Pharmaceutical composition comprising sclerostin inhibitor as active ingredient for prevention or treatment of diabetic eye disease

Abstract

The present invention relates to a pharmaceutical composition comprising a sclerostin inhibitor as an active ingredient for the prevention or treatment of diabetic eye disease, wherein the composition, by inhibiting sclerostin, not only reduces the expression levels of inflammatory cytokines significantly even under conditions of VEGF treatment and hyperglycemia, thereby exhibiting an excellent anti-inflammatory activity, but also is shown to have a significant angiogenic effect due to reduced expression of vascular adhesion markers and vascular endothelial growth factors, thereby inhibiting the principal characteristic of diabetic eye disease, the inflammatory response, and microvascular complications. In addition, the composition reduces oxidative stress caused by diabetes and does not induce cell death and thus can be useful as a preparation for the prevention or treatment of diabetic eye disease.

Description

Pharmaceutical composition for preventing or treating diabetic eye disease comprising a sclerostin inhibitor as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0073048, filed June 7, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] This application claims priority to Korean Patent Application No. 10-2024-0073989, filed June 5, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] Diabetes mellitus (DM) is a chronic, non-communicable, systemic metabolic disorder characterized by abnormally high blood glucose levels (sugar) that persist for a long period of time. Insulin, produced by the beta cells of the pancreas, plays a central role in the metabolism of glucose, which is produced through the digestion and absorption of carbohydrates. It is a hormone that regulates blood glucose levels within a certain range to prevent hyperglycemia.

[0005] Diabetes is the result of an absolute or relative insulin deficiency caused by autoimmune destruction of pancreatic beta cells, or, even if insulin is secreted, a decrease in its effective action in target organs (insulin resistance). Consequently, blood sugar levels remain persistently elevated, disrupting the body's metabolic functions, including glucose metabolism. Diabetics are unable to utilize sugar, the body's primary source of energy, disrupting the body's balance, leading to various complications and potentially life-threatening outcomes.

[0006] Chronic inflammation is a symptom of diabetes. Chronic inflammation involves mild elevations of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, triggered by endogenous or exogenous factors. Macrophages are a major source of inflammatory cytokines in chronic inflammation, and human macrophages have been shown to produce more proinflammatory cytokines in response to hyperglycemia. Chronic inflammation, a persistent, asymptomatic systemic inflammation, has been shown to promote vascular damage.

[0007] Furthermore, hyperglycemia is known to increase markers of inflammation and oxidative stress through a vicious cycle, which play a significant role in the development of diabetic complications. Beyond hyperglycemia, it induces numerous pathophysiological pathways associated with the development of diabetic complications, including the polyol pathway, oxidative stress, non-enzymatic glycation, and activation of protein kinase C.

[0008] Representative complications of diabetes include diabetic retinopathy, diabetic macular edema, glaucoma, and cataracts. Diabetic retinopathy (DR), the most common microvascular consequence of diabetes, is a leading cause of vision loss worldwide, affecting an estimated 93 million people, with another 21 million suffering from diabetic macular edema.

[0009] Although the need for research on the development of treatments for diabetic eye diseases is increasing, little is known about treatments for diabetic eye diseases using sclerostin.

[0010] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0011] Another object of the present invention is to provide a food composition for preventing or improving diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0012] Another object of the present invention is to provide a kit for preventing or treating diabetic eye disease, comprising a composition comprising a sclerostin inhibitor as an active ingredient and an instruction manual.

[0013]

[0014] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0015] The present invention provides a pharmaceutical composition for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0016] In one embodiment of the present invention, the inhibitor may be at least one of an expression inhibitor or an activity inhibitor, but is not limited thereto.

[0017] In one embodiment of the present invention, the expression inhibitor or activity inhibitor may be at least one selected from the group consisting of siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

[0018] In one embodiment of the present invention, the diabetic eye disease may be at least one selected from the group consisting of diabetic retinopathy, diabetic macular edema, glaucoma, and cataract, but is not limited thereto.

[0019] In one embodiment of the present invention, the composition may be in the form of any one formulation selected from the group consisting of a suspension, a gel, an ointment, an injectable solution, a spray, and an eye drop, but is not limited thereto.

[0020] In one embodiment of the present invention, the composition may suppress, but is not limited to, inflammatory responses and microvascular complications.

[0021] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0022] Inhibition of inflammatory cytokines in retinal cells;

[0023] Inhibition of angiogenesis;

[0024] Inhibition of vascular endothelial growth factor and vascular adhesion markers; and

[0025] Reduce oxidative stress.

[0026] The present invention provides a food composition for preventing or improving diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0027] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0028] The present invention provides a kit for preventing or treating diabetic eye disease, comprising a composition comprising a sclerostin inhibitor as an active ingredient and an instruction manual.

[0029]

[0030] In addition, the present invention provides a method for preventing or treating diabetic eye disease, comprising administering a composition containing a sclerostin inhibitor as an active ingredient in a pharmaceutically effective amount to a subject in need thereof.

[0031] In addition, the present invention provides a composition comprising a sclerostin inhibitor as an active ingredient for use in preventing or treating diabetic eye disease.

[0032] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating diabetic eye disease, comprising a composition comprising a sclerostin inhibitor as an active ingredient.

[0033] According to a pharmaceutical composition for preventing or treating diabetic eye disease containing a sclerostin inhibitor as an active ingredient, when sclerostin is inhibited, the expression level of inflammatory cytokines is significantly reduced even under conditions of hyperglycemia and VEGF treatment, so that not only is there excellent anti-inflammatory activity, but also a significant angiogenic effect due to a decrease in the expression of vascular endothelial growth factor and vascular adhesion markers, and it was confirmed that the composition can suppress the inflammatory response and microvascular complications, which are the most significant characteristics of diabetic eye disease. In addition, since it reduces oxidative stress caused by diabetes and does not induce cell death, it can be usefully utilized as a preparation for preventing or treating diabetic eye disease.

[0034] Figure 1a shows the results of mRNA expression and Western blot analysis for SOST expression after siRNA transfection at various concentrations (10, 30, and 50 nM siRNA) designated si10, si30, and si50 under HG treatment.

[0035] Figure 1b shows the results of Western blot analysis of mRNA expression and SOST expression after siRNA transfection at different concentrations (10, 30, 50 nM siRNA) under VEGF treatment (*P < 0.05, **P < 0.01, ***P < 0.001 control, #P < 0.05, ##P < 0.01, ##P < 0.001.01, ##P < 0.001.)

[0036] Figure 1c shows the results of immunofluorescence analysis of SOST expression under HG and VEGF treatment.

[0037] Figure 1d shows the results of analyzing HREC viability by MTT after transfecting siRNA at various concentrations (10 nM, 30 nM, and 50 nM siRNA) under high glucose conditions.

[0038] Figures 2a and 2b show the results of analyzing changes in mRNA and protein expression levels of pro-inflammatory cytokine gene markers induced by SOST inhibition, respectively. They show a tendency for high protein expression of pro-inflammatory cytokines under hyperglycemic conditions and low expression after SOST knockdown.

[0039] Figure 3a shows the mRNA level of the Wnt / β-catenin gene marker.

[0040] Figure 3b shows the protein expression level of Wnt / β-catenin after glucose treatment.

[0041] Figures 4a and 4b illustrate the elevated expression of pro-inflammatory genes following VEGF treatment and the lower expression observed following sclerostin / SOST knockdown. Figure 4a shows the mRNA levels of pro-inflammatory gene markers. Figure 4b shows the protein expression levels of pro-inflammatory chemokines and cytokines following VEGF treatment.

[0042] Figures 5a to 5c show the results of a study analyzing SOST overexpression in HRECs after CRISPR-SOST knockin. Figure 5a shows high expression levels of SOST mRNA and protein. Figure 5b shows the results of an immunofluorescence analysis of SOST overexpression. Figure 5c shows high expression levels of proinflammatory proteins after CRISPR-SOST knockin.

[0043] Figures 6a and 6b show the results of measuring the oxidative stress reduction effect in HRECs by SOST knockdown. Figure 6a shows the high oxidative stress observed after HG and VEGF treatment and the low oxidative stress observed after SOST knockdown. Figure 6b shows the results of measuring oxidative stress in HRECs after CRISPR-SOST knockdown.

[0044] Figures 7a and 7b show the results of analyzing the expression levels of vascular endothelial growth factor A (VEGFA) and blood adhesion factors ICAM-1 and VCAM-1 in HREC cells transfected with SOST siRNA. Figure 7a shows the mRNA and protein expression levels of VEGFA, ICAM-1, and VCAM-1 after HG treatment. Figure 7b shows the mRNA and protein expression levels of VEGFA, ICAM-1, and VCAM-1 after VEGF treatment.

[0045] Figures 8a and 8b show the results of analyzing angiogenic activity when SOST was silenced. Figure 8a shows a phase-contrast image of tube formation in HRECs treated with HG and VEGF. Figure 8b shows the results of analyzing the total tube length (pixels, px) and the number of branch points in tube formation using ImageJ densitometry.

[0046] Figures 8c and 8d show the results of analyzing angiogenic activity when SOST was overexpressed. Figure 8c shows a phase-contrast image of tube formation when CRISPR-SOST was knocked in. Figure 8d shows a phase-contrast image of tube formation in HREC after total tube length (pixels, px) and the number of branch points of tube formation, analyzed using ImageJ densitometry. Increased tube formation was observed in siCON + HG, siCON + VEGF, and SOST100 samples compared to siRNA transfected samples.

[0047] Figure 9 is a schematic diagram showing the role of sclerostin in activating angiogenesis and endothelial inflammation through the NF-B pathway under hyperglycemic conditions in HREC, and in antagonizing Wnt / β-catenin signaling by binding to (LRP) 5 / 6, and the inhibitory effect of sclerostin-siRNA on the inflammatory response.

[0048] The present invention provides a pharmaceutical composition for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0049] Since sclerostin is expressed by the SOST gene, the sclerostin inhibitor of the present invention may include a SOST expression inhibitor. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating diabetic eye disease, comprising a SOST expression inhibitor as an active ingredient.

[0050] In the present invention, the term "sclerostin inhibitor" may include either a sclerostin expression inhibitor or a sclerostin activity inhibitor. Accordingly, in one embodiment of the present invention, the inhibitor may be at least one of an expression inhibitor or an activity inhibitor, but is not limited thereto.

[0051] In the present invention, "expression suppression" may include not only expression suppression but also expression reduction. "Expression reduction" refers to any act that reduces gene expression, and specifically, it may be achieved by gene knockout, knockdown, gene interference, or introducing mutations such as deletion, duplication, inversion, or replacement into the gene DNA sequence, and includes all cases in which expression is reduced by any of the methods exemplified in the present invention.

[0052] In the present invention, “activity inhibition” may include activity reduction in addition to activity inhibition. “Activity reduction” means that one or more functions caused by sclerostin are inhibited, and the functions may be functions directly or indirectly related to the development / worsening of symptoms of diabetic eye disease, such as causing diabetic eye disease, progressing or worsening symptoms of diabetic eye disease, or increasing the rate of progression of diabetic eye disease, but are not limited thereto.

[0053] In addition, in the present invention, it was confirmed that when sclerostin is overexpressed, diabetic eye disease is induced or symptoms are worsened, and when the SOST gene is knocked down, the symptoms are reversed and a therapeutic effect for diabetic eye disease is shown. In this case, the knockdown of the SOST gene may mean that the level of sclerostin is reduced below the average level generally understood in the art. Therefore, the activity inhibitor or expression inhibitor of the present invention does not mean that the expression of the SOST gene is completely suppressed or the level of sclerostin is completely reduced. The sclerostin inhibitor or SOST expression inhibitor in the present invention may include any substance that reduces the amount / activity level of sclerostin below the level generally understood in the art.

[0054] The characteristic of the present invention is that the symptoms of diabetic eye disease that appear under hyperglycemic conditions or VEGF treatment conditions are treated when sclerostin expression is suppressed, and is not due to the specific type of sclerostin inhibitor. Therefore, it is self-evident that those skilled in the art will understand that the same diabetic eye disease treatment activity as the present invention will be exhibited by applying general techniques capable of suppressing sclerostin expression or activity.

[0055] In one embodiment of the present invention, the material may be at least one selected from the group consisting of siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

[0056] In the present invention, “polynucleotide” or “nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

[0057] In the present invention, “siRNA” means, but is not limited to, an RNA molecule, generally 17-24 bp in length, capable of inhibiting expression by interfering with the expression of a gene containing a nucleic acid sequence complementary to the sequence of the siRNA in a process called RNA interference.

[0058] In the present invention, siRNA may include both single-stranded siRNA and double-stranded siRNA. In this case, double-stranded siRNA is derived from a longer double-stranded RNA molecule (dsRNA), which is cleaved by endo-ribonuclease (Dicer) to form double-stranded siRNA, and single-stranded siRNA may be formed in the nuclear protein complex (RISC), but is not limited thereto.

[0059] In the present invention, methods for synthesizing or purifying siRNA, methods for delivering siRNA, etc. can be applied using methods generally used in the art, and for example, non-viral vectors such as various plasmids and liposomes that can be expressed in eukaryotic cells can be used, but are not limited thereto.

[0060] In the present invention, “shRNA” refers to a substance that induces the NAi phenomenon by binding complementarily to a target mRNA and inducing mRNA degradation through a short hairpin RNA with a loop structure. shRNA may be, but is not limited to, microRNA-adapted shRNAs or simple hairpin shRNAs, and includes all forms modified to enhance the function of shRNA.

[0061] In the present invention, “miRNA” refers to a fine regulator that is a single-stranded RNA molecule generally having a length of ~22 nucleotides (nt) and binds to the 3'UTR of a target mRNA in a complex with an Argonaute protein (AGO) to suppress the expression of the corresponding gene.

[0062] In the present invention, “antisense oligonucleotides” are drugs with the potential to realize personalized medicine together with RNAi therapeutics. They are single-stranded nucleic acid polymers synthesized with an average of 12 to 25 nucleotides, have specific sequence complementarity to target RNA, and can regulate protein expression through various mechanisms. In addition, antisense oligonucleotides, single-stranded ASOs, can regulate gene expression or affect mRNA splicing through various mechanisms depending on the chemical or binding site and target. When an ASO complementarily binds to a pre-mRNA target site, Ribonuclease H (RNase H), which hydrolyzes the RNA strand of an RNA-DNA heteroduplex, is activated, thereby inhibiting target mRNA degradation and protein expression, but is not limited thereto. ASOs can directly inhibit the target site, or ASOs can interfere with the interaction with RNA-binding proteins by inducing steric hindrance through their binding to mRNA. In the present invention, the antisense oligonucleotide may be used after being modified with phosphorothioate (PS), phosphorodiamidate morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), locked nucleic acid (LNA), ribose modifications, etc., but is not limited thereto, and includes all modifications to which techniques that can be generally applied in the art are applied to improve functionality.

[0063] In the present invention, “CRISPR / Cas” may mean a ‘guide RNA (gRNA)’ made of RNA and an enzyme that cuts DNA, ‘Cas’, and may be, for example, Cas9, Cas11, or Cas12, but is not limited thereto, and Cas may have a broad meaning that includes all forms of enzymes that can be used in the CRISPR system.

[0064] In the present invention, “Cas9” refers to “CRISPR-Cas9,” and CRISPR-Cas9 is a third-generation genetic scissors that recognizes, cuts, and edits a specific base sequence to be used, and can be usefully used to simply, quickly, and efficiently perform operations to insert a specific gene into a target location in the genome or to stop the activity of a specific gene.

[0065] Cas9 protein or genetic information can be obtained from publicly available databases, such as GenBank at the National Center for Biotechnology Information (NCBI), but is not limited thereto. Furthermore, those skilled in the art can appropriately link additional domains to the Cas9 protein, depending on the intended purpose.

[0066] In the present invention, the Cas9 protein may include not only wild-type Cas9 but also all variants of Cas9 as long as they have the function of a nuclease for gene editing, and the origin of the Cas9 protein is not limited, and non-limiting examples thereof include those derived from Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, or Streptococcus mutans, and those skilled in the art may appropriately select and use them, but are not limited thereto.

[0067] In the present invention, the “gRNA” refers to a single-stranded RNA that serves to find the position of a specific target DNA to be edited and guide the Cas protein to the target DNA. The gRNA may be adjacent to a PAM (protospacer adjacent motif) site and include a sequence complementary to a 10 to 25 bp base sequence of the DNA to be edited. The guide RNA may have 1 to 3 additional nucleotides, more specifically 2 or 3 nucleotides, in front of the 5' portion of the sequence capable of forming base pairs with the complementary strand of the target DNA sequence. In addition, the guide RNA may include a portion having a sequence complementary to a sequence in the target DNA (also referred to as a spacer region, target DNA recognition sequence, base pairing region, etc.) and a hairpin structure for Cas protein binding. More specifically, it may include, but is not limited to, a portion having a sequence complementary to a sequence in the target DNA, a hairpin structure for Cas protein binding, and a terminator sequence.

[0068] The gRNA of the present invention may target the sclerostin gene, and may be configured in pairs to simultaneously target different regions of the gene, but is not limited thereto.

[0069] In the present invention, “natural product” refers to a product derived from a living organism, such as a plant or animal living on land or in the sea, or a cell or tissue culture product of a living organism, and may mean a natural product without any artificial processing, and its origin is not particularly limited as long as it is an extract of a natural product, and may be, for example, a plant extract, an animal extract, a microbial extract, or a mineral extract. In addition, a natural product may refer to itself, but is not limited thereto, and may refer to all modified forms containing a pharmacologically active ingredient of the natural product. For example, it may be, but is not limited to, an extract, a fraction thereof, a fermented product, or juice thereof.

[0070] In the present invention, the conditions of the solvent, etc. of the extract are not particularly limited, and all conditions that can be generally used by those skilled in the art can be applied depending on the characteristics of the natural product or so that a pharmacologically active ingredient can be included.

[0071] In the present invention, the method of fractionating into fractions is not particularly limited as long as it is a method of fractionating a commonly used extract, and non-limiting examples thereof include a chromatographic separation method, a liquid separation method using a difference in solubility, a separation method using a difference in specific gravity, a centrifugal separation method using a difference in density, a solid sample extraction method, and a separation method using a difference in color, and may be fractionated using at least one method selected from the group consisting of:

[0072] In the present invention, “protein” is used interchangeably with “polypeptide” or “peptide” and refers to a polymer of amino acid residues, such as those commonly found in proteins in their natural state, and may include, but is not limited to, proteins produced by recombinant expression vectors.

[0073] In the present invention, “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector can be used as long as it can replicate and be stabilized in the host. The recombinant expression vector of the present invention may preferably include a promoter, which is a transcription initiation factor to which RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence for regulating the termination of transcription and translation, a terminator, etc., and the recombinant expression vector and the expression vector including a suitable transcription / translation control signal can be constructed by a method well known to those skilled in the art.

[0074] In addition, it may further include a selection marker gene such as a tag gene for increasing the production amount of the recombinant protein, a tag gene for maintaining the structural stability of the recombinant protein, a tag gene for easily separating the recombinant protein, an antibiotic resistance gene for selecting a transformant, etc., and the tag for easy separation may include, but is not limited to, an Avi tag, a Calmodulin tag, a polyglutamate tag, an E tag, a FLAG tag, an HA tag, a His tag, a Myc tag, an S tag, an SBP tag, an IgG-Fc tag, a CTB tag, a Softag 1 tag, a Softag 3 tag, a Strep tag, a TC tag, a V5 tag, a VSV tag, an Xpress tag, etc., and the selection marker gene may include, representatively, a herbicide resistance gene such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, Antibiotic resistance genes such as chloramphenicol, aadA gene, etc. may be included, and the promoters may include, for example, pEMU promoter, MAS promoter, histone promoter, Clp promoter, cauliflower mosaic virus-derived 35S promoter, cauliflower mosaic virus-derived 19S RNA promoter, plant actin protein promoter, ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, EF-1α (Elongation factor-1 alpha) promoter, etc., and the terminators may include, for example, nopaline synthase (NOS), rice amylase RAmy1 A terminator,Phaseolin terminator, terminator of the Octopine gene of Agrobacterium tumefaciens, rrnB1 / B2 terminator of Escherichia coli, etc., but there is no limitation on the type of the added gene as long as it is a type that has been used in the production of recombinant proteins.

[0075] In the present invention, any method known in the art for transforming with a recombinant vector, producing a target protein from a transformant transformed with a recombinant vector, etc. may be used. At this time, depending on the characteristics of the recombinant vector, the transformant may be an animal or a plant, but is not limited thereto. Any method may be applied as long as it is for obtaining a protein with excellent functionality from the transformant at a high yield.

[0076] As used herein, the term "peptidomimetic" refers to something designed to mimic a biologically active peptide, including but not limited to, a non-natural amino acid or other unique compound to stabilize the structure or alter the biological activity. Peptidomimetics may be modified to enhance the target specificity, stability, or reduce the likelihood of degradation of the peptide. For example, bending may be restricted to limit the conformational mobility of the peptide, or non-natural compounds may be incorporated into the structure to reduce the likelihood of enzymatic degradation, but are not limited thereto.

[0077] In the present invention, “antibody” is a term that is compatible with “antibody fragment” and refers to, but is not limited to, an antibody that naturally exists in the body, and includes all antibodies obtained from human antibody phage libraries and human antibody-producing transgenic animals produced by advances in genetic engineering, cell engineering, and developmental engineering technologies. In addition, an antibody fragment refers to a part of an antibody that can perform the same function as an antibody, and may be, for example, Fab, Fab', F(ab')2, scFv, diabody, dsFv, and a peptide including a CDR, but is not limited thereto. The antibody fragment can be produced by applying a method generally performed in the art.

[0078] In the present invention, “exosome” may refer to, but is not limited to, those secreted from somatic cells derived from human tissues, including neurospheres, fibroblasts, epithelial cells, muscle cells, cardiac cells, kidney cells, nerve cells, hair cells, hair root cells, hair follicle cells, epithelial cells, beta cells, gastric mucosal cells, goblet cells, G cells, immune cells, pericytes, mast cells, endothelial cells, etc. In addition, exosomes may refer to, but are not limited to, those secreted from cells extracted from solutions secreted from the human body, such as urine, saliva, sweat, and blood, or those secreted from bone marrow-derived stem cells such as neural cord blood, adipose-derived stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells such as iPSCs, placental stem cells, etc. In addition, in order to improve exosome secretion or functionality, such as facilitating exosome release or increasing the number of exosomes released, physical stimuli such as electrical stimulation, heat, sound waves, laser, oxidative stress, and LED light, chemical stimuli such as chemical substance treatment, and biological stimuli such as substance treatment with enzymes that act on in vivo mechanisms, may be applied, but are not limited thereto. In the present invention, the size of the exosome may be the size of a general exosome, but is not limited thereto, and may vary depending on the release method, stimulation method, or culture method, and may vary depending on the amount and type of the material to be loaded.

[0079] In the present invention, any method for stimulating, secreting, and obtaining exosomes can be applied as long as it is obvious to those skilled in the art, and is not limited thereto.

[0080] In the present invention, "compound" refers to a substance formed by chemically combining two or more different elements, including both organic and inorganic compounds. The compound of the present invention may refer to any chemical substance capable of inhibiting the expression or activity of sclerostin.

[0081] In the present invention, the "sclerostin inhibitor" can be delivered to the target cell via a method or delivery vehicle commonly used in the art. For example, delivery may be via a vector, but is not limited thereto. Furthermore, the substance of the present invention may be delivered via a method or delivery vehicle generally applicable to the dosage, cycle, age, sex, clinical symptoms, disease progression, etc. of the substance, but is not limited thereto.

[0082] In one embodiment of the present invention, the diabetic eye disease may be at least one selected from the group consisting of diabetic retinopathy, diabetic macular edema, glaucoma, and cataract, but is not limited thereto.

[0083] In the present invention, “diabetic retinopathy” is a retinal disease caused by diabetes, and is classified into non-proliferative and proliferative.

[0084] In the present invention, “non-proliferative diabetic retinopathy” is the most common type of diabetic retinopathy, and is characterized by retinal capillaries growing like small balloons and becoming deformed into an auricle. The progression of non-proliferative diabetic retinopathy is divided into three stages, and retinal vascular occlusion tends to become increasingly severe. Non-proliferative retinopathy usually does not cause blindness, but when the capillary walls weaken and fluid leaks out, the macula swells, causing visual impairment. Non-proliferative retinopathy usually does not require treatment, but if macular edema occurs, treatment is essential.

[0085] Additionally, in some diabetic patients, retinopathy can progress to a more severe form, "proliferative diabetic retinopathy," years later. In proliferative diabetic retinopathy, new blood vessels grow from the retina toward the vitreous, as retinal blood vessels become blocked or damaged. These new blood vessels are fragile and can easily burst, leaking blood and obscuring vision, a condition called a vitreous hemorrhage. Furthermore, the new blood vessels grow alongside fibrous tissue, and when these tissues contract and pull, they can wrinkle or detach the retina, causing a retinal detachment. New blood vessels can also form in the iris, leading to serious glaucoma. Vision changes may not be noticeable even when significant retinal damage has already occurred. While most patients with nonproliferative diabetic retinopathy experience no symptoms, some patients with proliferative diabetic retinopathy may remain unaware of the condition until it is too late to treat.

[0086] Blood sugar control, blood pressure, duration of diabetes, and physical characteristics are closely related to the development of diabetic retinopathy. Long-term diabetes is known to increase the risk of developing retinopathy. Most patients with type 1 diabetes eventually develop non-proliferative diabetic retinopathy, and the same holds true for type 2 diabetes.

[0087] In the present invention, “diabetic macular edema (DME)” is a disease in which swelling occurs in the macula, the center of the retina, and is the most significant cause of vision loss in patients with diabetic retinopathy. Diabetic macular complications increase the permeability of retinal blood vessels, causing microaneurysms, which in turn cause blood plasma proteins and lipid components to leak into the retinal tissue. These substances accumulate in the macula, the center of the retina that plays the most important role in vision, causing retinal thickness to increase and damaging neural connections, leading to vision loss.

[0088] In the present invention, "glaucoma" or "cataract" may be diabetic glaucoma or cataract induced by diabetes. Therefore, in the present invention, the glaucoma or cataract may be diabetic glaucoma or diabetic cataract. Diabetic patients are known to have a 40% higher risk of developing glaucoma than the general population, and the risk increases with duration of diabetes and age. Furthermore, cataracts are also more common, and are characterized by developing at a younger age and progressing rapidly.

[0089] In one embodiment of the present invention, the composition may be in the form of any one formulation selected from the group consisting of a suspension, a gel, an ointment, an injectable solution, a spray, and an eye drop, but is not limited thereto.

[0090] In one embodiment of the present invention, the composition may suppress, but is not limited to, inflammatory responses and microvascular complications.

[0091] The primary purpose of the present invention may be to suppress inflammatory responses and microvascular complications by reducing inflammatory cytokines or angiogenesis by inhibiting sclerostin in hyperglycemia, thereby treating diabetic eye disease. The inflammatory cytokines may be increased by diabetes. In one embodiment of the present invention, it was confirmed that inflammatory cytokines were significantly increased by hyperglycemic conditions or VEGF treatment, and that the expression level of inflammatory cytokines was reduced by inhibiting sclerostin. In addition, it was confirmed that angiogenesis was suppressed by reducing angiogenic activity and suppressing the expression of vascular endothelial markers, thereby inhibiting tube formation.

[0092] Hyperglycemia causes microvascular damage and induces the release of inflammatory cytokines, a hallmark of vasculopathy. In this study, we induced hyperglycemic conditions in HRECs using high glucose concentrations (30 mM) and assessed changes in molecular expression. Hyperglycemia can induce inflammatory conditions in both type 1 and type 2 DM, and elevated cytokine levels can lead to pancreatic beta cell degradation. Inflammatory cytokines are significantly elevated in the serum of DM patients. In vivo studies have shown that high glucose exposure significantly enhanced TNF-α mRNA and protein expression, which further increased sclerostin expression in an NF-κB-dependent manner under hyperglycemic conditions.

[0093] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0094] Inhibition of inflammatory cytokines in retinal cells;

[0095] Inhibition of angiogenesis;

[0096] Inhibition of vascular endothelial growth factor and vascular adhesion markers; and

[0097] Reduce oxidative stress.

[0098] In the present invention, "sclerostin (hereinafter used interchangeably with SOST)" is a protein encoded by the SOST gene. As a secreted glycoprotein primarily produced by osteocytes, SOST is known to inhibit bone formation.

[0099] Dickkopf family protein (DKK) and sclerostin are two additional Wnt antagonists that bind to the co-receptor LDL receptor-related protein (LRP) 5 / 6 and inhibit Wnt signaling, respectively. Sclerostin, a well-known antagonist of Wnt signaling, promotes angiogenesis through LRP6. Furthermore, it increases VEGF and PIGF, which further promote angiogenesis. In one embodiment of the present invention, silencing sclerostin was shown to decrease retinal endothelial angiogenic activity and angiogenic capacity. Furthermore, treatment with SOST siRNA was shown to decrease tube formation, thereby reducing angiogenesis. Furthermore, the present invention demonstrated that sclerostin reduces inflammation by minimally inhibiting Wnt / β-catenin.

[0100] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0101] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0102] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0103] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0104] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0105] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0106] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0107] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0108] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0109] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0110] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0111] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0112] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. Therefore, in the present invention, "pharmaceutically effective amount" can be used interchangeably with "therapeutically effective amount." The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity and drug sensitivity, administration time, route and excretion rate, treatment duration, concurrent medications, and other factors well known in the medical field.

[0113] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0114] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0115] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0116] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0117] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0118] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0119]

[0120] The present invention provides a food composition for preventing or improving diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

[0121] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0122] In one embodiment of the present invention, the material may be at least one selected from the group consisting of siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds, but is not limited thereto.

[0123] In one embodiment of the present invention, the diabetic eye disease may be at least one selected from the group consisting of diabetic retinopathy, diabetic macular edema, glaucoma, and cataract, but is not limited thereto.

[0124] The health food and health functional food compositions containing the active ingredient according to the present invention can be added directly to foods or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. Furthermore, the amount of active ingredient mixed can be appropriately determined depending on the intended use (e.g., for prevention or improvement).

[0125] In general, the amount of the composition in health foods and health functional foods may be added in an amount of 0.1 to 90 parts by weight based on the total food weight. However, for long-term intake for the purpose of maintaining or regulating health, the amount may be below the above range. For safety reasons, the effective ingredient according to the present invention may also be used in an amount exceeding the above range.

[0126] The health food and health functional food composition of the present invention contains the effective substance of the present invention as an essential ingredient in the indicated proportion, and other ingredients are not particularly limited, and various flavoring agents or natural carbohydrates, etc. may be included as additional ingredients, as in conventional beverages. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. The proportion of the natural carbohydrate may be generally about 1 to 20 parts by weight, preferably about 5 to 12 parts by weight, per 100 parts by weight of the health functional food composition of the present invention, but is not limited thereto.

[0127] In addition to the above, the health food and health functional food composition containing the effective substance of the present invention may include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health food and health functional food composition of the present invention may include fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

[0128] These components may be used independently or in combination, and are generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the effective substance of the present invention, but are not limited thereto.

[0129]

[0130] The present invention provides a kit for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor; or a composition comprising a sclerostin inhibitor as an active ingredient; and instructions.

[0131] In addition to the above components, the "kit" of the present invention may include other components, devices, materials, etc. that are typically necessary for the storage, management, and enhancement of the effectiveness of the composition of the present invention. In addition, all components included in the kit may be used more than once without limitation on the number of times, there is no restriction on the order in which each substance is used, and the application of each substance may be performed simultaneously or in microseconds.

[0132] The kit of the present invention may include a container in addition to the formulation and instructions. The container may serve to package the composition, and may also serve to store and secure the composition. The container may be formed of a material such as a bottle, a tub, a sachet, an envelope, a tube, an ampoule, or the like, and may be formed partially or wholly from plastic, glass, paper, foil, wax, or the like. The container may be equipped with a completely or partially detachable stopper, which may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper that allows access to the contents by means of a syringe needle. The kit may include an outer package, which may include, but is not limited to, instructions for the use of the components.

[0133]

[0134] The terms used in this invention have been selected from widely used, current terms, taking into account the functions of the invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, in which case their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the invention.

[0135] Throughout the specification of the present invention, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not exclude other components, but rather means that other components may be included. The terms "about," "substantially," and the like used throughout the specification of the present invention are used in a meaning at or close to the numerical value when manufacturing and material tolerances inherent to the meaning stated are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure, which states precise or absolute values ​​to aid understanding of the present invention.

[0136] Throughout the specification of the present invention, the term “combination thereof” included in the expressions in the Makushi format means a mixture or combination of one or more selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of the components.

[0137] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0138]

[0139] [Example]

[0140]

[0141] Antibodies and Chemical Reagents

[0142] A list of antibodies and chemical reagents used in the present invention is shown in Table 1 below.

[0143]

[0144] Product nameCompany nameFetal bovine serumWelgene(Gyeongsan-si, Gyeongsangbuk-do, South Korea)Dulbecco’s phosphate-buffered saline1X Trypsin-EDTA solutionDimethyl sulfoxide(DMSO)Sigma-Aldrich(St. Louis, MO, USA)3-[4, 5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazoliumbromide(MTT)Paraformaldehyde(4%)Biosesang(Seongnam-si, Gyeonggi-do, South Korea)MatrigelBD Biosciences(Bedford, MA, USA)Human retinal endothelial primary cells(HREC, 36205-33)Celprogen Inc,(Torrance, CA, USA).Human retinal endothelial cell culture complete media with serum and antibioticHuman retinal endothelial cell culture complete media without serum and antibioticAntibody against β-CateninCell Signaling Technology(Danvers, Massachusetts, USA)Halt™ Protease and Phosphatase Inhibitor CocktailThermo Fisher Scientific(Rockford, IL, USA)Pierce™ RIPA BufferPierce™ BCA Protein Assay KitOpti-MEM™ reduced serum mediumThermo Fisher Scientific(Grand Island, NY, USA).Phenylmethylsulphonyl fluorideRoche(Mannheim, Germany)Human VEGF165 (PeproTech, Cranbury, NJ, USA)Mounting Medium with 4’,6-diamidino-2-phenylindole(DAPI)-Aqueous FluoroshieldAbcam(Cambridge, MA, USA)Antibodies against VEGFA, LRP6, Wnt3a, and COX-2Antibodies against TNF-α and Wnt5aR&D Systems(Minneapolis, MN, USA)Sclerostin siRNA(h)Santa Cruz Biotechnology(Dallas, TX, USA).Sclerostin-CRISPR activation plasmid(h)UltraCruz Transfection ReagentPlasmid Transfection MediumAntibodies against SOST, ICAM-1, IL-6, and β-actinMouse anti-goat IgG-HRPMouse anti-rabbit IgG-HRPGoat anti-mouse IgG- conjugated with HRPLipofectamine RNAiMAX reagentInvitrogen(Carlsbad, CA, USA)Alexa Fluor 594 donkey anti-goat IgG(H&L)Alexa Fluor 594 goat anti-mouse IgG(H&L)Antibodies against MCP-1, NFκB p65, and iNOSTB Green® Premix Ex Taq™Takara Bio Inc.,(Kusatsu, Shiga, Japan)The protein loading bufferBio-Rad Laboratories, Inc.(Hercules, CA, USA)iScript cDNA synthesis kitThe RNA extraction kit Ribospin IIGeneAll Biotechnology(South Korea)

[0145]

[0146] Cell culture

[0147] Human retinal endothelial cells (HREC) were grown in human retinal endothelial cell culture complete medium (Celprogen Inc, Torrance, CA, USA) containing serum and antibiotics. Mononuclear cells were seeded at a specific density of 1.0 × 10 in extracellular matrix-coated T75 culture flasks (Celprogen Inc, Torrance, CA, USA). 6 mL -1 Mononuclear cells were quantified using the trypan blue exclusion technique before inoculation. The cell seeding density was maintained the same in subsequent experiments. HRECs were maintained in an incubator set at 37°C and 5% CO2.

[0148]

[0149] Treatment of HRECs with high glucose level and VEGF

[0150] HRECs were plated onto 6-well tissue culture dishes coated with extracellular matrix and maintained at 37°C in a humidified environment in human retinal endothelial cell culture complete medium containing serum and antibiotics from Celprogen Inc. A glucose concentration of 30 mM was prepared in the cell growth medium and applied to 80% confluent cells for hyperglycemic treatment and maintained for 24 h.

[0151] For VEGF treatment of HREC, 50 ng Human VEGF165 (PeproTech, Cranbury, NJ, USA) was prepared with cell growth medium and used after 80% cell confluence and stored for 24 hours.

[0152]

[0153] Small interfering RNA (siRNA) transfection

[0154] HRECs were seeded in serum- and antibiotic-free human retinal endothelial cell culture complete medium (Celprogen Inc, Torrance, CA, USA) onto 6-well tissue culture plates coated with extracellular matrix. When HRECs reached 50–60% confluence, cells were transfected with Sclerostin siRNA(h) (sc-61503, a pool of three target-specific 19–25 nt siRNAs from Santa Cruz Biotechnology) and control siRNA at final concentrations of 10 nM, 30 nM, and 50 nM using Lipofectamine RNAiMAX transfection reagent according to the manufacturer's instructions. After 24 h of transfection, HRECs were separately treated with 30 mM glucose and 50 ng VEGF for 24 h before further experiments. The control siRNA used was sc-37007 (Santa Cruz, Dallas, TX, USA).

[0155]

[0156] Cell viability assay

[0157] The MTT assay was performed using the standard test protocol [ISO10993-5:2009I] to assess in vitro cell viability after high glucose (HG) and siRNA transfection. The MTT test was performed after 24 h of glucose treatment and siRNA transfection. A previous protocol was used to determine cell viability (measured by optical density) (Sayed, S., et al., Thermal cycling effect on osteogenic differentiation of MC3T3-E1 cells loaded on 3D-porous Biphasic Calcium Phosphate (BCP) scaffolds for early osteogenesis. Mater Sci Eng C Mater Biol Appl, 2019. 105: p. 110027.). To generate formazan crystals, MTT solution (5 mg / mL in PBS) was added to the cell culture medium at a ratio of 1:9, and the mixture was incubated at 37°C for 4 h. To extract formazan crystals, DMSO was added and dissolved for 1 hour. Finally, the absorbance was measured at 595 nm using an ELISA reader (EL,312, Biodynamics Reader; Bio-Tek Instruments, Winooski, VT, USA). Triplicate tests were performed for each sample.

[0158]

[0159] Clustered regularly interspaced short palindromic repeats (CRISPR)-SOST knockin

[0160] For sclerostin CRISPR activation, HRECs were plated in serum- and antibiotic-free human retinal endothelial cell culture complete medium (HREC) in 6-well tissue culture plates with extracellular matrix coating. After 50–60% confluence of HRECs, sclerostin-CRISPR activating plasmids (h) from Santa Cruz Biotechnology and the SAM transcriptional activation system (three target-specific 19–25 nt pools) from the control CRISPR were used at final concentrations of 10 ng, 30 ng, 50 ng, and 100 ng, respectively, using UltraCruz Transfection Reagent and Plasmid Transfection Medium, respectively, according to the manufacturer's instructions. Additional testing was performed 24 h later.

[0161]

[0162] Quantitative RT-PCR

[0163] After cell treatment, total RNA was extracted using the Ribospin II RNA extraction kit (GeneAll Biotechnology). RNA concentration was measured using a NanoDrop-One spectrophotometer (Thermo Fisher Scientific, Cambridge, MA, USA), and RNA was reverse transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA) in a SimpliAmp Thermal Cycler (Applied Biosystems, Singapore). All cDNA samples were stored at -20°C for subsequent gene expression studies. qRT-PCR was performed using TB Green ®The assay was performed using Premix Ex TaqTM (Takara Bio Inc., Kusatsu, Shiga, Japan) and the CFX96TM RT-PCR Detection System (Bio-Rad, Singapore). Real-time PCR tests were performed in triplicate. After normalizing the data to GAPDH levels, the relative mRNA expression levels were estimated using the 2-ΔΔCt method. The qRT-PCR primer sequences used in the present invention are shown in Table 2 below.

[0164]

[0165] Seq.NameSequence 5’-3’Seq. 1SOST_FF- GAGCCTGTGCTACTGGAAGGSeq. 2SOST_RR- TGATTTCCGTGGCATCATTSeq. 3TNF-α_FF-GGTGCCCGCTGCTGTCTAATSeq. 4TNF-α_RR- TGCAACGCGAGTCTGTGTTTSeq. 5TNF-α_FF-AAGCTGAGGGGCAGCTCCAGTSeq. 6TNF-α_RR- TCTGGTAGGAGACGGCGATGCSeq. 7ICAM-1_FF- CTTCGTGTCCTGTATGGCCCSeq. 8ICAM-1_RR- CACATTGGAGTCTGCTGGGASeq. 9VCAM-1_FF- GTCAATGTTGCCCCCAGAGATASeq. 10VCAM-1_RR- ACAGGATTTTCGGAGCAGGASeq. 11iNOS_FF- GCTCTACACCTCCAATGTGACCSeq. 12iNOS_RR- CTGCCGAGATTTGAGCCTCATGSeq. 13NFκB_FF- CACTTATGGACAACTATGAGGTCTCTGGSeq. 14NFκB_RR-CTGTCTTGTGGACAACGCAGTGGAATTTTAGGSeq. 15Cox-2_FF- TTGCTGGCAGGGTTGCTGGTGGTASeq. 16Cox-2_RR- CATCTGCCTGCTCTGGTCAATGGAASeq. 17IL-1β_FF-TTGCTCAAGTGTCTGAAGCAGCSeq. 18IL-1β_RR-CTTGCTGTAGTGGTGGTCGGSeq. 19IL-6_FF- GGATTCAATGAGGAGACTTGCCSeq. 20IL-6_RR- GGGTCAGGGGTGGTTATTGCSeq. 21MCP-1_FF- GGCTGAGACTAACCCAGAAACSeq. 22MCP-1_RR- GAATGAAGGTGGCTGCTATGASeq. 23LRP5_FF-GTGACAATGGTGGCTGCTCSeq. 24LRP5_RR-TCCACAGGTCAGCAGGTTCTSeq. 25LRP6_FF-TCAATGATCAGCTCCCTCAGSeq.26LRP6_RR-TAAGTGCCTTTGGTGCTTGASeq. 27CTNNB1_FF-TCTCCCATTGAAAACATCCASeq. 28CTNNB1_RR-CTGTGGCTCCTCAGCTTCSeq. 29Wnt3a_FF-TGTTGGGCCACAGTATTCCTSeq. 30Wnt3a_RR-CACTCCTGGATGCCAATCTTSeq. 31Wnt5a_FF-TTAAGCCCAGGAGTTGCTTTSeq. 32Wnt5a_RR-CAATTACAACCTGGGGCGAAGSeq. 33GAPDH_FF- GAGAAACCTGCCAAGTATGATGATGACSeq. 34GAPDH_RR- AGAGTGGGAGTTGCTGTTGAAG.

[0166]

[0167] Western blot (WB) analysis

[0168] For WB analysis, total protein was obtained using RIPA lysis buffer containing 1X protease and phosphatase inhibitor cocktail. Total protein concentration was determined using the BCA protein assay kit from Thermo Scientific (Rockford, Illinois, USA). Protein samples were separated using 8% and 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then treated with specific primary antibodies overnight and then incubated with specific secondary antibodies (HRP-conjugated). Bands were identified using chemiluminescent ECL reagents, and densitometry analysis was performed using the ChemiDocTM XRS+ system (Bio-Rad, Hercules, CA, USA). Anti-β-actin antibody served as a loading control.

[0169]

[0170] Immunocytochemical analysis

[0171] After cell treatment, HRECs transfected with SOST siRNA were fixed in 4% paraformaldehyde solution. Cells were permeabilized with 0.25% Triton X-100 and then blocked with 2.5% bovine serum albumin (BSA). Cells were then immunostained overnight at 4°C with an antibody against mouse sclerostin (1:50) according to the previous protocol. Cells were incubated with the primary antibody and then with the secondary antibody (Alexa Fluor 594, 1:1000). After rinsing, cells were mounted with Fluoroshield-DAPI aqueous solution. Images were observed using a fluorescence microscope (Leica DMi8) and Leica LAS AF software.

[0172]

[0173] Tube formation assay

[0174] Matrigel (without phenol red) was coated on a pre-chilled 48-well plate. The coating process was completed on ice. Then, it was kept in an incubator at 37°C for 30 minutes. Pretreated HRECs were collected and diluted in serum- and antibiotic-free medium (4 × 10 5 After collection, the treated HRECs were seeded in triplicate on Matrigel-coated 48-well plates and cultured at 37°C. HREC tube formation occurred after 8 hours. Phase-contrast images of tube formation were captured using a Leica DMi8 microscope.

[0175]

[0176] Statistical analyses

[0177] All studies were performed at least three times, and all data were expressed as mean SD (standard deviation). Significant differences were determined using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test between the control and other groups. Statistically significant differences were identified through multiple comparisons and are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001 versus the control group, respectively, for HG-treated or VEGF-treated samples. All statistical evaluations were completed using GraphPad Prism 8.

[0178]

[0179] Example 1. Effect of SOST knockdown on HRECs: Reduced SOST expression and non-toxicity.

[0180] The effect of SOST knockdown on human retinal endothelial cells (HRECs) was confirmed under hyperglycemic (HG) conditions. For HG conditions, HRECs were treated with 30 mM glucose and 50 ng VEGF for 24 h. The 30 mM glucose treatment to create a hyperglycemic condition was selected from a previous experiment (Int J Mol Sci. 2023 Mar 12;24(6):5428. doi: 10.3390 / ijms24065428.). According to the study experiment, more than 80% of cells survived after 24 h of 30 mM glucose treatment, whereas less than 80% of cells survived under 60 mM glucose treatment. Treatment with 30 mM glucose for 24 h induced the expression of TNF-α, NF-κB, MCP-1, IL-1β, IL-6, COX-2, iNOS, VEGFA, and adhesion markers VCAM-1 and ICAM-1. After treatment of HRECs with various concentrations of siRNA (10 nM, 30 nM, and 50 nM), the SOST knockdown effect was evaluated using qRT-PCR, WB, and immunostaining.

[0181]

[0182] As a result, SOST expression was found to be higher when treated with control siRNA (siCON+HG) compared to the control, confirming that SOST increases under hyperglycemic conditions (Fig. 1a). In addition, after treatment with 10 nM, 30 nM, and 50 nM siRNA (denoted as si10+HG, si30+HG, and si50+HG, respectively), SOST expression was found to decrease in a dose-dependent manner in the HG sample.

[0183]

[0184] When VEGF was treated, a higher SOST expression level was observed compared to the hyperglycemic condition, and a high tendency for siRNA dose-dependent reduction was also confirmed (Fig. 1b).

[0185]

[0186] Additionally, the level of SOST expression when SOST siRNA was transfected was confirmed through immunofluorescence staining analysis.

[0187] As a result, SOST expression levels decreased after treatment with 30 nM siRNA. Furthermore, immunostained values ​​showed that SOST expression was observed in HRECs treated with HG and VEGF, regardless of whether SOST siRNA was transfected (Fig. 1c). These results reconfirmed the trend that SOST expression significantly increased after HG and VEGF treatment, but decreased significantly after SOST knockdown.

[0188]

[0189] Additionally, the cytotoxic effect of SOST knockdown was confirmed. Specifically, the cytotoxic effect of SOST knockdown was investigated by transfecting HRECs with 10 nM, 30 nM, and 50 nM siRNA, regardless of HG treatment, and then examining the cell death effect.

[0190]

[0191] As a result, nearly 90% of cells remained viable 24 hours after transfection (Fig. 1d). This demonstrates that siRNA transfection does not induce cell death, as the 30 mM HG-treated sample (siCON+HG) also exhibited 80% cell viability.

[0192]

[0193] Example 2. Confirmation of the effect of reducing hyperglycemia-induced inflammatory activity by SOST inhibition.

[0194] We investigated whether SOST inhibition suppresses hyperglycemia-induced inflammatory activity. Specifically, we analyzed whether silencing the SOST gene reduces cytokine expression in HRECs. To this end, we analyzed the expression of proinflammatory cytokines at the gene and protein levels using qRT-PCR and WB.

[0195]

[0196] As a result, in contrast to the control sample (siCON), the expression of proinflammatory cytokines was increased in the HG-treated samples (siCON+HG). On the other hand, the expression of all investigated gene markers was confirmed to be decreased in a concentration-dependent manner by SOST siRNA. Specifically, the expression levels of proinflammatory cytokines were significantly downregulated starting from the sample transfected with 10 nM SOST siRNA (si10+HG), which was the lowest concentration among the treatment concentrations, and this effect was confirmed to be statistically significant (Fig. 2a and Fig. 2b).

[0197]

[0198] Example 3. Confirmation of the effect of reducing Wnt signaling increased by hyperglycemia through SOST inhibition.

[0199] Sclerostin, expressed by the SOST gene, is known to be a Wnt antagonist. Since Wnt signaling is known to be increased under the influence of hyperglycemia and to induce retinal vascular dysfunction, we analyzed whether suppression of the SOST gene could indirectly induce retinal vascular dysfunction in association with Wnt signaling. Specifically, the effects of sclerostin / SOST knockdown on Wnt / β-catenin were confirmed by qRT-PCR and WB analysis after glucose treatment.

[0200]

[0201] As a result, after silencing sclerostin, the gene and protein expression levels of β-catenin, Wnt3a, and Wnt5a were found to decrease in HRECs (Fig. 3a and 3b). First, proteins related to Wnt signaling were found to be expressed at higher levels after HG treatment, demonstrating that Wnt signaling is increased by hyperglycemia. Furthermore, when the Wnt antagonist SOST was inhibited, the Wnt signaling increased by hyperglycemia was confirmed to be statistically significantly reduced.

[0202] That is, according to these results, silencing SOST, a Wnt antagonist, has minimal effects on Wnt, and thus, it has been proven that SOST inhibition does not induce retinal vascular dysfunction, etc., in relation to Wnt signaling increased by hyperglycemia.

[0203]

[0204] Example 4. Confirmation of the effect of reducing VEGF-induced inflammatory activity by SOST inhibition.

[0205] We investigated whether suppressing sclerostin expression by SOST inhibition could reduce the expression levels of inflammatory cytokines in HRECs following VEGF treatment. The gene and protein expression of inflammatory cytokines was analyzed using qRT-PCR and WB.

[0206]

[0207] As a result (Figs. 4a and 4b), all inflammatory cytokines were significantly upregulated after VEGF treatment compared to the control group at both the gene and protein levels. In addition, all cytokine levels were confirmed to be downregulated in a dose-dependent manner by siRNA after sclerostin / SOST knockdown. In particular, as shown in qRT-PCR and WB analyses, the expression of proinflammatory cytokines was significantly reduced in samples treated with 30 nM and 50 nM SOST siRNA (si30+VEGF and si50+VEGF), and this anti-inflammatory effect was confirmed to be statistically significant.

[0208]

[0209] Example 5. Confirmation of the effect of SOST overexpression in HREC.

[0210] To determine the effects of sclerostin overexpression on HRECs and proinflammatory cytokines, qRT-PCR, WB, and immunostaining analyses were performed. SOST overexpression in HRECs was evaluated using CRISPR-SOST knock-in at various concentrations (10 ng, 30 ng, 50 ng, and 100 ng).

[0211]

[0212] As a result, SOST gene and protein expression increased depending on the dose (Fig. 5a). Immunostaining analysis results also showed that SOST expression was higher when treated with 100 ng CRISPR-SOST (SOST100) compared to the control group (Fig. 5b). Furthermore, all inflammatory proteins, including VEGFA and NF-κB, were significantly enhanced after treatment with 100 ng of SOST (Fig. 5c).

[0213] These results suggest that the presence of sclerostin in HRECs can induce inflammatory cytokine expression via the NF-κB pathway.

[0214]

[0215] Example 6. Confirmation of the effect of reducing oxidative stress by SOST inhibition.

[0216] When oxidative stress was induced by treatment with high concentrations of glucose and VEGF, the green fluorescence expression of the oxidative stress marker increased, and when sclerostin expression was suppressed using siRNA, the expression of the oxidative stress marker decreased (Fig. 6a).

[0217] Additionally, when sclerostin was overexpressed using CRISPR, the fluorescence expression of the oxidative stress marker increased (Fig. 6b).

[0218]

[0219] Example 7. Confirmation of the effect of SOST inhibition on reducing the expression of vascular endothelial growth factor and vascular adhesion markers.

[0220] The effects of SOST inhibition on the expression of vascular endothelial growth factor and vascular adhesion markers were investigated. Specifically, gene expression of vascular adhesion markers VCAM-1 and ICAM-1, and vascular endothelial growth factor A (VEGFA) was analyzed after HG treatment or VEGF treatment, respectively, using qRT-PCR.

[0221]

[0222] As a result, VEGFA, VCAM-1, and ICAM-1 were found to be highly expressed after HG treatment or VEGF treatment, and were significantly downregulated after SOST knockdown (Fig. 7a). Reduced protein expression was detected after SOST knockdown, and the WB data on protein expression were largely consistent with the gene expression data (Fig. 7b). In particular, when 30 and 50 nM siRNA were transfected, the expression levels of ICAM-1 and VCAM-1 were found to be almost non-existent.

[0223]

[0224] Examples 2, 4, and 6 demonstrated that HG and VEGF stimulated the expression of inflammatory cytokines, and that SOST silencing significantly reduced the inflammatory response. Furthermore, in siRNA-transfected samples, sclerostin knockdown significantly reduced the expression of VEGFA, ICAM-1, and VCAM-1, as well as the protein transcription factor nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS). In contrast, greater expression was observed only in samples treated with HG and VEG.

[0225]

[0226] Example 8. Confirmation of the effect of reducing angiogenesis by SOST inhibition.

[0227] To evaluate the silencing effect of SOST siRNA on angiogenesis and vascular remodeling, pretreated HRECs were seeded on Matrigel and tube formation assays were performed.

[0228]

[0229] As a result (Figs. 8a and 8b), hyperglycemia induced intensive tube formation in siCON+HG and siCON+VEGF samples. However, SOST knockdown prevented tube formation even under hyperglycemic conditions. Specifically, angiogenesis was significantly inhibited by SOST siRNA treatment in si30+HG and si30+VEGF samples, and this effect was confirmed to be statistically significant.

[0230] In addition, the SOST overexpression sample (SOST100) treated with 100 ng CRISPR-SOST also showed a higher tube formation ability of sclerostin compared to the control group (Fig. 8c and Fig. 8d), reproving that sclerostin expressed by SOST induces angiogenesis and that tube formation is significantly inhibited when it is inhibited.

[0231]

[0232] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0233] According to a pharmaceutical composition for preventing or treating diabetic eye disease comprising a sclerostin inhibitor as an active ingredient, when sclerostin is inhibited, the expression level of inflammatory cytokines is significantly reduced even under conditions of hyperglycemia and VEGF treatment, so that not only is the inflammatory activity excellent, but also the angiogenic effect due to the reduced expression of vascular endothelial growth factor and vascular adhesion markers is remarkable, and it was confirmed that the inflammatory response and microvascular complications, which are the most significant characteristics of diabetic eye disease, can be suppressed. In addition, since oxidative stress caused by diabetes is reduced and cell death is not induced, it can be usefully utilized as a preparation for preventing or treating diabetic eye disease, and thus its industrial applicability is recognized.

Claims

1. A pharmaceutical composition for preventing or treating diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

2. A pharmaceutical composition according to claim 1, wherein the inhibitor is at least one of an expression inhibitor and an activity inhibitor.

3. In paragraph 1 or 2, A pharmaceutical composition, wherein the expression inhibitor or activity inhibitor is at least one selected from the group consisting of siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (micro RNA), antisense oligonucleotides (ASO), CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats), natural products, proteins, peptidomimetics, antibodies, exosomes, and compounds.

4. In any one of paragraphs 1 to 3, A pharmaceutical composition, wherein the diabetic eye disease is at least one selected from the group consisting of diabetic retinopathy, diabetic macular edema, glaucoma, and cataract.

5. In any one of paragraphs 1 to 4, A pharmaceutical composition, wherein the composition is for use in any one formulation selected from the group consisting of a suspension, a gel, an ointment, an injectable solution, a spray, and an ointment.

6. In any one of paragraphs 1 to 5, A pharmaceutical composition wherein the composition suppresses inflammatory response and microvascular complications.

7. In any one of paragraphs 1 to 6, A pharmaceutical composition characterized in that the composition comprises at least one selected from the group consisting of: Inhibition of inflammatory cytokines in retinal cells; Inhibition of angiogenesis; Inhibition of vascular endothelial growth factor and vascular adhesion markers; and Reduce oxidative stress.

8. A food composition for preventing or improving diabetic eye disease, comprising a sclerostin inhibitor as an active ingredient.

9. In paragraph 8, A composition wherein the above food is a health functional food.

10. A kit for preventing or treating diabetic eye disease, comprising a composition containing a sclerostin inhibitor as an active ingredient and an instruction manual.

11. A method for preventing or treating diabetic eye disease, comprising administering a pharmaceutically effective amount of a composition containing a sclerostin inhibitor as an active ingredient to a subject in need thereof.

12. Use of a composition containing a sclerostin inhibitor as an active ingredient for preventing or treating diabetic eye disease.

13. Use for manufacturing a preparation for preventing or treating diabetic eye disease comprising a composition containing a sclerostin inhibitor as an active ingredient.

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