KR102593936B1 - Composition for treating macular degeneration comprising novel peptides - Google Patents

Abstract

The present invention relates to a composition for preventing or treating macular degeneration containing a novel peptide. Specifically, the peptide of the present invention has an excellent effect of inhibiting choroidal neovascularization, a major cause of macular degeneration, and exhibits excellent stability and solubility, so it can be developed as an excellent treatment for macular degeneration.

Description

Composition for the treatment of macular degeneration comprising a novel peptide {COMPOSITION FOR TREATING MACULAR DEGENERATION COMPRISING NOVEL PEPTIDES}

The present invention relates to a composition for preventing or treating macular degeneration containing a novel peptide. Specifically, the peptide of the present invention has the effect of inhibiting choroidal neovascularization, a major cause of macular degeneration, and has excellent stability and solubility.

The macula is the part that plays the most important role in the neural layer within the eye called the retina. It is the central part of the retina with a radius of about 1.5 mm, and photoreceptors that can sense light are concentrated in this area. Therefore, the macula is the center of the retina and is responsible for most of vision. Macular degeneration is a disease in which the macula degenerates due to aging, genetic factors, toxicity, inflammation, etc. As the macula degenerates, vision decreases, and in severe cases, vision may be lost completely.

Macular degeneration is largely divided into non-exudative (dry) and exudative (wet). Non-exudative macular degeneration accounts for 80-90% of all macular degeneration and does not significantly affect vision in most cases, except in the later stages when retina and choroidal atrophy appear. No. However, caution is needed as it can progress to exudative macular degeneration. Exudative macular degeneration accounts for approximately 10-20% of all macular degeneration, but if left untreated, vision deteriorates rapidly and many patients become blind within two years after diagnosis.

Exudative macular degeneration is a stage in which choroidal neovascularization occurs and has a very poor vision prognosis, making it the disease with the highest incidence of blindness in people over 65 years of age. The vascular layer called the choroid serves the role of supplying nutrients to the retinal layer and removing metabolites from retinal cells. However, due to causes such as aging, the blood vessels of the choroid may penetrate into the retinal cells and form abnormally. In this case, the abnormal blood vessels are called choroidal neovascularization. Because these blood vessels are abnormal, they are very weak and prone to bursting, causing exudate and blood to flow out and damage the macular area.

Treatment of macular degeneration includes taking antioxidant vitamins known to slow the progression of macular degeneration in the case of non-exudative macular degeneration and treating high blood pressure and hyperlipidemia, which are risk factors for macular degeneration. For exudative macular degeneration, there are thermal laser photocoagulation, photodynamic therapy, antibody injection, and vitrectomy, but there is still no complete treatment and active research is in progress. Recently, the method of injecting anti-vascular endothelial growth factor antibodies (Anti-VEGF antibodies) such as aflibercept, ranibizumab, and bevacizumab into the eye has been mainly used, but has disadvantages such as repeated administration and high cost. There is.

Accordingly, as a result of research on substances that can effectively treat macular degeneration, the present inventors identified a novel peptide that not only has excellent stability and solubility, but also has an excellent effect of inhibiting choroidal neovascularization, and completed the present invention.

Korean Patent Publication No. 10-2020-0060397

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating macular degeneration containing the peptide represented by Formula 1.

In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating macular degeneration containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to 46.

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

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

In the _above formula, D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro-L-proline) , 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline ( It is an amino acid selected from the group consisting of 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 is one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester,

Here, if there is no X ₆ , there is also no X ₇ .

In _one embodiment, in the above formula, 4-hydroxy-D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro- L-proline), 4-methylene-L-proline (4-methylene-L-proline), 4,4-dimethyl-L-proline (4,4-dimethyl-L-proline), and trans-4-amino- It is an amino acid selected from the group consisting of L-proline (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 Glycine, phenyl-Glycine, Alanine, Valine, Leucine, tert-Leucine and 2-aminoisobutyric acid),

X ₆ is absent, Leucine or D-Leucine,

X ₇ may be absent or may be one or more amino acids selected from the group consisting _of Alanine, D-Alanine, and Alanine-isopropyl ester, where , there is no X ₇ either.

In _one embodiment, in the above formula, 4-hydroxy-D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro- L-proline), 4-methylene-L-proline (4-methylene-L-proline), 4,4-dimethyl-L-proline (4,4-dimethyl-L-proline), and trans-4-amino- It is an amino acid selected from the group consisting of L-proline (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 may be one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

In _one embodiment, in the above formula, 4-hydroxy-D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro- L-proline), 4-methylene-L-proline (4-methylene-L-proline), 4,4-dimethyl-L-proline (4,4-dimethyl-L-proline), and trans-4-amino- It is an amino acid selected from the group consisting of L-proline (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 Glycine, phenyl-Glycine, Alanine, Valine, Leucine, tert-Leucine and 2-aminoisobutyric acid),

X ₆ is Leucine or D-Leucine,

X ₇ may be one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

In _one embodiment, in the above formula, 4-hydroxy-D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro- L-proline), 4-methylene-L-proline (4-methylene-L-proline), 4,4-dimethyl-L-proline (4,4-dimethyl-L-proline), and trans-4-amino- It is an amino acid selected from the group consisting of L-proline (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 may be one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester.

The composition of the present invention may contain any one peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 to 46, and preferably may include a peptide consisting of the amino acid sequence of SEQ ID NO: 44.

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

The macular degeneration of the present invention may be one or more selected from the group consisting of wet macular degeneration, dry macular degeneration, and age-related macular degeneration.

The present invention provides a method of treating macular degeneration comprising administering a peptide represented by the following formula (1) to a subject with macular degeneration disease.

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

In the _above formula, D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro-L-proline) , 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline ( It is an amino acid selected from the group consisting of 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 is one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester,

Here, if there is no X ₆ , there is also no X ₇ .

The present invention provides a composition for the treatment or prevention of macular degeneration containing the peptide represented by Formula 1 above.

The present invention provides the use of a composition for the treatment or prevention of macular degeneration containing the peptide represented by Formula 1 above.

The peptide of the present invention has excellent stability and solubility and has an excellent effect of inhibiting choroidal neovascularization, which is the main cause of macular degeneration, and can be usefully used for the prevention or treatment of macular degeneration.

Figures 1 and 2 show the results of FFA (Fundus Fluorescein Angiogrpahy) measurement and CTF (Corrected Total Fluorescence) calculation for CNV of the peptide of the present invention.
Figures 3 and 4 show the results of OCT (optical coherence tomography) measurement and CNV lesion size calculation for CNV of the peptide of the present invention.
Figures 5 and 6 show electroretinography results for CNV of the peptide of the present invention.
Figures 7 and 8 show the results of FFA (Fundus Fluorescein Angiogrpahy) measurement and vascular leakage quantification for CNV of the peptide of Example 44 (002-175) of the present invention.
Figures 9 and 10 show the results of OCT (optical coherence tomography) measurement and CNV lesion size calculation for CNV of the peptide of Example 44 (002-175) of the present invention.

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

Throughout the specification of the present application, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary.

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

[Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇

In the _above formula, D-proline), cis-4-fluoro-L-proline (Cis-4-fluoro-L-proline), 4,4,-difluoro-L-proline (4,4-difluoro-L-proline) , 4-methylene-L-proline, 4,4-dimethyl-L-proline, and trans-4-amino-L-proline ( It is an amino acid selected from the group consisting of 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 is one or more amino acids selected from the group consisting of alanine, D-alanine, and alanine-isopropyl ester,

Here, if there is no X ₆ , there is also no X ₇ .

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

Example peptide number amino acid sequence sequence number One 002-053 Hyp Gly Gln Asp Val One 2 002-074 Hyp Gly Gln Asp Leu 2 3 002-075 Hyp Gly Gln Asp Ala 3 4 002-103 Hyp Gly D-Gln Asp Gly 4 5 002-104 Hyp Gly Gln D-Asp Gly 5 6 002-085 D-Hyp Gly Gln Asp Gly 6 7 002-076 Hyp Gly Gln Asp Val Leu 7 8 002-077 Hyp Gly Gln Asp Leu Leu 8 9 002-078 Hyp Gly Gln Asp Ala Leu 9 10 002-105 Hyp Gly D-Gln Asp Gly Leu 10 11 002-106 Hyp Gly Gln D-Asp Gly Leu 11 12 002-107 Hyp Gly Gln Asp Gly D-Leu 12 13 002-090 D-Hyp Gly Gln Asp Gly Leu 13 14 002-055 Hyp Gly Gln Asp Val Leu Ala 14 15 002-080 Hyp Gly Gln Asp Leu Leu Ala 15 16 002-081 Hyp Gly Gln Asp Ala Leu Ala 16 17 002-084 Hyp Gly Gln Asp Gly Leu Ala-IPE 17 18 002-108 Hyp Gly D-Gln Asp Gly Leu Ala 18 19 002-109 Hyp Gly Gln D-Asp Gly Leu Ala 19 20 002-110 Hyp Gly Gln Asp Gly D-Leu Ala 20 21 002-111 Hyp Gly Gln Asp Gly Leu D-Ala 21 22 002-086 cis-4F-Pro Gly Gln Asp Gly 22 23 002-087 trans-4NH ₂ -Pro Gly Gln Asp Gly 23 24 002-088 4,4-difluoro-Pro Gly Gln Asp Gly 24 25 002-089 4-methylene-Pro Gly Gln Asp Gly 25 26 002-100 4,4-dimethyl Pro Gly Gln Asp Gly 26 27 002-091 cis-4F-Pro Gly Gln Asp Gly Leu 27 28 002-092 trans-4NH ₂ -Pro Gly Gln Asp Gly Leu 28 29 002-093 4,4-difluoro-Pro Gly Gln Asp Gly Leu 29 30 002-094 4-methylene-Pro Gly Gln Asp Gly Leu 30 31 002-101 4,4-dimethyl Pro Gly Gln Asp Gly Leu 31 32 002-095 D-Hyp Gly Gln Asp Gly Leu Ala 32 33 002-096 cis-4F-Pro Gly Gln Asp Gly Leu Ala 33 34 002-099 4-methylene-Pro Gly Gln Asp Gly Leu Ala 34 35 002-102 4,4-dimethyl Pro Gly Gln Asp Gly Leu Ala 35 36 002-130 Hyp Gly Gln Glu Gly 36 37 002-169 Hyp Gly Gln Glu Leu 37 38 002-170 Hyp Gly Gln Glu Val 38 39 002-167 Hyp Gly Gln Glu Leu Leu 39 40 002-168 Hyp Gly Gln Glu Val Leu 40 41 002-132 Hyp Gly Gln Glu Gly Leu Ala 41 42 002-165 Hyp Gly Gln Glu Leu Leu Ala 42 43 002-166 Hyp Gly Gln Glu Val Leu Ala 43 44 002-175 Hyp Gly Gln Glu Aib Leu Ala 44 45 002-176 Hyp Gly Gln Glu tert-Leu Leu Ala 45 46 002-177 Hyp Gly Gln Glu phenyl-Gly Leu Ala 46

The term “macular degeneration” used in the present invention refers to a disease in which vision decreases as the macula deteriorates due to aging, genetic factors, toxicity, inflammation, etc., and in severe cases, vision may be completely lost.

In the present invention, the macular degeneration may include wet and dry macular degeneration, and age-related macular degeneration, but is not limited thereto.

The term “choroidal neovascularization (CNV)” used in the present invention refers to the process of generating new choroidal capillaries. The choroid is a highly vascular membrane of the eye and refers to 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 is the structural deformation of abnormal blood vessels and Bruch's membrane with stems formed from the choroid. This means that the subretinal space is violated due to destruction.

The term "angiogenesis" used in the present invention refers to the process of forming new blood vessels, that is, the development of new blood vessels into cells, tissues, or organs, and "new blood vessels" refers to blood vessels newly created through the angiogenesis process. it means. As used herein, “angiogenesis” and “new blood vessel” may be used interchangeably.

The CNV animal model used in the present invention is a mouse model in which lesions are induced by irradiating a laser to the choroid of C57BL/6 mice using a laser slit system. The laser-induced CNV model, first described in 1979, uses photocoagulation to disrupt Bruch's membrane to induce the growth of choroidal neovascularization into the retina area, and is similar to the human retina in that neovascularization arises from the choroid. It is similar in development to that of wet macular degeneration (wet AMD), a neovascular disease that occurs within the body. In addition, the laser-induced CNV model was successful in predicting the clinical efficacy of anti-vascular endothelial growth factor (VEGF) treatment for neovascular AMD, and is an animal model used in the development stage of existing treatments (Gong Y, Li J, Sun Y, Fu Z, Liu C-H, Evans L, et al. (2015) Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS ONE 10(7)).

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-4NH ₂ -Pro is trans-4-amino-L-proline,

Gly is glycine,

Gln is glutamine,

D Gln is D-Glutamine,

Ala is alanine,

Val is valine,

Leu is leucine,

Ile is Isoleucine,

Met is methionine,

Tert-Leu or Tert-Leucine is L-α-tert-Butylglycine,

Aib is 2-aminoisobutyric acid,

Isopropyl ester (IPE) is a derivative in which the terminal amino acid group is replaced with 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.

In the present invention, polar amino acid refers to an amino acid whose side chain is polar in the chemical structure of the amino acid, and includes and is limited to Thr, Ser, Cys, Asn, Gln, Asp, Glu, His, Lys, and Arg. It doesn't work.

In the present invention, non-polar amino acid refers to an amino acid whose side chain does not have polarity due to the chemical structure of the amino acid, and includes, but is limited to, Met, Leu, Pro, Ala, Gly, Val, Ile, and Aib. It doesn't work.

As used in the present invention, the term "prevention" refers to any act of suppressing or delaying the onset of a disease by administering a composition, and "treatment" refers to improving or beneficially improving the symptoms of an individual suspected of having a disease or developing a disease by administering a composition. It means any action that changes.

The present invention will be described in more detail through examples below. However, the examples below are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Manufacturing example]

The peptide of the present invention was synthesized based on the previously known solid phase peptide synthesis (SPPS) method, and the manufacturing process included the following steps.

Step 1: Resin soaking and loading

Step 2: Solid Phase Peptide Synthesis (SPPS)

Step 3: Deprotected peptide synthesis (global cleavage)

Step 4: Primary purification and concentration/Secondary purification and concentration

Step 5: Freeze drying

The solid-phase peptide synthesis method (SPPS) involves loading the first amino acid into a resin, deprotecting the N-terminus of the amino acid with Fmoc, performing amino acid coupling according to the amino acid sequence, and synthesizing the protected peptide in a solid-phase reactor. Additionally, the amino acid coupling includes loading the first amino acid into the resin and then removing Fmoc from the N-terminus of the amino acid; After completion of the reaction, removing the solvent and washing the resin; Coupling the next amino acid according to the sequence; After completion of the reaction, removing the solvent and washing the resin; This included repeating the above process until the final amino acid sequence was generated.

The alpha amine group of each amino acid is protected with a base-vulnerable Fmoc group, and the side chain functional group is protected with an acid-vulnerable group. All amino acids except Gly and Aib are in the L-configuration, and among them, several amino acids Hyp(tBu), Glu(tBu), and Gln(Trt) have unique protecting groups as follows. And amino acids such as Ala, Leu, Aib, and Gly do not have side chain protecting groups. Therefore, after completing amino acid coupling according to the sequence, the resin and protecting group were removed from the peptide to obtain a crude peptide. Thereafter, the peptide compounds of Examples 1 to 55 were obtained through purification, concentration, and freeze-drying.

[Test Example 1]

Evaluation of the efficacy of peptides to inhibit choroidal neovascularization: Measurement of fluorescence value (CTF; Corrected total fluorescence) at the CNV site

To evaluate the choroidal neovascularization (CNV) inhibitory effect of the peptide of the present invention, an experiment was performed as follows.

7-8 week old C57BL/6 mice were instilled with mydriatic to dilate the pupils and anesthetized by intraperitoneal injection of Ketamine (50 mg/Kg) and Rompun (23.32 mg/Kg). Using a slit-lamp laser injection system, the laser was irradiated to the choroid of each eye of the mouse four times in the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions. At this time, the laser irradiation conditions were 532 nm, 80 msec, and 240 mW. Immediately after laser irradiation, 1 ul of each peptide dissolved in PBS at a concentration of 10 mg/mL or aflibercept at a concentration of 20 mg/mL was injected into the vitreous body using a syringe. Ten days after drug administration, mice were anesthetized and 2% fluorescein was administered to the abdominal cavity to stain blood vessels. The retina was photographed 2 to 3 minutes after fluorescein administration. The size and fluorescence of CNV lesions induced by the laser were measured using the imageJ program, and the background was removed to reduce inter-individual differences to calculate CTF (Corrected total fluorescence) and compare the values of each group. The results of all tests are expressed as mean ± standard deviation, and significance was verified using one-way ANOVA and Kruskal-wallis test.

The results of the experiment are shown in Table 2 below and Figures 1 and 2.

Example peptide number amino acid sequence CTF* One 002-053 Hyp Gly Gln Asp Val +++ 2 002-074 Hyp Gly Gln Asp Leu + 3 002-075 Hyp Gly Gln Asp Ala + 4 002-103 Hyp Gly D-Gln Asp Gly + 5 002-104 Hyp Gly Gln D-Asp Gly + 6 002-085 D-Hyp Gly Gln Asp Gly + 7 002-076 Hyp Gly Gln Asp Val Leu ++ 8 002-077 Hyp Gly Gln Asp Leu Leu ++ 9 002-078 Hyp Gly Gln Asp Ala Leu ++ 10 002-105 Hyp Gly D-Gln Asp Gly Leu + 11 002-106 Hyp Gly Gln D-Asp Gly Leu + 12 002-107 Hyp Gly Gln Asp Gly D-Leu + 13 002-090 D-Hyp Gly Gln Asp Gly Leu ++ 14 002-055 Hyp Gly Gln Asp Val Leu Ala ++ 15 002-080 Hyp Gly Gln Asp Leu Leu Ala ++ 16 002-081 Hyp Gly Gln Asp Ala Leu Ala + 17 002-084 Hyp Gly Gln Asp Gly Leu Ala-IPE ++ 18 002-108 Hyp Gly D-Gln Asp Gly Leu Ala + 19 002-109 Hyp Gly Gln D-Asp Gly Leu Ala + 20 002-110 Hyp Gly Gln Asp Gly D-Leu Ala + 21 002-111 Hyp Gly Gln Asp Gly Leu D-Ala + 22 002-086 cis-4F-Pro Gly Gln Asp Gly + 23 002-087 trans-4NH2-Pro Gly Gln Asp Gly ++ 24 002-088 4,4-difluoro-Pro Gly Gln Asp Gly + 25 002-089 4-methylene-Pro Gly Gln Asp Gly + 26 002-100 4,4-dimethyl Pro Gly Gln Asp Gly + 27 002-091 cis-4F-Pro Gly Gln Asp Gly Leu +++ 28 002-092 trans-4NH2-Pro Gly Gln Asp Gly Leu + 29 002-093 4,4-difluoro-Pro Gly Gln Asp Gly Leu + 30 002-094 4-methylene-Pro Gly Gln Asp Gly Leu + 31 002-101 4,4-dimethyl Pro Gly Gln Asp Gly Leu + 32 002-095 D-Hyp Gly Gln Asp Gly Leu Ala + 33 002-096 cis-4F-Pro Gly Gln Asp Gly Leu Ala + 34 002-099 4-methylene-Pro Gly Gln Asp Gly Leu Ala + 35 002-102 4,4-dimethyl Pro Gly Gln Asp Gly Leu Ala + 36 002-130 Hyp Gly Gln Glu Gly + 37 002-169 Hyp Gly Gln Glu Leu - 38 002-170 Hyp Gly Gln Glu Val + 39 002-167 Hyp Gly Gln Glu Leu Leu + 40 002-168 Hyp Gly Gln Glu Val Leu + 41 002-132 Hyp Gly Gln Glu Gly Leu Ala + 42 002-165 Hyp Gly Gln Glu Leu Leu Ala + 43 002-166 Hyp Gly Gln Glu Val Leu Ala + 44 002-175 Hyp Gly Gln Glu Aib Leu Ala ++ 45 002-176 Hyp Gly Gln Glu tert-Leu Leu Ala + 46 002-177 Hyp Gly Gln Glu phenyl-Gly Leu Ala + PBS - aflibercept + *CTF value + : 300,000 ~ 800,000 ++ : 200,000 ~ 300,000 +++ : 100,000 ~ 200,000

As shown in Table 2 and Figures 1 and 2, as a result of measuring the fluorescence value of the CNV site, it was confirmed that the peptide of the present invention has a CNV inhibitory effect, especially Example 1 (002-053) and Example 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 44 (002-175) peptides showed very excellent inhibitory efficacy. Therefore, it can be seen that the peptide of the present invention has excellent CNV inhibition efficacy.

[Test Example 2]

Evaluation of the efficacy of peptides to inhibit choroidal neovascularization: Optical Coherence Tomography

To evaluate the CNV (choroidal neovascularization) inhibitory effect of the peptide of the present invention, an experiment was performed as follows.

7-8 week old C57BL/6 mice were instilled with mydriatic to dilate the pupils and anesthetized by intraperitoneal injection of Ketamine (50 mg/Kg) and Rompun (23.32 mg/Kg). Using a slit-lamp laser injection system, the laser was irradiated to the choroid of each eye of the mouse four times in the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions. At this time, the laser irradiation conditions were 532 nm, 80 msec, and 240 mW. Immediately after laser irradiation, 1 ul of each peptide dissolved in PBS at a concentration of 10 mg/mL or aflibercept at a concentration of 20 mg/mL was injected into the vitreous body using a syringe. 10 days after drug administration, the mouse was anesthetized, and the CNV lesion induced by the laser was calculated and expressed as μm ² using the ImageJ program by transmitting an OCT beam for each burn. The results of all tests are expressed as mean ± standard deviation, and significance was verified using one-way ANOVA and Kruskal-wallis test.

The experimental results are shown in Table 3 below and Figures 3 and 4.

Example peptide number amino acid sequence Size of lesion (μm ² ) One 002-053 Hyp Gly Gln Asp Val 5,636 3 002-075 Hyp Gly Gln Asp Ala 6,491 7 002-076 Hyp Gly Gln Asp Val Leu 6,046 13 002-090 D-Hyp Gly Gln Asp Gly Leu 6,395 14 002-055 Hyp Gly Gln Asp Val Leu Ala 5,407 15 002-080 Hyp Gly Gln Asp Leu Leu Ala 6,672 17 002-084 Hyp Gly Gln Asp Gly Leu Ala-IPE 5,978 22 002-086 cis-4F-Pro Gly Gln Asp Gly 10,100 24 002-088 4,4-difluoro-Pro Gly Gln Asp Gly 9,301 29 002-093 4,4-difluoro-Pro Gly Gln Asp Gly Leu 9,669 34 002-099 4-methylene-Pro Gly Gln Asp Gly Leu Ala 8,603 38 002-170 Hyp Gly Gln Glu Val 5,775 42 002-165 Hyp Gly Gln Glu Leu Leu Ala 4,960 43 002-166 Hyp Gly Gln Glu Val Leu Ala 6,102 44 002-175 Hyp Gly Gln Glu Aib Leu Ala 5,393 PBS 10,428 aflibercept 5,437

As shown in Table 3 and Figures 3 and 4, the size of the CNV lesion was measured, and it was confirmed that the size of the CNV lesion was reduced when the peptide of the present invention was injected. Therefore, it can be seen that the peptide of the present invention has excellent CNV inhibition efficacy.

[Test Example 3]

Evaluation of efficacy in inhibiting choroidal neovascularization: Electroretinography

To evaluate the CNV (choroidal neovascularization) inhibitory effect of the peptide of the present invention, an experiment was performed as follows.

7-8 week old C57BL/6 mice were instilled with mydriatic to dilate the pupils and anesthetized by intraperitoneal injection of Ketamine (50 mg/Kg) and Rompun (23.32 mg/Kg). Using a slit-lamp laser injection system, the laser was irradiated to the choroid of each eye of the mouse four times in the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions. At this time, the laser irradiation conditions were 532 nm, 80 msec, and 240 mW. Immediately after laser irradiation, 1 μL of 10 mg/mL peptide or 20 mg/mL aflibercept dissolved in PBS was injected into the vitreous body using a syringe. For electroretinogram analysis, dark adaptation was performed for more than 12 hours. After the mouse was dilated and anesthetized in a dark room, an electroretinogram was performed by contacting the ground electrode to the tail, the reference electrode to the head, and the corneal electrode to the cornea. Response values were obtained by stimulating the retina with a single white light of 0.9 cdㆍsec/m ² flash intensity, and the amplitude determined from the trough of the a-wave to the peak of the b-wave was measured and evaluated as an indicator of retinal function. The results of all tests are expressed as mean ± standard deviation, and significance was verified using one-way ANOVA and Kruskal-wallis test.

The experimental results are shown in Table 4 below and Figures 5 and 6.

Example peptide number amino acid sequence b-wave amplitude (μV) 38 002-170 Hyp Gly Gln Glu Val 400 42 002-165 Hyp Gly Gln Glu Leu Leu Ala 510 43 002-166 Hyp Gly Gln Glu Val Leu Ala 380 44 002-175 Hyp Gly Gln Glu Aib Leu Ala 371 Naive 353 PBS 240 aflibercept 398

As shown in Table 4 and Figures 5 and 6, 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. Therefore, it can be seen that the peptide of the present invention has excellent CNV inhibition efficacy.

[Test Example 4]

Stability evaluation of peptides

To evaluate the stability of the peptide of the present invention, an experiment was performed as follows.

The stability of the peptide was confirmed through content analysis in an aqueous solution at 40°C, 75% humidity, and harsh conditions, and was analyzed using HPLC under the following conditions.

Analytical column: C18 (4.6 Υ 250 mm, 5 μm, YMC Hydrosphere)

Analysis wavelength: ultraviolet absorption spectrophotometer (210 nm)

Injection volume/flow rate: 10 μL / 0.8 mL/min

Column temperature: 45℃

Mobile phase: 0.1M phosphate buffer methanol mixture (83:17)

The experimental results are shown in Table 5 below.

Example peptide number Initial content (%) Content after 7 days (%) Decrease rate (%) 41 002-132 96.123 95.685 0.438 42 002-165 98.155 97.555 0.600 44 002-175 96.917 96.562 0.355 45 002-176 98.390 97.785 0.605 46 002-177 98.586 98.147 0.439

As shown in Table 5 above, as a result of evaluating the stability, the content of all peptides of the present invention was maintained at about 95% or more after 7 days, and the reduction rate after 7 days was also less than about 1%, confirming that the stability was very excellent. . In particular, Example 44 (002-175) showed the best stability. Therefore, it can be seen that the peptide of the present invention has excellent stability.

[Test Example 5]

Solubility assessment of peptides

To evaluate the solubility of the peptide of the present invention, an experiment was performed as follows.

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

The experimental results are shown in Table 6 below.

Example peptide number Solubility (mg/ml) 41 002-132 330.0 44 002-175 500.0 45 002-176 100.0

As shown in Table 6, as a result of measuring solubility, the peptide of the present invention showed excellent solubility, and in particular, Example 44 (002-175) showed the best solubility. Therefore, it can be seen that the peptide of the present invention has excellent solubility.

[Test Example 6]

Evaluation of the efficacy of peptide-containing eye drops to inhibit choroidal neovascularization

An experiment was performed as follows to evaluate the CNV (choroidal neovascularization) inhibitory effect of the eye drop containing Example 44 (002-175), which was confirmed to be an excellent peptide through the above test examples.

7-8 week old C57BL/6 mice were instilled with mydriatic to dilate the pupils and anesthetized by intraperitoneal injection of Ketamine (50 mg/Kg) and Rompun (23.32 mg/Kg). Using a slit-lamp laser injection system, the laser was irradiated to the choroid of each eye of the mouse four times in the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions. At this time, the laser irradiation conditions were 532 nm, 80 msec, and 240 mW. Three days after laser irradiation, before drug administration, 1 μL of aflibercept dissolved in PBS at a concentration of 20 mg/mL was injected into the vitreous body using a syringe. Drugs prepared at each concentration were injected twice a day (5 μL/mL). eye, AM: 9:00 FM 04:00) was instilled in both eyes for 11 days. After 6, 10, and 14 days of laser induction, mice were anesthetized and 2% fluorescein was administered to the abdominal cavity to stain blood vessels. The retina was photographed 2 to 3 minutes after fluorescein administration. The size and fluorescence of CNV lesions induced by the laser were measured using the imageJ program, and the background was removed to reduce inter-individual differences to calculate CTF (Corrected total fluorescence) and compare the values of each group. In addition, mice were anesthetized 6, 10, and 14 days after laser induction, and the CNV lesions induced by the laser were calculated and expressed as μm ² using the ImageJ program by transmitting an OCT beam for each burn. The results of all tests are expressed as mean ± standard deviation, significance was verified using one-way ANOVA and Kruskal-wallis test, and the experimental results are shown in Figures 7 to 10.

As shown in Figures 7 and 8, as a result of measuring the fluorescence value of the CNV site, the eye drop containing Example 44 (002-175) of the present invention showed excellent CNV inhibition efficacy, especially after 14 days of administration, the control group It showed better inhibitory efficacy compared to aflibercept.

In addition, as shown in Figures 9 and 10, as a result of measuring the size of the CNV lesion, it was confirmed that the size of the CNV lesion decreased when the eye drop containing Example 44 (002-175) of the present invention was administered. In particular, it showed a similar level compared to the control group, aflibercept.

Therefore, it can be seen that the eye drop containing the peptide of Example 44 (002-175) of the present invention has excellent CNV inhibition efficacy.

Claims (9)

delete delete delete delete delete delete A pharmaceutical composition for preventing or treating macular degeneration, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 44.
The pharmaceutical composition for preventing or treating macular degeneration according to claim 7, wherein the peptide inhibits choroidal neovascularization (CNV).
The pharmaceutical composition for preventing or treating macular degeneration according to claim 7, wherein the macular degeneration is one or more selected from the group consisting of wet macular degeneration, dry macular degeneration, and age-related macular degeneration.