WO2022086039A1 - Composition for preventing or treating macular degeneration comprising peptide for inhibiting fas signaling - Google Patents

Abstract

The present invention relates to a composition for preventing or treating macular degeneration, comprising a peptide for inhibiting Fas signaling. More specifically, the composition of the present invention has no side effects as an ocular dosage form, and controls macular degeneration through the inhibition of intraocular inflammation and apoptosis according to disease progression, and thus can be used to treat patients who do not respond to angiogenesis inhibitors. In addition, according to the present invention, disease progression can be controlled even in early-stage macular degeneration.

Description

Composition for preventing or treating macular degeneration comprising a peptide for inhibiting FAS signaling

The present invention relates to a composition for preventing or treating macular degeneration comprising a peptide for inhibiting Fas signaling.

Retinal pigment epithelium (Retinal Pigment Epithelium) plays an important role in maintaining the normal visual function of the retina, and degeneration of the retinal pigment epithelium causes various retinal degenerative diseases. The macula, a neural tissue in the central part of the retina, has a dense concentration of photoreceptors and plays an important role in vision as an object image. However, when retinal degeneration occurs due to various causes such as aging, genetic factors, inflammation, stress, and excessive UV exposure, vision loss occurs. This macular degeneration (Macular Degeneration, MD), in particular, mainly occurs in elderly patients due to aging, which is called age-related macular degeneration (Age-related Macular Degeneration, AMD).

Although technologies for treating the age-related macular degeneration are being studied worldwide, it is difficult to expect a therapeutic effect with the current technology. In particular, dry macular degeneration accounts for 90% of macular degeneration, but there is no treatment. In addition, the intraocular injection treatment method has concerns about side effects, is complicated to use, and has poor accessibility. Existing angiogenesis inhibitors are drugs applicable only to wet macular degeneration and are ineffective in dry macular degeneration, an initial stage in which neovascularization does not occur.

It is an object of the present invention to provide a composition for preventing or treating macular degeneration comprising a peptide for inhibiting Fas signaling that controls apoptosis caused by Fas-FaL signal.

Another object of the present invention is to provide a method for treating macular degeneration using the Fas signaling inhibitory peptide.

In order to achieve the above object, the present invention provides a composition for preventing or treating macular degeneration comprising a peptide represented by the following general formula 1 as an active ingredient:

[General formula 1]

A-B

In the above formula,

A represents a peptide for inhibiting Fas signaling consisting of an amino acid sequence represented by the following general formula 2 (Fas Blocking Peptide),

B is absent or represents a cell penetrating peptide

[General formula 2]

Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

In the above formula,

Xaa ₁ and Xaa ₃ are each independently absent or any amino acid,

Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, Ile, Met, Pro, Ser, Cys, Thr, Asn and Gin.

The present invention also provides a method for treating macular degeneration comprising administering a therapeutically effective amount of the composition for preventing or treating macular degeneration to a subject in need thereof.

The present invention is an ocular dosage form, and there are no side effects such as proteinuria, hypertension, and bleeding, which are seen with conventional angiogenesis inhibitors. In addition, the present invention has the potential to treat patients who do not respond to angiogenesis inhibitors by controlling macular degeneration through inhibition of intraocular inflammation and apoptosis according to disease progression. Additionally, the present invention can control disease progression even in dry macular degeneration, which is an initial stage in which no new blood vessels are formed.

In addition, the present invention can be applied to the development of complex formulations using water-soluble peptides to various existing water-soluble eye drop formulations or injection formulations.

1 shows the results of confirming the apoptosis inhibitory effect of Fas-binding peptides through flow cytometry.

2 shows the results of confirming the apoptosis inhibitory effect of Fas-binding peptides through flow cytometry.

3 is a schematic diagram of an experiment for confirming whether or not the Fas-binding peptide is delivered to the eye using a fluorescent material.

4 shows the results of confirming whether or not the Fas-binding peptide was delivered to the eye using a fluorescent substance.

5 is a schematic diagram of an experiment for confirming the cytotoxicity of Fas-binding peptides through TUNEL assay in normal mouse eyes.

6 shows the results of confirming the cytotoxicity of Fas-binding peptides through TUNEL assay in normal mouse eyes.

7 shows the results of confirming the cytotoxicity of Fas-binding peptides through TUNEL assay in normal mouse eyes.

8 is an experimental schematic diagram for verifying the FBP eye drop effect through qPCR and TUNEL assay in a macular degeneration model.

9 shows the results of confirming the therapeutic effect of Fas-binding peptide through Real Time PCR.

10 shows the results of confirming the therapeutic effect of Fas-binding peptide through TUNEL assay.

11 shows the results of confirming the therapeutic effect of Fas-binding peptide through TUNEL assay.

12 shows vitreous turbidity on days 0 and 7 after eye drop administration of Fas-binding peptide in a chinchilla rabbit macular degeneration model.

FIG. 13 shows fundus imaging results at 7 days ( FIG. 13a ) and 14 days ( FIG. 13b ) after eye drop administration of Fas-binding peptide in a chinchilla rabbit dry macular degeneration model.

14 shows the therapeutic effect of macular degeneration after administration of FBP peptide by eye drop or intravitreal injection in a rat macular degeneration model (H&E staining and TUNEL assay).

15 shows the results of H&E staining and TUNEL assay after administration of FBP peptide by eye drop or intravitreal injection in a rat macular degeneration model, and statistically analyzed through One-way anova analysis by measuring through ImageJ.

16 shows the results of qPCR analysis of inflammatory genes after administration of FBP peptide by eye drop or intravitreal injection in a rat macular degeneration model.

The present inventors administered a peptide (Blocking Peptide (FBP), 8mer) for inhibiting Fas signaling, which binds to Fas expressed on the cell membrane during apoptosis and controls apoptosis, as an eye drop formulation, thereby reducing retinal cells in the eyeball in an animal model of macular degeneration. A new drug was developed to treat macular degeneration caused by retinal cell death and ocular inflammation by controlling apoptosis and inflammation caused by the combination of Fas-FasL (soluble form). In particular, it was confirmed that the FBP peptide that controls Fas-FasL binding was made as an eye drop and had therapeutic efficacy by inhibiting apoptosis of retinal cells around the macula when administered to the eye.

According to one embodiment of the present invention, when a test substance is administered intravitreally and repeatedly instilled in chinchilla rabbits induced with dry macular degeneration, the retinal pigment epithelial cell layer degeneration area level, retinal thickness measurement, and histopathological examination results are tested It was found that the administration of the substance slowed the progression of dry macular degeneration induced by sodium iodate compared to the induced control group.

According to another embodiment of the present invention, it was confirmed that apoptosis was inhibited by intravitreal injection of FBP peptide into the lens of the eye in a rat model induced with dry macular degeneration, and retinal thickness was well preserved.

Prototype of eye drops can be developed as an easy-to-use eye drop formulation to increase patient accessibility and minimize side effects. And it was confirmed that there is a control effect on inflammation.

In addition, according to another embodiment of the present invention, the FBP peptide is a cell penetrating peptide (CPP) and / or polyarginine, such as 3, 4, 6 or 9-arginine (R) to facilitate penetration of the ocular barrier in an animal model. can be combined.

Accordingly, the present invention relates to a composition for preventing or treating macular degeneration comprising a peptide represented by the following general formula 1 as an active ingredient:

[General formula 1]

A-B

In the above formula,

A represents a peptide for inhibiting Fas signaling consisting of an amino acid sequence represented by the following general formula 2 (Fas Blocking Peptide),

B is absent or represents a cell penetrating peptide

[General formula 2]

Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

In the above formula,

Xaa ₁ and Xaa ₃ are each independently absent or any amino acid,

Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, Ile, Met, Pro, Ser, Cys, Thr, Asn and Gin.

The peptide represented by Formula 1 may be a single peptide for Fas signaling inhibition alone or a fusion peptide of Fas signaling inhibition peptide and cell-penetrating peptide.

Specifically, in the general formula 2,

Xaa ₁ and Xaa ₃ are each independently absent or selected from the group consisting of Tyr, Phe and Trp,

Xaa ₂ is absent or selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.

More specifically, in the general formula 2,

Xaa ₁ and Xaa ₃ are each independently selected from the group consisting of Tyr, Phe and Trp,

Xaa ₂ is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.

As used herein, the term "Fas" is also called Fas, FasR (Fas receptor), APO-1 (apoptosis antigen 1), or CD95 (cluster of differentiation 95), a tumor necrosis factor that regulates apoptosis It is a type of receptor (tumor necrosis factor, TNF). When Fas binds to a ligand, it is activated through multimerization, and as a result, several adapter proteins bind to Fas. The bound adapter proteins activate various apoptosis signaling systems, and representative signaling regulators include caspase, NF-κB, stress-activated protein kinase (SAPK), and the Bcl-2 family.

As used herein, the term "peptide for inhibiting Fas signaling" refers to a peptide having an activity of binding to Fas described above and inhibiting the lower signaling system of Fas as opposed to a conventional Fas ligand, and apoptosis inhibitory activity. .

In one embodiment of the present invention, in Formula 2, Xaa ₁ and Xaa ₃ may each independently not exist or may be any amino acid, and preferably each independently be selected from the group consisting of Tyr, Phe and Trp. can

In addition, in Formula 2, Xaa ₂ is absent or may be selected from the group consisting of Ala, Gly, Val, Leu, Ile, Met, Pro, Ser, Cys, Thr, Asn and Gin, preferably Gly, It may be selected from the group consisting of Ala, Ser, Thr and Cys.

For example, 1) Xaa ₁ and Xaa ₃ may not exist and only Xaa ₂ may exist, 2) Xaa ₂ may be present while any one of Xaa ₁ and Xaa ₃ is present, 3) Xaa ₁ to Xaa ₃ Various combinations are possible, such as none of them.

In one embodiment of the present invention, the amino acid sequence of Formula 2 may be a sequence listed in Table 1 below.

SEQ ID NO: Amino acid sequence (5'-3') One CDEHF 2 YCDEHF 3 YCDEHFY 4 YCDEHFA 5 YCDEHFC 6 YCDEHFM 7 FCDEHFC 8 YCDEHFCY 9 YCDEHFMY 10 YCDEHFCF

On the other hand, the peptide for inhibiting Fas signaling used in the present invention has improved stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.) of the peptide, altered specificity (eg, broad spectrum of biological activity), reduced antigen To obtain sex, the N- and/or C-terminus of the peptide may be modified. The formula may be in the form of an acetyl group, a fluorenyl methoxycarbonyl group, an amide group, a formyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG) bonded to the N- and/or C-terminus of the peptide. However, if it is a component capable of improving the modification of the peptide, particularly the stability of the peptide, it may be included without limitation. As used herein, the term "stability" refers to storage stability (eg, room temperature storage stability) as well as in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo. In another embodiment of the present invention, the peptide of Formula 1 may be a fusion peptide of a Fas signaling inhibitory peptide and a cell-penetrating peptide. The cell-penetrating peptides may be those listed in Table 2 below.

designation Amino acid sequence (5'-3') SEQ ID NO: TAT GRKKRRQRRRPPQ 11 Penetratin RQIKIWFQNRRMKWKK 12 Polyarginines R _n (n=3, 4, 6, 9) 13 DPV1047 VKRGLKLRHVRPRVTRMDV 14 pVEC LLIILRRRIRKQAHAHSK 15 M918 MVTVLFRRLRIRRACGPPRVRV 16 M1073 MVRRFLVTLRIRRACGPPRVRV 17 BPrPr (1-28) MVKSKIGSWILVLFVAMWSDVGLCKKRP 18 MPG GALFLGFLGAAGSTMGAWSQPKKKRKV 19 Pep-1 KETWWETWWTEWSQPKKKRKV 20 transportan GWTLNSAGYLLGKINLKALAALAKKIL 21 MAP KLALKLALKALKAALKLA 22 MAP12 LKTLTETLKELTKTLTEL 23 MAP17 QLALQLALQALQAALQLA 24 GALA WEAALAEALAEALAEHLAEALAEALEALAA 25 p28 LSTAADMQGVVTDGMASGLDKDYLKPDD 26 PreS2 PLSSIFSRIGDP 27 VT5 DPKGDPKGVTVTVTVTVTGKGDPKPD 28 Bac 7 [(Bac (1-24)] RRIRPRPPRLPRPRPRPLPFPRPPG 29 PPR and PRR PPRn and PRRn (n = 3-6) 30 SAP VRLPPPVRLPPPVRLPPP 31 SAP(E) VELPPPVELPPPVELPPP 32 CyLoP-1 CRWRWKCCKK 33 gH 625 HGLASTLTRWAHYNALIRAF 34 CPP-C PIEVCMYREP 35 C105Y CSIPPEVKFNKPFVYLI 36 Pep-7 SDLWEMMMVSLACQY 37 SG3 RLSGMNEVLSFRW 38

As used herein, the term "treatment" refers to any action in which the symptoms of macular degeneration are improved or beneficially changed by administration of the peptide for inhibiting Fas signaling according to the present invention or a pharmaceutical composition comprising the same. Accordingly, the macular degeneration may be either dry macular degeneration or wet macular degeneration. As used herein, the term "administration" refers to a predetermined substance, i.e., a peptide for inhibiting Fas signaling according to the present invention, or a fusion peptide of a peptide for inhibiting Fas signaling and a cell-penetrating peptide according to the present invention to a subject by any suitable method. It means introducing a pharmaceutical composition that does. Preferably, injection into the vitreous cavity or topical administration in the form of eye drops may be used.

On the other hand, the composition of the present invention can be prepared in dosage forms such as injections, solutions, ointments, etc., and when used as injections, if necessary, it can be used as an aqueous suspension injection together with a suspending agent, a stabilizer, a buffer, a preservative, a thickener, an isotonic agent, etc. can When prepared as a solution, any dosage form used as an ophthalmic solution, for example, an aqueous ophthalmic solution, such as an aqueous ophthalmic solution, an aqueous emulsion ophthalmic solution, a viscous ophthalmic solution, and a dissolved ophthalmic solution; or non-aqueous ophthalmic solutions such as non-aqueous ophthalmic solutions and non-aqueous emulsion ophthalmic solutions.

When prepared as an aqueous emulsion ophthalmic solution, various additives known in the art may be included as long as the purpose of the present invention is not impaired, for example, isotonic agents, buffers, stabilizers, pH adjusters, thickeners, preservatives, chelating agents, solubilizers and solvents, etc. may be included. The buffer may be selected from, but not limited to, the group consisting of phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer (eg sodium acetate), tromethamine and amino acids. Preferably, a phosphate buffer may be used. The isotonic agent may be selected from, but not limited to, the group consisting of sorbitol, sugars such as glucose erythritol and mannitol, polyhydric alcohols such as glycerin, polyethylene glycol and polypropylene glycol, and salts such as sodium chloride. Preservatives include benzalkonium chloride, benzethonium chloride, alkyl paraoxybenzoates such as methyl paraoxybenzoate and ethyl paraoxybenzoate, benzyl alcohol, phenethyl alcohol, sorbic acid and its salts, thimerosal, polyquaternium , may be selected from the group consisting of benzododecinium bromide, oxychloro complex and chlorobutanol, but is not limited thereto. Stabilizers include cyclodextrins and derivatives thereof, water-soluble polymers such as poly(vinylpyrrolidone), and surfactants such as polysorbate 80 (Tween ^80® ), polysorbate 20, tyloxapol, tromethamine It may be selected from, but is not limited to, tromethamine may be preferably used. The pH adjusting agent may be selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, monoethanolamine, aqueous ammonia and ammonium hydroxide, but is not limited thereto. The thickener may be selected from the group consisting of hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose, carbomer, povidone, poloxamer, polycarbophil and salts thereof, but is limited thereto it doesn't happen The chelating agent may be selected from, but not limited to, the group consisting of sodium edetate, sodium citrate and condensed sodium phosphate. The solubilizing agent or solvent may be selected from glycerin, DMSO, DMA, N-methylpyrrolidone, ethanol, benzyl alcohol, isopropyl alcohol, polyethylene glycol or propylene glycol of various molecular weights, but is not limited thereto. There may be some overlap between components that can be used as solvents or solubilizers, and any component can be used as either a solvent or a solubilizer. If not, it can be regarded as a solubilizer. Alternatively, the solubilizer may be a surfactant in some variations. Combinations of surfactants including various types of surfactants may be used. For example, nonionic, anionic (ie soap, sulfonate), cationic (ie CTAB), zwitterionic, polymeric, amphoteric surfactants can be used. For example, surfactants that may be used include, but are not limited to, those having an HLB of 10, 11, 12, 13, or 14 or higher. Examples of surfactants are polyoxyethylene products of hydrogenated vegetable oils, polyethoxylated castor oil or polyethoxylated hydrogenated castor oil, polyoxyl castor oil or derivatives thereof, polyoxyethylene-sorbitan-fatty acid esters , including polyoxyethylene castor oil derivatives and the like. However, the present invention is not limited thereto.

In the present invention, the term "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, and activity of the drug in the patient. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The peptide according to the present invention or a pharmaceutical composition comprising the same may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, or may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. The dosage and frequency of the pharmaceutical composition of the present invention is determined according to the type of active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

Suitable dosages of the compositions of the present invention will be routinely determined by the medical practitioner or one of ordinary skill in the art. For example, when the composition according to the present invention is used as an eye drop for an adult patient with macular degeneration, the preferred dosage of the composition is, for example, 1 to 4 drops (about 0.025 to 0.1 mL) per day for 1 to 4 drops (about 0.025 to 0.1 mL) of the composition. It can be administered up to 10 times, but is not limited thereto, and a medical practitioner or a person skilled in the art may determine the most suitable actual dosage according to the age, weight, sex and response of the patient to be treated, as well as the circumstances to be treated.

The composition according to the present invention may be provided by filling in a sterile container, and may be provided including instructions on its use, wherein the instructions are physically placed in a container filled with the composition or a second container packaging the container. It can be attached as a hood or packaged together inside a second container.

The present invention also relates to a method for treating macular degeneration, comprising administering a therapeutically effective amount of the composition for preventing or treating macular degeneration to a subject in need thereof.

The subject may be a human or non-human organism, for example, a non-human animal such as a cow, a monkey, a bird, a cat, a mouse, a rat, a hamster, a pig, a dog, a rabbit, a sheep, a horse.

In the treatment method of the present invention, the formulation and administration method of the composition are the same as described above.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example 1> Confirmation of the apoptosis inhibitory effect of Fas-binding peptide through flow cytometry

AnnexinV/7AAD staining was performed through flow cytometry to confirm the apoptosis inhibitory effect of the therapeutic peptide. AnnexinV/7AAD staining is a commonly used staining method to confirm apoptosis. AnnexinV is used as a marker to confirm the initial stage of apoptosis by binding to phosphatidyl serine, which is mainly present inside the cell membrane and is exposed to the outside at the start of apoptosis. 7AAD is a chemical that is fluorescent and strongly binds to DNA, and is used as a late-apoptotic marker by staining fragmented DNA at the late stage of apoptosis. AnnexinV/7-AAD staining utilizes these two substances and a flow cytometer to divide the cells into 4 fractions. Both unstained AnnexinV(-)/7AAD(-) cells are live cells, and AnnexinV(+)/7AAD(-) cells stained only with AnnexinV are early apoptosis cells. Cells stained for both AnnexinV(+)/7AAD(+) were analyzed as late apoptosis cells (Late Apoptosis), and AnnexinV(-)/7AAD(+) stained only with 7AAD was classified as apoptosis (Necrosis).

For the experiment, it is divided into normal, Mock, and FBP-treated groups, and the Mock group is stained with Annexin V & 7-AAD after 12 hrs after treating Jurkat cells with 0.25 μg/mL of Fas L, and the FBP group is Fas L in Jurkat cells. After treatment with 0.25 μg/mL and 300 μM of FBP, 12 hours later, the cells were stained with Annexin V & 7-AAD.

As a result, as in FIGS. 1 and 2, it was confirmed that the Fas-binding peptide had an apoptosis inhibitory effect through AnnexinV staining.

<Example 2> Confirmation of intraocular delivery of Fas-binding peptide using a fluorescent substance

In order to check whether the fluorescent material actually reaches the retinal pigment epithelial layer, the target location inside the eye, Alexa488, a fluorescent material, is bound to Fas-binding FBP peptide (FBP:Alexa488=20:1(w/w), conjugation condition: room temperature 4 hours) after administration to the eye, 12 hours later, the eyeball was removed and cryosectioned samples were confirmed through a fluorescence microscope.

For the experiment, the mice were divided into normal (n=3), 9R-FBP (n=3), TAT-FBP (n=3) treatment groups, and 5 mg/day for 3 days as an eye drop into the eyes of the mice once a day. mL of FBP was administered at 5 μl/eye (total of 25 μg/eye) (see FIG. 3 ).

As shown in FIG. 4 , it was confirmed that the Fas-binding peptide bound to the fluorescent material actually reached the retinal pigment epithelial layer, which is the target location inside the eye. The blue fluorescence you can see in the picture is the staining of the cell nucleus, and the green fluorescence is the Fas-binding peptide to which Alexa488 is bound. Both the cell-permeable 9R-peptide and TAT-peptide passed through the ocular barrier to reach the target site.

<Example 3> Confirmation of cytotoxicity of Fas-binding peptide through TUNEL assay in normal rat eye

In order to check whether the prepared peptide shows cytotoxicity in experimental animals, 20 μg peptide was instilled 3 times a day for 3 days, and the dead cells were confirmed by TUNEL assay.

Specifically, the mice were divided into groups treated with FBP (Sham, n=2), FBP 9R (n=2), and FBP 9R/siRNA (25: 1 complex preparation, n=2), and the mice were placed in the eyes at 8 hour intervals. As an eye drop 3 times a day, 2 μl/eye (total of 20 μg/eye) of 10 mg/mL FBP 9R was administered after eye drop 9 times, and dead cells were confirmed by TUNEL assay 8 hours later (Fig. 5).

As shown in Figures 6 and 7, it was confirmed that the same cytotoxicity as the Fas-binding peptide without cell permeability, and the peptide complexed with the siRNA also showed the same cytotoxicity.

<Example 4> Confirmation of therapeutic effect of Fas-binding peptide through real-time PCR

Inducing macular degeneration in mice through NaIO ₃ (NaIO ₃ IP 50 mg/kg) and 3 days later, 2 mg/mL of this peptide was administered at 5 μl/eye 3 times a day for 3 days (a total of 9 eye drops, A total of 10 μg/eye) treatment effect was confirmed. For the positive control, dexamethasone, a steroidal anti-inflammatory, was used, and for the negative control, a Fas-binding peptide consisting of a random sequence and a peptide without cell permeability were used. The above experiment is to confirm whether the actually produced peptide has an anti-inflammatory effect, the peptides and drugs corresponding to each experimental group were treated, and the gene expression level was checked through real-time PCR by removing the eyes.

First, the expression level was taken as a standard through the eyes of normal rats (n=2) that were not administered with anything, and macular degeneration was induced in other rats by intraperitoneal injection of chemicals. The group that was not treated with any drug after induction was Mock (n=2), and a positive control group (n=2) (intraocular injection, 10 μg (10 μl)), and A cell-impermeable peptide (FBP, eye drop 10 μg (5 μl)) and a cell-permeable but random SCR-peptide (ScrFBP-9R, eye drop 10 μg (5 μl)) were each treated as a negative control ( n=2). TAT-peptide (TAT-FBP, eye drop 10 μg (5 μl)) and 9R-peptide (9R-FBP, eye drop 10 μg (5 μl)) are cell-permeable therapeutic peptides and target groups ( each n=3) (see FIG. 8).

As shown in Figure 9, it can be seen that the level of TNF-a, which is a representative cytokine representing inflammation in the mock compared to normal, increased more than 3 times. The positive control, dexamethasone, showed lower TNF-a levels compared to Mock, and the negative control peptide and SCR-peptide showed similar levels to Mock. On the other hand, the TAT-peptide and 9R-peptide, which are the modeled target groups, decreased to a level almost similar to the TNF-a level of the positive control dexamethasone, and the target group administered to the macular degeneration-induced mice had an inhibitory effect on inflammation. It can be said that there is

<Example 5> Confirmation of therapeutic effect of Fas-binding peptide through Tunel assay

In order to test whether the actually produced peptide has an inhibitory effect on retinal apoptosis, the peptides and drugs corresponding to each experimental group were treated as in Example 4, and the number of cells killed through Tunnel assay after making a cryoblock by excising the eyes Confirmed.

As shown in FIGS. 10 and 11 , it was confirmed that the part stained red due to cell death showed a decrease compared to the negative control in both Tat-peptide and 9R-peptide, which are Fas-binding peptide groups. In particular, it was confirmed that the prepared peptide group showed the same efficacy as the anti-inflammatory dexamethasone, and it was confirmed that there was no significant difference when compared with the normal group.

<Example 6> Therapeutic effect in rabbit macular degeneration model

In the sodium iodate (NaIO ₃ )-induced chinchilla rabbit macular degeneration model, the effects of intravitreal and eye drop administration of a test substance were evaluated (G1: induced control group, G2: TA1 intravitreal group, G3: TA2 eye drop group, G4: TA3 eye drop administration group, G4: TA4 eye drop administration group). NaIO ₃ is a material that has been widely used for making preclinical experimental models of Dry Aged-related Macular Degeneration (Dry AMD). In this test, after intravenous administration of NaIO ₃ into the ear veins of rabbits, the retinal pigment epithelial cell layer and photoreceptor cells were degenerated to produce a dry macular degeneration disease model. ) measurement, OCT (Optical Coherence Tomography) imaging, and histopathological examination were used to evaluate the efficacy of the test substance.

Specifically, the test is set at a temperature of 23±3 ℃, relative humidity of 55±15%, the number of ventilation 10-20 times/hr, illumination time of 12 hours (lights on at 8 am to off at 8 pm), and illuminance of 150-300 Lux. was performed. For the feed, the animal feed produced by Cargill Agripurina was supplied from Dream Bio (507 Gwangnaru-ro, Gwangjin-gu, Seoul) and freely consumed, and purified water was freely ingested using a polycarbonate drinking bottle. . During the period of acclimatization, administration and observation, rabbits were bred in a stainless steel box (W 500 x L 800 x H 500 mm) at 1 animal/bring box. For group separation, the weights of animals determined to be healthy during the acclimatization period were measured and randomly distributed so that the average weight of each group was distributed as uniformly as possible according to the ranked weights. The test group is configured as shown in Table 3 below.

army gender Number of animals (animals) animal
number cause
Whether substance to be administered administration
Route Dosage
(μg/eye) dose amount
(μL/eye) G1 M 4 1-4 Y Vehicle drip - 50 G2 M 4 5-8 Y TA1 I.Vt. 250 40 G3 M 4 9-12 Y TA2 drip 250 50 G4 M 4 13-16 Y TA3 drip 250 50 G5 M 4 17-20 Y TA4 drip 250 50

Administration The test substance was prepared by diluting an appropriate amount of the test substance in an excipient to a concentration of 5 mg/mL. For the administration route, intravitreal and eye drop administration were selected, and in the case of eye drop administration, the test substance and excipient were administered twice/day for 14 days from the scheduled start date of the test substance administration (Day 0), and repeated administration for a total of 28 times. In the case of intravitreal administration, the test substance was administered once on the G2 administration day (Day 1). The dose was administered at 5 mg/mL, that is, 250 μg/50 μL at one time in the case of the eye drop administration group, and in the case of intravitreal administration, 40 μL of the supernatant of the preparation was administered at one time of administration. In the case of eye drop administration, the assistant took the animal out of the cage and calibrated it naturally, and then the administerer instilled the test substance corresponding to each test group into the center of the cornea of the right eye using a pipette. In the case of intravitreal administration, the test substance was administered to the right eye of the animal using a syringe equipped with a 31 gauge needle while the animal was anesthetized. The macular degeneration model was produced by administering a single intravenous administration of NaIO ₃ (Sigma-Aldrich Co.) at a dose of 60 mg/kg to an animal that would induce macular degeneration on the induction day (Day 0).

(1) turbidity confirmation test

By administering the test substance to New Zealand White Rabbit (Hallym laboratory animal, Yuri, Bongdam-eup, Hwaseong-si, Gyeonggi-do) of 2.0 kg or more, anesthetize the animal before (Day 0), 3rd and 7th days after administration, and then fundus camera ( TRC-50IX, TOPCON, Japan) was used to perform fundus imaging to confirm ocular opacity.

As shown in FIG. 12 , as a result of confirming ocular clouding on the 3rd and 7th days after administration of the test substance, no adverse reactions were observed in all subjects.

(2) fundus photography

Before NaIO ₃ administration and before the end of the experiment, mydriatic drug (midriacil 1% eye drop) was instilled into the right eye of the animal, the animal was anesthetized, and fundus photography was performed using a fundus camera (TRC-50IX, TOPCON, Japan). was carried out. The fundus image was analyzed using Image J (NIH, Bethesda, MD) to analyze the area of the retinal pigment epithelium degenerated.

In the fundus image, the retinal area excluding the optic nerve area was defined as the total area, and the degenerated area of the retinal pigment epithelium was defined as the total area, and the ratio was calculated as a percentage (%).

Degenerated area (%) = Degenerated area / total area

As shown in FIGS. 13a-b and Table 4, as a result of analysis of the area of retinal pigment epithelial cell layer degeneration, the level of retinal pigment epithelial cell layer degeneration area level of all test substance-administered groups (G2-5) on the 7th day after induction of macular degeneration was higher than that of the induced control group. It was observed to be significantly low, and on the 14th day after macular degeneration induction, the level of retinal pigment epithelial cell layer degeneration in the TA2 administration group was observed to be significantly lower than that of the induced control group. Although there was no statistically significant difference, the TA1 administration group and TA3 administration group And the level of the retinal pigment epithelial cell layer degeneration of the TA4 administration group showed a lower tendency than that of the induced control group.

RPE regression RPE regression (UNIT: %) time
(One) G1 G2 G3 G4 G5 7 70.51±2.80 54.09±6.62 ^** 42.19±7.50 ^*** 56.63±7.28 ^* 53.78±5.84 ^** 14 80.16±6.62 65.52±8.27 53.98±5.44 ^** 66.89±7.32 76.68±10.71 N 4 4 4 4 4 Set NaIO ₃ administration day to 0.
Data are expressed as mean±SD.
G1: vehicle, G2: TA1, G3: TA2, G4: TA3. G5: TA4
***/**/* Significant difference at p<0.001/p<0.01/p<0.05 level compared to G1

(3) Electroretinogram (ERG) measurement experiment

Before termination, electroretinalography was performed on the right eye of all surviving animals. After the animals were anesthetized, they were acclimatized in a dark room for 60 minutes, and the retinal potential difference was measured using ERG equipment (HMs-ERG, Ocuscience, USA). The retinal potential difference was evaluated using the retinal function evaluation protocol of ISCEV (International Society for Clinical Electrophysiology of Vision), and the A-wave amplitude indicating photoreceptor activity and B-wave amplitude indicating the activity of glial cells such as Muller cells were evaluated. did The retinal potential was measured by flash in a dark room.

ERG (Electroretinogram) is a test that evaluates retinal function by recording the retina's response to photostimulation as a waveform of an electrical signal. As shown in Table 5, as a result of ERG analysis on the 14th day after induction of macular degeneration, a characteristic waveform of ERG was not observed in all subjects. is believed to have been lost.

Electroretinogram (ERG) ERG (UNIT: μV) exam G1 G2 G3 G4 G5 a wave 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 b wave 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 N 4 4 4 4 4 Set NaIO ₃ administration day to 0.
Data are expressed as mean±SD.
G1: vehicle, G2: TA1, G3: TA2, G4: TA3. G5: TA4

(4) Optical Coherence Tomography (OCT)

Before the end of the imaging experiment, OCT imaging was performed on the right eye for all animals, and after taking 2 parts of the upper and lower parts based on the optic nerve, the retinal thickness of 3 points in each image was analyzed to determine the average of the total of 6 values. calculated.

As a result of measuring the thickness of the retina by taking Optical Coherence Tomography (OCT), it was found that the retinal thickness level of the TA1 administration group and the TA2 administration group was significantly higher than that of the induced control group (Table 6).

Optical Coherence Tomography (OCT) OCT (UNIT: μm) time (days) G1 G2 G3 G4 G5 14 113.8±9.7 132.0±4.8 ^* 132.8±9.0 ^* 120.1±7.3 122.9±10.1 N 7 7 7 7 7 Set NaIO ₃ administration day to 0.
Data are expressed as mean±SD.
G1: vehicle, G2: TA1, G3: TA2, G4: TA3. G5: TA4
* There is a significant difference at the p<0.05 level compared to G1

(5) Autopsy and histopathological examination

On the day of termination, all surviving animals were anesthetized and euthanized, the induced eyeballs were excised and puncture was performed in the center of the cornea using an injection needle, followed by fixation in 10% neutral buffered formalin solution.

The fixed tissue undergoes general tissue processing such as trimming, dehydration, paraffin embedding, and sectioning to prepare a specimen for histopathological examination, then hematoxylin & eosin staining is performed, and an optical microscope (Olympus BX53, Japan) is used. Calculate the ratio (%) of the thickness of the outer nuclear layer (ONL) to the total retinal thickness. Two slides were made on both sides of the optic nerve per eye, and 3 points were measured for each slide, and the average of a total of six values was calculated.

Data normality was assumed for the results of this test, and significance was tested between test groups using parametric one-way ANOVA. was carried out.

Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and when the p-value was less than 0.05, it was determined to be statistically significant.

As a result of calculating the ratio of the thickness of the outer nuclear layer (ONL) to the thickness of the retina by H&E staining of the eye tissue, it was found that the ratio level to the total retinal thickness of all test substance-administered groups was significantly higher than that of the induced control group (Table 7).

ONL ratio ONL ratio (UNIT: %) time (days) G1 G2 G3 G4 G5 14 20.38±2.75 25.17±1.58 ^** 27.26±1.20 ^*** 24.54±1.67 ^* 24.04±1.09 N 7 7 7 7 7 Set NaIO ₃ administration day to 0.
Data are expressed as mean±SD.
G1: vehicle, G2: TA1, G3: TA2, G4: TA3. G5: TA4
* There is a significant difference at the p<0.05 level compared to G1

As described above, when the test substance was administered intravitreally and repeated eye drops were administered to chinchilla rabbits induced with dry macular degeneration, TA1, TA2, TA3 in the level of retinal pigment epithelial cell layer degeneration area, retinal thickness measurement, and histopathological examination results. And it was found that administration of the TA4 test substance delayed the progression of dry macular degeneration induced by sodium iodate compared to the induced control group.

<Example 7> Therapeutic effect in a rat macular degeneration model

In a rat macular degeneration model, the therapeutic effect of FBP peptide by eye drops or intravitreal injection was confirmed.

For histological analysis (H&E / TUNEL assay), after sacrificing an animal, the eyeball was removed, and the eyeball was fixed and frozen using a substance called OCT compound, sliced using a cryosectioning machine, and then the experiment was carried out. Histopathology was analyzed by staining the nucleus and cytoplasm (H&E staining), and the degree of apoptosis was analyzed by TUNEL assay.

The animals used in the experiment were rats, and the number of animals in each group was 3 animals. Details for each group are as follows.

Normal: Normal group to which no substance was administered;

Mock(6G) (negative control group): Mock(6G) administration group in which hexaglycine (6G), which does not play any role experimentally in the eye, was administered in the form of eyedrops;

Dexamethasone (positive control group): Dexamethasone-treated group, which is a corticosteroid-based drug that reduces intraocular inflammation and suppresses apoptosis through anti-inflammatory effects;

FBP(ED): FBP Eyedrop (hereinafter FBP(ED)) administration group in which FBP, a therapeutic peptide having an apoptosis inhibitory effect, was administered in the form of eye drops;

FBP(Ivt): FBP intravitreal injection (hereinafter FBP(Ivt)) group in which FBP, a therapeutic peptide with apoptosis inhibitory effect, was administered through intravitreal injection

As shown in Figure 14, as macular degeneration progresses, cells present in the retina die and the thickness of the retina becomes thinner. As a result, when each group was compared with the negative control Mock(6G) group, the dexamethasone administration group did not show a statistically significant difference (Mock(6G) vs dexamethasone: ns (not significant)), and the FBP Eyedrop administration group also showed a statistically significant difference. There was no difference (Mock(6G) vs FBP ED: ns (not significant)). On the other hand, it was confirmed that the FBP intravitreal injection group showed a statistically significant difference (Mock(6G) vs FBP Ivt: * (Significant)). Through this, it was confirmed that apoptosis was suppressed in the FBP intravitreal injection group, and the retinal thickness was well preserved.

In addition, after TUNEL assay, the ratio of red-stained dead cells in the total cell nuclei stained with blue was measured through ImageJ, an image analysis program, and statistically analyzed through one-way anova analysis, as shown in FIG. 15 . , When compared with the Mock(6G) group, which is each negative control group, the dexamethasone administration group did not show a statistically significant difference (Mock(6G) vs dexamethasone: ns (not significant)), and the FBP Eyedrop administration group also showed no statistically significant difference. (Mock(6G) vs FBP ED: ns (not significant)). On the other hand, it was confirmed that the FBP intravitreal injection group showed a statistically significant difference (Mock(6G) vs FBP Ivt: *** (Significant)). Through this, it was confirmed that apoptosis was inhibited in the FBP intravitreal injection group, and the percentage of TUNEL-positive cells, which are red cells, was reduced.

Next, qPCR analysis of the inflammatory gene was performed, and the qPCR analysis performed a real-time polymerase chain reaction using the cDNA synthesized after sacrificing the animal, removing the eyeballs, separating the RNA, and determining the expression level of the target gene. Confirmed. The details of each target gene are as follows.

TNF-α: TNF-α is a representative cytokine (signaling substance that controls and stimulates the immune system) that appears in the acute inflammatory response, and the higher the expression level, the higher the degree of inflammation.

IL-1β: IL-1β is a representative cytokine that appears in chronic inflammatory responses, and the higher the expression level, the higher the level of inflammation.

Fas: Fas is a representative receptor involved in apoptosis through Fas-Fas ligand interaction. When the level is increased, it means that apoptosis is actively proceeding.

Caspase3: Caspase3 is a protein that plays a pivotal role in the apoptosis reaction, and when the expression level increases, it means that apoptosis proceeds actively.

As a result of statistically analyzing the expression level of the target gene measured through qPCR through one-way anova analysis, as shown in FIG. 16, when each group was compared with the negative control Mock (6G) group, the expression of all target genes In common, it was confirmed that the amount of dexamethasone and FBP Ivt administration group decreased statistically significantly.

Through this, it was confirmed that the expression of inflammatory factors and apoptosis factors were significantly reduced in the dexamethasone-administered group and the FBP Ivt-administered group.

The present invention can be applied to the field of prevention or treatment of macular degeneration.

Claims (10)

A composition for preventing or treating macular degeneration comprising a peptide represented by the following general formula 1 as an active ingredient: [General formula 1] A-B In the above formula, A represents a peptide for inhibiting Fas signaling consisting of an amino acid sequence represented by the following general formula 2 (Fas Blocking Peptide), B is absent or represents a cell penetrating peptide [General formula 2] Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃ In the above formula, Xaa ₁ and Xaa ₃ are each independently absent or any amino acid, Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, Ile, Met, Pro, Ser, Cys, Thr, Asn and Gin. According to claim 1, wherein in the general formula 2, Xaa ₁ and Xaa ₃ are each independently absent or selected from the group consisting of Tyr, Phe and Trp, Xaa ₂ is absent or selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys, a composition for preventing or treating macular degeneration. According to claim 1, wherein in the general formula 2, Xaa ₁ and Xaa ₃ are each independently selected from the group consisting of Tyr, Phe and Trp, Xaa ₂ is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys, a composition for preventing or treating macular degeneration. According to claim 1, The amino acid sequence represented by Formula 2 is any one selected from the group consisting of SEQ ID NOS: 1 to 10, a composition for preventing or treating macular degeneration. The method of claim 1, The cell-penetrating peptide is any one selected from the group consisting of SEQ ID NOS: 11 to 38, a composition for preventing or treating macular degeneration. According to claim 1, A composition for preventing or treating macular degeneration is a composition for preventing or treating macular degeneration comprising a single peptide of a peptide for inhibiting Fas signaling as an active ingredient. According to claim 1, A composition for preventing or treating macular degeneration is a composition for preventing or treating macular degeneration, comprising, as an active ingredient, a fusion peptide of a Fas signaling inhibitory peptide and a cell-penetrating peptide. According to claim 1, Macular degeneration is either dry macular degeneration or wet macular degeneration, a composition for preventing or treating macular degeneration. According to claim 1, The composition for preventing or treating macular degeneration is an eye drop formulation or an injection formulation, a composition for preventing or treating macular degeneration. A method of treating macular degeneration comprising administering a therapeutically effective amount of the composition for preventing or treating macular degeneration of claim 1 to a subject in need thereof.

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