically, delivering an M2 macrophage polarization inducer to the choroid of the subject, or administering a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. In addition, one aspect of the present disclosure relates to a screening method for searching for a substance that modulates the function of choroidal resident immune cells, such as a component that promotes polarization into M2 macrophages, for controlling choroidal thinning, which is a mechanism of myopia onset, and a screening method for searching for an ingredient effective for the prevention and treatment of myopia.

[0017] <Substance for modulating the function of choroidal resident immune cells> 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 to suppress inflammation in the choroid and substances that regulate to create an anti-inflammatory environment in the choroid. 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 exhibit specific functions. Therefore, the M2 macrophage polarization inducers in the present disclosure include various substances involved in signal transduction that induce polarization into 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] Examples of the M2 macrophage polarization inducer 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, bosutinib, and the like.

[0020] As the M2 macrophage polarization inducer, agonists of the above substances can also be used. Those skilled in the art can culture cells such as monocytic cell lines such as RAW264 cells, J774 cells, and U937 cells, mouse peritoneal macrophages, and bone marrow-derived macrophages in a medium containing the substance to be evaluated, and use an in vitro evaluation system for evaluating the expression of M2 macrophage markers to evaluate whether the substance has the ability to induce M2 macrophage polarization. In addition to the M2 macrophage polarization inducers specifically described in this specification, 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 them. The nucleic acid encoding the M2 macrophage polarization inducer may be administered to a subject using a plasmid or an expression vector. The expression vector can be, for example, a viral vector, particularly an adenoviral vector, but is not limited thereto. Other usable viral vectors include, for example, retroviruses, adeno-associated viruses, poxviruses, baculoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, geminiviruses, and calimoviruses vectors. The nucleic acid encoding the M2 macrophage polarization inducer may contain regulatory elements for specifically expressing the protein in the choroid or RPE. That is, the nucleic acid encoding the M2 macrophage polarization inducer may be operably linked to regulatory elements such as promoters and enhancers.

[0022] <Mast cell stabilizer> A mast cell stabilizer is also referred to as a mast cell stabilizer or a mast cell stabilizing agent. A mast cell stabilizer is, for example, a drug that suppresses the release of allergic substances from mast cells, and is, for example, a drug that stabilizes the cell membrane of mast cells and suppresses the release of allergic substances from mast cells. Examples of mast cell stabilizers include azelastine hydrate solution (Zepelin), anlelexanox (Elex), pemirolast potassium (Allegasal), pemirolast potassium (Pemirolaston), sodium cromoglycate (Intal), tranilast (Rizaben), tranilast (Trameras), ibudilast (Ketas), β2-adrenergic agonist, cromolyn sodium, cromoglycic acid, ketotifen, methylxanthine, omalizumab, pemirolast, quercetin, etc., and preferably cromoglycic acid or pemirolast or a salt thereof, etc.

[0023] <Chemical mediator release inhibitor> A chemical mediator release inhibitor is a drug that suppresses allergic reactions, for example, by suppressing the release of chemical mediators from immune cells such as mast cells. It is a drug that stabilizes the cell membrane of mast cells to prevent the release of substances such as histamine to the outside. It is a drug that suppresses the release of allergic substances (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 azelastine hydrate solution (Zepelin), anlexanox (Elex), pemirolast potassium (Allegasal), pemirolast potassium (Pemirolaston), sodium cromoglycate (Intal), tranilast (Rizaben), tranilast (Trameras), ibudilast (Ketas), β2 - adrenergic agonist, cromolyn sodium, cromoglycic acid, ketotifen, methylxanthine, omalizumab, pemirolast, quercetin, etc. Preferably, cromoglycic acid or pemirolast or its salts, etc. are included.

[0024] <Lactic acid bacteria> Examples of lactic acid bacteria include homofermentative lactic acid bacteria and heterofermentative lactic acid bacteria. Examples of lactic acid bacteria also include spherical lactic acid cocci and rod-shaped lactic acid bacilli. Examples of lactic acid bacteria include bacteria that are Gram-positive, bacilli or cocci, have no spores, are non-motile, produce 50% or more lactic acid with respect to consumed glucose, and require niacin (B3) as an essential nutrient. 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 Actinobacteria. Examples of lactic acid bacteria of the order 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, and lactic acid bacteria of the genus Streptococcus (Streptococcus). Examples of lactic acid bacteria of the phylum Actinobacteria include lactic acid bacteria of the genus Bifidobacterium. 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, Enterococcus faecium, etc.Examples of lactic acid bacteria belonging to the genus Lactococcus include, for example, Lactococcus lactis and Lactococcus cremoris. Examples of lactic acid bacteria belonging to the genus Pediococcus include, for example, Pediococcus damnosus. Examples of lactic acid bacteria belonging to the genus Leuconostoc include, for example, Leuconostoc mesenteroides. Examples of lactic acid bacteria belonging to the genus Streptococcus include, for example, Streptococcus thermophiles and Streptococcus mutans. Examples of lactic acid bacteria belonging to the genus Bifidobacterium include, for example, Bifidobacterium bifidum and Bifidobacterium adolescentis. It is known that administration of Lactobacillus paracasei bacteria improves the symptoms of non-alcoholic steatohepatitis by shifting Kupffer cells (resident liver macrophages) in the liver to M2 (Sohn et al, Dig Dis Sci, 2015 Nov;60(11):3340-50). Administration of Lactobacillus paracasei bacteria is known to alleviate retinal degeneration caused by blue light by activating M2 macrophages (Morita et al, Nutrients, 2018 Dec 15;10(12):1991). Administration of Lactobacillus plantarum bacteria is known to alleviate colitis by promoting the polarization of M1 macrophages to M2 macrophages (Jang et al, Int Immunopharmacol, 2014 Jul;21(1):186-92). Administration of Lactobacillus brevis bacteria is known to alleviate colitis by promoting the polarization of M1 macrophages to M2 macrophages (Jang et al, J Appl Microbiol, 2013 Sep;115(3):888-96). Thus, lactic acid bacteria of the order Lactobacillales such as the genus Lactobacillus are known to induce and activate polarization to M2 macrophages by their administration. 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 for delivery to the choroid of substances that regulate the functions of choroid-resident immune cells, such as M2 macrophage polarization-inducing substances, mast cell stabilizers, and chemical mediator release inhibitors. For example, when a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor is a nucleic acid, DNA or RNA (messenger RNA) encoding a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor may be administered to a subject using a DDS such as a liposome. Other available DDSs include charged lipids, nucleic acid-protein complexes, and biopolymers. Further, a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor may be administered to a subject in the form of a protein. Administration can be performed, for example, by local administration to the choroid.

[0026] Delivery of a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor to the choroid of a subject may be performed by transplanting cells that secrete a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator to the subject. Cells that secrete a choroid resident immune cell function regulator 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, it may be a cell genetically engineered to secrete a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator.

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

[0028] [Composition for Suppressing or Treating Myopia] One aspect of the present disclosure relates to a composition for suppressing, improving, or treating myopia using a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. More specifically, one of the embodiments of the present disclosure relates to a composition for suppressing or treating myopia, containing a therapeutically effective amount of a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, a chemical mediator release inhibitor, or lactic acid bacteria. Such a composition can be used to deliver a choroid resident immune cell function regulator such as an M2 macrophage polarization inducer, a mast cell stabilizer, 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, or a supplement.

[0029] The pharmaceutical composition according to the present disclosure is administered, for example, locally to the eye. Examples of the administration form of the present composition include, for example, instillation (including instillation of an eye ointment and eye washing), subconjunctival administration, intracorneal sac administration, sub-Tenon's capsule administration, and the like.

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

[0031] Eye drops can be prepared by selectively using, as necessary, tonicity agents such as sodium chloride, potassium chloride, and concentrated glycerin; buffering agents 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; preservatives such as parabens, etc. The pH may 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] In addition, the pharmaceutical composition according to the present disclosure is not limited to local ocular administration, and can also be administered by any administration route such as enteral administration (oral, transcatheter, injection, etc.), parenteral administration (intravenous, intraarterial, transdermal, intramuscular injection, etc.). The dosage form of the composition used in these administration forms can be appropriately selected. For example, solid preparations such as tablets, granules, powders, capsules, chewable tablets, etc., and liquid preparations such as solutions, syrups, injections, drip infusions, etc. can be used. In the pharmaceutical composition according to the present disclosure, other components can be appropriately blended within a range that does not impair the effects of the present invention. Examples of other components include any carrier, buffer, diluent, excipient, suspending agent, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrating substance, absorbent, preservative, surfactant, coloring agent, flavor, or sweetening agent, etc. These components can be blended alone or in appropriate combinations of two or more.

[0033] The content ratio of the choroid resident immune cell function regulator such as the M2 macrophage polarization inducer, mast cell stabilizer, chemical mediator release inhibitor, etc., or lactic acid bacteria in 100% by weight of the pharmaceutical composition can be appropriately set, for example, in 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 according to the administration form, the age, weight of the administration subject, the degree of the desired effect, etc. The dosage of the choroid resident immune cell function regulator such as the M2 macrophage polarization inducer, mast cell stabilizer, chemical mediator release inhibitor, etc., or lactic acid bacteria is, for example, 100 to 1,000,000 nmol per day, preferably 150 to 100,000 nmol, 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 a food or a supplement. As the form of the food or supplement, for example, liquid, solid, tablet, granule, powder, capsule, paste, gel, etc., for example, the above-mentioned solid or liquid preparations can be arbitrarily selected. Specific examples of foods include, for example, beverages such as fruit juice drinks, vegetable juices, soft drinks, and tea, soups, puddings, yogurts, cake mix products, various general processed foods such as confectioneries, cookies, candies, gummies, gums, etc., as well as special-purpose foods, foods for specified health use, foods with nutritional functions, functional foods, dietary supplements, health supplements, fortified foods, nutritional adjustment foods, etc., for example, supplements, drink agents, etc.

[0036] The food or supplement according to the present disclosure can contain any functional materials (vitamins, minerals, etc.), any excipients, and any additives (flavoring agents, sweeteners, acidulants, colorants, thickeners, binders, fortifiers, disintegrants, buffers, surfactants, solubilizers, resorption promoters, dispersants, stabilizers, gelling agents, emulsifiers, antioxidants, surfactants, preservatives, moisture-proof agents, pH adjusters, colorants, soothing agents, isotonic agents, etc.).

[0037] One aspect of the present disclosure relates to the use of substances that regulate the functions of choroidal resident immune cells, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, for suppressing or treating myopia, or lactic acid bacteria. Further, another aspect of the present disclosure relates to the use of substances that regulate the functions of choroidal resident immune cells, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, for the production of pharmaceuticals used for suppressing or treating myopia, or lactic acid bacteria.

[0038] [Screening method] One aspect of the present disclosure relates to a screening method for searching for a component that promotes the polarization of macrophages into M2 macrophages for controlling the thinning of the choroid, which is a mechanism of myopia onset, and a screening method for searching for a component effective for the prevention and treatment of myopia. In some embodiments, the screening method includes: (i) administering a candidate substance to a model animal; and (ii) measuring the axial length of the eye, the thickness of the choroid, and the refractive power in the model animal. In some embodiments, the screening method includes: (i) administering a candidate substance to a model animal; and (ii) measuring the expression of a choroid resident immune cell marker such as an M2 macrophage marker in a sample derived from the model animal. In some embodiments, the screening method further includes (iii) measuring the expression of a choroid resident immune cell marker such as an M1 macrophage marker. In addition, in some embodiments, in vitro screening methods are also included, in which cell lines such as monocytic cell lines including RAW264 cells, J774 cells, U937 cells, 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 a choroid resident immune cell marker such as an M2 macrophage marker is evaluated. In some embodiments, the model animal can be an animal during myopia induction treatment. In some embodiments, the choroid resident immune cell markers such as M2 macrophage markers can be CD163, CD206, arginase, and IL-10. Examples of the method for measuring the choroid resident immune cell markers such as macrophage markers include quantitative PCR, flow cytometry, immunostaining, luciferase assay, arginase activity staining, and the like.

[0039] The substance identified by such a screening method can be used to suppress or treat myopia.

[0040] This specification shows preferred embodiments of the present invention, but it is obvious to those skilled in the art that such embodiments are provided for illustrative purposes only. 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 in this specification can be used when implementing the present invention. In addition, this application claims the benefit of priority of a Japanese patent application with the application number of Japanese Patent Application No. 2022-72596. The content of the Japanese patent application, as well as the content described in all publications including the patents and patent application documents referred to in this specification, should be construed as incorporated herein by reference in the same manner as the content specified herein.

Example

[0041] Hereinafter, the present invention will be described in more detail with reference to experimental examples.

[0042] <Test Example 1 Choroidal thinning and myopia induction by LPS administration> To confirm whether polarization into M1 macrophages is involved in choroidal thinning and myopia induction, the expression of polarization marker genes in the choroid after LPS administration and the measurement of choroidal thickness, axial length, and refractive value were performed.

[0043] The choroidal thickness, axial length, and refractive value were measured in mice administered LPS for 2 weeks and control mice administered PBS, and the amount of change was calculated (Figs. 1A, B). Then, the eyeballs were enucleated, and the expression of polarization marker genes was measured by quantitative PCR (Fig. 2).

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

[0045] (Measurement of axial length, choroidal thickness, and refractive power) The axial length, choroidal thickness, and refractive power of the mice in each group were measured. The axial length and choroidal thickness were measured using spectral domain optical coherence tomography (Envisu R4310, manufactured by Leica). The refractive power was measured using an infrared photorefractor for mice (developed by Professor Schaeffel of the University of Tubingen).

[0046] (Observation of the choroid by electron microscope) The eyes of the mice in each group were collected, fixed overnight at 4°C with 2.5% glutaraldehyde in PBS (Phosphate Buffered Saline), and rinsed with 0.1 M sodium cacodylate buffer for 1 hour. Next, after fixation with 1% OsO4 in 0.1 M cacodylate buffer for 2 hours, dehydration was performed using a graded ethanol solution. Furthermore, the eyes were infiltrated overnight in a 1:2 mixture of propylene oxide and Epon-Araldite and embedded in 100% resin. The block was sectioned and observed at an accelerating voltage of 100 kV using a transmission electron microscope (JEM1400 plus; JEOL).

[0047] (Evaluation of gene marker expression) Choroid, retina, and sclera samples were collected from the mice administered LPS for 2 weeks as described above, and the expression analysis of M1 macrophage marker genes and oxidative stress-related genes was performed by quantitative PCR method.

[0048] (Results) Compared with the control group administered PBS, in the LPS administration group, a decrease in refraction (left in Figure 1B), elongation of the eye axis (center in Figure 1B), and thinning of the choroid (right in Figure 1B) were observed during both the 1-week and 2-week administration periods.

[0049] Also, as a result of gene expression analysis, an increase in the expression of M1 marker genes and oxidative stress-related genes was particularly observed in the choroid (Figure 2).

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

[0051] <Test Example 2 Polarization into M2 Macrophages by Administration of IL-4 and IL-13 and Myopia Suppression Effect> 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 a myopia-inducing effect, 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 3B).

[0052] <IL-4 Administration> IL-4 solution (0.1 μg / 100 μl, 10 μg / kg BW) was intraperitoneally administered to C57BL6J mice (n = 4 per group). Choroid, retina, and liver samples were collected at 0 h (before administration) and 4, 24, and 48 h after administration, and the expression of M2 macrophage marker genes was analyzed by Western blot and real-time PCR (Fig. 3). Also, choroid and retina samples were collected at 0 h (before administration) and 4 and 24 h 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 myopia-induced mice to evaluate whether there was a myopia-suppressing effect (Fig. 4). IL-4 solution (0.1 μg / 100 μl, 10 μg / kg BW) was intraperitoneally administered to C57BL6 mice (-30D lens, n = 4) during myopia induction 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, 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). These results suggest that IL-4 administration induces polarization into M2 macrophages, suppresses the expression of oxidative stress-related genes, and inhibits the progression of myopia (Fig. 5).

[0055] Furthermore, administration of IL-4 during the myopia induction period was confirmed to suppress the decrease in refraction (left in Fig. 5B), suppress the elongation of the eye axis (center in Fig. 5B), and suppress the thinning of the choroid (right in Fig. 5B).

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

[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 eye axis, myopic shift in refraction, and thinning of the choroid caused by wearing a minus lens were suppressed, and polarization into M2 macrophages was induced (Fig. 6). Note that blood flow changes and measurement of the number of macrophages were performed as follows. Blood flow changes were measured using a wavelength-swept optical coherence tomography (OCT S-1, Canon). The number of macrophages was measured by staining with F4 / 80 antibody and CD11b antibody after digestion of the choroid and using a flow cytometer (CytoFLEX S, Beckman Coulter). The number of M2 macrophages was measured in the same manner as the measurement of the number of macrophages, except that the staining process with CD206 antibody was added.

[0058] As in the results of Test Examples 1 and 2, it was confirmed that myopia can be induced or suppressed, particularly by controlling the state of macrophages in the choroid. From the perspective of suppressing and treating myopia progression, it has been shown that promoting polarization into M2 macrophages is particularly effective for its achievement.

[0059] <Test Example 3 Suppression of Myopization by Mast Cell Stabilizer Eye Drops> To verify the inhibitory effect of mast cell stabilizer eye drops on myopia progression, a lens-induced myopia model in which myopia was induced by fitting a minus lens to mice was used, and cromolyn acid solution (4% solution) or pemirolast potassium (0.1% solution) was administered by eye drops once a day during the myopia induction period.

[0060] In accordance with the above test method, the axial length, refractive value, and choroid thickness were measured in mice and control mice that had undergone 3 weeks of myopia induction and eye drop administration of cromolyn acid solution or pemirolast potassium solution, and the amount of change was calculated (Figs. 7A, B, C).

[0061] As a result, in the minus lens-wearing eyes of the control group administered with PBS, elongation of the eye axis (Fig. 7A), myopization of refraction (Fig. 7B), and thinning of the choroid (Fig. 7C) were observed compared to the control eyes. On the other hand, the above-described changes observed in the PBS administration group were not observed in the administration groups administered with cromolyn acid solution or pemirolast potassium solution by eye drops.

[0062] As in these results, it was confirmed that mast cell stabilizer eye drop administration suppresses myopia progression.

[0063] <Test Example 4 Comparison of Myopia Inhibitory Effects between Mast Cell Stabilizer Eye Drops and Histamine Receptor Inhibitor Eye Drops> Antihistamines are roughly classified into two types: mast cell stabilizers that inhibit mast cell degranulation and histamine receptor antagonists that inhibit the action of histamine secreted by mast cell degranulation. In order to clarify whether the myopia inhibitory effect of the mast cell stabilizer eye drops in Test Example 3 is due to the action of the antihistamine or due to the inhibition of mast cell degranulation, pemirobast potassium (0.1% solution) was instilled into the eyes of mice induced with myopia by LIM as a mast cell stabilizer, and levocabastine solution (0.025% solution) was instilled as a histamine receptor antagonist, and their myopia inhibitory effects were compared. The results are shown in Fig. 8.

[0064] As shown in Fig. 8, as a result, similar to Test Example 3, in the pemirobast potassium administration group, elongation of the eye axis, myopic shift of refraction, and thinning of the choroid observed in the control group were not observed. On the other hand, in the levocabastine administration group, elongation of the eye axis, myopic shift of refraction, and thinning of the choroid were observed as in the control group.

[0065] From the above results, among antihistamines, the myopia inhibitory effect of mast cell stabilizers was confirmed.

[0066] <Test Example 5 Inhibition of Myopia Progression by Administration of Lactobacillus paracasei Since the induction of M2 macrophages can suppress myopia progression, Lactobacillus paracasei contained in Yakult (Yakult Honsha Co., Ltd.) as lactic acid bacteria was cultured and proliferated, administered to myopia model mice, and the eye axis length, refractive value, and choroid thickness were measured in the same manner as in Test Examples 3 to 4, and the amount of change was calculated (Fig. 9). As a result, it was confirmed that the administration of lactic acid bacteria suppressed myopia progression (Fig. 9).

Claims

**Claim 1** A composition for suppressing, improving or treating myopia, comprising a therapeutically effective amount of lactic acid bacteria, wherein the lactic acid bacteria are Lactobacillus paracasei bacteria. **Claim 2** The composition according to claim 1, wherein a decrease in refraction, elongation of the eye axis, and / or thinning of the choroid are suppressed. **Claim 3** A supplement for suppressing, improving or treating myopia, comprising an effective amount of lactic acid bacteria, wherein the lactic acid bacteria are Lactobacillus paracasei bacteria. **Claim 4** The supplement according to claim 3, wherein a decrease in refraction, elongation of the eye axis, and / or thinning of the choroid are suppressed. **Claim 5** A food for suppressing, improving or treating myopia, comprising an effective amount of lactic acid bacteria, wherein the lactic acid bacteria are Lactobacillus paracasei bacteria. **Claim 6** The food according to claim 5, wherein a decrease in refraction, elongation of the eye axis, and / or thinning of the choroid are suppressed. **Claim 7** Use of lactic acid bacteria in the manufacture of a medicament for suppressing, improving or treating myopia, wherein the lactic acid bacteria are Lactobacillus paracasei bacteria. **Claim 8** The use according to claim 7, wherein a decrease in refraction, elongation of the eye axis, and / or thinning of the choroid are suppressed.

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