JP2024032794A - Protective agent for retinal nerve cells - Google Patents

Abstract

[Problem] To provide a protective agent for retinal nerve cells, such as retinal ganglion cells and retinal photoreceptor cells. [Solution] The active ingredient is a compound represented by the general formula: R1-(CH2)m-SR2 or a pharmaceutically acceptable salt thereof (wherein R1 is -NR3R4 or an alkyl group having 1 to 6 carbon atoms). group.R2 represents a hydrogen atom or -S-(CH2)n-NR5R6.R3 to R6 are the same or different and represent a hydrogen atom or an acetyl group.m and n are the same or different and are integers of 2 to 4 ). [Selection diagram] Figure 1

Description

The present invention relates to a protective agent for retinal nerve cells, such as retinal ganglion cells and retinal photoreceptor cells.

Glaucoma is the number one cause of premature blindness in Japan. It has been reported that one in 20 people over the age of 40 suffers from glaucoma, and it is expected that the number of glaucoma patients will continue to increase as the population ages. Glaucoma is caused by an increase in intraocular pressure (21 mmHg or higher), which causes the death of retinal ganglion cells (RGCs) and causes abnormalities in the optic nerve, leading to decreased visual acuity and narrowing of the visual field, and ultimately It is a disease that can lead to blindness. Therefore, in the treatment of glaucoma, a method of lowering the intraocular pressure by instilling an intraocular hypotensive drug into the eye is mainly adopted. However, it is known that some patients develop glaucoma even with intraocular pressure within the normal range (less than 21 mmHg), and approximately 70% of all glaucoma patients have normal tension glaucoma (NTG). ). In normal tension glaucoma, the intraocular pressure is within the normal range. Therefore, even if treatment for lowering intraocular pressure is performed for normal-tension glaucoma, the effect is not necessarily sufficient. From this clinical background, attention has been paid to methods that protect retinal ganglion cells as a treatment method for glaucoma. For example, Patent Document 1 reports that pyrroloquinoline quinone has a protective effect on retinal ganglion cells. has been done. However, a method for treating glaucoma by protecting retinal ganglion cells has not yet been established. Furthermore, retinitis pigmentosa and age-related macular degeneration are retinal neurodegenerative diseases that originate in retinal photoreceptor cells, and the same situation exists regarding methods of treating these by protecting retinal photoreceptor cells.

Japanese Patent Application Publication No. 2018-035076

Therefore, an object of the present invention is to provide a protective agent for retinal nerve cells, such as retinal ganglion cells and retinal photoreceptor cells.

The present inventors conducted intensive studies in view of the above points, and found that cystamine and cysteamine have a protective effect on nerve cells in the retina. The researchers found that this method is effective in preventing and treating retinal neurodegenerative diseases such as retinitis pigmentosa and age-related macular degeneration.

The retinal nerve cell protective agent of the present invention, which has been developed based on the above findings, is, as described in claim 1, a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or its pharmaceutical (wherein R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. R ₂ represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different and represent a hydrogen atom or an acetyl group. m and n are the same or different and represent an integer of 2 to 4).
Further, the retinal nerve cell protective agent according to claim 2 is the retinal nerve cell protective agent according to claim 1, wherein the compound is
(a) Cystamine where R ₁ is -NH ₂ , R ₂ is -S-(CH ₂ ) ₂ -NH ₂ and m is 2 (b) R ₁ is -NH ₂ , R ₂ is a hydrogen atom and m is 2 Cysteamine (c) is any N-acetylcysteamine in which R ₁ is -NHAc (Ac: acetyl group), R ₂ is a hydrogen atom, and m is 2.
Furthermore, in the retinal nerve cell protective agent according to claim 3, in the retinal nerve cell protective agent according to claim 1, the retinal nerve cells are retinal ganglion cells and/or retinal photoreceptor cells.
Furthermore, as described in claim 4, the preventive and/or therapeutic agent for retinal neurodegenerative diseases of the present invention is a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable compound thereof. (wherein R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. _{R 2} represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different and represent a hydrogen atom or an acetyl group. m and n are the same or different and represent an integer of 2 to 4).
The preventive and/or therapeutic agent for a retinal neurodegenerative disease according to claim 5 is the preventive and/or therapeutic agent for a retinal neurodegenerative disease according to claim 4, wherein the retinal neurodegenerative disease is glaucoma, retinitis pigmentosa, At least one type of age-related macular degeneration.
Furthermore, as described in claim 6, the transcription factor C/EBP homologous protein (CHOP) expression inhibitor of the present invention is a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or its pharmaceutical (wherein R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. R ₂ represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different and represent a hydrogen atom or an acetyl group. m and n are the same or different and represent an integer of 2 to 4).

According to the present invention, it is possible to provide a protective agent for retinal nerve cells, such as retinal ganglion cells and retinal photoreceptor cells.

1 is a graph showing the protective effect of cystamine on nerve cells (HT22 cells, a nerve cell line derived from the mouse hippocampus) in Example 1. 3 is a graph showing that retinal ganglion cells are protected by instilling cystamine into normal tension glaucoma model mice in Example 4. It is a graph showing that thinning of the inner plexiform layer is suppressed in the same example. This is a graph showing that retinal thinning is suppressed. 3 is a graph showing that retinal ganglion cells are protected by instilling cysteamine into normal tension glaucoma model mice in Example 5. It is a graph showing that thinning of the inner plexiform layer is suppressed in the same example. This is a graph showing that retinal thinning is suppressed. 12 is a graph showing that thinning of the outer nuclear layer of the retina is suppressed by instilling cystamine into the eyes of a retinitis pigmentosa model mouse in Example 6. 7 is a graph showing that the increase in the number of deposits derived from the retinal pigment epithelial cell layer is suppressed by administering cystamine to age-related macular degeneration model mice in Example 7. This is a graph showing that retinal thinning is suppressed. 12 is a graph showing that the increase in the number of deposits derived from the retinal pigment epithelial cell layer is suppressed by administering cystamine to age-related macular degeneration model mice in Example 8. 12 is a graph showing that the increase in the number of deposits derived from the retinal pigment epithelial cell layer is suppressed by applying cysteamine to age-related macular degeneration model mice in Example 9. 10 is a graph showing that retinal ganglion cells are protected by instilling a long-term stored solution of cystamine or cysteamine into normal tension glaucoma model mice in Example 10. It is a graph showing that thinning of the inner plexiform layer is suppressed in the same example. FIG. 2 is a fluorescent immunohistological image showing that the expression of CHOP is suppressed by instilling cystamine into a normal-tension glaucoma model mouse in Example 11. FIG. 12 is a graph showing that cystamine suppresses the expression of CHOP in nerve cells induced by thapsigargin in Example 12.

The retinal nerve cell protective agent of the present invention contains a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof as an active ingredient (in the formula, R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. _{R 2} represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different represents a hydrogen atom or an acetyl group. m and n are the same or different and represent an integer from 2 to 4).

Here, the alkyl group having 1 to 6 carbon atoms may be linear or branched, and specifically includes a methyl group, ethyl group, n-propyl group, isopropyl group, Examples include n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, and n-hexyl group.

In addition, when the compound is acidic, pharmaceutically acceptable salts include ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, arginine salts and lysine salts. Examples include amino acid salts such as salt. If the compound is basic, inorganic acid salts (hydrochloride, sulfate, nitrate, phosphate, etc.), organic acid salts (acetate, lactate, citrate, tartrate, maleate, fumarate, etc.) , oxalate, etc.).

Specific examples of the compound represented by the above general formula include cystamine where R ₁ is -NH ₂ , R ₂ is -S-(CH ₂ ) ₂ -NH ₂ and m is 2; R ₁ is -NH ₂ Examples include cysteamine in which R ₂ is a hydrogen atom and m is 2, and N-acetylcysteamine in which R ₁ is -NHAc (Ac: acetyl group), R ₂ is a hydrogen atom, and m is 2. It may also be 3-amino-1-propanethiol, where R ₁ is -NH ₂ , R ₂ is a hydrogen atom, and m is 3.

The objects to be protected by the retinal nerve cell protective agent of the present invention are retinal nerve cells involved in the transmission of visual signals to the brain, specifically retinal ganglion cells, retinal photoreceptor cells, and the like. Therefore, the agent for protecting retinal nerve cells of the present invention can be used as a prophylactic and/or therapeutic agent for retinal neurodegenerative diseases originating from such retinal nerve cells. Specific examples of retinal neurodegenerative diseases include glaucoma, which originates in retinal ganglion cells, and retinitis pigmentosa and age-related macular degeneration, which originate in retinal photoreceptor cells. The agent for protecting retinal nerve cells of the present invention may be administered parenterally or orally to humans or non-human animals, and may be administered parenterally or orally using methods known per se. However, it is preferable that the formulation is in the form of eye drops.

Eye drops contain a compound represented by the above general formula or a pharmaceutically acceptable salt thereof as an active ingredient, as well as a buffer, a solubilizer, an isotonizing agent, a stabilizer, a preservative, and a viscosity agent. It can be prepared by blending various additives such as chelating agents, pH adjusters, and cooling agents with a solvent, sterile filtration in a sterile environment, and filling into a suitable sterilized container. Specific examples of buffering agents include boric acid and its salts (borax, etc.), citric acid and its salts (sodium citrate, etc.), phosphoric acid and its salts (sodium monohydrogen phosphate, etc.), and tartaric acid and its salts (sodium citrate, etc.). examples include sodium tartrate, etc.), gluconic acid and its salts (sodium gluconate, etc.), acetic acid and its salts (sodium acetate, etc.), various amino acids, and combinations thereof. Specific examples of solubilizing agents include polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil 60, etc.), polyethylene glycol (macrogol 4000, etc.), polyoxyethylene sorbitan higher fatty acid ester (polysorbate 80, etc.), polyoxyethylene Examples include ethylene (POE)-polyoxypropylene (POP) block copolymers (poloxamer 407, etc.) and propylene glycol. Specific examples of tonicity agents include inorganic salts (sodium chloride, potassium chloride, calcium chloride, etc.), sugars (mannitol, glucose, etc.), and polyhydric alcohols (glycerin, propylene glycol, etc.). Specific examples of the stabilizer include sodium edetate, cyclodextrin, sulfites, citric acid and its salts, and butylated hydroxytoluene. Specific examples of preservatives include benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, Examples include polyquartenium-1, polyaminopropylbiguanide, alkylaminoethylglycine hydrochloride, cetylpyridinium chloride, and thimerosal. Specific examples of thickeners include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethylcellulose, hydroxypropylmethylcellulose, 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, Examples include sodium hydrogen carbonate. Specific examples of refreshing agents include menthol, borneol, camphor, geraniol, limonene, eugenol, peppermint oil, eucalyptus oil, fennel oil, and bergamot oil. Specific examples of the solvent include sterile purified water, purified water, and physiological saline. The pH of the 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 active ingredient, the compound represented by the above general formula or a pharmaceutically acceptable salt thereof, is blended in an amount of 0.00001 to 100 mg per mL of eye drops, and 1 to several drops are administered per day. By protecting nerve cells in the retina, the eye drops have preventive and therapeutic effects on retinal neurodegenerative diseases such as glaucoma, retinitis pigmentosa, and age-related macular degeneration.

The eye drops may contain other pharmaceutical ingredients, such as prostaglandin preparations such as latanoprost and travoprost, beta blockers such as timolol maleate and carteolol hydrochloride, and carbonic anhydrase inhibitors such as dorzolamide hydrochloride and brinzolamide. It may also be combined with eye drops such as intraocular hypotensive drugs.

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to examples, but the present invention should not be interpreted as being limited to the following description.

Example 1: Protective effect of cystamine on nerve cells (experimental method)
The protective effect of cystamine on neurons was evaluated using HT22 cells, a mouse hippocampus-derived neuronal cell line whose apoptosis (cell death) is induced by glutamic acid. Specifically, cystamine was added to DMEM medium supplemented with 10% FBS at final concentrations of 10 μM, 5 μM, 2 μM, and 1 μM to HT22 cells (1.2×10 ³ cells per well) seeded in a 96-well plate. added to. After 4 hours, glutamic acid was added to a final concentration of 4 mM, and the cells were cultured for 20 hours at 37° C. in a 5% CO ₂ incubator. Next, 10 μL of cytotoxicity assay reagent (Cell Counting Kit-8, Dojindo) was added to each well to generate a color reaction, and the absorbance (490 nm) was measured 3 hours later to determine the survival of control cells without the addition of glutamate. The cell survival rate (%) was calculated with the rate as 100%.

(Experimental result)
Shown in Figure 1. As is clear from FIG. 1, cystamine suppressed apoptosis of HT22 cells in a concentration-dependent manner, indicating that it has a protective effect on nerve cells. In addition, separate experiments confirmed that cystamine inhibits the nuclear translocation of apoptosis-inducing factor (AIF) induced by glutamate, indicating that cystamine suppresses apoptosis in HT22 cells. It has been speculated that one of the mechanisms of action is to inhibit the translocation of apoptosis-inducing factors to the nucleus.

Example 2: Protective effect of cysteamine on nerve cells The same experiment as in Example 1 was conducted and evaluated. The results are as follows; cysteamine also inhibited apoptosis of HT22 cells in a concentration-dependent manner, confirming the protective effect of cysteamine on nerve cells.
Cell viability (%)
Cysteamine 2μM 82.76 ^*
Cysteamine 5 μM 104.17 ^*
Cysteamine 10μM 110.30 ^*
PBS (-) 65.01
* Wilcoxon-test ^* P<0.01, n=12

Example 3: Protective effect of N-acetylcysteamine on nerve cells The same experiment as in Example 1 was conducted and evaluated. The results are as follows, and since N-acetylcysteamine also inhibited apoptosis of HT22 cells, we were able to confirm the protective effect of N-acetylcysteamine on nerve cells (significance test was performed due to insufficient number of n). ).
Cell viability (%)
N-acetylcysteamine 10μM 94.51
PBS (-) 35.24

Example 4: Effect of instilling cystamine into normal tension glaucoma model mice (experimental method)
Using N-methyl-D-aspartate (NMDA)-administered mice (Investigative Ophthalmology & Visual Science, 40, 1004-1008, 1999), which is widely used as a model experimental system for normal-tension glaucoma, we investigated the ophthalmic effects of cystamine. was evaluated. Specifically, NMDA was dissolved in physiological saline to a concentration of 40 mM and sterilized by filtration, and 2 μL of the solution was administered into the vitreous of both eyes of a mouse (C57BL/6J, 7 weeks old) to maintain normal intraocular pressure. A glaucoma model mouse was created. To administer the NMDA solution into the vitreous, mice were anesthetized using isoflurane according to the method of Shimazawa et al. The test was carried out from the sclera of the limbus using a 10 μL microsyringe connected to 33G). After the needle was removed, Cravit eye drops, an anti-inflammatory drug, were instilled into the eyes. Immediately after the intravitreal administration of the NMDA solution, a solution of cystamine dissolved in physiological saline to a concentration of 10 mM and sterilized by filtration was instilled into both eyes in 2 μL doses three times a day for 7 days. Seven days later, the mice were euthanized, both eyeballs were enucleated and fixed with 4% paraformaldehyde, and the anterior segment and crystalline lens were removed. Next, frozen section slides of the tissue were prepared, stained with hematoxylin and eosin, and observed with a phase contrast microscope to count the number of cells present in the retinal ganglion cell layer, measure the thickness of the inner plexiform layer, and measure the retinal structure. The thickness was measured.

(Experimental result)
Figure 2 shows the measurement results of the number of cells present in the retinal ganglion cell layer (GCL), Figure 3 shows the measurement results of the inner plexiform layer (IPL) thickness, and Figure 4 shows the measurement results of the retina thickness. The results are shown respectively (cystamine/NMDA). Figures 2 to 4 show the results (saline/NMDA) when filter-sterilized physiological saline was instilled into both eyes at a rate of 2 μL three times a day for 7 days immediately after intravitreal administration of the NMDA solution. Also shown are the results (control) when filter-sterilized physiological saline was instilled into both eyes of healthy mice at a rate of 2 μL three times a day for 7 days (all results were obtained from both eyes of three mice). (average value of samples (n=6)). As is clear from FIG. 2, cystamine effectively suppressed the decrease in the number of cells present in the retinal ganglion cell layer, indicating that cystamine has a protective effect on retinal ganglion cells. Furthermore, as is clear from FIGS. 3 and 4, cystamine had the effect of suppressing the thinning of the inner plexiform layer and suppressed the thinning of the retina. From the above results, it was confirmed that cystamine eye drops are effective in preventing and treating normal tension glaucoma.

Example 5: Effect of instilling cysteamine into normal tension glaucoma model mice The same experiment as in Example 4 was conducted and evaluated. FIG. 5 shows the measurement results of the number of cells present in the retinal ganglion cell layer, FIG. 6 shows the measurement results of the inner plexiform layer thickness, and FIG. 7 shows the measurement results of the retinal thickness. As is clear from FIG. 5, cysteamine also effectively suppressed the decrease in the number of cells present in the retinal ganglion cell layer, indicating that it has a protective effect on retinal ganglion cells. Furthermore, as is clear from FIGS. 6 and 7, cysteamine also had the effect of suppressing the thinning of the inner plexiform layer and suppressed the thinning of the retina. From the above results, it was confirmed that cysteamine eye drops are also effective in preventing and treating normal tension glaucoma.

Example 6: Effect of eye drops of cystamine on retinitis pigmentosa model mice (experimental method)
The efficacy of cystamine eye drops was evaluated using methylnitrosourea (MNU)-administered mice (Experimental Eye Research, 167, 145-151, 2018), which is widely used as a model experimental system for retinitis pigmentosa. Specifically, mice (C57BL/6J, 7 weeks old) were intraperitoneally administered with a filter-sterilized solution of MNU dissolved in physiological saline, and 60 mg/kg of MNU was administered to induce retinitis pigmentosa. A model mouse was created. Immediately after administering MNU, a solution of cystamine dissolved in physiological saline to a concentration of 40 mM and sterilized through filtration was instilled into both eyes in 2 μL doses three times a day for 7 days. Seven days later, the mice were euthanized, frozen section slides of retinal tissue were prepared in the same manner as in Example 4, stained with hematoxylin and eosin, and observed with a phase contrast microscope. The thickness of the granular layer was measured.

(Experimental result)
FIG. 8 shows the measurement results of the thickness of the outer retinal nuclear layer (ONL) (cystamine/MNU). Figure 8 shows the results (saline/MNU) when filter-sterilized physiological saline was instilled into both eyes three times a day for 7 days immediately after MNU was administered, and filter-sterilized physiological saline was instilled into both eyes for 7 days. The control results are also shown when 2 μL of water was instilled into both eyes of healthy mice three times a day for 7 days. ). As is clear from FIG. 8, cystamine suppressed the thinning of the outer nuclear layer of the retina due to degeneration of retinal photoreceptor cells due to administration of MNU. From the above results, it was confirmed that cystamine eye drops are effective in preventing and treating retinitis pigmentosa.

Example 7: Effect of instilling cystamine on age-related macular degeneration model mice (Part 1)
(experimental method)
The efficacy of cystamine eye drops was evaluated using sodium iodate (SI)-treated mice (Investigative Ophthalmology & Visual Science, 58, 2239-2249, 2017), which is widely used as a model experimental system for dry age-related macular degeneration. . Specifically, a solution of cystamine dissolved in physiological saline to a concentration of 1 mM and sterilized by filtration was injected into both eyes of a mouse (C57BL/6J, 8 weeks old) in an amount of 2 μL three times a day for 7 days. I put it in my eyes. After pre-instillation, 25 mg/kg of SI was administered by intraperitoneally administering a filter-sterilized solution of SI dissolved in physiological saline, and cystamine was further dissolved in physiological saline to a concentration of 1 mM. The filter-sterilized solution was instilled into both eyes in 2 μL doses three times a day for 7 days. Seven days later, the mice were euthanized, frozen section slides of retinal tissue were prepared in the same manner as in Example 4, stained with hematoxylin and eosin, and observed with a phase contrast microscope to determine whether deposits derived from the retinal pigment epithelial cell layer were present. The number of pigments (a collection of degenerated retinal pigment epithelial cells that appears as pigmentation) was counted. In addition, images were obtained at positions approximately 300, 600, 900, 1200, and 1500 μm from the optic disc center on the nasal and temporal sides, respectively, and the thickness of the retina was measured.

(Experimental result)
Figure 9 shows the measurement results of the number of deposits derived from the retinal pigment epithelial cell layer (1mM cystamine/SI treatment). Figure 9 shows the results of instilling 2 μL of filter-sterilized saline into both eyes three times a day for 7 days before and after administering SI (physiological saline/SI treatment) and the results of filter-sterilized saline. The results are also shown (no treatment) when 2 μL of saline was instilled into both eyes of healthy mice three times a day for 7 days (all results are based on samples from both eyes of 5 mice). (average value of n=10). As is clear from Figure 9, cystamine effectively suppressed the increase in the number of deposits derived from the retinal pigment epithelial cell layer, which is a characteristic finding of age-related macular degeneration, and thus protects the retinal pigment epithelial cells. It was found that it has a protective effect on retinal photoreceptor cells. Further, FIG. 10 shows the measurement results of the retinal thickness at positions approximately 300, 600, 900, 1200, and 1500 μm from the center of the optic disc on the nasal and temporal sides, respectively (1 mM cystamine). Figure 10 shows the results (physiological saline) when filter-sterilized physiological saline was instilled into both eyes three times a day for 7 days before and after administering SI, and the filter-sterilized physiological saline The results are also shown when 2 μL of water was instilled into both eyes of healthy mice three times a day for 7 days (no treatment) (all results are based on samples from both eyes of 5 mice (n = 10)). As is clear from FIG. 10, cystamine suppressed retinal thinning. From the above results, it was confirmed that cystamine eye drops are effective in preventing and treating age-related macular degeneration.

Example 8: Effect of instilling cystamine on age-related macular degeneration model mice (Part 2)
The same experiment as in Example 7 was conducted and evaluated, except that the dose of SI to mice was 12.5 mg/kg. FIG. 11 shows the measurement results of the number of deposits derived from the retinal pigment epithelial cell layer. As is clear from FIG. 11, even when the dose of SI to mice was 1/2 of the dose in Example 7, cystamine was deposited from the retinal pigment epithelial cell layer, which is a characteristic finding of age-related macular degeneration. This effectively suppressed the increase in the number of objects.

Example 9: Effect of eye drops of cysteamine on age-related macular degeneration model mice The same experiment as in Example 8 was conducted and evaluated. FIG. 12 shows the measurement results of the number of deposits derived from the retinal pigment epithelial cell layer. As is clear from Figure 12, cysteamine also effectively suppressed the increase in the number of deposits derived from the retinal pigment epithelial cell layer, which is a characteristic finding of age-related macular degeneration, thereby protecting retinal pigment epithelial cells. It was found that it has a protective effect on retinal photoreceptor cells. From the above results, it was confirmed that cysteamine eye drops are also effective in preventing and treating age-related macular degeneration.

Example 10: Effect of instilling a long-term stored solution of cystamine or cysteamine on normal-tension glaucoma model mice A solution obtained by dissolving cystamine or cysteamine in physiological saline to a concentration of 10 mM and sterilizing it by filtration was added to a polypropylene tube. The solution was placed in a 1.5 mL microtube, sealed with a lid and wrapped with parafilm, and stored at room temperature for 6 months. The same experiment as in Example 4 was conducted and evaluated, except that the solution was instilled into the eyes of mice. FIG. 13 shows the results of measuring the number of cells present in the retinal ganglion cell layer, and FIG. 14 shows the results of measuring the thickness of the inner plexiform layer. As is clear from Figures 13 and 14, both cystamine and cysteamine solutions stored for long periods of time effectively suppressed the decrease in the number of cells present in the retinal ganglion cell layer and also prevented the thinning of the inner plexiform layer. It had a suppressive effect. From the above results, we were able to confirm the convenience of cystamine and cysteamine eye drops in the prevention and treatment of normal-tension glaucoma.

Example 11: Examination of the mechanism of protective effect of cystamine on retinal ganglion cells in normal-tension glaucoma model mice (experimental method)
In the retinal ganglion cell death induced in NMDA-treated mice, transcription factor C/EBP homologous protein (CHOP), an endoplasmic reticulum stress-related protein, is expressed, and suppressing CHOP expression leads to retinal ganglion cell death. (Journal of Neurochemistry, 96, 43-52, 2006). Therefore, an immunohistochemical experiment was conducted to confirm whether cystamine suppresses retinal ganglion cell death induced in NMDA-administered mice by suppressing CHOP expression. Specifically, a solution obtained by dissolving cystamine in physiological saline to a concentration of 10 mM and sterilizing it by filtration was applied to both eyes of a normal-tension glaucoma model mouse prepared in the same manner as in Example 4. Immediately after administration into the body, 4 hours after intravitreal administration of the NMDA solution, and 8 hours after intravitreal administration of the NMDA solution, 2 μL was instilled into the eye three times in total. Twelve hours after the intravitreal administration of the NMDA solution, the mice were euthanized, and frozen section slides of retinal tissue were prepared in the same manner as in Example 4. After performing cell membrane permeabilization on the prepared frozen section slides, anti-CHOP/GADD153 polyclonal antibody 15204-1-AP (Thermo), which is an anti-CHOP antibody, was used as a primary antibody, and Polyclonal Goat Anti-Rabbit was used as a secondary antibody. Antibody Alexa Fluor 546 A11035 (Thermo) was reacted and observed using a fluorescence microscope.

(Experimental result)
FIG. 15 shows a fluorescent immunohistological image (Cystamine 10mM/NMDA). Figure 15 shows that filter-sterilized physiological saline was administered three times in total: immediately after the intravitreal administration of the NMDA solution, 4 hours after the intravitreal administration of the NMDA solution, and 8 hours after the intravitreal administration of the NMDA solution. , Results when 2 μL of eye drops (saline/NMDA) were applied to both eyes, and filter-sterilized physiological saline was applied to both eyes of a healthy mouse, 12 hours, 8 hours, and 4 hours before euthanizing the mouse. The results (control) when 2 μL of each eye was instilled three times in total are also shown. As is clear from FIG. 15, when cystamine was instilled into the eye, fluorescence due to CHOP expression in the retinal ganglion cell layer, which was observed when physiological saline was instilled, was not observed. From the above results, we were able to clarify the inhibitory effect on CHOP expression as the mechanism of action of cystamine's protective effect on retinal ganglion cells.

Example 12: Confirmation of the inhibitory effect of cystamine on CHOP expression in nerve cells (experimental method)
The inhibitory effect of cystamine on CHOP expression was confirmed using HT22 cells, a neuronal cell line derived from the mouse hippocampus. Specifically, HT22 cells (8.0 x 10 ⁵ cells/mL) seeded in a 100 mm dish were cultured in a DMEM medium supplemented with 10% FBS for 24 hours at 37°C in a 5% CO ₂ incubator. The medium was replaced with cystamine-added medium to a final concentration of 5 μM, and cultured for an additional 24 hours. Next, thapsigargin, an endoplasmic reticulum stress inducer, was added to the medium to a final concentration of 0.1 μM, and after culturing for 6 hours, the cells were collected. After performing sonication for protein solubilization on the collected cells, 20 μg of the centrifugal supernatant (solubilized protein) obtained by centrifugation was subjected to Western blotting using the anti-CHOP antibody Anti-CHOP/ This was carried out using GADD153 polyclonal antibody 15204-1-AP (Thermo), and the expression level of CHOP was investigated by image analysis of the obtained signal.

(Experimental result)
FIG. 16 shows the signal intensity of CHOP (relative value with the signal intensity of CHOP in HT22 cells treated with Cystamine/thapsigargin and untreated as 1 (Control)). FIG. 16 also shows the results when the same volume of filter-sterilized physiological saline as the filter-sterilized physiological saline used for adding cystamine was added instead of cystamine (Saline/thapsigargin treatment). As is clear from FIG. 16, cystamine effectively attenuated the increase in CHOP signal intensity caused by thapsigargin. From the above results, it was possible to confirm the inhibitory effect of cystamine on CHOP expression in nerve cells.

The present invention has industrial applicability in that it can provide a protective agent for retinal nerve cells, such as retinal ganglion cells and retinal photoreceptor cells.

Claims (6)

A retinal nerve cell protective agent containing a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof as an active ingredient (wherein R ₁ is -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. _{R 2} represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different and represent a hydrogen atom or an acetyl group. (m and n are the same or different and represent integers of 2 to 4). The compound is
(a) Cystamine where R ₁ is -NH ₂ , R ₂ is -S-(CH ₂ ) ₂ -NH ₂ and m is 2 (b) R ₁ is -NH ₂ , R ₂ is a hydrogen atom and m is 2 Protection of retinal nerve cells according to claim 1, wherein cysteamine (c) R ₁ is -NHAc (Ac: acetyl group), R ₂ is a hydrogen atom, and m is 2. agent. The agent for protecting retinal nerve cells according to claim 1, wherein the retinal nerve cells are retinal ganglion cells and/or retinal photoreceptor cells. A prophylactic and/or therapeutic agent for retinal neurodegenerative diseases containing a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof as an active ingredient (wherein R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. _{R 2} represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or different; Represents a hydrogen atom or an acetyl group. m and n are the same or different and represent integers of 2 to 4). The preventive and/or therapeutic agent for retinal neurodegenerative disease according to claim 4, wherein the retinal neurodegenerative disease is at least one of glaucoma, retinitis pigmentosa, and age-related macular degeneration. A transcription factor C/ _{EBP homologous} protein ₍ CHOP) expression inhibitor (in the formula _, R ₁ represents -NR ₃ R ₄ or an alkyl group having 1 to 6 carbon atoms. _{R 2} represents a hydrogen atom or -S-(CH ₂ ) _n -NR ₅ R _6. R ₃ to R ₆ are the same or (differently represent a hydrogen atom or an acetyl group; m and n are the same or different and represent an integer from 2 to 4).

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