JP7832745B2 - Protective agent for retinal pigment epithelial cells - Google Patents

Description

This invention relates to a protective agent for retinal pigment epithelial cells.

When we see, the light entering our eyes is received as a light stimulus by the retina, and this signal is transmitted to the optic nerve to the brain. Age-related macular degeneration (AMD), which affects the macula, located in the center of the retina that plays a crucial role in vision, and causes vision loss, is one of the most common age-related eye diseases. The number of patients worldwide is approximately 170 million, and this number is expected to continue to rise. There are two types of age-related macular degeneration: exudative (wet) age-related macular degeneration and atrophic (dry) age-related macular degeneration. Although the mechanisms of onset differ between the two, in both cases, functional damage to retinal pigment epithelial cells (RPE cells) located beneath the macula is a factor in vision loss. Currently, treatment for age-related macular degeneration (AMD) involves injecting anti-VEGF drugs into the vitreous humor to suppress neovascularization from the choroid, which causes functional damage to retinal pigment epithelial cells, or photodynamic therapy for exudative AMD. However, there is currently no effective treatment for atrophic AMD. Given this clinical background, methods that protect retinal pigment epithelial cells have attracted attention as a way to prevent or treat AMD. For example, Patent Document 1 reports that (S)-4-fluoro-5-(2-methyl-1,4-diazepane-1-ylsulfonyl)isoquinoline has a protective effect on retinal pigment epithelial cells. However, a method for preventing or treating AMD by protecting retinal pigment epithelial cells has not yet been established, and the search for new components that have a protective effect on retinal pigment epithelial cells remains a significant effort.

Japanese Patent Publication No. 2015-229671

Therefore, the present invention aims to provide a protective agent for retinal pigment epithelial cells that is useful for the prevention and treatment of age-related macular degeneration.

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 excellent protective effects on retinal pigment epithelial cells.

Based on the above findings, the present invention provides a protective eye drop for retinal pigment epithelial cells, as described in claim 1, which contains as an active ingredient a peptide selected from peptides consisting of amino acid sequences shown in SEQ ID NOs: 6, 8 to 16, having an inhibitory effect on mitochondrial calpain, to which a protein transfer domain is added to the N-terminus .

According to the present invention, a protective agent for retinal pigment epithelial cells, useful for the prevention and treatment of age-related macular degeneration, can be provided.

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. This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. This graph shows that thinning of the retinal photoreceptor cell layer is suppressed. 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. This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. This graph shows that thinning of the retinal photoreceptor cell layer is suppressed. This is a histological photograph of a retinal section showing that the retinal pigment epithelial cell layer is protected by instilling 200 μM Tat-μCL peptide into the eye during an experiment using an age-related macular degeneration model mouse in Example 3. This graph shows that the increase in the number of deposits originating from the retinal pigment epithelial cell layer is suppressed. This graph shows that thinning of the retinal photoreceptor cell layer is suppressed.

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.

Among peptides that inhibit mitochondrial calpain, those having a protein transfer domain added to the N-terminus of a peptide, include the peptide consisting of the amino acid sequence (PDALCSRTJR) shown in SEQ ID NO: 1 (where J is L or I). The peptide consisting of 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 consisting of 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 five 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 five 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 five 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 five amino acids added to 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.

The protein transfer domain (PTD) attached to the N-terminus of a peptide that inhibits mitochondrial calpain can be a known cell-penetrating peptide that allows not only 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 arginine molecules, peptides consisting of 3 to 12 lysine molecules, peptides consisting of 3 to 16 histidine molecules, PEP-1 peptide, ANTP, VP22 protein, etc. (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 themselves but also other oligonucleotides, peptides, proteins, oligosaccharides, and other compounds bound to them to pass through the cell membrane phospholipid bilayer; and peptides consisting of 3 to 16 histidines. 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.

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.

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, where functional damage to retinal pigment epithelial cells is a factor in vision loss. When administering peptides that inhibit mitochondrial calpain to humans or non-human animals, the method of administration is not particularly limited; they can be formulated using known methods in a form suitable for the administration method. However, the formulation should preferably be a non-invasive eye drop.

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 viscosity modifiers include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, sodium chondroitin sulfate, sodium hyaluronate, carboxymethylcellulose, and carboxyvinyl polymer. Specific examples of chelating agents include sodium edetate and sodium citrate. Specific examples of pH adjusters 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 bicarbonate. Specific examples of cooling agents include menthol, borneol, camphor, geraniol, limonene, eugenol, peppermint oil, eucalyptus oil, fennel oil, and bergamot oil. Specific examples of solvents include sterile purified water, purified water, and physiological saline solution. The pH of eye drops is not particularly limited as long as it is within an ophthalmologically acceptable range, and is usually in the range of pH 4 to 9, preferably in the range of pH 5 to 8.

The peptide, which has an inhibitory effect on mitochondrial calpain (the active ingredient) and has a protein transfer domain added to its N-terminus, is included in 0.00001 to 100 mg per 1 mL of eye drops. By instilling one to several drops at a time, one to several times a day, it protects retinal pigment epithelial cells and exerts preventive and therapeutic effects against age-related macular degeneration.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following description.

Example 1: 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 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, we evaluated the eye drop effect of a peptide in which a protein transfer domain was added to the N-terminus of a peptide that inhibits mitochondrial calpain. Specifically, a peptide consisting of the amino acid sequence shown in Sequence ID No. 6 (GRKKRRQRRRRPQPDALKSRTLR), which has an inhibitory effect on mitochondrial calpain and is the same as the μ-N2-10-2 (HIV-Nμ) peptide described in Japanese Patent No. 6183879, was dissolved in physiological saline to a concentration of 20 mM, filtered, and sterilized. This solution was then instilled into both eyes of mice (C57BL/6J, 8 weeks old) at a concentration of 2 μL three times a day for 7 days. After the initial instillation, SI was dissolved in physiological saline and filtered, and a solution of SI was administered intraperitoneally, administering 12.5 mg/kg. Furthermore, a solution of Tat-μCL peptide dissolved in physiological saline to a concentration of 20 mM and filtered, was instilled into both eyes at a concentration of 2 μL three times a day for 7 days. Seven days later, 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 to count the number of deposits derived from the retinal pigment epithelial layer (RPE) (degenerated retinal pigment epithelial cells that appear as pigment deposits). Furthermore, images were acquired at approximately 300, 600, 900, 1200, and 1500 μm nasally and temporally from the center of the optic disc, and the thickness of the retinal photoreceptor layer (OS + IS) was measured.

(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 from the center of the optic nerve head, both nasally and temporally (20 mM Tat-μCL; the measurement results in Figure 2 and Figure 3 are the average values of samples from both eyes of three 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 (GRKKRRQRRRRPPQASLRLDRPTK), in which the amino acid sequence of the μCL portion of the Tat-μCL peptide was randomly rearranged, in a solution prepared by dissolving it in physiological saline to a 20 mM concentration and sterilizing it (hereinafter abbreviated as "Tat-μCL scrambled peptide"), and sterilizing it by filtering it, instead of a solution prepared by dissolving the Tat-μCL peptide in physiological saline to a 20 mM concentration and sterilizing it. The results are also shown for healthy mice that received no administration to the eyeball (none). As is clear from Figures 1 and 2, the Tat-μCL peptide effectively protected the retinal pigment epithelial cell layer when administered as eye drops at a 20 mM concentration. The suppression of thinning of the retinal photoreceptor layer by administration of Tat-μCL peptide, as is evident from Figure 3, is considered to be a secondary effect due to the protection of the retinal pigment epithelial cell layer by Tat-μCL peptide.

Example 2: 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 mitochondrial calpain (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 indicates results from 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.

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 indicates results from 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.

(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 the 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 a peptide that has 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.

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.
- A peptide consisting of the amino acid sequence shown in SEQ ID NO: 8 (GRKKRRRQRRRRPQICNLTPDALKSRTLR), 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) - A peptide consisting of the amino acid sequence shown in SEQ ID NO: 9 (GRKKRRRQRRRRPQPDALKSRTLRNW), 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 amino acid sequence of the μCL portion of the Tat-μCL peptide, consisting of NTT, is modified to an amino acid sequence in which five amino acids are added to the N-terminus and C-terminus of the amino acid sequence (the amino acid sequence shown in SEQ ID NO: 4), resulting in the amino acid sequence shown in SEQ ID NO: 10 (GRKKRRRQRRRPPQICNLTPDALKSRTLRRNWNTT). The amino acid sequence of the μCL portion of the Tat-μCL peptide, consisting of NTT, is modified to an amino acid sequence in which the two amino acids at the N-terminus of the amino acid sequence are deleted, resulting in the amino acid sequence shown in SEQ ID NO: 11 (GRKKRRRQRRRPPQA The peptide consisting of LKSRTLR) and the amino acid sequence of the μCL portion of the Tat-μCL peptide, modified to an amino acid sequence in which the two amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO: 12 (GRKKRRQRRRRPQPDALKSRT) and the amino acid sequence of the μCL portion of the Tat-μCL peptide, modified to an amino acid sequence in which the two amino acids at the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 13 (GRKKRRQRRRRPQALKSRT) are obtained by modifying the amino acid sequence of the μCL portion of the Tat-μCL peptide, modified to an amino acid sequence in which the two amino acids at the N-terminus and 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: 14 (GRKKRRQRRRRPPQPDALKSRTAR), in which alanine is added to the C-terminus of the amino acid sequence; a peptide consisting of the amino acid sequence shown in SEQ ID NO: 15 (GRKKRRQRRRRPPQPDALKSRTIR), in which the second leucine from the C-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with isoleucine; and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 (HHHHHHPDALCRTLR), in which the amino acid sequence of the Tat portion of the Tat-μCL peptide is changed to an amino acid sequence consisting of six histidines.

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 (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 .