Compositions for treating diabetic retinopathy comprising peptides

Abstract

The present invention relates to a composition for preventing or treating diabetic retinopathy, comprising a peptide having an excellent effect of inhibiting the generation of neovascularization in the retina, the loss of perivascular cells, and the expansion of blood vessels. The present invention can be effectively used in the prevention or treatment of retinopathy by inhibiting increase in vascular permeability, inflammation, and the occurrence of neovascularization.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating diabetic retinopathy, comprising a peptide consisting of seven amino acids. Specifically, the peptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 and exhibits excellent therapeutic effects on retinopathy by inhibiting retinal neovascularization, perivascular cell loss, and vascular dilation. Background Art

[0002] Diabetic retinopathy is one of the three major microvascular complications of diabetes. It is a disease in which persistent high blood sugar and the resulting metabolic abnormalities damage capillaries, leading to overall retinal damage, inflammation, and angiogenesis, resulting in visual impairment.

[0003] Diabetic retinopathy, which occurs in approximately 60% to 70% of patients with diabetes mellitus lasting about 15 years, is divided into nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) based on symptoms and progression.

[0004] Retinal blood vessels primarily have a selectively permeable blood-retinal barrier (BRB) structure that protects retinal nerve cells from external substances. In the early stages of diabetic retinopathy, high blood sugar levels damage tight junctions and perivascular astrocytes (glia), damaging the BRB and allowing blood components to leak into the retina, resulting in retinal microvascular abnormalities such as hard exudates, retinal edema, and vascular abnormalities. Furthermore, plasma leaking from blood vessels due to microvascular obstruction causes retinal edema in the macula, leading to decreased vision.

[0005] Proliferative diabetic retinopathy (PDR), a late stage of diabetic retinopathy, is a condition in which new blood vessels form in the retina, causing severe visual impairment. Retinal neovascularization and fibrous tissue proliferation are observed. Neovascularization begins within the retina and propagates to the back of the vitreous. If bleeding from these new vessels occurs, severe vision loss and even blindness can occur, necessitating immediate treatment, such as intravitreal administration of anti-vascular endothelial growth factor (anti-VEGF).

[0006] Treatments for diabetic retinopathy include risk factor adjustment, laser therapy, intraocular injection therapy, and surgical treatment (vitrectomy). Recently, injections of anti-vascular endothelial growth factor (anti-VEGF) drugs such as aflibercept (Eylea), ranibizumab (Luzid), and bevacizumab (Avastin) have been used. However, these methods have disadvantages such as side effects, high costs, repeated administration, and the risk of rapid deterioration if the injection is interrupted.

[0007] The present inventors have continued their research on eye disease treatment targeting peptides with advantages such as ease of mass production and excellent bioavailability, and have identified a peptide with excellent effects in treating diabetic retinopathy, thereby completing the present invention.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Korean Patent Publication No. 10-2020-0134175 Summary of the Invention

[0011] Technical issues

[0012] The object of the present invention is to provide 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 90% or greater homology to the sequence.

[0013] Furthermore, the present invention aims to provide a pharmaceutical composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 for preventing or treating nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR).

[0014] Technical Solution

[0015] 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 with the sequence.

[0016] The peptide can inhibit the formation of new blood vessels, the loss of pericytes or vasodilation, the expression of vascular endothelial growth factor, the phosphorylation of protein kinase B (Akt), the expression of cyclooxygenase-2 (COX-2) or the phosphorylation of c-Jun N-terminal kinase (JNK), and can also restore or improve ZO-1, a tight junction protein.

[0017] The retinopathy may be diabetic retinopathy, retinopathy of prematurity, diabetic macular edema or retinal vein occlusion, preferably, diabetic retinopathy.

[0018] The diabetic retinopathy may be non-proliferative diabetic retinopathy or proliferative diabetic retinopathy.

[0019] The present invention provides a 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 with the sequence.

[0020] The present invention provides a composition for preventing or treating retinopathy, wherein the composition comprises 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 the sequence.

[0021] The present invention provides a method for treating retinopathy by administering to a subject suffering from 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 greater homology thereto.

[0022] Effects of the Invention

[0023] The peptide of the present invention has an excellent effect of inhibiting the formation of neovascularization, loss of perivascular cells, and vascular dilation in the retina, and can be effectively used in the prevention or treatment of retinopathy by inhibiting increased vascular permeability, inflammation, and the occurrence of neovascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 and Figure 2 Shown are fluorescent images of retinal blood vessels in an oxygen-induced retinopathy mouse model following administration of the peptide of the present invention, and calculation results of the number and area of ​​neovascularization.

[0025] Figure 3 The results of electroretinogram examination in a streptozotocin-induced diabetic retinopathy mouse model following administration of the peptide of the present invention are shown.

[0026] Figure 4 Shown are the results of electroretinography in the db / db mouse model following administration of the peptide of the present invention.

[0027] Figure 5 and Figure 6 Shown are PAS staining images of retinal blood vessels in the db / db mouse model following administration of the peptide of the present invention, and the quantitative results of the numbers of vascular endothelial cells and perivascular cells.

[0028] Figure 7 The results of confirming protein expression in retinal tissue of a streptozotocin-induced diabetic retinopathy mouse model following administration of the peptide of the present invention are shown.

[0029] Figure 8 The results are shown, which confirm the expression of vascular endothelial growth factor (VEGF) in the retinal tissue of the db / db mouse model following administration of the peptide of the present invention.

[0030] Figure 9 The results are shown for confirming the phosphorylation level of Akt in the retinal tissue of the db / db mouse model following administration of the peptide of the present invention.

[0031] Figure 10 The results of confirming the expression of COX-2 in the retinal tissue of the db / db mouse model following administration of the peptide of the present invention are shown.

[0032] Figure 11 The results are shown for confirming the phosphorylation level of JNK in the retinal tissue of the db / db mouse model following administration of the peptide of the present invention.

[0033] Figure 12 and Figure 13 Shown are fluorescent images of retinal blood vessels (*: optic nerve) and calculation results of blood vessel diameters in a zebrafish diabetic retinopathy model following administration of the peptide of the present invention.

[0034] Figure 14 and Figure 15 The results of confirming gene expression in retinal tissue of a zebrafish diabetic retinopathy model following administration of the peptide of the present invention are shown.

[0035] Figure 16 and Figure 17Shown are the results of confirming the fluorescence images of the retinal tissue of the zebrafish diabetic retinopathy model following administration of the peptide of the present invention and the expression of cells and proteins. DETAILED DESCRIPTION

[0036] Hereinafter, the embodiments and examples of the present application will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement the present invention. However, the present application can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0037] Throughout this specification, when it is mentioned that a part “comprises” a certain structural element, unless there is any special description to the contrary, it means that other structural elements may also be included, rather than excluding other structural elements.

[0038] The present invention relates to a composition for preventing, improving or treating retinopathy, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0039] The composition may be a pharmaceutical composition for preventing or treating diabetic retinopathy.

[0040] The amino acid sequence of SEQ ID NO: 1 may include a sequence consisting of "Hyp-Gly-Gln-Glu-Aib-Leu-Ala", or include a sequence having 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more homology to the amino acid sequence of SEQ ID NO: 1.

[0041] 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, and Ala is alanine.

[0042] For the purposes of this invention, "diabetic retinopathy" refers to an ophthalmic complication of diabetes that causes impaired peripheral circulation and retinal microcirculation, leading to decreased vision. Initially, there may be no symptoms or only mild vision problems, but it can eventually lead to blindness. Diabetic retinopathy can develop in any patient with type 1 or type 2 diabetes.

[0043] In the present invention, the diabetic retinopathy may be non-proliferative diabetic retinopathy or proliferative diabetic retinopathy, but is not limited thereto.

[0044] As used herein, the term "angiogenesis" refers to the process of new blood vessel formation, i.e., the development of new blood vessels within cells, tissues, or organs. "Neovascularization" refers to newly generated blood vessels through the angiogenesis process. Throughout this application, "angiogenesis" and "neovascularization" are used interchangeably.

[0045] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a gastrointestinal disease, etc., by administering the composition of the present invention. "Treatment" refers to any action that improves or cures the symptoms of a subject suspected of having a gastrointestinal disease or suffering from the disease by administering the composition. The term "amelioration" as used herein refers to any action that at least reduces a parameter related to the condition being treated, such as the degree of symptoms, by administering a composition containing the extract of the present invention.

[0046] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are for illustrative purposes only and are not intended to limit the present invention.

[0047] Preparation Example

[0048] Preparation of peptides consisting of 7 amino acids (7mer)

[0049] The peptides of the present invention are synthesized based on the well-known solid phase peptide synthesis (SPPS) method. The preparation process includes the following steps: step 1, resin swelling and loading; step 2, solid phase peptide synthesis (SPPS); step 3, synthesis of deprotected peptides (global cleavage); step 4, primary purification and concentration / secondary purification and concentration; and step 5, freeze drying.

[0050] The solid phase peptide synthesis (SPPS) method includes the steps of loading a first amino acid onto a resin, removing the Fmoc protection at the amino acid N-terminus, and then coupling amino acids according to the amino acid sequence, and synthesizing the protected peptide in a solid phase reactor. Furthermore, the amino acid coupling includes the steps of: loading the first amino acid onto the resin, removing the Fmoc protection at the amino acid N-terminus; removing the solvent and washing the resin after the reaction; coupling the next amino acid according to the sequence; removing the solvent and washing the resin after the reaction; and repeating the above process until the final amino acid sequence is generated.

[0051] The α-amine group of each amino acid is protected by a weakly alkaline Fmoc group, while the side chain functional groups are protected by weakly acidic groups. All amino acids except Gly and Aib are in the L-configuration. Among these, the following amino acids, Hyp (tBu), Glu (tBu), and Gln (Trt), possess unique protecting groups. However, amino acids such as Ala, Leu, Aib, and Gly do not possess side chain protecting groups. Therefore, to obtain a crude peptide after sequence-coupled amino acids, the resin and protecting groups are removed from the peptide. Then, purification, concentration, and freeze-drying are performed to obtain a peptide compound consisting of the amino acid sequence of SEQ ID NO: 1.

[0052] Example 1

[0053] Evaluation of neovascularization inhibitory efficacy in a mouse model of oxygen-induced retinopathy

[0054] In order to evaluate the neovascularization inhibitory effect of the peptide of the present invention in an oxygen-induced retinopathy (OIR) mouse model, the following experiment was performed.

[0055] The oxygen-induced retinopathy (OIR) mouse model is a model of ischemic retinopathy such as proliferative diabetic retinopathy (PDR), retinopathy of prematurity, and retinal vein occlusion, and shows active neovascularization in the retina.

[0056] After stabilization for approximately 3-4 days, the mother and newborn mice were housed under 75% hyperoxia for 5 days, starting from postnatal day 7 to day 11. They were then housed under normoxia until postnatal day 17. During normoxia, 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 intravitreally (IVT) once on postnatal day 12 in the positive control group. The mouse eyeballs were then enucleated, fixed in 10% formalin for 1 hour, and flat-mounted. The retinas were cut into four equal sections centered on the optic nerve and treated with isolectin B4 (1 mM CaCl2, 1:200) overnight (O / N). The sample was then spread on a slide, covered with mounting solution, and covered with a cover glass. Fluorescence images of retinal blood vessels were obtained and the number, area, and vascular area of ​​new blood vessels were calculated using the imageJ program.

[0057] Results, such as Figure 1 and Figure 2 As shown, the oxygen-induced retinopathy mouse model showed neovascularization areas, whereas the group administered with the peptide of the present invention showed a reduction in neovascularization areas at a level similar to that of the group administered with aflibercept.

[0058] Therefore, it was found that the peptide of the present invention has an effect of inhibiting neovascularization.

[0059] Example 2

[0060] Evaluation of neovascularization inhibitory efficacy in a hyperglycemia-induced diabetic retinopathy mouse model

[0061] In order to evaluate the neovascularization inhibitory effect of the peptide of the present invention in a high glucose induced diabetic retinopathy mouse model, the following experiment was performed.

[0062] 2-1. STZ-induced diabetic retinopathy mouse model

[0063] The streptozotocin-induced mouse model is a type 1 diabetes model in which diabetes is induced by intraperitoneal administration of the antibiotic streptozotocin (STZ) to damage the insulin-producing β cells in the pancreas. As one of the models used in diabetic retinopathy research, it is a model that allows for the identification of diabetic retinopathy from the onset of diabetes to the early stages of the disease.

[0064] After fasting 3-4 hours before administration, 8-week-old C57BL / 6 mice were intraperitoneally administered with sodium citrate buffer and a buffer solution containing 50 mg / kg of streptozotocin (STZ) for 5 days. In this case, the drugs used were prepared immediately before administration and used quickly. Blood glucose was measured at a time point one week after the last intraperitoneal administration of STZ, and only mice with blood glucose exceeding 250 mg / dL were judged as diabetic mice and used in the experiment. Starting from the second day after the model was prepared, the drug was administered by eye drops (ED) twice a day for a total of about 17 weeks, and intraocular administration (IVT) was administered once a month at 10 μg. In order to evaluate retinal function, the mice were dark-adapted for more than 12 hours before electroretinogram (ERG) measurement. Then, the mice were dilated and anesthetized, and electrodes were placed in contact with the skin, tail, and cornea to measure ERG. The retina is stimulated with monochromatic white light to obtain a response value, and the amplitude from the trough of the a wave to the apex of the b wave is measured and evaluated as an indicator of retinal function.

[0065] Results, such as Figure 3 As shown, the amplitudes of a-wave and b-wave decreased in the STZ-induced diabetic retinopathy mouse group, whereas the group administered with the peptide of the present invention showed an increase in the amplitudes of a-wave and b-wave at a level similar to that of the group administered with aflibercept.

[0066] Therefore, it was found that the peptide of the present invention has an effect of inhibiting neovascularization.

[0067] db / db mouse model

[0068] The db / db mouse model, in which the leptin receptor, a hormone that suppresses appetite, is mutated, exhibits phenotypes such as severe obesity, bulimia, insulin resistance, and diabetes. It is widely used as a model for type 2 diabetes and is a model that can identify the early stages of diabetic retinopathy.

[0069] The drug was administered to 12-week-old db / db mice by eye drops (ED) twice a day (BID) or three times a day (TID) for approximately 12 weeks. To assess retinal function, mice were dark-adapted for at least 12 hours before electroretinogram (ERG) measurement. The mice were then anesthetized and dilated, and electrodes were placed in contact with the skin, tail, and cornea to measure ERGs. The retina was stimulated with monochromatic white light to obtain a response value, and the amplitude from the trough of the a wave to the apex of the b wave was measured and evaluated as an indicator of retinal function.

[0070] Results, such as Figure 4 As shown, the amplitudes of the a- and b-waves decreased in the db / db mouse group, whereas they increased in the group administered with the peptide of the present invention. In particular, the group administered with the peptide three times a day (TID) showed a superior effect compared to the group administered with the peptide twice a day (BID).

[0071] Therefore, it was found that the peptide of the present invention has an effect of inhibiting neovascularization.

[0072] Example 3

[0073] Evaluation of the efficacy of pericyte loss inhibition in a hyperglycemia-induced diabetic retinopathy mouse model

[0074] In the early stages of nonproliferative diabetic retinopathy (NPDR), loss of pericytes, the cells that surround capillaries, leads to blood infiltration into the tissue, causing macular edema. Macular edema causes a loss of visual focus and blurring of the visual field, resulting in a temporary reduction in vision and is a typical symptom of nonproliferative diabetic retinopathy. Therefore, to evaluate the efficacy of the peptides of the present invention in inhibiting pericyte loss, the following experiment was conducted.

[0075] Twelve-week-old db / db mice were administered the drug by eye drops (ED) twice daily (BID) or three times daily (TID) for approximately 12 weeks. To assess the status of retinal blood vessels, the enucleated mouse eyeballs were fixed in 10% formalin for one week. The fixed eyeballs were then treated with elastase to isolate retinal vessels, followed by PAS staining. The permeability of retinal blood vessels was assessed by quantifying the ratio of endothelial cells to pericytes present in the vascular wall, as well as the number of acellular vessels.

[0076] Results, such as Figure 5 and Figure 6As shown, the db / db mouse group had a decrease in pericytes, whereas the group administered with the peptide of the present invention had a suppressed decrease in pericytes. In particular, the group administered with the peptide three times a day (TID) showed a superior effect compared to the group administered with the peptide twice a day (BID).

[0077] Therefore, it was found that the peptide of the present invention has the effect of inhibiting the disappearance of vascular pericytes.

[0078] Example 4

[0079] Evaluation of retinal protein expression levels in a hyperglycemia-induced diabetic retinopathy mouse model

[0080] In order to evaluate the expression level of proteins in retinal tissue following administration of the peptide of the present invention in a high glucose induced diabetic retinopathy mouse model, the following experiment was performed.

[0081] 4-1. Streptozotocin-induced diabetic retinopathy mouse model

[0082] After fasting 3-4 hours prior to dosing, 8-week-old C57BL / 6 mice were intraperitoneally administered with sodium citrate buffer and a buffer containing 50 mg / kg of streptozotocin (STZ) for 5 days. In this case, all drugs were prepared immediately before dosing and administered promptly. Blood glucose was measured one week after the final intraperitoneal administration of STZ, and only mice with blood glucose levels exceeding 250 mg / dL were considered diabetic and used in the experiment. Starting from the day after model establishment, the drug was administered by eye drop (ED) twice daily for approximately 17 weeks, and intraocularly (IVT) was administered once a month at a dose of 10 μg. To assess retinal protein expression levels, retinal tissue was isolated from enucleated mouse eyeballs and treated with RIPA solution to extract retinal proteins. Western blot was performed to assess the expression of factors associated with retinal angiogenesis and vascular permeability.

[0083] Results, such as Figure 7 As shown, the STZ-induced diabetic retinopathy mouse group showed increased expression of vascular endothelial growth factor (VEGF) associated with angiogenesis, whereas the group administered with the peptide of the present invention showed decreased expression of VEGF.

[0084] Furthermore, the STZ-induced diabetic retinopathy mouse group showed an increased phosphorylation level of Akt, a signaling factor associated with vascular endothelial growth factor, whereas the Akt phosphorylation level was decreased in the group administered with the peptide of the present invention.

[0085] Therefore, it was found that the peptide of the present invention inhibits the expression of proteins related to neovascularization and permeability of vascular cells in the retina.

[0086] 4-2.db / db mouse model

[0087] Twelve-week-old db / db mice were administered the drug by eye drop (ED) twice daily (BID) or three times daily (TID) for approximately 12 weeks. To assess retinal protein expression levels, retinal tissue was isolated from enucleated mouse eyeballs and treated with RIPA solution to extract retinal proteins. Western blotting was performed to assess the expression of factors associated with retinal angiogenesis and vascular permeability.

[0088] Results, such as Figure 8 As shown, 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.

[0089] And, as Figure 9 As shown, the phosphorylation level of Akt increased in the db / db mouse group, whereas the phosphorylation level of Akt decreased in the group to which the peptide of the present invention was administered three times a day (TID).

[0090] And, as Figure 10 As shown, the db / db mouse group showed increased expression of COX-2, an inflammation-related factor, whereas the group to which the peptide of the present invention was administered three times a day (TID) showed decreased expression of COX-2.

[0091] And, as Figure 11 As shown, the phosphorylation level of JNK, a factor involved in cell apoptosis, increased in the db / db mouse group, whereas the phosphorylation level of JNK decreased in the group administered with the peptide of the present invention.

[0092] Therefore, it was found that the peptide of the present invention inhibits the expression of proteins related to neovascularization and permeability of vascular cells in the retina, and inhibits the expression of factors related to inflammation.

[0093] Example 5

[0094] Evaluating retinal vasodilation inhibitory efficacy in a zebrafish model of diabetic retinopathy

[0095] Embryos obtained by mating live zebrafish Tg[flk:EGFP] were screened for those expressing the fluorescent protein. Six days after fertilization, embryos were treated with glucose and the peptide of the present invention (002-175) for four days before being euthanized by placing them in 4 mg / ml tricaine. After fixation with 4% paraformaldehyde for three days, the eyeballs were isolated using 3% trypsin, and changes in the blood vessels within the isolated eyeballs were observed using a fluorescence microscope.

[0096] Results, such as Figure 12 and Figure 13 As shown, compared with the normal group, the diameter of the zebrafish retinal blood vessels in the group treated with high blood sugar (130mM glucose) increased by more than 1.5 times. In contrast, the diameter of the retinal blood vessels in the group treated with the peptide of the present invention decreased, especially in the 100μg / ml and 200μg / ml treatment groups, the diameter of the retinal blood vessels decreased to a level similar to that of the normal group.

[0097] Therefore, it was found that the peptide of the present invention has the effect of inhibiting retinal vasodilation.

[0098] Example 6

[0099] Profiling gene expression in a zebrafish model of diabetic retinopathy

[0100] Embryos expressing fluorescent protein were screened from embryos obtained by mating living zebrafish Tg[flk:EGFP] and used. On the 6th day after fertilization, the embryos were treated with glucose and the peptide of the present invention (002-175) for 4 days, and then euthanized by placing 4 mg / ml of tricaine. RNA extraction and microarray analysis were performed after the eyeballs were removed. In addition, Trizol was placed in the removed eyeballs to homogenize the tissue and extract mRNA. The extracted mRNA was quantified using a NanoDrop spectrophotometer, and cDNA was synthesized using RNA with a purity of 1.8 or more. Using the synthesized cDNA as the object, a polymerization reaction of SYBR Green master mix, primers, and genes was performed, and the concentration of each gene was divided by GAPDH to obtain the concentration of the corrected gene.

[0101] Results, such as Figure 14 As shown in the results of microarray analysis, the group treated with the peptide of the present invention showed a decrease in the gene expression of IL-1β, MMP9, and NF-κB.

[0102] And, as Figure 15As shown in the results of real-time polymerase chain reaction (PCR), the group treated with the peptide of the present invention showed decreased gene expression of ikkα, ikkβ, ikkγ, NF-kB, IL-1β, and MMP9.

[0103] Il-1β is an inflammatory factor associated with angiogenesis, increasing vascular permeability. Under hyperglycemic conditions, it promotes the natural apoptosis of retinal capillary cells by activating NF-κB. Furthermore, NF-κB present in the retina and microvasculature forms the central axis of the intracellular signaling system, inducing programmed cell death by increasing the expression of multiple cytokines, such as IL-1β, IL-6, and IL-8, in the vitreous humor and serum. Furthermore, matrix metalloproteinases (MMPs) play a crucial role in the progression of diabetic retinopathy, with MMP9, in particular, being associated with angiogenesis in retinal capillary cells. Activation of NF-κB, TNF-α, and interleukin promotes the development of diabetic retinopathy by increasing MMP9.

[0104] Therefore, it can be seen that the peptide of the present invention has the effect of inhibiting inflammatory factors in the retina and the pathogenesis of diabetic retinopathy.

[0105] Example 7

[0106] Analyzing Histological Changes in a Zebrafish Diabetic Retinopathy Model

[0107] Zebrafish larvae (6 days post-fertilization, 6 dpf) were fixed with 4% paraformaldehyde for 3-4 hours, then treated with 30% sucrose and incubated at 4°C overnight. OCT-embedded blocks were prepared and sectioned, followed by TUNEL and immunofluorescence (IF) staining.

[0108] Results, such as Figure 16 and Figure 17 As shown in the results of TUNEL assay in retinal tissue, the group treated with hyperglycemia showed an increase in TUNEL-positive cells in the inner nuclear layer (INL) of the retina, while the group treated with the peptide of the present invention (002-175) showed a decrease in TUNEL-positive cells.

[0109] Furthermore, immunofluorescence staining analysis of the expression of VEGF, GFAP, and ZO-1 in the zebrafish eyeball revealed increased expression of VEGF and GFAP, inflammatory factors, in the outer nuclear layer (ONL) of the retina in the hyperglycemic group, while the group treated with the peptide of the present invention (002-175) showed decreased expression of VEGF and GFAP. Furthermore, expression of ZO-1, a tight junction protein, decreased in the hyperglycemic group but increased in the group treated with the peptide of the present invention (002-175), indicating a restored state.

[0110] Therefore, it can be seen that the peptides of the present invention have the effect of inhibiting inflammatory factors in the retina and the pathogenesis of diabetic retinopathy. In particular, in the zebrafish diabetic retinopathy model induced by hyperglycemia, it was confirmed that ZO-1, a tight junction marker, was reduced, resulting in damage to the blood-retinal barrier (BRB). This increased vascular permeability and vascular edema were observed. This is the same mechanism as clinical diabetic macular edema. The peptides of the present invention can effectively inhibit diabetic retinopathy and diabetic macular edema by inhibiting retinal neovascularization and inflammatory factors to reduce vascular permeability.

Claims

1. A pharmaceutical composition for preventing or treating retinopathy, characterized in that: A peptide consisting of a sequence having 90% or more homology to the amino acid sequence of SEQ ID NO: 1 is included.

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

3. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, characterized in that It comprises a peptide consisting of the amino acid sequence of SEQ ID NO:

1.

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

5. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, characterized in that The retinopathy is diabetic retinopathy.

6. The pharmaceutical composition for preventing or treating retinopathy according to claim 5, characterized in that 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, characterized in that The peptide inhibits angiogenesis.

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

9. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, characterized in that The peptide inhibits vasodilation.

10. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, characterized in that The peptide inhibits at least one of the expression of vascular endothelial growth factor, phosphorylation of protein kinase B, expression of cyclooxygenase-2, and phosphorylation of c-Jun amino-terminal kinase.

11. The pharmaceutical composition for preventing or treating retinopathy according to claim 1, characterized in that The peptide restores the tight junction protein ZO-1.

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