Treatment agent for severe ocular surface disease for topical administration

Abstract

The purpose of the present invention is to develop a novel method for treating diseases leading to severe corneal and conjunctival epithelial disorder from dysfunction of corneal / conjunctival epithelial stem cells, such as severe ocular surface diseases caused by chronic graft-versus-host disease.　The present invention provides a prophylactic or therapeutic agent for severe ocular surface disease for topical administration, the agent containing apocynin as an active ingredient.

Description

Topically administered agent for treating severe ocular surface diseases

[0001] The present invention relates to a topical agent for treating severe ocular surface diseases.

[0002] Non-Patent Document 1 describes that treatment with apocynin limits the onset of acute graft-versus-host disease in mice. However, Non-Patent Document 1 neither describes nor suggests the relationship between apocynin and chronic graft-versus-host disease. Furthermore, Non-Patent Document 1 only describes the relationship between apocynin and systemic acute graft-versus-host disease, and neither describes nor suggests the relationship between apocynin and ocular surface disease of chronic GVHD. Furthermore, Non-Patent Document 1 neither describes nor suggests topical administration.

[0003] Non-Patent Document 2 describes that the 2014 National Institutes of Health (NIH) Consensus Conference proposed new criteria for diagnosing and scoring the severity of chronic graft-versus-host disease (GVHD), which is positioned as a distinct disease from acute GVHD. Furthermore, acute GVHD does not exhibit ocular surface disease, whereas chronic GVHD does, and this is a crucial difference between acute and chronic GVHD.

[0004] Dry eye is a typical ocular surface disease. Non-Patent Document 3 describes a crucial difference between the ocular surface of dry eye, including Sjögren's syndrome, and that of chronic GVHD. Specifically, cellular fibrosis is observed on the ocular surface of chronic GVHD, but not on the ocular surface of dry eye. Conjunctival fibrosis is a more severe and intractable ocular surface disease, making it difficult to treat severe ocular surface disease caused by chronic GVHD with keratoconjunctival protective agents, moisturizing agents, or lacrimal secretion promoters, which are used to treat dry eye.

[0005] J Immunol Res. 2019 Nov 3:2019:9015292.Biol Blood Marrow Transplant. 2015 Mar;21(3):389-401.Investigative Ophthalmology & Visual Science November 2018, Vol.59, DES71-DES79.

[0006] Therefore, an object of the present invention is to develop a novel method for treating diseases such as ocular chronic graft-versus-host disease, which causes severe conjunctival fibrosis due to breakdown of immune function.

[0007] The present inventors have found that instillation of apocynin has an effect of suppressing inflammatory cells in the cornea and conjunctiva in a mouse model of chronic graft-versus-host disease.

[0008] That is, the present invention provides the following.

[0009] [Aspect A-1] A topically administered treatment for severe ocular surface disease, comprising apocynin as an active ingredient. [Aspect A-2] The treatment according to Aspect A-1, wherein the severe ocular surface disease is a disease resulting in severe conjunctival fibrosis due to immune dysfunction. [Aspect A-3] The treatment according to Aspect A-1, wherein the severe ocular surface disease is ocular chronic graft-versus-host disease. [Aspect A-4] The treatment according to Aspect A-1, wherein the severe ocular surface disease is a disease resulting in severe corneal and / or conjunctival epithelial stem cell dysfunction resulting in severe corneal and / or conjunctival epithelial damage. [Aspect A-5] The treatment according to Aspect A-4, wherein the disease resulting in severe corneal and / or conjunctival epithelial stem cell dysfunction resulting in corneal and / or conjunctival epithelial stem cell exhaustion. [Aspect A-6] An eye drop for preventing or treating chronic graft-versus-host disease, comprising apocynin as an active ingredient. [Aspect B-1] A topically administered therapeutic agent for severe ocular surface disease, comprising apocynin as an active ingredient. [Aspect B-2] The therapeutic agent according to Aspect B-1, wherein the severe ocular surface disease is a disease resulting in severe corneal epithelial damage due to dysfunction of corneal or conjunctival epithelial stem cells. [Aspect B-3] The therapeutic agent according to Aspect B-2, wherein the disease resulting in severe corneal epithelial damage due to dysfunction of corneal or conjunctival epithelial stem cells is ocular chronic graft-versus-host disease. [Aspect B-4] A therapeutic agent for chronic graft-versus-host disease, comprising apocynin as an active ingredient. [Aspect B-5] An agent for suppressing inflammatory cells that infiltrate into the cornea, comprising apocynin as an active ingredient. [Aspect B-6] An agent for suppressing the infiltration of inflammatory cells that infiltrate into the conjunctiva and are positive for oxidative stress markers, comprising apocynin as an active ingredient. [Aspect B-7] A tissue stem cell preservation agent for conjunctiva, corneal epithelium, or meibomian gland, comprising apocynin as an active ingredient. [Aspect B-8] An agent for maintaining COL17A expression, which contains apocynin as an active ingredient. [Aspect B-9] An agent for suppressing ocular GVHD, which contains apocynin as an active ingredient. [Aspect B-10] An agent for promoting wound healing of corneal epithelium, which contains apocynin as an active ingredient. [Aspect B-11] An agent for maintaining the number of conjunctival goblet cells, which contains apocynin as an active ingredient. [Aspect B-12] An agent for maintaining COL17A expression in the corneal fornix, which contains apocynin as an active ingredient. [Aspect B-13] An agent for maintaining epithelial stem cells in the corneal limbus or fornix, which contains apocynin as an active ingredient.[Aspect B-14] An agent for suppressing the expression of senescent cell markers in epithelial stem cells of the corneal limbus or fornix, which contains apocynin as an active ingredient. [Aspect B-15] An agent for maintaining the function of corneal and conjunctival epithelial stem cells, which contains apocynin as an active ingredient. [Aspect B-16] The agent according to any one of Aspects B-1 to B-15, wherein the apocynin is in the form of apocynin eye drops.

[0010] According to the present invention, severe ocular surface diseases can be treated by topical administration. According to the present invention, chronic graft-versus-host disease can be treated. According to the present invention, corneal epithelial damage can be suppressed. According to the present invention, inflammatory cells infiltrating the cornea can be suppressed. According to the present invention, infiltration of oxidative stress marker-positive inflammatory cells infiltrating the conjunctiva can be suppressed. According to the present invention, tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland can be maintained. According to the present invention, COL17A expression can be maintained. According to the present invention, ocular GVHD can be suppressed. According to the present invention, wound healing of the corneal epithelium can be promoted. According to the present invention, the number of conjunctival goblet cells can be maintained. According to the present invention, COL17A expression in the corneal fornix can be maintained. According to the present invention, epithelial stem cells of the corneal limbus or fornix can be maintained. According to the present invention, the expression of senescent cell markers in epithelial stem cells of the corneal limbus or fornix can be suppressed. According to the present invention, the function of corneal and conjunctival epithelial stem cells can be maintained.

[0011] Figure 1 shows the corneal fluorescein staining score (CFS score) measurement results for the syngeneic control group, the apocynin solution-treated group, and the vehicle-treated group 28 days after radiation exposure. Figure 2 shows the conjunctival goblet cell count measurement results for the syngeneic control group, the apocynin-treated group, and the vehicle-treated group 28 days after radiation exposure. Figure 3 shows the analysis results for inflammatory cells infiltrating the cornea in the apocynin-treated group and the vehicle-treated group 28 days after radiation exposure. Figure 4 shows the analysis results for the expression of COL17A, which has the effect of preserving the function of tissue stem cells, in the apocynin-treated group and the vehicle-treated group 28 days after radiation exposure. Figure 5 shows the analysis results for the expression of COL17A, which has the effect of preserving the function of tissue stem cells, in the apocynin-treated group and the vehicle-treated group 28 days after radiation exposure. Figure 6 shows the results of analysis of inflammatory cells (CD45+ cells) expressing oxidative stress markers 8-OHdG and 4HNE in the conjunctival fornix in the syngeneic control group, apocynin-treated group, and vehicle-treated group 28 days after radiation. Figure 7 shows the results of analysis of inflammatory cells (CD45+ cells) expressing oxidative stress markers 8-OHdG and 4HNE in the conjunctival fornix in the syngeneic control group, apocynin-treated group, and vehicle-treated group 28 days after radiation. Figure 8 shows the results of analysis of the expression of the tissue stem cell marker SLC1A3 in the conjunctival fornix in the syngeneic control group, apocynin-treated group, and vehicle-treated group 28 days after radiation. Figure 9 shows the results of analysis of the expression of the tissue stem cell marker ABCB5 in the corneal limbus in the syngeneic control group, apocynin-treated group, and vehicle-treated group 28 days after radiation. FIG. 10 shows the results of expression analysis of COL17A and tissue stem cell marker p63 in the meibomian glands in the syngeneic control group, apocynin administration group, and vehicle administration group 28 days after radiation exposure.

[0012] The agent of the present invention contains apocynin as an active ingredient. Apocynin is 4-hydroxy-3-methoxyacetophenone and is represented by the following formula: Apocynin can be produced by known methods, and commercially available products can also be used.

[0013] By administering the agent of the present invention to a subject, it is possible to treat severe ocular surface disease by topical administration, treat chronic graft-versus-host disease, suppress corneal and conjunctival epithelial damage, suppress corneal infiltration of inflammatory cells, suppress infiltration of oxidative stress marker-positive inflammatory cells into the conjunctiva, maintain tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland, maintain COL17A expression, suppress ocular GVHD (graft-versus-host disease), promote corneal epithelial wound healing, maintain the number of conjunctival goblet cells, maintain COL17A expression in the corneal fornix, maintain epithelial stem cells in the corneal limbus or fornix, suppress the expression of senescent cell markers in epithelial stem cells in the corneal limbus or fornix, and maintain the function of corneal and conjunctival epithelial stem cells. Severe ocular surface disease is, for example, a disease in which severe corneal epithelial damage occurs due to dysfunction of corneal or conjunctival epithelial stem cells. Examples of diseases that cause severe corneal epithelial damage due to dysfunction of corneal or conjunctival epithelial stem cells include ocular chronic graft-versus-host disease. Disease treatments include, for example, disease therapy and disease prevention. Disease treatments include, for example, complete cure of the disease as well as alleviation of disease symptoms.

[0014] The subject may be, for example, a vertebrate. The vertebrate may be, for example, a mammal such as a mouse, a rat, a rabbit, a pig, a cow, a monkey, or a human. The mammal is preferably a human. The subject may be of any age, including an infant, a juvenile, an adolescent, an adult, or an elderly person.

[0015] Prior to administering the agent of the present invention to a subject, the subject may be diagnosed as having a severe ocular surface disease; the subject may be diagnosed as having chronic graft-versus-host disease; the subject may be diagnosed as having corneal epithelial damage; inflammatory cells infiltrating the cornea of ​​the subject may be measured; inflammatory cell infiltration positive for oxidative stress markers infiltrating the conjunctiva of the subject may be measured; tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland of the subject may be measured; COL17A expression may be measured in the subject; the subject may be diagnosed as having ocular GVHD (graft-versus-host disease); corneal epithelial wound healing may be measured in the subject; the number of conjunctival goblet cells may be measured in the subject; COL17A expression in the corneal fornix of the subject may be measured; epithelial stem cells in the corneal limbus or fornix of the subject may be measured; senescent cell marker expression in epithelial stem cells in the corneal limbus or fornix of the subject may be measured; or the function of corneal and conjunctival epithelial stem cells may be measured in the subject.

[0016] Examples of subjects include subjects who have been diagnosed with a severe ocular surface disease and identified as having a severe ocular surface disease; subjects who are predicted to have a severe ocular surface disease; subjects who have been diagnosed with chronic graft-versus-host disease and identified as having chronic graft-versus-host disease; subjects who are predicted to have chronic graft-versus-host disease; subjects who have been diagnosed with a corneal epithelial disorder and identified as having a corneal epithelial disorder; and subjects who are predicted to have a corneal epithelial disorder. Examples of such subjects include subjects in whom inflammatory cells infiltrating the cornea are measured and identified as having an excess of inflammatory cells infiltrating the cornea; subjects in whom it is predicted that there will be an excess of inflammatory cells infiltrating the cornea; subjects in whom it is predicted that there will be an excess of inflammatory cells infiltrating the conjunctiva that are positive for oxidative stress markers are measured and identified as having an excess of inflammatory cells infiltrating the conjunctiva that are positive for oxidative stress markers; subjects in whom it is predicted that there will be ... are measured and tissue stem cells in the conjunctiva, corneal epithelium, or meibomian gland are measured and Examples of such subjects include subjects who have been identified as having a deficiency in tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland; subjects who are predicted to have a deficiency in tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland; subjects who have been measured for COL17A expression and identified as having a deficiency in COL17A expression; subjects who are predicted to have a deficiency in COL17A expression; subjects who have been diagnosed for ocular GVHD (graft versus host disease) and identified as having ocular GVHD (graft versus host disease); and subjects who have been diagnosed for ocular GVHD (graft versus host disease). corneal epithelial wound healing is measured to identify a subject having insufficient corneal epithelial wound healing; a subject predicted to have insufficient corneal epithelial wound healing; a subject having conjunctival goblet cell count measured to identify a subject having insufficient conjunctival goblet cell count; a subject predicted to have insufficient conjunctival goblet cell count; a subject having corneal fornix COL17A expression measured to identify a subject having insufficient corneal fornix COL17A expression;Examples include subjects predicted to have insufficient COL17A expression in the corneal fornix; subjects identified as having insufficient limbal or fornix epithelial stem cells by measuring epithelial stem cells in the limbus or fornix; subjects predicted to have insufficient limbal or fornix epithelial stem cells; subjects identified as having excessive expression of senescent cell markers in epithelial stem cells in the limbus or fornix by measuring expression of senescent cell markers in epithelial stem cells in the limbus or fornix; subjects predicted to have excessive expression of senescent cell markers in epithelial stem cells in the limbus or fornix; subjects identified as having insufficient corneal and conjunctival epithelial stem cell function by measuring function of corneal and conjunctival epithelial stem cells; and subjects predicted to have insufficient corneal and conjunctival epithelial stem cell function.

[0017] The agent of the present invention may consist of apocynin, or may be formulated as a pharmaceutical composition in which it is mixed with a pharmaceutically acceptable carrier.

[0018] The pharmaceutical composition may be formulated into a dosage form for parenteral use such as an injection, ointment, or patch, or may be formulated into an ophthalmic dosage form such as eye drops, eye wash, or eye ointment.

[0019] Examples of pharmaceutically acceptable carriers include solvents such as sterilized water and physiological saline; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as crystalline cellulose; and leavening agents such as corn starch, gelatin, and alginic acid.

[0020] Pharmaceutically acceptable carriers include additives such as lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, and saccharin, flavorings such as peppermint and rhododendron oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, preservatives, surfactants, and emulsifiers.

[0021] Pharmaceutical compositions can be formulated by combining the above-mentioned carriers appropriately and mixing them in unit dosage forms required for generally accepted pharmaceutical practice.

[0022] When the pharmaceutical composition is an injection, examples of the solvent for the injection include isotonic solutions containing adjuvants such as physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. The solvent for the injection may contain alcohol such as ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactants such as Polysorbate 80 (trademark), HCO-50, etc.

[0023] The administration of the agent of the present invention to a subject is not particularly limited and can be appropriately selected depending on the target organ in which the symptom is manifested, but is preferably carried out by local administration. Examples of local administration include parenteral administration such as ocular administration, intraocular administration, periocular administration, intrapalpebral administration, subcutaneous administration, intradermal administration, and transdermal administration. When the target organ in which the symptom is manifested is the eye, the agent of the present invention is preferably administered by local administration such as subconjunctival injection or ocular administration.

[0024] When the agent of the present invention is for topical administration such as eye drops, the concentration of apocynin contained in the agent of the present invention is, for example, 0.000001 w / v% or more, 0.00001 w / v% or more, 0.0001 w / v% or more, 0.001 w / v% or more, 0.002 w / v% or more, 0.003 w / v% or more, 0.004 w / v% or more, 0.005 w / v% or more, 0.006 w / v% or more, 0.007 w / v% or more. or more, 0.008 w / v% or more, or 0.009 w / v% or more, and 10 w / v% or less, 1 w / v% or less, 0.1 w / v% or less, 0.09 w / v% or less, 0.08 w / v% or less, 0.07 w / v% or less, 0.06 w / v% or less, 0.05 w / v% or less, 0.04 w / v% or less, 0.03 w / v% or less, or 0.02 w / v% or less, preferably about 0.01 w / v% or less.

[0025] The present invention provides a method for treating severe ocular surface disease in a subject, which comprises topically administering apocynin to the subject; a method for treating chronic graft-versus-host disease in a subject, which comprises administering apocynin to the subject; a method for suppressing corneal epithelial damage in a subject, which comprises administering apocynin to the subject; a method for suppressing inflammatory cells infiltrating the cornea in a subject, which comprises administering apocynin to the subject; a method for suppressing inflammatory cell infiltration positive for oxidative stress markers infiltrating the conjunctiva in a subject, which comprises administering apocynin to the subject; a method for maintaining tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland in a subject, which comprises administering apocynin to the subject; a method for maintaining COL17A expression in a subject, which comprises administering apocynin to the subject; a method for suppressing ocular GVHD in a subject; a method for promoting wound healing of the corneal epithelium in a subject, which comprises administering apocynin to the subject; a method for maintaining the number of conjunctival goblet cells in a subject, which comprises administering apocynin to the subject; a method for maintaining COL17A expression in the corneal fornix in a subject, which comprises administering apocynin to the subject; a method for maintaining epithelial stem cells in the corneal limbus or fornix in a subject, which comprises administering apocynin to the subject; a method for suppressing the expression of senescent cell markers in epithelial stem cells in the corneal limbus or fornix in a subject, which comprises administering apocynin to the subject; a method for maintaining corneal and conjunctival epithelial stem cell function in a subject, which comprises administering apocynin to the subject; and a method for protecting the corneal epithelium in a subject, which comprises administering apocynin to the subject.

[0026] The present invention provides a pharmaceutical composition containing apocynin as an active ingredient for use in the treatment of severe ocular surface disease in a subject by topical administration; a pharmaceutical composition containing apocynin as an active ingredient for use in the treatment of chronic graft-versus-host disease in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in suppressing corneal epithelial damage in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in suppressing inflammatory cells infiltrating the cornea in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in suppressing inflammatory cell infiltration that is positive for oxidative stress markers infiltrating the conjunctiva in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland in a subject; and a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining COL17A expression in a subject. a pharmaceutical composition containing apocynin as an active ingredient for use in suppressing ocular GVHD in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in promoting wound healing of the corneal epithelium in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining the number of conjunctival goblet cells in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining COL17A expression in the corneal fornix in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining epithelial stem cells in the corneal limbus or fornix in a subject; a pharmaceutical composition containing apocynin as an active ingredient for use in suppressing the expression of senescent cell markers in epithelial stem cells in the corneal limbus or fornix in a subject; and a pharmaceutical composition containing apocynin as an active ingredient for use in maintaining corneal conjunctival epithelial stem cell function in a subject.

[0027] The present invention relates to the use of apocynin in the manufacture of a pharmaceutical composition for use in treating severe ocular surface diseases by topical administration; the use of apocynin in the manufacture of a pharmaceutical composition for use in treating chronic graft-versus-host disease; the use of apocynin in the manufacture of a pharmaceutical composition for use in suppressing corneal epithelial damage; the use of apocynin in the manufacture of a pharmaceutical composition for use in suppressing inflammatory cells infiltrating the cornea; the use of apocynin in the manufacture of a pharmaceutical composition for use in suppressing inflammatory cell infiltration positive for oxidative stress markers infiltrating the conjunctiva; the use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining tissue stem cells of the conjunctiva, corneal epithelium, or meibomian gland; and the use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining the expression of COL17A. Use of apocynin in the manufacture of a pharmaceutical composition for use in suppressing ocular GVHD; Use of apocynin in the manufacture of a pharmaceutical composition for use in promoting wound healing of corneal epithelium; Use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining the number of conjunctival goblet cells; Use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining COL17A expression in the corneal fornix; Use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining epithelial stem cells in the corneal limbus or fornix; Use of apocynin in the manufacture of a pharmaceutical composition for use in suppressing the expression of senescent cell markers in epithelial stem cells in the corneal limbus or fornix; Use of apocynin in the manufacture of a pharmaceutical composition for use in maintaining corneal conjunctival epithelial stem cell function.

[0028] Preparation of test compound solution: Apocynin was mixed with phosphate-buffered saline (PBS) to prepare a 0.01% apocynin ophthalmic solution. PBS was used as the vehicle solution.

[0029] Creation of a chronic graft-versus-host disease (chronic GVHD) animal model and observation of corneal epithelium: Spleen cells and bone marrow cells from major histocompatibility complex (MHC)-compatible, minor histocompatibility antigen (miHA)-mismatched male mice (B10.D2, 7-9 weeks old; Sankyo Research Institute) were transplanted into female mice (BALB / c, 7-9 weeks old; Sankyo Research Institute). As a control, female mice (BALB / c) that had received allogeneic spleen and bone marrow cell transplants were used (syngeneic control). The transplantation method involved irradiating the recipient with 7 Gy of radiation, followed by 1×10 6 of bone marrow cells and 2 x 10 6 Spleen cells from the mice were dissolved in 0.1 mL of Roswell Park Memorial Institute medium (RPMI medium), and a total of 0.2 mL was administered via the tail vein. The model mice were maintained in a specific pathogen-free (SPF) environment with sterilized water and food for 7 days, after which drug administration began. A 0.01% apocynin solution was administered by eye drop twice daily for 21 consecutive days. For comparison, vehicle solution was administered in the same manner. Twenty-eight days after irradiation, the condition of the corneal epithelium was observed using corneal fluorescein staining, and the mice were sacrificed and tissue samples were collected.

[0030] Figure 1 shows the results of measuring the corneal fluorescein staining score (CFS score) 28 days after radiation in the syngeneic control group, the apocynin solution-administered group, and the vehicle-administered group. The ocular surface was stained with fluorescein, and the stained image was scored on a three-point scale. The CFS score was calculated as the mean ± standard error of the total scores for the upper, middle, and lower corneal regions. First, pathological analysis of the ocular surface of the vehicle-administered mice revealed clear fibrosis beneath the conjunctival epithelium. On the other hand, the CFS score was 0.38±0.15 in the apocynin eye drop group and 1.36±0.19 in the vehicle eye drop group, with the apocynin solution group showing a significantly better CFS score (p = 0.0032, n = 5-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P<0.05, **P<0.01, ***P<0.001). These results demonstrate that 0.01% apocynin eye drop is effective against corneal damage caused by chronic GVHD. The scoring criteria are as follows: 0: No staining 1: Slight staining 2: Moderate staining 3: Severe staining

[0031] Figure 2 shows the results of measuring the number of conjunctival goblet cells in the syngeneic control group, apocynin-treated group, and vehicle-treated group 28 days after radiation. Ocular samples were stained with PAS and the number of goblet cells in the conjunctiva was measured. The mean ± standard error of the mean was calculated for each group. The apocynin-treated group had 49.5 ± 4.35 goblet cells, while the control group had 26.7 ± 4.10 goblet cells. Significantly more goblet cells were preserved in the apocynin-treated group (p = 0.015, n = 6-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). These results suggest that 0.01% apocynin ophthalmic solution is effective in treating conjunctival damage caused by chronic GVHD.

[0032] Figure 3 shows the analysis of inflammatory cells infiltrating the cornea in the apocynin-treated and vehicle-treated groups 28 days after radiation. CD45-positive inflammatory cells were labeled using immunohistochemistry throughout the cornea, and the CD45-positive area was quantified using ImageJ image analysis software. The mean ± standard error of the CD45-positive area relative to the entire cornea was used as the quantification value. CD45-positive cell infiltration was significantly suppressed in the apocynin-treated group (3.21 ± 0.39%) and in the vehicle-treated group (5.98 ± 0.38%) (p = 0.0004, n = 7-8 per group, Unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001). These results suggest that 0.01% apocynin eye drops are effective against corneal inflammation due to chronic GVHD.

[0033] Figures 4 and 5 show the results of analysis of COL17A expression, which has the effect of preserving tissue stem cell function, in the apocynin-treated and vehicle-treated groups 28 days after radiation. COL17A-positive areas were analyzed in the limbus and fornix, where epithelial tissue stem cells reside on the ocular surface, using immunohistochemistry and the image analysis software ImageJ. Results were calculated as the percentage of COL17A-positive areas relative to nuclear staining (DAPI) within the visual field ± standard error. In the limbus, COL17A expression was significantly higher in the apocynin-treated group (195 ± 22%) than in the control solution-treated group (100 ± 28%) (p = 0.028, n = 5-8 per group, Unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001). In the conjunctival fornix, COL17A expression remained significantly higher in the apocynin-treated group (67.2±7.4% vs. 28.5±7.13%) than in the vehicle-treated group (p=0.0075, n=5-8 per group, Unpaired t-test, *P<0.05, **P<0.01, ***P<0.001). These results suggest that 0.01% apocynin eye drops are effective against the decline in stem cell function in the corneal limbus and conjunctival fornix due to chronic GVHD.

[0034] Figures 6 to 7 show the analysis results of inflammatory cells (CD45-positive cells) expressing oxidative stress markers 8-OHdG and 4HNE in the conjunctival fornix in the syngeneic control group, apocynin-administered group, and vehicle-administered group 28 days after radiation. + 8 - OHdG + cells, CD45 + 4HNE + The cells were analyzed using immunohistochemistry and image analysis software ImageJ. + 8 - OHdG +The number of cells ± standard error was used as the measurement result. + 8-OHdG + The number of cells was 4.6±0.63 cells in the apocynin-treated group and 9.0±2.1 cells in the vehicle-treated group. + 8 - OHdG + The apocynin-treated group showed a significant reduction in CD45 cells in the conjunctival fornix (p = 0.026, n = 5-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). + 4HNE + The number of cells was 48.1±6.7 cells in the vehicle-administered group and 78.3±9.14 cells in the apocynin-administered group. + 8 - OHdG + The number of cells was suppressed (p = 0.027, n = 6-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). These results suggest that 0.01% apocynin eye drops are effective against inflammation of the conjunctival fornix caused by chronic GVHD.

[0035] Figure 8 shows the expression of the tissue stem cell marker SLC1A3 in the conjunctival fornix 28 days after radiation in the syngeneic control group, apocynin-treated group, and vehicle-treated group. Analysis was performed using image analysis software ImageJ and immunohistochemistry. Results were calculated as the mean ± standard error of the SLC1A3-positive area per visual field. SLC1A3 expression in the conjunctival fornix was 16.1 ± 1.8% in the apocynin group and 4.3 ± 1.3% in the vehicle-treated group, demonstrating significant maintenance of SLC1A3 expression in the apocynin-treated group (p = 0.0023, n = 5-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). These results suggest that 0.01% apocynin eye drops are effective against the reduction of stem cells in the conjunctival fornix due to chronic GVHD.

[0036] Figure 9 shows the results of expression analysis of the tissue stem cell marker ABCB5 in the corneal limbus in the syngeneic control group, the apocynin-treated group, and the vehicle-treated group 28 days after radiation. Analysis was performed using the image analysis software ImageJ and immunohistochemistry. Measurement results were calculated as the mean ± standard error of the ABCB5-positive area in the visual field. In the apocynin-treated group, the corneal limbus expression was 17.5 ± 3.3%, while in the vehicle-treated group it was 5.2 ± 0.51%. Significantly higher expression of the tissue stem cell marker ABCB5 was maintained in the apocynin-treated group (p = 0.0069, n = 5-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). These results suggest that 0.01% apocynin eye drops are effective against the loss of stem cells in the limbus due to chronic GVHD.

[0037] It is known that the pathology of meibomian gland dysfunction is related to the exhaustion and decrease of tissue stem cells in the meibomian gland. Meibomian gland dysfunction also occurs in the eyelids of cGVHD model mice. Figure 10 shows the results of expression analysis of COL17A and tissue stem cell marker p63 in the meibomian gland in the syngeneic control group, apocynin-administered group, and vehicle-administered group 28 days after radiation. COL17A in the meibomian gland + p63 + The number of positive cells was analyzed using immunohistochemistry. + p63 + The number of cells ± standard error was used as the measurement result. The number of cells in the meibomian glands of the apocynin solution-administered group was 23.1 ± 2.2 cells, and that of the vehicle-administered group was 12.1 ± 1.6 cells. The apocynin-administered group showed a significant increase in COL17A. + p63 + (p = 0.0053, n = 6-7 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001) These results suggest that 0.01% apocynin eye drops are effective against the decline in meibomian gland stem cell function caused by chronic GVHD.

[0038] In summary, in chronic GVHD model mice, apocynin eye drops suppressed inflammatory cell infiltration into the cornea, suppressed oxidative stress marker-positive inflammatory cell infiltration into the conjunctiva, preserved tissue stem cells in the conjunctiva, corneal epithelium, and meibomian gland, and maintained the expression of COL17A, which plays an important role in maintaining tissue stem cell function. These findings suggest that apocynin is effective in treating severe ocular surface disease caused by chronic GVHD. Furthermore, because apocynin was effective in preserving stem cells and maintaining their function, it may also be effective in corneal and conjunctival epithelial stem cell exhaustion.

[0039] Apocynin eye drops have been suggested to suppress severe ocular surface disease (ocular GVHD) caused by chronic GVHD by preserving epithelial stem cells. Until now, ocular GVHD has been prevented and treated, as with regular dry eye, using tear stability-improving drugs and steroid eye drops, but many cases have been resistant to treatment. These conventional treatments lack pharmacological activity on corneal and conjunctival epithelial stem cells, which is thought to be why they have been insufficiently effective in treating ocular GVHD. Apocynin eye drops are highly useful as a therapeutic agent for ocular GVHD based on a novel mechanism of epithelial stem cell preservation, and therefore have potential for industrial applicability.

Claims

1. A topical agent for treating severe ocular surface diseases, containing apocynin as an active ingredient.

2. The treatment agent according to claim 1, wherein the severe ocular surface disease is a disease that causes severe conjunctival fibrosis due to a breakdown of immune function.

3. The treatment of claim 1, wherein the severe ocular surface disease is ocular chronic graft-versus-host disease.

4. The treatment agent according to claim 1, wherein the severe ocular surface disease is a disease resulting in severe corneal and / or conjunctival epithelial damage due to dysfunction of corneal and / or conjunctival epithelial stem cells.

5. The treatment agent according to claim 4, wherein the disease causing severe corneal and / or conjunctival epithelial damage due to dysfunction of corneal and / or conjunctival epithelial stem cells is corneal and conjunctival epithelial stem cell exhaustion syndrome.

6. Eye drops for preventing or treating chronic graft-versus-host disease, containing apocynin as an active ingredient.

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