Therapeutic agent or prophylactic agent for dry eye

Abstract

The purpose of the present invention is to provide a novel therapeutic agent or prophylactic agent for dry eye, a therapeutic agent or prophylactic agent for a graft-versus-host disease, a corneal epithelial wound healing promoter, an inflammatory cell inhibitor that infiltrates the cornea, a tear fluid amount retaining agent, a corneal epithelial disorder inhibitor, a cell proliferation marker-positive cell inhibitor, and an inhibitor of lymphocyte differentiation into effector T cells. The present invention provides the following, each of which contains, as an active ingredient, a substance derived from a culture supernatant of stem cells: a therapeutic agent or prophylactic agent for dry eye; a therapeutic agent or prophylactic agent for a graft-versus-host disease; a corneal epithelial wound healing promoter; an inflammatory cell inhibitor infiltrating into the cornea; a tear fluid amount retaining agent; a corneal epithelial disorder inhibitor; a cell proliferation marker-positive cell inhibitor; or an inhibitor of lymphocyte differentiation into effector T cells.

Description

Treatment or prevention of dry eye

[0001] The present invention relates to a therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft versus host disease, an inhibitor of inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an inhibitor of corneal epithelial damage, an inhibitor of cells positive for cell proliferation markers, or an inhibitor of lymphocyte differentiation into effector T cells.

[0002] Dry eye is a chronic disease or symptom that results from abnormalities in tear volume and composition due to various factors, causing dryness, discomfort, and visual impairment, and can lead to damage to the cornea and conjunctiva on the surface of the eye. It is said that people obtain more than 80% of their information through their eyes, and dry eye, which causes chronic eye symptoms, limits activity, reduces motivation, and reduces quality of life (Non-Patent Document 1).

[0003] Dry eye is a highly prevalent disease or symptom. In recent years, the number of dry eye patients has rapidly increased due to factors such as the widespread use of air conditioning and an increase in the number of people working with visual display terminals (VDTs), with an estimated 8 million patients in Japan and over 90 million worldwide, creating a growing need for medical care. In addition, it has been reported that the prevalence of dry eye in Asia, including Japan, is higher than in Europe and the United States.

[0004] Furthermore, the number of people suing due to risk factors for dry eye, such as aging, contact lens wear, and prolonged computer work, is expected to continue to increase in the future. Continuously looking at a monitor screen causes tears to dry out more easily, leading to eye strain and a decline in visual function known as evening presbyopia. Dry eye has a wide range of other causes, and it is also known to occur as a side effect of anticancer drug treatment. Anticancer drugs in particular are thought to affect corneal cells, which are actively dividing, and dry eye is considered a frequent side effect of many molecularly targeted drugs (Non-Patent Document 2).

[0005] In dry eye, impaired tear film stability is one of the core mechanisms. As the condition progresses and becomes more severe, corneal and conjunctival epithelial damage becomes more pronounced, resulting in a variety of subjective symptoms. Dry eye is classified into aqueous-deficient and evaporative types based on its etiology. Meibomian gland dysfunction (MGD) is believed to be the cause of evaporative dry eye, and MGD is the cause in a significant proportion of patients who visit ophthalmologists complaining of dry eye symptoms such as ocular discomfort (Non-Patent Document 1). Furthermore, short BUT (tear film break-up time) dry eye is characterized by a shortened BUT, normal tear secretion, and severe subjective symptoms despite minor ocular surface epithelial damage.

[0006] The meibomian glands are sebaceous glands located within the tarsal plate, with openings at the upper and lower eyelid margins. The lipids secreted by the meibum are distributed along the eyelid margin and in the outermost layer of the tear film, where they play a role in inhibiting tear evaporation, promoting tear stability, promoting tear spreading over the ocular surface, and inhibiting tear outflow onto the skin at the eyelid margin (Non-Patent Document 3).

[0007] The term meibomian gland dysfunction (MGD) is clinically used to refer to a condition in which meibomian gland function is abnormal (Non-Patent Document 4). However, it has not received much attention in general ophthalmology clinical practice for several reasons: (1) the clinical manifestations are diverse, with or without the involvement of inflammation and resident bacteria; (2) the severity ranges widely from mild to severe; (3) there has been no established definition or diagnostic criteria; and (4) there are few effective treatments. However, MGD is actually the cause of a significant proportion of patients who visit ophthalmologists complaining of symptoms such as ocular discomfort, and it is thought to cause a decline in quality of life in many patients. Despite its clinical importance, research into MGD has not progressed significantly for the above reasons. Aside from a treatment for meibomian gland dysfunction containing activated vitamin D3 or its derivatives as an active ingredient (Patent Document 1), no effective treatment has yet been found. MGD is currently treated by thermotherapy or physical expression to alleviate blockage of the meibomian glands. In recent years, methods have also been developed that evaluate the tear lipid layer using interference images and treat the eyelids from the inside with heat and massage.However, these methods all require treatment in a medical institution, so a simpler treatment method is eagerly awaited.

[0008] As described above, dry eye is a chronic eye disease in which tear film destabilization is one of the core mechanisms, and corneal epithelial damage and meibomian gland dysfunction mutually influence and contribute to a vicious cycle. Although stratified ocular surface treatments have been proposed to treat dry eye by supplementing deficient components on the ocular surface to improve the stability of the tear film, agents effective only on certain layers are insufficient, and a therapeutic agent that is multifaceted and effective against the core mechanism of dry eye is desired. Although a therapeutic agent containing apocynin is known (Patent Document 2), new therapeutic agents are also needed.

[0009] Hematopoietic stem cell transplantation (HSCT) is a treatment for hematologic malignancies caused by abnormalities in hematopoietic stem cells, in which healthy donor HSCT cells are transplanted to a recipient, potentially curing leukemia. However, in recent years, graft-versus-host disease (GVHD) has become a problem, occurring when the donor's bone marrow, peripheral blood, or umbilical cord blood graft attacks the recipient's target organs through an immune response.

[0010] Previously, using a mouse model exhibiting a pathology closely resembling human chronic GVHD, it has been reported that fresh donor bone marrow mesenchymal stem cells are immunogenic, engraft in the exocrine glands and mucosa of GVHD, and are involved in the onset of the disease (Non-Patent Document 7). It has also been reported that hematopoietic stem cells transplanted from bone marrow undergo telomere shortening after transplantation, resulting in stem cell senescence (Non-Patent Document 8). It has also been reported that mesenchymal stem cells similarly undergo stem cell senescence (Non-Patent Document 9).

[0011] It has also been reported that infiltration of senescent macrophages is observed in the lacrimal glands of GVHD model mice at the early stage of the disease (Non-Patent Document 10). Furthermore, it has been shown that the local tissue renin-angiotensin system (RAS) is involved in immune-mediated fibrosis in GVHD lacrimal glands and other target organs, and that its inhibitors alleviate the pathology (Non-Patent Document 11). RAS is believed to cause mitochondrial dysfunction associated with the progression of aging, suggesting that age-related changes are involved in the pathogenesis of GVHD. Furthermore, it has been reported that endoplasmic reticulum stress, which is associated with aging, is excessively elevated in various target organs of chronic GVHD, and that administration of the endoplasmic reticulum stress inhibitor phenylbutyric acid suppresses inflammation and fibrosis in target organs (Non-Patent Document 12). These reports suggest that age-related changes are involved in the pathogenesis of GVHD.

[0012] After hematopoietic stem cell transplantation, senescent cells accumulate due to excessive stress, such as radiation exposure, and a weakened immune system (Non-Patent Document 13). Recently, a mechanism by which senescent cells continuously release humoral factors, promoting the activation of neighboring cells and leading to chronic inflammation, has been reported, known as the senescence-associated secretory phenotype (SASP) (Non-Patent Documents 14-15). Furthermore, it has been reported that senescent cells are actively involved in the pathogenesis of age-related diseases such as macular degeneration (Non-Patent Document 16).

[0013] Immunosuppressants are known as treatments for chronic GVHD, but although they are partially effective, there are many intractable cases.

[0014] For example, rebamipide and diquafosol are known as therapeutic agents for GVHD for treating ocular surface conditions, and it has been disclosed that the combined use of rebamipide and diquafosol improves dry eye, one of the symptoms of GVHD. However, while rebamipide and diquafosol can be used to treat patients with mild to moderate GVHD, they cannot be used to treat patients with severe GVHD. Furthermore, systemic treatments have been performed using immunosuppressants such as cyclosporine and tacrolimus, as well as steroids. However, the effects of these treatments are still insufficient, and there are currently no effective therapeutic agents for GVHD.

[0015] Mesenchymal stem cell (MSC) culture supernatant is known to contain various growth factors and cytokines, and is currently expected to be useful in a wide range of fields, from cosmetics to regenerative medicine. MSCs can be easily extracted from various tissues, such as bone marrow, dental pulp, adipose tissue, and umbilical cord. However, deciduous dental pulp stem cells, which can be extracted from deciduous teeth, are particularly promising as they can be easily prepared from deciduous teeth that have traditionally been discarded, and they also have high proliferation potential.

[0016] It has been reported that culture supernatants of deciduous dental pulp stem cells are useful for repairing damaged tissues such as those caused by cerebral infarction and for protecting skin tissue (Patent Documents 4 and 5). In addition, in an attempt to treat dry eye using cell culture supernatants, Patent Document 6 describes a pharmaceutical composition for use in treating dry eye disease, which contains amniotic fluid stem cell secretome prepared by a method comprising culturing amniotic fluid stem cells in a basal medium for 24 to 72 hours to obtain a medium, recovering the culture medium supernatant after centrifugation, and filtering the supernatant to obtain the secretome.

[0017] Furthermore, Patent Documents 7 and 8 describe a regenerative therapy composition comprising a dental pulp-derived stem cell culture supernatant but not containing dental pulp-derived stem cells themselves, characterized in that the concentration of vascular endothelial growth factor (VEGF), a cytokine contained in the dental pulp-derived stem cell culture supernatant, is 150 to 5,000 pg / ml, and describe that the regenerative therapy composition is used to improve or prevent dry eye. However, even if an invention can be understood by a person skilled in the art from the matters described in the publication or equivalent matters, an invention of a product that is not clearly capable of being made by a person skilled in the art based on the description in the publication and the common general technical knowledge at the time of filing cannot be claimed as a cited invention (Examination Guidelines). Patent Documents 7 and 8 do not describe examples showing that the regenerative therapy composition can be used to improve or prevent dry eye, and do not clearly state that the regenerative therapy composition for use in improving or preventing dry eye can be made. Therefore, Patent Documents 7 and 8 were not eligible to recognize the invention of a dental pulp-derived stem cell culture supernatant for use in improving or preventing dry eye as a cited invention.

[0018] Furthermore, Patent Document 9 describes a sex hormone secretagogue that contains an umbilical cord-derived stem cell culture supernatant obtained by culturing umbilical cord-derived stem cells and promotes the secretion of sex hormones, and describes that the sex hormone secretagogue is used to improve or prevent dry eye. However, like Patent Documents 7 and 8, Patent Document 9 also does not describe any examples showing that the sex hormone secretagogue can be used to improve or prevent dry eye, and does not state clearly that the sex hormone secretagogue can be made to improve or prevent dry eye. Therefore, Patent Document 9 was not eligible to recognize an invention of an umbilical cord-derived stem cell culture supernatant used to improve or prevent dry eye as a cited invention.

[0019] International Publication No. 2014 / 208709 International Publication No. 2022 / 065436 JP 2020-111548 A International Publication No. 2011 / 118795 International Publication No. 2014 / 126176 JP 2022-184814 A Japanese Patent Application Laid-Open No. 2021-038268 International Publication No. 2020 / 130038 JP 2017-119646 A

[0020] Kojima et al. Progress in Retinal and Eye Research, p. 100842, 2020 Borkar et al. Support Care Cancer 21:1167-1174(2013)Yang et al. Int J Mol Med 41:1427-1436 (2018) Lin et al. Mol Vis 17:257-264(2011)Foulks GN, Bron AJ: Ocul Surf 1: 107-126, 2003.Gutgesell VJ, Stern GA, Hood CI: Am J Ophthalmol 94: 383-387, 1982.Chang J, et al. Nat Med 2016 Jan;22(1):78-83. doi: 10.1038 / nm.4010. Epub 2015 Dec 14.FASEB J. 2020 Aug;34(8):10778-10800. doi: 10.1096 / fj.201900218R. Epub 2020 Jul 3.Ogawa Y, et al. eLife, 2016 Jan 26;5:e09394. doi: 10.7554 / eLife.09394.Seligman SJ, Lancet,1998 Apr 25;351(9111):1287-8.Sethe S, Aging Res Rev, 2006 Feb;5(1):91-116. Epub 2005 Nov 28.Kawai M, Ogawa Y, et al. Sci Rep, 2013;3:2455. doi: 10.1038 / srep02455.Yaguchi S, Ogawa Y, et al. PLoS One, 2013 Jun 6;8(6):e64724. doi: 10.1371 / journal.pone.0064724. Print 2013.Mukai S, Ogawa Y, et al. Sci Rep 2017, Royal Society Open Science, 2018 Oct 17;5(10):181067. doi: 10.1098 / rsos.181067. eCollection 2018 Oct.Munoz-Espin D, Nat Rev Mol Cell Biol, 2014 Jul;15(7):482-96. doi: 10.1038 / nrm3823.Rodier F, J Cell Biol. 2011 Feb 21;192(4):547-56. doi: 10.1083 / jcb.201009094. Epub 2011 Feb 14.Ohtani N, Nature 2013 Jul 4;499(7456):97-101. doi: 10.1038 / nature12347. Epub 2013 Jun 26.Apte RS, Cell Metab. 2013 Apr 2;17(4):549-61. doi: 10.1016 / j.cmet.2013.03.009.

[0021] Therefore, an object of the present invention is to provide a novel therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft-versus-host disease, an agent for promoting corneal epithelial wound healing, an agent for suppressing inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an agent for suppressing corneal epithelial damage, an inhibitor of cell proliferation marker-positive cells, or an inhibitor of lymphocyte differentiation.

[0022] After extensive research, the present inventors have found that substances derived from stem cell culture supernatants can be used as active ingredients in therapeutic or preventive agents for dry eye, therapeutic or preventive agents for graft versus host disease, agents that promote corneal epithelial wound healing, agents that suppress inflammatory cells that infiltrate the cornea, agents that maintain tear volume, agents that suppress corneal epithelial damage, agents that suppress cells that are positive for cell proliferation markers, or agents that suppress the differentiation of lymphocytes into effector T cells.

[0023] That is, the present invention provides the following. [Aspect 1] A therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft versus host disease, an agent for promoting corneal epithelial wound healing, an agent for suppressing inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an agent for suppressing corneal epithelial damage, an inhibitor of cells positive for cell proliferation markers, or an inhibitor of lymphocyte differentiation into effector T cells, which comprises a substance derived from a stem cell culture supernatant as an active ingredient. [Aspect 2] The agent according to Aspect 1, wherein the graft versus host disease is ocular graft versus host disease. [Aspect 3] The agent according to Aspect 1, wherein the stem cells are dental pulp stem cells. [Aspect 4] The agent according to Aspect 3, wherein the dental pulp stem cells are deciduous tooth pulp stem cells. [Aspect 5] The agent according to Aspect 1, wherein the stem cells are primary culture cells. [Aspect 6] The agent according to Aspect 1, wherein the stem cells are immortalized cells. [Aspect 7] The agent according to Aspect 1, wherein the substance derived from stem cell culture supernatant is obtained by a method comprising the step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant. [Aspect 8] The agent according to Aspect 7, wherein the method comprising the step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant is a method using a dialysis membrane. [Aspect 9] The agent according to Aspect 1, wherein the substance derived from stem cell culture supernatant does not contain components with a molecular weight of less than 3.5 kDa. [Aspect 10] The agent according to Aspect 1, wherein the substance derived from stem cell culture supernatant has a protein concentration of 30 μg / mL to 100 μg / mL. [Aspect 11] The agent according to Aspect 1, wherein the substance derived from stem cell culture supernatant comprises extracellular vesicles. [Aspect 12] The agent according to Aspect 1, wherein the substance derived from stem cell culture supernatant comprises exosomes.

[0024] According to the present invention, it is possible to suppress the differentiation of lymphocytes into effector T cells, suppress cell proliferation marker-positive cells, suppress corneal epithelial damage, maintain tear volume, suppress inflammatory cells infiltrating the cornea, promote the healing of corneal epithelial wounds, treat or prevent graft-versus-host disease, and treat or prevent dry eye.

[0025] Figure 1 shows the corneal epithelial damage scores measured 28 days after radiation exposure in the control mouse group, the 3.5 kD Hi eye drop group, the 3.5 kD Lo eye drop group, and the control solution group. Figure 2 shows the tear volume measurements measured 28 days after radiation exposure in the control mouse group, the 3.5 kD Hi, 3.5 kD Lo eye drop group, and the control solution group. Figure 3 shows the number of inflammatory cells infiltrating the cornea in the control mouse group, the 3.5 kD Hi, 3.5 kD Lo eye drop group, and the control solution group 28 days after radiation exposure. Figure 4 shows the analysis of the area of ​​corneal epithelial defects in the 3.5 kD Hi, 3.5 kD Lo eye drop group, and the control solution group 72 hours after corneal epithelial abrasion. Figure 5 shows the results of a lymphocyte activation experiment using spleen-derived immune cells. Figure 5(a) shows the inhibitory effect of this drug on proliferation marker-positive CD4 T cells. FIG. 5(b) shows the inhibitory effect of this agent on the differentiation of lymphocytes into IFNγ-positive effector Th1 cells.

[0026] The present invention provides a therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft versus host disease, a promoter of corneal epithelial wound healing, an inhibitor of inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an inhibitor of corneal epithelial damage, an inhibitor of cells positive for cell proliferation markers, or an inhibitor of lymphocyte differentiation into effector T cells, each of which contains as an active ingredient a substance derived from the culture supernatant of stem cells.

[0027] In the substance derived from the stem cell culture supernatant, the stem cells are preferably dental pulp stem cells, and the dental pulp stem cells are preferably deciduous tooth pulp stem cells. Deciduous tooth pulp stem cells are, for example, mesenchymal stem cells collected from the dental pulp of a deciduous tooth. Methods for preparing deciduous tooth pulp stem cells are already known (see, for example, WO 2011 / 118795), and for example, deciduous tooth pulp stem cells can be isolated and prepared from deciduous tooth pulp according to methods known in the art.

[0028] The stem cells are preferably mesenchymal stem cells. The stem cells may be primary cultured cells or immortalized cells. Immortalized cells are, for example, cells that remain proliferative even after a certain number of divisions, e.g., cells that have the ability to proliferate indefinitely. Methods for preparing immortalized cells have already been established, and known techniques can be used, for example. For example, immortalized cells can be prepared by introducing the SV40 T antigen gene or the telomerase reverse transcriptase (TERT) gene into cells.

[0029] Examples of stem cells include stem cells derived from any vertebrate, preferably stem cells derived from mammals such as mice, rats, rabbits, pigs, cows, monkeys, and humans, and more preferably stem cells derived from humans.

[0030] In the method for preparing the stem cell culture supernatant, stem cells can be cultured, for example, according to a method known in the art (see, for example, International Publication No. 2011 / 118795). Specifically, stem cells are cultured at a concentration of, for example, 5 × 10 in a medium containing, as appropriate, a basal medium such as DMEM, IMDM, Ham's F-12, or RPMI-1640, or a mixture thereof, supplemented with serum such as FBS or a serum substitute such as KSR, glucose, amino acids, vitamins, etc. 4 ~2×10 5 Stem cells may be cultured by seeding them at a concentration of 0 to 100 cells / mL. For example, when the stem cells are primary culture cells, the stem cells are preferably passaged 0 to 15 times, and when the stem cells are immortalized cells, the stem cells are preferably passaged 30 to 100 times. The stem cell culture supernatant is preferably a culture supernatant obtained by culturing stem cells in a serum-free medium. Stem cell culture in a serum-free medium is carried out for, for example, 24 to 72 hours. The stem cell culture supernatant can be obtained, for example, by recovering the medium in which the stem cells were cultured and filtering it using a separation membrane. The separation membrane preferably has a pore size of 0.1 μm to 1 μm, more preferably 0.1 μm to 0.5 μm, and even more preferably 0.1 μm to 0.3 μm.

[0031] The substance derived from stem cell culture supernatant may be the stem cell culture supernatant itself, or may be a concentrated version of the stem cell culture supernatant. When the substance derived from stem cell culture supernatant is a concentrated version of the stem cell culture supernatant, the substance is preferably a 2- to 100-fold concentrated version of the stem cell culture supernatant, more preferably a 3- to 50-fold concentrated version of the stem cell culture supernatant, and even more preferably a 5- to 20-fold concentrated version of the stem cell culture supernatant. The stem cell culture supernatant is concentrated, for example, by ultrafiltration. The substance derived from stem cell culture supernatant is preferably a substance obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant. Here, kDa may be calculated, for example, based on SDS-PAGE or ultrafiltration. When the culture supernatant is purified using a commercially available ultrafiltration filter unit, it may be the nominal molecular weight cutoff (NMWL) or molecular weight cutoff (MWCO) value labeled on the product. A method including a step of removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant is, for example, a method using a dialysis membrane. Examples of dialysis membranes include dialysis membranes with a MWCO of 1,000 to 10,000, preferably a 3,500 MWCO. When a substance derived from stem cell culture supernatant is obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant, the substance derived from stem cell culture supernatant may be a substance obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant, which is further concentrated. Here, the concentration is preferably 2-fold to 100-fold, more preferably 3-fold to 50-fold, and even more preferably 5-fold to 20-fold. Concentration is performed, for example, by ultrafiltration.

[0032] When a substance derived from stem cell culture supernatant is obtained by a method including a step for removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant, the substance derived from stem cell culture supernatant may be a substance obtained by a method including a step for the purpose of removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant. Therefore, the fact that components with a molecular weight of less than 3.5 kDa remain after the method, for example, due to technical limitations of the method, does not negate the fact that the substance derived from stem cell culture supernatant is a substance obtained by a method including a step for removing components with a molecular weight of less than 3.5 kDa from stem cell culture supernatant. Furthermore, when a substance derived from stem cell culture supernatant is obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant, the substance may be obtained by adding, as appropriate, components with a molecular weight of less than 3.5 kDa to the substance obtained by the method including that step. Therefore, the mere detection of a component with a molecular weight of less than 3.5 kDa does not negate the fact that the substance derived from stem cell culture supernatant is a substance obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant. However, the substance derived from stem cell culture supernatant is preferably substantially free of components with a molecular weight of less than 3.5 kDa, and more preferably free of components with a molecular weight of less than 3.5 kDa.

[0033] The protein concentration in the substance derived from the stem cell culture supernatant is preferably 5 μg / mL to 1,000 μg / mL, more preferably 5 μg / mL to 500 μg / mL, even more preferably 5 μg / mL to 300 μg / mL, even more preferably 5 μg / mL to 200 μg / mL, and even more preferably 5 μg / mL to 100 μg / mL. The protein concentration in the substance derived from the stem cell culture supernatant is preferably 10 μg / mL to 1,000 μg / mL, more preferably 10 μg / mL to 500 μg / mL, even more preferably 10 μg / mL to 300 μg / mL, even more preferably 10 μg / mL to 200 μg / mL, and even more preferably 10 μg / mL to 100 μg / mL. Furthermore, the protein concentration in the substance derived from the stem cell culture supernatant is preferably 15 μg / mL to 1,000 μg / mL, more preferably 15 μg / mL to 500 μg / mL, even more preferably 15 μg / mL to 300 μg / mL, even more preferably 15 μg / mL to 200 μg / mL, and even more preferably 15 μg / mL to 100 μg / mL. The protein concentration in the substance derived from the stem cell culture supernatant is preferably 20 μg / mL to 1,000 μg / mL, more preferably 20 μg / mL to 500 μg / mL, even more preferably 20 μg / mL to 300 μg / mL, even more preferably 20 μg / mL to 200 μg / mL, and even more preferably 20 μg / mL to 100 μg / mL. Furthermore, the protein concentration in the substance derived from the stem cell culture supernatant is preferably 30 μg / mL to 1,000 μg / mL, more preferably 30 μg / mL to 500 μg / mL, even more preferably 30 μg / mL to 300 μg / mL, even more preferably 30 μg / mL to 200 μg / mL, and even more preferably 30 μg / mL to 100 μg / mL. Here, the protein concentration in the substance derived from the stem cell culture supernatant is measured using a protein assay kit.

[0034] The substance derived from stem cell culture supernatant preferably contains extracellular vesicles (EVs). Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. The molecular weight of EVs is 100 kDa or greater, and the size of EVs is approximately 50 nm to 200 nm. EVs are molecular complexes, and their components include various proteins as well as nucleic acids such as microRNA and mRNA. Therefore, even if the substance derived from stem cell culture supernatant is obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant, it still contains extracellular vesicles (EVs) such as exosomes.

[0035] As shown in the examples below, administering a substance derived from stem cell culture supernatant to a subject can inhibit the differentiation of lymphocytes into effector T cells in the subject, inhibit proliferation marker-positive cells in the subject, inhibit corneal epithelial damage in the subject, maintain tear volume in the subject, inhibit inflammatory cells infiltrating the cornea in the subject, promote corneal epithelial wound healing in the subject, treat or prevent graft versus host disease in the subject, and treat or prevent dry eye in the subject. Examples of inhibiting the differentiation of lymphocytes into effector T cells in the subject include inhibiting the differentiation of lymphocytes into effector helper T cells in the subject. Examples of inhibiting the differentiation of lymphocytes into effector helper T cells in the subject include inhibiting the differentiation of lymphocytes into IFNγ-positive effector Th1 cells in the subject. IFNγ is a cytokine that induces inflammation. Examples of inhibiting proliferation marker-positive cells in the subject include inhibiting proliferation marker-positive CD4-positive T cells in the subject. Furthermore, examples of suppressing proliferation marker-positive cells in a subject include suppressing proliferation marker-positive lymphocytes in a subject, etc. Graft versus host disease is, for example, ocular graft versus host disease.

[0036] The subject may be, for example, any vertebrate, but is preferably a mammal such as a mouse, rat, rabbit, pig, cow, monkey, or human, and more preferably a human. The subject may be of any age, including infants, juveniles, adolescents, adults, and geriatric subjects.

[0037] The therapeutic or preventive agent for dry eye, therapeutic or preventive agent for graft versus host disease, agent for promoting corneal epithelial wound healing, agent for suppressing inflammatory cells infiltrating the cornea, agent for maintaining tear volume, agent for suppressing corneal epithelial damage, agent for suppressing cell proliferation marker-positive cells, or agent for suppressing lymphocyte differentiation into effector T cells (hereinafter referred to as "agent of the present invention") of the present invention may consist of, for example, only stem cell culture supernatant itself or a fraction purified from stem cell culture supernatant, but may also contain pharmaceutically acceptable carriers and additives as optional components.

[0038] The agent of the present invention may be prepared as a liquid or solid, and may be formulated into various dosage forms such as injections, jellies, sprays, tablets, and granules, but is preferably prepared as a liquid. When prepared as a liquid, sterile water, physiological saline, an aqueous glucose solution, or the like may be used as a carrier, and if desired, a bactericide, an isotonic agent, a stabilizer, or the like may be added. When prepared as a solid, additives such as starch, lactose, mannitol, and inorganic salts may be added, and if desired, a binder, a disintegrant, a lubricant, or the like may be added.

[0039] The content of substances derived from stem cell culture supernatant in the agent of the present invention (converted to protein weight) may be, for example, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, preferably 60 to 70% by weight, when the total mass of the agent of the present invention is taken as 100%.

[0040] The agent of the present invention may be administered by any administration method appropriate for the dosage form, for example, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, nasal administration, intraperitoneal administration, direct injection into target tissue, inhalation administration, etc. The dosage of the agent of the present invention may vary depending on the age, weight, health condition, etc. of the subject, but may be, for example, 0.1 to 10 mg / kg (body weight), preferably 0.5 to 2 mg / kg (body weight). The dosage may be administered once or multiple times.

[0041] The present invention provides a method for treating or preventing dry eye, a method for treating or preventing graft versus host disease, a method for promoting corneal epithelial wound healing, a method for suppressing inflammatory cells infiltrating the cornea, a method for maintaining tear volume, a method for suppressing corneal epithelial damage, a method for suppressing proliferation marker-positive cells, or a method for suppressing lymphocyte differentiation into effector T cells in a subject, which comprises administering to the subject a substance derived from stem cell culture supernatant.The present invention also provides a pharmaceutical composition for use in a therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft versus host disease, a corneal epithelial wound healing promoter, an inhibitor of inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an inhibitor of corneal epithelial damage, an inhibitor of proliferation marker-positive cells, or an inhibitor of lymphocyte differentiation into effector T cells, which comprises a substance derived from stem cell culture supernatant as an active ingredient. Furthermore, the present invention provides use of a substance derived from a stem cell culture supernatant in the manufacture of a pharmaceutical composition for use as a therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft versus host disease, an agent for promoting corneal epithelial wound healing, an agent for suppressing inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an agent for suppressing corneal epithelial damage, an agent for suppressing cells positive for cell proliferation markers, or an agent for suppressing the differentiation of lymphocytes into effector T cells.

[0042] Materials and Reagents: NIH3T3 cells were purchased from the JCRB Cell Bank. Human umbilical vein endothelial cells (HUVECs) were purchased from EBM. TM -2 basal medium (CC-3156) and EGM TMThe α-2 supplement kit (CC-4176) was purchased from Lonza. Dulbecco's Modified Eagle Medium (DMEM) was purchased from GIBCO. Bovine Calf Serum (hereinafter referred to as "CS") and antimycotic solution (100x) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and 0.05% trypsin / EDTA solution were purchased from Invitrogen. Charcoal (powder, activated) (hereinafter referred to as "activated charcoal") was purchased from Nacalai Tesque. Dulbecco's phosphate buffer solution (PBS), RIPA buffer, and quick CBB staining solution were purchased from Fujifilm Wako Pure Chemical Industries. An ultrafiltration unit was purchased from SARTORIUS. Millex-GV low protein binding durapore (PVDF) membrane 0.22 μm was purchased from Merck Millipore. Spectra / Por TM 7 Dialysis Membrane (MWCO: 3.5 kD) was purchased from REPLIGEN. PD-10 columns were purchased from Cytiva. Micro BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 was purchased from Dojindo Laboratories. Acrylamide, protein standard markers, and Clarity Western ECL Substrate were purchased from Bio-Rad. Protease inhibitor complete (#11 697 498 001) was purchased from Roche. Anti-phospho p44 / 42 antibody (#9106) and anti-phospho Akt antibody (#9271) were purchased from Cell Signaling Technology. Anti-mouse IgG-HRP antibody (#62-6520) and anti-rabbit IgG-HRP antibody (#65-6120) were purchased from Invitrogen.

[0043] Example 1: Preparation of deciduous tooth pulp-derived stem cell culture supernatant: (1) Preparation of human deciduous tooth pulp-derived stem cells (SHED). After detached or extracted human deciduous teeth were disinfected with 5% chlorhexidine solution or 10% povidone-iodine solution, the crowns were divided, and dental pulp tissue was collected using a dental reamer. The dental pulp tissue was suspended in DMEM containing 10% FBS by volume, and 2 mg / ml collagenase and dispase were added. The mixture was incubated at 37°C for 1 hour. The supernatant was removed and dental pulp cells were collected by centrifugation at 777 × g for 5 minutes. Dental pulp cells were suspended in DMEM (4 mL) supplemented with 10% FBS by volume and an antifungal agent and seeded onto a 6-well plate. The cells were cultured at 37°C under 5% CO2 conditions until subconfluent. After adding 0.05% trypsin / EDTA solution, the cells were incubated at 37°C for 5 minutes, and then collected and seeded onto a 10 cm dish for adherent cells (Violamo). After that, they were subcultured three times, resulting in approximately 1 × 10 7 The cells were grown to a density of 1000 cells / mL. 0.05% trypsin / EDTA solution was added, and the cells were incubated at 37°C for 5 minutes. The collected cells were named "SHED (Stem cells from Human Exfoliated Deciduous Teeth)."

[0044] (2) Preparation of immortalized human deciduous dental pulp-derived stem cells (IM-SHED) Immortalized SHED (IM-SHED) was prepared by introducing the SV40 gene into the stem cells using a viral vector.

[0045] (3) Preparation of culture supernatant. SHED or IM-SHED prepared above were subcultured in DMEM containing 10% FBS by volume. Culture supernatants were prepared using SHED cultures that had been subcultured up to 8 times, and IM-SHED cultures that had been subcultured 50–55 times. The medium was replaced with serum-free DMEM and cultured for 48 hours. The medium was then collected and filtered through a separation membrane (Stericup Quick Release-GP, PVDF, 0.22 μm; Merck Millipore) to obtain the culture supernatant (conditioned medium: CM). Hereafter, the culture supernatant derived from SHED is referred to as "SHED-CM," and the culture supernatant derived from IM-SHED is referred to as "IM-SHED-CM."

[0046] [Example 2] Preparation of test solution: IM-SHED-CM (protein concentration: 107 μg / mL) was dissolved in Spectra / Por TM The solution was sealed in a dialysis membrane (MWCO: 3.5 kDa) and dialyzed against 100 times the volume of Milli-Q water for 72 hours. The Milli-Q water was exchanged eight times during this period. The dialyzed solution was lyophilized, dissolved in 1 / 10 the starting volume of PBS (on ice for >30 minutes), and filtered through a 0.22 μm PVDF filter. This resulted in a fraction of ≥3.5 kDa. In this manner, a fractionated extract with a molecular weight of less than 3.5 kDa was prepared using the culture supernatant of immortalized deciduous tooth-derived dental pulp stem cells (IM-SHED-CM). A 10x concentrated extract (protein concentration: 427 μg / mL) (3.5 kD Hi) and a concentration equivalent to the original solution (protein concentration: 42.7 μg / mL) (3.5 kD Lo) of the fractionated extract with a molecular weight of less than 3.5 kDa were administered intraocularly to the following animal models. Protein concentration was measured using a protein assay kit. The extract was diluted with PBS. PBS was also instilled as a control solution.

[0047] Creation of a chronic graft-versus-host disease model animal: 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 control mice, female mice (BALB / c) that had received allogeneic spleen and bone marrow cell transplants were used. 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 6Spleen 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. Model mice were housed in a specific pathogen-free (SPF) environment with sterilized water and food for 7 days, after which drug administration began. The above 3.5 kD Hi and 3.5 kD Lo eye drops were administered twice daily for 21 consecutive days, at a dose of one drop (approximately 5 μL). A control solution (PBS) was administered in the same manner for comparison. Twenty-eight days after irradiation, the condition of the corneal epithelium was observed using corneal fluorescein staining, and the mice were sacrificed for tissue collection.

[0048] Observation of wound healing following corneal epithelial abrasion in mice: The corneal epithelium of female mice (BALB / c, 12 weeks old; Sankyo Research Institute) was abraded over a 3 mm diameter area, and the above-mentioned 3.5 kDa Lo ophthalmic solution was administered in a single drop (approximately 5 μL) three times daily for three consecutive days. For comparison, a control solution (PBS) was administered in the same manner. The state of the corneal epithelium was observed using corneal fluorescein staining, and the process of corneal epithelial wound healing was observed.

[0049] The corneal epithelial damage score was measured 28 days after radiation exposure in the control mouse group, the 3.5 kD Hi eye drop group, the 3.5 kD Lo eye drop group, and the control solution administration group. The results are shown in Figure 1. The ocular surface was stained with fluorescein, and the stained image was scored on a four-point scale. The corneal epithelial damage score was calculated as the mean ± standard error of the total scores for the four corneal sites: the nasal dorsal side, nasal ventral side, temporal dorsal side, and temporal ventral side. The corneal epithelial damage scores were 5.5±0.77 in the 3.5kD Hi group, 5.7±0.54 in the 3.5kD Lo group, and 9.5±1.5 in the control solution group. The 3.5kD Hi and 3.5kD Lo groups had significantly better corneal epithelial damage scores than the control solution group (p = 0.015, 0.032, n = 3-5 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P<0.05, **P<0.01, ***P<0.001). The scoring criteria are as follows: 0: No staining; 1: Mild punctate corneal epithelial damage (fewer than 30); 2: Diffuse punctate corneal epithelial damage; 3: Corneal plaque formation.

[0050] The tear volume measurements for the control mouse group, 3.5 kD Hi, 3.5 kD Lo eye drops, and control solution groups 28 days after irradiation are shown in Figure 2. A phenol red thread was inserted into the outer canthus of the eye for 15 seconds, the length of the stained area was measured, and the mean ± standard error was used as the tear volume for each group. The corneal epithelial damage score was 1.56 ± 0.20 in the 3.5kD Hi group, 2.19 ± 0.73 in the 3.5kD Lo group, and 0.55 ± 0.23 in the control solution group. The 3.5kD Hi group tended to have a better score than the control solution group (p = 0.82, n = 3-5 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). The 3.5kD Lo group had a significantly better corneal epithelial damage score than the control solution group (p = 0.023, n = 3-5 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

[0051] Figure 3 shows the results of measuring the number of inflammatory cells infiltrating the cornea in the control mouse group, and the groups treated with 3.5kD Hi, 3.5kD Lo eye drops, and control solution 28 days after radiation exposure. The inflammatory cell marker CD45 was immunohistochemically labeled using the entire cornea, and the CD45-positive area relative to the entire cornea was analyzed using the image analysis software ImageJ. The CD45-positive area was calculated as the mean ± standard error for each group. The corneal epithelial damage score was 2.0±0.54% in the 3.5kD Hi group, 1.63±0.41% in the 3.5kD Lo group, and 3.9±0.60% in the control solution group. The 3.5kD Hi group tended to have a better result than the control solution group (p = 0.087, n = 3-5 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P<0.05, **P<0.01, ***P<0.001). The 3.5kD Lo group had a significantly better corneal epithelial damage score than the control solution group (p = 0.046, n = 3-5 per group, one-way analysis of variance (ANOVA) with Tukey-Kramer's post hoc test, *P<0.05, **P<0.01, ***P<0.001).

[0052] Figure 4 shows the results of an analysis of the area of ​​corneal epithelial defects in mice treated with 3.5kD Lo ophthalmic solution and the control solution 72 hours after corneal epithelial abrasion. The ocular surface was stained with fluorescein, and the percentage of corneal epithelial defect area relative to the entire cornea was analyzed using the image analysis software ImageJ. The mean ± standard error of the mean for each group was calculated. The area of ​​corneal epithelial defect was 1.21% ± 0.39% in the 3.5kD Lo group and 22.7% ± 5.5% in the control solution group, demonstrating a significant reduction in the area of ​​corneal epithelial defect in the 3.5kD Lo group (p = 0.015, n = 5 per group, unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

[0053] In summary, in chronic GVHD model mice, IM-SHED-CM extract was shown to have inhibitory effects on corneal epithelial damage, tear volume maintenance, inhibitory effects on inflammatory cells infiltrating the cornea, and promotion of corneal epithelial wound healing, suggesting that it has inhibitory effects on ocular GVHD and dry eye.

[0054] [Example 3] Lymphocyte activation experiment using spleen-derived immune cells: 6 x 10 6 Immune cells from each spleen were collected and lymphocytes were activated with anti-CD3 and anti-CD28 antibodies. PBS and 3.5 kDa Lo solution were added to the medium and cultured for 3 days. The effects of each on lymphocyte proliferation and differentiation into effector helper T cells were examined, and the results are shown in Figure 5(a) and (b), respectively.

[0055] Analysis of cell proliferation marker Ki67-positive CD4-positive T cells using flow cytometry showed that the Ki67-positive CD4-positive cells in the group to which half the total volume of 3.5 kD Lo solution was added to the medium were 3.83%.

[0056] On the other hand, the Ki67-positive CD4-positive cells in the control group (the group to which half the volume of PBS was added to the medium) was 6.37%, and the proliferation of CD4-positive T cells was suppressed in the 3.5kD Lo group (Figure 5(a)).

[0057] The percentage of IFNγ-positive CD4-positive Th1 cells in the group in which half the total volume of 3.5 kD Lo solution was added to the medium was 0.09%, while the percentage of IFNγ-positive CD4-positive Th1 cells in the control group (group in which half the volume of PBS was added to the medium) was 0.26%, indicating that differentiation of lymphocytes into Th1 cells was suppressed in the 3.5 kD Lo group (Figure 5(b)).

[0058] The compound of the present invention exhibits an effect of suppressing corneal epithelial damage, an effect of maintaining tear volume, an effect of suppressing inflammatory cells infiltrating the cornea, and an effect of promoting corneal epithelial wound healing, and is useful as a new agent for treating ocular GVHD and dry eye, and therefore has industrial applicability.

Claims

1. A therapeutic or preventive agent for dry eye, a therapeutic or preventive agent for graft-to-host disease, a promoter of corneal epithelial wound healing, an inhibitor of inflammatory cells infiltrating the cornea, an agent for maintaining tear volume, an inhibitor of corneal epithelial damage, an inhibitor of cells positive for cell proliferation markers, or an inhibitor of lymphocyte differentiation into effector T cells, which contains a substance derived from the culture supernatant of stem cells as an active ingredient.

2. The agent according to claim 1, wherein the graft-versus-host disease is ocular graft-versus-host disease.

3. The agent according to claim 1, wherein the stem cells are dental pulp stem cells.

4. The agent according to claim 3, wherein the dental pulp stem cells are deciduous tooth pulp stem cells.

5. The agent according to claim 1, wherein the stem cells are primary cultured cells.

6. The agent according to claim 1, wherein the stem cells are immortalized cells.

7. The agent described in claim 1, wherein the substance derived from stem cell culture supernatant is obtained by a method including a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant.

8. The agent described in claim 7, wherein the method comprising a step of removing components with a molecular weight of less than 3.5 kDa from the stem cell culture supernatant is a method using a dialysis membrane.

9. The agent described in claim 1, wherein the substance derived from the stem cell culture supernatant does not contain any components with a molecular weight of less than 3.5 kDa.

10. The agent according to claim 1, wherein the protein concentration of the substance derived from the stem cell culture supernatant is 30 μg / mL to 100 μg / mL.

11. The agent described in claim 1, wherein the substance derived from the stem cell culture supernatant comprises extracellular vesicles.

12. The agent described in claim 1, wherein the substance derived from the stem cell culture supernatant contains exosomes.

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