Composition for treating macular degeneration comprising a novel peptide

Abstract

The present invention relates to a composition for preventing or treating macular degeneration, comprising a novel peptide. Specifically, the peptide of the present invention has excellent inhibitory effect on choroidal neovascularization, which is the main cause of macular degeneration, and has excellent stability and solubility, and therefore can be developed as an excellent therapeutic agent for macular degeneration.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing or treating macular degeneration, comprising a novel peptide. Specifically, the peptide of the present invention has an effect of inhibiting choroidal neovascularization, which is the main cause of macular degeneration, and has excellent stability and solubility. [Background technology]

[0002] The macula is the most important part of the nerve layer inside the eyeball called the retina. It is the central part of the retina with a radius of about 1.5 mm, and is densely packed with photoreceptors that can detect light. Therefore, the macula is the center of the retina and is responsible for most of the vision. Macular degeneration is a disease in which the macula degenerates due to aging, genetic factors, toxicity, inflammation, etc. When the macula degenerates, vision is reduced, and in severe cases, vision can be completely lost.

[0003] Macular degeneration can be broadly divided into non-exudative (atrophic; dry) and exudative (wet) types. Non-exudative macular degeneration accounts for 80-90% of all cases of macular degeneration, and in most cases does not significantly affect vision, except in the later stages when atrophy of the retina and choroid appears. However, it may progress to exudative macular degeneration, so caution is required. Exudative macular degeneration accounts for about 10-20% of all cases of macular degeneration, and if left untreated, vision will rapidly deteriorate, and many patients will become blind within two years of diagnosis.

[0004] Exudative macular degeneration is a disease in which the prognosis for vision is very poor once choroidal neovascularization has developed, and the rate of blindness is the highest in people over the age of 65. The vascular layer called the choroid supplies nutrients to the retina and removes metabolic substances released by retinal cells. However, due to aging and other factors, choroidal blood vessels may invade the retinal cells and be abnormally generated. These abnormal blood vessels are called choroidal neovascularization. These abnormal blood vessels are very weak and easily ruptured, which allows exudate and blood to leak out and damage the macula.

[0005] In the case of non-exudative macular degeneration, treatment involves taking antioxidant vitamins, which are known to slow the progression of macular degeneration, and treating risk factors for macular degeneration such as high blood pressure and hyperlipidemia. In the case of exudative macular degeneration, there are thermal laser photocoagulation, photodynamic therapy, antibody injections, and vitrectomy, but there is still no perfect treatment, and research on this is being actively conducted. In recent years, the most commonly used method is to inject anti-vascular endothelial growth factor antibodies (anti-VEGF antibodies) such as aflibercept, ranibizumab, and bevacizumab into the eye, but this method has disadvantages such as the need for repeated administration and high cost.

[0006] Therefore, as a result of research into substances that can effectively treat macular degeneration, the present inventors have identified a novel peptide that not only has excellent stability and solubility but also has an excellent inhibitory effect on choroidal neovascularization, leading to the completion of the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Publication No. 10-2020-0060397 [Non-patent literature]

[0008] [Non-Patent Document 1] Gong Y, Li J, Sun Y, Fu Z, Liu CH, Evans L, et al. (2015) Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.PLoS ONE 10(7) 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 macular degeneration, which comprises a peptide represented by general formula (1).

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide consisting of the amino acid sequence of SEQ ID NO:1-45. [Means for solving the problem]

[0011] The present invention provides a pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide represented by the following general formula (1):

[0012] X1-X2-X3-X4-X5-X6-X7...(1)

[0013] In the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is a non-polar amino acid; X6 is absent, leucine or D-leucine; X7 is absent or is an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester; Here, if X6 is absent, then X7 is also absent.

[0014] In one embodiment, in the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is an amino acid selected from the group consisting of glycine, phenyl-glycine, alanine, valine, leucine, tert-leucine, and 2-aminoisobutyric acid; X6 is absent, leucine or D-leucine; X7 may be absent or an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester, where if X6 is absent, then X7 is also absent.

[0015] In one embodiment, in the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is a non-polar amino acid; X6 is leucine or D-leucine, X7 may be an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

[0016] In one embodiment, in the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is an amino acid selected from the group consisting of glycine, phenyl-glycine, alanine, valine, leucine, tert-leucine, and 2-aminoisobutyric acid; X6 is leucine or D-leucine, X7 may be an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

[0017] In one embodiment, in the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is Glutamate or D-Glutamate, X5 is 2-aminoisobutyric acid, X6 is leucine or D-leucine, X7 may be an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

[0018] The composition of the present invention may contain any one peptide selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 45, and preferably contains a peptide consisting of the amino acid sequence of SEQ ID NO: 43.

[0019] The peptide of the present invention has the effect of inhibiting choroidal neovascularization (CNV).

[0020] The macular degeneration of the present invention may be at least one selected from the group consisting of wet macular degeneration, dry macular degeneration, and age-related macular degeneration.

[0021] The present invention provides a method for treating macular degeneration, which comprises administering a peptide represented by the following general formula (1) to a subject suffering from macular degeneration.

[0022] X1-X2-X3-X4-X5-X6-X7...(1)

[0023] In the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is a non-polar amino acid; X6 is absent, leucine or D-leucine; X7 is absent or is an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester; Here, if X6 is absent, then X7 is also absent.

[0024] The present invention provides a composition for use in treating or preventing macular degeneration, comprising a peptide represented by the above general formula (1).

[0025] The present invention provides use of a composition comprising a peptide represented by the general formula (1) for treating or preventing macular degeneration. Effect of the Invention

[0026] The peptide of the present invention has excellent stability and solubility, and has an excellent inhibitory effect on choroidal neovascularization, which is the main cause of macular degeneration, and therefore can be useful for preventing or treating macular degeneration. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and calculating CTF (Corrected Total Fluorescence) for CNV of the peptide of the present invention. [Diagram 2]FIG. 1 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and calculating CTF (Corrected Total Fluorescence) for CNV of the peptide of the present invention. [Diagram 3] FIG. 1 shows the results of OCT (Optical coherence tomography) measurements of CNV of the peptide of the present invention and calculation of the CNV lesion size. [Figure 4] FIG. 1 shows the results of OCT (Optical coherence tomography) measurements of CNV of the peptide of the present invention and calculation of the CNV lesion size. [Diagram 5] FIG. 1 shows the results of electroretinography for CNV of the peptide of the present invention. [Figure 6] FIG. 13 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and quantifying vascular leakage in relation to CNV for the peptide of Example 43 (002-175) of the present invention and an eye drop solution containing the same. [Figure 7] FIG. 13 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and quantifying vascular leakage in relation to CNV for the peptide of Example 43 (002-175) of the present invention and an eye drop solution containing the same. [Figure 8] FIG. 1 shows the results of OCT (Optical coherence tomography) measurements of CNV for the peptide of Example 43 (002-175) of the present invention and an ophthalmic solution containing the same, and the results of calculation of the CNV lesion size. [Figure 9] FIG. 1 shows the results of OCT (Optical coherence tomography) measurements of CNV for the peptide of Example 43 (002-175) of the present invention and an ophthalmic solution containing the same, and the results of calculation of the CNV lesion size. [Figure 10]FIG. 13 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and calculating the neovascular area for CNV in an eye drop containing the peptide of Example 43 (002-175) of the present invention. [Figure 11] FIG. 13 shows the results of measuring FFA (Fundus Fluorescein Angiogrpahy) and calculating the neovascular area for CNV in an eye drop containing the peptide of Example 43 (002-175) of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Hereinafter, the present application will be described in detail with reference to the accompanying drawings so that a person having ordinary skill in the art to which the present invention pertains can easily carry out the present application. However, since the present application can be carried out in various forms, it is not limited to the following embodiments and examples.

[0029] 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.

[0030] The present invention provides a pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide represented by the following general formula (1):

[0031] X1-X2-X3-X4-X5-X6-X7...(1)

[0032] In the formula, X1 is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X2 is glycine; X3 is glutamine or D-glutamine, X4 is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X5 is a non-polar amino acid; X6 is absent, leucine or D-leucine; X7 is absent or is an amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester; Here, if X6 is absent, then X7 is also absent.

[0033] The peptide of the present invention may be a peptide selected from the group consisting of the peptides in Table 1 below.

[0034] [Table 1(1)] [Table 1(2)]

[0035] The term "macular degeneration" used in the present invention refers to a disease in which the macula becomes less functional due to aging, genetic factors, toxicity, inflammation, etc., causing a decrease in vision and, in severe cases, complete loss of vision.

[0036] In the present invention, the macular degeneration may be, but is not limited to, wet macular degeneration, atrophic macular degeneration, and age-related macular degeneration.

[0037] The term "choroidal neovascularization (CNV)" used in the present invention refers to the process of new choriocapillary generation. The choroid is a hypervascular membrane of the eyeball, a complex network of fine capillaries that supply nutrients to the iris and retina located in the periphery where they are found. Pathologically, choroidal neovascularization refers to the invasion of the subretinal space by abnormal blood vessels stalked from the choroid and structural deformation and destruction of Bruch's membrane.

[0038] The term "angiogenesis" used in the present invention means the process of new blood vessels being formed, i.e., new blood vessels are generated in cells, tissues or organs, and the term "neovascularization" means blood vessels newly generated by the angiogenesis process. In the present application, "angiogenesis" and "neovascularization" may be described interchangeably.

[0039] The CNV animal model used in the present invention is a mouse model in which a lesion is induced by irradiating a laser to the choroid of a C57BL / 6 mouse using a laser slit system. The laser-induced CNV model, which was first reported in 1979, uses photocoagulation to disrupt Bruch's membrane and induce the growth of choroidal neovessels in the retinal region, and is similar in development to neovascular disease that occurs in the human retina, i.e., wet AMD, in that neovessels originate from the choroid. In addition, the laser-induced CNV model has been successful in predicting the clinical efficacy of anti-vascular endothelial growth factor (VEGF) treatment for neovascular AMD, and is an animal model that is used in the development stage of existing therapeutic agents (Non-Patent Document 1).

[0040] In the present invention, Pro is proline, Hyp is trans-4-hydroxy-L-proline, D Hyp is trans-4-hydroxy-D-proline, cis-4F-Pro is cis-4-fluoro-L-proline; 4,4-difluoro-Pro is 4,4-difluoro-L-proline; 4-methylene-Pro is 4-methylene-L-proline, 4,4-dimethyl Pro is 4,4-dimethyl-L-proline; trans-4NH2-Pro is trans-4-amino-L-proline, Gly is glycine, Gln is glutamine, D-Gln is D-Glutamine, Ala is alanine, D-Ala is D-Alanine, Val is valine. Leu is leucine, D-Leu is D-Leucine, Ile is isoleucine, Met is methionine, Tert-Leu is tert-leucine and L-α-tert-butylglycine. Aib is 2-aminoisobutyric acid, Isopropyl ester (IPE) is a derivative in which the amino acid terminal group is substituted with an isopropyl ester. Ser is serine, Thr is threonine, Cys is cysteine, Asn is asparagine, Phe is phenylalanine, Tyr is tyrosine, Trp is tryptophan, Lys is lysine, Arg is arginine, His is histidine, Asp is aspartate or aspartic acid, D-Asp is D-aspartate or D-aspartic acid, Glu is glutamate or glutamic acid, D-Glu is D-glutamate or D-glutamic acid.

[0041] In the present invention, a polar amino acid refers to an amino acid whose side chain has a polarity in terms of its chemical structure, and includes, but is not limited to, Thr, Ser, Cys, Asn, Gln, Asp, Glu, His, Lys, and Arg.

[0042] In the present invention, a non-polar amino acid refers to an amino acid whose side chain does not have polarity in terms of its chemical structure, and includes, but is not limited to, Met, Leu, Pro, Ala, Gly, Val, Ile, and Aib.

[0043] As used herein, the term "prevention" refers to any action of administering a composition to inhibit or delay the onset of a disease, and the term "treatment" refers to any action of administering a composition to improve or beneficially alter the symptoms of an individual suspected of or suffering from a disease.

[0044] The present invention will be described in more detail below with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0045] [Production Example] The peptide of the present invention was synthesized based on the known solid phase peptide synthesis (SPPS) method, and the production process included the following steps.

[0046] Step 1: Resin softening and loading Step 2: Solid Phase Peptide Synthesis (SPPS) Step 3: Synthesis of deprotected peptide (Global cleavage) Step 4: Primary purification and concentration / secondary purification and concentration Step 5: Freeze-drying

[0047] The solid phase peptide synthesis method (SPPS) includes the steps of loading the first amino acid onto a resin, deprotecting the N-terminus of the amino acid with Fmoc, coupling the amino acids according to the amino acid sequence, and synthesizing the protected peptide in a solid phase reactor. The amino acid coupling 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 completed, coupling the next amino acid according to the sequence, removing the solvent and washing the resin after the reaction is completed, and repeating the above process until the final amino acid sequence is produced.

[0048] The alpha amine group of each amino acid is protected with a base-sensitive Fmoc group, and the functional group of the side chain is protected with an acid-sensitive group. All amino acids except Gly and Aib are in the L-configuration, and among them, some amino acids Hyp(tBu), Glu(tBu), and Gln(Trt) have their own protective groups, as follows. And amino acids such as Ala, Leu, Aib, and Gly do not have protective groups on their side chains. Therefore, after completing the amino acid coupling according to the sequence, the resin and protective groups were removed from the peptide to obtain the Crude peptide. Then, the peptide compounds of Examples 1 to 45 were obtained by purification, concentration, and lyophilization.

[0049] [Test Example 1] Evaluation of the inhibitory effect of peptides on choroidal neovascularization: Measurement of fluorescence value (CTF; corrected total fluorescence) in CNV area In order to evaluate the efficacy of the peptide of the present invention in inhibiting choroidal neovascularization (CNV), the following experiment was carried out.

[0050] C57BL / 6 mice aged 7-8 weeks were anesthetized by intraperitoneal injection of Ketamine (50mg / Kg) and Rompun (23.32mg / Kg). The choroid of each mouse eye was irradiated with a laser beam using a Slit-lamp laser injection system four times at the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions. The laser irradiation conditions were 532nm, 80msec, and 240mW. Immediately after the laser irradiation, 10mg / mL peptide or 20mg / mL aflibercept dissolved in PBS was injected into the vitreous cavity using a syringe. Ten days after drug administration, the mice were anesthetized and 2% fluorescein was administered intraperitoneally to stain the blood vessels. The retinas were photographed 2-3 minutes after fluorescein administration. The size and fluorescence value of the laser-induced CNV lesions were measured using the imageJ program, and the corrected total fluorescence (CTF) was calculated by removing background to reduce interindividual differences, and the values ​​of each group were compared. All test results were presented as mean ± standard deviation, and significance was verified using one-way ANOVA and Kruskal-Wallis test.

[0051] The results of the experiment are shown in Table 2 below and in FIGS.

[0052] [Table 2(1)] [Table 2(2)]

[0053] As shown in Table 2 and Figures 1 and 2, the fluorescence values ​​of the CNV site were measured, and it was confirmed that the peptides of the present invention have a CNV inhibitory effect. In particular, the peptides of Example 1 (002-053), Example 7 (002-076), Example 8 (002-077), Example 9 (002-078), Example 13 (002-090), Example 14 (002-055), Example 15 (002-080), Example 17 (002-084), Example 23 (002-087), Example 27 (002-091), and Example 43 (002-175) showed very excellent inhibitory effects.

[0054] Therefore, it is clear that the peptide of the present invention has excellent CNV inhibitory effects.

[0055] [Test Example 2] Evaluation of the inhibitory effect of peptides on choroidal neovascularization: Optical Coherence Tomography In order to evaluate the inhibitory effect of the peptide of the present invention on CNV (choroidal neovascularization), the following experiment was carried out.

[0056] C57BL / 6 mice aged 7-8 weeks were anesthetized by intraperitoneal injection of Ketamine (50mg / Kg) and Rompun (23.32mg / Kg). The choroid of each mouse eye was irradiated with a laser beam at 12, 3, 6, and 9 o'clock directions four times each using a Slit-lamp laser injection system. The laser irradiation conditions were 532nm, 80msec, and 240mW. Immediately after the laser irradiation, 10mg / mL peptide or 20mg / mL aflibercept dissolved in PBS was injected into the vitreous cavity using a syringe. Ten days after drug administration, the mice were anesthetized, and the CNV lesions induced by the laser were visualized by measuring the micrometer scale using the ImageJ program by passing an OCT beam through each burn. 2The results of all tests were expressed as mean ± standard deviation, and significance was verified using one-way ANOVA and Kruskal-Wallis test.

[0057] The results of the experiment are shown in Table 3 below, and in Figures 3 and 4.

[0058] [Table 3]

[0059] As shown in Table 3 and Figures 3 and 4, the size of CNV lesions was measured and it was confirmed that the size of CNV lesions was reduced when the peptide of the present invention was injected, indicating that the peptide of the present invention has an excellent CNV inhibitory effect.

[0060] [Test Example 3] Evaluation of the inhibitory effect of peptides on choroidal neovascularization: Electroretinography In order to evaluate the inhibitory effect of the peptide of the present invention on CNV (choroidal neovascularization), the following experiment was carried out.

[0061] C57BL / 6 mice (7-8 weeks old) were anesthetized by intraperitoneal injection of Ketamine (50mg / Kg) and Rompun (23.32mg / Kg). The mouse choroid was irradiated with a laser beam from a slit-lamp laser injection system at 12, 3, 6, and 9 o'clock directions four times each. The laser irradiation conditions were 532nm, 80msec, and 240mW. Immediately after the laser irradiation, 10mg / mL peptide or 20mg / mL aflibercept dissolved in PBS was injected into the vitreous using a syringe. The mice were dark-adapted for more than 12 hours for electroretinogram analysis. After dilating the pupils of the mice and anesthetizing them in a dark room, the ground electrode was placed on the tail, the reference electrode on the head, and the corneal electrode on the cornea to perform electroretinogram examinations. 0.9cd sec / m 2 The retina was stimulated with a single white light of 10 ...

[0062] The experimental results are shown in Table 4 below and FIG.

[0063] [Table 4]

[0064] As shown in Table 4 and Figure 5, the b-wave amplitude was measured and it was confirmed that the b-wave amplitude increased when the peptide of the present invention was injected, indicating that the peptide of the present invention has an excellent CNV inhibitory effect.

[0065] [Test Example 4] Assessment of peptide stability To evaluate the stability of the peptides of the present invention, the following experiment was carried out.

[0066] The stability of the peptide was confirmed by content analysis under strict conditions of 40℃ and 75% humidity in an aqueous solution. The analysis was performed using HPLC under the following conditions.

[0067] Analysis column: C18 (4.6Υ 250mm, 5μm, YMC Hydrosphere) Analysis wavelength: Ultraviolet absorption photometer (210nm) Injection volume / flow rate: 10μL / 0.8mL / min Column temperature: 45℃ Mobile phase: 0.1M phosphate buffer methanol mixture (83:17)

[0068] The experimental results are shown in Table 5 below.

[0069] [Table 5]

[0070] As shown in Table 5, the stability of the peptides of the present invention was evaluated, and the content was maintained at about 95% or more after 7 days, and the decrease rate after 7 days was also less than about 1%, confirming that the peptides have excellent stability. In particular, Example 43 (002-175) showed the best stability. Therefore, it can be seen that the peptides of the present invention have excellent stability.

[0071] [Test Example 5] Assessment of peptide solubility To evaluate the solubility of the peptides of the present invention, an experiment was carried out as follows.

[0072] The solubility test was conducted according to the Korean Pharmacopoeia method, and the solubility was determined by the degree to which the peptide dissolved within 30 minutes when 0.1 g was placed in 10 mL of water and vigorously shaken for 30 seconds every 5 minutes at 20 ± 5°C.

[0073] The experimental results are shown in Table 6 below.

[0074] [Table 6]

[0075] As shown in Table 6, the results of the solubility measurement showed that the peptides of the present invention exhibited excellent solubility, and in particular, Example 43 (002-175) had the highest solubility. This shows that the peptides of the present invention have excellent solubility.

[0076] [Test Example 6] Evaluation of the inhibitory effect of peptide-containing eye drops on choroidal neovascularization In order to evaluate the inhibitory effect of the eye drop containing Example 43 (002-175), which was confirmed to be an excellent peptide in the above test examples, on CNV (choroidal neovascularization), the following experiment was carried out.

[0077] C57BL / 6 mice aged 7-8 weeks were anesthetized by intraperitoneal injection of Ketamine (50mg / Kg) and Rompun (23.32mg / Kg). The mouse choroid was irradiated with a laser beam from a Slit-lamp laser injection system at 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions four times each. The laser irradiation conditions were 532nm, 80msec, and 240mW. Three days after laser irradiation, 1μL of aflibercept dissolved in PBS at 20mg / mL was injected into the vitreous cavity using a syringe before drug administration. The drugs prepared according to the concentration were instilled into both eyes twice a day (5μL / eye, AM:9:00 FM04:00) for 11 days. Six, 10, and 14 days after laser induction, mice were anesthetized and 2% fluorescein was administered intraperitoneally to stain blood vessels. Retinas were photographed 2-3 minutes after fluorescein administration. The size and fluorescence value of the laser-induced CNV lesions were measured using the ImageJ program, and CTF (Corrected Total Fluorescence) was calculated by removing background to reduce individual differences, and the values ​​for each group were compared. Additionally, 6, 10, and 14 days after laser induction, mice were anesthetized and the laser-induced CNV lesions were imaged in μm using the ImageJ program by passing an OCT beam through each burn. 2 The results of all tests were shown as mean ± standard deviation, and the significance was verified using one-way ANOVA and Kruskal-Wallis test. The experimental results are shown in Figures 6 to 9.

[0078] As shown in Figures 6 and 7, the fluorescence value at the CNV site was measured, and the ophthalmic solution containing the peptide of Example 43 (002-175) of the present invention showed an excellent CNV inhibitory effect, and in particular, it showed an even more excellent inhibitory effect than the positive control group, aflibercept, 14 days after administration.

[0079] In addition, as shown in Figures 8 and 9, the size of CNV lesions was measured and it was confirmed that the size of CNV lesions was reduced when an eye drop containing the peptide of Example 43 (002-175) of the present invention was administered, and in particular, the level was similar to that of the positive control group, aflibercept.

[0080] Therefore, it is evident that the eye drops containing the peptide of Example 43 (002-175) of the present invention have an excellent CNV inhibitory effect.

[0081] [Test Example 7] Evaluation of the angiogenesis inhibitory effect of peptide-containing eye drops A symptom of neovascular macular degeneration is retinal angiomatous proliferation (RAP), which occurs in approximately 12-15% of newly diagnosed neovascular macular degeneration patients. Meanwhile, Vldlr is known to be one of the functional candidate genes for human macular degeneration, and retinal angiomatous proliferation occurs along with subretinal neovascularization in Vldlr knockout (- / -) mice. Therefore, the following experiment was performed to evaluate the neovascularization inhibitory effect of an ophthalmic solution containing Example 43 (002-175), which was confirmed to be an excellent peptide in the above test example, using Vldlr knockout (- / -) mice, an animal model of neovascular macular degeneration.

[0082] Vldlr from 6 weeks old (P42) to 14 weeks old (P98) - / - Peptide eye drops containing Example 43 (002-175) were instilled into the eyes of mice twice a day, and 1 μL of aflibercept at a concentration of 20 mg / mL was injected intravitreally into 6-week-old mice as a positive control group. After instilling mydriatics into the eyes of the mice to dilate the pupils, the mice were anesthetized by intraperitoneal injection of Ketamine (50 mg / Kg) and Rompun (23.32 mg / Kg), and blood vessels were stained by intraperitoneal injection of 2% fluorescein. Images were obtained by retinal fluorescence photography, and the lesion area was calculated using the imageJ program. The experimental results are shown in Figures 10 and 11.

[0083] As shown in Figures 10 and 11, the neovascular area was calculated, and the lesion size in the positive control group administered with aflibercept (AF) dropped rapidly one week after administration (P49), but gradually increased again from the second week, and after eight weeks, it showed a similar size to the negative control group administered with the vehicle (P98). On the other hand, the lesion size in the group administered with the eye drops containing the peptide of Example 43 (002-175) of the present invention gradually decreased, and after eight weeks, it was shown that the size of the lesion was suppressed (P98).

[0084] Therefore, it is evident that the eye drop containing the peptide of Example 43 (002-175) of the present invention has an excellent neovascularization inhibitory effect.

Claims

1. A pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide represented by the following general formula (1): X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ ・・・(1) In the formula, X １ is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X ２ is glycine, X ３ is glutamine or D-glutamine, X ４ is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X ５ is a non-polar amino acid, X ６ is absent, leucine or D-leucine; X ７ is absent or at least one amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester; Here, X ６ If absent, then X ７ is also absent.

2. In the formula, X １ is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X ２ is glycine, X ３ is glutamine or D-glutamine, X ４ is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X ５ is an amino acid selected from the group consisting of glycine, phenyl-glycine, alanine, valine, leucine, tert-leucine and 2-aminoisobutyric acid; X ６ is absent, leucine or D-leucine; X ７ The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, wherein is absent or is at least one amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

3. In the formula, X １ is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X ２ is glycine, X ３ is glutamine or D-glutamine, X ４ is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X ５ is a non-polar amino acid, X ６ is leucine or D-leucine, X ７ The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, wherein is at least one amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

4. In the formula, X １ is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X ２ is glycine, X ３ is glutamine or D-glutamine, X ４ is an amino acid selected from the group consisting of aspartate, D-aspartate, glutamate, and D-glutamate; X ５ is an amino acid selected from the group consisting of glycine, phenyl-glycine, alanine, valine, leucine, tert-leucine and 2-aminoisobutyric acid; X ６ is leucine or D-leucine, X ７ The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, wherein is at least one amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

5. In the formula, X １ is an amino acid selected from the group consisting of proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline, cis-4-fluoro-L-proline, 4,4-difluoro-L-proline, 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline; X ２ is glycine, X ３ is glutamine or D-glutamine, X ４ is glutamate or D-glutamate, X ５ is 2-aminoisobutyric acid, X ６ is leucine or D-leucine, X ７ The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, wherein is at least one amino acid selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

6. The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, comprising any one peptide selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 45.

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

43.

8. The pharmaceutical composition for preventing or treating macular degeneration according to any one of claims 1 to 7, wherein the peptide inhibits choroidal neovascularization (CNV).

9. The pharmaceutical composition for preventing or treating macular degeneration according to claim 1, characterized in that the macular degeneration is at least one selected from the group consisting of exudative macular degeneration, atrophic macular degeneration and age-related macular degeneration.

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