ophthalmic medicine

Abstract

To provide agents for optic nerve axonal regeneration.SOLUTION: Provided is an agent for optic nerve axon regeneration containing ripasudil or a salt thereof or a solvate thereof as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a drug for optic nerve axon regeneration.

Background Art

[0002] It is known that optic nerve damage causes a decrease in retinal ganglion cells (RGC) and atrophy of the optic nerve. In a rodent optic nerve crush (ONC) model, a neuroprotective effect and an optic nerve axon regeneration effect have been confirmed by treating optic nerve damage. It has been found that the Rho / Rho-associated protein kinase (ROCK) signal is involved in the central axon regeneration mechanism and is regulated by axon regeneration inhibitors related to myelin such as Nogo. By inhibiting the Rho / ROCK signal, the inhibitory effect by axon regeneration inhibitors such as Nogo is released, axon regeneration is promoted, and functional recovery has been observed in an animal model of axonal spinal cord injury (Non-Patent Document 1). In the ONC model, it has been reported that the optic nerve regeneration effect was examined by intravitreal injection of the ROCK inhibitor Y-27632 or fasudil (Non-Patent Documents 2 and 3). Y-27632 significantly increased the optic nerve fibers in rats and cats, but the effect was not observed with fasudil, which is also known as a ROCK inhibitor. From these results, it was suggested that the axon regeneration promoting effect differs depending on the type of ROCK inhibitor.

[0003] Ripasudil eye drops were approved in 2014 for the treatment of glaucoma and ocular hypertension, and their Rho kinase inhibitory effect is potent compared to fasudil and Y-27632. Ripasudil lowers intraocular pressure by promoting aqueous humor outflow from the main outflow pathway via the trabecular meshwork-Schlemm's canal, based on its Rho kinase inhibitory effect. The inventors confirmed the neuroprotective effect of ripasudil eye drops using EAAC1-deficient mice, a normal-tension glaucoma (NTG) model (Non-Patent Literature 4). In EAAC1-deficient mice, ripasudil strongly suppressed the phosphorylation of p38 MAPK (a factor that promotes RGC death) induced by oxidative stress. These results suggest that the RGC protective effect of ripasudil is not only intraocular pressure-dependent but also intraocular pressure-independent. Furthermore, it has been reported that oral administration of ripasudil partially suppressed RGC death by ONC through the suppression of oxidative stress via Nox1 downregulation (Non-Patent Literature 5). However, the axon regeneration-promoting effect of lipasudil has not been known until now. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Neurosci. 23, 1416-1423 (2003) [Non-Patent Document 2] J. Neurochem. 103, 181-189 (2007). [Non-Patent Document 3] Brain Res. 1201, 23-33 (2008). [Non-Patent Document 4] Invest. Ophthalmol. Vis. Sci. 59, 2080-2089 (2018). [Non-Patent Document 5] Invest. Ophthalmol. Vis. Sci. 55, 7126-7136 (2014). [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention relates to providing ophthalmic agents, particularly novel agents for optic nerve axon regeneration. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors of the present invention unexpectedly discovered that lipasudil ((S)-(-)-1-(4-fluoro-5-isoquinoline sulfonyl)-2-methyl-1,4-homopiperazine) or its salts or solvates thereof are useful for optic nerve axon regeneration, and thus completed the present invention.

[0007] In other words, the present invention relates to the following invention. 1) An optic nerve axon regeneration agent comprising lipasudil or its salts or solvates thereof as an active ingredient. 2) An optic nerve axon regeneration agent as described in 1) above, which is an eye drop. 3) A pharmaceutical composition for optic nerve axon regeneration comprising lipasudil or a salt thereof or a solvate thereof, and a pharmaceutically acceptable carrier. 4) The pharmaceutical composition of 3) above, which is an eye drop. 5) A method for regenerating optic nerve axons, characterized by administering lipasudil or a salt thereof or a solvate thereof. 6) Use of lipasudil or its salts or solvates thereof for the manufacture of preparations for optic nerve axon regeneration. [Effects of the Invention]

[0008] According to the present invention, a drug for optic nerve axon regeneration can be provided. The pharmaceutical composition of the present invention is effective even when administered as eye drops in small amounts, and is extremely useful as an optic nerve axon regeneration agent because it can be administered non-invasively without causing significant physical or mental suffering to the patient, and can be easily administered to the elderly and children. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the effect of lipasudil eye drops on RGC degeneration after ONC. a: Experimental protocol, b: Schematic diagram of the observed retinal area, c: Immunological histological findings 7 days after OCN (scale bar: 50 μm), df: Quantification of RGC in each retinal area 7 days after OCN, g: Immunological histological findings 14 days after OCN (scale bar: 50 μm), hj: Quantification of RGC in each retinal area 14 days after OCN, n=4 for each group, means±SEM. *P<0.05, **P<0.01 [Figure 2] This figure shows the effect of lipasudil eye drops on p38 phosphorylation in the retina after ONC. a: Experimental protocol, b: Immunoblot of total p38, phosphorylated p38 (p-p38), and actin in the retina 3 hours and day 1 after ONC, c: Immunoblot of total p38, p-p38, and actin in PBS or lipasudil 3 hours after ONC, d: Quantitative evaluation of c, the ratio of p-p38 to total p38, with the ratio of PBS set to 1. e: Double immunostaining using antibodies against p-p38 and RNA-binding protein with multiple splicing (RBPMS), scale bar: 50 μm, f: Quantitative evaluation of e, the intensity of p-p38 in GCL, with the ratio of PBS set to 1. Each group n=4, means±SEM. *P<0.05 [Figure 3] This figure shows the effect of ripasudil eye drops on optic nerve regeneration after ONC. a: Experimental protocol, b: Longitudinal section of axons treated with PBS or ripasudil (labeled with CTB, * indicates injury site), scale bar 200 μm, ce: Number of regenerated axons, distance from injury site: c 100 μm, d 250 μm, e 500 μm, n=4 in each group, means ± SEM. **P<0.01, ***P<0.001 [Figure 4]This figure shows the expression of phosphorylated CRMP2 and cofilin after ONC. a: Immunoblot of phosphorylated CRMP2 (p-CRMP2), total CRMP2, phosphorylated cofilin, total cofilin, and actin in the retina at 3 hours, 1 day, 3 days, 7 days, and 14 days after ONC. b: Quantification of a, with the ratio of p-CRMP2 to total CRMP2, where the proportion of control mice was set to 1. c: Quantification of a, with the ratio of p-cofilin to total cofilin, where the proportion of control mice was set to 1. n=4 (control group), 3 (3 hours, 1 day, 3 days), 10 (7 days, 14 days), means±SEM. *P<0.05 [Figure 5] This figure shows the effect of lipasudil on the expression of phosphorylated CRMP2 after ONC. a: Immunoblot of phosphorylated CRMP2 (p-CRMP2), total CRMP2, and actin in the retina 3 hours after ONC, b: Quantification of a, c: Immunoblot of p-CRMP2, total CRMP2, and actin on day 3 of ONC, d: Quantification of c, e: Immunoblot of p-CRMP2, total CRMP2, and actin on day 7 of ONC, f: Quantification of e, the ratio of p-CRMP2 to total CRMP2, with the proportion of control mice set to 1. g: Double immunostaining with anti-p-CRMP2 antibody and anti-RBPMS antibody 3 hours after ONC, scale bar 50 μm, h: Quantification of g, intensity of p-CRMP2 in GCL, with PBS set to 1. Each group n=4 (control group), means±SEM. *P<0.05 [Figure 6]This figure shows the effect of lipasudil on the expression of phosphorylated cofilin after ONC. a: Immunoblot of phosphorylated cofilin (p-cofilin), total cofilin, and actin in the retina 3 hours after ONC; b: Quantification of a; c: Immunoblot of p-cofilin, total cofilin, and actin on day 3 of ONC; d: Quantification of c; e: Immunoblot of p-cofilin, total cofilin, and actin on day 7 of ONC; f: Quantification of e, with the proportion of control mice set to 1 based on the ratio of p-cofilin to total cofilin. g: Double immunostaining using anti-p-cofilin antibody and anti-RBPMS antibody 3 hours after ONC. Scale bar 50 μm; h: Quantification of g, intensity of p-cofilin in GCL, with PBS set to 1. Each group n=4 (control group), means±SEM. *P<0.05 [Modes for carrying out the invention]

[0010] Lipasdil ((S)-(-)-1-(4-fluoro-5-isoquinoline sulfonyl)-2-methyl-1,4-homopiperazine) used in the present invention is a compound that acts as a cerebrovascular treatment agent and has substance P antagonistic activity, leukotriene D4 antagonistic activity, and Rho kinase inhibitory activity, and can be produced by known methods, for example, the method described in International Patent Publication No. 99 / 20620.

[0011] Examples of salts of lipasudil include salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, and hydrobromic acid, or salts with organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, and camphorsulfonic acid, with hydrochloride salts being particularly preferred.

[0012] Lipasdil or its salts can exist not only in an unsolvated form but also as a hydrate or solvate. Hydrates are preferred, but in this invention, all crystalline forms and hydrates or solvates are included.

[0013] When using repasodil or a salt thereof or a solvate thereof, as shown in the following examples, it exhibits a strong optic nerve axon regeneration effect and is thus useful as an optic nerve axon regenerating agent.

[0014] When formulating repasodil or a salt thereof or a solvate thereof, a formulation can be prepared according to a known method. For example, the formulation of repasodil or a salt thereof or a solvate thereof can be prepared by referring to the formulation examples described in International Patent Publication Gazette (such as WO00 / 09162, WO97 / 23222, etc.).

[0015] When preparing a formulation of repasodil or a salt thereof or a solvate thereof, it can also be prepared according to a known method. For example, an eye drop can be prepared by using an isotonic agent, a buffer, a surfactant, a preservative, etc. as needed. The pH may be within the range acceptable for ophthalmic preparations, and a range of pH 4 - 8 is preferred.

[0016] The formulation of the present invention is preferably used as an ophthalmic preparation, particularly for eye drops. Such eye drops may be any of aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, eye ointments, etc. Such a formulation can be manufactured by a method known to those skilled in the art as a composition suitable for the administration form, and by blending pharmaceutically acceptable carriers as needed, such as an isotonic agent, a chelating agent, a stabilizer, a pH adjuster, a preservative, an antioxidant, a solubilizing agent, a thickening agent, etc.

[0017] When preparing eye drops, for example, the desired components can be dissolved or suspended in an aqueous solvent such as sterile purified water or physiological saline, or in a non-aqueous solvent such as a vegetable oil such as cottonseed oil, soybean oil, sesame oil, or peanut oil, adjusted to a predetermined osmotic pressure, and then sterilized by filtration sterilization or other methods. When preparing eye ointments, an ointment base may be included in addition to the various components mentioned above. The ointment base is not particularly limited, but preferred examples include oily bases such as petrolatum, liquid paraffin, and polyethylene; emulsion bases obtained by emulsifying the oil phase and aqueous phase with a surfactant or the like; and water-soluble bases consisting of hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, etc.

[0018] When lipasudil, its salts, or their solvates are used for the prevention or treatment of retinal diseases, particularly diabetic retinopathy or age-related macular degeneration, the dosage varies depending on the patient's weight, age, sex, symptoms, form of administration, and frequency of administration. However, for adults, the usual daily dose is 0.025 to 10,000 μg of lipasudil, its salts, or their solvates, preferably 0.025 to 2,000 μg, more preferably 0.1 to 2,000 μg, and even more preferably 0.025 to 200 μg, or 0.025 to 100 μg.

[0019] Furthermore, while there are no particular limitations on the number of administrations, it is preferable to administer it once or in several divided doses. In the case of liquid eye drops, it is sufficient to instill one to several drops at a time. The present invention will be described in more detail below, but the present invention is not limited thereto. [Examples]

[0020] Example 1 RGC protective effect of lipasudil After optic nerve injury (ONC) of 8-10 week old C57BL6 / J mice, 2.0% ripasudil or solvent (PBS) was instilled once daily at a dose of 5 μL for 14 days. Eyeballs were extracted on day 7 or 14 after ONC and immunostaining was performed. Anti-RBPMS antibody was used to detect RGCs. (Figure 1a)

[0021] ONC significantly reduced the density of RGCs (Figure 1c), and improvements were observed in all retinal areas on day 7 of lipasudil instillation (Figures 1d-f). Similar results were observed on day 14 (Figures 1g-j).

[0022] Example 2 Lipasdil's p38 phosphorylation inhibitory effect After optic nerve injury (ONC) of 8-10 week old C57BL6 / J mice, 5 μL of 2.0% ripasudil or solvent (PBS) was instilled once daily as a single eye drop. Eyeballs were extracted 3 hours or 1 day after ONC and immunoblotting was performed (Figure 2a).

[0023] We evaluated the protein levels of total p38 and phosphorylated p38 (p-p38) after ONC. Similar to our previous report (Non-Patent Literature 4), p-p38 expression increased 3 hours after ONC, but no increase was observed after 1 day. On the other hand, total p38 remained unchanged (Figure 2b). The ratio of p-p38 to total p38 3 hours after ONC was significantly reduced in the ripasudil group compared to the PBS group (Figure 2c,d). There was no difference in total p38 protein levels between the two groups. p-p38 was mainly found in the retinal ganglion cell layer (GCL) and inner granular layer of mice administered with PBS, and p-p38 in the GCL was consistent with staining with anti-RBPMS antibody (Figure 2e). On the other hand, p-p38 expression in the GCL of mice administered with ripasudil was low (Figure 2e). Quantitative analysis of p38 phosphorylation in GCL showed a significant decrease in the lipasudil-treated group compared to the PBS-treated group (Figure 2f).

[0024] Example 3 Ripasil's axonal regeneration effect After injuring the optic nerve of 8-10 week old C57BL6 / J mice, 2.0% ripasudil or a solvent (PBS) was instilled once daily at a dose of 5 μL for 14 days. On day 12 after optic nerve injury, a labeling agent (CTB) was injected intravitreously, and on day 14, the eyeballs were extracted and histologically examined. (Figure 3a)

[0025] While intravitreal injection of Y-27632 has been reported to regenerate the optic nerve in rats and cats (Non-Patent Literature 2, 3), this study investigated whether the same effect could be observed by instilling lipasudil. On day 14 after ONC, the number of regenerated axons in mice administered lipasudil was higher than in mice administered PBS (Figure 3b). At a distance of 100 μm from the injury site, the number of regenerated axons was 47 ± 10 in the control group, compared to 110 ± 13 in the lipasudil group (Figure 3c). At a distance of 250 μm from the injury site, the number of regenerated axons was 25 ± 4 in the control group, compared to 62 ± 8 in the lipasudil group (Figure 3d). At a distance of 500 μm from the injury site, the number of regenerated axons was 21 ± 3 in the lipasudil group (Figure 3e). These results suggest that lipasudil eye drops have an axon regeneration effect.

[0026] Example 4 Inhibitory effect of lipasudil on the phosphorylation of CRMP2 and cofilin in RGCs. After damaging the optic nerve of 8-10 week old C57BL6 / J mice, 2.0% lipasudil or solvent (PBS) was instilled into the eyes at a dose of 5 μL once daily for 7 days. Immunoblotting was performed after enucleation 3 hours, 1 day, 3 days, and 7 days after ONC. Double immunostaining was performed after enucleation 3 hours after ONC.

[0027] Downstream of the Rho / ROCK signaling pathway lies the phosphorylation (inactivation) of the microtubule-binding protein Collapsin Response Mediator Protein 2 (CRMP2). CRMP2, which interacts with tubulin heterodimers and promotes microtubule polymerization, is inactivated by phosphorylation, thereby suppressing nerve extension. Therefore, we compared the expression level of phosphorylated CRMP2 (p-CRMP2) in ONC mice with that of mice that did not undergo ONC. The expression level was stable at 3 hours, 1 day, and 3 days after ONC, but appeared to decrease at 7 days and 14 days (Figure 4a). Quantitative evaluation of p-CRMP2 expression revealed a significant decrease at 7 days and 14 days after ONC (Figure 4b). Furthermore, it is known that inactivating cofilin with LIM kinase stabilizes actin filaments and suppresses nerve extension. Similar experiments were conducted on cofilin phosphorylation, as with CRMP2. The results showed that the expression level remained unchanged at all points (Figure 4a,c). Next, we investigated the effect of lipasudil on CRMP2. Lipasudil significantly reduced the ratio of p-CRMP2 to total CRMP2 at 3 hours and 3 days after ONC compared to PBS. This effect was not observed after 7 days of ONC (Figure 5a-f). There was no significant difference in total CRMP2 expression levels between the two groups. Next, double immunohistochemistry was performed 3 hours after ONC, and lipasudil suppressed p-CRMP2 expression in GCL (Figure 5g,h). Furthermore, the effect of ripasudil on cofilin was investigated. Ripasudil significantly reduced the ratio of p-cofilin to total cofilin at ONC 3 hours, day 3, and day 7 compared to PBS (Figure 6a-f). There was no significant difference in total cofilin expression levels between the two groups. Immunostaining revealed that p-cofilin expression was observed in the inner retinal layer in PBS-treated mice, but this was suppressed by ripasudil (Figure 6g). Quantitative analysis of cofilin expression in GCLs also showed that it was significantly suppressed by ripasudil (Figure 6h).

[0028] These results demonstrate that lipasudil possesses excellent optic nerve protective and optic nerve axon regeneration effects. [Industrial applicability]

[0029] The lipasudil or its salts or solvates of the present invention have excellent optic nerve axon regeneration properties and are useful as pharmaceuticals.

Claims

1. An optic nerve axon regeneration agent comprising lipasudil or its salts or solvates thereof as an active ingredient.

2. The optic nerve axon regeneration agent according to claim 1, which is an eye drop.

3. A pharmaceutical composition for optic nerve axon regeneration comprising lipasudil or a salt thereof or a solvate thereof, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, which is an eye drop.

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