Novel peptide derivative and pharmaceutical composition for preventing or treating macular degeneration comprising same

Abstract

The present invention provides: a novel peptide derivative or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition comprising the novel peptide derivative or the pharmaceutically acceptable salt thereof as an active ingredient for the prevention or treatment of macular degeneration.

Description

Novel peptide derivative and pharmaceutical composition containing the same for preventing or treating macular degeneration

[0001] The present invention relates to a novel peptide derivative or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for preventing or treating macular degeneration comprising the novel peptide derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

[0002] Macular degeneration (AMD) is a medical condition characterized by blurred vision or complete loss of vision in the central part of the retina. It is caused by damage to the macula, the area of ​​the retina. It primarily affects people over 50 years of age and is on the rise worldwide. While the exact cause of macular degeneration remains unknown, known risk factors include age, smoking, high blood pressure, obesity, genetic predisposition, excessive UV exposure, and low blood antioxidant levels.

[0003] For the treatment or improvement of macular degeneration, aflibercept (Eylea) TM ) are being used clinically. However, treatment with these anti-VEGF antibodies requires repeated invasive injections into the eye (i.e., intravitreal injections). Direct injection into the eye has many disadvantages, such as limited capacity, very low patient compliance due to invasive repeated injections, high risk of bleeding, pain, infection, retinal detachment, and high treatment costs. Therefore, many patients are unable to receive repeated intraocular / intravitreal injections, and gradually become blind.

[0004] Therefore, there is a need in the art to develop therapeutic agents and / or formulations that can effectively treat or improve macular degeneration and also avoid invasive injections.

[0005] The present inventors conducted various studies on derivatives based on small peptides to develop compounds that can effectively treat or improve macular degeneration. As a result, the present inventors discovered that certain peptide derivatives possess excellent therapeutic and improvement activities against macular degeneration. In particular, the present inventors discovered that these specific peptide derivatives can be used in the form of eye drops, fundamentally avoiding invasive intraocular / intravitreal injections.

[0006] Accordingly, the present invention aims to provide a specific peptide derivative having excellent therapeutic and improving activity against macular degeneration.

[0007] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating macular degeneration, which comprises the specific peptide derivative as an active ingredient.

[0008] According to one aspect of the present invention, a peptide derivative of the following chemical formula 1 or a pharmaceutically acceptable salt thereof is provided.

[0009] <Chemical Formula 1>

[0010]

[0011] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating macular degeneration is provided, comprising a peptide derivative of the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] The present invention has revealed that a specific peptide derivative, i.e., the peptide derivative of the above chemical formula 1 or a pharmaceutically acceptable salt thereof, has excellent therapeutic and ameliorating activity against macular degeneration. Furthermore, the present invention has revealed that the peptide derivative of the above chemical formula 1 or a pharmaceutically acceptable salt thereof can be used in the form of eye drops, thereby fundamentally avoiding invasive intraocular / intravitreal injections. Therefore, the pharmaceutical composition according to the present invention can be usefully used for the prevention or treatment of macular degeneration.

[0013] Figure 1 shows the results of evaluating the cytotoxicity of the peptide (SE101B2L) of the present invention in HUVECs.

[0014] Figure 2 shows the results of evaluating the angiogenesis inhibitory activity of the peptide (SE101B2L) of the present invention in HUVECs. Figures 2a, 2b, and 2c show the results of evaluating the effects of the peptide (SE101B2L) of the present invention on tube formation, cell migration, and phosphorylation of signaling molecules in HUVECs, respectively.

[0015] Figure 3a shows the results of evaluating the regulatory ability of the peptide (SE101B2L) of the present invention on membrane proteins related to vascular stability and substance permeation in HUVECs.

[0016] Figure 3b shows the results of evaluating the inhibitory activity of the peptide of the present invention (SE101B2L) on substance permeation caused by changes in membrane proteins related to vascular stability in HUVECs.

[0017] Figure 4 shows the results of evaluating the enhancing activity of the peptide (SE101B2L) of the present invention on cell viability decreased by oxidative stress in ARPE19.

[0018] Figure 5 shows the results of evaluating the inhibitory activity of the peptide of the present invention (SE101B2L) against cell senescence induced by hydrogen peroxide in ARPE19 (Figure 5a: optical micrograph, Figure 5b: quantitative analysis results).

[0019] Figure 6 shows the results of evaluating the inhibitory activity of the peptide (SE101B2L) of the present invention against apoptosis induced by hydrogen peroxide in ARPE19 (Figure 6a: fluorescence micrograph, Figure 6b: quantitative analysis results of apoptotic cells, Figure 6c: quantitative analysis results of living cells).

[0020] Figure 7 shows the results of evaluating the pharmacological activity of the peptide (SE101B2L) of the present invention in an animal model of macular degeneration (CNV-induced chinchilla rabbit). Figures 7a and 7b show the results of analyzing retinal fluorescence intensity on the 7th and 14th days, respectively.

[0021] Figure 8 is a spectrum obtained by analyzing the peptide (SE101B2L) of the present invention manufactured in Example 1 using a mass spectrometer.

[0022] The present invention provides a peptide derivative having excellent therapeutic and improving activity for macular degeneration, i.e., a peptide derivative of the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0023] <Chemical Formula 1>

[0024]

[0025] The peptide derivative of the above chemical formula 1 may also be expressed as "decylbenzoyl-Cys-Phe-Lys-amide". In the peptide derivative of the above chemical formula 1, the amino acids constituting the peptide moiety (i.e., the Cys-Phe-Lys moiety) may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of the above chemical formula 1 include, but are not limited to, acid addition salts, for example.

[0026] The peptide derivative of the above chemical formula 1 can be prepared by various methods. For example, the peptide moiety (i.e., Cys-Phe-Lys moiety) can be synthesized using a general FMOC solid-phase peptide synthesis method (e.g., Adam, GK; Patrick, JS; Yang, H.; Gretchen, G. Standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory (Version 1.7.2), University of California, Irvine, CA, US, 2020) using a reactor (Polypropylene LibraTube, HipepKorea, Korea) and a shaker (EUROSTAR digital, IKA, Germany), or using an automatic peptide synthesizer (ASP48S, Peptron, Daejeon, Korea). Introduction of decylbenzoyl to the N-terminus can be carried out by coupling decylbenzoyl chloride (4-n-Decylbenzoyl chloride) to the N-terminus of the peptide moiety on the solid polymer support in the presence of diisopropylethylamine (DIEA) in dimethylformamide (DMF), or alternatively, by coupling decylbenzoic acid to the N-terminus of the peptide moiety on the solid polymer support using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HBTU) and 1-hydroxybenzotriazole (HOBt) as coupling agents in the presence of diisopropylethylamine (DIEA) in dimethylformamide (DMF). Here, if the solid polymer support is Rink Amide MBHA Resin, the C-terminus can be obtained in the amide form later. The obtained peptide derivative of chemical formula 1 is, for example, Inspire TM(C18 column (S-5 μm / 12nm.5 μm (20 x 250 mm), Dikma, USA) or Vydac Everest C18 column (250 mm x 22 mm, 10 μm) can be used for purification and analysis by reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan). Elution in chromatography can be performed with a water-acetonitrile linear gradient (5-90% (v / v) acetonitrile) containing 0.1% (v / v) trifluoroacetic acid, and the molecular weight of the obtained product can be determined by mass spectrometry (AXIMA Assurance TM , MALDI-TOF, Shimadzu, Japan) or LC / MS (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

[0027] The present invention also provides a pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide derivative of the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0028] In the pharmaceutical composition of the present invention, the macular degeneration includes, but is not limited to, age-related macular degeneration (AMD), macular dystrophy due to genetic causes, myopic macular degeneration due to high myopia, macular degeneration caused by inflammatory diseases, and trauma. Age-related macular degeneration is a disease in which the macula degenerates due to aging of the retina. Age-related macular degeneration includes both dry (non-exudative) macular degeneration and wet (exudative) macular degeneration. Dry (non-exudative) macular degeneration is associated with drusen, which are cellular debris in the macula that gradually damage light-sensitive cells and cause vision loss. Wet (exudative) macular degeneration is caused by blood vessels growing under the macula, leaking blood and fluid into the retina. As used herein, 'macular degeneration' includes all forms of macular degeneration mentioned above.

[0029] Preferably, the pharmaceutical composition according to the present invention may have the formulation of an eye drop. The eye drop may be in the form of a solution or a suspension. In one embodiment, the eye drop may include pharmaceutically acceptable additives such as a solubilizer, a stabilizer, a buffer, a pH adjuster, etc. If necessary, the pharmaceutical composition in the form of an eye drop may include a buffer such as boric acid; a pH adjuster such as hydrochloric acid or sodium hydroxide; a preservative, etc., and may be sterilized according to a conventional method.

[0030] In the pharmaceutical composition according to the present invention, the active ingredient, the peptide derivative of chemical formula 1 or a pharmaceutically acceptable salt thereof, may be administered at a concentration of about 0.1 to 200 w / v %, preferably about 1 to 20 w / v %, preferably via eye drops, but may vary depending on the patient's age, sex, health condition, severity of disease, etc. The administration (eye drops, etc.) may be performed at appropriate intervals, and may be administered, for example, once a day or in divided doses, according to the instructions of a doctor or pharmacist.

[0031] Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are intended to illustrate the present invention, and the present invention is not limited to these examples and test examples.

[0032] Example

[0033] A peptide having the sequence of Cys-Phe-Lys was synthesized by the FMOC solid-phase method using Rink Amide MBHA Resin (385 mg, 0.2 mmol) in a reactor (Polypropylene LibraTube®, HipepKorea, Korea) and a shaker (EUROSTAR digital, IKA, Germany).

[0034] Afterwards, diisopropylethylamine (0.348 ml) and decylbenzoyl chloride (0.28 ml) were sequentially added to the peptide moiety on the solid polymer support suspended in dimethylformamide (DMF, 8 ml), and stirred at room temperature for 1 hour to introduce a decylbenzoyl group at the N-terminus. Afterwards, the peptide derivative with the decylbenzoyl group introduced was liberated in the form of an amide from Rink Amide MBHA Resin and Inspire TM Decylbenzoyl-Cys-Phe-Lys-amide of chemical formula 1 (hereinafter referred to as 'SE101B2L') was prepared with a purity of 95% or more (230 nm) by reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan) using a C18 column (S-5 μm / 12nm.5 um (20 x 250 mm), Dikma, USA). The obtained SE101B2L was analyzed by mass spectrometry (AXIMA Assurance TM , MALDI-TOF, Shimadzu, Japan) was used to confirm the results, and the results are shown in Fig. 8. (MS cal.: 639.4, MS observed: [M+H] + = 640.2)

[0035] Test Example 1: Evaluation of angiogenesis inhibition activity using HUVECs

[0036] The angiogenesis inhibitory activity of the test substance (SE101B2L) was evaluated using human umbilical vein endothelial cells (HUVECs). HUVECs were cultured using CEFOgro-HUVEC basal medium (Cell Bio Co., Ltd.) supplemented with growth supplements (HUVECs supplements, Cell Bio Co., Ltd.) containing vascular endothelial cell growth-related factors and serum, as a vascular endothelial cell growth medium (hereinafter referred to as “growth medium”).

[0037] (1) Cytotoxicity of test substance

[0038] HUVECs were seeded at 5 x 10 per well in a 96-well plate. 3 The cells were seeded with 0.1 ml of growth medium. After culturing for one day, the test substance (SE101B2L) was treated at a final concentration of 0.001 nM (noted; log -3) and increased by 10-fold to 10,000 nM (noted; log 4). The test groups were then cultured for 24, 48, and 72 hours, and treated with Cell Counting Kit-8 (CCK-8) at the designated times, followed by an additional 2-hour culture, and the absorbance was measured at 450 nm. The viability of the cells was calculated relatively based on the absorbance value of the untreated group at 24 hours. The results are shown in Figure 1. From the results in Figure 1, there was no significant difference in cell viability at each time point up to a concentration of 1,000 nM (1 μM), and therefore, it can be confirmed that the test substance did not exhibit cytotoxicity against HUVECs up to the corresponding concentration.

[0039] (2) Inhibition of angiogenesis

[0040] The test was conducted by dividing the subjects into an untreated group, a group treated with vascular endothelial cell growth factor (VEGF) alone, and a group treated with VEGF and the test substance (SE101B2L) simultaneously.

[0041] First, the tube formation ability was confirmed to confirm whether the test substance inhibited angiogenesis induced by VEGF. HUVECs were seeded at 5 x 10 per well in a 48-well plate coated with Matrigel. 4The cells were seeded with 0.5 ml of growth medium. VEGF was administered to each VEGF-treated group to a final concentration of 10 ng / ml, and simultaneously, the test substance was administered to the test substance-treated groups to final concentrations of 0.1, 1, 10, and 100 nM. The test plates were cultured for 24 h in a 5% CO2, 37°C incubator. After 24 h, the degree of tube formation was confirmed under a microscope and photographs were taken. The total tube length was measured for each photograph using the ImageJ program. The results were converted into relative values ​​based on the tube length of the untreated group. The results are shown in Figure 2a.

[0042] In addition, it was confirmed whether the test substance could inhibit the change in cell migration ability of HUVECs by VEGF. Cell migration ability was confirmed by checking the change in cell migration by chemotaxis using a transwell chamber (24-well plate type). After coating 10 μg of gelatin on the lower surface of the membrane of a transwell insert with a diameter of 6.5 mm and a pore size of 8.0 μm, 5 X 10 4Cultured HUVECs were dispensed using 0.1 ml of HUVEC basal medium. The test substances were treated to final concentrations of 0.1, 1, 10, and 100 nM for the test substance treatment groups, and VEGF was treated to a final concentration of 10 ng / ml together with HUVEC basal medium in the lower well of a 24-well plate format. The cells were then incubated for 3 hours in a 5% CO2, 37°C incubator. The medium in the inserts of the test groups was then removed, and the inserts were fixed using a 10% formalin solution. The cells attached to the lower part of the insert chamber membrane were stained using a 0.5% crystal violet solution, and any unmigrated cells remaining inside the inserts were removed using a cotton swab. The migrated cells were photographed for each test group, and the number of cells was counted. The number of counted cells was calculated as a relative value based on the untreated group. The results are as shown in Fig. 2b.

[0043] Additionally, we tested whether the test substance could regulate the phosphorylation level of AKT and ERK molecules, which are important in signaling related to angiogenesis by VEGF. 2 X 10 in a 6-well plate 5HUVECs cells were seeded in 2 ml of growth medium and cultured for 1 day in a 5% CO2, 37℃ incubator. Afterwards, the cells were washed with phosphate-buffered saline (PBS) and cultured for an additional 6 hours using CEFOgro-HUVEC medium containing 1% fetal bovine serum. The prepared cells were divided into an untreated group, a VEGF-only group, and a VEGF and test substance co-treatment group (0.1, 1, 10, and 100 nM) and treated with the test substance. After an additional 10 min of incubation, the cells were lysed in NP-40 cell lysis buffer containing protease inhibitors (0.5 mM AEBSF hydrochloride, 150 nM aprotinin, 1 μM E-64, 1 μM leupeptin, and 1 mM PMSF) and phosphatase inhibitors (50 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na3VO4). The cell lysate was separated using SDS-PAGE after measuring the protein amount using the Bradford assay. After protein transfer to a PVDF membrane, immunoassay was performed using antibodies that recognize each target protein. The antibodies used were anti-phospho-AKT (S473) [sc-7985, Santa Cruz Biotech.], anti-phospho-ERK (T202 / Y204) [#4370, Cell Signaling Tech.], and anti-AKT [sc-271149, Santa Cruz Biotech.], and anti-ERK [sc-135900, Santa Cruz Biotech.]. The PVDF membrane onto which each prepared protein was transferred was blocked for 1 hour with a Tris-buffered saline solution (TBST) containing 3% bovine serum albumin (BSA), and then reacted with antibodies at 4°C for approximately 16 hours. Afterwards, the residual antibodies were removed, the membrane was washed with a TBST solution, and then reacted with an antibody conjugated with horseradish peroxidase (HRP), which recognizes the antibody, at room temperature for 1 hour. Afterwards, the residual antibodies were removed in the same manner, and washing was performed with a TBST solution. The prepared membrane was treated with an ECL solution and confirmed using a DaVinci-Chemi fluoro imager system. The results are shown in Fig. 2c.

[0044] From the results of Figures 2a, 2b, and 2c, it can be confirmed that the test substance concentration-dependently reduces the degree of tube formation, cell migration, and phosphorylation of signaling molecules. Therefore, it can be confirmed that the test substance can suppress the angiogenesis in macular degeneration by suppressing the increase in angiogenesis induced by VEGF.

[0045] (3) Confirmation of membrane protein stability and changes in material permeability of vascular endothelial cells

[0046] We confirmed whether the test substance (SE101B2L) could control the change in material permeability according to the change in stability of membrane proteins of vascular endothelial cells caused by VEGF. The expression level and pattern of proteins related to vascular stability and material permeability, i.e. junctional proteins that connect cells, VE-cadherin and ZO-1, were confirmed. One circular cover slip with a diameter of 12 mm was placed per well in a 24-well plate, and gelatin was applied using a 2% gelatin solution to help cell attachment. For the prepared wells, 1 X 10 per well 5HUVECs cells were seeded. They were cultured for 4 days in a 5% CO2, 37℃ incubator, changing the growth medium every other day. After confirming that the cells were fully confluent, they were cultured for 1 hour using serum-free CEFOgro-HUVECs medium. During this time, the test substances were treated to the corresponding wells at final concentrations of 10 and 100 nM. When the incubation was complete, VEGF was treated at a final concentration of 50 ng / ml and incubated for an additional 90 minutes. The cultured samples were fixed for 15 minutes at room temperature using 10% formalin solution. The fixed samples were washed with phosphate-buffered saline (PBS), and then perforated and blocked using PBS (containing 0.2% Triton X-100) containing 3% bovine serum albumin (BSA). Afterwards, the reaction was carried out overnight at 4°C using a blocking solution containing anti-VE-cadherin (#2158, Cell Signaling Tech.) or anti-ZO-1 (#40-2200, Invitrogen) antibody. After washing with PBS, the reaction was carried out for 2 hours at room temperature in the dark (blocking external light) using antibodies conjugated with Rhodamine Red. After completing the antibody reaction, the sample was washed with PBS, and the sample was mounted on a slide glass using a mounting solution containing DAPI. The sample was photographed using a fluorescence microscope. The results are shown in Fig. 3a.

[0047] In addition, in order to confirm the effect of the test substance in relation to the phenomenon of increased substance permeability due to the change in stability of vascular endothelial cell membrane protein triggered by VEGF, an in vitro endothelial cell substance permeation test was performed. The experiment was performed using a 24-well plate type transwell plate containing an insert with a diameter of 6.5 mm and a pore size of 0.1 μm. To increase cell adhesion to the cell attachment area inside the insert, gelatin was applied using a 2% gelatin solution. 1 ml of HUVECs cell growth medium was added to the lower part corresponding to the well of the plate, and 1 X 10 50.3 ml of cell growth medium containing the cells was dispensed. The cells were cultured in a 5% CO2, 37℃ incubator, and the medium in the insert and wells was replaced every two days for 4 days. To check the confluency of the cells, the inserts were transferred to new empty wells, and the flow of medium from the inserts to the wells was checked for 5 minutes at room temperature. If no medium flowed out, the medium in the inserts and the lower wells was replaced with serum-free basal medium, and the test substance was treated in the test substance treatment group at a final concentration of 1 nM and 10 nM in the inserts and wells. After culturing for 1 hour in a 5% CO2, 37℃ incubator, the medium in the well was replaced with new serum-free medium, and the inserts were treated with serum-free medium containing HRP-streptavidin conjugate to the untreated group, VEGF with a final concentration of 50 ng / mL, and VEGF and test substance co-treatment (1 and 10 nM) groups, respectively, and incubated for 30 minutes in a 5% CO2, 37℃ incubator. Afterwards, the inserts were removed, and the serum-free medium in the wells was collected. The collected medium was dispensed into a 96-well plate at 20 μL per well, and 50 μL of TMB solution was treated per well to induce a color reaction, and then the color reaction was stopped using a 1 M hydrochloric acid solution. The absorbance was then measured at 450 nm. The measured values ​​were converted to relative values ​​based on the absorbance of the untreated group sample. The results are shown in Figure 3b.

[0048] From the results of Figures 3a and 3b, it can be confirmed that when the test substance was treated, the expression and pattern of the protein induced by VEGF in the cell-to-cell junction protein expression level were similar to those of the untreated group in a concentration-dependent manner. In addition, it can be confirmed that the phenomenon of increased substance penetration due to decreased vascular stability by VEGF was reduced by treating the test substance. These results show that the test substance can inhibit the penetration of intravascular substances into the ocular tissue caused by uncontrolled neovascularization in macular degeneration.

[0049] Test Example 2: Evaluation of inhibitory activity against cell senescence and apoptosis using human retinal pigment epithelial cells.

[0050] (1) Cell culture

[0051] Human retinal pigment epithelial cells (ARPE19) were cultured in a humidified incubator at 37°C and 5% CO2. The medium used was DMEM / F12 (Gibco) containing 10% fetal bovine serum and penicillin (100 U / ml) / streptomycin (100 μm / ml). Cultures were performed with fresh medium replaced every two days, and passages were performed using 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid (EDTA).

[0052] (2) Enhancement of cell viability of SE101B2L under oxidative stress

[0053] After inducing oxidative stress in human retinal pigment epithelial cells using hydrogen peroxide (H2O2), changes in cell viability were confirmed by treating the cells with test substances (SE101B2L) at various concentrations. Cell counting kit-8 (CCK-8) was used to confirm cell viability.

[0054] 1×10 ARPE19 in a 96-well plate 4Cells were seeded at 10 cells / well and cultured in an incubator for 24 hours. The treatment group was treated with 400 μM hydrogen peroxide and the test substance at concentrations of 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 2 μM, respectively, and cultured for 24 hours. In addition, to confirm the change when the test substance was treated alone, the test substance was treated at concentrations of 0.01 μM, 0.1 μM, 1 μM, and 2 μM, respectively, without hydrogen peroxide treatment and cultured for 24 hours. The untreated group (denoted as “No treat”) was not treated and cultured for 24 hours. After culture, 96-well plates containing 90 μL of cell suspension per well were inoculated with 10 μL of CCK-8 pre-filled solution. The plates were incubated for 2 hours, and cell viability was confirmed through quantification by measuring the OD450 of the supernatant. The results are shown in Figure 4. From the results in Figure 4, it can be confirmed that cell viability was significantly reduced in the group treated with only hydrogen peroxide, and when hydrogen peroxide and the test substance were treated simultaneously, cell viability was confirmed to increase in a concentration-dependent manner. These results demonstrate that the test substance effectively increases cell viability, which is reduced by oxidative stress induced by hydrogen peroxide.

[0055] (3) Inhibition of cellular senescence induced by hydrogen peroxide

[0056] After oxidative stress-induced cell senescence in ARPE19 cells was promoted using hydrogen peroxide (H2O2), the test substance (SE101B2L) was treated at various concentrations to evaluate its anti-cellular senescence effect. The anti-cellular senescence activity was evaluated using SA-β-galactosidase activity staining (senescence-associated β-galactosidase; SA-β-Gal), a well-known marker of cellular senescence.

[0057] 1×10 ARPE19 in a 96-well plate 5Cells were seeded at 1 / well and cultured in a 37°C, 5% CO2 incubator for 24 hours, reaching approximately 80% confluency. The treatment group was treated with 200 μM hydrogen peroxide and the test substance at concentrations of 0 μM, 0.01 μM, 0.1 μM, and 1 μM, respectively, and cultured for 24 hours. In addition, to confirm the change when the test substance was treated alone, the test substance was treated at a concentration of 1 μM without hydrogen peroxide treatment and cultured for 24 hours. The untreated group (indicated as “No treat”) was cultured for 24 hours without any treatment. After incubation, the cells in each well were washed with PBS and fixed with 2% formaldehyde for 15 minutes. After additional washing with PBS three times, the staining solution was added according to the manufacturer's instructions using the Senescence β-Galactosidase Staining kit (Cell Signaling Technology) and the staining step was performed at 37℃ overnight. The blue-stained cells were observed under an optical microscope, and the degree of SA-β-gal activity was expressed as a percentage by measuring the number of cells with blue staining in the cytoplasm among a total of 50 to 100 cells. The degree of senescence was quantified through the measurement, and the results are as shown in Figures 5a and 5b. From the results of Figures 5a and 5b, it can be confirmed that SA-β-gal activity decreased in a concentration-dependent manner when hydrogen peroxide and the test substance were treated simultaneously. These results demonstrate that the test substance effectively inhibits cell senescence induced by hydrogen peroxide.

[0058] (4) Inhibition of apoptosis induced by hydrogen peroxide

[0059] After inducing apoptosis in ARPE19 cells using hydrogen peroxide (H2O2), the inhibitory effect of the test substance (SE101B2L) was evaluated by treating the cells at various concentrations. The inhibitory effect on apoptosis was measured using phosphatidylserine (PS), which is exposed on the surface of apoptotic cells.

[0060] ARPE19 was seeded at 1 × 10 in a 24-well microplate containing a coverslip. 6Cells were seeded at 1 / well and cultured in a 37°C, 5% CO2 incubator for 24 hours, reaching approximately 80% confluency. The treatment group was treated with 200 μM hydrogen peroxide and the test substance at concentrations of 0 μM, 0.01 μM, 0.1 μM, and 1 μM, respectively, and cultured for 24 hours. In addition, to confirm the change when the test substance was treated alone, the test substance was treated at a concentration of 1 μM without hydrogen peroxide treatment and cultured for 24 hours. The untreated group (indicated as “No treat”) was cultured for 24 hours without any treatment. After culture, the cells were washed twice with the washing solution provided by the manufacturer according to the manufacturer's instructions using an apoptosis assay kit (abcam) for phosphatidylserine (PS) measurement, and the staining step was performed simultaneously at room temperature for 1 hour with phosphatidylserine (green) for apoptotic cell staining and CytoCalcein Violet 450 (blue), a cytoplasmic marker dye for live cell staining, without fixing the cells. After fixation by treating with 2% formaldehyde for 15 minutes and washing three times with PBS, the mounting step was performed. The luminescence signal detected in each cell was measured with a digital fluorescence microscope (CELENA S digital imaging system, Logos Biosystems, Korea) to quantify apoptotic and live cells. The results are shown in Figs. 6a, 6b, and 6c. From the results in Figures 6a, 6b, and 6c, it can be confirmed that the number of cells induced to undergo apoptosis (green) by hydrogen peroxide increases and the number of living cells (blue) decreases, and in the case of cells treated with hydrogen peroxide and the test substance, the number of cells induced to undergo apoptosis (green) decreases in a concentration-dependent manner and the number of living cells (blue) increases. These results demonstrate that the test substance effectively inhibits apoptosis induced by hydrogen peroxide.

[0061] Test Example 3: Evaluation of Pharmacological Activity for Macular Degeneration

[0062] (1) Test method

[0063] As an animal model of macular degeneration, male chinchilla rabbits with induced choroidal neovascularization (CNV) were used. The efficacy test on the CNV animal model was performed by HLB Biostep Co., Ltd. (formerly Notus Co., Ltd.). To induce CNV, a mydriatic agent (1% Midriacyl eye drop) was instilled into the right eye of the chinchilla rabbit, and after anesthesia was administered, a laser (LIGHTLas 532, LIGHTMED, USA) was irradiated to the right eye at 532 nm, power 150 mW for 0.1 s, creating six wounds at the 6 o'clock position centered on the optic nerve.

[0064] The test group was divided into four groups (n=8) according to Table 1 below. The provocation control group (G1) received repeated eye drops of 0.9% sodium chloride solution (vehicle) twice a day. The positive control group (G2) received aflibercept (Eylea), a treatment for macular degeneration. TM ) was administered intravitreously once on the day of CNV induction. The test substance treatment groups (G3 and G4) used solutions prepared by dissolving the test substance (SE101B2L) in a 0.9% sodium chloride aqueous solution as a vehicle to a concentration of 3750 ppm (G3) and 187.5 ppm (G4), respectively, and administered repeatedly by eye drop twice a day (50 μL per time). In the case of intravitreous administration, after anesthetizing the animal, intravitreous administration was performed using a syringe equipped with a 31-gauge needle into the right eye of the animal. In the case of eye drop administration, the test substance was instilled into the center of the cornea of ​​the right eye using a pipette.

[0065] Animal number, administered substance, route of administration, amount of solution (㎕ / eye), number of administrations, G18 vehicle, eye drops, 502 times / day, G28 Eye drops ®Intravitreal injection 50 times per day G38 test substance (3750 ppm) Eye drop 502 times / day G48 test substance (187.5 ppm) Eye drop 502 times / day

[0066] After the start of administration to each group, on days 0, 7, and 14, the animals were anesthetized after instilling a mydriatic agent (1% Midriacyl eye drops) into the right eye. Approximately 1 ml of a 2% fluorescein sodium salt solution was injected into the intraocular vein, and images were taken within approximately 2 minutes using a fundus camera (TRC-50IX, TOCON, Japan). Retinal CNV confirmation and drug efficacy evaluation were performed using retinal fluorescein fundus photographs, and image analysis was performed using ImageJ software (NIH, Bethesda, MD) to analyze the fluorescence intensity of the irradiated area.

[0067] The results of this study assumed normality and tested the significance between test groups using parametric one-way ANOVA. Statistical analysis was performed using Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA), and a p value less than 0.05 was considered statistically significant.

[0068] (2) Test results

[0069] The results of analyzing retinal fluorescence intensity on the 7th and 14th days after the start of test substance administration are shown in Figures 7a and 7b, respectively. As can be confirmed from the results of Figures 7a and 7b, the retinal fluorescence intensity levels of the positive control group (G2) and test substance administration groups (G3 and G4) were statistically significantly lower (p<0.001) than those of the induced control group (G1). These results demonstrate that the ocular administration of the peptide derivative according to the present invention exhibits pharmacological activity equivalent to that of conventional macular degeneration treatments requiring invasive intravitreal injection.

Claims

1. A peptide derivative of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <Chemical formula 1> 2. A pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide derivative of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Chemical formula 1> 3. A pharmaceutical composition having the formulation of an eye drop according to claim 2.

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