Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• 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.
• 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.
• 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.
• 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 .
• MHC major histocompatibility complex
• 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.
• a 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.
• a 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.
• 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.
• MMD meibomian gland dysfunction
• 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.
• 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.
• 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.
• MGD meibomian gland dysfunction
• (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.
• a method for evaluating the efficacy of a test substance as a therapeutic and/or preventive agent for an eye disease 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.
• step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
• 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
• 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.
• step (2) 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.
• step (2) 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.
• 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.
• a method for evaluating the efficacy of a test substance as a therapeutic and/or prophylactic agent for dry eye 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.
• MHC major histocompatibility complex
• (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.
• a method for screening a therapeutic and/or preventive agent for an eye disease 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.
• step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
• 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.
• step (2) 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.
• step (2) 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.
• step (2) 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.
• a method for screening a therapeutic and/or prophylactic agent for dry eye 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.
• MHC major histocompatibility complex
• (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 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.
• the method comprises evaluating at least one characteristic associated with meibomian gland dysfunction.
• step (2) 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.
• step (2) 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.
• 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.
• 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.
• 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.
• the eye disease is meibomian gland dysfunction.
• eye disease is dry eye.
• the allogeneic animal cells are immune cells.
• a method for screening a therapeutic and/or preventive agent for an eye disease 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.
• a method for screening an agent for treating and/or preventing meibomian gland dysfunction 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.
• MHC major histocompatibility complex
• a method for screening a therapeutic and/or prophylactic agent for dry eye 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.
• MHC major histocompatibility complex
• (Item B1) 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.
• step (2) comprises a step of evaluating characteristics associated with dry eye.
• step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
• 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.
• step (2) 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.
• step (2) 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.
• 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 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.
• 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.
• MHC major histocompatibility complex
• a use of an eye disease model animal in a method for screening a therapeutic and/or preventive agent for an eye disease 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.
• step (2) comprises a step of evaluating characteristics associated with dry eye.
• step (2) comprises a step of evaluating characteristics associated with meibomian gland dysfunction.
• 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
• 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.
• step (2) 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.
• 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.
• 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.
• a use of a dry eye model animal in a method for screening a therapeutic and/or preventive agent for dry eye 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.
• MHC major histocompatibility complex
• 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)
• step (2) comprises evaluating at least one characteristic associated with meibomian gland dysfunction.
• step (3) 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.
• step (2) 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.
• step (2) 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.
• 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.
• 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.
• step (3) includes at least a step of evaluating blockage of the meibomian gland orifice, abnormalities in tear film breakup time, and abnormalities in blink frequency.
• the eye disease is meibomian gland dysfunction.
• eye disease is dry eye.
• the allogeneic animal cells are immune cells.
• a use of an eye disease model animal in a method for screening a therapeutic and/or preventive agent for an eye disease 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.
• 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
• a use of a dry eye model animal in a method for screening a therapeutic and/or preventive agent for dry eye 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.
• MHC major histocompatibility complex
• 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.
• 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
• 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.
• the step (2) 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.
• 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.
• 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.
• MHC major histocompatibility complex
• 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.
• MHC major histocompatibility complex
• (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.
• 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.
• 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
• 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.
• 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.
• 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.
• a dry eye model animal for use in a method for screening a therapeutic and/or preventive agent for dry eye 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.
• MHC major histocompatibility complex
• (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.
• 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.
• the step (2) 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.
• the step (2) 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.
• 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.
• 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.
• 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.
• MHC major histocompatibility complex
• 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.
• a meibomian gland dysfunction model animal for use in a method for screening for a therapeutic and/or preventive agent for meibomian gland dysfunction 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
• MHC major histocomp
• a dry eye model animal for use in a method for screening a therapeutic and/or preventive agent for dry eye 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.
• MHC major histocompatibility complex
• 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.
• 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.
• MHC major histocompatibility complex
• (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 .
• 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.
• FIG. 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.
• 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.
• 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.
• BUT head break-up time
• 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.
• 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.
• 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.
• BUT tear breakup time
• 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.
• meibomian glands refers to sebaceous glands located within the tarsal plate with openings at the upper and lower eyelid margins.
• Meibum a lipid
• tears 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.
• 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.
• MSD Meibomian gland dysfunction
• Meibomian gland dysfunction 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).
• 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.
• "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.
• 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.
• 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.
• TERT head 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.
• number of blinks refers to the number of times you blink within a certain period of time.
• Numberer 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.
• 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.
• a dye e.g., fluorescein
• 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.
• tissue 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.
• the certain period of time is, for example, 15 seconds.
• the term “tear film optical interference score” is an index for evaluating tear abnormalities (e.g., lipid abnormalities in tears).
• Tea 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).
• 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.
• treatment means curing, improving, or suppressing or alleviating a disease or symptom.
• Treatment an eye disease includes treating objective or subjective symptoms.
• 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.
• subjective symptoms refers to symptoms of a disease that can be perceived by a patient suffering from the disease.
• objective symptoms refers to disease symptoms that can be objectively proven by findings (objective findings) such as imaging findings or numerical test results.
• animal refers to animals used as model animals, including mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.).
• mammals e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.
• 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.
• allogeneic refers to individuals of the same species but genetically distinct.
• 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.
• the major histocompatibility complex (MHC) type is H-2D d
• H-2D b refers to the homozygous b type of H-2D, a subclass that constitutes MHC class I.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a rodent such as a mouse, rat, or hamster
• 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.
• 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.
• Prkdc protein kinase, DNA activated, catalytic polypeptide
• the model animal of the present disclosure may be a SCID mouse or a NOD-SCID mouse.
• the model animal of the present disclosure can be CB17/Icr-Prkdc scid /CrlCrlj.
• the allogeneic animal may have a different major histocompatibility complex (MHC) type from the model animal.
• MHC major histocompatibility complex
• the MHC class I type of the model animal of the present disclosure is d
• the MHC class I type of the allogeneic animal may be a, b, k, q, or s other than d, but is preferably b.
• the subclass H-2D constituting the MHC class I of the model animal of the present disclosure is d
• 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 ).
• the model animal of the disclosure can be a SCID or NOD-SCID mouse with major histocompatibility complex (MHC) type H-2D d .
• the model animal of the disclosure can be a SCID or NOD-SCID mouse with major histocompatibility complex (MHC) type H-2D d
• the allogeneic animal can be a mouse with MHC type H-2D b .
• the allogeneic animal may be a C57BL/6 mouse, a C57BL/10 mouse, a C57L/J mouse, or a BXSB/Mp mouse.
• 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.
• 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").
• non-model animals e.g., animals that have not been administered allogeneic cells to the model animal
• the evaluation of "obstruction of meibomian gland orifices" can be performed using the number of obstructions of the meibomian gland orifices per individual.
• Atrophy of the meibomian glands can be confirmed, whereas no atrophy of the meibomian glands is observed in non-model animals.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• the model animals of this specification have a reduced BUT compared to non-model animals.
• the evaluation of "BUT” can be performed using the BUT per individual of a group of model animals under the same conditions.
• the model animals of this specification have an increased number of blinks compared to non-model animals.
• the "number of blinks" can be evaluated using the number of blinks per individual in a group of model animals under the same conditions.
• the corneal staining spot score is increased in the model animals of this specification compared to non-model animals.
• 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.
• the model animals of this specification have reduced tear volume compared to non-model animals.
• the "tear volume" can be evaluated using the tear volume per individual of a group of model animals under the same conditions.
• the tear layer optical interference score is increased in the model animals of this specification compared to non-model animals.
• 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.
• 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.
• 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.
• 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 .
• 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.
• 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.
• 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.
• 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 .
• MHC major histocompatibility complex
• 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.
• 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.
• model animals of the present disclosure may develop features associated with meibomian gland dysfunction after 4, 5, or 6 weeks of age.
• 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.
• 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.
• 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.
• 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.
• 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.
• MGD meibomian gland dysfunction
• 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.
• MGD meibomian gland dysfunction
• 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.
• 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 .
• 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.
• 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.
• 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.
• the ocular disease may be dry eye and/or meibomian gland dysfunction.
• 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.
• 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.
• the method of administration of the test substance is oral administration, eye drop administration, intraocular injection administration, etc.
• the dosage form of the administered test substance may be a tablet, eye drop, injection, etc.
• the test substance may be administered by eye drop administration.
• 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.
• the efficacy of the test substance can be evaluated by changing the administration route, formulation composition, and formulation form and concentration.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• step (2) at least the obstruction of the meibomian gland orifice, the tear film breakup time, and the blink rate may be evaluated.
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ L of PBS (manufactured by Thermo Fisher Scientific) into the tail vein.
• the spleen-derived cell administration group received 200 ⁇ L 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.
• 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.
• 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 ⁇ m-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.
• 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.
• the tear film breakdown time was measured using a fluorescein sodium solution, 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.
• mice 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).
• 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.
• 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 ).
• Example 2 Observation of localization of spleen-derived cells
• MMD meibomian gland dysfunction
• DE dry eye
• 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.
• PKH reagent Sigma-Aldrich
• 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.
• PKH26 does not show fluorescence, and PKH67 shows fluorescence.
• PKH26 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.
• fluorescence was observed in the meibomian gland tissue, as well as fluorescent secretions from the meibomian glands.
• 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.
• tissues that contribute to the homeostasis of tears such as the meibomian glands.
• the spleen-derived cells recognize them as non-self, causing an immune reaction and damaging the meibomian glands.
• the cell membrane components of the spleen-derived cells 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
• 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.
• mice were prepared in the same manner as in Example 1. 200 ⁇ L/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.
• mice 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.
• 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 ⁇ L 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 ⁇ L 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the eyelids were harvested and embedded and frozen in OCT compound (Sakura Finetech Japan), and 10 ⁇ m-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).
• anti-CD3 T cell surface antigen
• anti-CD19 B cell surface antigen
• anti-F4/80 macrophage surface antigen
• anti-CD11 dendritic cell surface antigen
• anti-NK1.1 NK cell surface antigen
• anti-CD31 endothelial cell marker
• anti-Claudin-5 endothelial cell marker
• SCID mouse tissue marker anti-mouse MHC Class I H2-D d antibody
• 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.
• 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.

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• 2023
  • 2023-06-16 WO PCT/JP2023/022377 patent/WO2024257328A1/ja not_active Ceased
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