TW202312986A - Protective agent for nerve cell in retina - Google Patents

Abstract

An object of the present invention is to provide a protective agent for retinal neurons exemplified by retinal ganglion cells and retinal photoreceptor cells. The protective agent for nerve cells of the retina of the present invention contains a compound represented by the general formula: R1- (CH2)m-SR2 or a pharmaceutically acceptable salt thereof as an active ingredient. (In the formula, R1 represents -NR3R4 or an alkyl group having 1 to 6 carbon atoms, R2 represents a hydrogen atom or -S-(CH2)n-NR5R6, R3 to R6 represent the same or different hydrogen atoms or acetyl groups, m and n indicate the same or different integers of 2 to 4).

Description

Protective agent for retinal nerve cells

The present invention relates to a protective agent for retinal nerve cells represented by retinal ganglion cells or retinal visual cells.

In Japan, glaucoma is the number one cause of halfway blindness. There is also a report that 1 out of 20 people over the age of 40 suffers from glaucoma. It is expected that as the aging society proceeds, the number of glaucoma patients will increase in the future. Glaucoma is a disease in which retinal ganglion cells (RGC) are necrotic due to elevated intraocular pressure (above 21mmHg), resulting in optic nerve abnormalities, reduced vision or narrowed field of vision, and ultimately blindness. Therefore, in the treatment of glaucoma, the method of lowering intraocular pressure is mainly adopted by instilling ocular hypotensive drugs. However, it is known that there are patients who develop glaucoma even in the normal range of intraocular pressure (less than 21mmHg), and about 70% of all glaucoma patients become normal tension glaucoma (Normal Tension Glaucoma: NTG). In normal intraocular pressure glaucoma, the intraocular pressure is within the normal range. Therefore, for normal tension glaucoma, the effect may not be sufficient even if the intraocular pressure is lowered. From these clinical backgrounds, the treatment of glaucoma focuses on the method of protecting retinal ganglion cells. For example, it is reported in Patent Document 1 that pyrroloquinoline quinone has a protective effect on retinal ganglion cells. However, it has not yet been established that by protecting retinal ganglion The treatment method of glaucoma. Also, retinitis pigmentosa and age-related macular degeneration are retinal neurodegenerative diseases originating from retinal retinal cells, and the same applies to the treatment of these diseases by protecting retinal cells.

[Prior Art Literature]

[Patent Document]

[Patent Document 1] Japanese Patent Laid-Open No. 2018-035076

The object of the present invention is to provide a protective agent for retinal nerve cells represented by retinal ganglion cells or retinal visual cells.

The inventors of the present invention have conducted in-depth examinations based on the above points and found that cystamine or cysteamine has a protective effect on retinal nerve cells, and is effective for glaucoma originating from retinal ganglion cells and retinitis pigmentosa originating in retinal visual cells. It is effective in the prevention or treatment of retinal neurodegenerative diseases such as age-related macular degeneration.

Based on the above findings, the protective agent for retinal nerve cells of the present invention is as described in claim 1, and the compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof is used as Active ingredients (wherein, R ₁ represents -NR ₃ R ₄ or an alkyl group with 1 to 6 carbons, 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, represent a hydrogen atom or acetyl group, m and n are the same or different, represent an integer of 2 to 4).

Also, the protective agent for retinal nerve cells described in claim 2 is the protective agent for retinal nerve cells described in claim 1, and the compound is any one of the following (a) to (c)

(a) R ₁ is -NH ₂ , R ₂ is -S-(CH ₂ ) ₂ -NH ₂ , m is cystamine of 2,

(b) R ₁ is -NH ₂ , R ₂ is a hydrogen atom, m is cysteamine of 2,

(c) R ₁ is -NHAc (Ac: acetyl), R ₂ is a hydrogen atom, and m is 2 N-acetyl cysteamine.

Also, the protective agent for retinal nerve cells described in Claim 3 is the protective agent for retinal nerve cells described in Claim 1, and the retinal nerve cells are retinal ganglion cells and/or retinal visual cells.

In addition, the preventive and/or therapeutic agent for retinal neurodegenerative diseases of the present invention is as described in claim 4, the compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof as Active ingredients (wherein, R ₁ represents -NR ₃ R ₄ or an alkyl group with 1 to 6 carbons, 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, represent a hydrogen atom or acetyl group, m and n are the same or different, represent an integer of 2 to 4).

Also, the preventive and/or therapeutic agent for retinal neurodegenerative diseases described in claim 5 is the preventive and/or therapeutic agent for retinal neurodegenerative diseases described in claim 4, and the retinal neurodegenerative diseases are glaucoma, retinitis pigmentosa, At least one of age-related macular degeneration.

In addition, the transcription factor C/EBP homologous protein (CHOP) expression inhibitor of the present invention is as described in Claim 6, a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or its pharmaceutically acceptable (In the formula, R ₁ represents -NR ₃ R ₄ or an alkyl group with 1 to 6 carbons, 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, representing a hydrogen atom or an acetyl group, m and n are the same or different, representing an integer of 2 to 4).

According to the present invention, a protective agent for retinal nerve cells represented by retinal ganglion cells or retinal visual cells can be provided.

FIG. 1 is a graph showing the protective effect of cystamine on nerve cells (HT22 cells belonging to a nerve cell line derived from the mouse hippocampus) in Example 1. FIG.

Fig. 2 is a graph showing the protection of retinal ganglion cells by administering cystamine to normal-tension glaucoma model mice in Example 4.

FIG. 3 is a graph showing suppression of thinning of the inner network layer in Example 4. FIG.

FIG. 4 is a graph showing suppression of retinal thinning in Example 4. FIG.

Fig. 5 is a graph showing the protection of retinal ganglion cells by administering cysteamine to normal-tension glaucoma model mice in Example 5.

FIG. 6 is a graph showing suppression of thinning of the inner network layer in Example 5. FIG.

FIG. 7 is a graph showing suppression of retinal thinning in Example 5. FIG.

Fig. 8 is a graph showing that in Example 6, the thinning of the retinal extragranular layer was suppressed by administering cystamine to the retinitis pigmentosa model mice.

9 is a graph showing that in Example 7, by administering cystamine to age-related macular degeneration model mice, the increase in the number of deposits derived from the retinal pigment epithelial cell layer was suppressed.

FIG. 10 is a graph showing suppression of retinal thinning in Example 7. FIG.

Fig. 11 shows that in embodiment 8, by injecting eye bladder to age-related macular degeneration model mice Amine, a graph that inhibits the increase in the number of deposits originating from the retinal pigment epithelial layer.

Fig. 12 is a graph showing that in Example 9, by administering cysteamine to age-related macular degeneration model mice, the increase in the number of deposits derived from the retinal pigment epithelial cell layer was suppressed.

Fig. 13 is a graph showing the protection of retinal ganglion cells by administering long-term preserved cystamine or cysteamine solution to the eyes of normal-tension glaucoma model mice in Example 10.

Fig. 14 is a graph showing suppression of thinning of the inner network layer in Example 10.

Fig. 15 is a fluorescence immunohistograph showing inhibition of CHOP expression by injecting cystamine into the eyes of normal-tension glaucoma model mice in Example 11.

Fig. 16 is a graph showing that in Example 12, cystamine inhibits the expression of CHOP in nerve cells induced by thapsigargin.

The protective agent for retinal nerve cells of the present invention is 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 with 1 to 6 carbons, 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, representing a hydrogen atom or an acetyl group, m and n are the same or different, and represent an integer of 2 to 4).

Here, the alkyl group having 1 to 6 carbon atoms may be linear or branched, and specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, and isobutyl Base, second butyl, third butyl, n-pentyl, isopentyl, n-hexyl, etc.

In addition, when the compound is acidic, pharmaceutically acceptable salts include: alkali metal salts such as ammonium salts, sodium salts, or potassium salts; alkaline earth metal salts such as calcium salts or magnesium salts; acid salts etc. When the compound is alkaline, it can be enumerated: inorganic acid salt (hydrochloride, sulfate, nitric acid salt, phosphate, etc.); organic acid salts (acetate, lactate, citrate, tartrate, maleate, fumarate, oxalate, etc.), etc.

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

The protective agent for retinal nerve cells of the present invention protects retinal nerve cells involved in the transmission of visual signals to the brain, specifically retinal ganglion cells or retinal visual cells. Therefore, the protective agent for retinal nerve cells of the present invention can be used as a preventive and/or therapeutic agent for retinal neurodegenerative diseases originating from these retinal nerve cells. Specific examples of retinal neurodegenerative diseases include glaucoma originating in retinal ganglion cells, retinitis pigmentosa originating in retinal optic cells, and age-related macular degeneration. Administration of the protective agent for retinal nerve cells of the present invention to humans or animals other than humans may be administered parenterally or orally, and can be administered in a form suitable for the administration method by a known method per se. Formulation, the preferred form of preparation is eye drops.

Eye drops are compounds represented by the above general formula or their pharmaceutically acceptable salts, buffers, dissolution aids, tonicity agents, stabilizers, preservatives, viscous agents, chelating agents, Various additives such as pH regulators and cooling agents are prepared together, filtered under sterile conditions, and filled in appropriate sterile containers for preparation. Specific examples of buffering agents include: boric acid or its salts (borax, etc.), citric acid or its salts (sodium citrate, etc.), phosphoric acid or its salts (sodium monohydrogen phosphate, etc.), tartaric acid or its salts (sodium tartrate, etc.) , gluconic acid or its salts (sodium gluconate, etc.), acetic acid or its salts (sodium acetate, etc.), various amino acids, or combinations thereof. Tool for dissolving supplements Examples include: polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil 60, etc.), polyethylene glycol (macroglycol 4000, etc.), polyoxyethylene sorbitan higher fatty acid ester (polysorbate 80, etc.) , Polyoxyethylene (POE)-polyoxypropylene (POP) block copolymer (Poloxamer (Poloxamer) 407, etc.), propylene glycol. Specific examples of tonicity agents include inorganic salts (sodium chloride, potassium chloride, calcium chloride, etc.), sugars (mannitol, glucose, etc.), polyhydric alcohols (glycerol, propylene glycol, etc.). Specific examples of the stabilizer include sodium edetate, cyclodextrin, sulfite, citric acid or its salt, and butylhydroxytoluene. Specific examples of preservatives include: benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, p-hydroxybenzoic acid Methylparaben, Ethylparaben, Propylparaben, Butylparaben, Polyquaternium-1, Polyaminopropylbiguanide, Alkylamine ethylglycine hydrochloride, cetylpyridinium chloride, thimerosal). Specific examples of viscous agents include: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, sodium chondroitin sulfate, sodium hyaluronate, Carboxymethylcellulose, 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 or its salt, boric acid or its salt, phosphoric acid or its salt, acetic acid or its salt, tartaric acid or its salt, sodium hydroxide, potassium hydroxide, sodium carbonate, bicarbonate sodium. Specific examples of cooling agents include menthol, borneol, camphor, geraniol, limonene, eugenol, peppermint oil, eucalyptus oil, anise oil, and bergamot oil. Specific examples of the solvent include sterilized purified water, purified water, and physiological saline. The pH value of the eye drops is not particularly limited as long as it is within the range allowed by ophthalmology, and it is usually in the range of pH 4 to 9, preferably in the range of pH 5 to 8.

The compound represented by the above general formula or its pharmaceutically acceptable salt as an active ingredient Mix 0.00001 to 100mg in 1mL of each eye drop, 1 to several drops each time, 1 to several times a day, by protecting the nerve cells of the retina, for glaucoma, retinitis pigmentosa, age-related macular degeneration and other retinal neurodegeneration The disease exerts a preventive effect or a therapeutic effect.

In addition, eye drops can also be formulated with other pharmaceutical ingredients, such as prostaglandin preparations such as latanoprost or travoprost, timolol maleate or carteolol hydrochloride (Carteolol) and other β-receptor blockers, ophthalmic ocular hypotensive drugs such as Dorzolamide HCl or Brinzolamide and other carbonic anhydrase inhibitors, etc.

[Example]

Hereinafter, the present invention will be described in detail by means of examples, but the present invention is not limited to the following descriptions.

Example 1: Cystamine's protective effect on nerve cells

(experimental method)

The protective effect of cystamine on nerve cells was evaluated using HT22 cells derived from a mouse hippocampal neural cell line that induced apoptosis (cell necrosis) by glutamate. Specifically, for HT22 cells (1.2×10 ³ cells per well) seeded in a 96-well plate, the final concentration was 10 μM, 5 μM, 2 μM, and 1 μM in DMEM medium supplemented with 10% FBS. Add cystamine. After 4 hours, glutamic acid was added so that the final concentration would be 4 mM, and cultured in a 37° C., 5% CO ₂ incubator for 20 hours. Next, 10 μL of cytotoxicity assay reagent (Cell Counting Kit-8, manufactured by Dojindo Co., Ltd.) was added to each well to generate a color reaction, and the absorbance (490 nm) was measured after 3 hours. The cell survival rate (%) of the glutamic acid control cell was calculated as 100%.

(experimental results)

Shown in Figure 1. It can be seen from Figure 1 that cystamine inhibits the apoptosis of HT22 cells in a concentration-dependent manner, and thus has a protective effect on nerve cells. In addition, it can be confirmed by other experiments that cystamine hinders the movement of Apoptosis-inducing Facto (AIF) induced by glutamate to the nucleus. One of the mechanisms by which amines inhibit apoptosis in HT22 cells.

Example 2: Protective effect of cysteamine on nerve cells

The same experiment as in Example 1 was carried out and evaluated. As a result, as follows, since cysteamine also inhibited the apoptosis of HT22 cells in a concentration-dependent manner, the protective effect of cysteamine on nerve cells was confirmed.

Example 3: The protective effect of N-acetyl cysteamine on nerve cells

The same experiment as in Example 1 was carried out and evaluated. The results are as follows, since N-acetyl cysteamine also inhibits the apoptosis of HT22 cells, it can be confirmed that N-acetyl cysteamine has a protective effect on nerve cells (due to insufficient n numbers, no significant difference was performed test).

Example 4: Effects of eye cystamine on normal intraocular pressure glaucoma model mice

(experimental method)

The widely used N-methyl-D-aspartic acid (NMDA) was administered to mice in the normal tension glaucoma model experiment (Investigative Ophthalmology & Visual Science, 40, 1004-1008, 1999) to evaluate cystamine The dot effect. Specifically, NMDA was dissolved in physiological saline so that it became 40 mM, and 2 μL of the solution sterilized by filtration was administered into the vitreous of both eyes of a mouse (C57BL/6J, 7 weeks old) to prepare normal intraocular pressure. Glaucoma model mice. The administration of NMDA solution into the vitreous is according to the method of Shimazawa et al. (Nippon Yakuru, 129, 445-450, 2007), using isoflurane to induce anesthesia on mice, and using a dental injection needle (33G ) with a 10 μL microsyringe from the sclera of the corneal wheel. After pulling out the needle, apply Cravit eyedrops, an anti-inflammatory agent. Immediately after injecting NMDA solution into the vitreous, instill 2 μL of the solution in the eyes three times a day for 7 days. The solution is dissolved cystamine in normal saline and sterilized by filtration in such a way that it becomes 10 mM. . After 7 days, the mice were euthanized, and the two eyeballs were taken out, fixed with 4% paraformaldehyde, and the anterior eye and lens were excised. Next, make slides of frozen sections of the tissue, stain with Hematoxylin/Eosin, observe with a phase-contrast microscope, and measure the number of cells present in the retinal ganglion cell layer and the inner reticular layer. Determination of thickness, measurement of retinal thickness.

(experimental results)

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 thickness of the inner reticular layer (IPL), and Figure 4 shows the measurement results of the retinal (Retina) thickness (cystamine /NMDA). In Figs. 2 to 4, the combined results of intravitreal administration of NMDA solution in both eyes, 3 times a day, 2 μL per eye for 7 days, and the results of filter-sterilized normal saline (normal saline/ NMDA), and in the two eyes of healthy mice, the results of instilling 2 μL of saline sterilized by filtration for 7 days (control group) (any one of the results were from 3 mice) Mean value of specimens (n=6) of both eyes of mice). It can be seen from Figure 2 that cystamine has a protective effect on retinal ganglion cells because it effectively inhibits the reduction of the number of cells present in the retinal ganglion cell layer. Also, as can be seen from Fig. 3 and Fig. 4, cystamine has the effect of suppressing the thinning of the inner reticular layer and suppressing the thinning of the retina. From the above results, it can be confirmed that eye drops of cystamine are effective in the prevention or treatment of normal tension glaucoma.

Example 5: Effects of eye cysteamine on normal intraocular pressure glaucoma model mice

The same experiment as in Example 4 was carried out 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 thickness of the inner reticular layer, and Fig. 7 shows the measurement results of the retinal thickness. It can be seen from Figure 5 that cysteamine also effectively inhibits the reduction of the number of cells present in the retinal ganglion cell layer, and has a protective effect on retinal ganglion cells. Also, from Figures 6 and 7, it can be seen that cysteamine also has the effect of inhibiting the thinning of the inner reticular layer and inhibiting the thinning of the retina. From the above results, it can be confirmed that cysteamine instillation is also effective in the prevention or treatment of normal tension glaucoma.

Example 6: Effects of Cystamine on Retinitis Pigmentosa Model Mice

(experimental method)

Mice administered with methylnitrosourea (MNU), which is widely used as a model experiment for retinitis pigmentosa (Experimental Eye Research, 167, 145-151, 2018), was used to evaluate the eye drop effect of cystamine. Specifically, mice (C57BL/6J, 7 weeks old) were intraperitoneally administered a solution in which MNU was dissolved in physiological saline and sterilized by filtration to produce retinitis pigmentosa administered with 60 mg/kg of MNU. Model mice. Immediately after the administration of MNU, instill in both eyes 3 times a day, 2 μL each time for 7 days. The solution was dissolved cystamine in normal saline so that it was 40 mM and sterilized by filtration. After 7 days, the mice were euthanized, and frozen section slides of retinal tissues were made in the same way as in Example 4, stained with hematoxylin/eosin, and observed with a phase-contrast microscope to detect the presence of extraretinal granules in retinal visual cells. Determination of layer thickness.

(experimental results)

Figure 8 shows the results of measurement of retinal extragranular layer (ONL) thickness (cystamine/MNU). In Figure 8, it is shown that the NMDA solution was administered to both eyes immediately, 3 times a day, each time 2 μ L was instilled in the eyes for 7 days, and the results of filter-sterilized normal saline were applied to both eyes, 3 times a day (physiological salt water/NMDA), and in the eyes of healthy mice, 3 times a day, each time 2μL instilled in the eyes of 7 days of filter-sterilized physiological saline (control group) (any one of the results are from Average of specimens (n=12) from both eyes of 6 mice). It can be seen from FIG. 8 that cystamine inhibits the thinning of the retinal extragranular layer caused by the degeneration of retinal optic cells caused by the administration of MNU. From the above results, it can be confirmed that eye drops of cystamine are effective in the prevention or treatment of retinitis pigmentosa.

Embodiment 7: The effect (part 1) of injecting cystamine on age-related macular degeneration model mice

(experimental method)

Using mice administered with sodium iodate (SI), which is widely used as a model experiment for atrophic age-related macular degeneration (Investigative Ophthalmology & Visual Science, 58, 2239-2249, 2017), to evaluate the effect of cystamine eyedrops . Specifically, the two eyes of mice (C57BL/6J, 8 weeks old) were instilled with 2 μL each time three times a day for 7 days. The solution was added to 1 mM in physiological saline Cystamine dissolved in water and sterilized by filtration. After instilling before the eyes, intraperitoneally administer a solution of SI dissolved in normal saline and sterilized by filtration, so as to administer SI 25mg/kg, and further, 3 times a day, each time 2μL for each point on both eyes Eye solution for 7 days, the solution is dissolved cystamine in physiological saline in a manner of 1 mM and sterilized by filtration. After 7 days, the mice were euthanized, and frozen section slides of retinal tissues were prepared in the same manner as in Example 4, stained with hematoxylin/eosin, observed with a phase-contrast microscope, and precipitated from the retinal pigment epithelial cell layer Measurement of the number of substances (degenerated retinal pigment epithelial cells concentrated and manifested as pigmentation). In addition, the images of the positions of about 300, 600, 900, 1200, and 1500 μm from the center of the optic nerve head to the nasal side and the ear side were obtained, and the retinal thickness was measured.

(experimental results)

Fig. 9 shows the measurement results of the number of precipitates derived from the retinal pigment epithelial cell layer (1 mM cystamine/SI treatment). In Fig. 9, the results of filter-sterilized normal saline (normal saline/SI treatment) were instilled in both eyes before and after SI administration, and 2 μL each time for 7 days, and the results in healthy For the two eyes of the mice, the results (without treatment) when 2 μL of each eye was instilled with filtered sterilized saline for 7 days three times a day (any one of the results are from the two eyes of 5 mice) The average value of samples (n=10)). It can be seen from Figure 9 that cystamine effectively inhibits the increase in the number of precipitates derived from the retinal pigment epithelial cell layer, which is a characteristic marker of age-related macular degeneration, and has a protective effect on retinal visual cells by protecting the retinal pigment epithelial cells. Also, Figure 10 shows from the optic nerve The measurement results of retinal thickness (1mM cystamine) in the center of the nipple at the positions of about 300, 600, 900, 1200, and 1500 μm on the nose side and ear side respectively. In Fig. 10, before and after SI administration, the results of filter-sterilized saline (physiological saline) were instilled in both eyes with 2 μL each time for 7 days, and in healthy children. For the two eyes of the mice, the result (no treatment) when 2 μL of each eye was instilled with filtered sterilized saline for 7 days three times a day (the result of any one is from the examination of the two eyes of 5 mice) Individual (n=10) mean). From Fig. 10, it is known that cystamine suppresses retinal thinning. From the above results, it can be confirmed that eye drops of cystamine are effective in the prevention or treatment of age-related macular degeneration.

Embodiment 8: The effect (part 2) of injecting cystamine on age-related macular degeneration model mice

The same experiment as in Example 7 was performed and evaluated except that the dose of SI administered to mice was 12.5 mg/kg. Fig. 11 shows the measurement results of the number of precipitates derived from the retinal pigment epithelial cell layer. As can be seen from Figure 11, even if the dose of SI administered to mice is 1/2 of the dose in Example 7, cystamine can effectively inhibit the retinal pigment epithelium-derived cells that are characteristic markers of age-related macular degeneration. The increase in the number of layers of sediment.

Example 9: Effects of eye cysteamine on age-related macular degeneration model mice

The same experiment as in Example 8 was carried out and evaluated. Fig. 12 shows the measurement results of the number of precipitates derived from the retinal pigment epithelial cell layer. It can be seen from Figure 12 that cysteamine also effectively inhibits the increase in the number of precipitates derived from the retinal pigment epithelial cell layer, which is a characteristic marker of age-related macular degeneration, and has a protective effect on retinal visual cells by protecting the retinal pigment epithelial cells. From the above results, it can be confirmed that eye drops of cysteamine are also effective for the prevention or treatment of age-related macular degeneration.

Example 10: Effects of long-term preservation of cystamine or cysteamine solution on normal intraocular tension glaucoma model mice

In addition to making a solution of 10mM, dissolve cystamine or cysteamine in physiological saline and sterilize it by filtration to make a solution, put the solution into a 1.5mL microtube made of polypropylene, cover it, and wrap it with a parafilm to seal That is, except that the solution stored at room temperature for 6 months was instilled in the eyes of mice, the rest were subjected to the same experiment as in Example 4 and evaluated. Fig. 13 shows the results of measurement of the number of cells present in the retinal ganglion cell layer, and Fig. 14 shows the results of measurement of the thickness of the inner reticular layer. From Figure 13 and Figure 14, it can be seen that long-term storage of cystamine and any solution of cysteamine effectively inhibits the reduction of the number of cells present in the retinal ganglion cell layer, and has the effect of inhibiting the thinning of the inner reticular layer . From the above results, it can be confirmed that cystamine and cysteamine eyedrops are convenient for the prevention or treatment of normal tension glaucoma.

Example 11: Review of the mechanism of action of cystamine on the protective effect of retinal ganglion cells in normal tension glaucoma model mice

(experimental method)

Known to express the transcription factor C/EBP homologous protein (CHOP), which is an endoplasmic reticulum stress-related protein, in retinal ganglion cell necrosis induced by NMDA-administered mice, and to inhibit retinal ganglion cell necrosis by inhibiting the expression of CHOP Ganglion cell necrosis (Journal of Neurochemistry, 96, 43-52, 2006). Here, an immunohistochemical experiment was performed to confirm whether the inhibition of CHOP expression by cystamine inhibited retinal ganglion cell necrosis induced in NMDA-administered mice. Specifically, in both eyes of a normal-tension glaucoma model mouse prepared in the same manner as in Example 4, a solution in which 10 mM cystamine was dissolved in physiological saline and sterilized by filtration was added at the following times for a total of 3 Each time, 2μL in each eye: Immediately after the NMDA solution was administered into the vitreous, 4 hours after the NMDA solution was administered into the vitreous, and 8 hours after the NMDA solution was administered into the vitreous. The mice were euthanized 12 hours after the NMDA solution was administered into the vitreous, and frozen section glass slides of retinal tissues were prepared in the same manner as in Example 4. After permeabilizing the frozen section slides, they were mixed with Anti-CHOP/GADD153 polyclonal antibody 15204-1-AP (Thermo) which is an anti-CHOP antibody as the primary antibody and Polyclonal as the secondary antibody. Goat Anti-Rabbit antibody Alexa Fluor 546 A11035 (Thermo) was used for the reaction and observed with a fluorescent microscope.

(experimental results)

Figure 15 shows fluorescent immunohistographs (cystamine 10 mM/NMDA). As shown in Fig. 15 , a total of 3 times immediately after the administration of the NMDA solution into the vitreous, 4 hours after the administration of the NMDA solution into the vitreous, and 8 hours after the administration of the NMDA solution into the vitreous, the filter-sterilized The result of instilling 2 μL of normal saline in each eye (normal saline/NMDA), and the two eyes of healthy mice, 12 hours before, 8 hours before, and 4 hours before the mice were euthanized for 3 times, The results when 2 μL of filtered sterilized physiological saline was instilled in each eye (control group). From Fig. 15, it can be seen that the fluorescence caused by the expression of CHOP in the retinal ganglion cell layer observed when injecting physiological saline was not observed when cystamine was instilled. From the above results, it can be known that the expression-inhibiting effect of CHOP is the mechanism for the protective effect of cystamine on retinal ganglion cells.

Example 12: Confirmation of the inhibitory effect of cystamine on the expression of CHOP in nerve cells

(experimental method)

Using HT22 cells belonging to a neural cell line derived from the mouse hippocampus, the inhibitory effect of cystamine on CHOP expression was confirmed. Specifically, HT22 cells (8.0×10 ⁵ cells/mL) seeded on a 100 mm dish were cultured in DMEM medium supplemented with 10% FBS for 24 hours at 37°C in a 5% CO ₂ incubator, and then mixed with The medium was exchanged with cystamine at a final concentration of 5 μM and cultured for an additional 24 hours. Next, thapsigargin, which is an endoplasmic reticulum stress-inducing substance, was added to the medium at a final concentration of 0.1 μM, and cells were collected after culturing for 6 hours. After performing sonication to solubilize the recovered cells, 20 μg of the centrifugation supernatant (soluble protein) obtained by centrifugation was subjected to western blotting using Anti-CHOP, which is an anti-CHOP antibody. /GADD153 polyclonal antibody 15204-1-AP (Thermo), and the expression of CHOP was investigated by image analysis of the obtained signal.

(experimental results)

Fig. 16 shows the signal intensity of CHOP (the signal intensity of CHOP in cystamine/thapsigargin treated and untreated HT22 cells is taken as the relative value of 1 (control group)). The filter-sterilized saline used for the addition of cystamine and the results when the same volume of filter-sterilized saline was added instead of cystamine are shown in Figure 16 (saline/thapsigargin treatment). It can be seen from FIG. 16 that cystamine effectively attenuates the increase of CHOP signal intensity induced by thapsigargin. From the above results, it was confirmed that cystamine exhibits an inhibitory effect on CHOP in nerve cells.

[Industrial availability]

The present invention is industrially applicable in that it can provide a protective agent for retinal nerve cells typified by retinal ganglion cells and retinal visual cells.

Claims (6)

A protective agent for retinal nerve cells, the active ingredient is a compound represented by the general formula: R ₁ -(CH ₂ ) _m -SR ₂ or a pharmaceutically acceptable salt thereof, where R ₁ represents -NR ₃ R ₄ Or an alkyl group with 1 to 6 carbons, 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, representing a hydrogen atom or an acetyl group, m and n is the same or different, and represents an integer of 2 to 4. The protective agent for retinal nerve cells according to claim 1, wherein the compound is any one of the following (a) to (c), (a) R ₁ is -NH ₂ , R ₂ is -S-(CH ₂ ) ₂ -NH ₂ , m is cystamine of 2, (b) R ₁ is -NH ₂ , R ₂ is a hydrogen atom, m is cysteamine of 2, (c) R ₁ is -NHAc, Ac: acetyl group, R ₂ is a hydrogen atom, m is 2 N-acetyl cysteamine. The protective agent for retinal nerve cells according to claim 1, wherein the retinal nerve cells are retinal ganglion cells and/or retinal visual cells. A preventive and/or therapeutic agent for retinal neurodegenerative diseases, which uses 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 with 1 to 6 carbons, 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, representing a hydrogen atom or acetyl base, m and n are the same or different, and represent an integer of 2 to 4. The preventive and/or therapeutic agent for retinal neurodegenerative diseases according to claim 4, wherein the retinal neurodegenerative diseases are at least one of glaucoma, retinitis pigmentosa, and age-related macular degeneration. A transcription factor C/EBP homologous protein (CHOP) expression inhibitor, which uses 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 with 1 to 6 carbons, 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, representing a hydrogen atom or acetyl, m and n are the same or different, and represent an integer of 2 to 4.

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