Composition for treating diabetic retinopathy containing peptide

Abstract

The present invention relates to a composition for preventing or treating diabetic retinopathy, which contains a peptide. The peptide has excellent effects of suppressing the development of neovascularization in the retina, the loss of vascular pericytes, and vascular dilation, and can therefore be usefully used for the prevention or treatment of retinopathy by suppressing increased vascular permeability, inflammation, and the development of neovascularization.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating diabetic retinopathy, which comprises a peptide consisting of seven amino acids. Specifically, the peptide of the present invention is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, and has excellent therapeutic effects for retinopathy by suppressing the development of neovascularization in the retina, the loss of vascular pericytes, and vasodilation. [Background technology]

[0002] Diabetic retinopathy is one of the three major microvascular complications of diabetes. It is a disease in which capillaries are damaged by persistent hyperglycemia and the metabolic abnormalities that accompany it, resulting in damage, inflammation, and neovascularization throughout the retina, leading to visual impairment.

[0003] Diabetic retinopathy, which develops in approximately 60-70% of patients with a diabetes duration of around 15 years, is divided into nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) depending on the symptoms and progression.

[0004] Retinal blood vessels primarily have a selectively permeable blood-retinal barrier (BRB) structure, which protects retinal neurons from external substances. However, in the early stages of diabetic retinopathy, hyperglycemia damages tight junctions and perivascular glial cells (glia), disrupting the blood-retinal barrier and allowing components in the blood to leak into the retina, resulting in retinal microvascular abnormalities such as hard exudates, retinal edema, and vascular abnormalities. Furthermore, microvascular damage and blockage can cause plasma leaking from blood vessels to cause retinal edema in the macula, potentially leading to vision loss.

[0005] Proliferative diabetic retinopathy (PDR), the later stage of diabetic retinopathy, is a condition characterized by the development of new blood vessels in the retina, potentially leading to severe visual impairment and manifesting as retinal neovascularization and fibrous tissue proliferation. Neovascularization originates from the inner retinal vessels and grows toward the posterior surface of the vitreous. Hemorrhage from these new vessels can cause severe vision loss and even blindness, necessitating prompt treatment, such as intravitreal administration of anti-vascular endothelial growth factor (anti-VEGF).

[0006] Treatments for diabetic retinopathy include risk factor adjustment, laser treatment, intraocular injection treatment, and surgical treatment (vitrectomy). Recently, injections of anti-vascular endothelial growth factor (anti-VEGF) drugs such as aflibercept (Eylea), ranibizumab (Lucentis), and bevacizumab (Avastin) have been used, but these have drawbacks such as side effects, high costs, repeated administration, and the risk of sudden worsening if injections are discontinued.

[0007] Therefore, the present inventors continued their research into the treatment of ocular diseases, focusing on peptides that have advantages such as ease of mass production and excellent bioavailability, and identified peptides that have excellent effects in the treatment of diabetic retinopathy, thereby completing the present invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 10-2020-0134175 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a pharmaceutical composition for preventing or treating retinopathy, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having 90% or more homology to said sequence.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1. [Means for solving the problem]

[0011] The present invention provides a pharmaceutical composition for preventing or treating retinopathy, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having 80%, 90%, 95%, 97%, 98%, or 99% or more homology to said sequence.

[0012] The peptides can inhibit the development of new blood vessels, the loss of pericytes, or vascular dilation, and may inhibit the expression of vascular endothelial growth factor, the phosphorylation of Akt (Protein kinase B), the expression of COX-2, or the phosphorylation of JNK (c-Jun N-terminal kinase), and may restore or improve the tight junction protein ZO-1.

[0013] The retinopathy may be diabetic retinopathy, retinopathy of prematurity, diabetic retinal edema, or retinal vein occlusion, and preferably diabetic retinopathy.

[0014] The diabetic retinopathy may be non-proliferative diabetic retinopathy or proliferative diabetic retinopathy.

[0015] The present invention provides a composition for use in preventing or treating retinopathy, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having 80%, 90%, 95%, 97%, 98%, or 99% or more homology to said sequence.

[0016] The present invention provides use of a composition for preventing or treating retinopathy, the composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having 80%, 90%, 95%, 97%, 98%, or 99% or more homology to said sequence.

[0017] The present invention provides a method for treating retinopathy by administering to a subject with retinopathy a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having 80%, 90%, 95%, 97%, 98%, or 99% or more homology thereto. [Effects of the Invention]

[0018] The peptide of the present invention has an excellent effect of suppressing the development of neovascularization in the retina, the loss of vascular pericytes, and vascular dilation, and can therefore be useful for the prevention or treatment of retinopathy by suppressing increased vascular permeability, inflammation, and the development of neovascularization. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows fluorescent photographic images of retinal blood vessels in a mouse model of oxygen-induced retinopathy administered with the peptide of the present invention, and calculation results of the number and area of ​​new blood vessels. [Figure 2] FIG. 1 shows fluorescent photographic images of retinal blood vessels in a mouse model of oxygen-induced retinopathy administered with the peptide of the present invention, and calculation results of the number and area of ​​new blood vessels. [Figure 3] FIG. 1 shows the results of electroretinography in a mouse model of streptozotocin-induced diabetic retinopathy administered with the peptide of the present invention. [Figure 4] FIG. 1 shows the results of electroretinography in a db / db mouse model administered with the peptide of the present invention. [Figure 5] FIG. 1 shows PAS staining images of retinal blood vessels in a db / db mouse model administered with the peptide of the present invention, and the results of quantification of the numbers of vascular endothelial cells and vascular pericytes. [Figure 6]FIG. 1 shows PAS-stained images of retinal blood vessels in a db / db mouse model administered with the peptide of the present invention, and the results of quantification of the numbers of vascular endothelial cells and vascular pericytes. [Figure 7] FIG. 1 shows the results of confirming protein expression in retinal tissue of a mouse model of streptozotocin-induced diabetic retinopathy after administration of the peptide of the present invention. [Figure 8] FIG. 1 shows the results of confirming the expression of vascular endothelial growth factor (VEGF) in the retinal tissue of a db / db mouse model after administration of the peptide of the present invention. [Figure 9] FIG. 1 shows the results of examining the degree of Akt phosphorylation in the retinal tissue of a db / db mouse model after administration of the peptide of the present invention. [Figure 10] FIG. 1 shows the results of confirming COX-2 expression in the retinal tissue of a db / db mouse model after administration of the peptide of the present invention. [Figure 11] FIG. 1 shows the results of examining the degree of JNK phosphorylation in the retinal tissue of a db / db mouse model after administration of the peptide of the present invention. [Figure 12] FIG. 1 shows fluorescent photographs (*: optic nerve) of retinal blood vessels in a zebrafish diabetic retinopathy model administered with the peptide of the present invention, and the results of calculating the blood vessel diameter. [Figure 13] FIG. 1 shows fluorescent photographs (*: optic nerve) of retinal blood vessels in a zebrafish diabetic retinopathy model administered with the peptide of the present invention, and the results of calculating the blood vessel diameter. [Figure 14] FIG. 1 shows the results of confirming gene expression in retinal tissue of a zebrafish diabetic retinopathy model after administration of the peptide of the present invention. [Figure 15] FIG. 1 shows the results of confirming gene expression in retinal tissue of a zebrafish diabetic retinopathy model after administration of the peptide of the present invention. [Figure 16] FIG. 1 shows fluorescent photographs of retinal tissues in a zebrafish diabetic retinopathy model administered with the peptide of the present invention, and the results of confirming cell and protein expression. [Figure 17]FIG. 1 shows fluorescent photographs of retinal tissues in a zebrafish diabetic retinopathy model administered with the peptide of the present invention, and the results of confirming cell and protein expression. DETAILED DESCRIPTION OF THE INVENTION

[0020] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will now be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily understand and practice the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples set forth herein.

[0021] Throughout this specification, when a part is said to "comprise" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0022] The present invention relates to a composition for preventing, ameliorating or treating retinopathy, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0023] The composition may be a pharmaceutical composition for preventing or treating diabetic retinopathy.

[0024] The amino acid sequence of SEQ ID NO: 1 may include a sequence consisting of "Hyp-Gly-Gln-Glu-Aib-Leu-Ala" or a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence of SEQ ID NO: 1.

[0025] In the present invention, Hyp is trans-4-hydroxy-L-proline, Gly is glycine, Gln is glutamine, Glu is glutamate or glutamic acid, Aib is 2-aminoisobutyric acid, Leu is leucine, Ala is alanine.

[0026] In the present invention, "diabetic retinopathy" refers to an ocular complication in which diabetes causes peripheral circulatory disorders, which in turn disrupt retinal microcirculation and result in decreased vision. In the early stages, the condition may not cause any symptoms or may only cause minor visual impairment, but may ultimately lead to blindness. Diabetic retinopathy can occur in any patient with type 1 or type 2 diabetes.

[0027] In the present invention, the diabetic retinopathy may be, but is not limited to, non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR).

[0028] The term "angiogenesis" as used herein refers to the process of new blood vessel formation, i.e., the generation of new blood vessels within cells, tissues, or organs, and "neovascularization" refers to blood vessels newly generated by the angiogenic process. In the present invention, "angiogenesis" and "neovascularization" can be used interchangeably.

[0029] The term "prevention" as used herein means any action of suppressing or delaying gastrointestinal disorders by administering the composition according to the present invention, and the term "treatment" means any action of improving or favorably altering the symptoms of individuals suspected of or suffering from gastrointestinal disorders by administering the composition. The term "amelioration" as used herein means any action of at least reducing the parameters related to the condition being treated, e.g., the severity of symptoms, by administering the composition containing the extract of the present invention.

[0030] The present invention will be described in more detail below with reference to examples. However, these examples are merely for the purpose of illustrating the present invention and are not intended to limit the present invention. [Example]

[0031] [Manufacturing example] Preparation of peptides consisting of seven amino acids (7mer) The peptides of the present invention are synthesized based on the known solid phase peptide synthesis (SPPS) method, and the production process includes the following steps: Step 1: Resin swelling and loading Step 2: Solid Phase Peptide Synthesis (SPPS) Step 3: Deprotected peptide synthesis (Global cleavage) Step 4: Primary purification and concentration / Secondary purification and concentration Step 5: Freeze-drying

[0032] The solid phase peptide synthesis (SPPS) method includes the steps of loading a first amino acid onto a resin, deprotecting the N-terminus of the amino acid with Fmoc, and then coupling amino acids according to the amino acid sequence, and synthesizing the protected peptide in a solid phase reactor. The amino acid coupling step also includes the steps of loading the first amino acid onto a resin, removing the Fmoc from the N-terminus of the amino acid, removing the solvent and washing the resin after the reaction is complete, coupling the next amino acid according to the sequence, removing the solvent and washing the resin after the reaction is complete, and then repeating the above process until the final amino acid sequence is produced.

[0033] The alpha amine group of each amino acid was protected with a base-labile Fmoc group, and the side chain functional groups were protected with acid-labile groups. All amino acids except Gly and Aib were in the L-configuration. Among them, several amino acids, such as Hyp(tBu), Glu(tBu), and Gln(Trt), had unique protecting groups. Other amino acids, such as Ala, Leu, Aib, and Gly, did not have side chain protecting groups. After amino acid coupling was completed according to the sequence, the resin and protecting groups were removed from the peptide to obtain the crude peptide. The peptide was then purified, concentrated, and lyophilized to obtain the peptide compound with the amino acid sequence of SEQ ID NO: 1.

[0034] [Example 1] Evaluation of the inhibitory effect on neovascularization in a mouse model of oxygen-induced retinopathy In order to evaluate the angiogenesis-inhibiting effect of the peptide of the present invention in a mouse model of oxygen-induced retinopathy (OIR), the following experiment was carried out.

[0035] The oxygen-induced retinopathy (OIR) mouse model is a model of ischemic retinopathies such as proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and retinal vein occlusion (RVO), and shows active neovascularization in the retina.

[0036] After stabilizing for approximately 3–4 days, newborn mice were housed under 75% hyperoxic conditions for 5 days from postnatal day 7 to 11. They were then housed under normoxic conditions until postnatal day 17. During this period, drugs were administered. The peptide of the present invention (002-175) was administered by eye drops (ED) for 5 days from postnatal day 12 to 16. Aflibercept was administered intraocularly (IVT) once on postnatal day 12 as a positive control. The mouse eyes were then enucleated, fixed in 10% formalin for 1 hour, and flat-mounted. The retinas were incised into four equal sections centered on the optic nerve and treated overnight with Isolectin B4 (1 mM CaCl2, 1:200). The specimen was then spread onto a glass slide, a drop of mounting solution was added, and the specimen was covered with a cover glass and photographed under a fluorescence microscope. Fluorescent images of the intraretinal blood vessels were obtained, and the number and area of ​​neovascularization and the vascular area were calculated using the ImageJ program.

[0037] As a result, as shown in Figures 1 and 2, neovascular areas were observed in the mouse model of oxygen-induced retinopathy, whereas the group administered with the peptide of the present invention showed a reduction in neovascular areas to the same extent as the group administered with aflibercept.

[0038] Therefore, it is clear that the peptide of the present invention has an inhibitory effect on neovascularization.

[0039] [Example 2] Evaluation of the inhibitory effect on neovascularization in a mouse model of hyperglycemia-induced diabetic retinopathy In order to evaluate the angiogenesis-inhibiting effect of the peptide of the present invention in a mouse model of high glucose-induced diabetic retinopathy, the following experiment was carried out.

[0040] 2-1. Streptozotocin-induced diabetic retinopathy mouse model The streptozotocin-induced mouse model is a type 1 diabetes model in which the antibiotic streptozotocin (STZ) is administered intraperitoneally to mice, damaging the beta cells that produce insulin in the pancreas and inducing diabetes.It is one of the models used in research on diabetic retinopathy, and is a model that can confirm the early stages of diabetic retinopathy from the time diabetes occurs.

[0041] Eight-week-old C57BL / 6 mice were fasted for 3–4 h before administration and then intraperitoneally administered streptozotocin (STZ) at a dose of 50 mg / kg in sodium citrate buffer or buffer over a 5-day period. The drugs were prepared immediately before administration and used promptly. Blood glucose was measured one week after the final STZ intraperitoneal administration, and only mice with blood glucose levels above 250 mg / dL were identified as diabetic and used in the experiment. The drugs were administered by eye drop (ED) twice daily and intraocular (IVT) at 10 μg doses once a month for approximately 17 weeks, starting the day after model creation. To assess retinal function, mice were dark-adapted for more than 12 h before electroretinogram (ERG) measurements. Afterward, mice were anesthetized and pupils were dilated, and ERGs were measured using electrodes attached to the skin, tail, and cornea, respectively. The retina was stimulated with a single white light to obtain a response value, and the amplitude from the trough of the a wave to the peak of the b wave was measured and evaluated as an index of retinal function.

[0042] As a result, as shown in Figure 3, the amplitude of the a and b waves was reduced in the STZ-induced diabetic retinopathy mouse group, whereas the amplitude of the a and b waves was increased to the same extent as in the aflibercept-administered group in the group administered with the peptide of the present invention.

[0043] Therefore, it is clear that the peptide of the present invention has an inhibitory effect on neovascularization.

[0044] 2-2.db / db mouse model The db / db mouse model is a mouse with a mutation in the leptin receptor, an appetite-suppressing hormone. It exhibits phenotypes such as severe obesity, hyperphagia, insulin resistance, and diabetes. It is widely used as a model for type 2 diabetes and can be used to identify the early stages of diabetic retinopathy.

[0045] Twelve-week-old db / db mice were administered drugs by eye drop (ED) twice daily (BID) or three times daily (TID) for approximately 12 weeks. To assess retinal function, mice were dark-adapted for at least 12 hours before electroretinogram (ERG) measurements. Afterward, the mice were anesthetized and pupils were dilated, and ERGs were recorded using electrodes attached to the skin, tail, and cornea. The retina was stimulated with a single white light, and the amplitude from the trough of the a wave to the peak of the b wave was measured and evaluated as an index of retinal function.

[0046] As a result, as shown in Figure 4, the db / db mouse group showed a decrease in the amplitude of the a and b waves, whereas the group administered with the peptide of the present invention showed an increase in the amplitude of the a and b waves. In particular, it was confirmed that the group administered three times a day (TID) showed a superior effect compared to the group administered twice a day (BID).

[0047] Therefore, it is clear that the peptide of the present invention has an inhibitory effect on neovascularization.

[0048] [Example 3] Evaluation of the inhibitory effect of pericyte loss in a mouse model of hyperglycemia-induced diabetic retinopathy The initial step in non-proliferative diabetic retinopathy is the loss of vascular pericytes surrounding capillaries, which causes blood flow into the tissue and swelling of the macula. Macular retinal edema is a typical symptom of non-proliferative diabetic retinopathy, characterized by a temporary decrease in vision due to blurred focus and darkening of the visual field. Therefore, the following experiment was conducted to evaluate the inhibitory effect of the peptide of the present invention on vascular pericyte loss.

[0049] Twelve-week-old db / db mice were administered drugs by eye drop (ED) twice daily (BID) or three times daily (TID) for approximately 12 weeks. To evaluate the state of intraretinal vasculature, the mouse eyes were enucleated and fixed in 10% formalin for one week. The fixed ocular tissue was then treated with elastase to isolate retinal vessels, which were then stained with PAS. The ratios of endothelial cells and pericytes in the vascular wall, as well as the number of acellular vessels, were quantified to evaluate intraretinal vascular permeability.

[0050] As a result, as shown in Figures 5 and 6, the db / db mouse group showed a decrease in vascular pericytes, whereas the group administered with the peptide of the present invention suppressed the decrease in vascular pericytes. In particular, it was confirmed that the group administered three times a day (TID) showed a superior effect compared to the group administered twice a day (BID).

[0051] Therefore, it is clear that the peptide of the present invention has an inhibitory effect on the loss of vascular pericytes.

[0052] [Example 4] Evaluation of intraretinal protein expression levels in a mouse model of hyperglycemia-induced diabetic retinopathy In order to evaluate the expression level of proteins in retinal tissues as a result of administration of the peptide of the present invention in a mouse model of high glucose-induced diabetic retinopathy, the following experiment was carried out.

[0053] 4-1. Streptozotocin-induced diabetic retinopathy mouse model Eight-week-old C57BL / 6 mice were fasted for 3–4 hours before administration and then intraperitoneally administered streptozotocin (STZ) at a dose of 50 mg / kg in sodium citrate buffer over a 5-day period. The drug was prepared immediately before administration and used promptly. Blood glucose was measured one week after the final STZ intraperitoneal administration, and only mice with blood glucose levels above 250 mg / dL were considered diabetic and used in the experiment. The drug was administered by eye drop (ED) twice daily for approximately 17 weeks, starting the day after model creation, and intraocular administration (IVT) at 10 μg doses once a month. To assess the expression levels of intraretinal proteins, retinal tissue was isolated from the excised mouse eyes, treated with RIPA solution to extract intraretinal proteins, and Western blot analysis was performed to evaluate the expression of factors related to retinal angiogenesis and vascular permeability.

[0054] As a result, as shown in Figure 7, the group of mice with STZ-induced diabetic retinopathy showed increased expression of vascular endothelial growth factor (VEGF), which is associated with the development of neovascularization, whereas the group administered the peptide of the present invention showed decreased expression of VEGF.

[0055] Furthermore, it was found that the group of mice with STZ-induced diabetic retinopathy had an increased level of phosphorylation of Akt, a signaling factor related to vascular endothelial growth factor, whereas the group administered the peptide of the present invention had a decreased level of phosphorylation of Akt.

[0056] Therefore, it is clear that the peptide of the present invention suppresses the expression of proteins related to neovascularization and vascular cell permeability in the retina.

[0057] 4-2.db / db mouse model Twelve-week-old db / db mice were administered drugs twice daily (BID) or three times daily (TID) via eye drop (ED) for approximately 12 weeks. To assess the expression levels of intraretinal proteins, retinal tissue was isolated from the excised mouse eyes, treated with RIPA solution, and Western blot analysis was performed to assess the expression of factors related to intraretinal angiogenesis and vascular permeability.

[0058] As a result, as shown in Figure 8, the db / db mouse group showed increased expression of vascular endothelial growth factor (VEGF), whereas the group to which the peptide of the present invention was administered three times a day (TID) showed decreased expression of vascular endothelial growth factor.

[0059] Furthermore, as shown in Figure 9, the level of Akt phosphorylation increased in the db / db mouse group, whereas the level of Akt phosphorylation decreased in the group administered the peptide of the present invention three times a day (TID).

[0060] Furthermore, as shown in Figure 10, the db / db mouse group showed increased expression of COX-2, an inflammation-related factor, whereas the group administered the peptide of the present invention three times a day (TID) showed decreased expression of COX-2.

[0061] Furthermore, as shown in Figure 11, the level of phosphorylation of JNK, a factor related to cell death, increased in the db / db mouse group, whereas the level of JNK phosphorylation decreased in the group administered the peptide of the present invention.

[0062] Therefore, it is clear that the peptide of the present invention suppresses the expression of proteins related to neovascularization and vascular cell permeability in the retina, and suppresses the expression of factors related to inflammation.

[0063] [Example 5] Evaluation of the inhibitory effect on retinal vasodilation in a zebrafish diabetic retinopathy model Embryos expressing fluorescent proteins were selected from embryos obtained by mating adult zebrafish Tg[flk:EGFP]. Six days after insemination, the embryos were treated with glucose and the peptide of the present invention (002-175) for four days, and then euthanized by adding 4 mg / mL of Tricane. After fixation with 4% paraformaldehyde for three days, the eyeballs were isolated using 3% trypsin, and changes in the isolated intraocular blood vessels were observed under a fluorescence microscope.

[0064] As a result, as shown in Figures 12 and 13, the diameter of zebrafish retinal blood vessels in the group treated with high blood glucose (130 mM glucose) increased by more than 1.5 times compared to the normal group, whereas the diameter of retinal blood vessels in the group treated with the peptide of the present invention was observed to decrease. In particular, the diameter of retinal blood vessels in the 100 and 200 μg / mL treatment groups decreased to the same level as that in the normal group.

[0065] Therefore, it is clear that the peptide of the present invention has the effect of suppressing retinal vascular dilation.

[0066] [Example 6] Analysis of gene expression in a zebrafish diabetic retinopathy model Embryos expressing fluorescent proteins were selected from embryos obtained by mating adult zebrafish Tg[flk:EGFP]. Six days after insemination, the embryos were treated with glucose and the peptide of the present invention (002-175) for 4 days, followed by euthanasia with 4 mg / mL Tricane. Eyeballs were enucleated for RNA extraction and microarray analysis. The enucleated eyeballs were homogenized with Trizol and mRNA was extracted. The extracted mRNA was quantified using a NanoDrop spectrophotometer, and cDNA was synthesized using RNA with a purity of 1.8 or higher. The synthesized cDNA was subjected to gene polymerization using SYBR Green Master Mix, primers, and gene cleavage. The concentration of each gene was normalized for GAPDH and then determined.

[0067] As a result, as shown in FIG. 14, the microarray analysis revealed that the gene expression of IL-1β, MMP9, and NF-kB was reduced in the group treated with the peptide of the present invention.

[0068] Furthermore, as shown in Figure 15, the results of real-time PCR showed that the group treated with the peptide of the present invention had reduced gene expression of ikkα, ikkβ, ikkγ, NF-kB, IL-1β, and MMP9.

[0069] IL-1β increases vascular permeability by activating inflammatory factors associated with angiogenesis and promotes apoptosis of retinal capillary cells in a hyperglycemic environment through activation of NF-kB. NF-kB, present in the retina and microvessels, plays a central role in intracellular signaling, increasing the expression of various cytokines, such as IL-1β, IL-6, and IL-8, in the vitreous humor and serum, thereby inducing inflammatory apoptosis. Matrix metalloproteinases (MMPs) also play an important role in the progression of diabetic retinopathy. MMP9, in particular, is involved in the angiogenesis of retinal capillary cells. Activation of NF-kB, TNF-α, and interleukin (IL) increases MMP9, leading to the development of diabetic retinopathy.

[0070] Therefore, it is clear that the peptide of the present invention has the effect of suppressing intraretinal inflammatory factors and the mechanism of development of diabetic retinopathy.

[0071] [Example 7] Analysis of histological changes in a zebrafish diabetic retinopathy model Zebrafish fry 6 days post-fertilization (6 dpf) were fixed in 4% paraformaldehyde for 3–4 hours, then treated with 30% sucrose and incubated overnight at 4°C. OCT blocks were prepared and sectioned, followed by TUNEL staining and immunofluorescence (IF) staining.

[0072] As a result, as shown in Figures 16 and 17, the results of the TUNEL assay in retinal tissue showed that the hyperglycemia-treated group had an increased number of TUNEL-positive cells in the inner nuclear layer (INL) of the retina, whereas the group treated with the peptide of the present invention (002-175) had a decreased number of TUNEL-positive cells.

[0073] Furthermore, analysis of the expression of VEGF, GFAP, and ZO-1 in the zebrafish eye by immunofluorescence (IF) staining revealed that the hyperglycemic group showed increased expression of inflammatory factors, VEGF and GFAP, in the outer nuclear layer (ONL) of the retina, whereas the peptide (002-175)-treated group showed decreased expression of VEGF and GFAP. Furthermore, the expression of the tight junction protein ZO-1 was decreased in the hyperglycemic group, whereas it increased in the peptide (002-175)-treated group, indicating a tendency toward recovery.

[0074] Therefore, it can be seen that the peptides of the present invention have the effect of suppressing intraretinal inflammatory factors and the mechanism of diabetic retinopathy. In particular, in the hyperglycemia-induced zebrafish diabetic retinopathy model, it was confirmed that the tight junction marker ZO-1 was reduced and the blood-retinal barrier (BRB) was damaged, and angioedema was observed due to the accompanying increased vascular permeability. This is the same mechanism as clinical diabetic retinal edema, and it can be seen that the peptides of the present invention reduce vascular permeability by suppressing intraretinal neovascularization and inflammatory factors, thereby effectively suppressing diabetic retinopathy and diabetic retinal edema.

Claims

1. A pharmaceutical composition for preventing or treating retinopathy, comprising a peptide having a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO:

1.

2. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, comprising a peptide consisting of a sequence having 99% or more homology with the amino acid sequence of SEQ ID NO:

1.

3. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, comprising a peptide consisting of the amino acid sequence of SEQ ID NO:

1.

4. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the retinopathy is at least one selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, diabetic retinal edema, and retinal vein occlusion.

5. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the retinopathy is diabetic retinopathy.

6. 6. The pharmaceutical composition for preventing or treating retinopathy according to claim 5, wherein the diabetic retinopathy is non-proliferative diabetic retinopathy or proliferative diabetic retinopathy.

7. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the peptide inhibits the development of neovascularization.

8. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the peptide inhibits loss of vascular pericytes.

9. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the peptide inhibits vasodilation.

10. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the peptide inhibits at least one of the expression of vascular endothelial growth factor, phosphorylation of Akt (protein kinase B), expression of COX-2, and phosphorylation of JNK (c-Jun N-terminal kinase).

11. 2. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, wherein the peptide restores ZO-1, a tight junction protein.

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