APPLICATION OF REGULATION OF EYE SCLERA LIPID METABOLISM TO INHIBIT MYOPIA

Abstract

The present invention relates to an application of inhibiting myopia by regulating eye scleral lipid metabolism. The present discloses a new mechanism leading to myopia, i.e., a close relationship between abnormal eye scleral lipid metabolism and myopia, thus revealing a new target for prevention and control of myopia; meanwhile, also provided is an eye drop that can effectively prevent and control myopia while avoiding eye allergies.

Claims

1-72. (canceled)

73. A method of regulating eye scleral lipid metabolism in an individual, wherein the method comprises administering to said individual a ω-3 polyunsaturated fatty acid.

74. The method according to claim 73, wherein ω-3 polyunsaturated fatty acid is an oral product, a health care product, food, a dietary supplement, a nutritional product, or a cosmetic.

75. The method according to claim 73, wherein ω-3 polyunsaturated fatty acid is an injection, a tablet, a lyophilized powder injection, a capsule, an effervescent tablet, a chewable tablet, a buccal tablet, a granule, an ointment, a syrup, an oral liquid, an aerosol, a nasal drop, an external preparation, or an ophthalmic dosage form.

76. The method according to claim 75, wherein ophthalmic dosage form including but not limited to an eye drop, an eye ointment, an eye spray, an implant, an ophthalmic gel, an eye patch, an ophthalmic microsphere, an ophthalmic sustained-release preparation, a periocular injection, or an intraocular injection.

77. The method according to claims 73, wherein ω-3 polyunsaturated fatty acid is administered systemically, and/or topically, and/or parenterally.

78. The method according to claims 73, wherein ω-3 polyunsaturated fatty acid is administered in combination with other drugs, and wherein other drugs are drugs for preventing and controlling and/or treating myopia, vasodilators, smooth muscle relaxers, drugs for preventing vasospasm, drugs for regulating collagen metabolism, Piracetam, antiallergic drugs, liver-protecting drugs, or combinations thereof.

79. The method according to claims 73, wherein ω-3 polyunsaturated fatty acid forms a composition with other ophthalmic preparations, wherein ophthalmic preparations including but not limited to drugs for treating myopia, M receptor blockers, dibazole, polyunsaturated fatty acids, salidroside, prazosin, homatropine, anisodamine (racemic), topicamide, 7-methyl xanthine, nicotinic acid, Piracetam, a red sage root extract, a safflower extract, fish oil, a bear bile extract, vitamins, ATP, and adjuvants for ophthalmic diseases.

80. The method according to claims 73, wherein the ω-3 polyunsaturated fatty acid is DHA alone or a composition of DHA and EPA.

81. A method of preventing, delaying, inhibiting and/or treating myopia and myopia-related diseases in a subject, wherein method comprises administering to said subject a substance for regulating eye scleral lipid metabolism, wherein the substance for regulating eye scleral lipid metabolism is a ω-3 polyunsaturated fatty acid, wherein the ω-3 polyunsaturated fatty acid is a composition of DHA and EPA, in the composition of DHA and EPA, EPA is a predominant component, EPA:DHA>1:1.

82. The method according to claims 81, wherein the ω-3 polyunsaturated fatty acid is an oral product, a health care product, food, a dietary supplement, a nutritional product, a drug or a cosmetic.

83. The method according to claims 81, wherein the ω-3 polyunsaturated fatty acid is an injection, a tablet, a lyophilized powder injection, a capsule, an effervescent tablet, a chewable tablet, a buccal tablet, a granule, an ointment, a syrup, an oral liquid, an aerosol, a nasal drop, an external preparation, or an ophthalmic dosage form.

84. The method according to claims 83, wherein ophthalmic dosage form including but not limited to an eye drop, an eye ointment, an eye spray, an implant, an ophthalmic gel, an eye patch, an ophthalmic microsphere, an ophthalmic sustained-release preparation, a periocular injection, or an intraocular injection.

85. The method according to claims 81, wherein the ω-3 polyunsaturated fatty acid is administered systemically, and/or topically, and/or parenterally.

86. The method according to claims 81, wherein the ω-3 polyunsaturated fatty acid is administered in combination with other drugs, and wherein the other drugs are drugs for preventing or treating myopia, vasodilators, smooth muscle relaxers, drugs for preventing vasospasm, drugs for regulating collagen metabolism, Piracetam, antiallergic drugs, liver-protecting drugs, or combinations thereof.

87. The method according to claims 81, wherein ω-3 polyunsaturated fatty acid is in conjunction with other ophthalmic preparations, wherein ophthalmic preparations including but not limited to drugs for treating myopia, M receptor blockers, dibazole, polyunsaturated fatty acids, salidroside, prazosin, homatropine, anisodamine (racemic), topicamide, 7-methyl xanthine, a nicotinic acid, Piracetam, a red sage root extract, a safflower extract, fish oil, a bear bile extract, vitamins, ATP, and adjuvants for ophthalmic diseases.

88. The method according to claims 81, wherein the myopia is refractive myopia and/or axial myopia; congenital myopia, early-onset myopia, delayed myopia, late-onset myopia; low myopia, moderate myopia, high myopia, pseudomyopia, true myopia, myopia in minors, myopia in adults, and myopia in the elderly; simple myopia, and pathological myopia, primary myopia, secondary myopia or progressive myopia.

89. The method according to claims 81, wherein subject refer to people whose eyes are still in the stage of growth and development.

90. A drug, preparation or device for regulating eye scleral lipid metabolism, comprising a ω-3 polyunsaturated fatty acid, wherein the ω-3 polyunsaturated fatty acid is a composition of DHA and EPA, in the composition of DHA and EPA, EPA is a predominant component, EPA:DHA>1:1.

91. The drug, preparation or device according to claim 90, wherein the drug or the preparation is an injection, a tablet, a lyophilized powder injection, a capsule, an effervescent tablet, a chewable tablet, a buccal tablet, a granule, an ointment, a syrup, an oral liquid, an aerosol, a nasal drop, an external preparation, or an ophthalmic dosage form.

92. The drug, preparation or device according to claim 91, wherein ophthalmic dosage form including but not limited to an eye drop, an eye ointment, an eye spray, an implant, an ophthalmic gel, an eye patch, an ophthalmic a microsphere, an ophthalmic sustained-release preparation, a periocular injection, or an intraocular injection.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 is a sclera transcriptome sequencing map of mice with form-deprivation myopia, wherein, “Treat” indicates a form-deprivation treated eye; “Fellow” indicates a fellow eye of the form-deprived eye; “Control” indicates an untreated control eye.

[0074] FIG. 2 is an electron microscopy of sclera in mice with form-deprivation myopia.

[0075] FIG. 3 is a diagram showing refraction difference between an experimental eye and a fellow eye in intragastric administration group. Refraction: diopter; FD+vehicle: form deprivation+edible olive oil treatment group (solvent group); FD+ω-3: form deprivation+(DHA 300 mg, EPA 60 mg) treatment group (administration group).

[0076] FIG. 4 is diagram showing a vitreous chamber depth difference between an experimental eye and a fellow eye in intragastric administration group, VCD: vitreous chamber depth; FD+vehicle: form deprivation+edible olive oil treatment group (solvent group); FD+ω-3: form deprivation+ω-3 polyunsaturated fatty acid (DHA 300 mg, EPA 60 mg) treatment group (drug administration group).

[0077] FIG. 5 is a diagram showing an axis length difference between an experimental eye and a fellow eye in the intragastric administration group, AL: axis length; FD+vehicle: form deprivation+edible olive oil treatment group (solvent group); FD+ω-3: form deprivation+ω-3 polyunsaturated fatty acid (DHA 300 mg, EPA 60 mg) treatment group (drug administration group).

[0078] FIG. 6 is a diagram showing vitreous chamber depth difference, vitreous chamber depth difference and axis length difference between an experimental eye and a fellow eye in peribulbar injection group, wherein, refraction: diopter; VCD: vitreous chamber depth; AL: axis length; FIG. 6A shows binocular refraction difference among low-dose and high-dose DHA groups, Vehicle group and 0.1% atropine group (positive control group) by injection; FIG. 6B shows binocular difference in VCD among low-dose and high-dose DHA groups, Vehicle group and 0.1% atropine group by injection; FIG. 6C shows binocular difference in AL among low-dose and high-dose DHA groups, Vehicle group and 0.1% atropine group by injection; FIG. 6D shows binocular difference in refraction among low-dose and high-dose EPA groups, Vehicle group and 0.1% atropine group (positive control group) by injection; FIG. 6E shows binocular difference in VCD among low-dose and high-dose EPA groups, Vehicle group and 0.1% atropine group by injection; FIG. 6F shows binocular difference in AL among low-dose and high-dose EPA injection groups, Vehicle group and 0.1% atropine group by injection.

[0079] FIG. 7 is a diagram showing difference in ChT and ChBP between an experimental eye and a fellow eye after different treatments as well as the detection results of HIF-1α protein expression, wherein, FIG. 7A is a schematic diagram of OCT assay, where lines AB and CD show the inner surface of the choroid and O represents the optic disc; AA′ and BB′ indicate nasal/inferior choroidal thickness; and CC′ and DD′ indicate temporal/superior choroidal thickness. FIG. 7B is an OCTA image, where the bright part indicates the perfusion signal point; FIG. 7C shows ChT difference between the experimental eye and the fellow eye after FD treatment plus 2 weeks of feeding olive oil and ω-3 polyunsaturated fatty acids; FIG. 7D shows ChBP difference between the experimental eye and the fellow eye after RD treatment plus 2 weeks of feeding olive oil and ω-3 polyunsaturated fatty acids; FIG. 7E shows ChT difference between the experimental eye and the fellow eye after LIM treatment plus 2 weeks of feeding olive oil and ω-3 polyunsaturated fatty acids; FIG. 7F shows ChBP difference between the experimental eye and the fellow eye after LIM treatment plus 2 weeks of feeding olive oil and ω-3 polyunsaturated fatty acids; FIG. 7G shows interocular ChT difference after 2 weeks of injection on FD eyes with Vehicle, 1.0 μg DHA, 3.0 μg DHA, or 0.1% atropine, respectively; FIG. 7H shows interocular ChBP difference after 2 weeks of injection on FD eyes with Vehicle, 1.0 μg DHA, 3.0 μg DHA, or 0.1% atropine; FIG. 7I shows difference in ChT between the experimental eye and the fellow eye after 2 weeks of injection on FD eyes with Vehicle, 1.0 μg EPA, 3.0 μg EPA or 0.1% atropine; FIG. 7J shows difference in ChBP between the experimental eye and the fellow eye after 2 weeks of injection on FD eyes with Vehicle, 1.0 μg EPA, 3.0 μg EPA or 0.1% atropine; FIG. 7K and FIG. 7L show the results of western blotting on the interocular HIF-1α protein and related protein expression after FD treatment plus 2 weeks of feeding olive oil and ω-3 polyunsaturated fatty acids, where FD-F indicates the fellow eye, and FD-T indicates the FD experimental eye; FIG. 7M and FIG. 7N show the results of western blotting on the interocular HIF-1α protein expression after 2 weeks of injection on FD eyes with Vehicle, DHA or EPA, where FD-F indicates the fellow eye, and FD-T indicates the FD experimental eye.

[0080] FIG. 8 shows a clinical trial of the effect of oral administration of cod liver oil on ChBP before and after near-distance work in humans, wherein, FIG. 8A indicates an operational flow of near-distance work; FIG. 8B indicates changes in ChT; FIG. 8C indicates changes in a stomal zone; FIG. 8D indicates changes in a vascular luminal zone; FIG. 8E indicates changes in a choroidal vascularity index; FIG. 8F indicates changes in the area of a choroidal non-perfused zone.

[0081] FIG. 9 shows a safety study on the effects of intragastric administration of ω-3 polyunsaturated fatty acids on anterior chamber depth (ACD), crystal thickness (LT) and body weight (Weight).

[0082] FIG. 10 shows a safety study on the effects of peribulbar injection of DHA and EPA on anterior chamber depth (ACD), lens thickness (LT), and radius of corneal curvature (RCC).

[0083] FIG. 11 shows an allergy study: A: form deprivation+fish oil group; B: form deprivation+drug group (DHA alone or DHA+EPA).

[0084] FIG. 12 shows a study on the effectiveness of an optimal treatment scheme, wherein the effects of peribulbar injection of polyunsaturated fatty acids with different proportional formulations on refraction (FIG. A), vitreous chamber depth (FIG. B) and eye axis length (FIG. C) are studied.

[0085] FIG. 13 shows a study on the safety of an optimal treatment scheme, wherein the effects of peribulbar injection of polyunsaturated fatty acids with different proportional formulations on refraction (FIG. A), crystal thickness (FIG. B) and radius of corneal curvature (FIG. C) are studied.

[0086] In the above figures, “difference” refers to the difference in refraction or eye axial parameters between the experimental eye and the fellow eye; variance analysis based on repeated measurement data are used for comparison between solvent and drug administration groups: “*” indicates P<0.05; “**” indicates P<0.01; “***” indicates P<0.001, * denotes a statistical difference between ω-3 polyunsaturated fatty acids/DHA/EPA treatment and the solvent control; # denotes a statistical difference between atropine treatment and solvent control.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 Close Connection Between Abnormal Scleral Metabolism and Myopia

[0087] The test animals were C57/BL6 mice aged 3 weeks, and subjected to monocular form deprivation (FD) by an eyeshade method, one group of animals were anesthetized and killed after experiment for 2 days, and the binocular scleras were taken for transcriptome sequencing, while another group of animals were taken for electron microscopic observation after experiment for 2 weeks.

[0088] As seen from FIG. 1, after form deprivation for 2 days, a scleral lipid metabolism signaling pathway in myopic eyes of mice show significant changes, as compared to the fellow eyes, and a key lipid metabolism enzyme at this site, carnitine palmitoyl transferase 2 (Cpt2), is significantly reduced, indicating the downregulation of the scleral lipid metabolism pathway in myopic eyes.

[0089] As seen from FIG. 2, after form deprivation for 2 weeks, scleral lipid deposition increases in myopic eyes of mice as compared to fellow eyes, indicating abnormal scleral lipid metabolism in myopic eyes.

Example 2 A Scleral Metabolism Regulating Substance is Capable of Inhibiting Myopia

[0090] The test animals were British tricolored short-haired guinea pigs aged 3 weeks. The guinea pigs were subjected to monocular form deprivation (FD) by a mask method, and allowed to an intragastric administration of ω-3 polyunsaturated fatty acid. The animals were randomly divided into 2 groups: FD+solvent control group (FD+vehicle) (a solvent here was edible olive oil); and FD+drug group (FD+ω-3 (DHA 300 mg, EPA 60 mg)). Intragastric administration was performed at 9 a.m., continuing for 2 weeks. Before test, and administration for 1 week and 2 weeks, respectively, refraction was measured by an eccentric infrared refractometer (EIR), ocular axis parameters such as vitreous chamber depth and axial length were measured by A-scan (11 MHz), and scleral lipid metabolism was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0091] Comparing the measured parameters before and after the experiment, it was found that FD eyes, degrees of refractive myopia, vitreous cavity elongation and eye axis elongation in administration group are smaller than those in FD control group and solvent administration group, and were statistically significant as compared to solvent control group, moreover, the scleral lipid metabolism level was partly restored or basically restored to be normal. Therefore, feeding ω-3 polyunsaturated fatty acids can inhibit the formation of FD myopia in guinea pigs or slow down the development of FD myopia in guinea pigs.

[0092] As shown in FIG. 3, after 2 weeks of experiment, the degree of refractive myopia in administration group is less than that in solvent group, and there is a temporal effect, indicating that the ω-3 polyunsaturated fatty acid can inhibit the progression of FD myopia.

[0093] As shown in FIG. 4, after 2 weeks of experiment, the vitreous cavity elongation in administration group is significantly less that in solvent group, and there is a temporal effect, indicating that the ω-3 polyunsaturated fatty acid can inhibit the vitreous cavity elongation in FD treated eyes.

[0094] As shown in FIG. 5, after 2 weeks of experiment, the ocular axis elongation in administration group is significantly less that in solvent group, and there is a temporal effect, indicating that the ω-3 polyunsaturated fatty acid can inhibit the ocular axis elongation in FD treated eyes.

[0095] The above experiments prove that the ω-3 polyunsaturated fatty acid can significantly play a role in delaying negative refraction and eye axis elongation.

Example 3 A Scleral Metabolism Regulating Substance Can Inhibit Negative Refraction and Eye Axis Elongation of Myopic Eyes

[0096] The test animals were British tricolored short-haired guinea pigs aged 3 weeks. The animals were subjected to monocular form deprivation (FD) by a mask method and were randomly divided into 6 groups, which were treated by peribulbar injection with the following different substances: (1) ethanol solvent group (Vehicle); (2) low-dose DHA group (1.0 μg); (3) high-dose DHA group (3.0 μg): (4) low-dose EPA group (1.0 μg); (5) high-dose EPA group (3.0 μg); and (6) 0.1% atropine group.

[0097] The measurement methods of refraction, vitreous chamber depth and eye axis length were the same as those in Example 2.

[0098] As seen from FIG. 6, form deprivation successfully induces myopia in guinea pigs after two weeks of injection, and the myopia progression of the treated guinea pigs in high-dose DHA group is reduced by 35.3% compared to that in Vehicle group (FIG. 6A), accompanied by significant reduction in both VCD and AL elongation, and the effect is more apparent with the extension of time; likewise, similar results are obtained in high-dose EPA group.

[0099] In summary, it can be seen from the above experiments that peribulbar injection with high-dose ω-3 polyunsaturated fatty acids (3 μg/day) can play roles in inhibiting the negative refraction and eye axis elongation. Topical administration of ω-3 polyunsaturated fatty acids can delay myopia progression.

Example 4 ω-3 Polyunsaturated Fatty Acids Inhibit Myopia By Inhibiting ChBP Reduction and Sclera Hypoxia Cascade Reaction

[0100] ChT and ChBP of guinea pigs were detected by optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA), and the HIF-1α protein expression levels in different treatments were detected by western blotting.

[0101] The test animals were British tricolored short-haired guinea pigs aged 3 weeks, and were subjected to monocular form deprivation (FD) by a mask method or subjected to monocular lens induction (L1), and divided into 3 groups for test: (1) the FD treated guinea pigs were fed with ω-3 polyunsaturated fatty acids and olive oil control, and the interocular (between the experimental eye and the fellow eye, similar hereinafter) differences in ChT and ChBP were compared, respectively; (2) the L1 treated guinea pigs were fed with ω-3 polyunsaturated fatty acids and olive oil control, and the interocular (between the experimental eye and the fellow eye, similar hereinafter) differences in ChT and ChBP were compared, respectively; (3) the FD treated guinea pigs were subjected to peribulbar injection, and randomly divided into 6 groups: (a) ethanol solvent group (Vehicle); (b) low-dose DHA group (1.0 μg) (c) high-dose DHA group (3.0 μg); (d) low-dose EPA group (1.0 μg; (e) high-dose EPA group (3.0 μg); (f) 0.1% atropine group, and the interocular differences in ChT and ChBP were compared, respectively.

[0102] As seen from FIG. 7C and FIG. 7D, the ChT of the FD treated guinea pigs fed with olive oil treatment group is significantly reduced accompanied by reduction in ChBP, however, compared to olive oil treatment group, feeding with ω-3 polyunsaturated fatty acids can significantly inhibit reduction in both ChT and ChBP. As seen from FIG. 7E and FIG. 7F, the L1 treated guinea pigs also show a similar inhibitory effect.

[0103] As seen from FIG. 7G and FIG. 7H, treatment groups injected with DHA, regardless of high-dose group and low-dose group, significantly inhibit the reduction in both ChT and ChBP, as compared to Vehicle group. As shown in FIG. 7I and FIG. 7J, treatment groups injected with EPA have no significant effects, but according to data in the figures, both high-dose group and low-dose group show a certain inhibitory effect.

[0104] It is reported that increasing ChT and ChBP can inhibit the development of myopia (X. Zhou et al., Increased Choroidal Blood Perfusion Can Inhibit Form Deprivation Myopia in Guinea Pigs. Invest. Ophthalmol. Vis. Sci. 61, 25 (2020)). Combined with the above experimental results, it can be seen that ω-3 polyunsaturated fatty acids can delay and inhibit the development of myopia by inhibiting reduction in ChT and ChBP.

[0105] Scleral hypoxia and upregulation of HIF-1α expression promote transdifferentiation of myofibroblasts and remodeling of extracellular matrix (ECM), leading to the occurrence and development of myopia (H. Wu et al., Scleral hypoxia is a target for myopia control. Proc. Natl. Acad. Sci. U.S.A. 115, E7091-E7100 (2018); F. Zhao et al., Scleral HIF-1alpha is a prominent regulatory candidate for genetic and environmental interactions in human myopia pathogenesis. EBioMedicine 57, 102878 (2020)). As shown from FIG. 7K and FIG. 7L, in guinea pigs fed with olive oil, the HIF-1α protein level in the sclera of the experimental eyes (FD-T) is higher than that in the fellow eyes (FD-F), while the increase of HIF-1α protein level in the sclera of the experimental eyes in guinea pigs fed with ω-3 polyunsaturated fatty acids is inhibited. Similarly, according to FIG. 7M and FIG. 7L, in peribulbar injection treatment group, the increase of the HIF-1α protein level in guinea pigs injected with DHA or EPA is inhibited. Combined with the above experiments, it can be seen that ω-3 polyunsaturated fatty acids can inhibit the scleral hypoxic cascade reaction during the myopia development, and then inhibits the development of myopia.

Example 5 ω-3 Polyunsaturated Fatty Acid Can Improve Reduction in ChBP Caused By Human's Near-Distance Work

[0106] To verify the effect of ω-3 polyunsaturated fatty acid on human myopia, a clinical trial was implemented. The clinical trial was approved by the Ethics Committee of the Eye Hospital of Wenzhou Medical University, and the participants were first-year college students in Wenzhou Medical University. Experimental procedure: first, participants were allowed to watch TV at 3 meter distance for 15 minutes, and then subjected to OCTA measurements on choroidal thickness and the areas of the choroidal stomal area, vascular area, and non-perfused area, after which the participants were allowed to read with an electronic display at 33 cm distance for 40 minutes, and then subjected to the above OCTA measurements; after that, the participants were asked to take a fish oil capsule containing 600 mg DHA and 120 mg EPA daily, continuing for 14 days; on day 15, the choroidal data after reading 40 minutes were detected, respectively (FIG. 8A).

[0107] As can be seen from analysis, near-distance reading has no significant effect on changes in ChT and the stomal area (FIG. 8B and FIG. 8C), but shows a significant decrease in the alteration of the vascular luminal area and the alteration of the choroidal vascularity index (FIG. 8D and FIG. 8E), moreover, the near-distance reading has a significant effect on the area of the choroidal nonperfused zone, implying an increase in an area without a blood flow signal (FIG. 8F), all of which indicate that near-distance reading can reduce ChBP. Supplementation of fish oil can significantly delay the decrease in choroidal vascular index (FIG. 8E) and improve the reduction in the vascular luminal zone (FIG. 8D) and the increase in the area of nonperfused zone (FIG. 8F) to some extents. The above results indicate that ω-3 polyunsaturated fatty acids can inhibit the reduction in ChBP caused by human's near-distance work.

Example 6 High-Dose DHA is Significantly Superior to EPA, Resulting in Unpredicted Efficacy

[0108] Peribulbar injection of either DHA or EPA can inhibite the development of myopia in guinea pigs. At the same dose, DHA shows a stronger inhibitory effect than EPA.

[0109] FD induces significant myopia in both solvent-and DHA-treated eyes of guinea pigs, including low-dose (1 μg/day) and high-dose DHA (3 μg/day) treatments (FIG. 6A). However, after two weeks of treatment, myopia progression in high-dose DHA treatment group is 35.3% less than that in solvent control group (PP<0.01, FIG. 6A). This inhibitory effect is accompanied by significant reduction in VCD and AL elongation (FIG. 6B and FIG. 6C). Administration of atropine is a widely accepted drug treatment, which can inhibit the progression of myopia in human. Atropine (0.1%) treatment, as a positive control, has a reduced myopia rate of 35.6%, as compared with drug treatment group (PP<0.01, FIG. 6A). Thus, it is proved that the inhibitory effect of atropine is similar to that of high-dose DHA. That is, these results suggest that peribulbar injection of guinea pigs with DHA can inhibit the development of FDM.

[0110] The trend for peribulbar injection of EPA is similar to that of DHA, but with a relatively weak inhibitory effect. After two weeks of treatment, administration of high-dose EPA (3.0 μg/day) shows 29.6% inhibition on the development of FD induced myopia, which has no statistically difference compared to negative control group, and its efficacy is lower than that of 0.1% atropine (FIG. 6D). Two doses of EPA have no significant effects on VCD or AL elongation (FIG. 6E and FIG. 6F).

Example 7 Good Saftey

[0111] All grouped animals in Example 2 were subjected to safety test. The results show that there are no significant differences in ACD, LT and body weight between eye group fed with ω-3 polyunsaturated fatty acids and Vehicle control group (FIG. 9), indicating that ω-3 (DHA 300 mg, EPA 60 mg) is not potentially toxic to other organs, and has a good safety.

[0112] Similarly, there are no significant ocular differences in ACD, LT and body weight between eyes treated with DHA alone in Example 6 and Vehicle control group (FIG. 10), indicating that even a high dose of DHA (3 μg/day) is not potentially toxic to eyeballs, and bas a good safety.

Example 8 Avoidance of Allergies

[0113] Animals were randomly divided into three groups: FD+cod liver oil (commercially available ω-3 polyunsaturated fatty acids) group; FD+high-purity drug group 1 (FD+DHA 3.0 μg); and FD+high-purity drug group 2 (FD+“DHA 3.0 μg+EPA 3.0 μg”), and both high-purity drug groups were at a high dose.

[0114] After applying a topical anesthetic (one drop of 0.5% propazocaine hydrochloride, Alcon Laboratories, Inc., Puurs, Belgium), 100 μL of drug was daily administered at the surrounding area of FD eyeballs at 9:00 a.m., continuing for 2 weeks. All injections were completed within 10 seconds under dark red light to minimize any possible impact of red light on induced myopic recovery.

[0115] It is found that periocular swelling occurs after 1-3 days of injection on animals in FD+cod liver oil group (FIG. 11A), and conjunctivitis occurs after 1 week; whereas animals in high-purity groups (DHA alone or DHA+EPA) have no adverse reactions such as allergies (FIG. 11B).

Example 9 Optimal Treatment Scheme

[0116] The optimal treatment protocol or scheme applying ω-3 by means by of feeding and peribulbar injection were investigated, respectively, and some critical findings were shown, especially when the eye was subjected to topical administration, unexpected results were obtained by using DHA alone or a composition of DHA and EPA. The basic experimental procedures were as described above.

[0117] Regardless of the economy, “the composition of DHA and EPA containing EPA as a predominant component” is significantly more effective than ω-3 polyunsaturated fatty acids in other forms, if only considering the most critical therapeutic effects. That is unexpected. Based on previous experimental results and experience (e.g., Example 6), the myopic inhibition effect of DHA alone was stronger than that of EPA alone. Therefore, for a mixture of DHA and EPA, it should be reasonable to conclude that, the higher the proportion of DHA (e.g., 99% or more), the better the effect.

[0118] However, the inventors surprisingly find that in young guinea pig models with FD myopia, the mypia inhibition effect of DHA alone is still superior to that of EPA alone, and the treatment efficacy of DHA alone is reduced after mixing of EPA in DHA (the total mass of ocular topical administration is 3 μg in each case). However, as the content of EPA increases to become the predominant component (i.e., DHA:EPA<1:1), the efficacy of the mixture of DHA and EPA (the total mass of ocular topical administration is 3 μg in each case) is even better than that of DHA alone, EPA alone, or a mixture of DHA and EPA containing DHA as the main component (i.e., DHA:EPA>1:1, for example, DHA:EPA=1:5, or DHA:EPA=1:9) and is statistically significant (Table 1 and FIG. 12). It can be seen that the mixture of DHA and EPA that uses EPA as the predominant component or the predominant active ingredient has the optimal inhibition effect of myopia and myopia-related symptoms. Overall, all the treatment protocols in this example are very effective in inhibiting the negative refraction, eye axis elongation, and vitreous cavity depth increase.

[0119] Meanwhile, there are no significant interocular differences or statistical differences in ACD, LT and RCC between eye groups treated with polyunsaturated fatty acids in different proportional formulations and Vehicle control group (FIG. 13). It can be seen that ocular topical administration of various mixture forms of DHA and EPA (e.g., 3 μg/day) is not potentially toxic to eyes, and is very safety.

TABLE-US-00001 TABLE 1 Sample size 12 21 12 12 12 11 25 Veh DHA DHA:EPA DHA:EPA DHA:EPA DHA:EPA EPA 5:1 1:1 1:5 1:9 Refraction 0 W 0.02 −0.23 −0.20 −0.21 −0.06 0.17 −0.40 1 W −4.33 −2.95 −3.06 −3.19 −2.67 −2.27 −3.42 Myopia 31.8% 29.4% 26.3% 38.3% 47.5% 21.0% inhibition rate Vitreous 0 W 0.00 0.00 0.00 0.00 0.00 0.00 0.00 chamber depth 1 W 0.07 0.06 0.06 0.07 0.06 0.05 0.07 Eye axis 0 W 0.01 −0.01 −0.01 0.00 −0.01 −0.01 0.01 length 1 W 0.08 0.06 0.04 0.07 0.06 0.04 0.07

[0120] As known to those skilled in the art, since human eyes are much larger than animal (such as guinea pigs) eyes and specific ocular tissue structures are different, it is predicted that the dose for optimal myopic treatment in human is 5-1000 times the dose specifically administered in the above examples, which can be achieved by increasing the single dose and/or increasing the frequency of daily dose clinically.

[0121] Therefore, the above description of the specific embodiments of the present invention discloses the technical details of the present invention in detail, exemplarily gives the technical thinking of the present invention, and is intended to satisfy the authorization provision of the patent law, but should not be considered as limiting the scope of protection of the present invention. Various changes or deformations can be made by researchers in the light of the present application in combination with the knowledge and technology at that time, and shall fall within the protection of the appended claims without departing from the core ideas and spirit of the present application.