JP7279890B2 - Graft-versus-host disease therapeutic or prophylactic agent - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Publication name Cornea Conference 2018 (42nd Annual Meeting of the Japanese Corneal Society / 34th Annual Meeting of the Japanese Cornea Transplantation Society) Program/Abstract Collection Page 60 Japanese Corneal Society, Japanese Cornea Transplantation Issued by the Society Date of distribution: January 18, 2018 ▲2 ▼Meeting name, Venue Corneal Conference 2018 (42nd Annual Meeting of the Japanese Corneal Society/34th Annual Meeting of the Japanese Cornea Transplantation Society) 1st Venue General Performance 10 Grand Prince Hotel Hiroshima Date: February 17, 2018 ▲3▼ Name of publication Journal of the Japanese Ophthalmological Society Vol. June 10 ▲ 4 ▼ Publication name Cornea Conference 2018 (42nd Annual Meeting of the Japanese Corneal Society / 34th Annual Meeting of the Japanese Cornea Transplantation Society) Proceedings of the Conference Published by Otsuka Pharmaceutical Co., Ltd. Date of distribution: July 3, 2018 ( Created June 2018)

TECHNICAL FIELD The present invention relates to therapeutic or prophylactic agents for graft-versus-host disease.

Hematopoietic stem cell transplantation is performed as a therapeutic method for eradicating leukemia by transplanting other's healthy hematopoietic stem cells for hematological malignancies caused by abnormal hematopoietic stem cells. On the other hand, in recent years, graft-versus-host disease develops when bone marrow cells, peripheral blood or umbilical cord blood grafts provided by donors attack target organs of recipients by immune response. hereinafter sometimes referred to as GVHD) is a problem.

So far, using a mouse model that shows a pathology that closely resembles human chronic GVHD, we have found that fresh donor bone marrow mesenchymal stem cells are immunogenic, engrafted in the exocrine glands and mucosa of GVHD, and are involved in the onset of the disease. has been reported (Non-Patent Document 1). In addition, it has been reported that bone marrow-transplanted hematopoietic stem cells undergo telomere shortening after transplantation, resulting in stem cell senescence (Non-Patent Document 2). There is also a report that mesenchymal stem cells also cause stem cell senescence (Non-Patent Document 3).

In addition, it is known that infiltration of senescent macrophages is observed in the lacrimal gland at the early stage of onset of GVHD model mice (Non-Patent Document 4). In addition, it has been shown that the local tissue renin-angiotensin system (RAS) is involved in immune fibrosis of GVHD lacrimal glands and other target organs, and its inhibitors alleviate the pathology (Non-Patent Document 5). RAS is believed to induce mitochondrial dysfunction associated with the progression of aging, and it has been suggested that aspects of age-related changes are involved in the pathogenesis of GVHD. Furthermore, it was found that endoplasmic reticulum stress associated with aging is excessively enhanced in chronic GVHD target organs, and administration of the endoplasmic reticulum stress inhibitor phenylbutyric acid suppresses inflammation and fibrosis in target organs. reported (Non-Patent Document 6). These reports suggest that age-related changes are involved in the pathogenesis of GVHD.

After hematopoietic stem cell transplantation, senescent cells accumulate due to excessive stress such as radiation exposure and immune dysfunction (Non-Patent Document 7).
In recent years, senescence associated secretory phenotype (SASP) has been reported as a mechanism by which senescent cells continuously release humoral factors to promote the activation of adjacent cells and lead to chronic inflammation. (Non-Patent Documents 8 and 9). It has also been reported that senescent cells are actively involved in pathogenesis of age-related diseases such as macular degeneration (Non-Patent Document 10).

Immunosuppressants are known as therapeutic agents for chronic GVHD. Although immunosuppressants are partially effective, there are many intractable cases.

As therapeutic agents for GVHD, for example, rebamipide and diquafosol are known for treatment of the ocular surface, and it is disclosed that dry eye, which is one of the symptoms of GVHD, can be improved by using rebamipide and diquafosol in combination. ing. However, although rebamipide and diquafosol can be used to treat mild and moderate GVHD patients, they cannot be used to treat severe GVHD patients.
In addition, systemic treatment with immunosuppressive drugs such as cyclosporine and tacrolimus and steroids is performed. However, the effect of treatment with the above drugs is still insufficient, and the actual situation is that there is no effective therapeutic drug for GVHD.

ABT-263, one of the senolytic agents, is a drug that targets the apoptotic Bcl-2 family. (Non-Patent Document 11), but its effect on GVHD is unknown.

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. Chang J, et al. Nat Med 2016 Jan;22(1):78-83. doi: 10.1038/nm.4010. Epub 2015 Dec 14.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel therapeutic or preventive agent for GVHD that can be used for treatment of GVHD patients.

The present invention includes the following aspects.
[1] A therapeutic or preventive agent for graft-versus-host disease, containing a senolytic agent as an active ingredient.
[2] The therapeutic or preventive agent according to [1], wherein the senolytic agent is ABT-263 represented by the following formula (I).

[3] The therapeutic or prophylactic agent of [1] or [2], wherein the graft-versus-host disease is post-bone marrow transplantation graft-versus-host disease.
[4] The therapeutic or prophylactic agent according to any one of [1] to [3], wherein the graft-versus-host disease is ocular graft-versus-host disease.
[5] The therapeutic or prophylactic agent of [4], wherein the graft-versus-host disease is dry eye.
[6] The therapeutic or prophylactic agent according to any one of [1] to [5], wherein the graft-versus-host disease is accompanied by inflammation and pathological fibrosis.

INDUSTRIAL APPLICABILITY According to the present invention, a novel therapeutic or preventive agent for GVHD that can be used for treatment or prevention of GVHD patients can be provided.

FIG. 1 shows whole-body imaging in chronic GVHD (hereinafter also referred to as cGVHD) mice and control mice. FIG. 2 shows images of hematoxin-eosin staining (HE staining) and Maloney staining (Mallory staining) in the lacrimal glands of cGVHD model mice and control mice. FIG. 2 shows images of lacrimal glands of cGVHD model mice and control mice observed with an electron microscope. FIG. 10 is a graph showing post-bone marrow transplantation levels of IL-6 in the serum of cGVHD model mice and control mice. γ-H2Ax and 53BP1, which are DNA damage markers, and p16, which is a senescence-related marker, in formalin-fixed, paraffin-embedded tissue sections of the lacrimal glands of cGVHD model mice and control mice. It is a graph showing the number of cells. Macrophage marker CD68, SASP major factor IL-6, CXCL1, and CXCL9 in formalin-fixed, paraffin-embedded tissue sections of lacrimal glands of cGVHD model mice and control mice. It is a graph showing the number of cells. Fig. 10 is an image of immunostaining of senescence-related marker p16, cell proliferation marker Ki-67, and macrophage marker CD68-positive cells in formalin-fixed, paraffin-embedded tissue sections of lacrimal glands of cGVHD model mice. Fig. 3 shows images of immunostaining of fibrosis marker HSP47-positive cells in formalin-fixed, paraffin-embedded tissue sections of lacrimal glands of cGVHD model mice and control mice, and a graph showing the number of each positive cell. FIG. 10 is a graph showing the appearance and fluorescein-stained images of the eyes of senolytic agent ABT-263-administered mice and control mice, and the tear volume in each mouse 4 weeks after bone marrow transplantation. In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice. 1 is a graph showing observed images of immunostaining of lacrimal glands of senolytic agent ABT-263-administered mice and control mice, and fibrosis area per unit area. In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice. Fig. 2 shows electron micrographs of lacrimal glands of senolytic agent ABT-263-administered mice and control mice, and a graph showing the number of inflammatory cells per unit area. In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice. 1 is a graph showing the expression of p16 gene and CXCL9 gene in lacrimal glands of senolytic agent ABT-263-administered mice and control mice. In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice. Images of immunostaining of cells positive for macrophage marker CD68, DNA damage marker 53BP1, senescence-related markers p16 and p21 in lacrimal glands of senolytic agent ABT-263-administered mice and control mice, and the number of each positive cell per unit area. is a graph showing In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice. FIG. 4 is a graph showing immunostaining images of CXCL9-positive cells, which are major factors of SASP in the lacrimal gland of senolytic agent ABT-263-administered mice and control mice, and the number of CXCL9-positive cells per unit area. In the figure, Vehicle indicates the results of control mice, and ABT-263 indicates the results of ABT-263-administered mice.

[Senescent cell removing agent]
In the present invention, the senolytic agent is not particularly limited as long as it is a substance that can specifically remove senescent cells, and examples thereof include substances that specifically induce cell death in senescent cells. Specific senolytic agents include, for example, ABT-263, FOXO4-DRI, Pipelongamine, A1331852, A1155436, Fisetin, a combination drug of dasatinib and quercetin, etc. ABT-263 is preferred. ABT-263 is a compound represented by the following formula (I). These senolytic agents may be used alone or in combination.

Regarding the fact that senolytic agents can remove senescent cells, for example, expression of markers such as senescence markers p16, p53, p21, DNA damage marker BP53, SASP-related molecules IL-6, CXCL9 is reduced, osteopontin It can be confirmed by a decrease in CD68-positive senescent macrophages that are double-positive with the positive cells and the above markers, a decrease in senescence marker CD153-positive T cells, and the like.

Senescent cells can be identified by DNA damage, expression of senescence-related markers, irreversible arrest of cell proliferation, increased expression of SASP-related molecules, and the like. DNA damage can be confirmed by the expression of DNA damage markers such as γ-H2Ax and 53BP1. Senescence-related markers include p16, p21, and the like. Irreversible arrest of cell proliferation can be confirmed by expression of cell proliferation marker Ki-67 or the like. SASP-related molecules include inflammasome factor Caspase-1, SASP-induced upstream factors IL-1β, IL-8, SASP major factors CXCL1, IL-6, CXCL9, and the like.

(GVHD)
The type of GVHD in the present invention is not particularly limited, and examples thereof include those manifesting symptoms in organs having exocrine glands such as eyes, oral cavity, liver, gastrointestinal tract, skin, and lungs. GVHD in the present invention includes those accompanied by inflammation and pathological fibrosis.
Among these, the therapeutic or prophylactic agent of the present invention is preferably used for GVHD in eyes.

Examples of GVHD in the eye include, but are not limited to, conjunctival fibrosis associated with dry eye, such as dry eye, conjunctivitis sicca, and corneal injury. Among these, dry eye is preferred as the eye GVHD disease to be treated or prevented, because it has particularly excellent therapeutic or preventive effects.

Examples of GVHD in the oral cavity include, but are not limited to, xerostomia and trismus caused by hardened skin.

Examples of GVHD in the liver include, but are not limited to, acute hepatitis, liver cirrhosis, and the like.

GVHD in the gastrointestinal tract includes, but is not limited to, diarrhea, anorexia, vomiting, upper gastrointestinal stricture and restriction, and the like.

GVHD in the skin includes, but is not limited to, dry skin disease, scleroderma, and the like.

Examples of GVHD in the lung include, but are not limited to, interstitial pneumonia, obstructive pneumonia, and the like.

Organs to be transplanted that cause GVHD are not particularly limited, and examples thereof include bone marrow, blood (blood transfusion), and the like. In GVHD, it has been pointed out that, particularly after bone marrow transplantation, bone marrow-derived hematopoietic stem cells differentiate into fibroblasts, which are the cause of GVHD, and infiltrate into exocrine tissue via the blood. . However, since the therapeutic or prophylactic agent of the present invention is excellent in the effect of suppressing fibroblast infiltration, it exhibits a particularly excellent therapeutic or prophylactic effect on such GVHD after bone marrow transplantation. For this reason, the GVHD targeted by the therapeutic or prophylactic agent of the present invention is preferably GVHD after bone marrow transplantation.

The GVHD may be chronic GVHD or acute GVHD. Chronic GVHD usually refers to GVHD that develops after a certain period of time has passed after transplantation. For example, in the classical classification, it refers to GVHD that develops after 100 days after transplantation. Acute GVHD refers to GVHD that develops within a certain period of time after transplantation (within a period shorter than the period until chronic GVHD develops), for example, refers to GVHD that develops classically within 100 days after transplantation. . The current classification does not classify according to time, but distinguishes between acute and chronic according to typical and characteristic findings. The therapeutic or prophylactic agent for GVHD of the present invention can be suitably used particularly for chronic GVHD. The reason for this is presumed to be that it is effective in suppressing fibrosis.

The therapeutic or prophylactic agent of the present invention can be applied to any of mild, moderate or severe GVHD, and is particularly suitable for treating or preventing severe GVHD. The reason is that fibrosis is prominent in severe cases of dry eye. Severe GVHD usually refers to ocular GVHD in which lacrimal epithelial cells, keratoconjunctival mucin-secreting epithelial cells, and meibomian gland epithelial cells, which are the cell sources of tear secretion, are severely damaged.

[Evaluation of therapeutic or preventive effect of GVHD]
GVHD therapeutic or preventive effects can be evaluated using GVHD model animals. Methods for producing GVHD model animals are known, see Sci Rep, 2013;3:2455. doi: 10.1038/srep02455. It can be produced by the method described in et al. For example, when preparing a mouse GVHD model animal, male B10. D2 (H-2d) mouse bone marrow can be generated by transplanting into female BALB/c (H-2d) mice.
For the therapeutic or preventive effect of GVHD, for example, using the GVHD model mouse, after bone marrow transplantation, a group administered with a test drug is compared with a group not administered with the test drug, and it is confirmed whether the pathology of GVHD is suppressed. can be evaluated by
Suppression of the pathology of GVHD is achieved, for example, by suppressing the infiltration of inflammatory cells such as lymphocytes in the lacrimal gland of GVHD model mice, decreasing the area occupied by collagen fibers per unit area, and interleukin (IL)-1 and IL as inflammatory markers. -6, IL-17, NF-kB, type 1 collagen as an index of fibrosis, type 3 collagen, connective tissue growth factor, decreased gene expression of heat shock protein 47, serum IL-6, IL-17 It can be evaluated by the decrease of

The therapeutic or preventive effect of GVHD can also be evaluated by confirming whether the expression of aging-related molecules is suppressed in various tissues.
For example, suppression of protein expression or gene expression of p16, p21, p53, BP53, IL-6, CXCL9, and osteopontin can be evaluated by confirming target organ tissue using immunohistochemical staining and quantitative PCR. .

[Pharmaceutical composition]
The therapeutic or prophylactic agent for GVHD described above may be administered as such, or may be administered as a pharmaceutical composition mixed with a pharmaceutically acceptable carrier.

The pharmaceutical composition may be formulated into dosage forms for oral use such as tablets, capsules, elixirs and microcapsules, and parenterally such as injections, ointments and patches. It may be formulated into the dosage form used.

Examples of pharmaceutically acceptable carriers include solvents such as sterile water and physiological saline; binders such as gelatin, cornstarch, tragacanth gum and gum arabic; excipients such as crystalline cellulose; cornstarch, gelatin, alginic acid and the like. and the like.

The pharmaceutical composition may contain additives. Additives include lubricating agents such as magnesium stearate; sweeteners such as sucrose, lactose and saccharin; flavoring agents such as peppermint and red leaf oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphate and sodium acetate. agents; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives; surfactants;

The pharmaceutical composition can be formulated by appropriately combining the above carriers and excipients and admixing them in a unit dose form generally required for pharmaceutical practice.

When the pharmaceutical composition is an injection, examples of solvents for injection include isotonic solutions containing adjuvants such as physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. be done. Solvents for injections may contain alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; nonionic surfactants such as polysorbate 80 (trademark) and HCO-50;

The administration of a therapeutic or prophylactic agent for GVHD to a patient is not particularly limited, and can be appropriately selected according to the target organ in which symptoms appear. In addition to subcutaneous injection, intranasal, transbronchial, intramuscular, percutaneous, or oral administration can be performed by methods known to those skilled in the art. In addition, when the target organ that develops symptoms is the eye, administration may be performed by subconjunctival injection or instillation.

The dose of the therapeutic or prophylactic agent for GVHD varies depending on the symptoms, but in the case of oral administration, it is generally 0.1 to 100 mg, for example 1 to 100 mg per day for an adult (with a body weight of 60 kg). 50 mg, such as 1 to 20 mg.

In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, and administration method. For example, about 0.01 to 30 mg, eg 0.1 to 20 mg, eg 0.1 to 10 mg may be administered by intravenous injection or local injection.

Next, the present invention will be described in more detail by showing experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental example 1]
Using a cGVHD model mouse model, whether chronic graft-versus-host disease (cGVHD) immune response is associated with SASP was confirmed as follows.
To create cGVHD model mice, allogeneic hematopoietic stem cell transplantation was performed to 7-9 week old male B10/D2 mice as donors and female BALB/c mice as recipients. This transplantation is MHC-matched, minor histocompatibility antigen (MiHC)-mismatched hematopoietic stem cell transplantation. As a control, a non-cGVHD model was also generated in which allogeneic hematopoietic stem cell transplantation was performed (both donor and recipient BALB/c mice). As a transplantation method, the recipient was irradiated with 7 Gy, then 1×10 ⁶ bone marrow cells and 2×10 ⁶ spleen cells were dissolved in 0.1 mL of RPMI solution, and a total of 0.2 mL was passed through. It was administered via the tail vein. These transplantations were performed in an SPF (specific pathogen free) environment, and all experimental animals were similarly given sterilized water and food. cGVHD model mice and non-cGVHD model mice were analyzed 3, 4 and 8 weeks after transplantation, respectively.

cGVHD model mice (allogeneic hematopoietic stem cell transplantation) were treated with ABT-263 administration group and control solution [10% ethanol, 30% polyethylene 994 glycol 400 (HR2-603, manufactured by Hampton Research), 60% Phosal50PG995 (H. Holstein company)] administration group. ABT-263 was adjusted to a concentration of 50 mg/kg and orally administered for 7 consecutive days starting 10 days after hematopoietic stem cell transplantation, and analyzed 28 days after transplantation.

To detect the myeloperoxidase activity of activated phagocytic cells, i.e. macrophages in vivo, we performed in vivo imaging using an electron multiplying CCD camera (manufactured by Hamamatsu Photonics). Mice were intraperitoneally injected with 200 mg/kg XenoLight Rediject Inflammation Probe (manufactured by PerkinElmer) 10 minutes before the start of photon recording, and imaging was performed in a dark room. Imaging was performed 35 days after bone marrow transplantation.

Next, tissue sections of lacrimal glands of GVHD model mice were fixed with formalin 4% paraformaldehyde and embedded with paraffin wax. Sections of this paraffin-embedded tissue were stained with hematoxylin and eosin staining (HE staining) and Mallory staining. Lacrimal gland sections subjected to hematoxylin and eosin staining and Mallory staining were observed under a microscope. Staining patterns were semi-quantitatively graded according to labeling intensity and segmentation.

Specifically, using a Nikon Coolscope II (manufactured by Nikon Corporation) as a microscope, four or more randomly selected fields were captured at 200X magnification for each section, and Image J, a computerized image analysis system, was used. manufactured by NIH (National Institutes of Health)]. Observation by staining was performed by preparing a tissue section from the tissue collected 28 days after the bone marrow transplantation and analyzing the histological image after Mallory staining.

As a result, in the GVHD model mouse, inflammatory cells infiltrated into the site corresponding to the target organ of the mouse compared with the control mouse (Fig. 1A). In addition, inflammatory cell infiltration and severe fibrosis were formed, which closely resembled human GVHD lacrimal glands (Fig. 1B). Electron microscopy showed an image of interaction between macrophages infiltrating the stroma and activated fibroblasts (Fig. 1C). In FIG. 1B, “Du” indicates ducts, “I” indicates inflammatory cells, and “F” indicates fibrosis. In FIG. 1C, “Ac” indicates acinus, “mt” indicates mitochondria, Macrophages, 'f' indicate fibroblasts, respectively.

In contrast, control mice showed poorly activated fibroblasts (Fig. 1C). In addition, it was confirmed that IL-6, a major molecule of senescence-associated secretory trait (SASP), was significantly elevated in cGVHD model mice in serum over time compared to control mice (Fig. 1D). . These findings are a good reflection of human GVHD.

[Experimental example 2]
Senescent cells are characterized by DNA damage, expression of senescence-associated markers, irreversible arrest of cell proliferation, and elevated expression of key SASP factors. Therefore, formalin-fixed, paraffin-embedded tissue sections of the lacrimal glands of cGVHD model mice and control mice were treated with γ-H2Ax (manufactured by Cell Signaling Technology), 53BP1 (manufactured by Santa Cruz Biotechnology), which are DNA damage markers, as primary antibodies. Aging-related marker p16 (manufactured by Santa Cruz Biotechnology), cell proliferation marker Ki-67 (manufactured by Abcam), SASP major factor CXCL1 (manufactured by Abcam), IL-6 (manufactured by Abcam), CXCL9 (manufactured by Abcam), Immunostaining was performed using macrophage marker CD68 (manufactured by Bio-Rad Laboratories) and fibrosis marker HSP47 (manufactured by Stressgen Biotechnologies).
The results are shown in FIGS. As shown in FIGS. 2-5, DNA damage markers γ-H2Ax, 53BP1, senescence-associated marker p16, cell proliferation marker Ki-67, SASP major factors CXCL1, IL-6, and CXCL9 were significantly higher in GVHD lacrimal glands than in control mice. was observed to increase the expression of
Increased expression of macrophage marker CD68 and fibrosis marker HSP47 as indicators of inflammatory cells was observed in GVHD lacrimal glands compared to control mice.
The above findings suggest that expression of aging-related molecules is increased in the lacrimal gland, which is a GVHD target organ, and that macrophages produce one of SASP major factors.

[Experimental example 3]
Allogeneic bone marrow transplantation recipient mice were divided into two groups, one group receiving the senolytic agent ABT-263 (manufactured by Navioclax) and the other group receiving control solution alone. ABT-263 was orally administered once a day for seven days from day 10 to day 16 after bone marrow transplantation (hereinafter abbreviated as BMT). There was no difference in body weight between the two groups.

To assess lacrimal gland function in cGVHD, tear volume was measured. Tear volume measurements were made by temporarily placing a phenol red thread on the lower eyelid margin for 15 seconds and measuring the length of the wet thread from the end of the thread. The tear volume was measured in both eyes of each mouse, and each average value was used as the measured value.

As a result, lacrimal secretion was significantly increased in ABT-263-administered mice compared with control mice 4 weeks after BMT (FIG. 6, right panel).

Four weeks after transplantation, 1 μl of 0.5% fluorescein sodium solution (Fluorescite, Novartis Pharma) was added to the conjunctival sac of the recipient mouse without anesthesia. The ocular surface of the mouse was observed with a microscope (SZ61, manufactured by Olympus) equipped with cobalt blue light. The results are shown in the left diagram of FIG. Compared to control mice, ABT-263-treated mice had less blepharitis and diffuse corneal epithelialitis.

In addition, as shown in the left panel of FIG. 7, pathological changes such as inflammatory cell infiltration in cGVHD lacrimal glands and skin were less observed in ABT-263-administered mice than in control mice. As shown in the right panel of FIG. 7, the fibrotic area per unit area in the lacrimal tissue section was significantly smaller in ABT-263-administered mice than in control mice.

As shown in FIG. 8, transmission electron microscope images show that inflammatory cell infiltration and collagen fiber bundles are reduced in the interstitium in the lacrimal glands of ABT-263-administered mice (arrows in the figure). Mitochondria in the tuft epithelium were destroyed in the control mice (asterisks in the figure), but maintained their structure. This result suggests that ABT-263, a senolytic agent, is effective in suppressing pathologic effects in cGVHD lacrimal glands.

[Experimental example 4]
Total RNA of the extraorbital lacrimal gland was extracted using miRNeasy mini kit (manufactured by Qiagen) according to the protocol. Complementary DNA was synthesized using ReverTra Ace qPCR RT Kit (FSQ-101, manufactured by Toyobo). Real-time qPCR was performed using the StepOnePlus system (manufactured by Thermo Fisher Scientific). The housekeeping gene GAPDH was used as an endogenous control to examine the mRNA expression of the samples.
As a result, the expression of Cdkn2a (p16) gene and Cxcl9 gene was significantly suppressed in the lacrimal gland of ABT-263-administered mice as compared with control mice (Fig. 9).

In addition, as shown in FIG. 10, the number of cells positive for each of the DNA damage marker 53BP1, the senescence-related markers p16 and p21, and the macrophage marker CD68 in the lacrimal gland stroma per unit area was determined by ABT-263. It was significantly reduced in treated mice compared to control mice. These results suggest that removal of senescent cells by senolytic agents suppresses lacrimal pathology in cGVHD.

In addition, CXCL9-positive sites in the vascular lumen of GVHD lacrimal glands were significantly suppressed in ABT-263-administered mice compared to control mice (FIG. 11, left). This is probably because CXCL9 attracts macrophages and T cells to cGVHD lesions.
Furthermore, the area of CXCL9 in the vascular lumen per unit area of the lacrimal gland analyzed using Image J analysis software was significantly decreased in ABT-263-administered mice compared to control mice (Fig. 11, right). figure).
From the above, senescent macrophages produce IL-6 and CXCL9, CXCL9 attracts adjacent T cells to the microenvironment of target organs, and IL-6 and CXCL9 individually or cooperatively cause injury due to various stresses. It is thought that it promotes the proliferation of diseased fibroblasts. Senolytic agents are thought to eliminate pathological fibroblasts and suppress the pathology of GVHD by eliminating the senescent macrophages and senescent T cells.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel therapeutic or preventive agent for graft-versus-host disease that can be used for the treatment or prevention of GVHD patients.

Claims (4)

A therapeutic or preventive agent for ocular graft-versus-host disease, comprising a senolytic agent as an active ingredient ,
The therapeutic or preventive agent , wherein the senolytic agent is ABT-263 represented by the following formula (I) .

The therapeutic or prophylactic agent according to claim 1 , wherein the graft-versus-host disease is post-bone marrow transplantation graft-versus-host disease. 3. The therapeutic or prophylactic agent according to claim 1 or 2 , wherein said graft-versus-host disease is dry eye. The therapeutic or preventive agent according to any one of claims 1 to 3 , wherein the graft-versus-host disease is accompanied by inflammation and pathological fibrosis.

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