Composition containing crocetin

Abstract

The purpose of the present invention is to provide a composition for preventing, treating, or ameliorating diseases or conditions associated with excessive axial elongation occurring not in childhood but in adulthood. Provided is a composition for preventing, treating, or ameliorating diseases or conditions associated with excessive axial elongation, or for preventing, treating, or ameliorating diseases or conditions associated with decreases in the function of the inner layer of the retina, said composition containing at least one component selected from the group consisting of crocetin and pharmaceutically acceptable salts thereof as an active ingredient.

Description

Composition containing crocetin

[0001] The present invention relates to a composition containing crocetin. In particular, it relates to a composition for the prevention, treatment, or improvement of retinal diseases or conditions that occur in adults, unlike progressive myopia in children.

[0002] The eye axis of infants elongates anteroposteriorly as they grow and approaches emmetropia from a hyperopic state. This is normal eye axis elongation. If there is an abnormality in this normal eye axis elongation, it may result in insufficient elongation and hyperopia, or excessive elongation and myopia. When this excessive elongation reaches an abnormal level, the retinal tissue in the posterior segment of the eye is physically compressed, leading to various posterior segment diseases. One of the retinal diseases caused by this excessive eye axis elongation is myopic retinopathy, which is a serious posterior segment disease that ranks among the top causes of blindness in developed countries. In Japan, in the Taji Study (large-scale epidemiological survey), myopic macular degeneration is reported to be the third leading cause of visual impairment and the first leading cause of blindness (Non-Patent Document 1).

[0003] These retinal diseases caused by excessive eye axis elongation initially present as just a decrease in visual acuity. Since visual acuity can be corrected with glasses or contact lenses, there is no extreme decrease in QOL, and thus patients have a low level of problem awareness. Also, it is difficult to detect the initial stage of retinal dysfunction caused by excessive eye axis elongation, making early diagnosis and treatment of the disease difficult. Although there are diagnostic devices such as OCT (optical coherence tomography) or ERG (electroretinogram) that can measure organic and functional abnormalities of the retina, patients do not undergo diagnosis with these devices unless they visit an ophthalmologist. Eventually, there is a problem that many patients notice the posterior segment disease only when they become aware of the abnormality through their own symptoms.

[0004] Treatment options are limited at the stage where abnormalities are felt through subjective symptoms as described above. For example, in myopic choroidal neovascularization (myopic CNV), a retinal disease caused by excessive elongation of the eyeball, there are photodynamic therapy (PDT) and fundus injections of ranibizumab (VEGF antibody). However, PDT is invasive and has problems with postoperative visual acuity, and ranibizumab requires fundus injections under local anesthesia at least once a month for three times (sometimes for a year), which inevitably results in high medical costs. In addition, in myopic traction maculopathy, surgical intervention is the main treatment, and although various surgical methods have been proposed, there are challenges such as poor prognosis, which necessitates careful application.

[0005] Thus, in order to treat retinal diseases caused by excessive axial elongation while keeping patient burden and medical costs down, early diagnosis and intervention at the point when slight retinal dysfunction begins, even before any noticeable symptoms appear, are extremely important. However, although early diagnosis is technically possible, it is not yet widespread, and there is a strong need for effective early intervention (prevention) methods.

[0006] Japanese Patent Publication No. 2018-193357

[0007] Iwase A, et al. , “Prevalence and causes of low vision and blindness in a Japanese adult population: The Tajima Study”, Ophthalmology 2006, 113:1354-1362.

[0008] Therefore, the present invention aims to provide a composition for preventing, treating, or improving diseases or conditions associated with excessive axial elongation of the eye that occur in adulthood rather than in childhood.

[0009] Based on this background, the inventors diligently studied and established an animal model that can reproduce the initial lesions of retinal disease caused by excessive axial elongation. This animal model is particularly capable of reproducing the functional decline of the inner retina and is also an animal model that can evaluate the decline in photoreceptor cell function. In particular, by using ERG for measurement, even subtle functional changes in the retina can be measured with high sensitivity.

[0010] The inner retina processes light information received by photoreceptor cells and ultimately transmits it to the brain via the optic nerve. A decline in this function can lead to various diseases, including decreased visual acuity, constricted visual field, color vision deficiencies, and reduced contrast sensitivity. The inventors have found that this mouse model can be used to screen for components that can prevent and treat such retinal diseases.

[0011] The inventors then used this model to search for safe food materials suitable for prevention and treatment, and confirmed that crocetin, which is widely used as a food material, has the effect of preventing and treating the initial lesions of these retinal diseases (deterioration of retinal potential), thus completing the present invention.

[0012] Furthermore, while Patent Document 1 discloses an ophthalmic composition containing crocetin used to inhibit axial elongation, it is intended for children in their growth phase, and the tests were conducted only on mice of the same age as children in their growth phase.

[0013] In other words, the present invention provides: [1] A composition comprising as an active ingredient at least one component selected from the group consisting of crocetin and pharmaceutically acceptable salts thereof for preventing, treating or improving a disease or condition involving impaired function of the inner retina. [2] The composition according to claim 1, wherein the disease or condition is due to excessive elongation of the eyeball. [3] The composition according to claim 1 or claim 2, for use in adults. [4] The composition according to any one of claims 1 to 3, wherein the disease is at least one selected from the group consisting of pathological myopia, including diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, glaucoma-ischemic optic neuropathy, retinal vasculitis, retinopathy of prematurity, and myopic traction maculopathy. [5] The composition according to any one of claims 1 to 4, wherein the condition is at least one selected from the group consisting of decreased visual acuity, visual field defects, decreased dynamic visual acuity, decreased contrast sensitivity, glare, photophobia, visual fatigue, decreased retinal blood flow, and electroretinogram abnormalities. [6] The composition according to any one of claims 1 to 5, which is a food composition. [7] A food composition according to item 6, provided in the form of a beverage, supplement, health food, functional food, health supplement, nutritional functional food, special dietary food, or food for specified health use. [8] A food composition according to item 6 or 7 for maintaining or improving retinal function. [9] A food composition according to any one of items 6 to 8, bearing a statement to the effect that it will make the outlines of objects appear clearer, that it will make moving objects appear clearer, that it will be possible to distinguish patterns with little difference in shade, that it will maintain retinal health, that it will be useful for people who are concerned about blurred vision, those who are concerned about blurriness, those who want to be able to focus, those who are concerned about eye health, or those who want to live a clear life.

[0014] The present invention makes it possible to provide a composition that has preventive, therapeutic, or ameliorative effects on retinal diseases or conditions caused by excessive axial elongation that develop in adults, unlike progressive myopia in children. Furthermore, the present invention makes it possible to provide a composition that prevents, treats, or ameliorates diseases or conditions associated with the decline in function of the inner retina.

[0015] Figure 1 shows an image of the model mouse used in the present embodiment. The joint allows for precise adjustment of the left and right lens frames. The control group was fitted with 0D lenses in both eyes, and the test group was fitted with -30D lenses in both eyes. Figure 2 shows the test schedule for the present embodiment. Myopia induction was performed from 3 weeks of age, with 0D lenses fitted in both eyes of the control group and -30D lenses fitted in both eyes of the test group. From 6 weeks of age, the control group was given normal feed, and the test group was given normal feed or crocetin-mixed feed. The test continued until 9 weeks of age, after which various parameters were measured. Figure 3 shows the change of refractive error in the test of the present embodiment. The vertical axis represents the change of refractive error, and the horizontal axis represents the induction time. Figure 4 shows the data for the control group (0D) + normal diet, the test group (30D) + normal diet, and the test group (30D) + crocetin diet. Figure 4 shows the change of axial length in the test of the present invention. The vertical axis represents the change in refractive index, and the horizontal axis represents the induction time. Data for the control group + normal diet, the test group + normal diet, and the test group + crocetin diet are shown. Figure 5 shows the change of total retinal thickness in the test of the present invention. The vertical axis represents the change in total retinal thickness, and the horizontal axis represents the induction time. Data for the control group + normal diet, the test group + normal diet, and the test group + crocetin diet are shown. Figure 6 shows the change of outer retinal thickness in the test of the embodiment of the present invention. The vertical axis represents the change in outer retinal thickness, and the horizontal axis represents the induction time. Data for the control group + normal diet, test group + normal diet, and test group + crocetin mixed diet are shown. Figure 7 shows the change of inner retinal thickness in the test of the embodiment of the present invention. The vertical axis represents the change in inner retinal thickness, and the horizontal axis represents the induction time. Data for the control group + normal diet, test group + normal diet, and test group + crocetin mixed diet are shown. Figure 8 shows the change of choroid thickness in the test of the embodiment of the present invention.The vertical axis represents the change in choroidal thickness, and the horizontal axis represents the induction time. Data for the control group + normal diet, test group + normal diet, and test group + crocetin mixed diet are shown. Figure 9 compares the results for refractive error, axial length, thickness of the inner retina, thickness of the outer retina, and choroidal thickness between the test group + normal diet (30 control) and the test group + crocetin mixed diet (30 crocetin). Figure 10 shows, on the left, the electroretinogram measurement process. In the middle, it shows the OP wave in normal (control) mice and diabetic mice (Source: Kurihara, et al. Diabetes, 2008). On the right, it shows which cells in the retina the a-wave, b-wave, and OP-wave reflect the function of (Source: Fu, Tomita, Smith, et al. iScience, 2021). Figure 11 shows the a-wave amplitude, b-wave amplitude, and op-wave amplitude in the control group + normal diet, test group + normal diet, and test group + crocetin-mixed diet. Figure 12A shows the mean blood flow velocity (MV) including tissue components, mean blood flow velocity (MT) in tissues, and mean blood flow velocity excluding tissue components (MV-MT) in blood vessels, expressed as the mean blur rate (MBR), three weeks after myopia induction (before crocetin intake). Figure 12B shows the MV, MT, and MV-MT in MBR six weeks after myopia induction (including three weeks of crocetin intake). G1: control group + normal diet, G2: test group + normal diet, G3: test group + crocetin-mixed diet.

[0016] In one embodiment, the present invention provides a composition comprising at least one component selected from the group consisting of crocetin and pharmaceutically acceptable salts thereof as an active ingredient for preventing, treating, or improving diseases or conditions accompanied by impaired function of the inner retina. In one embodiment, the disease or condition accompanied by impaired function of the inner retina is a disease or condition accompanied by thinning of the inner retina. In another embodiment, the disease or condition accompanied by impaired function of the inner retina is a disease or condition accompanied by attenuation of rhythmic small waves (op waves) in ERG. In yet another embodiment, the disease or condition accompanied by impaired function of the inner retina is a disease or condition accompanied by decreased retinal blood flow velocity and / or decreased vascular density.

[0017] Crocetin is a natural pigment and one of the components classified as a carotenoid. Crocetin is a component with strong antioxidant properties and can be extracted in high purity from the fruit of gardenia (Rubiaceae family, Gardenia genus; Gardenia augusta MERRIL var., grandiflora HORT., Gardenia jasminoides ELLIS) and the stigma of saffron (Iridaceae family, Crocus genus; Crocus sativus).

[0018] Crocetin is usually obtained by hydrolyzing crocin (digenthiobiose ester of crocetin), a carotenoid yellow pigment. Crocin is found in gardenia fruit, dried saffron stigmas, etc., but gardenia fruit is preferred as an industrial raw material for obtaining crocin.

[0019] The method for extracting crocin from plant sources is not particularly limited, and known methods can be used, such as extracting from crushed dried gardenia fruit with water or alcohol (e.g., methanol, ethanol, etc.), or a mixture thereof. When using a water-alcohol mixture, the extraction conditions are preferably 0 to 50°C for 1 to 18 hours, and more preferably 30 to 40°C for 2 to 4 hours. The extraction operation is usually repeated multiple times.

[0020] Industrially, hydrolysis of crocin is preferably carried out by alkaline hydrolysis. Hydrolysis may also be carried out under stirring and / or heating. Hydrolysis is preferably carried out under stirring at 20 to 70°C, and at 40 to 60°C for 1 to 24 hours, preferably 3 to 5 hours.

[0021] When crocin hydrolysis is alkaline, after the hydrolysis is complete, an appropriate amount of an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid, or phosphoric acid, or an organic acid such as citric acid, is usually added to the reaction solution to bring the pH down to 4.0 or lower, preferably 1.0 to 3.0, thereby precipitating crocetin. Subsequently, the mixture containing the precipitated crocetin can be recovered as a paste-like solid by centrifugation or filtration.

[0022] The resulting crocetin usually has acids, neutralized salts, and impurities derived from the raw materials adhering to its solid surface; therefore, a washing treatment is performed to remove these impurities. This treatment may be carried out using known methods, such as washing the paste-like solid with a sufficient amount of water. Next, the washed solid can be dried, for example, using a shelf-type ventilated dryer or a vacuum dryer, preferably under a nitrogen gas atmosphere at a temperature not exceeding approximately 50°C, to remove any remaining water from the solid.

[0023] Crocetin should preferably have a purity of 50% by mass or higher from the viewpoint of having fewer impurities, but the purity is not limited as long as a sufficient crocetin content for effective use is obtained. The purity of crocetin can be calculated based on the color value of pure crocetin. The color value can be calculated according to the standard method, referring to the "Voluntary Standards for Food Additives Other Than Chemically Synthesized Products (Second Edition)" compiled by the Japan Food Additives Association, and "Gardenia Yellow Pigment".

[0024] Crocetin can be obtained by hydrolyzing crocin, which is found in natural products such as plants, as described above, or it can be chemically synthesized. Alternatively, crocetin found in natural products such as gardenia fruit, gardenia extract, and saffron extract may be used. From the standpoint of safety and purity, it is more desirable to obtain crocetin from gardenia yellow pigment (example of food labeling).

[0025] Examples of pharmacologically acceptable salts of crocetin include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium and calcium; and salts of pharmaceutically acceptable organic amino compounds such as pyridine, dimethylamine, diethylamine, and ethanolamine.

[0026] Examples of commercially available crocetin preparations include Crovit® manufactured by Riken Vitamin Co., Ltd. "Crovit P (product name)" is a powder with a crocetin content of 75% or more, and "Crovit 2.5WD (product name)" is a water-dispersible preparation with a crocetin content of 2.5% or more.

[0027] The qualitative and quantitative methods for crocetin or its pharmaceutically acceptable salts in the composition of the present invention are not particularly limited as long as they are scientifically valid. For example, the sample can be separated by high-performance liquid chromatography using an ODS column and a concentration gradient mobile phase (TFA aqueous solution → TFA aqueous-methanol solution), and the test substance can be qualitatively and quantitatively determined using a photodiode array detector with all-trans-crocetin or 13-cis-crocetin as standard substances.

[0028] In this specification, the inner retina refers to the region of the retina that includes the nerve fiber layer, ganglion cell layer, and internal plexiform layer. The inner retina contains bipolar cells, amacrine cells, ganglion cells, etc., which are involved in processing visual information. The inner retina plays a role in transmitting and integrating light information received by photoreceptor cells among nerve cells and ultimately sending it to the brain via the optic nerve. For example, a decrease in the function of amacrine cells may lead to a decrease in contrast sensitivity.

[0029] In one embodiment, dysfunction of the inner retina is accompanied by a decrease in op waves in ERG. Op waves reflect the activity of amacrine cells and can be an important indicator in the assessment of retinal function. Therefore, in a more specific embodiment, dysfunction of the inner retina is a decrease in the function of amacrine cells.

[0030] In one embodiment, impaired function of the inner retina is accompanied by a decrease in retinal blood flow velocity and / or vascular density. More specifically, the decrease in retinal blood flow velocity is a decrease in the mean blood flow velocity within the vessels, excluding the tissue blood flow component. Retinal blood flow velocity can be measured, for example, using a laser speckle flowgraphy system. Vascular density, more specifically vascular density in the mid-periphery of the retina, can be analyzed, for example, by IB4 staining.

[0031] The effects of the composition of the present invention can be specifically evaluated using the model mouse described in Japanese Patent Application No. 2024-029565. Unlike conventional model mice, which could only be evaluated up to the age corresponding to childhood and pre-adulthood (up to 6 weeks of age), this model mouse can be evaluated from 6 weeks of age onward, corresponding to adulthood, for example, at 9 weeks of age.

[0032] This mouse model is not primarily intended to evaluate the suppression of myopia progression in childhood, but rather to evaluate the preventive effect on the early symptoms of retinal diseases, specifically those that begin with very mild functional abnormalities (abnormalities in retinoid potential) without subjective symptoms, before posterior segment diseases such as retinal choroidal atrophy and neovascularization develop. Therefore, in specific embodiments, the present invention relates to the preventive, therapeutic, or ameliorative effects of crocetin on diseases or conditions associated with functional decline of the inner retina, as confirmed in this mouse model, and more specifically, to the preventive, therapeutic, or ameliorative effects on posterior segment diseases.

[0033] In this mouse model, it was possible to sustain myopia induction even after 6 weeks of age. As a result, it was confirmed that axial elongation persists not only in childhood, where the eyeball elongates and progresses from hyperopia to myopia, but also into adulthood. Furthermore, it was confirmed that crocetin has an inhibitory effect on such excessive axial elongation. Therefore, in one embodiment, the disease or condition of the present invention is caused by excessive axial elongation.

[0034] In some embodiments, the present invention is intended for adults. More specifically, adults as defined herein are those 18 years of age or older, for example, 19 years of age or older, 20 years of age or older, 21 years of age or older, 22 years of age or older, and 40 years of age or younger, for example, 30 years of age or younger.

[0035] Specific examples of diseases that are the target of the compositions of the present invention, which involve impaired function of the inner retina or diseases caused by excessive elongation of the eyeball, include pathological myopia, such as diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, glaucoma, ischemic optic neuropathy, retinal vasculitis, retinopathy of prematurity, and myopic traction maculopathy.

[0036] Specific examples of conditions that the compositions of the present invention are intended for, which include conditions involving impaired function of the inner retina or conditions caused by excessive elongation of the eyeball, include decreased visual acuity, visual field defects, night blindness, distorted vision, decreased dynamic visual acuity, decreased contrast sensitivity, glare, photophobia, visual fatigue, decreased retinal blood flow, and abnormalities in electroretinography.

[0037] Furthermore, subjective symptoms that may arise from these conditions include difficulty in distinguishing patterns that lack clear outlines and have little difference in shade, blurring, reduced clarity, and glare.

[0038] In one embodiment, the composition of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention can be prepared by adding, for example, pharmaceutically acceptable excipients. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, but it can be eye drops, oral preparations (solid preparations such as tablets, capsules, granules, fine granules, powders, chewables, lozenges, etc., or liquid preparations such as liquids, syrups, etc.), injections, etc. Of these, eye drops and oral preparations are preferred from the viewpoint of easily achieving the effects of the present invention. The pharmaceutical composition of the present invention may contain other additives other than the components described above, depending on the properties and uses of each.

[0039] (Eye drops) When using the composition of the present invention as eye drops, the solubility and stability in water of at least crocetin or a pharmaceutically acceptable salt thereof should be considered, and the composition should be selected from aqueous eye drops, eye drops that dissolve immediately before use, suspension eye drops, oily eye drops, eye ointments, etc. For example, since crocetin is an amphiphilic carotenoid that has both a water-soluble structure and an oil-soluble structure, the dosage form of the composition of the present invention can usually be an aqueous eye drop or a suspension eye drop.

[0040] In addition to the above-mentioned components, eye drops may contain other active ingredients (pharmacologically active ingredients, physiologically active ingredients, etc.). The types of such ingredients are not particularly limited, and examples include decongestant ingredients, ocular muscle stimulant ingredients, anti-inflammatory ingredients, astringent ingredients, antihistamine ingredients, anti-allergic ingredients, vitamins, amino acids, antibacterial ingredients, sugars, high molecular weight compounds or their derivatives, cellulose or its derivatives, local anesthetic ingredients, glaucoma treatment ingredients other than those listed above, cataract treatment ingredients other than those listed above, etc.

[0041] In the eye drops, within a range not impairing the effects of the present invention, various components and additives can be appropriately selected and contained singly or in combination in accordance with common methods according to their uses and forms. Examples of these components or additives include carriers, fragrances or cooling agents, preservatives, bactericides or antibacterial agents, pH adjusters, chelating agents, stabilizers, isotonic agents, buffers, thickening agents and other various additives generally used for the preparation of liquid preparations and the like. Representative components used in the eye drops are exemplified below, but are not limited thereto.

[0042] Examples of the carrier include aqueous solvents such as water and hydrous ethanol. When various components are hardly soluble in the aqueous solvent, a solubilizer may be used. Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polyoxyl 40 stearate, povidone, polysorbate 80 and the like.

[0043] Examples of the fragrance or cooling agent include terpenes (specifically, anethole, eugenol, camphor, geraniol, cineol, borneol, menthol, limonene, borneol, etc. These may be any of the d-form, l-form or dl-form.), essential oils (star anise oil, cool mint oil, cinnamon oil, spearmint oil, peppermint water, peppermint oil, bergamot oil, eucalyptus oil, rose oil, etc.) and the like.

[0044] Examples of the preservative, bactericide or antibacterial agent include poly(dimethyldiallylammonium chloride), alkyl diaminoglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, sodium dehydroacetate, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexamethylene biguanide or its hydrochloride, etc.), glochil (trade name manufactured by Rhodia) and the like.

[0045] Examples of the pH adjuster include hydrochloric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, phosphoric acid, and the like.

[0046] Examples of the chelating agent include ascorbic acid, tetrasodium edetate, sodium edetate, citric acid, and the like.

[0047] Examples of the stabilizer include sodium edetate hydrate, povidone, polysorbate 80, dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (rongalit), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glycerin monostearate, and the like.

[0048] Examples of the isotonic agent include potassium chloride, sodium chloride, concentrated glycerin, glucose, D-mannitol, and the like.

[0049] Examples of the buffer include sodium citrate hydrate, sodium acetate hydrate, sodium hydrogen carbonate, trometamol, boric acid, borax, disodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, and the like.

[0050] Examples of the thickening agent include carboxyvinyl polymer, povidone, polyvinyl alcohol (partially saponified product), hydroxyethyl cellulose, hypromellose, methyl cellulose, glycerin, and the like.

[0051] The eye drop of the present invention preferably contains 0.001 to 1% by mass of a component such as crocetin or a pharmaceutically acceptable salt thereof, and more preferably contains 0.01 to 0.1% by mass. Further, other additives can be blended within a range where the effects of the present invention are expected or not inhibited. The content thereof is not particularly limited, but it is preferably about 0.001 to 1% by mass in the composition.

[0052] The pH of the eye drop may be 3 to 10, preferably 4 to 9 from the viewpoint of the feeling of use, and more preferably 5 to 8.5 from the viewpoint of the feeling of use.

[0053] Any known eye drop container can be used without limitation as a container for filling the eye drops of the present invention. Typically, an eye drop container can be used that has a shape that allows the eye drops to be dropped into the eye, for example, one equipped with a nozzle and having a container opening at the tip of the nozzle. Furthermore, the eye drop container for the eye drops of the present invention may have a structure in which a nozzle, which is molded separately, is attached to the container, or a structure in which the nozzle part (liquid dispensing part) and the container body are integrally molded (for example, a single-use type eye drop container).

[0054] The container for the eye drops of the present invention may be made of plastic. The material of the plastic container is not particularly limited, but examples include one of polyethylene terephthalate, polyarylate, polyethylene naphthalate, polycarbonate, polyethylene, polypropylene, polyimide, copolymers thereof, or mixtures of two or more thereof. Polyethylene terephthalate, polyarylate, polyethylene naphthalate, copolymers thereof, or mixtures of two or more thereof are preferred, as they allow the effects of the present invention to be easily achieved by adjusting the extrusion process.

[0055] The eye drops of the present invention may be filled into a transparent container (a container with sufficient transparency to allow observation of foreign matter) whose main material is such a material, or into a light-shielded container. Light shielding may be achieved, for example, by adding a coloring agent to the transparent container material, or by covering the container with shrink film or an outer box. Furthermore, the capacity of the container is preferably about 0.5 to 50 mL, and more preferably about 3 to 20 mL, in order to further enhance the effects of the present invention through the degree of extrusion, etc.

[0056] Furthermore, the nozzle provided in the container for the eye drops of the present invention is not particularly limited in terms of its structure or constituent materials. The nozzle structure can be any structure commonly used for eye drop containers, and the constituent materials of the nozzle can be, for example, those similar to those used for the plastic container described above. From the viewpoint of further improving the liquid flow of the eye drops of the present invention and suppressing variations in the amount of drops dispensed, a nozzle containing polyethylene or polypropylene as a constituent material is preferred. Examples of polyethylene include high-density polyethylene and low-density polyethylene, but among these, a nozzle containing low-density polyethylene as a constituent material is preferred.

[0057] The eye drops of the present invention can be prepared by methods commonly used and known to those skilled in the art. For example, the components can be dispersed in a carrier such as water, a solubilizer can be added if necessary, the mixture can be heated as needed, homogenized, dissolved, or emulsified using a homogenizer or the like, and the pH can be adjusted with a pH adjusting agent. Furthermore, methods such as autoclaving or filtration sterilization can be selected as sterilization methods for the formulation.

[0058] The dosage and administration of the eye drops of the present invention may vary depending on the patient's symptoms, age, etc., but usually, it is sufficient to instill about 1 to 2 drops about 1 to 6 times a day.

[0059] (Oral preparations: Solid formulations) The compositions of the present invention can be made into solid formulations such as tablets, capsules, granules, and powders. Oral preparations have the advantage of being highly portable and allowing for easy administration of a fixed amount by mouth. The shape, weight, size, and color of oral preparations are designed with ease of handling and administration in mind. Depending on their use and form, solid formulations can contain one or more components and additives, selected appropriately according to conventional methods. In addition to antioxidant plant extracts or components derived therefrom, such as crocetin or its pharmaceutically acceptable salts mentioned above, carotenoids, and polyphenols, excipients, lubricants, binders, and disintegrants can be incorporated as components or additives. Furthermore, preservatives, antioxidants, colorants, sweeteners, and other additives can be used as needed. The following are examples of typical components used in solid formulations, but are not limited to these.

[0060] Examples of excipients include sugar alcohols such as D-sorbitol, mannitol, and xylitol; sugars such as glucose, sucrose, lactose, and fructose; crystalline cellulose; carmellose sodium; croscarmellose sodium; calcium hydrogen phosphate; wheat starch; rice starch; corn starch; potato starch; dextrin; β-cyclodextrin; light anhydrous silicic acid; titanium dioxide; magnesium aluminometasilicate; talc; kaolin; and olive oil.

[0061] Examples of binders include cellulose derivatives such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, acrylic acid polymers, gelatin, gum arabic, pullulan, pregelatinized starch, agar, tragacanth, sodium alginate, and propylene glycol alginate.

[0062] Examples of disintegrants include starch, low-substituted hydroxypropyl cellulose, carboxymethylcellulose calcium, croscarmellose sodium, hydroxypropyl starch, and partially pregelatinized starch.

[0063] Examples of lubricants include stearic acid, magnesium stearate, calcium stearate, polyoxyl stearate, cetanol, talc, hydrogenated oil, sucrose fatty acid ester, dimethylpolysiloxane, beeswax, bleached beeswax, and the like.

[0064] These additives can be incorporated within a range that does not impair the effects of the present invention.

[0065] The oral preparation (solid dosage form) of the present invention can be prepared by methods commonly used or known to those skilled in the art. Examples include a method of kneading a composition, forming an extruded granule by passing it through a screen, then crushing and sizing the granule; a method of adding kneading water to the composition and forming it with a vertical granulator, followed by stirring granulation, then crushing and sieving with a co-mill; a method of compressing the formulation composition with a roller compactor, then crushing and sieving with a roll granulator; and a method of stirring granulation followed by fluidized bed drying. Furthermore, for example, when manufacturing by direct compression, the composition can be mixed and then directly fed into a tablet press for tableting.

[0066] (Oral preparations: Liquid formulations) The composition of the present invention can also be made into a liquid formulation, such as a syrup or a drink. In addition to the components described above, the liquid formulation may contain solvents, solubilizers, suspending agents, isotonic agents, buffering agents, analgesics, etc. Additives such as preservatives, antioxidants, colorants, and sweeteners may also be used as needed. These additives may be included within a range that does not impair the effects of the present invention.

[0067] Examples of solvents include water, alcohol, propylene glycol, macrogol, sesame oil, and corn oil.

[0068] Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.

[0069] Examples of suspending and emulsifying agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and waxes such as shellac wax, beeswax, carnauba wax, whalebone wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, cyclic lanolin, lanolin wax, candelilla wax, Japanese wax, montan wax, shellac wax, and rice wax.

[0070] Examples of isotonic agents include sodium chloride, glycerin, and D-mannitol. Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of analgesics include benzyl alcohol. Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid.

[0071] When the composition of the present invention is prepared as a liquid formulation, conventional methods can be used. When preparing a liquid formulation, the solubility and stability in water of at least crocetin or a pharmaceutically acceptable salt thereof should be considered. For example, since crocetin is an amphiphilic carotenoid that possesses both a water-attracting structure and an oil-attracting structure, the dosage form of the liquid formulation can usually be an aqueous formulation or a suspension formulation.

[0072] The dosage of the oral preparation (solid dosage form, liquid dosage form) of the present invention can be appropriately set according to the target disease and condition, the degree of the disease and condition, the age and weight of the subject, etc. The amount of crocetin is 0.075 to 75 mg per day, more preferably 0.1 to 25 mg, and even more preferably 0.25 to 10 mg. The amount of extracts other than crocetin is 0.1 to 5000 mg per day, more preferably 1 to 1000 mg, and even more preferably 10 to 300 mg. The number of doses may be once a day, or multiple times a day, for example, two, three, or more times.

[0073] (Food) The composition of the present invention may be provided as a food composition, either as is or mixed with other ingredients, or incorporated into food, in the form of food. Examples of such functional foods include beverages, supplements, health foods, foods with functional claims, health supplements, nutritional functional foods, foods for special dietary uses, foods for specified health uses, or ordinary foods. Because these foods contain the composition of the present invention, they can be widely consumed with the expectation of preventing or improving retinal conditions caused by excessive axial elongation of the eye, or preventing or improving conditions associated with decreased function of the inner retina.

[0074] These foods can take the form of liquids such as juices, soft drinks, energy drinks, and tea; solids such as biscuits, tablets, granules, powders, and capsules; and semi-liquids such as pastes, jellies, soups, seasonings, and dressings.

[0075] Specifically, examples include rice; various types of noodles including soba, udon, vermicelli, Chinese noodles, instant noodles, and cup noodles; beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and sports drinks; curry roux, stews, and various soups; frozen desserts such as ice cream, ice sherbet, and shaved ice; confectionery such as candy, cookies, gum, chocolate, tablets, snacks, biscuits, jelly, jam, cream, and other baked goods; processed seafood and livestock products such as kamaboko, hanpen, ham, and sausages; dairy products such as processed milk and fermented milk; oils and processed oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces, dressings, miso, soy sauce, and tare; soups, stews, salads, prepared foods, furikake, and pickles; and various other forms of health and nutritional supplements.

[0076] Furthermore, foods that may be provided containing the composition of the present invention also include supplements (powder, granules, soft capsules, hard capsules, tablets, chewable tablets, fast-disintegrating tablets, syrups, liquids, etc.).

[0077] Furthermore, the composition of the present invention can be incorporated into animal feed, such as that for pets.

[0078] These foods can all be manufactured by adding crocetin or a pharmaceutically acceptable salt thereof, by methods known to those skilled in the art.

[0079] Other ingredients besides crocetin may be added to food products as needed. Examples of such ingredients include sugars such as glucose, fructose, sucrose, maltose, lactose, and corn syrup; polysaccharides such as dextrin and oligosaccharides; sugar alcohols such as sorbitol and mannitol; and stevioside, rubusoside, and sucralose. 登録商標Sweeteners such as acesulfame potassium and aspartame; acids such as citric acid, sodium citrate, tartaric acid, malic acid, succinic acid, and lactic acid; antioxidants such as L-ascorbic acid, dl-α-tocopherol, and sodium erythorbate; softeners such as glycerin and propylene glycol; emulsifiers such as glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, and sorbitan fatty acid esters; thickening polysaccharides such as gum arabic, carrageenan, pectin, and agar; proteins such as casein and gelatin; vitamins such as vitamin C, B vitamins, vitamin E, nicotinamide, and calcium pantothenate; alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium and calcium; amino acids; oils and fats; fruit juice; dietary fiber; color fixatives; pigments; flavorings; and preservatives.

[0080] The food product in this invention may be labeled with a statement indicating that it has an effect of preventing or improving retinal conditions caused by excessive axial elongation, or an effect of preventing or improving conditions associated with a decline in the function of the inner retina. For example, the food product in this invention is for maintaining and improving retinal function. More specifically, the food product in this invention may be labeled with statements such as, "You will be able to see the outlines of objects more clearly," "You will be able to see moving objects more clearly," "You will be able to distinguish patterns with little difference in shade," "Maintain retinal health," "For those who are concerned about blurred vision," "For those who are concerned about blurriness," "For those who want to focus," "For those who are concerned about eye health," or "For those who want to live a clear life." The food product in this invention may also be labeled as being for adults.

[0081] In one embodiment, the present invention provides a method for preventing, treating, or improving excessive axial elongation, particularly retinal diseases or conditions caused by excessive axial elongation that develop in adults, or diseases or conditions associated with impaired function of the inner retina, comprising administering crocetin or a pharmaceutically acceptable salt thereof to a subject including or not including humans. In another embodiment, the present invention provides the use of crocetin or a pharmaceutically acceptable salt thereof for producing a pharmacopoeia for preventing, treating, or improving excessive axial elongation, particularly retinal diseases or conditions caused by excessive axial elongation that develop in adults, or diseases or conditions associated with impaired function of the inner retina. The above description also applies to embodiments of the method and embodiments of use.

[0082] Next, the present invention will be specifically described with reference to examples and test cases, but the present invention is not limited to the following examples and test cases.

[0083] (Mouse Model) The test was conducted using a model mouse described in Japanese Patent Application No. 2024-029565. More specifically, three-week-old C57BL / 6J mice, shortly after weaning, were anesthetized with a three-part anesthetic mixture consisting of Domitol (Nippon Zenyaku Kogyo Co., Ltd.), Betolfar (Meiji Seika Pharma Co., Ltd.), and Midazolam (Sandoz Inc.), and their skulls were exposed with scissors. A support post 1 was erected on the skull and fixed with dental cement (Super-Bond, Sun Medical Co., Ltd.). The support post has screw threads so that the adjustment device described later can be fixed with a nut.

[0084] The mice described above were divided into two groups: a test group and a control group. Using the device shown in Figure 1, the test group wore -30 diopters (D) negative lenses (Rainbow Contact, Rainbow Optical Research Institute Co., Ltd.) in both eyes for six weeks. The control group wore 0D lenses in the same manner. To prevent the mice from damaging the lenses with their forelegs, etc., a protector 4 with a laterally protruding shape was attached to the frame at the bottom of the lens. The protector 4 prevented the mice from touching the lenses, thus preventing damage to the lenses. Note that -30D in mice is equivalent to -6D in humans.

[0085] An adjustment device 5 is attached to the frame above the lens to adjust the width and angle of the attached lens as the mouse grows. The adjustment device 5 is bent into a "V" shape, with the lens attached to one end and an elongated hole 6 on the other end so that it can be attached to a support column 1 erected on the head. By passing the elongated hole 6 through the support column 1 and screwing it in with a nut 7, the device can be fixed in close contact with the skin without compressing the edges of the mouse's eyes.

[0086] The adjustment mechanism, consisting of three parts—a support column 1, a nut 7, and an adjustment device 5—allowed the width and angle to be adjusted according to the mouse's growth, ensuring the lens was positioned at the mouse's eye level. During rearing, the lens was removed from the mounting device and cleaned every three days, and its position was adjusted to ensure the appropriate angle and width relative to the mouse's eye axis.

[0087] (Normal diet group and crocetin diet group) As shown in Figure 2, the control group and the test group + normal diet were given normal diet throughout the test period. In the test group + crocetin diet, from 3 weeks after lens implantation, crocetin was added to the normal diet, and the diet consisted of crocetin diet with 0.001% Crovit P (manufactured by Riken Vitamin Co., Ltd.) (0.00075% crocetin). 3 weeks after lens implantation refers to 6-week-old mice, which is equivalent to adult humans. In other words, this study was conducted to confirm the effect of crocetin on model mice after axial elongation during the growth period was complete.

[0088] (Measurement of refractive error, axial length, choroidal thickness, and retinal thickness) The refractive error, axial length, and choroidal and retinal (inner, outer, and full layers) thickness of the mouse eye were measured before lens implantation, 3 weeks after implantation, and 6 weeks after implantation, using the following methods (1) and (2), and the difference from the pre-implantation state was calculated. The results were analyzed using the Mann-Whitney U test.

[0089] (1) The refractive index was measured using a refractometer (Infrared photorefractor for mice, manufactured by Professor Schaeffel of the University of Tubingen).

[0090] (2) The axial length of the eye, as well as the thickness of the choroid and retina, were measured by imaging the entire eyeball using SD-OCT (Spectral-domain OCT, Envisu R4310, bioptigen Inc.), selecting a circular region with a radius of 0.5 mm from the optic nerve head, measuring and analyzing the posterior choroidal region using ImageJ, and then dividing the area by the circumference to obtain the average choroidal thickness. The same original image was used to measure the retinal thickness as for the choroidal thickness. The retina was divided into an inner layer and an outer layer with the outer plexiform layer (OPL) as the boundary, and the inner and outer regions of the posterior retina were measured and analyzed using ImageJ, and the average retinal thickness was obtained by dividing the area by the circumference.

[0091] (ERG Measurement) Electroretinogram (ERG) measurements were performed on the eyes of mice in each group six weeks after lens implantation. Using LED stimulation and a Ganzfeld Dome detector (PuREC; MAYO), measurements were taken at 0.5–10 cd·s / m². ２ ERGs in both eyes were measured after applying light stimulation. After more than 8 hours of dark adaptation, tropicamide and phenylephrine hydrochloride were administered, dilated the pupils of both eyes, and then light stimulation was applied. The active electrode was recorded using a contact electrode. The reference electrode was placed in the mouth. A clip electrode attached to the tail of the mouse was used as ground. The amplifier's low-pass filter was set to 30 Hz. Statistical analysis was performed using an unpaired t-test.

[0092] (Measurement of retinal blood flow velocity) Three weeks and six weeks after lens implantation, mice in the control group, test group + normal diet, and test group + crocetin diet were anesthetized by intraperitoneal injection of a triple anesthetic (MMB) after pupil dilation with tropicamide / phenylephrine hydrochloride eye drops (Midrin P, Santen Pharmaceutical), and retinal blood flow velocity was measured using a laser speckle flowgraphy system. The mean blur rate (MBR) was calculated as an index of relative blood flow velocity. Specifically, using the built-in analysis software, a circular region of interest (ROI) centered on the optic nerve head (ONH) was selected, the relative blood flow index (RBI) was set to 200, and the same analysis region was applied to all measurements. Within the selected retinal region, MBR values ​​were calculated for the vascular and tissue regions. The mean blood flow velocity (MV) within blood vessels, which includes tissue components, encompasses both vascular and background tissue components, while the mean blood flow velocity (MT) within tissue represents the blood flow velocity within the tissue region. Therefore, MV-MT was calculated to obtain the vascular blood flow component excluding the contribution of tissue blood flow. Data are presented as mean + standard deviation (SEM). Statistical analysis was performed using one-way analysis of variance (one-way ANOVA). Measurements from both eyes of each mouse were averaged and used as a single value. N = 6 / group.

[0093] (Analysis of vascular density) The retinal vascular structure was evaluated in mice of the control group, the test group + normal diet, and the test group + crocetin diet, 3 weeks and 6 weeks after lens implantation. Specifically, vascular density in the middle periphery was analyzed by IB4 staining.

[0094] (Refractive Index) In the control group, refractive index increased slightly three weeks after lens fitting before returning to its initial value. In the test group + normal diet, refractive index decreased sharply three weeks after lens fitting and then decreased gradually over six weeks. In the test group + crocetin diet, the refractive index that had decreased due to lens fitting recovered, showing a significant difference compared to the test group + normal diet (Figure 3). This confirmed that crocetin has an effect of suppressing myopia that progresses in adulthood.

[0095] (Axial length) Axial length tended to increase in all groups, but in the control group, it increased rapidly until 3 weeks of age, then gradually increased until 6 weeks of age. The test group + normal diet showed a significant increase in axial length compared to the control group. This trend continued after 3 weeks of age, suggesting that this model mouse reflects excessive axial lengthening in adulthood. In the test group + crocetin diet, axial lengthening was suppressed compared to the test group + normal diet (Figure 4). This confirmed that crocetin has the effect of suppressing excessive axial lengthening that progresses in adulthood.

[0096] (Retinal Thickness) In the control group, total retinal thickness decreased by 3 weeks of age, and then gradually decreased until 6 weeks of age. In the test group + normal diet, total retinal thickness decreased significantly compared to the control group. In the test group + crocetin diet, the decrease in total retinal thickness was suppressed compared to the test group + normal diet (Figure 5). There were no significant differences in the thickness of the outer retina among the control group, test group + normal diet, and test group + crocetin diet (Figure 6). In the control group, the thickness of the inner retina decreased by 3 weeks of age, and then gradually decreased until 6 weeks of age. In the test group + normal diet, total retinal thickness decreased significantly compared to the control group. In the test group + crocetin diet, the decrease in total retinal thickness was suppressed compared to the test group + normal diet (Figure 7). Therefore, it was confirmed that this model mouse reduces the thickness of the inner retina, and that crocetin can suppress the decrease in the thickness of the inner retina.

[0097] (Choroid Thickness) In the control group, choroid thickness increased with growth. In the test group + normal diet, choroid thickness decreased. In the test group + crocetin diet, the decrease in choroid thickness was suppressed compared to the test group + normal diet (Figure 8). Therefore, it was confirmed that this model mouse reduces choroid thickness, and that crocetin can suppress the decrease in choroid thickness.

[0098] In summary, as shown in Figure 9, crocetin was confirmed to be particularly effective in suppressing the decrease in refractive error and the decrease in the thickness of the inner retinal layer.

[0099] (ERG) Figure 10 left shows the measurement of an electroretinogram. As shown in Figure 10 middle, it is known that the op wave is attenuated by diabetes, etc. As shown in Figure 10 right, it has been shown that the a wave originates from photoreceptor cells, the b wave from bipolar cells, and the op wave from the inner retina. As shown in Figure 11, in the test group + normal diet, the a wave, b wave, and op wave were all significantly attenuated compared to the control group. In the test group + crocetin diet, the a wave and b wave showed an improving trend compared to the test group + normal diet, and the op wave showed a significant improvement compared to the test group + normal diet.

[0100] As shown in Figure 10 (right), the op wave originates from the inner retina, more specifically from amacrine cells, and this mouse model was shown to cause a decline in the function of the inner retina. Crocetin was shown to improve the decline in the function of the inner retina, more specifically from amacrine cells. The degree of attenuation of the op wave in the ERG shown in this model is a stage before the onset of subjective symptoms, and it was shown that administering crocetin can prevent or suppress the progression of diseases accompanied by a decline in the function of the inner retina.

[0101] (Retinal blood flow velocity) As shown in Figure 12, there was no significant difference in MBR among the groups 3 weeks after myopia induction (Figure 12A), but a significant decrease in MBR was observed in the test group + normal diet at 6 weeks (Figure 12B). In contrast, the test group + crocetin diet showed a significantly higher MBR compared to the test group + normal diet, and the decrease in MV and MV-MT was also significantly suppressed (Figure 12B).

[0102] (Vascular Density) Analysis using IB4 staining showed that vascular density in the middle circumferential region was significantly lower in the test group + normal diet compared to the control group, whereas this decrease was suppressed in the test group + crocetin diet, and vascular density was maintained.

[0103] Crocetin showed the effect of maintaining and improving perioptic nerve head blood flow and retinal vascular structure under myopia induction, suggesting its potential to suppress myopia progression and retinal microcirculatory disorders.

[0104] The present invention makes it possible to provide a composition that has therapeutic and preventive effects against retinal diseases or conditions caused by excessive axial elongation that develop in adults, unlike progressive myopia in children. Furthermore, the present invention makes it possible to provide a composition that treats or prevents diseases or conditions associated with the decline of function in the inner layers of the retina. Therefore, the present invention can be used in the medical, veterinary, food, and cosmetic fields, among others.

[0105] 1...Support column, 2...Negative lens, 3...Frame, 4...Protector, 5...Adjustment device, 6...Slotted hole, 7...Nut

Claims

1. A composition comprising, as an active ingredient, at least one component selected from the group consisting of crocetin and pharmaceutically acceptable salts thereof, for the prevention, treatment, or improvement of diseases or conditions involving impaired function of the inner retina.

2. The composition according to claim 1, wherein the disease or condition is due to excessive elongation of the eyeball.

3. The composition according to claim 1, intended for use by adults.

4. The composition according to claim 1, wherein the disease is at least one selected from the group consisting of pathological myopia, including diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, glaucoma, ischemic optic neuropathy, retinal vasculitis, retinopathy of prematurity, and myopic traction maculopathy.

5. The composition according to claim 1, wherein the condition is at least one selected from the group consisting of decreased visual acuity, visual field defects, decreased dynamic visual acuity, decreased contrast sensitivity, glare, photophobia, visual fatigue, decreased retinal blood flow, and electroretinogram abnormalities.

6. The composition according to claim 1, which is a food composition.

7. The food composition according to claim 6, provided in the form of a beverage, supplement, health food, functional food, health supplement, nutritional functional food, special dietary food, or specified health food.

8. The food composition according to claim 6 for maintaining and improving retinal function.

9. The food composition according to claim 6, which is labeled to the effect that it will make the outlines of objects appear clearer, that moving objects will appear clearer, that it will enable the identification of patterns with little difference in shade, that it will maintain retinal health, and that it will be beneficial for people who are concerned about blurred vision, people who are concerned about blurriness, people who want to be able to focus, people who are concerned about eye health, or people who want to live a clear life.

Images