Methods and compositions for preventing or treating myopia

Abstract

The present invention aims to provide a method for preventing and treating myopia and a composition for use therein. [Solution] The present invention provides a method and composition for suppressing, improving, or treating myopia by administering choroidal resident immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria. Inducing polarization of M2 macrophages in the choroid, or regulating the function of choroidal resident immune cells by administering mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria, is effective in preventing and treating myopia.

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 by inducing polarization to M2 macrophages or modulating the functions of choroidal 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 of the eye, 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 the progression of myopia. 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 the choroidal thickness and the axial length, it has been suggested that changes in the choroidal thickness may be a predictive biomarker for the elongation of the axial length of the eye. However, the detailed mechanism by which the choroid is involved in the onset and progression of myopia remains 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 the retinal cells, it has a function of supplying growth factors involved in the tissue remodeling of the sclera outside the eyeball and the 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 change in scleral structure in which the axial length increases. Therefore, maintaining and increasing the choroidal thickness and blood flow have attracted attention as new targets in the prevention and treatment of myopia.

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

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

[0006] The choroid contains 13 types of cells, four of which are immune cells. In addition to immune responses, tissue-resident immune cells are responsible for maintaining tissue homeostasis and structure. For example, it has been reported that the choroid thins in mice lacking macrophages or in mice in which mast cell degranulation is induced (Non-patent Literature 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] This disclosure aims to provide methods for preventing or treating myopia, and compositions for use therein, particularly compositions targeting choroidal resident immune cells, especially macrophages and mast cells. [Means for solving the problem]

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

[0010] Furthermore, mast cells are abundant around small blood vessels and are also found in the choroid, a tissue composed of blood vessels, suggesting their involvement in maintaining choroidal morphology. The inventors have discovered that myopia progression is suppressed by administering a mast cell degranulation inhibitor (mast cell stabilizer) as eye drops. Specifically, the inventors found that myopia progression is suppressed by administering a mast cell stabilizer (sodium cromoglycate, pemirolast potassium), a type of anti-allergic drug, as eye drops to myopic model mice wearing negative contact lenses. Notably, no suppression of myopia progression was observed when another anti-allergic drug, an antihistamine (levocabastine), was administered as eye drops to mice undergoing myopia induction by wearing negative contact lenses.

[0011] Furthermore, it is known that lactic acid bacteria of the Lactobacillus genus, such as Lactobacillus paracasei, induce and activate polarization to M2 macrophages upon administration. The present inventors have found that administration of lactic acid bacteria of the Lactobacillus genus, such as Lactobacillus paracasei, suppresses myopia progression. Considering these findings in conjunction with the aforementioned Non-Patent Documents 6-7, it is thought that if the choroidal structure (thickness) can be maintained through intervention targeting immune cells commensal to the choroid, it will have an inhibitory effect on myopia progression. Based on these findings, it is thought that compositions that target immune cells present in the choroid, such as macrophages and mast cells, and induce their respective properties to an appropriate state, have a myopia progression inhibitory effect. The present invention is based on these findings and encompasses the following embodiments.

[0012] [Aspect 1] A method for suppressing, improving, or treating myopia, comprising administering a therapeutically effective amount of a choroidal immune cell function modifier or lactic acid bacteria to a subject requiring treatment. [Aspect 2] The method according to embodiment 1, wherein the choroidal resident immune cell function modifier is an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor. [Aspect 3] The method according to embodiment 2, wherein the M2 macrophage polarization-inducing substance is selected from the group consisting of proteins, peptides, nucleic acids, small molecule compounds, and large molecule compounds. [Aspect 4] The method according to Embodiment 2, wherein the M2 macrophage polarization-inducing substance is selected from the group consisting of IL-4, IL-10, IL-13, TLR2,4,7,9 and their ligands, bisanthren 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 according to embodiment 2, wherein the mast cell stabilizer or chemical mediator release inhibitor is cromoglycic acid, pemirolast, or a salt thereof. [Aspect 6] The method according to Embodiment 1, wherein the lactic acid bacteria are of the genus Lactobacillus. [Aspect 7] The method according to embodiment 1, wherein the lactic acid bacteria is Lactobacillus paracasei. [Aspect 8] The method according to embodiment 1, wherein a choroidal immune cell function modifier or lactic acid bacteria is contained in a pharmaceutical composition, supplement, or food. [Aspect 9] The method according to embodiment 1, wherein a decrease in refraction, elongation of the axial length, and / or thinning of the choroid are suppressed. [Aspect 10] A composition for suppressing or treating myopia, containing a therapeutically effective amount of a choroidal immune cell function modifier or lactic acid bacteria. [Aspect 11] A supplement containing an effective amount of choroidal immune cell function modifier or lactic acid bacteria to suppress or improve myopia. [Aspect 12] A food for suppressing or improving myopia, containing an effective amount of a substance for regulating the function of choroidal resident immune cells or lactic acid bacteria. [Aspect 13] Use of a substance for regulating the function of choroidal resident immune cells or lactic acid bacteria in the manufacture of a medicament for suppressing or treating myopia. [Aspect 14] Use of a substance for regulating the function of choroidal resident immune cells or lactic acid bacteria for suppressing or treating myopia. [Effect of the Invention]

[0013] According to the present disclosure, it is possible to provide a new method for preventing and treating myopia by regulating 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] [Figure 1] Results of induction of myopia by LPS administration. (A) A diagram showing the experimental schedule. (B) A graph showing changes in refractive value (left), axial length (center), and choroid thickness (right). [Figure 2] A graph showing changes in the expression of M1 macrophage marker genes and oxidative stress-related genes by LPS administration. [Figure 3] Results of induction of M2 macrophage polarization by IL-4 administration. (A) A diagram showing the experimental schedule. (B) A graph showing changes in the expression of CD206 protein and phosphorylated STAT6 in the choroid by IL-4 administration. (C) A graph showing changes in the expression of CD163 and CD206 mRNAs by IL-4 administration. [Figure 4] A graph showing changes in the expression of M2 macrophage marker genes and oxidative stress-related genes by IL-4 administration. [Figure 5]These are the results of an experiment to suppress myopia development with IL-4 administration. (A) This figure shows the experimental schedule. (B) This figure shows the changes in refraction (left), axial length (center), and choroidal thickness (right) due to IL-4 administration during the myopia induction period. [Figure 6] These are the results of myopia suppression and M2 macrophage polarization induction by IL-13 administration. (A) This figure shows the experimental schedule. (B) This graph shows the changes in refraction (upper left), axial length (upper right), choroid thickness (lower left), and blood flow (lower right) due to IL-13 administration during the myopia induction period. (C) This graph shows the changes in macrophage number and M2 macrophage percentage due to myopia induction and IL-13 administration. [Figure 7] These results demonstrate the myopia-suppressing effect of mast cell stabilizer eye drops. (A) Graph showing the change in axial length. (B) Graph showing the change in refractive error. (C) Graph showing the change in choroid thickness. The left graph shows the control group vs. the pemirolast potassium eye drop group, and the right graph shows the control group vs. the cromoglycic acid eye drop group. *p<0.05, **p<0.01, ***p<0.001, student's t-test [Figure 8] This graph compares the myopia-suppressing effects of mastcell stabilizer eye drops and histamine receptor inhibitors. (A) Graph showing the change in axial length. (B) Graph showing the change in refractive error. (C) Graph showing the change in choroid thickness. From left to right, the graphs represent the control group, the levocabastine eye drop group (histamine receptor inhibitor), and the pemirolast potassium eye drop group (mastocell stabilizer). *p<0.05, **p<0.01, ***p<0.001, student's t-test [Figure 9] These results demonstrate the effect of administering Lactobacillus paracasei in suppressing myopia progression. (A) A graph showing the change in axial length. (B) A graph showing the change in refractive error. (C) A graph showing the change in choroid thickness. In each graph, the left shows the control group and the right shows the group treated with Lactobacillus paracasei. [Modes for carrying out the invention]

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

[0016] [Methods to suppress or treat myopia] As described above, in recent years, it has become clear that thinning of the choroid is involved in the development of myopia. The inventors of this invention have diligently researched the factors that control this thinning and have found that changes in the state (polarization) of macrophages in the choroid change the thickness of the choroid, thereby controlling the progression of myopia. Macrophages exist in two states: M1 macrophages and M2 macrophages. M1 macrophages are primarily involved in inducing inflammation, while M2 macrophages play an antagonistic role in ending or suppressing inflammation. The inventors have for the first time discovered that inducing polarization to M1 macrophages in the choroid leads to choroidal thinning and myopia progression, while inducing polarization to M2 macrophages suppresses choroidal thinning and myopia progression. Furthermore, mast cells are abundant around small blood vessels and are also found in the choroid, a tissue composed of blood vessels, suggesting their involvement in maintaining choroidal morphology. The inventors have found that eye drops containing a mast cell degranulation inhibitor (mast cell stabilizer) suppress myopia progression. Furthermore, it is known that lactic acid bacteria of the Lactobacillus genus, such as Lactobacillus paracasei, induce and activate polarization to M2 macrophages upon administration. The inventors have found that administration of lactic acid bacteria of the Lactobacillus genus, such as Lactobacillus paracasei, suppresses the progression of myopia. Thus, the inventors have found that the progression of myopia can be suppressed by regulating the function of choroidal resident immune cells. Therefore, one aspect of this disclosure relates to methods for suppressing, improving, 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 mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria. One aspect of this disclosure relates to methods 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 M2 macrophage polarization inducers, or by administering mast cell stabilizers, chemical mediator release inhibitors, or lactic acid bacteria. One aspect of this disclosure relates to a method for suppressing refractive error, axial lengthening, and / or choroidal thinning in a subject requiring treatment, including modulating the function of choroidal resident immune cells, 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. Furthermore, one aspect of this disclosure relates to a screening method for searching for substances that modulate the function of choroidal resident immune cells, such as components that promote polarization to M2 macrophages, in order to control choroidal thinning, which is a mechanism of myopia development, and to a screening method for searching for components that are effective in preventing and treating myopia.

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

[0018] <M2 macrophage polarization inducer substance> Macrophages are classified into inflammatory M1 macrophages and anti-inflammatory M2 macrophages with different functions, and respond to signals such as cytokines and stimulatory components to polarize and perform specific function expression. Therefore, the M2 macrophage polarization inducer substances in the present disclosure include various substances involved in signal transduction that induces 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. Also, it is known that there are several populations of M2 macrophages. In addition to the above-mentioned IL-4 and IL-13 that induce M2a macrophages, IL-10, glucocorticoid hormone (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, etc. can also be mentioned as M2 macrophage polarization inducer substances in the present disclosure.

[0019] Examples of M2 macrophage polarization-inducing substances in this disclosure include various small molecule compounds such as bisanthren 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, for example, culture monocyte cell lines such as RAW264 cells, J774 cells, and U937 cells, mouse intraperitoneal macrophages, and bone marrow-derived macrophages in a culture medium containing the substance to be evaluated, and use an in vitro evaluation system to assess the expression of M2 macrophage markers to determine whether the substance has M2 macrophage polarization-inducing ability. This will allow them to obtain M2 macrophage polarization inducers suitable for use in the methods and compositions disclosed herein, in addition to those specifically described herein.

[0021] If the M2 macrophage polarization inducer is a protein or peptide, it may be administered to the target in the form of the encoding DNA or RNA. The nucleic acid encoding the M2 macrophage polarization inducer may be administered to the target using a plasmid or expression vector. The expression vector may be, but is not limited to, a viral vector, particularly an adenovirus vector. Other usable viral vectors include, for example, retroviruses, adeno-associated viruses, pox, baculoviruses, vaccinia, herpes simplex, Epstein-Barr, geminivirus, and karimovirus vectors. The nucleic acid encoding the M2 macrophage polarization inducer may contain regulatory elements for choroidal or RPE-specific expression of the protein. 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> Mast cell stabilizers are also called mast cell stabilizers or mast cell stabilizers. Mast cell stabilizers are drugs that suppress the release of allergens from mast cells, for example, drugs that stabilize the cell membrane of mast cells to suppress the release of allergens from mast cells. Examples of mast cell stabilizers include acitazanolase hydrate (Zeppelin), anlexanox (Erix), pemirolast potassium (Allegisal), pemirolast potassium (Pemilaston), sodium cromoglycate (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonists, cromolin sodium, cromoglycic acid, ketotifen, methylxanthine, omalizumab, pemirolast, and quercetin, with cromoglycic acid or pemirolast or its salts being preferred.

[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 are drugs that stabilize the cell membrane of mast cells to prevent the release of histamine and other substances, and drugs that suppress 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 asitazanolase hydrate (Zeppelin), anlexanox (Erix), pemirolast potassium (Allegisal), pemirolast potassium (Pemilaston), sodium cromoglycate (Intal), tranilast (Rizaben), tranilast (Tramelas), ibudilast (Ketas), β2-adrenergic agonists, cromolyn sodium, cromoglycic acid, ketotifen, methylxanthine, omalizumab, pemirolast, and quercetin, with cromoglycic acid or pemirolast or its salts being preferred.

[0024] <Lactic acid bacteria> Examples of lactic acid bacteria include homolactic acid bacteria and heterolactic acid bacteria. Examples of lactic acid bacteria include spherical lactic acid cocci and rod-shaped lactic acid bacilli. Examples of lactic acid bacteria include Gram-positive bacteria that are rod-shaped or cocci, lack spores, are non-motile, produce lactic acid at a rate of 50% or more of consumed glucose, and require niacin (B3) as an essential nutrient. Examples of lactic acid bacteria include enteric lactic acid bacteria, animal lactic acid bacteria, plant lactic acid bacteria, and marine lactic acid bacteria. Examples of lactic acid bacteria include Lactobacillus and Actinomycetes. Examples of lactic acid bacteria belonging to the Lactobacillales order include lactic acid bacteria of the genera Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, and Streptococcus. Examples of lactic acid bacteria belonging to the Actinomycetes phylum include lactic acid bacteria of the genus Bifidobacterium. Examples of lactic acid bacteria belonging to 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, and Lactobacillus casei Shirota. Examples of lactic acid bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium.Examples of lactic acid bacteria belonging to the genus Lactococcus include Lactococcus lactis and Lactococcus cremoris. Examples of lactic acid bacteria belonging to the genus Pediococcus include Pediococcus damnosus. Examples of lactic acid bacteria belonging to the genus Leuconostoc include Leuconostoc mesenteroides. Examples of lactic acid bacteria belonging to the genus Streptococcus include Streptococcus thermophiles and Streptococcus mutans. Examples of lactic acid bacteria belonging to the genus Bifidobacterium include Bifidobacterium bifidum and Bifidobacterium adolescentis. It is known that administering Lactobacillus paracasei improves the symptoms of non-alcoholic steatohepatitis by shifting hepatic Kupffer cells (resident hepatic macrophages) to M2 (Sohn et al, Dig Dis Sci, 2015 Nov;60(11):3340-50). It is known that administering Lactobacillus paracasei alleviates blue light-induced retinal degeneration by activating M2 macrophages (Morita et al, Nutrients, 2018 Dec15;10(12):1991). It is known that administering Lactobacillus plantarum alleviates colitis by promoting polarization from M1 macrophages to M2 macrophages (Jang et al, Int Immunopharmacol, 2014 Jul;21(1):186-92). It is known that administration of Lactobacillus brevis bacteria alleviates colitis by promoting the polarization from M1 macrophages to M2 macrophages (Jang et al, J Appl Microbiol, 2013 Sep;115(3):888-96). Thus, it is known that lactic acid bacteria of the Lactobacillus order, such as those of the genus Lactobacillus, induce and activate polarization to M2 macrophages upon administration. Therefore, the lactic acid bacteria are preferably those of the Lactobacillus order, such as those of the genus Lactobacillus.

[0025] Drug delivery systems (DDS) may be used to deliver target choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, to the choroid. For example, if the choroidal immune cell function modifier, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, is a nucleic acid, the DNA or RNA (messenger RNA) encoding the choroidal immune cell function modifier, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, may be administered to the target using a DDS such as liposomes. Other available DDSs include charged lipids, nucleic acid-protein complexes, and biopolymers. Furthermore, the choroidal immune cell function modifier, such as an M2 macrophage polarization inducer, mast cell stabilizer, or chemical mediator release inhibitor, may be administered to the target in protein form. Administration can be performed, for example, by local administration to the choroid.

[0026] The delivery of choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, to the target choroid may be performed by transplanting cells that secrete choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediators. Cells that secrete choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediators, may be, for example, retinal pigment epithelial cells (RPE). Alternatively, these may be cells that have been genetically modified to secrete choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediators.

[0027] The subjects of the methods for suppressing or treating myopia of this disclosure may be mammals including humans, and non-human mammals including dogs, cats, cattle, and horses, but preferably humans.

[0028] [Composition for suppressing or treating myopia] One aspect of this disclosure relates to compositions for suppressing, improving, or treating myopia using choroidal immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria. More specifically, one aspect of this disclosure relates to compositions for suppressing or treating myopia containing therapeutically effective amounts of choroidal immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria. Such compositions may be used to deliver choroidal immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria to a target choroid. Such compositions may be pharmaceutical compositions, foods, or supplements.

[0029] The pharmaceutical composition relating to this disclosure is administered, for example, topically to the eye. Examples of administration methods for this composition include ophthalmic administration (including application of eye ointment and eye washing), subconjunctival administration, intraconjunctival sac administration, and sub-Tenon's capsule administration.

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

[0031] Eye drops can be prepared by selecting and using, as needed, isotonic 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; and preservatives such as parabens. The pH should be within the range acceptable for ophthalmic preparations, but is usually preferred to be 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 compositions relating to this disclosure are not limited to local administration to the eye, but can also be administered via any route of administration, such as enteral administration (oral, tube feeding, intravenous injection, etc.) or parenteral administration (intravenous, intraarterial, transdermal, intramuscular injection, etc.). The dosage form of the composition used for these administration methods can be appropriately selected, but may be solid preparations such as tablets, granules, powders, capsules, or chewable preparations, or liquid preparations such as solutions, syrups, injections, or intravenous drips. The pharmaceutical compositions relating to this disclosure may contain other components in appropriate amounts, provided that they do 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 decomposition product, absorbent, preservative, surfactant, colorant, fragrance, or sweetener. These components may be included individually or in appropriate combinations of two or more.

[0033] The content of choroidal commensal immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, 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] There are no particular restrictions on the dosage of the pharmaceutical composition relating to this disclosure, and it can be appropriately selected according to the form of administration, the age and weight of the recipient, the desired degree of effect, etc. The dosage of choroidal commensal immune cell function modifiers such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria, for example, is 100 to 1,000,000 nmol per day, preferably 150 to 1,000,000 nmol, and the frequency of administration can be, for example, 1 to 100 times per month.

[0035] Furthermore, the compositions relating to this disclosure may be foods or supplements. The form of the food or supplement can be arbitrarily selected, for example, liquid, solid, tablet, granule, powder, capsule, paste, gel, etc. Specific examples of foods include beverages such as fruit juices, vegetable juices, soft drinks, and tea; soups, puddings, yogurts, cake premixes, confectionery, cookies, candies, gummies, and gum; as well as foods for special dietary uses, foods for specified health uses, foods with nutritional function claims, functional foods, nutritional supplements, health supplements, fortified foods, nutritional adjustment foods, etc., such as supplements and drinks.

[0036] The foods or supplements relating to this disclosure may contain any functional ingredients (vitamins, minerals, etc.), any excipients, and any additives (flavoring agents, sweeteners, acidulants, colorants, thickeners, binders, fortifiers, disintegrants, buffers, surfactants, solubilizers, reabsorption enhancers, dispersants, stabilizers, gelling agents, emulsifiers, antioxidants, surfactants, preservatives, moisture-proofing agents, pH adjusters, colorants, analgesics, isotonic agents, etc.).

[0037] One aspect of this disclosure relates to the use of choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria, for the suppression or treatment of myopia. Furthermore, another aspect of this disclosure relates to the use of choroidal immune cell function modifiers, such as M2 macrophage polarization inducers, mast cell stabilizers, and chemical mediator release inhibitors, or lactic acid bacteria, in the manufacture of pharmaceuticals used for the suppression or treatment of myopia.

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

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

[0040] While preferred embodiments of the 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 they can be modified, altered, and substituted in various ways without departing from the invention. It should be understood that various alternative embodiments of the invention described herein can be used in carrying out the invention. Furthermore, this application claims priority to Japanese patent application No. 2022-72596, and the contents of said Japanese patent application, as well as the contents of all publications including patents and patent application documents referenced herein, should be construed as being incorporated by reference in the same way as the contents expressed herein. [Examples]

[0041] The present invention will be explained in more detail below with reference to experimental examples.

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

[0043] Choroid thickness, axial length, and refractive error were measured in mice treated with LPS for two weeks and control mice treated with PBS, and the changes were calculated (Figure 1A, B). Subsequently, the eyeballs were extracted, and the expression of polarization marker genes was measured by quantitative PCR (Figure 2).

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

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

[0046] (Observation of the choroid using an electron microscope) Eyes were collected from mice in each group, fixed overnight at 4°C with 2.5% glutaraldehyde in PBS (Phosphate Buffered Saline), and rinsed with 0.1M sodium cacodylate buffer for 1 hour. Next, they were fixed with 1% OsO4 in 0.1M cacodylate buffer for 2 hours, followed by dehydration with stepwise ethanol solutions. Furthermore, the eyes were immersed overnight in a 1:2 mixture of propylene oxide and epone-araldite and embedded in 100% resin. The blocks were cut and observed using a transmission electron microscope (JEM1400 plus; JEOL) at an accelerating voltage of 100kV.

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

[0048] (result) Compared to the control group administered PBS, the LPS-administered group showed a decrease in refractive error (Figure 1B left), elongation of the axial length (Figure 1B center), and thinning of the choroid (Figure 1B right) in 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 by Administration of IL-4 and Myopia Suppression Effect> M1 macrophages are mainly involved in the induction of inflammation, whereas 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 thought 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, B).

[0052] <Administration of IL-4> C57BL6J mice (n = 4 per group) were intraperitoneally administered 0.1 μg / 100 μl (10 μg / kg BW) of an IL-4 solution, and 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). Also, 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] IL-4 was also administered to myopia-induced mice to evaluate whether there was a myopia-suppressing effect (Fig. 4). C57BL6 mice (-30D lens, n = 4) during myopia induction were intraperitoneally administered 0.1 μg / 100 μl (10 μg / kg BW) of an IL-4 solution 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 after administration of IL-4 (Figs. 3B and C), and polarization into M2 macrophages was confirmed. In addition, 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 suppresses the progression of myopia (Fig. 5).

[0055] In addition, suppression of refractive reduction (left in Fig. 5B), suppression of axial 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 administration of IL-4 exhibits an anti-myopia effect.

[0057] <IL-13 Administration> By administering IL-13, which is known as a cytokine that polarizes macrophages into M2, during the myopia induction period, similar to IL-4, it was confirmed using C57BL6J mice that elongation of the axial length, myopic shift of refraction, and choroidal thinning caused by wearing a minus lens were suppressed, and polarization into M2 macrophages was induced (Fig. 6). Measurement of blood flow changes and the number of macrophages was 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). Measurement of the number of M2 macrophages was performed in the same manner as measurement of the number of macrophages, 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 inhibiting and treating myopia progression, promoting polarization to M2 macrophages is particularly effective in achieving this.

[0059] <Test Example 3: Suppression of Myopia Development by Mast Cell Stabilizer Eye Drops> To verify the effect of mast cell stabilizer eye drops on inhibiting myopia progression, a lens-induced myopia model was used in which myopia was induced in mice by fitting them with negative lenses. During the myopia induction period, cromoglycic acid solution (4% solution) or pemirolast potassium (0.1% solution) was administered as eye drops once daily.

[0060] Following the above test method, axial length, refractive error, and choroidal thickness were measured in mice that underwent myopia induction and eye drop administration of cromoglycic acid solution or pemirolast potassium solution for three weeks, as well as in control mice, and the changes were calculated (Figure 7A, B, C).

[0061] As a result, in the control group that received PBS, the eyes wearing negative lenses showed elongation of the axial length (Figure 7A), myopia (diminutive refractive error) (Figure 7B), and thinning of the choroid (Figure 7C) compared to the control eyes. On the other hand, the groups that received cromoglycic acid solution or pemirolast potassium solution eye drops did not show the above-mentioned changes observed in the PBS group.

[0062] As these results show, it was confirmed that mastcell stabilizer eye drops suppress myopia progression.

[0063] <Test Example 4: Comparison of Myopia-Inhibiting Effects of Mast Cell Stabilizer Eye Drops and Histamine Receptor Inhibitor Eye Drops> Antiallergic drugs are broadly classified into two types: mast cell stabilizers, which inhibit the degranulation of mast cells, and histamine receptor inhibitors, which inhibit the action of histamine secreted by the degranulation of mast cells. To clarify whether the myopia-suppressing effect of mast cell stabilizer eye drops in Test Example 3 was due to the action of the antiallergic drug or to the inhibition of mast cell degranulation, mice that had undergone myopia induction with LIM were administered pemirolast potassium (0.1% solution) as a mast cell stabilizer and levocabastine solution (0.025% solution) as a histamine receptor inhibitor as eye drops, and their myopia-suppressing effects were compared. The results are shown in Figure 8.

[0064] As shown in Figure 8, the results were similar to those in Test Example 3. In the pemirolast potassium administration group, the elongation of the eyeball, myopia, and choroidal thinning observed in the control group were not observed. On the other hand, in the levocabastine administration group, elongation of the eyeball, myopia, and choroidal thinning were observed, similar to the control group.

[0065] Based on these results, the myopia-suppressing effect of mast cell stabilizers was confirmed among anti-allergic drugs.

[0066] <Test Example 5: Suppression of Myopia Progression by Administration of Lactobacillus paracasei> Since induction of M2 macrophages can suppress myopia progression, Lactobacillus paracasei, a lactic acid bacterium found in Yakult (Yakult Honsha Co., Ltd.), was cultured and grown, and administered to myopic model mice. As in Test Examples 3 and 4, axial length, refractive error, and choroidal thickness were measured, and the changes were calculated (Figure 9). As a result, it was confirmed that administration of lactic acid bacteria suppresses myopia progression (Figure 9).

Claims

1. A method for suppressing, improving, or treating myopia, comprising administering a therapeutically effective amount of a choroidal immune cell function modifier or lactic acid bacteria to a subject requiring treatment.

2. The method according to claim 1, wherein the choroidal resident immune cell function modifier is an M2 macrophage polarization inducer, a mast cell stabilizer, or a chemical mediator release inhibitor.

3. The method according to claim 2, wherein the M2 macrophage polarization-inducing substance is selected from the group consisting of proteins, peptides, nucleic acids, small molecules, and large molecules.

4. The method according to claim 2, wherein the M2 macrophage polarization-inducing substance is selected from the group consisting of IL-4, IL-10, IL-13, TLR2,4,7,9 and their ligands, bisanthren 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.

5. The method according to claim 2, wherein the mast cell stabilizer or chemical mediator release inhibitor is cromoglycic acid, pemirolast, or a salt thereof.

6. The method according to claim 1, wherein the lactic acid bacteria are of the genus Lactobacillus.

7. The method according to claim 1, wherein the lactic acid bacteria is Lactobacillus paracasei.

8. The method according to claim 1, wherein a choroidal immune cell function modifier or lactic acid bacteria is contained in a pharmaceutical composition, supplement, or food.

9. The method according to claim 1, wherein a decrease in refraction, elongation of the axial length, and / or thinning of the choroid are suppressed.

10. A composition for suppressing or treating myopia, containing a therapeutically effective amount of a choroidal immune cell function modifier or lactic acid bacteria.

11. A supplement containing an effective amount of choroidal immune cell function modifier or lactic acid bacteria to suppress or improve myopia.

12. A food product containing an effective amount of choroidal immune cell function modifier or lactic acid bacteria for suppressing or improving myopia.

13. The use of choroidal immune cell function modifiers or lactic acid bacteria in the manufacture of pharmaceuticals for suppressing or treating myopia.

14. Use of choroidal immune cell function modifiers or lactic acid bacteria to suppress or treat myopia.

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