Protective agent for retinal pigment epithelial cells

Abstract

The present invention addresses the problem of providing a protective agent for retinal pigment epithelial cells, which is useful for the prevention or treatment of age-related macular degeneration. A protective agent for retinal pigment epithelial cells according to the present invention, which is a solution for the problem, contains, as an active ingredient, a peptide in which a protein transduction domain is attached to the N-terminus of a peptide having an inhibitory effect on mitochondrial calpains. A specific example of the peptide having an inhibitory effect on mitochondrial calpains is a peptide comprising the amino acid sequence (PDALKSRTJR) represented by SEQ ID NO: 1 (wherein J represents L or I). A specific example of the protein transduction domain is a peptide comprising an amino acid sequence that is at least a part of the amino acid sequence for the transduction domain of HIV-1 Tat.

Description

Technical Field

[0001] The present invention relates to a protective agent for retinal pigment epithelial cells.

Background Art

[0002] When we see an object, the light that enters our eyes is received by the retina as a light stimulus, and the signal is transmitted to the optic nerve to be sent to the brain. Age-related macular degeneration (AMD), which is located in the center of the retina and plays an important role in seeing objects, is damaged with aging and causes vision loss. It is one of the representative examples of age-related eye diseases, and the number of patients worldwide has reached about 170 million, and it is predicted that the number of patients will continue to increase in the future. There are two types of age-related macular degeneration: exudative (wet type) age-related macular degeneration and atrophic (dry type) age-related macular degeneration. Although the mechanisms of onset are different for both, functional damage to retinal pigment epithelial cells (Retinal Pigment Epithelium (RPE) cells) existing below the macula is a factor in vision loss. Today, for exudative age-related macular degeneration, treatment is carried out by injecting an anti-VEGF drug into the vitreous body in the eye to suppress the generation of new blood vessels from the choroid that causes functional damage to retinal pigment epithelial cells, or by photodynamic therapy. However, at present, there is no effective method for treating atrophic age-related macular degeneration. Against this clinical background, a method of protecting retinal pigment epithelial cells has attracted attention as a method for preventing and treating age-related macular degeneration. For example, in Patent Document 1, it has been reported that (S)-4-fluoro-5-(2-methyl-1,4-diazepan-1-ylsulfonyl)isoquinoline has a protective effect on retinal pigment epithelial cells. However, a method of preventing and treating age-related macular degeneration by protecting retinal pigment epithelial cells has not yet been established, and the search for new components having a protective effect on retinal pigment epithelial cells is still in a significant situation.

Prior Art Documents

Patent Documents

[0003] [Patent Document 1] Japanese Patent Publication No. 2015-229671 [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, the present invention aims to provide a protective agent for retinal pigment epithelial cells that is useful for preventing and treating age-related macular degeneration. [Means for solving the problem]

[0005] In view of the above points, the present inventors conducted diligent studies and found that a peptide having a protein transfer domain added to the N-terminus of a peptide that inhibits mitochondrial calpain, which the present inventors' research group reported in Japanese Patent No. 6183879 as having a protective effect on retinal photoreceptor cells, has an excellent protective effect on retinal pigment epithelial cells.

[0006] Protection of retinal pigment epithelial cells based on the above findings: eye drops The agent is as described in claim 1, Selected from peptides consisting of the amino acid sequences shown in SEQ ID NOs: 6, 8-16 The active ingredient is a peptide in which a protein transfer domain is added to the N-terminus of a peptide that has an inhibitory effect on mitochondrial calpain. 。 [Effects of the Invention]

[0007] According to the present invention, a protective agent for retinal pigment epithelial cells that is useful for the prevention and treatment of age-related macular degeneration can be provided. [Brief explanation of the drawing]

[0008] [Figure 1] This is a histological photograph of a retinal section showing that the retinal pigment epithelial cell layer is protected by instilling 20 mM Tat-μCL peptide into the eye during an experiment using an age-related macular degeneration model mouse in Example 1. [Figure 2]This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. [Figure 3] This graph shows that thinning of the retinal photoreceptor cell layer is suppressed. [Figure 4] This is a histological photograph of a retinal section showing that the retinal pigment epithelial cell layer is protected by instilling 2 mM Tat-μCL peptide into the eye during an experiment using an age-related macular degeneration model mouse in Example 2. [Figure 5] This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. [Figure 6] This graph shows that thinning of the retinal photoreceptor cell layer is suppressed. [Figure 7] This is a histological photograph of a retinal section from an experiment using an age-related macular degeneration model mouse in Example 3, showing that the retinal pigment epithelial cell layer is protected by instilling 200 μM Tat-μCL peptide into the eye. [Figure 8] This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. [Figure 9] This graph shows that thinning of the retinal photoreceptor cell layer is suppressed. [Modes for carrying out the invention]

[0009] The protective agent for retinal pigment epithelial cells of the present invention contains as an active ingredient a peptide in which a protein transfer domain is added to the N-terminus of a peptide that has an inhibitory effect on mitochondrial calpain. The peptide in which a protein transfer domain is added to the N-terminus of a peptide that has an inhibitory effect on mitochondrial calpain is the peptide that the present inventors' research group reported in Japanese Patent No. 6183879 to have a protective effect on retinal photoreceptor cells.

[0010] Among peptides that inhibit mitochondrial calpain, those having a protein transfer domain added to the N-terminus of a peptide, include the peptide with the amino acid sequence (PDALKSRTJR) shown in SEQ ID NO: 1 (where J is L or I). The peptide with the amino acid sequence where J is L is included in the amino acid sequence of domain III of the active subunit of rat μ-calpain (also called CAPN1 or μCL), identified by accession number: NP_062025.1. Domain III of the active subunit of μ-calpain is a functional domain also called the C2L (C2-like) domain. The peptide with the amino acid sequence where J is I is included in the amino acid sequence of domain III of the active subunit of human μ-calpain, identified by accession number: NP_005177. In a peptide having an inhibitory effect on mitochondrial calpain, the peptide having an inhibitory effect on mitochondrial calpain may be an amino acid sequence having up to 5 amino acids added to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and which has an inhibitory effect on mitochondrial calpain. Specific examples include a peptide having an amino acid sequence having up to 5 amino acids added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 (for example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2), a peptide having an amino acid sequence having up to 5 amino acids added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 1 (for example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3), and a peptide having an amino acid sequence having up to 5 amino acids added to both the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 1 (for example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4).Furthermore, in a peptide in which a protein transfer domain is added to the N-terminus of a peptide that inhibits mitochondrial calpain, the peptide that inhibits mitochondrial calpain may be an amino acid sequence in which up to two amino acids at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1 are substituted with other amino acids or deleted, and which is a peptide that inhibits mitochondrial calpain.

[0011] The protein transduction domain (PTD) attached to the N-terminus of a peptide that inhibits mitochondrial calpain can be a known cell-penetrating peptide that not only allows itself but also other oligonucleotides, peptides, proteins, oligosaccharides, and other compounds bound to it to pass through the cell membrane phospholipid bilayer. Specific examples include peptides consisting of at least a portion of the amino acid sequence of the transduction site of HIV-1 Tat, peptides consisting of 3 to 12 arginines, peptides consisting of 3 to 12 lysines, peptides consisting of 3 to 16 histidines, PEP-1 peptide, ANTP, and VP22 protein (see Morris et al., Nat. Biotechnol., 19:1173-1175, 2001, Schwarze et al., Trends Cell Biol., 10:290-295, 2000, Vives et al., J. Biol. Chem., 272:16010-16017, 1997, etc., if necessary). Examples of peptides that have an inhibitory effect on mitochondrial calpain and for which a protein transfer domain is desirable to be added to the N-terminus include peptides consisting of the amino acid sequence shown in SEQ ID NO: 5, which corresponds to amino acid positions 49-57 of the transduction site amino acid sequence of HIV-1 Tat; peptides consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added (maximum of 5 substitutions and deletions, and maximum of 10 additions), and which have the effect of allowing not only itself but also other oligonucleotides, peptides, proteins, oligosaccharides, and other compounds bound to it to pass through the cell membrane phospholipid bilayer; and peptides consisting of 3 to 16 histidine molecules. A specific example of a peptide that has an inhibitory effect on mitochondrial calpain with a protein transfer domain added to the N-terminus is the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0012] Furthermore, peptides having an inhibitory effect on mitochondrial calpain, with a protein transfer domain added to the N-terminus, can be prepared by known methods, such as chemical synthesis using a peptide synthesizer, or by genetic engineering techniques.

[0013] Peptides that inhibit mitochondrial calpain and have a protein transfer domain added to their N-terminus exhibit excellent protective effects on retinal pigment epithelial cells. Therefore, they are useful in preventing and treating age-related macular degeneration, in which functional damage to retinal pigment epithelial cells is a factor in vision loss. When administering peptides that inhibit mitochondrial calpain and have a protein transfer domain added to their N-terminus to humans or non-human animals, there are no particular restrictions on the method of administration. They can be formulated in a form suitable for the administration method using known methods, but it is desirable that the formulation be a non-invasive eye drop.

[0014] Eye drops can be prepared by mixing a peptide, which has an inhibitory effect on mitochondrial calpain as its active ingredient, with a protein transfer domain attached to the N-terminus of the peptide, along with various additives such as buffers, solubilizers, isotonic agents, stabilizers, preservatives, viscosity enhancers, chelating agents, pH adjusters, and cooling agents, in a solvent, aseptically filtering the mixture in a sterile environment, and filling it into a suitable sterile container. Specific examples of buffers include boric acid and its salts (such as borax), citric acid and its salts (such as sodium citrate), phosphoric acid and its salts (such as sodium monohydrogen phosphate), tartaric acid and its salts (such as sodium tartrate), gluconic acid and its salts (such as sodium gluconate), acetic acid and its salts (such as sodium acetate), various amino acids, and combinations thereof. Specific examples of solubilizers include polyoxyethylene hydrogenated castor oil (such as polyoxyethylene hydrogenated castor oil 60), polyethylene glycol (such as macrogol 4000), polyoxyethylene sorbitan higher fatty acid esters (such as polysorbate 80), polyoxyethylene (POE)-polyoxypropylene (POP) block copolymer (such as poloxamer 407), and propylene glycol. Specific examples of isotonic agents include inorganic salts (such as sodium chloride, potassium chloride, and calcium chloride), sugars (such as mannitol and glucose), and polyhydric alcohols (such as glycerin and propylene glycol). Specific examples of stabilizers include sodium edetate, cyclodextrin, sulfites, citric acid and its salts, and butylhydroxytoluene. Specific examples of preservatives include benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, polyquartenium-1, polyaminopropylbiguanide, alkylaminoethylglycine hydrochloride, cetylpyridinium chloride, and thimerosal.Specific examples of the thickening agent include polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, sodium chondroitin sulfate, sodium hyaluronate, carboxymethyl cellulose, carboxyvinyl polymer. Specific examples of the chelating agent include sodium edetate and sodium citrate. Specific examples of the pH adjuster include hydrochloric acid, citric acid and its salts, boric acid and its salts, phosphoric acid and its salts, acetic acid and its salts, tartaric acid and its salts, sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydrogen carbonate. Specific examples of the cooling agent include menthol, borneol, camphor, geraniol, limonene, eugenol, peppermint oil, eucalyptus oil, star anise oil, and bergamot oil. Specific examples of the solvent include sterilized purified water, purified water, and physiological saline. The pH of the eye drops is not particularly limited as long as it is within the ophthalmologically acceptable range, and is usually in the range of pH 4 to 9, preferably in the range of pH 5 to 8.

[0015] A peptide obtained by adding a protein transduction domain to the N-terminus of a peptide having an inhibitory effect on mitochondrial calpain, which is an active ingredient, is formulated at 0.00001 to 100 mg per 1 mL of the eye drops, and 1 to several drops are instilled 1 to several times a day, thereby protecting retinal pigment epithelial cells, and exerting a preventive effect and a therapeutic effect against age-related macular degeneration.

Example

[0016] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not construed as being limited to the following description.

[0017] Example 1: Protective effect of a peptide obtained by adding a protein transduction domain to the N-terminus of a peptide having an inhibitory effect on mitochondrial calpain on retinal pigment epithelial cells (effect by instilling into age-related macular degeneration model mice) Part 1 (Experimental method) Using sodium iodate (SI)-administered mice (Investigative Ophthalmology & Visual Science, 58, 2239-2249, 2017), a widely used model system for atrophic age-related macular degeneration, the ophthalmic effect of a peptide having a protein transfer domain added to the N-terminus of a peptide that inhibits mitochondrial calpain was evaluated. Specifically, the peptide having a protein transfer domain added to the N-terminus of a peptide that inhibits mitochondrial calpain was the peptide with the amino acid sequence shown in Sequence ID No. 6 (GRKKRRQRRRPPQPDALKSRTLR) (hereinafter abbreviated as "Tat-μCL peptide," the same as the μ-N2-10-2 (HIV-Nμ) peptide described in Japanese Patent No. 6183879). This peptide was dissolved in physiological saline to a 20 mM solution, filtered and sterilized, and 2 μL was instilled into both eyes of mice (C57BL / 6J, 8 weeks old) three times a day for 7 days prior to administration. After initial eye drops, SI was dissolved in physiological saline, filtered, and sterilized before being administered intraperitoneally to administer SI at a dose of 12.5 mg / kg. Furthermore, a solution of Tat-μCL peptide dissolved in physiological saline to a concentration of 20 mM, filtered, and sterilized was instilled into both eyes three times daily at a dose of 2 μL for 7 days. After 7 days, the mice were euthanized, both eyeballs were removed, fixed with 4% paraformaldehyde, and the anterior segment and lens were excised. Frozen tissue sections were then prepared, stained with hematoxylin and eosin, and observed under a phase-contrast microscope. The number of deposits derived from the retinal pigment epithelial layer (RPE) (aggregates of degenerated retinal pigment epithelial cells appearing as pigment deposits) was measured. Additionally, images were acquired at approximately 300, 600, 900, 1200, and 1500 μm nasally and temporally from the center of the optic disc, respectively, and the thickness of the retinal photoreceptor layer (OS+IS) was measured.

[0018] (Experimental results) Figure 1 shows histological images of retinal sections, Figure 2 shows the measurement results of the number of deposits derived from the retinal pigment epithelial cell layer, and Figure 3 shows the measurement results of the thickness of the retinal photoreceptor layer at approximately 300, 600, 900, 1200, and 1500 μm nasally and temporally from the center of the optic nerve head (20 mM Tat-μCL; the measurement results in Figure 2 and Figure 3 are the average values ​​of samples from both eyes of 3 mice (n=6)). Figures 1-3 show the results of a similar experiment using a solution prepared by dissolving a peptide consisting of the amino acid sequence shown in Sequence ID No. 7 (GRKKRRQRRRPPQASLRLDRPTK), in which the amino acid sequence of the μCL portion of the Tat-μCL peptide was randomly rearranged (hereinafter abbreviated as "Tat-μCL scrambled peptide"), in a solution prepared by dissolving it

[0019] Example 2: Protective effect on retinal pigment epithelial cells of a peptide having an inhibitory effect on mitochondrial calpain, with a protein transfer domain added to the N-terminus (effect by instillation into age-related macular degeneration model mice) Part 2 The same experiment as in Example 1 was performed using a solution of Tat-μCL peptide dissolved in physiological saline to a concentration of 2 mM and filtered and sterilized, and a solution of Tat-μCL scrambled peptide dissolved in physiological saline to a concentration of 2 mM and filtered and sterilized. Figure 4 shows tissue images of retinal sections, Figure 5 shows the results of measuring the number of deposits derived from the retinal pigment epithelial cell layer, and Figure 6 shows the results of measuring the thickness of the retinal photoreceptor layer at positions of approximately 300, 600, 900, 1200, and 1500 μm nasally and temporally from the center of the optic nerve head, respectively (none represents the results of healthy mice in which no administration was performed on the eyeball). As is clear from Figures 4 to 6, Tat-μCL peptide effectively protected the retinal pigment epithelial cell layer and suppressed thinning of the retinal photoreceptor layer, even with 2 mM eye drops.

[0020] Example 3: Protective effect on retinal pigment epithelial cells of a peptide having an inhibitory effect on mitochondrial calpain, with a protein transfer domain added to the N-terminus (effect by instillation into age-related macular degeneration model mice) Part 3 The same experiment as in Example 1 was performed using a solution of Tat-μCL peptide dissolved in physiological saline to a concentration of 200 μM and filtered and sterilized, and a solution of Tat-μCL scrambled peptide dissolved in physiological saline to a concentration of 200 μM and filtered and sterilized. Figure 7 shows tissue images of retinal sections, Figure 8 shows the results of measuring the number of deposits derived from the retinal pigment epithelial cell layer, and Figure 9 shows the results of measuring the thickness of the retinal photoreceptor layer at positions of approximately 300, 600, 900, 1200, and 1500 μm nasally and temporally from the center of the optic nerve head, respectively (none represents the results of healthy mice in which no administration was performed on the eyeball). As is clear from Figures 7 to 9, Tat-μCL peptide effectively protected the retinal pigment epithelial cell layer and suppressed thinning of the retinal photoreceptor layer, even with 200 μM eye drops.

[0021] (Summary of experimental results for Examples 1-3) In Japanese Patent No. 6183879, our research group reported that Tat-μCL peptide has a protective effect on retinal photoreceptor cells, but the concentration at which this effect could be confirmed by eye instillation was 40 mM. In light of this, the fact that we were able to confirm that Tat-μCL peptide has a protective effect on retinal pigment epithelial cells by eye instillation at a concentration less than 1 / 100th of the concentration at which the protective effect on retinal photoreceptor cells could be confirmed by eye instillation is a surprising result that we had not anticipated. This demonstrates that peptides having a protein transfer domain added to the N-terminus of peptides that have an inhibitory effect on mitochondrial calpain, as exemplified by Tat-μCL peptide, are useful for preventing or treating age-related macular degeneration in a non-invasive manner.

[0022] Example 4: Protective effect on retinal pigment epithelial cells of a peptide having a protein transfer domain added to the N-terminus of a peptide that inhibits various mitochondrial calpains (effect obtained by instilling into age-related macular degeneration model mice). The following nine peptides, prepared by chemical synthesis using a peptide synthesizer, were dissolved in physiological saline to a concentration of 200 μM each, and the resulting solutions were filtered and sterilized. The same experiment as in Example 1 was then performed using these solutions. As a result, it was confirmed that all of the peptides, like the Tat-μCL peptide, had a protective effect on retinal pigment epithelial cells when instilled at a concentration of 200 μM. The peptide consists of the amino acid sequence shown in SEQ ID NO: 8 (GRKKRRQRRRPPQICNLTPDALKSRTLR), which is obtained by changing the amino acid sequence of the μCL portion of the Tat-μCL peptide to an amino acid sequence in which five amino acids are added to the N-terminus of that amino acid sequence (the amino acid sequence shown in SEQ ID NO: 2). The peptide consists of the amino acid sequence shown in SEQ ID NO: 9 (GRKKRRQRRRPPQPDALKSRTLRNWNTT), which is obtained by changing the amino acid sequence of the μCL portion of the Tat-μCL peptide to an amino acid sequence in which five amino acids are added to the C-terminus of that amino acid sequence (the amino acid sequence shown in SEQ ID NO: 3). The peptide consists of the amino acid sequence shown in SEQ ID NO: 10 (GRKKRRQRRRPPQICNLTPDALKSRTLRNWNTT), which is obtained by changing the amino acid sequence of the μCL portion of the Tat-μCL peptide to an amino acid sequence in which five amino acids are added to the N-terminus and C-terminus of that amino acid sequence (the amino acid sequence shown in SEQ ID NO: 4). A peptide consisting of the amino acid sequence shown in SEQ ID NO: 11 (GRKKRRQRRRPPQALKSRTLR), obtained by changing the amino acid sequence of the μCL portion of the Tat-μCL peptide to an amino acid sequence in which the two amino acids at the N-terminus of that amino acid sequence are deleted. The peptide consists of the amino acid sequence shown in SEQ ID NO: 12 (GRKKRRQRRRPPQPDALKSRT), which is obtained by changing the amino acid sequence of the μCL portion of the Tat-μCL peptide to an amino acid sequence in which the two C-terminal amino acids of that amino acid sequence are deleted. The peptide consists of the amino acid sequence shown in SEQ ID NO: 13 (GRKKRRQRRRPPQALKSRT), which is obtained by modifying the amino acid sequence of the μCL portion of the Tat-μCL peptide by deleting two amino acids each from the N-terminus and C-terminus of that amino acid sequence. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 14 (GRKKRRQRRRPPQPDALKSRTA), in which alanine is added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 12. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 15 (GRKKRRQRRRPPQPDALKSRTIR), in which the second leucine from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with isoleucine. • A peptide consisting of the amino acid sequence shown in SEQ ID NO. 16 (HHHHHHPDALKSRTLR), which is obtained by changing the amino acid sequence of the Tat portion of the Tat-μCL peptide to an amino acid sequence consisting of six histidines. [Industrial applicability]

[0023] The present invention has industrial applicability in that it can provide a protective agent for retinal pigment epithelial cells that is useful for the prevention and treatment of age-related macular degeneration.

Claims

[Claim 1] A protective eye drop for retinal pigment epithelial cells, comprising as an active ingredient a peptide selected from peptides consisting of the amino acid sequences shown in SEQ ID NOs: 6, 8 to 16, wherein a protein transfer domain is added to the N-terminus of a peptide that has an inhibitory effect on mitochondrial calpain.

Images