WO2024257328A1 - Eye disease model animal - Google Patents

Abstract

The present disclosure provides an eye disease model animal, a method for producing the eye disease model animal, and a method for screening therapeutic agents and/or prophylactic agents for dry eye. One aspect of the present disclosure provides a dry eye model animal that has a characteristic associated with meibomian gland dysfunction. Another aspect of the present disclosure provides a method for producing the dry eye model animal, the method including a step for transplanting cells from an animal that is allogeneic to the dry eye model animal into an animal that has reduced or deficient immune function.

Description

Eye disease model animals

The present disclosure relates to model animals for eye diseases, in particular model animals for meibomian gland dysfunction, model animals for dry eye, and related techniques.

Dry eye is classified into aqueous-deficient and evaporative types, but there are also combinations of these. Causes of evaporative dry eye include meibomian gland dysfunction and lipid abnormalities. It is thought that 89% of dry eye patients also have meibomian gland dysfunction. However, there are no clinically available dry eye treatments that target the improvement of meibomian gland dysfunction. There is a need for treatment and prevention agents for dry eye that is accompanied by meibomian gland dysfunction, including evaporative dry eye.

In order to develop therapeutic and preventive agents, screening using model animals that allow evaluation of therapeutic and preventive effects is essential. However, there are few model animals that can evaluate the therapeutic and preventive effects of candidate compounds against meibomian gland dysfunction. Furthermore, there were no model animals that could be used to confirm the effects on dry eye symptoms in addition to the effects on meibomian gland dysfunction.

The present inventors have unexpectedly found that by administering cells from an allogeneic animal to an immunodeficient animal, the immunodeficient animal has meibomian gland dysfunction, and furthermore has both meibomian gland dysfunction and dry eye. Thus, in one aspect, the present disclosure provides a model animal with meibomian gland dysfunction and a method for producing such a model animal, or a dry eye model animal with meibomian gland dysfunction and a method for producing such a dry eye model animal. In another aspect, the present disclosure provides a method for evaluating the efficacy of a therapeutic and/or preventive agent for dry eye or meibomian gland dysfunction using such a model animal, and a method for screening a therapeutic and/or preventive agent for dry eye or meibomian gland dysfunction.

The present disclosure provides, for example, the following:
(Item 1)
A dry eye model animal having characteristics associated with meibomian gland dysfunction.
(Item 2)
The model animal according to the above item, wherein the model animal is an animal with reduced or deficient immune function to which cells from an animal allogeneic to the model animal have been transplanted.
(Item 3)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 4)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item 5)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item 6)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item 7)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are spleen-derived immune cells.
(Item 8)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 9)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item 10)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 11)
The model animal described in any one of the above items, wherein the characteristic associated with meibomian gland dysfunction is at least one selected from the group consisting of blockage of meibomian gland orifices, atrophy of meibomian glands, and eyelid margin irregularities.
(Item 12)
The model animal described in any one of the above items, wherein the characteristic associated with meibomian gland dysfunction is blockage of the meibomian gland orifice.
(Item 13)
The model animal described in any one of the above items, further having a characteristic related to dry eye selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film photointerference score, and abnormal blink rate.
(Item 14)
The model animal according to any one of the preceding items, further having a characteristic associated with dry eye selected from the group consisting of an abnormality in tear film breakup time and an abnormality in corneal staining score.
(Item 15)
A dry eye model animal,
the dry eye model animal is a dry eye model animal having reduced or deficient immune function to which immune cells from an allogeneic animal have been transplanted,
and at least one characteristic selected from the following characteristics associated with meibomian gland dysfunction:
the features associated with meibomian gland dysfunction are selected from obstruction of meibomian gland orifices, atrophy of the meibomian glands, and irregular eyelid margins;
the dry eye associated features are selected from abnormal tear film break-up time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate;
Dry eye model animal.
(Item 15A)
The dry eye model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 15B)
The dry eye model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item 15C)
The dry eye model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item 15D)
The dry eye model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 15E)
The dry eye model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item 15F)
The dry eye model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 16)
A dry eye model animal,
The dry eye model animal is an animal having a reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
have obstructed meibomian gland orifices, abnormal tear film break-up times, and abnormal corneal staining scores;
Dry eye model animal.
(Item 16A)
The dry eye model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 16B)
The dry eye model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item 16C)
The dry eye model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item 16D)
The dry eye model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are spleen-derived immune cells.
(Item 16E)
The dry eye model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 16F)
The dry eye model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 17)
A dry eye model animal characterized in that it develops features associated with meibomian gland dysfunction by 40 weeks of age.
(Item 17A)
The dry eye model animal according to any one of the preceding items, wherein the characteristic associated with meibomian gland dysfunction is at least one selected from the group consisting of blockage of meibomian gland orifices, atrophy of meibomian glands, and eyelid margin irregularities.
(Item 18)
A model animal characterized in that the meibomian gland contains cells derived from the spleen.
(Item 18A)
The model animal according to any one of the preceding items, wherein the cells are cells of an animal allogeneic to the model animal.
(Item 19)
The model animal according to any one of the preceding items, wherein the model animal is a dry eye model animal.
(Item 20)
The model animal according to any one of the preceding items, wherein the model animal is a meibomian gland dysfunction (MGD) model animal.
(Item 21)
A dry eye model animal comprising at least one type of cell selected from the group consisting of T cells, B cells, macrophages, dendritic cells, venous sinus endothelial cells, and NK cells in the meibomian gland.
(Item 22)
A dry eye model animal, which is an animal having a reduced or defective immune function and into which cells of an allogeneic animal are transplanted.
(Item 23)
1. A meibomian gland dysfunction (MGD) model animal, the model animal being an animal with reduced or defective immune function into which cells from an allogeneic animal are transplanted.
(Item 23A)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 23B)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item 23C)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item 23D)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item 23E)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 23F)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item 23G)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 23H)
The model animal described in any one of the above items, wherein the model animal has at least one characteristic associated with meibomian gland dysfunction selected from obstruction of the meibomian gland orifice, atrophy of the meibomian gland, and eyelid margin irregularity.
(Item 23I)
The model animal described in any one of the above items, further having a characteristic related to dry eye selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film photointerference score, and abnormal blink rate.
(Item 24)
A meibomian gland dysfunction (MGD) model animal having a blockage of the orifice of the meibomian gland, the model animal being an animal with reduced or insufficient immune function and into which cells from an allogeneic animal have been transplanted.
(Item 24A)
The MGD model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 24B)
The MGD model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item 24C)
The MGD model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item 24D)
The MGD model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item 24E)
The MGD model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 24F)
The MGD model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item 24G)
The MGD model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 25)
A method for producing a dry eye model animal, the method comprising a step of transplanting cells from an animal allogeneic to the dry eye model animal into an animal with reduced or deficient immune function.
(Item 26)
A method for producing an MGD model animal, the method comprising a step of transplanting cells from an animal allogeneic to the MGD model animal into an animal with reduced or deficient immune function.
(Item 26A)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 26B)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item 26C)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item 26D)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item 26E)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item 26F)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item 26G)
The method of any one of the preceding claims, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 26H)
The method according to any one of the above items, wherein the model animal has at least one characteristic associated with meibomian gland dysfunction selected from obstruction of the meibomian gland orifice, atrophy of the meibomian glands, and irregular eyelid margins.
(Item 26I)
The method according to any one of the preceding items, wherein the model animal further has a characteristic associated with dry eye selected from the group consisting of an abnormality in tear film breakup time, an abnormality in corneal staining score, a decrease in tear volume, an abnormality in tear film photointerference score, and an abnormality in blink rate.
(Item 27)
A method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method.
(Item 28)
The method according to any one of the preceding items, wherein the eye disease is dry eye, and the step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item 29)
The method according to any one of the preceding items, wherein the eye disease is meibomian gland dysfunction, and step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item 30)
The method according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of blockage of meibomian gland orifices, atrophy of the meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from the group consisting of abnormal tear film break-up time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.
(Item 31)
The method according to any one of the preceding items, wherein in step (2), the features associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item 32)
The method according to any one of the preceding items, wherein in step (2), the method comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item 33)
The method according to any one of the preceding items, wherein the step (2) includes a step of evaluating at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in blink frequency.
(Item 34)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item 35)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item 36)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are spleen-derived cells.
(Item 37)
The method according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item 38)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 39)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item 40)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item 41)
A method for evaluating the efficacy of a test substance as a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering the test substance to a dry eye model animal; and (2) evaluating blockage of meibomian gland orifices, abnormalities in tear film breakup time, and abnormalities in the number of blinks in the dry eye model animal, wherein the dry eye model animal is an animal with reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
The MHC type of the allogeneic animal is H- ^2Db ;
Methods for assessing drug efficacy.
(Item 41A)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item 41B)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item 41C)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 42)
A method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method.
(Item 42A)
The method according to any one of the preceding items, wherein the eye disease is dry eye, and the step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item 42B)
The method according to any one of the preceding items, wherein the eye disease is meibomian gland dysfunction, and step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item 42C)
The method according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from blockage of meibomian gland orifices, atrophy of the meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from abnormal tear film breakup time, abnormal corneal staining spot score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.
(Item 42D)
The method according to any one of the preceding items, wherein in step (2), the features associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item 42E)
The method according to any one of the preceding items, wherein in step (2), the method comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item 42F)
The method according to any one of the preceding items, wherein in the step (2), at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in blink frequency are evaluated.
(Item 42G)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item 42H)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item 42I)
The method according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item 42J)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 42K)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item 42L)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item 43)
A method for screening a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Screening methods.
(Item 43A)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item 43B)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item 43C)
The method according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item 43D)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item 43E)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item 43F)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item A1)
A method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method.
(Item A2)
The method according to any one of the preceding items, wherein in step (2), the method comprises evaluating at least one characteristic associated with meibomian gland dysfunction.
(Item A3)
The method according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of obstruction of the meibomian gland orifice, atrophy of the meibomian gland, and irregular eyelid margins.
(Item A4)
The method according to any one of the preceding items, wherein in step (2), the features associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item A5)
The method according to any one of the preceding items, wherein in step (2), the method comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item A6)
The method according to any one of the preceding items, wherein the step (2) includes a step of evaluating at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in blink frequency.
(Item A7)
The method according to any one of the preceding claims, wherein the eye disease is meibomian gland dysfunction.
(Item A8)
The method according to any one of the above items, wherein the eye disease is dry eye.
The method according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item A10)
The method of any one of the preceding claims, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item A11)
The method according to any one of the preceding claims, wherein the allogeneic animal cells are spleen-derived cells.
(Item A12)
The method according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item A13)
The method according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item A14)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item A15)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item A16)
A method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method.
(Item A17)
A method for screening an agent for treating and/or preventing meibomian gland dysfunction, the method comprising:
(1) administering a test substance to a meibomian gland dysfunction model animal;
(2) evaluating blockage of the meibomian gland orifice in a meibomian gland dysfunction animal model; and (3) identifying the test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction when blockage of the meibomian gland orifice is improved and/or progression of blockage of the meibomian gland orifice is inhibited in the meibomian gland dysfunction animal model,
the meibomian gland dysfunction model animal is an animal having a reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the meibomian gland dysfunction model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Screening methods.
(Item A18)
A method for screening a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Screening methods.
(Item B1)
1. Use of an ophthalmic disease model animal in a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an ophthalmic disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
use.
(Item B2)
The use according to any one of the preceding claims, wherein the eye disease is dry eye, and step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item B3)
The use according to any one of the preceding claims, wherein the eye disease is meibomian gland dysfunction, and step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item B4)
The use according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from blockage of meibomian gland orifices, atrophy of the meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from abnormal tear film break-up time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.
(Item B5)
The use according to any one of the preceding items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item B6)
The use according to any one of the preceding items, wherein in step (2), the method includes a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item B7)
The use according to any one of the preceding items, wherein the step (2) includes at least a step of evaluating blockage of the meibomian gland orifice, abnormalities in tear film breakup time, and abnormalities in blink frequency.
(Item B8)
The use according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item B9)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item B10)
The use according to any one of the preceding claims, wherein the allogeneic animal cells are spleen-derived cells.
(Item B11)
The use according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item B12)
The use according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item B13)
The use according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item B14)
The use according to any one of the preceding items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass that constitutes MHC class I, is b.
(Item B15)
1. Use of a dry eye model animal in a method for evaluating the efficacy of a test substance as a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering the test substance to a dry eye model animal; and (2) evaluating blockage of meibomian gland orifices, abnormalities in tear film breakup time, and abnormalities in the number of blinks in the dry eye model animal, wherein the dry eye model animal is an animal with reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
The MHC type of the allogeneic animal is H- ^2Db ;
use.
(Item B16)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells.
(Item B17)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item B18)
The use according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item B19)
A use of an eye disease model animal in a method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
use.
(Item B20)
The use according to any one of the preceding claims, wherein the eye disease is dry eye, and step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item B21)
The use according to any one of the preceding claims, wherein the eye disease is meibomian gland dysfunction, and step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item B21)
The use according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from blockage of meibomian gland orifices, atrophy of the meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from abnormal tear film break-up time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.
(Item B22)
The use according to any one of the preceding items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item B23)
The use according to any one of the preceding items, wherein in step (2), the method includes a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item B24)
The use according to any one of the preceding items, wherein the step (2) includes at least a step of evaluating blockage of the meibomian gland orifice, abnormalities in tear film breakup time, and abnormalities in blink frequency.
(Item B25)
The use according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item B26)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item B27)
The use according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item B28)
The use according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item B29)
The use according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item B30)
The use according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass that constitutes MHC class I, is b.
(Item B31)
A use of a dry eye model animal in a method for screening a therapeutic and/or preventive agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when the blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
use.
(Item B32)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells.
(Item B33)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item B34)
The use according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item B35)
The use according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item B36)
The use according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item B37)
The use according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass that constitutes MHC class I, is b.
(Item C1)
1. Use of an ophthalmic disease model animal in a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an ophthalmic disease, the method comprising:
(1) administering a test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
use.
(Item C2)
The use according to any one of the preceding items, wherein step (2) comprises evaluating at least one characteristic associated with meibomian gland dysfunction.
(Item C3)
The use according to any one of the above items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of obstruction of the meibomian gland orifice, atrophy of the meibomian gland, and irregular eyelid margins.
(Item C4)
The use according to any one of the preceding items, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item C5)
The use according to any one of the preceding items, wherein in step (2), the method includes a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item C6)
The use according to any one of the preceding items, wherein the step (2) includes at least a step of evaluating blockage of the meibomian gland orifice, abnormalities in tear film breakup time, and abnormalities in blink frequency.
(Item C7)
The use according to any one of the preceding claims, wherein the eye disease is meibomian gland dysfunction.
(Item C8)
The use according to any one of the above items, wherein the eye disease is dry eye.
The use according to any one of the preceding claims, wherein the allogeneic animal cells are immune cells.
(Item C10)
The use according to any one of the preceding paragraphs, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes.
(Item C11)
The use according to any one of the preceding claims, wherein the allogeneic animal cells are spleen-derived cells.
(Item C12)
The use according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item C13)
The use according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item C14)
The use according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC type from the model animal.
(Item C15)
The use according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass that constitutes MHC class I, is b.
(Item C16)
A use of an eye disease model animal in a method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
use.
(Item C17)
1. Use of a meibomian gland dysfunction model animal in a method for screening for a therapeutic and/or preventive agent for meibomian gland dysfunction, the method comprising:
(1) administering a test substance to a meibomian gland dysfunction model animal;
(2) evaluating blockage of the meibomian gland orifice in a meibomian gland dysfunction animal model; and (3) identifying the test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction when blockage of the meibomian gland orifice is improved and/or progression of blockage of the meibomian gland orifice is inhibited in the meibomian gland dysfunction animal model,
the meibomian gland dysfunction model animal is an animal having a reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the meibomian gland dysfunction model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
use.
(Item C18)
A use of a dry eye model animal in a method for screening a therapeutic and/or preventive agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
use.
(Item D1)
An eye disease model animal for use in a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
Model animals.
(Item D2)
The model animal according to any one of the preceding items, wherein the eye disease is dry eye, and the step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item D3)
The model animal according to any one of the preceding items, wherein the eye disease is meibomian gland dysfunction, and the step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item D4)
The model animal according to any one of the above items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of blockage of meibomian gland orifices, atrophy of meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining spot score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink count.
(Item D5)
The model animal according to any one of the preceding items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item D6)
The model animal according to any one of the preceding items, wherein the step (2) comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item D7)
The eye disease model animal according to any one of the above items, wherein the step (2) includes a step of evaluating at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the number of blinks.
(Item D8)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item D9)
The eye disease model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item D10)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are cells derived from the spleen.
(Item D11)
The eye disease model animal according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item D12)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item D13)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having an MHC type different from that of the model animal.
(Item D14)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item D15)
A dry eye model animal for use in a method for evaluating the efficacy of a test substance as a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering the test substance to a dry eye model animal; and (2) evaluating blockage of meibomian gland orifices, abnormalities in tear film breakup time, and abnormalities in the number of blinks in the dry eye model animal, wherein the dry eye model animal is an animal with reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
The MHC type of the allogeneic animal is H- ^2Db ;
Model animals.
(Item D16)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item D17)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item D18)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item D19)
An eye disease model animal for use in a method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
Model animals.
(Item D20)
The model animal according to any one of the preceding items, wherein the eye disease is dry eye, and the step (2) comprises a step of evaluating characteristics associated with dry eye.
(Item B21)
The model animal according to any one of the preceding items, wherein the eye disease is meibomian gland dysfunction, and the step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
(Item D21)
The model animal according to any one of the above items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of blockage of meibomian gland orifices, atrophy of meibomian glands, and eyelid margin irregularities, and the characteristics associated with dry eye include at least one selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining spot score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink frequency.
(Item D22)
The model animal according to any one of the preceding items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item D23)
The model animal according to any one of the preceding items, wherein the step (2) comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item D24)
The model animal according to any one of the preceding items, wherein the step (2) includes a step of evaluating at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the number of blinks.
(Item D25)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item D26)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item D27)
The model animal according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item D28)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item D29)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having an MHC type different from that of the model animal.
(Item D30)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item D31)
A dry eye model animal for use in a method for screening a therapeutic and/or preventive agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Model animals.
(Item D32)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item D33)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item D34)
The model animal according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item D35)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item D36)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having an MHC type different from that of the model animal.
(Item D37)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item E1)
An eye disease model animal for use in a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
Model animals.
(Item E2)
The model animal described in any one of the preceding items, wherein the step (2) includes a step of evaluating at least one characteristic associated with meibomian gland dysfunction.
(Item E3)
The model animal described in any one of the above items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of blockage of the meibomian gland orifice, atrophy of the meibomian gland, and eyelid margin irregularity.
(Item E4)
The model animal according to any one of the preceding items, wherein in the step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice.
(Item E5)
The model animal according to any one of the preceding items, wherein the step (2) comprises a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye.
(Item E6)
The model animal according to any one of the preceding items, wherein the step (2) includes a step of evaluating at least blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the number of blinks.
(Item E7)
The model animal according to any one of the preceding items, wherein the eye disease is meibomian gland dysfunction.
(Item E8)
The model animal according to any one of the preceding items, wherein the eye disease is dry eye.
(Item E9)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item E10)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item E11)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are cells derived from the spleen.
(Item E12)
The model animal according to any one of the above items, wherein the model animal has a type d of H-2D, a subclass that constitutes major histocompatibility complex (MHC) class I.
(Item E13)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item E14)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having an MHC type different from that of the model animal.
(Item E15)
The model animal according to any one of the above items, wherein the allogeneic animal is a mouse whose H-2D type, a subclass constituting MHC class I, is b.
(Item E16)
An eye disease model animal for use in a method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal; (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the eye disease model animal; and (3) identifying the test substance as an agent for treating and/or preventing an eye disease when the evaluated characteristic improves or inhibits progression,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
Model animals.
(Item E17)
A meibomian gland dysfunction model animal for use in a method for screening for a therapeutic and/or preventive agent for meibomian gland dysfunction, the method comprising:
(1) administering a test substance to a meibomian gland dysfunction model animal;
(2) evaluating blockage of the meibomian gland orifice in a meibomian gland dysfunction animal model; and (3) identifying the test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction when blockage of the meibomian gland orifice is improved and/or progression of blockage of the meibomian gland orifice is inhibited in the meibomian gland dysfunction animal model,
the meibomian gland dysfunction model animal is an animal having a reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the meibomian gland dysfunction model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Model animals.
(Item E18)
A dry eye model animal for use in a method for screening a therapeutic and/or preventive agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the major histocompatibility complex (MHC) type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Model animals.
(Item F1)
A dry eye model animal is prepared by transplanting cells from an animal of the same species as the dry eye model animal into an animal having a reduced or deficient immune function.
(Item F2)
An MGD model animal, which is prepared by transplanting cells from an animal allogeneic to the MGD model animal into an animal with reduced or deficient immune function.
(Item F3)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse.
(Item F4)
The model animal according to any one of the preceding items, wherein the model animal is a SCID mouse or a NOD-SCID mouse whose major histocompatibility complex (MHC) type is H- ^2Dd .
(Item F5)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells.
(Item F6)
The model animal according to any one of the preceding items, wherein the cells of the allogeneic animal are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph node.
(Item F7)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a mouse having a different MHC class I type from the model animal.
(Item F8)
The method according to any one of the preceding items, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db .
(Item F9)
The model animal according to any one of the preceding items, wherein the allogeneic animal is a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
(Item 26H)
The model animal described in any one of the above items, wherein the model animal has at least one characteristic associated with meibomian gland dysfunction selected from obstruction of the meibomian gland orifice, atrophy of the meibomian gland, and eyelid margin irregularity.
(Item F10)
The model animal described in any one of the above items, further having a characteristic related to dry eye selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film photointerference score, and abnormal blink rate.

It is contemplated that in the present disclosure, one or more of the above features may be provided in combinations in addition to those combinations explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present disclosure provides an eye disease model animal with meibomian gland dysfunction, in particular a dry eye model animal with meibomian gland dysfunction. According to the present disclosure, it is possible to develop therapeutic and/or preventive agents for dry eye associated with meibomian gland dysfunction and therapeutic and/or preventive agents for meibomian gland dysfunction, which have been difficult to develop until now.

Figure 1 shows photographs of eyelid sections stained with Oil Red O and hematoxylin. The dotted line indicates the border between the meibomian glands and other tissues. FIG. 2 shows an image of eyelid margin irregularity. 3 shows fluorescent photographs of eyelids taken two days after administration of fluorescently labeled spleen-derived cells. Photographs of mice administered PKH26 (excitation wavelength 551 nm, fluorescence wavelength 567 nm)-labeled spleen-derived cells at the time of opening and eversion of the eyelids, and of mice administered PKH67 (excitation wavelength 490 nm, fluorescence wavelength 502 nm)-labeled spleen-derived cells at the time of opening and eversion of the eyelids. Observations were performed with an excitation wavelength bandpass of 460-480 nm and a fluorescence wavelength bandpass of 495-540 nm.

The present disclosure is described below. Throughout this specification, singular expressions should be understood to include the concept of the plural unless otherwise specified. Thus, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural unless otherwise specified. In addition, terms used in this specification should be understood to be used in the sense commonly used in the field unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence. In this specification, "about" means ±10% of the value that follows.

(Definition)
As used herein, the term "ocular disease" refers to a disease that presents symptoms in the eyes, including dry eye and meibomian gland dysfunction, and is a disease that presents subjective and objective symptoms in the eyes.

In this specification, "dry eye" refers to a disease diagnosed as "dry eye" according to clinical diagnostic criteria, and is defined as "a multifactorial disease of the tears and ocular surface caused by various factors, with ocular discomfort, abnormal visual function, instability of the tear film, or damage to the ocular surface." Dry eye is broadly classified into "aqueous tear-deficient" dry eye and "evaporative" dry eye. In recent years, a classification called "tear film break-up time (BUT) shortened dry eye" (hereinafter also referred to as "BUT shortened dry eye") has also been used.

In this specification, "aqueous-deficient dry eye" refers to dry eye that develops due to tissue destruction of the lacrimal gland or impaired tear conduction from the lacrimal gland to the ocular surface.

In this specification, "evaporative dry eye" refers to dry eye that develops when tear secretion is normal but excessive water loss from the ocular surface occurs due to various causes. Causes of evaporative dry eye include meibomian gland dysfunction (MGD) and tear lipid abnormalities, and the presence of either or both of these.

In this specification, "short BUT dry eye" refers to dry eye in which the tear breakup time (BUT) is short and there are subjective symptoms of dry eye, but tear secretion and the corneal and conjunctival epithelium are almost normal. There is also a view that a shortened BUT can be interpreted as being linked to meibomian gland dysfunction, which is thought to be one of the causes of evaporative dry eye.

In this specification, "meibomian glands" refers to sebaceous glands located within the tarsal plate with openings at the upper and lower eyelid margins. Meibum (a lipid) is secreted from the meibomian glands. The presence of lipids in tears exerts effects such as inhibiting tear evaporation, promoting tear stability, and promoting the spreading of tears over the ocular surface. Although there are individual differences, humans have approximately 50 meibomian glands in the upper eyelid and approximately 25 in the lower eyelid, while mice have approximately 11 on each side.

In this specification, "Meibomian gland dysfunction (MGD)" refers to a disease defined as "a state in which the function of the meibomian glands becomes diffusely abnormal due to various causes, and is accompanied by chronic ocular discomfort." Meibomian gland dysfunction is also widely recognized as one of the causes of dry eye. Meibomian gland dysfunction (MGD) is broadly classified into hyposecretory type, in which the secretion of oil from the meibomian glands is reduced, and hypersecretory type, in which the secretion of oil from the meibomian glands is excessive, but hyposecretory type is overwhelmingly more common (Definition and diagnostic criteria for meibomian gland dysfunction, Atarashii Ophthalmology 27 (5): 627-631, 2010). Hyposecretory type MGD leads to evaporative dry eye due to a decrease in the tear oil phase. Hyposecretory MGD is characterized by subjective symptoms including ocular discomfort and foreign body sensation, abnormal findings around the meibomian gland orifices including eyelid margin irregularities, and obstruction of the meibomian gland orifices (Table 4 in Atarashii Ophthalmology 27 (5): 627-631, 2010).

In this specification, "characteristics associated with meibomian gland dysfunction" refers to characteristics in the meibomian glands associated with meibomian gland dysfunction. The characteristics may be symptoms of meibomian gland dysfunction or may cause the onset of meibomian gland dysfunction.

In this specification, "obstruction of the meibomian gland orifice" refers to a state in which meibum or components of the meibomian gland remain in the meibomian gland orifice without being secreted or excreted. "Obstruction of the meibomian gland orifice" refers to a state in which, when the eyelid is everted under a stereomicroscope, a white to yellow protrusion is observed at the meibomian gland orifice, or a cylindrical or rice-grain-shaped mass is observed within the orifice along the meibomian gland duct, or the meibomian gland orifice is pushed open by the contents.

In this specification, "atrophy of the meibomian glands" refers to a state in which the acini of the meibomian glands have shrunk and the amount of meibum stored in the meibomian glands has decreased. "Atrophy of the meibomian glands" refers to a state in which the white reflex of visible light is reduced when the eyelid is everted under a stereomicroscope, or the oily reflex of infrared light is reduced.

In this specification, "irregular eyelid margin" refers to the line where the upper or lower eyelid meets the cornea (hereinafter referred to as "eyelid margin") being a line that is concave in places. "Irregular eyelid margin" is observed with the naked eye or under a stereomicroscope either without treatment or after instillation or instillation of fluorescein.

In this specification, "tear film breakup time (BUT)" is an index of the stability of the tears covering the cornea. "BUT" refers to the time it takes to observe "tear film breakup," which occurs when the tear film on the cornea is broken down and a dry spot appears and gradually expands, when blinking is suppressed and the eyelids are forcibly opened for a certain period of time (10 seconds). It can be measured as the time it takes for the tear film to be opened from the state in which reflected light from the tear film, interference light, or the fluorescence of fluorescein molecules contained in the tear film after administration of fluorescein is uniformly observed on the cornea to the appearance of an area where the reflected light or fluorescence disappears.

In this specification, "number of blinks" refers to the number of times you blink within a certain period of time. "Number of blinks" is one of the characteristics related to dry eye, and is the number of times you blink within a certain period of time (e.g., one minute), and can be observed visually.

In this specification, the term "corneal staining score" refers to the score of staining spots that are seen when the corneal surface is damaged in a punctate manner. The corneal surface can be visualized as staining spots by the incorporation of a dye (e.g., fluorescein) into corneal epithelial cells or by the accumulation of a dye (e.g., fluorescein) in areas where corneal epithelial cells have fallen off. The corneal staining spot is observed, for example, under a stereomicroscope using a fluorescence wavelength of 480 to 680 nm with an excitation wavelength of 400 to 540 nm after instillation or instillation of fluorescein. The corneal staining spot score is calculated by dividing the cornea into three quadrants, upper, middle, and lower, and judging the number of punctate corneal staining spots in each quadrant as none (0 points), sparse (1 point), intermediate between sparse and dense (2 points), and dense (3 points), for a total of 9 points. The corneal staining spot score is also called the superficial punctate keratopathy (SPK) score.

In this specification, "tear volume" refers to the amount of liquid secreted from the lacrimal gland, and refers to the mixed amount of tears stored in the meniscus (the tear pool above the lower eyelid) and tears secreted during the measurement period. One end of a Schirmer test paper or cotton thread is inserted into the conjunctival fornix of the upper and lower or inner and outer corners without irritating the cornea and can be removed after a certain period of time and measured as the wet length of the Schirmer test paper or cotton thread. For mice, the certain period of time is, for example, 15 seconds.

In this specification, the term "tear film optical interference score" is an index for evaluating tear abnormalities (e.g., lipid abnormalities in tears). "Tear film optical interference" refers to the phenomenon in which the phase of the light reflected from water and the light reflected from the oil changes due to differences in the refractive index, content, and/or mixing ratio of the water and oil components of tears (which are composed of three layers: lipid layer (oil layer), aqueous layer, and mucin layer), and certain wavelengths are reinforced, resulting in the observation of rainbow-colored interference fringes and areas with little reflected light. The "tear film optical interference score" is observed using a stereomicroscope and scored from 1 to 5 points based on the presence or absence of interference and the amount of reflected light. The presence or absence of interference is visualized by the number of rainbow-colored fringes, and the amount of reflected light is visualized by the brightness of the light reflected from the entire tear film. The evaluation can be made, for example, as follows: uniform white to silvery reflection (1 point), non-uniform white to silvery reflection (2 points), stripes of two to three colors (3 points), rainbow stripes (4 points), or exposed areas of the cornea (5 points).

As used herein, the term "subject" refers to the subject to which the therapeutic and preventive medicine or method disclosed herein is administered. Subjects include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.), with primates being preferred, and humans being particularly preferred.

In this specification, "treatment" means curing, improving, or suppressing or alleviating a disease or symptom. "Treating an eye disease" includes treating objective or subjective symptoms.

In this specification, "prophylaxis" means to prevent the onset of a disease or symptom, and this concept also includes minimizing the onset of a disease or symptom by delaying the onset of the disease or symptom or treating before the onset of the disease or symptom. "Treatment and prevention of eye disease" includes prevention of objective or subjective symptoms.

In this specification, "subjective symptoms" refers to symptoms of a disease that can be perceived by a patient suffering from the disease.

In this specification, "objective symptoms" refers to disease symptoms that can be objectively proven by findings (objective findings) such as imaging findings or numerical test results.

In this specification, "animal" refers to animals used as model animals, including mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.).

In this specification, the term "model animal" refers to an animal that has been created to produce symptoms similar to those of a human disease, and is used to elucidate mechanisms, develop preventive or therapeutic agents, or evaluate efficacy. If an animal develops the disease of interest and exhibits the characteristics of the disease, but it is not possible to elucidate mechanisms, develop preventive or therapeutic agents, or evaluate efficacy because the characteristics are irreversible or it takes a considerable amount of time for the disease to develop or the characteristics to appear (e.g., approximately 50 weeks of age or older), such an animal is not a model animal.

As used herein, "allogeneic" refers to individuals of the same species but genetically distinct.

As used herein, "reduced or impaired immune function" refers to a state characterized by a lack of immune cell function, in which an immune response to allogeneic cells is reduced or not elicited.

As used herein, "the major histocompatibility complex (MHC) type is H-2D ^d " refers to the homozygous d type of H-2D, a subclass that constitutes MHC class I. "The MHC type is H-2D ^b " refers to the homozygous b type of H-2D, a subclass that constitutes MHC class I.

As used herein, "immune cells" refers to cells that make up the immune system, including T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, and natural killer cells.

As used herein, "cells derived from the spleen, blood, bone marrow, thymus, or lymph node" refers to cells prepared and/or isolated from the spleen, blood, bone marrow, thymus, or lymph node, or cultured cells obtained by culturing cells prepared and/or isolated from the spleen, blood, bone marrow, thymus, or lymph node.

As used herein, "containing cells in the meibomian gland" refers to containing foreign cells in the meibomian gland that are not derived from the model animal of the present disclosure itself. Cells contained in the meibomian gland are identified by detecting the presence of foreign cells through a pathological test of the meibomian gland, or by using an antibody against a cell surface membrane protein or the like of the foreign cells.

In this specification, "developing characteristics associated with meibomian gland dysfunction by 40 weeks of age" refers to developing characteristics associated with meibomian gland dysfunction at any time before reaching 40 weeks of age and becoming usable as a model animal.

In this specification, the term "test substance" refers to a substance that is subjected to efficacy evaluation or screening using a model animal, and may be any substance, such as a low molecular weight compound, polysaccharide, protein, peptide, nucleic acid, or a fusion product thereof.

In this specification, "evaluation" means confirming the state of characteristics related to meibomian gland dysfunction and/or characteristics related to dry eye in an animal model, and may also include observing or measuring the characteristics as necessary, thereby determining whether or not the test substance has the desired property or effect, or the degree of the test substance's desired property or effect.

Preferred Embodiments
A description of preferred embodiments will be given below, but it should be understood that this embodiment is an example of the present invention, and the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present disclosure by referring to the following preferred examples. Regarding these embodiments, those skilled in the art can combine any embodiments as appropriate.

(Model Animals)
In one aspect, the present disclosure provides an ophthalmic disease model animal, particularly a meibomian gland dysfunction model animal or a dry eye model animal, having characteristics related to meibomian gland dysfunction. Most dry eye patients also suffer from meibomian gland dysfunction, so in the treatment of dry eye, it is desirable to treat both dry eye symptoms and meibomian gland dysfunction, but there is no model animal that suffers from both dry eye symptoms and meibomian gland dysfunction, making it difficult to develop a treatment or preventive agent. In addition, there is a problem that there are few meibomian gland dysfunction model animals that can be used to develop a treatment or preventive agent for meibomian gland dysfunction. The present inventors have newly discovered a model animal that has characteristics related to meibomian gland dysfunction, and further a model animal that suffers from both dry eye and meibomian gland dysfunction.

In some embodiments, the model animal of the present disclosure may be a non-human animal, preferably a non-human mammal, more preferably a rodent, such as a mouse, rat, or hamster, and most preferably a mouse.

In some embodiments, the model animal of the present disclosure may be an animal with reduced or deficient immune function transplanted with cells from an animal allogeneic to the model animal. Cells from an animal allogeneic to the model animal may include, but are not limited to, T cells, B cells, macrophages, dendritic cells, sinus endothelial cells, and NK cells. In some embodiments, the cells from an animal allogeneic to the model animal may be immune cells, such as immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes. The cells from the allogeneic animal may preferably be spleen cells.

A model animal with reduced or insufficient immune function may be deficient in T cells and/or B cells, and preferably in both T cells and B cells. The reduced or insufficient immune function is preferably a congenital condition, i.e., may be a condition caused by a genetic mutation.

Model animals with reduced or defective immune function may be in a state in which the function of the protein Prkdc (protein kinase, DNA activated, catalytic polypeptide) is deficient due to a mutation in the Prkdc gene, and may preferably have a mutation designated as Prkdc ^scid . Due to the lack of Prkdc function, gene rearrangement of B cells and T cells cannot be achieved, resulting in a deficiency in mature B cells and T cells.

In some embodiments, the model animal of the present disclosure may be a SCID mouse or a NOD-SCID mouse.

In some embodiments, the model animal of the present disclosure can be CB17/Icr-Prkdc ^scid /CrlCrlj.

In some embodiments, the allogeneic animal may have a different major histocompatibility complex (MHC) type from the model animal. The MHC class I type of the model animal of the present disclosure is d, and the MHC class I type of the allogeneic animal may be a, b, k, q, or s other than d, but is preferably b. In a specific embodiment, the subclass H-2D constituting the MHC class I of the model animal of the present disclosure is d, and the subclass H-2D constituting the MHC class I of the allogeneic animal may be a, b, k, q, or s other than d, but is preferably b. The subclass constituting the MHC class I may be homozygous or heterozygous. The H-2D of the model animal of the present disclosure may be homozygous for d (H-2D ^d ), and the H-2D of the allogeneic animal may be homozygous for b (H-2D ^b ).

In some embodiments, the model animal of the disclosure can be a SCID or NOD-SCID mouse with major histocompatibility complex (MHC) type H-2D ^d . In further embodiments, the model animal of the disclosure can be a SCID or NOD-SCID mouse with major histocompatibility complex (MHC) type H-2D ^d , and the allogeneic animal can be a mouse with MHC type H-2D ^b .

In some embodiments, the allogeneic animal may be a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.

In some embodiments, the model animal of the present disclosure may have at least one, at least two, or all of the characteristics associated with meibomian gland dysfunction selected from the group consisting of obstruction of meibomian gland orifices, meibomian gland atrophy, and eyelid margin irregularities.

Preferably, the model animal of the present disclosure has an increased number of obstructions of the meibomian gland orifices compared to healthy animals that do not develop eye diseases (e.g., animals that have not been administered allogeneic cells to the model animal) (hereinafter referred to as "non-model animals"). For example, the evaluation of "obstruction of meibomian gland orifices" can be performed using the number of obstructions of the meibomian gland orifices per individual.

Preferably, in the model animal of the present disclosure, atrophy of the meibomian glands can be confirmed, whereas no atrophy of the meibomian glands is observed in non-model animals. For example, the evaluation of "atrophy of the meibomian glands" can be performed using the number of individuals that exhibit atrophy of the meibomian glands relative to the total number of individuals in a model animal group under the same conditions (i.e., the ratio of the number of individuals that exhibit atrophy of the meibomian glands).

Preferably, the model animals of the present specification have a concave eyelid margin, i.e., an irregular eyelid margin, whereas non-model animals have a smooth eyelid margin line. For example, the evaluation of "irregular eyelid margin" can be performed using the number of individuals that exhibit irregular eyelid margins (i.e., the ratio of individuals that exhibit irregular eyelid margins) to the total number of individuals in a group of model animals under the same conditions. The model animals of the present disclosure are advantageous as model animals because they develop characteristics associated with meibomian gland dysfunction and/or dry eye characteristics relatively soon after administration of allogeneic cells. In some embodiments, the onset may occur on the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth day, or at the latest on the fifteenth to thirty-first day, after administration of allogeneic cells. In some embodiments, the model animals of the present disclosure may be characterized as having onset of features associated with meibomian gland dysfunction by 40 weeks, 35 weeks, 25 weeks, 20 weeks, 15 weeks, 14 weeks, 13 weeks, 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, or 7 weeks of age.

In a further embodiment, the model animal of the present disclosure may have at least one, at least two, at least three, at least four, or all of the characteristics associated with dry eye selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.

Preferably, the model animals of this specification have a reduced BUT compared to non-model animals. For example, the evaluation of "BUT" can be performed using the BUT per individual of a group of model animals under the same conditions.

Preferably, the model animals of this specification have an increased number of blinks compared to non-model animals. For example, the "number of blinks" can be evaluated using the number of blinks per individual in a group of model animals under the same conditions.

Preferably, the corneal staining spot score is increased in the model animals of this specification compared to non-model animals. For example, the "corneal staining spot score" can be evaluated using the corneal staining spot score per individual of a group of model animals under the same conditions.

Preferably, the model animals of this specification have reduced tear volume compared to non-model animals. For example, the "tear volume" can be evaluated using the tear volume per individual of a group of model animals under the same conditions.

Preferably, the tear layer optical interference score is increased in the model animals of this specification compared to non-model animals. For example, the "tear layer optical interference score" can be evaluated using the tear layer optical interference score per individual in a group of model animals under the same conditions.

In certain embodiments, the model animal of the present disclosure may have at least the characteristics associated with dry eye, such as abnormal tear film breakup time and/or abnormal corneal staining score, as symptoms of dry eye.

In a further aspect, the present disclosure provides an animal model for meibomian gland dysfunction, the animal being an immunodeficient animal with reduced or insufficient immune function, the animal being transplanted with immune cells from an allogeneic animal, the animal having at least one characteristic selected from characteristics associated with meibomian gland dysfunction, the characteristic associated with meibomian gland dysfunction being selected from obstruction of meibomian gland orifices, atrophy of meibomian glands, and irregular eyelid margins. In a particular embodiment, the animal model for meibomian gland dysfunction is an animal with reduced or insufficient immune function transplanted with cells from an allogeneic animal, the major histocompatibility complex (MHC) type being H-2D ^d , and the allogeneic animal may be a mouse with MHC type H-2D ^b . In a further embodiment, the meibomian gland dysfunction model animal may be an animal further having at least one characteristic selected from characteristics associated with dry eye, the characteristic associated with dry eye being selected from abnormal tear film breakup time, abnormal corneal staining spot score, decreased tear volume, abnormal tear film optical interference score, and abnormal blink frequency. In a further embodiment, the characteristic associated with meibomian gland dysfunction in the meibomian gland dysfunction model animal may be obstruction of the meibomian gland orifice, and the characteristic associated with dry eye may be abnormal tear film breakup time and/or abnormal corneal staining spot score.

In a further aspect, the present disclosure provides a dry eye model animal, the dry eye model animal being an immunodeficient animal with reduced or deficient immune function, into which immune cells of an animal allogeneic to the dry eye model animal have been transplanted, the dry eye model animal having at least one characteristic selected from the following (1) characteristics associated with meibomian gland dysfunction and (2) at least one characteristic selected from the characteristics associated with dry eye, wherein (1) the characteristics associated with meibomian gland dysfunction are selected from blockage of meibomian gland orifices, meibomian gland atrophy, and eyelid margin irregularity, and (2) the characteristics associated with dry eye are selected from abnormal tear film breakup time, abnormal corneal staining spot score, decreased tear volume, abnormal tear film optical interference score, and abnormal blink count. In a particular embodiment, the dry eye animal model is an animal with reduced or deficient immune function transplanted with cells from an allogeneic animal, and the major histocompatibility complex (MHC) type is H-2D ^d , and the allogeneic animal may be a mouse with MHC type H-2D ^b . In a further embodiment, the feature associated with meibomian gland dysfunction in the dry eye animal model may be obstruction of meibomian gland orifices, and the feature associated with dry eye may be abnormal tear film breakup time and/or abnormal corneal staining score.

In a further aspect, the present disclosure provides a model animal characterized by the onset of features associated with meibomian gland dysfunction by 40 weeks, 35 weeks, 25 weeks, 20 weeks, 15 weeks, 10 weeks, or 7 weeks of age.

In some embodiments, the model animals of the present disclosure may develop features associated with meibomian gland dysfunction after 4, 5, or 6 weeks of age.

In a further aspect, the present disclosure provides a model animal, characterized in that the meibomian gland contains cells derived from the spleen, blood, bone marrow, thymus, or lymph node, preferably cells derived from the spleen. The model animal may be a dry eye model animal or a meibomian gland dysfunction (MGD) model animal, or both.

In a further aspect, the present disclosure provides a model animal comprising at least one cell selected from the group consisting of T cells, B cells, macrophages, dendritic cells, sinus endothelial cells, and NK cells in the meibomian gland. The cells may be allogeneic cells.

The cells contained in the meibomian gland can be identified as follows.
(1) When the model animal is an immunodeficient mouse lacking immune cells, a pathological examination of the meibomian gland is performed using a marker (antibody, etc.) specific to the lacking immune cells, and if the lacking immune cells are detected in the meibomian gland, it can be confirmed that external cells have reached the meibomian gland of the model animal. Since immunodeficient mice such as SCID mice lack T cells and B cells, if T cells or B cells are present in a pathological examination of the meibomian gland, it can be confirmed that external cells have reached the meibomian gland.
(2) A pathological examination of the meibomian glands is performed using a marker (antibody, etc.) specific to an MHC type different from that of the model animal, and if an MHC type different from that of the model animal is detected in the meibomian glands, it can be confirmed that external cells have reached the meibomian glands of the model animal. In this case, it is desirable to confirm that the cells derived from the spleen have an MHC type different from that of the model animal.

In a further aspect, the present disclosure provides a dry eye model animal, the model animal being an animal with reduced or deficient immune function (preferably congenitally) and into which cells from an animal allogeneic to the model animal are transplanted.

In a further aspect, the present disclosure provides a meibomian gland dysfunction (MGD) model animal, the MGD model animal being an animal with (preferably congenitally) reduced or deficient immune function, into which cells from an allogeneic animal are transplanted to the model animal.

In a further aspect, the present disclosure provides a meibomian gland dysfunction (MGD) model animal having a blockage of the orifice of the meibomian gland, the MGD model animal being an animal with reduced or defective immune function and into which cells from an allogeneic animal are transplanted from the model animal.

(Method of producing model animals)
In a different aspect, the present disclosure provides a method for producing a dry eye model animal, the method comprising transplanting cells from an animal allogeneic to the dry eye model animal into an animal with reduced or compromised immune function.

In a further aspect, the present disclosure provides a method for producing an MGD model animal, the method comprising transplanting cells from an animal allogeneic to the MGD model animal into an animal with reduced or compromised immune function.

The production method of the present disclosure may include a step of transplanting cells into an animal with reduced or insufficient immune function, followed by rearing the mice for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at the latest 15 to 31 days. Rearing can be performed by a general method, but it is preferable to rear multiple mice in one space (e.g., in a cage, etc.) in an environment where the multiple mice are reared in an area of 50 to 200 ^cm2 or 130 to 180 ^cm2 .

Embodiments of model animals, allogeneic animals, cells of allogeneic animals, etc. are described above.

In clinical practice, meibomian gland dysfunction typically presents with symptoms such as abnormal findings around the meibomian gland orifice, obstruction of the meibomian gland orifice, and meibomian gland atrophy. Abnormal findings around the meibomian gland orifice include irregular eyelid margins. This model animal has the characteristics of meibomian gland dysfunction such as "obstruction of the meibomian gland orifice," "meibomian gland atrophy," and "irregular eyelid margins." These characteristics in this model animal are reversible, and this model animal can be used to develop therapeutic and preventive agents for eye diseases, particularly meibomian gland dysfunction.

In clinical practice, dry eye typically presents with objective symptoms such as tear breakup time (BUT) and subjective symptoms (eye discomfort or visual abnormality). BUT is an essential test item for diagnosing dry eye in Japan. The U.S. Food and Drug Administration (FDA) standard for developing a drug for treating dry eye requires that at least one objective finding be significantly improved, and BUT is included in the objective findings. Subjective symptoms (eye discomfort, visual abnormality, etc.) are also essential test items for diagnosing dry eye in Japan, and are scored by asking the patient about them in clinical practice. Subjective symptoms of human dry eye can be evaluated in model animals using the blink rate, with an increase in the blink rate indicating a worsening of subjective symptoms. This model animal has "BUT" or "blink rate" as a characteristic related to dry eye. This characteristic in this model animal is reversible, and this model animal can be used to develop treatments and preventive agents for eye diseases.

Dry eye may also be accompanied by objective symptoms such as "corneal and conjunctival epithelial damage," "decreased tear volume," and "abnormal tear oil layer." "Corneal and conjunctival epithelial damage" is a condition in which the cornea or conjunctiva is damaged and impaired, and is clinically evaluated by staining tests. Corneal and conjunctival epithelial damage includes superficial punctate keratopathy, which is a defect in the corneal epithelial layer down to the wing cells, corneal erosion, which is a defect in the entire epithelium including the basal cells but not the basement membrane, and corneal ulcer, which is a lesion that extends beyond the basement membrane to the parenchyma. Corneal and conjunctival epithelial damage is not essential for the diagnosis of dry eye, but it is desirable to show efficacy as a drug for treating dry eye. "Corneal and conjunctival epithelial damage" is scored by checking slit lamp microscope images stained/unstained with a dye (e.g. fluorescein) in a staining test. The "corneal staining spot score" in a model animal corresponds to the scoring of the corneal and conjunctival epithelial damage. "Decreased tear volume" is also not essential for the diagnosis of dry eye, but it is desirable to show efficacy as a drug for treating dry eye. Tear volume is one of the objective findings that must be improved in the FDA's standards for developing a drug for dry eye. Clinically, tear volume can be measured by the Schirmer test, and it can be measured using a similar method in model animals. In addition, although "abnormal tear lipid layer" is not essential for diagnosing dry eye, it is desirable to confirm its improvement as an indicator for developing a drug for dry eye. Abnormal tear lipid layer is an abnormality in the quantity and quality of lipids, which affects the stability of tears. Clinically, "abnormal tear lipid layer" is detected by observing "tear film optical interference," but it can also be scored and measured using a similar method in model animals. This model animal has "abnormal corneal staining spot score," "decreased tear volume," or "abnormal tear film optical interference score" as characteristics related to dry eye, and these characteristics in this model animal are reversible. Therefore, this model animal can be advantageously used in the development of treatments and preventive agents for eye diseases.

The model animal of the present disclosure can be used to develop therapeutic and/or preventive agents for meibomian gland dysfunction, and for dry eye associated with meibomian gland dysfunction, such as therapeutic and/or preventive agents for evaporative dry eye.

(Drug efficacy evaluation method)
In a different aspect, the present disclosure provides a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an ocular disease, the method comprising: (1) administering the test substance to an ocular disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye, wherein the ocular disease model animal is an animal with reduced or impaired immune function transplanted with cells from an allogeneic animal. In certain embodiments, the ocular disease may be dry eye and/or meibomian gland dysfunction.

In a further aspect, the present disclosure provides a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction, the method comprising: (1) administering the test substance to an animal model of meibomian gland dysfunction; and (2) evaluating characteristics associated with meibomian gland dysfunction.

In a further aspect, the present disclosure provides a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for dry eye, the method comprising: (1) administering the test substance to a dry eye model animal; and (2) evaluating characteristics associated with meibomian gland dysfunction and characteristics associated with dry eye.

In some embodiments, in step (1), the method of administration of the test substance is oral administration, eye drop administration, intraocular injection administration, etc., and the dosage form of the administered test substance may be a tablet, eye drop, injection, etc. In a preferred embodiment, the test substance may be administered by eye drop administration. For example, the test substance may be administered 1 to 3 days, preferably 1 to 2 days, after the onset of characteristics associated with meibomian gland dysfunction or characteristics associated with dry eye, or 6 to 9 days, preferably 7 to 8 days, after transplantation of cells from an animal allogeneic to the model animal into the model animal.

The method for evaluating the efficacy of a drug in the present disclosure may include a step of evaluating the test substance as having a medicinal effect if the test substance improves or inhibits the progression of characteristics associated with meibomian gland dysfunction and/or characteristics associated with dry eye in a model animal to which the test substance is administered.

In some embodiments, the efficacy of the test substance can be evaluated by changing the administration route, formulation composition, and formulation form and concentration.

In some embodiments, in step (2), the characteristic associated with meibomian gland dysfunction may be at least one characteristic selected from obstruction of meibomian gland orifices, atrophy of meibomian glands, and eyelid margin irregularities, and the characteristic associated with dry eye may be at least one characteristic selected from abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate.

In some embodiments, step (2) may assess features associated with dry eye and/or features associated with meibomian gland dysfunction. In certain embodiments, step (2) may assess at least one, at least two, or three features associated with meibomian gland dysfunction and at least one, at least two, at least three, at least four, or five features associated with dry eye.

Preferably, the obstruction of the meibomian gland orifices in the model animal can be determined to be an improved characteristic, for example, if the number of obstructions is reduced compared to before administration of the test substance, or the progression of the characteristic can be determined to be inhibited, for example, if the number of obstructions is reduced when the test substance is administered compared to when a placebo is administered.

Preferably, the atrophy of the meibomian glands in the model animal can be determined to have improved as a characteristic when, for example, the size of the acini of the meibomian glands increases compared to before administration of the test substance, when the accumulation of meibum increases, or when the reflection of infrared light by the accumulated oil increases, or when the reflex that should be anatomically present is restored. Alternatively, the progression of the characteristic can be determined to have been inhibited, for example, when the acini of the meibomian glands are larger when the test substance is administered compared to when a placebo is administered, when the accumulation of meibum increases, or when the reflection of infrared light by the accumulated oil increases, or when the reflex that should be anatomically present is restored.

Preferably, the characteristic of eyelid margin irregularity in a model animal can be determined to have improved, for example, when the eyelid margin line becomes less concave and smoother compared to before administration of the test substance, or the progression of the characteristic can be determined to have been inhibited, for example, when the eyelid margin line becomes less concave and smoother when the test substance is administered compared to when a placebo is administered.

Preferably, for example, if the BUT in the model animal is increased compared to before administration of the test substance, it can be determined that the characteristic has been improved, or, for example, if the BUT is increased when the test substance is administered compared to when a placebo is administered, it can be determined that the progression of the characteristic has been inhibited.

Preferably, for example, if the number of blinks in the model animal is reduced compared to before administration of the test substance, it can be determined that the characteristic has been improved. Alternatively, for example, if the number of blinks is reduced when the test substance is administered compared to when a placebo is administered, it can be determined that the progression of the characteristic has been inhibited.

Preferably, for example, if the corneal staining spot score in the model animal is reduced compared to before administration of the test substance, it can be determined that the characteristic has been improved, or, for example, if the score is reduced when the test substance is administered compared to when a placebo is administered, it can be determined that the progression of the characteristic has been inhibited.

Preferably, if the tear volume in the model animal is increased compared to before administration of the test substance, it can be determined that the characteristic has been improved. Alternatively, for example, if the tear volume in the model animal is increased when the test substance is administered compared to when a placebo is administered, it can be determined that the progression of the characteristic has been inhibited.

Preferably, the characteristic can be determined to have improved when the tear film optical interference score in the model animal is reduced compared to before administration of the test substance, or the progression of the characteristic can be determined to have been inhibited when the tear film optical interference score in the model animal is reduced, for example, when the test substance is administered compared to when a placebo is administered.

Preferably, an improvement in each characteristic refers to a significant difference in the measured value of the characteristic after administration of the test substance compared to before administration of the test substance, or a difference of 10% or more, 20% or more, 30% or more, 40% or more, 45% or more, 46% or more, or 48% or more.

Preferably, inhibition of progression of each characteristic refers to a case where there is a significant difference in the measured value of the characteristic when the test substance is administered compared to when a placebo is administered, or there is a difference of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more.

Note that a significant difference can be confirmed using common statistical methods, and means that there is a statistically significant difference.

In a further embodiment, in step (2), at least the obstruction of the meibomian gland orifice, the tear film breakup time, and the blink rate may be evaluated.

Embodiments of model animals, allogeneic animals, cells of allogeneic animals, etc. are described above.

(Screening Method)
In a different aspect, the present disclosure provides a method for screening for a therapeutic and/or preventive agent for dry eye, the method comprising the steps of: (1) administering a test substance to a dry eye model animal; (2) evaluating characteristics associated with meibomian gland dysfunction and characteristics associated with dry eye; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when the evaluated characteristics are improved or progression is inhibited in the dry eye model animal.

In a different aspect, the present disclosure provides a method for screening for a therapeutic and/or preventive agent for meibomian gland dysfunction, the method comprising the steps of: (1) administering a test substance to an animal with meibomian gland dysfunction; (2) evaluating characteristics associated with meibomian gland dysfunction; and (3) identifying the test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction when the evaluated characteristics improve or progression is inhibited in the animal with meibomian gland dysfunction.

In a further aspect, the present disclosure provides a method for screening for a therapeutic and/or preventive agent for an eye disease, the method comprising: (1) administering a test substance to an eye disease model animal, the eye disease model animal being an immunodeficient animal transplanted with immune cells from an allogeneic animal; and (2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye; and (3) identifying the test substance as a therapeutic and/or preventive agent for an eye disease when the evaluated characteristic improves or progression is inhibited in the eye disease model animal.

　Embodiments of model animals, allogeneic animals, allogeneic animal cells, evaluation items, etc. are described above.

In a further aspect, the screening method may include a step (1) in which the test substance is two or more candidate substances, and a step (3) in which a therapeutic agent and/or a preventive agent for an eye disease is selected from the plurality of candidate substances.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

The present disclosure will be explained in more detail below based on examples, but these are intended to provide a better understanding of the present disclosure and are not intended to limit the scope of the present disclosure. In addition, this example was carried out with the approval of the Animal Experiment Ethics Committee of Senju Pharmaceutical Co., Ltd.

Example 1: Preparation and evaluation of eye disease model mice
1. Test Method 1-1. Preparation of Model Mice Five to seven week old female C.B17 ICR SCID mice (CB17/Icr-Prkdc ^scid /CrlCrlj, H-2D ^d : purchased from Charles River Japan, Inc.) were divided into a sham group and a spleen-derived cell administration group (six mice per group). Five mice were used for time-course observation and one mouse was used for pathological observation in each group.

The sham group received 200 μL of PBS (manufactured by Thermo Fisher Scientific) into the tail vein. The spleen-derived cell administration group received 200 μL of PBS suspension of spleen-derived cells (2.5×10 ⁷ cells/mL) into the tail vein. The PBS suspension of spleen-derived cells (hereinafter referred to as "spleen-derived cell suspension") was prepared by making an incision in the spleen taken from a C57BL/6 mouse (H-2D ^b ), dispersing the cells by pipetting several times, and suspending them in PBS. After administration of PBS or the spleen-derived cell suspension, the mice were kept in a cage (size: length 338 mm, width 225 mm, height 140 mm) in groups of five.

1-2. Evaluation of each characteristic (a) Evaluation of characteristics related to meibomian gland dysfunction (a1) Number of obstructed meibomian gland orifices The day of administration of the spleen-derived cell suspension was set as day 0, and the number of obstructed meibomian glands in the right eye of each group of mice was counted under anesthesia using a stereomicroscope on days 8, 15, and 21 after administration. The number of obstructed meibomian glands was counted under observation under a stereomicroscope by gently everting the upper and lower eyelids to expose the meibomian gland orifices, and counting the number of orifices that were found to be blocked from the medial canthus toward the lateral canthus.

The meibomian gland orifice was judged to be obstructed when, under a stereomicroscope, a white to yellow protrusion was observed at the orifice, the orifice was pushed open by the contents, or a cylindrical or rice-grain-shaped mass was observed within the orifice along the meibomian gland duct. The total number of obstructions observed on the upper and lower eyelids was used as the individual value for the number of obstructed meibomian glands, and the average value for five mice was calculated.

(a2) Atrophy of Meibomian Glands As in the confirmation of the number of obstructed meibomian glands, the day of administration of the spleen-derived cell suspension was set as day 0, and atrophy of the meibomian glands in the right eye was confirmed under anesthesia using a stereomicroscope on days 8, 15, and 21 after administration, and the number of mice with atrophy was counted.

　Meibomian gland atrophy was evaluated by observing the area of the meibomian glands visible under the conjunctiva by inverting the upper and lower eyelids under a stereomicroscope. Atrophy was determined when the area was smaller than the size of meibomian glands in a typical mouse, and the number of mice with one or more atrophied meibomian glands was counted.

Meibomian gland atrophy was also confirmed anatomically. 28 days after administration of the spleen-derived cell suspension, the mice were euthanized and the eyelids were harvested. The harvested eyelids were embedded and frozen in OCT compound (Sakura Finetech Japan), and 10 μm-thick sagittal frozen sections were prepared. The obtained sections were stained red for lipids in the meibomian glands using Oil Red O, and the cell nuclei were stained blue using hematoxylin dye. After staining, the condition of the meibomian glands in the sections was observed under a microscope.

(a3) Irregular eyelid margin The day of administration of the spleen-derived cell suspension was set as day 0, and on days 7, 14, and 21 after administration, irregular eyelid margins of the right eye were observed under anesthesia using a stereomicroscope.

　A sodium fluorescein solution (a solution made by dissolving uranine (manufactured by Fujifilm Wako Pure Chemical Industries) in physiological saline and filtering it) was instilled into the right eye of anesthetized mice and mixed well with the tears by forcing the mice to blink several times. The mice were then placed under fluorescent observation with a stereomicroscope (excitation wavelength bandpass 460-480 nm, fluorescence wavelength bandpass 495-540 nm), and fluorescent stereomicroscope images of the eyelid margin were taken and observed. A smooth curved line drawn by fluorescein at the edge of the eyelid was considered normal, and an irregular eyelid margin was judged to be present when an uneven surface was drawn.

(b) Evaluation of characteristics related to dry eye (b1) Tear film break-up time (BUT)
The day of administration of the spleen-derived cell suspension was set as day 0, and on days 7, 14, and 21 after administration, the tear film breakup time in the right eye was measured under anesthesia using a stereomicroscope.

The tear film breakdown time was measured using a fluorescein sodium solution, as in (a3). As in (a3), the fluorescein sodium solution was instilled into the right eye of anesthetized mice and mixed well with the tear fluid by forced blinking several times. The mice were then placed under fluorescent observation with a stereomicroscope (excitation wavelength bandpass 460-480 nm, fluorescence wavelength bandpass 495-540 nm). The mixed solution of tear fluid and fluorescein covered the cornea, and tear film breakdown could be evaluated by confirming the disappearance of the fluorescence of fluorescein. After placing the mice under fluorescent observation with a stereomicroscope, the right eye of the mice was forced to open, and the time from the moment the eyelids were opened to the appearance of the area where the fluorescence disappeared was measured three times, and the average of the three measurements was taken as the individual value. The average of the individual values of the five mice was calculated and used as the tear film breakdown time (BUT).

(b2) Number of blinks The day of administration of the spleen-derived cell suspension was set as day 0, and on the 4th, 11th, and 18th days after administration, the mice were transferred to small observation cages and isolated one by one, and the number of blinks of the left eye of the mice moving freely in the cage was measured. Measurements were performed 5 times for 1 minute per mouse, and the total of the 5 measurements was used as the individual value, and the average value of the 5 mice was calculated. After the measurements, the mice were returned to the breeding cage, and the 5 mice were kept in one cage as usual and continued to be bred.

(b3) Corneal staining spot score The day of administration of the spleen-derived cell suspension was set as day 0, and the corneal staining spot score (SPK score) of the right eye was measured under anesthesia using a stereomicroscope on days 7, 14, and 21 after administration.

(b1) After measuring the tear film breakup time, the eyes were washed with saline and observed for corneal staining. After washing the eyes, the mice were again placed under fluorescent observation with a stereomicroscope (excitation wavelength bandpass 460-480 nm, fluorescence wavelength bandpass 495-540 nm). Corneal staining was observed by confirming dot-like fluorescence due to fluorescein on the outermost layer of the cornea.

Observations were performed in five quadrants, the upper, right, lower, left and center of the cornea, and each was assessed as having no dot-like fluorescence (0 points), sparse (1 point), slightly dense (2 points) or dense (3 points). The total score for the five quadrants was calculated and used as an individual value for the corneal staining spot score (SPK score). The average value was calculated from the individual values obtained.

(b4) Tear volume The day of administration of the spleen-derived cell suspension was set as day 0, and tear volume in the right eye was measured under a stereomicroscope while the mouse was awake and restrained on days 7, 14, and 21 after administration.

One end of a cotton thread containing phenol red (Zone Quick, manufactured by Ayumi Pharmaceutical Co., Ltd.) was inserted into the conjunctival fornix of the right eye of a mouse and held there for 15 seconds, after which the thread was immediately removed. The length of discoloration of the phenol red contained in the cotton thread caused by tears was measured and used as an index of tear volume. The average value was calculated from the measured lengths.

(b5) Tear Layer Optical Interference Score The day of administration of the spleen-derived cell suspension was set as day 0, and on day 31 after administration, the tear layer optical interference of the right eye was observed under anesthesia using a stereomicroscope.

After forcibly opening the right eye of the mouse, the microscope was focused on the tear film, and the area was scored from 1 to 5 based on the number of iridescent stripes caused by optical interference and the brightness of the light reflection from the entire tear film. Scoring was performed according to the report by Yokoi et al. Am J Ophthalmol 1996 Dec;122(6):818-24. The average score was calculated from the obtained scores.

2. Results (a1) Number of obstructed meibomian gland orifices The number of obstructed meibomian gland orifices was significantly increased in the spleen-derived cell administration group compared to the sham group on days 8 and 15 after administration of the spleen-derived cell suspension, and this increasing trend continued until day 22 (Table 1).

(a2) Atrophy of the Meibomian Glands Meibomian gland atrophy developed in 4 out of 5 mice in the spleen-derived cell administration group from 8 days after administration of the spleen-derived cell suspension (Table 2). Furthermore, pathological eyelid sections taken from the spleen-derived cell administration group 28 days after administration of the spleen-derived cell suspension showed severe atrophy of the meibomian glands (FIG. 1).

(a3) Irregular eyelid margin Irregular eyelid margin was observed from day 7 after administration of the spleen-derived cell suspension. On day 21 after administration, the eyelid edge in the sham group was visualized as a smooth curve by fluorescein, whereas in the spleen-derived cell administration group, it was visualized as an uneven surface, confirming the irregular eyelid margin ( FIG. 2 ).

(b1) Tear film breakup time (BUT)
The tear film breakup time was significantly shortened in the spleen-derived cell administration group compared to the sham group on days 7, 14, and 21 after administration of the spleen-derived cell suspension (Table 3).

(b2) Number of Blinks The number of blinks tended to increase in the spleen-derived cell administration group on day 11 after administration of the spleen-derived cell suspension, and showed a significant increase compared to the sham group on day 18 after administration (Table 4).

(b3) Corneal staining spot score The corneal staining spot score was significantly increased in the spleen-derived cell administration group compared to the sham group on day 14 after administration of the spleen-derived cell suspension (Table 5).

(b4) Tear Volume Tear volume was significantly reduced in the spleen-derived cell administration group compared to the sham group on days 7 and 14 after administration of the spleen-derived cell suspension (Table 6).

(b5) Tear Film Optical Interference Score The tear film optical interference score was significantly increased in the spleen-derived cell administration group compared to the sham group on day 31 after administration of the spleen-derived cell suspension (Table 7).

These results indicate that C.B17 ICR SCID mice that were intravenously administered a suspension of spleen-derived cells collected from C57BL/6 mice exhibited symptoms of human meibomian gland dysfunction and human dry eye.

(Example 2: Observation of localization of spleen-derived cells)
The purpose of this example was to explore the mechanism of meibomian gland dysfunction (MGD) and dry eye (DE) symptoms in the model animal of the present disclosure. In the same manner as in Example 1, spleen-derived cells isolated from donor C57BL/6 mice (H-2D ^b ) were fluorescently labeled using PKH reagent (Sigma-Aldrich) and administered intravenously to C.B17 icr-SCID mice (CB17/Icr-Prkdc ^scid /CrlCrlj, H-2D ^d : purchased from Charles River Japan, Inc.), and the anterior segment was observed over time.

Specifically, the PKH reagent was added to the spleen-derived cell suspension prepared in the same manner as in Example 1, and mixed to perform labeling. The fluorescently labeled spleen-derived cell suspension (hereinafter, "fluorescently labeled spleen-derived cells") was then administered to C. B17 icr-SCID mice via the tail vein. Two days after administration of the fluorescently labeled spleen-derived cells, the cells were observed under a stereo microscope using an observation wavelength with an excitation wavelength bandpass of 460-480 nm and an emission wavelength bandpass of 495-540 nm. The PKH reagent binds to the lipid region of the cell membrane, and is thought to label T cells, B cells, and the like. The peak excitation wavelength of PKH26 is 551 nm, the peak emission wavelength is 567 nm, and the peak excitation wavelength of PKH67 is 490 nm, the peak emission wavelength is 502 nm. Therefore, at the above observation wavelengths, PKH26 does not show fluorescence, and PKH67 shows fluorescence. When PKH26 is used, only autofluorescence from the living body is observed, so the fluorescence seen only when PKH67 is used can be determined to be the localization of labeled cells derived from the spleen.

Figure 3 shows a photograph taken two days after administration of fluorescently labeled spleen-derived cells. In animals transplanted with PKH67 fluorescently labeled spleen-derived cells, fluorescence was observed in the meibomian gland tissue, as well as fluorescent secretions from the meibomian glands.

From these results, it was considered that the model animals of the present disclosure exhibited MGD and DE symptoms as a result of the administered spleen-derived cells themselves or components of the administered spleen-derived cells reaching tissues that contribute to the homeostasis of tears, such as the meibomian glands. Without wishing to be bound by theory, it is believed that when the spleen-derived cells themselves reach the meibomian glands of the host cells, the spleen-derived cells recognize them as non-self, causing an immune reaction and damaging the meibomian glands. Without wishing to be bound by theory, when the cell membrane components of the spleen-derived cells reach the meibomian glands, the cell membrane components of the spleen-derived cells are supplied to the meibomian glands via the blood and taken in as a raw material for meibum, but the properties of the synthesized meibum change, the openings are blocked, and the balance of differentiation from stem cells to ductal epithelial cells or acinar cells is shifted toward differentiation to ductal epithelial cells, causing the meibomian glands to shrink. The spleen-derived cells include the spleen itself and immune cells such as T cells and B cells contained in the spleen.

(Example 3: Evaluation of efficacy of test substance in model animals)
In this example, the efficacy of an azithromycin-containing eye drop as a test substance was evaluated using this model animal. Azithromycin is known to be effective against meibomian gland dysfunction. This example was conducted with the approval of the Animal Experiment Ethics Committee of Senju Pharmaceutical Co., Ltd.

(Generation and evaluation of eye disease model mice)
1. Test Method 1-1. Preparation of Model Mice Model mice were prepared in the same manner as in Example 1. 200 μL/animal of PBS suspension of spleen-derived cells (2.5×10 ⁷ cells/mL) was administered into the tail vein of 5-7 week-old female C.B17 ICR SCID mice (CB17/Icr-Prkdc ^scid /CrlCrlj: purchased from Charles River Japan, Inc.). The PBS suspension of spleen-derived cells (hereinafter referred to as "spleen-derived cell suspension") was prepared by making an incision in the spleen taken from a C57BL/6 mouse, pipetting several times, and dispersing in PBS.

After administration of the spleen-derived cell suspension, the mice were housed in cages (dimensions: length 338 mm, width 225 mm, height 140 mm) in groups of five or six.

1-2. Administration of test substance The day of administration of the spleen-derived cell suspension was set as day 0, and on the 10th day after administration, the mice were divided into a control eye drop administration group and an azithromycin-containing eye drop administration group (1% azithromycin eye drop, manufactured by Senju Pharmaceutical Co., Ltd.) (8 mice per group). From the 10th day after administration, the control eye drop or the azithromycin-containing eye drop was administered to both eyes twice a day, 2 μL per administration, for 2 days, and from the 12th day after administration, the control eye drop or the azithromycin-containing eye drop was administered once a day, 2 μL per administration, for 12 days. The control eye drop was an isotonic buffer solution. The day when the administration of the control eye drop or the azithromycin-containing eye drop was started (10th day after administration of the spleen-derived cell suspension) was counted as the 1st day of administration.

1-3. Confirmation of the number of obstructed meibomian gland orifices, BUT, and blink count On the 10th day (before the start of eye drop administration), the 15th day (6th day of eye drop administration), and the 22nd day (13th day of eye drop administration) after administration of the cell suspension, the number of obstructed meibomian gland orifices in the right eye of each mouse group was measured under anesthesia. The number of obstructed meibomian gland orifices was measured by gently everting the upper and lower eyelids of the mouse under observation with a stereomicroscope to expose the meibomian gland orifices, and the number of orifices that were found to be obstructed from the medial canthus to the lateral canthus was counted. Obstruction of the meibomian gland orifices was evaluated in the same manner as in Example 1.

The total number of blockages observed on the upper and lower eyelids was taken as the individual value for the number of blockages of the meibomian gland orifices, and the average of the individual values was calculated. BUT and blink count were also evaluated in the same manner as in Example 1.

2. Results The average number of blocked meibomian gland orifices in the control eye drop group changed from 10.8 before the start of eye drop administration to 15.0 on the 6th day and 17.8 on the 13th day, a change of +7.0. In the azithromycin-containing eye drop group, the average number of blocked meibomian gland orifices changed from 10.5 before the start of eye drop administration to 12.0 on the 6th day and 11.3 on the 13th day, showing a significant decrease in the number of blocked meibomian gland orifices in the azithromycin-containing eye drop group compared to the control eye drop group (p=0.00158, t-test, 2-sided).

The average BUT in the control eye drop group changed from 0.59 seconds before the start of eye drop administration to 0.7 seconds on the 6th day of eye drop administration and 0.38 seconds on the 13th day of eye drop administration. In the azithromycin eye drop group, the average BUT changed from 0.59 seconds before the start of eye drop administration to 0.76 seconds on the 6th day of eye drop administration and 1.10 seconds on the 13th day of eye drop administration. In the azithromycin eye drop group, a significant increase in BUT was observed on the 13th day of eye drop administration compared to the control eye drop group (p = 0.003122, t-test, 2-sided)

The average number of blinks in the control eye drop group changed from 2.79 before the start of eye drop administration to 2.25 on the 5th day of administration and 3.53 on the 12th day of administration, a change of +0.74 blinks. In the azithromycin eye drop group, the average number of blinks changed from 3.67 before the start of eye drop administration to 4.00 on the 5th day of administration and 2.84 on the 12th day of administration, a change of -0.83 blinks.

Therefore, it was found that in this animal model, the blockage of the meibomian gland orifice, BUT, and blink rate were reversible and can be used to evaluate drug efficacy.

Furthermore, using a similar method, it was confirmed that the tear film optical interference score in this model animal improved with the drug. It was also found that the corneal staining spot score and tear volume could be changed in this model animal. Therefore, these evaluation items are reversible and can be used to evaluate the efficacy of drugs.

(Example 4: Confirmation of distribution of immune cells in meibomian glands)
Arrival of immune cells (e.g., cells derived from the spleen) in the meibomian glands can be confirmed by the following technique.

After euthanizing the mice, the eyelids were harvested and embedded and frozen in OCT compound (Sakura Finetech Japan), and 10 μm-thick sagittal frozen sections were prepared.

Immunohistochemical staining is performed using anti-CD3 (T cell surface antigen) antibody, anti-CD19 (B cell surface antigen) antibody, anti-F4/80 (macrophage surface antigen) antibody, anti-CD11 (dendritic cell surface antigen) antibody, anti-NK1.1 (NK cell surface antigen) antibody, anti-CD31 (endothelial cell marker) or anti-Claudin-5 (endothelial cell marker), and anti-mouse MHC Class I H2-D ^d antibody (SCID mouse tissue marker).

If the mouse is a SCID mouse that lacks T cells and B cells, the presence or absence of CD3 or CD19 positive cells can be determined, and if positive cells are present, it can be determined that cells from an allogeneic animal have been mixed in.

Macrophages, dendritic cells, NK cells, and endothelial cells are included in the cells derived from the spleen, and these have MHC class I. Therefore, whether the mouse is a SCID mouse or not, the presence of spleen-derived cells in the eyelid of the mouse and whether the spleen-derived cells have a different MHC class I type from that of the mouse are detected to confirm the contamination with cells from an allogeneic animal. When the mouse is a SCID mouse, the presence or absence of cells that are MHC Class I H2-D ^d negative and F4/80, CD11, NK1.1, CD31, or Claudin-5 positive is determined, and if cells that satisfy both criteria are present, it can be determined that allogeneic animal cells have been contaminated.

As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted only in terms of the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth in this specification.

Claims (18)

A method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering the test substance to an ophthalmic disease model animal; and (2) evaluating at least one of a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in the ophthalmic disease model animal,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method. The method of claim 1, further comprising evaluating at least one characteristic associated with meibomian gland dysfunction in step (2). The method according to claim 1 or 2, wherein in step (2), the characteristics associated with meibomian gland dysfunction include at least one selected from the group consisting of obstruction of the meibomian gland orifice, atrophy of the meibomian gland, and eyelid margin irregularity, and the characteristics associated with dry eye include at least one selected from the group consisting of abnormal tear film breakup time, abnormal corneal staining score, reduced tear volume, abnormal tear film optical interference score, and abnormal blink rate. The method according to any one of claims 1 to 3, wherein in step (2), the characteristic associated with meibomian gland dysfunction includes at least blockage of the meibomian gland orifice. The method according to any one of claims 1 to 4, wherein step (2) includes a step of evaluating at least one characteristic associated with meibomian gland dysfunction and at least one characteristic associated with dry eye. In the step (2), the characteristics associated with meibomian gland dysfunction include at least blockage of the meibomian gland orifice,
6. The method of claim 5, wherein the features associated with dry eye include at least an abnormality in tear film break-up time and an abnormality in blink rate. The method according to any one of claims 1 to 6, wherein the eye disease is meibomian gland dysfunction. The method according to any one of claims 1 to 6, wherein the eye disease is dry eye. The method according to any one of claims 1 to 8, wherein the cells of the allogeneic animal are immune cells. The method of claim 9, wherein the allogeneic animal cells are immune cells derived from the spleen, blood, bone marrow, thymus, or lymph nodes. The method of claim 10, wherein the allogeneic animal cells are spleen-derived cells. The method according to any one of claims 1 to 11, wherein the eye disease model animal is a mouse whose MHC type is H- ^2Dd . The method according to any one of claims 1 to 12, wherein the eye disease model animal is a SCID mouse or a NOD-SCID mouse. The method according to any one of claims 1 to 13, wherein the allogeneic animal is a mouse having a different MHC type from that of the eye disease model animal. The method according to any one of claims 1 to 14, wherein the allogeneic animal is a mouse whose MHC type is H- ^2Db . A method for screening a therapeutic and/or preventive agent for an eye disease, the method comprising:
(1) administering a test substance to an eye disease model animal;
(2) evaluating a characteristic associated with meibomian gland dysfunction or a characteristic associated with dry eye in an ophthalmic disease model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for an ophthalmic disease when the evaluated characteristic is improved or progression is inhibited,
The eye disease model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted.
method. A method for screening a therapeutic and/or preventive agent for meibomian gland dysfunction, the method comprising:
(1) administering a test substance to a meibomian gland dysfunction model animal;
(2) evaluating blockage of the meibomian gland orifice in a meibomian gland dysfunction animal model; and (3) identifying the test substance as a therapeutic and/or preventive agent for meibomian gland dysfunction when blockage of the meibomian gland orifice is improved and/or progression of blockage of the meibomian gland orifice is inhibited in the meibomian gland dysfunction animal model,
the meibomian gland dysfunction model animal is an animal having a reduced or insufficient immune function to which cells from an allogeneic animal have been transplanted,
the MHC type of the meibomian gland dysfunction model animal is H-2D ^d ;
the MHC type of the allogeneic animal is H- ^2Db ;
Screening methods. A method for screening a therapeutic and/or prophylactic agent for dry eye, the method comprising:
(1) administering a test substance to a dry eye model animal;
(2) evaluating blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency in a dry eye model animal; and (3) identifying the test substance as a therapeutic and/or preventive agent for dry eye when blockage of the meibomian gland orifice, abnormality in tear film breakup time, and abnormality in the blink frequency are improved or inhibited from progressing in the dry eye model animal,
the dry eye model animal is an animal with reduced or deficient immune function to which cells from an allogeneic animal have been transplanted,
the MHC type of the dry eye model animal is H- ^2Dd ;
the MHC type of the allogeneic animal is H- ^2Db ;
Screening methods.