WO2018230712A1 - TGF-β阻害剤の新規スクリーニング法 - Google Patents
TGF-β阻害剤の新規スクリーニング法 Download PDFInfo
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- WO2018230712A1 WO2018230712A1 PCT/JP2018/022944 JP2018022944W WO2018230712A1 WO 2018230712 A1 WO2018230712 A1 WO 2018230712A1 JP 2018022944 W JP2018022944 W JP 2018022944W WO 2018230712 A1 WO2018230712 A1 WO 2018230712A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5064—Endothelial cells
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/495—Transforming growth factor [TGF]
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
Definitions
- the present invention relates to a method and a kit for screening a compound effective for a disease in corneal endothelium using a model system of Fuchs corneal endothelial dystrophy.
- Visual information is transmitted from the cornea, the transparent tissue in the foreground of the eyeball, to reach the retina and excite the neurons of the retina, and the generated electrical signals are transmitted to the visual cortex via the optic nerve.
- the cornea needs to be transparent. The transparency of the cornea is maintained by keeping the water content constant by the pump function and the barrier function of corneal endothelial cells.
- Fuchs corneal endothelial dystrophy is a disease in which endothelial cells inside the cornea become abnormal and cause corneal edema, and the details of the cause are unknown.
- an extracellular matrix such as collagen is deposited on a part of the posterior surface of the Descemet's membrane at the back of the cornea, forming a Corneal guttae or thickening of the Descemet's membrane. It becomes.
- Fuchs corneal endothelial dystrophy has no effective treatment other than corneal transplantation, but the cornea donation in Japan is insufficient, and it is conducted in Japan annually for about 2600 patients waiting for corneal transplantation.
- the number of corneal transplants is about 1700.
- an animal test is performed after testing in advance by a method using a primary cultured cell collected from a living tissue or an immortalized cell (established cell) system that grows indefinitely.
- primary cells proliferate well at the initial stage, but gradually stop growing (cell aging) or undergo transformation with subculture.
- primary cells change with passage in addition to the problem of heterogeneity that their characteristics differ each time they are collected from living tissue. In particular, when the growth rate is very slow or when it is derived from a micro-organ, it is very difficult to obtain primary cells that are sufficient for the test.
- Patent Document 1 discloses a method for producing immortalized cells of Fuchs corneal endothelial dystrophy.
- immortalized cells of Fuchs corneal endothelial dystrophy are used in order to confirm the cytostatic effect.
- a rapid, simple and reliable screening method for screening a compound that is effective and highly safe for diseases in the corneal endothelium has not been reported so far.
- the present inventors use TGF- ⁇ inhibitory activity when contacting a candidate compound and immortalized cells of Fuchs' corneal endothelial dystrophy in the presence of TGF- ⁇ as an index, and are effective for diseases in the corneal endothelium. It was found that it was possible to select a compound, and further safer selection was achieved by performing toxicity evaluation. In addition, the TGF- ⁇ inhibitory activity score and toxicity score were processed and ranked to enable more reliable selection of candidate compounds. A method for conducting such screening at high throughput was also found.
- the present invention provides the following inventions.
- (Item 1) A method of screening for a compound effective for a symptom, disorder or disease in the corneal endothelium, the method comprising: (A) contacting the candidate compound with immortalized cells of Fuchs corneal endothelial dystrophy in the presence of transforming growth factor (TGF) - ⁇ , and evaluating the inhibitory activity of the candidate compound on the TGF- ⁇ ; Selecting a compound having (B) evaluating the toxicity of the compound selected in step (a) to the cells; (C) optionally evaluating the inhibitory activity against the TGF- ⁇ of the compound selected in step (a); (D) selecting a compound that is evaluated as having low toxicity to the cell in the step (b) and having an inhibitory activity against the TGF- ⁇ in the step (a) or (c); Including a method.
- TGF transforming growth factor
- Steps (a) and (c) above comprise measuring caspase activity, wherein the inhibitory activity against TGF- ⁇ is caspase activity that is substantially reduced relative to the candidate compound non-contact group, 2.
- the method according to 2. (Item 4) The number of trials for evaluating the inhibitory activity against TGF- ⁇ in the step (c) is larger than the number of trials for evaluating the inhibitory activity against TGF- ⁇ in the step (a). The method according to item.
- Item (b) includes measuring cell viability, wherein if the cell viability is not substantially reduced, the toxicity is evaluated as low. The method described.
- Item 9 Item 1-8, wherein in the step (d), a compound is selected that exhibits a substantially unreduced cell viability and a substantially reduced caspase 3/7 activity relative to the candidate compound non-contact group. The method according to any one of the above. (Item 10) In the step (d), a compound is selected wherein the cell viability is about 90% or more and the ratio of caspase 3/7 activity (%) to the cell viability (%) is about 0.8 or less. 10. The method according to any one of items 1 to 9, wherein (Item 11) Item 11.
- (Item 13) 13 The method according to item 12, wherein the inhibitory activity against the endoplasmic reticulum-related stress inducer is an activity of inhibiting apoptosis of the immortalized human corneal endothelial cells caused by the endoplasmic reticulum-related stress inducer.
- the step (e) includes measuring caspase activity, wherein the caspase activity in which the inhibitory activity against the endoplasmic reticulum-related stress inducer is substantially reduced with respect to the candidate compound non-contact group is item 12 or 13 The method described in 1.
- (Item 15) Item 15. The method according to Item 14, wherein the caspase activity is caspase 3/7 activity.
- a compound showing the cell viability of about 90% or more and a ratio of caspase 3/7 activity (%) to the cell viability (%) of about 0.8 or less is selected.
- (Item 22) The method according to any one of Items 12 to 21, wherein the endoplasmic reticulum-related stress inducer is MG-132.
- (Item 23) A method of screening for a compound effective for a symptom, disorder or disease in the corneal endothelium, the method comprising: (A) contacting a candidate compound with immortalized cells of Fuchs corneal endothelial dystrophy in the presence of transforming growth factor (TGF) - ⁇ , evaluating the caspase activity of the candidate compound, Selecting a compound having substantially reduced caspase activity; (B) evaluating the toxicity of the compound selected in step (a) to immortalized cells of the Fuchs corneal endothelial dystrophy; (C) evaluating the caspase activity of the compound selected in step (a); (D) selecting a compound evaluated in the step (b) as having low toxicity to the immortalized cells of the Fuchs corneal endothelial dystrophy and having a substantially reduced caspase activity in the step
- TGF- ⁇ is TGF- ⁇ 2.
- Item 26 26.
- the immortalized cells of the Fuchs corneal endothelial dystrophy are 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide and 4 Derived from corneal endothelial cells of Fuchs corneal endothelial dystrophy patients cultured in medium containing [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine)
- the kit according to any one of items 24 to 26. (Item 28) 28.
- the kit according to any one of items 24 to 30, wherein the reagent or means for measuring the inhibitory activity against TGF- ⁇ comprises a luminescent reagent.
- (Item 32) The kit according to any one of Items 24 to 31, wherein the reagent or means for measuring cytotoxicity comprises a luminescent reagent.
- (Item 33) A method of screening for a compound effective for a symptom, disorder or disease in the corneal endothelium, the method comprising: (A) contacting a candidate compound with an immortalized human corneal endothelial cell in the presence of an endoplasmic reticulum-related stress inducer, evaluating the inhibitory activity of the candidate compound against the endoplasmic reticulum-related stress inducer, and having the inhibitory activity Selecting a compound; (B) evaluating the toxicity of the compound selected in step (a) to the cells; (C) optionally evaluating the inhibitory activity of the compound selected in step (a) on the endoplasmic reticulum-related stress inducer; (D) selecting a compound that is evaluated as having low toxicity to the cell in the step (b) and having an inhibitory activity against the endoplasmic reticul
- Steps (a) and (c) include measuring caspase activity, and the caspase activity is such that the inhibitory activity against the endoplasmic reticulum-related stress inducer is substantially reduced relative to the candidate compound non-contact group. 35. A method according to item 33 or 34.
- (Item 36) Item 33, wherein the number of trials for evaluating the inhibitory activity against the endoplasmic reticulum-related stress inducer in the step (c) is larger than the number of trials for evaluating the inhibitory activity against the endoplasmic reticulum-related stress inducer in the step (a).
- 36. The method according to any one of .about.35.
- the method according to any one of items 33 to 36, wherein the number of trials is n 1 to 3.
- Item 39 Item 33, wherein in the step (a), a compound having a caspase 3/7 activity of about 75% or less with respect to the candidate compound non-contact group is selected as a compound having an inhibitory activity against the endoplasmic reticulum-related stress inducer. 40.
- the step (b) includes measuring cell viability, and the toxicity is evaluated as low when the cell viability is not substantially reduced, according to any of items 33-39.
- Kit 46 A kit for screening a compound effective for a symptom, disorder or disease in corneal endothelium using the method according to any one of items 33 to 45, the kit comprising: Endoplasmic reticulum-related stress inducer, Immortalized human corneal endothelial cells, A reagent or means for measuring inhibitory activity against the endoplasmic reticulum-related stress inducer; A kit comprising a reagent or means for measuring cytotoxicity.
- Item 47 Item 47. The kit according to Item 46, wherein the immortalized human corneal endothelial cells are derived from normal corneal endothelial cells into which SV40 large T antigen and hTERT have been introduced.
- Said immortalized human corneal endothelial cells are 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide and 4- [ Item 46 or 47, derived from normal corneal endothelial cells cultured in a medium containing 4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine) The described kit. (Item 49) 49. The kit according to any one of items 46 to 48, wherein the reagent for measuring the inhibitory activity against TGF- ⁇ is a reagent for measuring caspase activity.
- (Item 50) The kit according to any one of items 46 to 49, wherein the caspase activity is caspase 3/7 activity.
- the kit according to any one of items 46 to 51, wherein the endoplasmic reticulum-related stress inducer is selected from the group consisting of MG-132, thapsigargin and tunicamycin.
- (Item 53) 53 The kit according to any one of items 46 to 52, wherein the endoplasmic reticulum-related stress inducer is MG-132.
- the kit according to any one of items 46 to 53, wherein the reagent or means for measuring the inhibitory activity against the endoplasmic reticulum-related stress inducer comprises a luminescent reagent. (Item 55) 55.
- the kit according to any one of items 46 to 54, wherein the reagent or means for measuring cytotoxicity comprises a luminescent reagent.
- a model system of Fuchs corneal endothelial dystrophy is provided, and a technique that can be used in industries (cell culture industry, pharmaceuticals, etc.) relating to techniques such as screening for Fuchs corneal endothelial dystrophy treatment and prevention drugs is provided. .
- FIG. 1 shows representative conceptual diagrams of a screening method using immortalized cells (iFECD) of Fuchs corneal endothelial dystrophy and a screening method using immortalized human corneal endothelial cells (iHCEC).
- FIG. 2 shows an outline of screening using immortalized cells (iFECD) of Fuchs corneal endothelial dystrophy.
- FIG. 3 shows representative results of caspase 3/7 activity measured by Caspase-Glo® 3/7 Assay in the primary screening. The vertical axis represents caspase activity (%) relative to the candidate compound non-contact group (TGF- ⁇ 2 added group).
- the horizontal axis represents the control group, the TGF- ⁇ 2 (5 ng / mL) addition group, the TGF- ⁇ 2 (5 ng / mL) + Z-VD-FMK addition group (10 ⁇ M), the TGF- ⁇ 2 (5 ng / mL) + SCREEN ⁇
- the plate position of each drug addition group (10 ⁇ M) of WELL (registered trademark) FDA Approved Drug Library V2, Japan version is shown.
- FIG. 4 shows the case where the control is 0%, the TGF- ⁇ 2 reaction is 100%, the TGF- ⁇ 2 reaction is 100%, and the positive control TGF- ⁇ 2 + Z-VD-FMK reaction is 0%.
- FIG. 5 shows representative results of measuring cell viability in the secondary screening.
- shaft shows the cell survival rate (%) with respect to a candidate compound non-contact group. From the left, the horizontal axis represents the control group, the TGF- ⁇ 2 (5 ng / mL) addition group, the TGF- ⁇ 2 (5 ng / mL) + Z-VD-FMK addition group (10 ⁇ M), the TGF- ⁇ 2 (5 ng / mL) + SCREEN ⁇
- the plate position of each drug addition group (10 ⁇ M) of WELL (registered trademark) FDA Approved Drug Library V2, Japan version is shown.
- FIG. 6 shows representative results of measuring caspase 3/7 activity in the secondary screening.
- the vertical axis shows caspase 3/7 activity (%) relative to the candidate compound non-contact group. From the left, the horizontal axis represents the control group, the TGF- ⁇ 2 (5 ng / mL) addition group, the TGF- ⁇ 2 (5 ng / mL) + Z-VD-FMK addition group (10 ⁇ M), the TGF- ⁇ 2 (5 ng / mL) + SCREEN ⁇
- the plate position of each drug addition group (10 ⁇ M) of WELL (registered trademark) FDA Approved Drug Library V2, Japan version is shown.
- FIG. 7 shows SCREEN-WELL (registered trademark) FDA Approved Drug Library V2, Japan version drug name, secondary screening result of caspase 3/7 activity in TGF- ⁇ 2 stimulation, cell viability result, and The result of the secondary screening in TGF- ⁇ 2 stimulation is shown.
- FIG. 7 shows drugs with a value of (caspase 3/7 activity) / (cell viability) (%) ranked 1-25.
- FIG. 7 shows SCREEN-WELL (registered trademark) FDA Approved Drug Library V2, Japan version drug name, secondary screening result of caspase 3/7 activity in TGF- ⁇ 2 stimulation, cell viability result, and The result of the secondary screening in TGF- ⁇ 2 stimulation is shown.
- FIG. 7 shows SCREEN-WELL (registered trademark) FDA Approved Drug Library V2, Japan version drug name, secondary screening result of caspase 3/7 activity in TGF- ⁇ 2 stimulation, cell viability result, and The result of the secondary screening in TGF- ⁇ 2 stimulation is shown.
- FIG. 7 shows drugs with a value of (caspase 3/7 activity) / (cell viability) (%) ranked 1-25.
- FIG. 8 shows SCREEN-WELL (registered trademark) FDA Applied Drug Library V2, Japan version drug name, secondary screening result of caspase 3/7 activity in TGF- ⁇ 2 stimulation, cell viability result, and The result of the secondary screening in TGF- ⁇ 2 stimulation is shown.
- FIG. 8 shows drugs with a value of (caspase 3/7 activity) / (cell viability) (%) ranked in the 26th to 47th positions.
- FIG. 8 shows SCREEN-WELL (registered trademark) FDA Applied Drug Library V2, Japan version drug name, secondary screening result of caspase 3/7 activity in TGF- ⁇ 2 stimulation, cell viability result, and The result of the secondary screening in TGF- ⁇ 2 stimulation is shown.
- FIG. 8 shows drugs with a value of (caspase 3/7 activity) / (cell viability) (%) ranked in the 26th to 47th positions.
- FIG. 9 shows an outline of screening using immortalized human corneal endothelial cells (iHCEC).
- FIG. 10 shows the results of screening for caspase 3/7 activity in each drug name, MG-132 (0.1 ⁇ M) stimulation, cell viability results in MG-132 (0.1 ⁇ M) stimulation, and MG-132. (0.1 ⁇ M) shows the value obtained by dividing the result of screening by stimulation by cell viability ((caspase 3/7 activity) / (cell viability) (%)).
- eye cell is used in a broad sense and means any cell present in the eye, and any cell present in the eyelid, sclera, cornea, uvea, lens, vitreous, retina, optic nerve. Cells are included.
- cornea endothelial cell is used in the usual meaning used in the art.
- the cornea is one of the layered tissues constituting the eye, is transparent, and is located in the portion closest to the outside world.
- the cornea is said to be composed of five layers in order from the outside (body surface) in humans, and is composed of the corneal epithelium, Bowman's membrane (outer boundary line), lamina intestinal, Descemet's membrane (inner boundary line), and corneal endothelium from the outside. Is done.
- portions other than the epithelium and endothelium are sometimes collectively referred to as “corneal stroma” and are referred to as such in this specification.
- iFECD immortalized Fuchs ’endothelial corneal dystrophy
- iFECD immortalized Fuchs ’endothelial corneal dystrophy
- HCEC human corneal endothelial cells
- IHCEC immortalized human corneal endothelial cells
- corneal tissue is used in a normal sense and refers to the tissue itself constituting the cornea.
- corneal tissue all of the corneal epithelium, Bowman's membrane, parenchyma, Descemet's membrane, and corneal endothelium that make up the cornea (in the case of humans, in the case of other animals, all depending on the corresponding category) May be included, may be partially missing, or may contain other tissues (sclera) in addition to the cornea, in which case it may be referred to as the cornea.
- undifferentiated cell refers to any cell capable of differentiation.
- undifferentiated cells include stem cells as well as cells that have differentiated to a certain extent but can still differentiate.
- immortalized cell refers to a cell line (lineage) that has an apparent infinite life during culture (has the ability to proliferate through an infinite number of divisions).
- Such immortalized cells can acquire the ability to proliferate indefinitely by avoiding cellular senescence in the course of repeated primary cultures, or immortalized by introducing specific genes into the target cells. Abilities can also be granted.
- established or “established” cells maintain certain properties (eg, pluripotency) and the cells continue to grow stably under culture conditions. State. Usually, the immortalized cells often have the desired characteristics and thus often correspond to established cells.
- Transformation means spontaneous conversion (cell) to an uncontrollable growth state.
- Transformed cells acquire the ability to grow through an infinite number of divisions in an incubator.
- Transformed cells may be referred to using terms such as neoplastic, undifferentiated and / or hyperplastic with respect to their lack of growth control.
- isolated means that a substance that naturally accompanies in a normal environment is at least reduced, preferably substantially free.
- an isolated cell, tissue, etc. refers to a cell that is substantially free of other materials (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment.
- thickening of the extracellular matrix layer refers to having an extracellular matrix layer thicker than the extracellular matrix layer in the normal form of the cell when referring to a certain cell.
- the thickness of the extracellular matrix layer is thicker than about 3 ⁇ m, and preferably the thickness is about 5 ⁇ m or more. Preferably, it may be about 6 ⁇ m or more.
- extracellular matrix is also referred to as “extracellular matrix” and refers to a substance that exists between somatic cells regardless of whether they are epithelial cells or non-epithelial cells.
- the extracellular matrix is usually produced by cells and is therefore one of the biological materials.
- the extracellular matrix is responsible not only for tissue support, but also for the construction of the internal environment necessary for the survival of all somatic cells.
- the extracellular matrix is generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.
- the fiber component is roughly classified into a fiber component and a matrix filling the space, and the fiber component includes collagen fiber and elastic fiber.
- the basic component of the substrate is glycosaminoglycan (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a polymer of proteoglycan (acid mucopolysaccharide-protein complex).
- laminin of the basement membrane, microfibril around the elastic fiber, fiber, and glycoprotein such as fibronectin on the cell surface are included in the substrate.
- the basic structure is the same in specially differentiated tissues, for example, hyaline cartilage produces chondrocytes that contain a large amount of proteoglycan, characteristically by chondroblasts, and bone produces bone matrix that causes calcification by osteoblasts. .
- a substance constituting a typical extracellular matrix for example, collagen I, collagen IV, collagen VIII, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (for example, Decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), but not limited to them, and various extracellular matrices that are responsible for cell adhesion may be relevant in the present invention. .
- extracellular matrix protein refers to a protein constituting the extracellular matrix.
- proteins include, but are not limited to, collagen I, collagen IV, collagen VIII, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, and thrombospondin.
- the properties of Fuchs corneal endothelial dystrophy are maintained with at least one of collagen I, collagen IV, collagen VIII and fibronectin as an index. It can be determined whether or not.
- extracellular matrix protein refers to the production of extracellular matrix proteins such as collagen I, collagen IV, collagen VIII, and fibronectin in the normal form when referring to certain cells. It means that it is produced more than the amount that has been.
- the production situation includes an increase in the expression level as required by a response to transforming growth factor (TGF) - ⁇ .
- TGF transforming growth factor
- the amount of extracellular matrix in normal is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times. Times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, or about 2.0 times or more.
- the difference from normal is preferably significant, but not necessarily significant.
- epithelial-mesenchymal transition (EMT) -related marker refers to a marker that serves as an index in epithelial-mesenchymal transition (EMT), and examples include ZEB1 and Snail1.
- epithelial-mesenchymal transition (EMT) -related marker expression enhancement selected from the group consisting of ZEB1 and Snail1 refers to a cell that is produced in ZEB1 or Snail1 in a normal form when referring to a certain cell. It means that it is produced more than the amount it has.
- the production situation includes an increase in the expression level as required by a response to transforming growth factor (TGF) - ⁇ .
- TGF transforming growth factor
- the difference from normal is preferably significant, but not necessarily significant.
- non-fibroblast morphology refers to the cell morphology, and means that it does not have fibroblasts. Specifically, it is a polygonal shape that maintains a polygonal shape as in normal cells, does not cause significant stratification, and has a pump function and a barrier function, but is not limited thereto. Fibroblasts do not have a function that is normal in normal tissues, but when damaged, they migrate to the damaged area and begin to produce extracellular matrix such as collagen, and update the extracellular matrix. To do.
- the “normal cell function” of a cell refers to a function inherent to the cell when referring to a specific cell such as a corneal endothelial cell.
- corneal endothelial cells such functions include ZO-1 and Na + / K + -ATPase, adaptability to corneal transplantation (Matsubara M, Tanishima T: Wound-healing of the corneal endothelium in the monkey: a morphometric study, Jpn J Ophthalmol 1982, 26: 264-273; Matsubara M, Tanishima T: Wound-healing of corneal endothelium in monkey: an autoradiographic study, Jpn J Ophthalmol 1983, 27: 444-450; Van Horn DL, Hyndiuk RA: Endothelial wound repair in primate cornea, Exp Eye Res 1975, 21: 113-124 and VanHorn DL, Sendele DD, Seideman S, Buco PJ: Regenerative capacity of the cornea
- ZO-1 and Na + / K + -ATPase can be evaluated by observing expression at the nucleic acid level such as immunological means or RT-PCR. Confirm whether the subject cell has normal function by confirming that Na + / K + -ATPase and ZO-1 are expressed and / or functioning to the same extent as normal cells Can do.
- the adaptability to corneal transplantation can be performed by transplanting cultured cells by mechanically scraping the corneal endothelium as a bullous keratosis model even in laboratory animals such as rabbits.
- rabbit corneal endothelial cells proliferate in vivo, the possibility of natural healing due to proliferation of host corneal endothelial cells cannot be denied (Matsubara M, et al., Jpn J Ophthalmol 1982, 26: 264-273; Matsubara M, et al., Jpn J Ophthalmol 1983, 27: 444-450; Van Horn DL, et al., Exp Eye Res 1975, 21: 113-124 and Van Horn DL, et al., Invest Ophthalmol Vis Sci 1977 16: 597-613).
- a primate cynomolgus monkey for example, at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 6 months, even more preferably Assess adaptability after at least 12 months. Confirmation of transplantation adaptability in primates such as monkeys is particularly important in human application.
- the “subject” refers to a subject (for example, a living organism such as a human or an organ (eye) or a cell taken out from a living organism) as a subject of diagnosis or detection of the present invention.
- sample refers to any substance obtained from a subject or the like, and includes, for example, eye cells. Those skilled in the art can appropriately select a preferable sample based on the description of the present specification.
- the gene (nucleic acid sequence) of the large T antigen (SV40 large T antigen) is used in transformation.
- marker refers to a certain state (eg, normal cell state, transformed state, disease state, disordered state, proliferative ability, differentiation state level, presence / absence, etc. ) Or a substance that serves as an indicator for tracking whether there is a danger or not.
- detection, diagnosis, preliminary detection, prediction or pre-diagnosis for a certain condition is a drug, agent, factor or means specific for the marker associated with the condition, or It can be realized by using a composition, kit or system containing them.
- gene product refers to a protein or mRNA encoded by a gene.
- a gene product ie, a molecule such as CD98
- TERT Telomerase Reverse Transcriptase
- hTERT telomerase reverse transcriptase
- TERC telomerase RNA component
- the IDs are Entrez Gene ID: 7015, NM_001193376 (human mRNA), NP_001180305 (human protein).
- hTERT includes not only a protein having an amino acid sequence described in a specific accession number (and a sequence number as specifically described herein) (or a nucleic acid encoding the same), but also functionally active.
- Snail1 is a protein of the C2H2 type zinc finger family that regulates transcription, and is one of epithelial-mesenchymal transition (EMT) -related markers. SLUGH2; SNA; SNAH; SNAIL; SNAIL1; also called dJ710H13.1. The IDs are Entrez Gene ID: 6615, NM_005985 (human mRNA), NP_005976 (human protein). Snail1 is not only a protein having an amino acid sequence described in a specific accession number (and a SEQ ID NO as specifically described herein) (or a nucleic acid encoding it), but also functionally active.
- EMT epithelial-mesenchymal transition
- ZEB1 refers to zinc finger E-box-binding homeobox 1 and is one of the markers related to epithelial-mesenchymal transition (EMT). AREB6; BZP; DELTAEF1; FECD6; NIL2A; PPCD3; TCF8; ZFHEP; also called ZFHX1A.
- the IDs are Entrez Gene ID: 6035, NM_001128128 (human mRNA) and NP_001121600 (human protein).
- ZEB1 is not only a protein having an amino acid sequence described in a specific accession number (and a SEQ ID NO as specifically described herein) (or a nucleic acid encoding it), but also functionally active.
- a “derivative” or “analog” or “variant” preferably includes, but is not intended to be limited to, a molecule comprising a region that is substantially homologous to a component protein, Such molecules may, in various embodiments, be at least 30%, 40% when compared to sequences that are aligned over amino acid sequences of the same size or aligned by computer homology programs known in the art. Nucleic acids that are 50%, 60%, 70%, 80%, 90%, 95% or 99% identical, or that encode such molecules, can be stringent, moderately stringent, or It can hybridize to a sequence encoding a component protein under non-stringent conditions.
- the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art.
- humans are mainly discussed, but it is understood that this applies to other species such as those of other species within primates or other species of animals, It is understood that it is within the scope of the invention.
- “functionally active” as used herein refers to a protein, such as a biological activity such as telomerase activity, according to the embodiment to which the polypeptide of the invention, ie, a fragment or derivative, relates.
- a polypeptide, ie, a fragment or derivative having a structural function, a regulatory function, or a biochemical function.
- polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art.
- a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe.
- hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained.
- a polynucleotide that can be identified by washing the filter under conditions of 65 ° C using an SSC (saline-sodium citrate) solution (composition of a 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate).
- SSC saline-sodium citrate
- composition of a 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate.
- Hybridization is described in experimental documents such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Prac1tical Approach, Second Edition, Oxford University Press (1995). It can be carried out according to the method.
- the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions.
- a polypeptide (for example, transthyretin) used in the present invention is a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide particularly described in the present invention.
- the polypeptide encoded by is also encompassed. These low stringency conditions are: 35% formamide, 5 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% polyvinylpyrrolidone (PVP), 0.02% BSA, 100 ⁇ g / ml denatured salmon sperm DNA , And 10% (weight / volume) dextran sulfate in buffer solution at 40 ° C.
- a “corresponding” gene eg, a polynucleotide sequence or molecule
- a gene for example, a polynucleotide sequence or a molecule
- a gene corresponding to a gene can be an ortholog of that gene. Therefore, molecules such as mouse and rat TERT can each find a corresponding molecule such as TERT in humans.
- Such corresponding genes can be identified using techniques well known in the art.
- a corresponding gene in an animal is obtained by using a sequence found in the gene accession number as a reference gene for the corresponding gene (eg, human) as a query sequence. It can be found by searching an animal sequence database.
- expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form.
- a gene, polynucleotide or the like is transcribed and translated into a polypeptide form.
- transcription and production of mRNA can also be an aspect of expression.
- polypeptide forms may be post-translationally processed (derivatives as referred to herein).
- transforming growth factor ⁇ transformed growth factor ⁇ ; also abbreviated as TGF- ⁇
- TGF- ⁇ transformed growth factor ⁇
- TGF- ⁇ signal refers to a signal mediated by TGF- ⁇ and triggered by TGF- ⁇ .
- the TGF- ⁇ signal includes, for example, a signal mediated by TGF- ⁇ 2, and other examples include signals mediated by TGF- ⁇ 1, TGF- ⁇ 3, and the like.
- TGF- ⁇ there are three isoforms with about 70% homology from TGF- ⁇ 1 to ⁇ 3 in humans, and their actions are similar.
- TGF- ⁇ is produced as an inactive latent form having a molecular weight of about 300 kD that cannot bind to the receptor, and is activated on the surface of the target cell or its surroundings to become an active form that can bind to the receptor and exerts its action.
- inhibitory activity against TGF- ⁇ or “inhibitory activity against endoplasmic reticulum-related stress inducer” refers to the activity of inhibiting apoptosis of cells caused by TGF- ⁇ / endoplasmic reticulum-related stress inducer. .
- Having inhibitory activity against TGF- ⁇ ” or “having inhibitory activity against an endoplasmic reticulum-related stress inducer” means that a cell (iFECD or iHCEC, etc.) is not contacted with a candidate compound (candidate compound non-contact group) ) Having substantially reduced caspase activity, and “substantially reduced caspase activity” refers to caspase activity that is statistically significantly reduced with respect to the non-contact group of candidate compounds.
- a symptom, disorder or disease in corneal endothelium caused by transforming growth factor (TGF) - ⁇ signal refers to any corneal endothelial symptom, disorder or condition induced by TGF- ⁇ in corneal endothelial cells. Refers to disease.
- TGF transforming growth factor
- Symptoms, disorders or diseases of corneal endothelium caused by TGF- ⁇ signal include, for example, symptoms, disorders or diseases in corneal endothelium caused by TGF- ⁇ include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorder, corneal endotheliitis, Injury after trauma, ophthalmic surgery, ophthalmic laser surgery, aging, posterior polymorphic corneal dystrophy (PPD), congenital hereditary corneal endothelial dystrophy (CHED), idiopathic corneal endothelial dysfunction, and cytomegalovirus corneal endotheliitis But are not limited to these.
- cell viability refers to the number of cells when iFCED or iHCEC is cultured in the absence of the candidate compound, when iFCED or iHCEC is cultured in the presence of the candidate compound. Refers to the percentage of cells.
- caspase activity refers to TGF- ⁇ for caspase activity in a TGF- ⁇ (eg, TGF- ⁇ 2) added group (no candidate compound) or an endoplasmic reticulum related stress inducer added group (no candidate compound).
- TGF- ⁇ eg, TGF- ⁇ 2
- ⁇ eg, TGF- ⁇ 2
- endoplasmic reticulum-related stress inducer e.g, TGF- ⁇ 2
- low toxicity refers to the fact that cell viability is not substantially reduced in the absence of TGF- ⁇ and endoplasmic reticulum-related stress inducers.
- Non-cell viability means that the cell viability is not significantly reduced as compared to a group (candidate compound non-contact group) in which cells (iFCED or iHCEC, etc.) are not contacted with a candidate compound. .
- a numerical value serving as a guide there can be mentioned a decrease of less than 10%, a decrease of less than 5%, and the like.
- Candidate compounds include, but are not limited to, small molecule compounds, peptides, proteins, nucleic acids, cells and the like, including any candidate substance that can achieve prevention or treatment of symptoms, disorders or diseases in the corneal endothelium. To do.
- endoplasmic reticulum (ER) -related stress inducer refers to a substance that can induce denatured protein. Endoplasmic reticulum stress is caused by accumulation of denatured protein, and when denatured protein accumulates excessively, apoptosis is induced. Endoplasmic reticulum (ER) -related stress inducers include MG-132, thapsigargin and tunicamycin.
- symptom, disorder or disease in corneal endothelium due to endoplasmic reticulum (ER) -related stress refers to those associated with endoplasmic reticulum (ER) stress, such as corneal endothelium in Fuchs corneal endothelium dystrophy.
- search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
- Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444- 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol.
- Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
- immortalized cells of Fuchs corneal endothelial dystrophy are used.
- the immortalized cells of Fuchs corneal endothelial dystrophy are reviewed in Patent Documents 1 and 2 and the like, and incorporated herein by reference as necessary.
- the immortalized cells of Fuchs corneal endothelial dystrophy used in the present invention are (1) thickening of extracellular matrix layer, (2) at least one cell selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin Overproduction of outer matrix protein, (3) enhanced expression of at least one epithelial-mesenchymal transition (EMT) related marker selected from the group consisting of ZEB1 and Snail1, (4) non-fibroblast morphology, (5) normal cell function ( For example, Na + / K + -ATPase and ZO-1 are expressed and / or functioning to the same extent as normal cells), preferably May have at least 2, at least 3, at least 4, or all features . More preferably, the cells of the invention have all of these characteristics.
- EMT epithelial-mesenchymal transition
- the enhanced expression of the extracellular matrix protein and / or EMT-related marker comprises responsiveness to transforming growth factor (TGF) - ⁇ .
- said normal cell function comprises expression of Na + / K + -ATPase and ZO-1.
- Fuchs corneal endothelial dystrophy dystrophy cells have characteristics that could not be achieved in the past, and suitable cells are provided for screening drugs for the treatment or prevention of Fuchs corneal endothelial dystrophy. is there.
- Each feature can be evaluated as follows: (1) Thickening of the extracellular matrix layer It is confirmed that it is thicker than normal cells (usually 2-4 ⁇ m). It is possible to determine whether the target cells are immortalized cells of Fuchs corneal endothelial dystrophy by observing that the thickness is preferably at least 1.5 times that of normal cells, more preferably at least 2 times that of normal cells. . (2) Overproduction of at least one extracellular matrix protein selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin. It is confirmed that the expression level is higher than that of normal cells.
- the expression level is preferably at least 1.5 times that of normal cells, more preferably at least 2 times, it is possible to determine whether the target cells are immortalized cells of Fuchs corneal endothelial dystrophy. . Alternatively, whether or not the expression of extracellular matrix protein is enhanced in response to TGF- ⁇ can also be used as a basis for judgment. (3) Enhanced expression of at least one epithelial-mesenchymal transition (EMT) -related marker selected from the group consisting of ZEB1 and Snail1 It is confirmed that the expression level is higher than that of normal cells.
- EMT epithelial-mesenchymal transition
- the expression level is preferably at least 1.5 times that of normal cells, more preferably at least 2 times, it is possible to determine whether the target cells are immortalized cells of Fuchs corneal endothelial dystrophy. . Alternatively, whether or not the expression of the EMT-related marker is enhanced in response to TGF- ⁇ can be used as a basis for judgment.
- Non-fibroblast morphology Judged from cell shape. It can be determined by maintaining a polygonal morphology as in normal cells and confirming that it exhibits a single epithelial cell-like morphology without significant stratification.
- Normal cell function The expression of Na + / K + -ATPase and ZO-1 can be confirmed by immunostaining and the like in the same manner as normal cells, and can be judged by the same level of expression as normal cells.
- the present invention provides a method for screening a drug for treating or preventing Fuchs corneal endothelial dystrophy using Fuchs corneal endothelial dystrophy immortalized cells of the present invention.
- the immortalized cells of Fuchs corneal endothelial dystrophy used in the present invention are produced by introducing SV40 large T antigen and hTERT into corneal endothelial cells derived from patients with Fuchs corneal endothelial dystrophy.
- the method for preparing immortalized cells of Fuchs corneal endothelial dystrophy is outlined in Patent Documents 1 and 2 and the like, and incorporated herein by reference as necessary.
- immortalized cells can acquire the ability to proliferate indefinitely by avoiding cellular senescence as the primary culture is subcultured, or immortalized by introducing a specific gene into the target cell Ability can also be granted.
- many of such immortalized cells lose part or all of the forms and functions originally possessed in the living body, in the test using such an immortalized cell line, It is said that it is difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived, and it has been difficult to provide immortalized cells that maintain the desired characteristics.
- the present invention maintains the desired characteristics, that is, the characteristics of Fuchs corneal endothelial dystrophy by using the above-described specific gene transfer technology, and retains the same function as that of normal cells (that is, has been transformed). Not) cells could be provided.
- telomerase reverse transcriptase TERT
- immortalization is maintained while maintaining the characteristics of Fuchs corneal endothelial dystrophy.
- TRET telomerase reverse transcriptase
- Such immortalized cells of Fuchs corneal endothelial dystrophy can withstand screening of useful substances such as pharmaceuticals targeting Fuchs corneal endothelial dystrophy.
- any known gene (nucleic acid molecule) introduction technique transformation, transduction, transfection, etc.
- gene product (protein) introduction method may be used.
- the introduction of these genes can be realized by introduction of nucleic acid molecules.
- “gene transfer” refers to introducing a foreign gene, preferably a functional gene, into a chromosome genome or the like, for example, by an appropriate transfer technique.
- an exogenous functional gene can be introduced by replacing the endogenous functional gene.
- the exogenous functional gene is a gene that does not originally exist in the chromosome genome of the organism, and may be a gene derived from another species, a synthetic gene prepared by PCR, or the like.
- a transfection technique is an example of such a gene introduction technique.
- a target gene in the present invention, SV40 large T antigen, hTERT gene and the like
- an appropriate virus vector for example, but not limited to, a lentivirus vector.
- an appropriate cell for example, 293T cell
- a helper plasmid as necessary (for example, pLP1, pLP2, pLP / VSVG, etc.).
- the culture supernatant containing the virus is recovered after infection, and the culture is added to the culture solution of the target cells (in the present invention, cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy patients).
- Kiyo can be added with an appropriate reagent (here, but not limited to polybrene) to produce an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs corneal endothelial dystrophy.
- exogenous DNA containing DNA encoding the transforming gene can be introduced into the cell of interest using techniques for transfection into retroviruses (eg, lentiviral vectors).
- recombinant retroviruses are produced in packaging cell lines and have high titer virus particles (typically but not limited to 10 5 to 10 6 pfu / ml).
- a recombinant virus particle is a medium containing a target cell culture in which the cell culture is a virus particle and a reagent such as polybrene at an appropriate concentration (for example, 5 to 10 ⁇ g / ml but not limited thereto). And used for infection by incubating for an appropriate time (for example, but not limited to about 1 to 12 hours). After infection with retrovirus, the cells are rinsed and cultured in standard medium. The infected cells are then analyzed for foreign DNA uptake and expression.
- the cells may be subjected to selective conditions that select for cells that have taken up and expressed the selectable marker gene.
- exogenous DNA may be introduced using calcium phosphate mediated transfection techniques.
- a calcium phosphate precipitate containing DNA encoding the gene of interest, eg, a transforming gene, may be prepared using the technique of Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1373-1376. .
- the cells are analyzed for foreign DNA uptake and expression.
- the cells may be subjected to selective conditions that select for cells that have taken up and expressed the selectable marker gene.
- Such techniques are exemplified in, but not limited to, examples 2013-509179 and JP 2004-254509 in addition to the examples specifically disclosed in the embodiments.
- the corneal endothelial cells from a patient with Fuchs corneal endothelial dystrophy are SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide) and SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine)
- Preferred cells can be obtained by culturing in the above.
- This medium may preferably be SB203580 + SB431542 + 3T3 conditioned medium used in the examples, but is not limited thereto.
- the present invention provides a method of screening compounds effective for diseases in the corneal endothelium using Fuchs corneal endothelial dystrophy immortalized cells (iFECD).
- the method of the present invention includes a primary screen for selecting a compound having an inhibitory activity against TGF- ⁇ and a secondary screen for selecting a compound having low toxicity to cells and having an inhibitory activity against TGF- ⁇ .
- the present invention provides a reliable screening method by performing both primary screening and secondary screening.
- the methods of the invention comprise (a) contacting a candidate compound with immortalized cells of Fuchs corneal endothelial dystrophy in the presence of transforming growth factor (TGF) - ⁇ , A step of evaluating inhibitory activity against - ⁇ , selecting a compound having the inhibitory activity, a step of (b) evaluating the toxicity of the compound selected in step (a) to the cells, and if necessary ( c) a step of evaluating the inhibitory activity against the TGF- ⁇ of the compound selected in step (a); (d) a step (b) that is evaluated as having low toxicity to the cell; and step (c) And selecting a compound evaluated to have an inhibitory activity against the TGF- ⁇ .
- TGF transforming growth factor
- the candidate compound is contacted with immortalized cells of Fuchs corneal endothelial dystrophy in the presence of transforming growth factor (TGF) - ⁇ , and the inhibitory activity of the candidate compound on the TGF- ⁇
- the secondary screening includes a step of (b) evaluating the toxicity of the compound selected in step (a) to the cells, and if necessary, a step of selecting a compound having the inhibitory activity.
- step (c) is not an essential step, it is preferable to include step (c) in order to improve the accuracy of screening. Surprisingly, unexpectedly, performing step (c) significantly increased the accuracy of the screening. It is understood that it is advantageous to include the step (c) because it is significantly increased even if only the tester's proficiency and other factors are taken into account. The effect of including such a step (c) is not expected from the conventional screening technique.
- the number of trials for evaluating the inhibitory activity against TGF- ⁇ in step (c) may be greater than the number of trials for evaluating the inhibitory activity against TGF- ⁇ in step (a). This is because a useful compound is selected with higher reliability by increasing the number of trials in step (c) in the secondary screening. There is also an advantage that a large number of candidate compounds can be processed by setting the number of trials of the primary screening to be small. Due to the small number of trials, the reliability may be lowered in the primary screening, but the reliability is ensured by the secondary screening. In fact, some compounds that were considered useful in the primary screen were excluded in the secondary screen.
- Table 1 shows an example in which, although the primary screening showed caspase activity of 75% or less, the secondary screening with an increased number of trials showed almost no decrease in caspase activity.
- the cell viability measured by CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega catalog number: G7570) was indicated by “ ⁇ ”, and the cell viability was 90%. What was less than is shown by "x”.
- the number of trials for evaluating inhibitory activity against TGF- ⁇ in step (a) may be 1, 2, 3, 4, 5, or more times,
- the number of trials for evaluating the inhibitory activity against TGF- ⁇ in (c) is 2, 3, 4, 5, 6, 7 and 8 so as to be greater than the number of trials in step (a). Times, 9, 10, or more times.
- a person skilled in the art can appropriately set the number of trials in consideration of the number of candidate compounds.
- the inhibitory activity against TGF- ⁇ can be evaluated based on the activity of inhibiting cell apoptosis caused by the TGF- ⁇ signal.
- the activity that inhibits apoptosis is indicated by, for example, caspase activity (for example, caspase 3/7 activity), exposure of phosphatidylserine on the outer surface of cell membrane, cleavage of Poly (ADP-ribose) Polymerase (PARP), or fragmentation of chromosomal DNA
- caspase activity against TGF- ⁇ may be assessed by measuring caspase activity (eg, caspase 3/7 activity), eg, substantially reduced relative to a candidate compound non-contact group
- the compound having caspase activity can be selected as a compound having inhibitory activity against TGF- ⁇ .
- the candidate compound non-contact group is a group to which no candidate compound is added (TGF- ⁇ added group).
- a caspase activity substantially reduced with respect to the candidate compound non-contact group is a caspase activity (eg, caspase 3) that is statistically significantly reduced with respect to the candidate compound non-contact group.
- a caspase activity eg, caspase 3/7 activity
- caspase 3/7 activity is a caspase activity that is statistically significantly reduced with respect to the candidate compound non-contact group.
- a compound that exhibits about 75% or less of caspase 3/7 activity relative to the non-contact group of candidate compounds can be selected as a compound having inhibitory activity against TGF- ⁇ .
- the toxicity of the candidate compound Fuchs corneal endothelial dystrophy to immortalized cells can be evaluated. By assessing toxicity, it is possible to select compounds that are useful as pharmaceuticals. Toxicity can be performed by assessing cell viability.
- step (b) includes measuring cell viability and may be assessed as low toxicity if cell viability is not substantially reduced.
- Cell viability is measured in the absence of TGF- ⁇ .
- the cell survival rate that is not substantially reduced means that the cell survival rate is not significantly reduced as compared to the candidate compound non-contact group, for example, about 90% of the candidate compound non-contact group.
- the above cell viability can be obtained.
- the cell viability threshold may be about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, about 100%.
- Caspase activity also decreases due to the decrease in the number of cells, so even if it seems to suppress caspase activity at first glance, it is clear that the number of cells may be significantly reduced due to toxicity became.
- the drug in addition to the measurement of caspase activity, the drug can be accurately selected by measuring the number of cells.
- the compounds as shown in Table 2 seemed to suppress caspase activity at first glance, but were excluded in the secondary screening because the cell viability was below 90% and the toxicity was strong.
- the cell viability measured by CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega catalog number: G7570) was indicated by “ ⁇ ”, and the cell viability was 90%. What was less than "x" is shown.
- compounds that exhibit substantially unreduced cell viability and substantially reduced caspase 3/7 activity relative to the candidate compound non-contact group can be selected.
- Such compounds have a ratio of caspase 3/7 activity (%) to cell viability (%) of about 0.9 or less, about 0.8 or less, about 0.75 or less, about 0.7 or less, about 0 or less. .65 or less, about 0.6 or less, about 0.55 or less, about 0.5 or less, about 0.45 or less, about 0.4 or less, about 0.35 or less, about 0.3 or less, about 0.25 Or a compound that exhibits about 0.2 or less.
- a compound may be selected that exhibits a ratio of caspase 3/7 activity (%) to cell viability (%) of about 0.8 or less.
- the concentration of the candidate compound can be any concentration. It is understood that all candidate compound concentrations may use the same concentration, or different concentrations may be used for each candidate compound. If a person skilled in the art knows a concentration that is toxic to cells (for example, 50% inhibitory concentration: IC50), the concentration of the candidate compound can be set in consideration of this. Examples of the concentration of the candidate compound used include, but are not limited to, about 1 nM, about 10 nM, about 100 nM, about 1 ⁇ M, about 10 ⁇ M, about 100 ⁇ M, about 1 mM, about 10 mM, about 100 mM.
- any numerical value between these concentrations can also be used. Any range of these values can also be employed as a preferred concentration range in the present invention.
- the concentration of candidate compound is about 10 ⁇ M. In another embodiment, the concentration of the candidate compound is about 1 ⁇ M.
- a compound having an inhibitory activity against TGF- ⁇ is selected. Therefore, the compound selected by the screening method of the present invention can prevent symptoms, disorders or diseases in the corneal endothelium caused by TGF- ⁇ signal. Can be prevented or treated.
- Symptoms, disorders or diseases in the corneal endothelium caused by TGF- ⁇ include Fuchs corneal endothelial dystrophy, corneal transplantation disorders, corneal endotheliitis, trauma, ophthalmic surgery, disorders after ophthalmic laser surgery, aging, posterior polymorphic cornea It can be selected from the group consisting of dystrophy (PPD), congenital hereditary corneal endothelial dystrophy (CHED), idiopathic corneal endothelial dysfunction, and cytomegalovirus corneal endotheliitis. Since the immortalized cells of Fuchs corneal endothelial dystrophy are used in the screening method of the present invention, the compound selected by the screening method of the present invention can particularly prevent or treat Fuchs corneal endothelial dystrophy.
- a compound selected by a screening method using immortalized cells of Fuchs corneal endothelial dystrophy may be further subjected to a screening method using immortalized human corneal endothelial cells described in detail below.
- a compound selected by a screening method using immortalized human corneal endothelial cells may be subjected to a screening method using immortalized cells of Fuchs corneal endothelial dystrophy.
- the primary screening may be omitted as necessary in the second screening system. This is because the number of candidate compounds is limited to some extent by the first screening.
- compounds having inhibitory activity against endoplasmic reticulum-related stress inducers are selected, so both immortalized cells of Fuchs corneal endothelial dystrophy and immortalized human corneal endothelial cells are used.
- the compounds selected by the screening method that had been used may have inhibitory activity against TGF- ⁇ and endoplasmic reticulum-related stress inducers. Since Fuchs corneal endothelial dystrophy is also considered to be caused by endoplasmic reticulum-related stress, compounds having inhibitory activity against TGF- ⁇ and endoplasmic reticulum-related stress inducers are particularly effective against Fuchs corneal endothelial dystrophy It is thought that.
- the present invention provides a method of screening for compounds effective for diseases in the corneal endothelium using immortalized human corneal endothelial cells (iHCEC).
- the method of the present invention comprises a primary screening for selecting a compound having an inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer, a low toxicity to cells, and an inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer.
- Secondary screening to select compounds with.
- iHCEC immortalized human corneal endothelial cells
- ER endoplasmic reticulum
- ER endoplasmic reticulum
- the method of the invention comprises (a) contacting a candidate compound with immortalized human corneal endothelial cells in the presence of an endoplasmic reticulum (ER) -related stress inducer, the endoplasmic reticulum of the candidate compound ( (ER) evaluating the inhibitory activity against a related stress inducer and selecting a compound having the inhibitory activity; (b) evaluating the toxicity of the compound selected in step (a) to the cells; c) evaluating the inhibitory activity of the compound selected in step (a) on the endoplasmic reticulum (ER) -related stress inducer, and (d) evaluating that the toxicity to the cell is low in step (b), And selecting a compound evaluated in step (c) as having an inhibitory activity against the endoplasmic reticulum (ER) -related stress inducer.
- ER endoplasmic reticulum
- ER-related stress inducers include MG-132, thapsigargin and tunicamycin.
- MG-132 is known as a substance capable of inducing protein folding abnormality (accumulation of denatured protein), which is one of the causes of endoplasmic reticulum-related stress.
- Fuchs corneal endothelial dystrophy is known to be caused by endoplasmic reticulum-related stress due to accumulation of denatured protein.
- endoplasmic reticulum-related stress induced by MG-132 is more It is thought to reflect the state of A screening method for evaluating inhibitory activity against MG-132 in iHCEC is a compound that suppresses Fuchs corneal endothelial dystrophy dystrophy (endoplasmic reticulum-related stress caused by accumulation of denatured protein) that cannot be evaluated by the above-described screening method using iFCED. It is advantageous to be able to screen. Although not wishing to be bound by theory, it has been confirmed that the compounds that have confirmed the inhibitory activity with MG-132 actually suppress the pathology of Fuchs corneal endothelial dystrophy advantageously. Can also be seen.
- a candidate compound is brought into contact with an immortalized human corneal endothelial cell in the presence of an endoplasmic reticulum (ER) -related stress inducer, and the endoplasmic reticulum (ER) -related stress induction of the candidate compound is performed.
- ER endoplasmic reticulum
- the secondary screening is a step of (b) evaluating the toxicity of the compound selected in step (a) to the cells; (C) evaluating the inhibitory activity of the compound selected in step (a) on the endoplasmic reticulum (ER) -related stress inducer, and (d) evaluating that the toxicity to the cell is low in step (b) And corresponds to the step of selecting a compound evaluated in step (c) as having an inhibitory activity against the endoplasmic reticulum (ER) -related stress inducer.
- the number of trials for evaluating the inhibitory activity against the endoplasmic reticulum (ER) -related stress inducer in step (c) is the number of trials for evaluating the inhibitory activity against the endoplasmic reticulum (ER) -related stress inducer in step (a). It may be more than the number of trials. This is because a useful compound is selected with higher reliability by increasing the number of trials in step (c) in the secondary screening. There is also an advantage that a large number of candidate compounds can be processed by setting the number of trials of the primary screening to be small. Due to the small number of trials, the reliability may be lowered in the primary screening, but the reliability is ensured by the secondary screening.
- the number of trials for evaluating inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer in step (a) is 1, 2, 3, 4, 5 or more times.
- the number of trials for evaluating the inhibitory activity against the endoplasmic reticulum (ER) -related stress inducer in step (c) is 2, 3, 4 so that the number of trials is larger than the number of trials in step (a). Times, 5, 6, 7, 8, 9, 10, or more times.
- a person skilled in the art can appropriately set the number of trials in consideration of the number of candidate compounds.
- the inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer can be assessed based on the activity of inhibiting cell apoptosis caused by the endoplasmic reticulum (ER) -related stress inducer.
- inhibitory activity against endoplasmic reticulum (ER) -related stress inducers may be assessed by measuring caspase activity (eg, caspase 3/7 activity), eg, in a candidate compound non-contact group
- caspase activity eg, caspase 3/7 activity
- a compound having a substantially reduced caspase activity can be selected as a compound having an inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer.
- the candidate compound non-contact group is a group to which no candidate compound is added (an endoplasmic reticulum (ER) -related stress inducer addition group).
- a caspase activity substantially reduced with respect to the candidate compound non-contact group is a caspase activity (eg, caspase 3) that is statistically significantly reduced with respect to the candidate compound non-contact group. For example, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, It can be 65% or less, about 60% or less, or about 55% or less of caspase activity (eg, caspase 3/7 activity).
- a compound that exhibits about 75% or less of caspase 3/7 activity relative to the candidate compound non-contact group can be selected as a compound having inhibitory activity against endoplasmic reticulum (ER) -related stress inducers.
- the toxicity of the candidate compound to immortalized human corneal endothelial cells can be evaluated.
- Toxicity can be performed by assessing cell viability.
- step (b) includes measuring cell viability and may be assessed as low toxicity if cell viability is not substantially reduced.
- Cell viability is measured in the absence of endoplasmic reticulum (ER) -related stress inducers.
- the cell survival rate that is not substantially reduced means that the cell survival rate is not significantly reduced as compared to the candidate compound non-contact group, for example, about 90% of the candidate compound non-contact group.
- the above cell viability can be obtained.
- the cell viability threshold may be about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, about 100%.
- Caspase activity also decreases due to the decrease in the number of cells, so even if it seems to suppress caspase activity at first glance, it is clear that the number of cells may be significantly reduced due to toxicity became. Therefore, in addition to the measurement of caspase activity, the drug can be accurately selected by measuring the number of cells.
- compounds that show substantially unreduced cell viability and substantially reduced caspase 3/7 activity relative to the candidate compound non-contact group can be selected.
- Such compounds have a ratio of caspase 3/7 activity (%) to cell viability (%) of about 0.9 or less, about 0.85 or less, about 0.8 or less, about 0.75 or less, about 0 0.7 or less, about 0.65 or less, about 0.6 or less, about 0.55 or less, about 0.5 or less, about 0.45 or less, about 0.4 or less, about 0.35 or less, about 0.3
- it may be a compound exhibiting about 0.25 or less, or about 0.2 or less.
- a compound in step (d), a compound may be selected that exhibits a ratio of caspase 3/7 activity (%) to cell viability (%) of about 0.8 or less.
- the screening method of the present invention selects a compound having an inhibitory activity against an endoplasmic reticulum (ER) -related stress inducer
- the compound selected by the screening method of the present invention results from endoplasmic reticulum (ER) -related stress.
- ER endoplasmic reticulum
- Symptoms, disorders or diseases in the corneal endothelium can be prevented or treated.
- Symptoms, disorders or diseases in the corneal endothelium that result from endoplasmic reticulum (ER) -related stress can result from abnormal protein folding.
- the symptom, disorder or disease in the corneal endothelium due to endoplasmic reticulum (ER) -related stress is corneal endothelial cell damage, corneal endothelial damage, corneal endothelial density reduction, gut formation, in Fuchs corneal endothelial dystrophy.
- the compounds selected by the screening method of the present invention can particularly prevent or treat Fuchs corneal endothelial dystrophy.
- kits In another aspect, the present invention provides a kit for screening a compound effective for a disease in corneal endothelium using the above screening method.
- kit refers to a unit provided with a portion to be provided (eg, drug, antibody, label, cell, instructions, etc.) usually divided into two or more compartments.
- This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
- Such a kit preferably comprises instructions or instructions that describe the provided part (eg how to use the drug or how to process the reagent).
- the kit when a kit is used as a reagent kit, the kit usually includes instructions describing how to use drugs, antibodies, and the like.
- the “instruction” describes the method for using the present invention for the user.
- this instruction manual a word indicating the screening method and the method of using the reagent of the present invention is described.
- the instruction sheet may include a word indicating a usage method (screening method).
- This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has been received.
- the instruction sheet is a so-called package insert, and may be provided in a paper medium or may be provided in a form such as an electronic medium (for example, a home page or an e-mail provided on the Internet).
- kits of the invention comprise TGF- ⁇ , immortalized cells of Fuchs corneal endothelial dystrophy, reagents or means for measuring inhibitory activity against TGF- ⁇ , and for measuring cytotoxicity.
- the TGF- ⁇ can be TGF- ⁇ 2.
- the method for producing immortalized cells of Fuchs corneal endothelial dystrophy is as described above.
- the kit of the present invention comprises an endoplasmic reticulum-related stress inducer, an immortalized human corneal endothelial cell, a reagent or means for measuring inhibitory activity against the endoplasmic reticulum-related stress inducer, and cytotoxicity.
- Reagents or means for measuring include MG-132, thapsigargin and tunicamycin.
- the endoplasmic reticulum-related stress inducer can be MG-132.
- Immortalized human corneal endothelial cells were prepared in the same manner as immortalized cells (iFECD) of Fuchs's corneal endothelial dystrophy using normal research corneas (SightLife).
- the kit of the present invention comprises TGF- ⁇ , endoplasmic reticulum-related stress inducer, immortalized cells of Fuchs corneal endothelial dystrophy, immortalized human corneal endothelial cells, and inhibitory activity against TGF- ⁇ . And a reagent or means for measuring inhibitory activity against an endoplasmic reticulum-related stress inducer, and a reagent or means for measuring cytotoxicity.
- the reagent or means of the present invention may contain a “label” for distinguishing the target molecule or substance from others.
- Label include substances (fluorescent substances, luminescent substances, etc.), energy, electromagnetic waves, radiation and the like. Examples of the labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. Any labeling that does not affect the function can be used.
- the reagent or means for measuring inhibitory activity against TGF- ⁇ or an endoplasmic reticulum-related stress inducer is a reagent or means for measuring caspase activity (eg, caspase 3/7 activity). possible.
- the reagent or means for measuring the inhibitory activity against TGF- ⁇ or endoplasmic reticulum-related stress inducer may comprise a luminescent reagent.
- the luminescent reagent can be a reagent for causing the subject to be measured (caspase in the case of caspase activity) to emit light.
- caspase activity for example, caspase 3/7 activity
- Caspase-Glo registered trademark 3/7 Assay
- CellEvent trademark
- Caspase-3 / 7 Green Detection Reagent SensoLite (registered) (Trademark) Reagents or means used in Homogeneous AFC Caspase-3 / 7 Assay Kit, etc.
- One skilled in the art can use known techniques, commercially available kits or reagents or means for measuring caspase activity (eg, caspase 3/7 activity).
- the reagent or means for measuring cytotoxicity can be a reagent or means for measuring cell viability.
- the reagent or means for measuring cytotoxicity can be a luminescent reagent.
- the luminescent reagent may be a reagent for causing the subject to be measured (live cell or dead cell, or both in the case of cell viability) to emit light.
- Reagents or means for measuring cell viability include CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, RealTime-Glo (registered trademark) MT Cell Viability Assay, CellTiter-Blue (registered trademark) Cell Visibility, Cell Visibility. Trademarks) Examples include, but are not limited to, reagents or means used in Cell Viability Reagent and the like.
- One skilled in the art can use known techniques, commercially available kits or reagents or means for measuring cell viability.
- Example 1 Production of an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs' corneal endothelial dystrophy and an immortalized cell of normal corneal endothelial cells (iHCEC))
- immortalized corneal endothelial cell lines iFECD and iHCEC were prepared from corneal endothelial cells derived from Fuchs corneal endothelial dystrophy patients and healthy individuals.
- SB431542 (1 ⁇ mol / l) and SB203580 (4- (4-fluorophenyl) -2- (4-methylsulfonylphenyl) -5 (4-pyridyl) imidazole ⁇ 4- [4- (4-fluoro (Phenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine) (1 ⁇ mol / l) was added (hereinafter referred to as “SB203580 + SB431542 + 3T3 conditioned medium”).
- corneal endothelial cells were enzymatically collected and cultured in SB203580 + SB431542 + 3T3 conditioned medium.
- Corneal endothelial cells derived from a patient with Fuchs corneal endothelial dystrophy were amplified by SV40 large T antigen and hTERT gene by PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc).
- the lentiviral vector was 293T cells (RCB2202; Riken) using three types of helper plasmids (pLP1, pLP2, pLP / VSVG; Life Technologies Inc.) and transfection reagent (Fugene HD; Promega Corp., Madison, WI). Bioresource Center, Ibaraki, Japan).
- the culture supernatant containing the virus was recovered and added to the culture medium of cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy using 5 ⁇ g / ml of polybrene to obtain SV40 large T antigen.
- the hTERT gene was introduced.
- iHCEC immortalized corneal endothelial cell line
- iFECD immortalized corneal endothelial cell line
- Example 2 Screening using immortalized cells of Fuchs corneal endothelial dystrophy
- immortalized cells of Fuchs' corneal endothelial dystrophy produced in Example 1 were used to screen for compounds effective for diseases in the corneal endothelium.
- TGF- ⁇ 2 was used as a substance that induces cytotoxicity of corneal endothelial cells.
- TGF- ⁇ 2 (manufacturer: R & D Systems, Inc., distributor: Wako Pure Chemical Industries, Ltd./manufacturer code 302-B2-002, 302-B2-010, distributor code: 553-62881, 559-62883 ) (5 ng / mL) alone or both TGF- ⁇ 2 (5 ng / mL) and SCREEN-WELL® FDA Applied Drug Library V2, Japan version of each drug (10 ⁇ M) and 5% CO 2 at 37 ° C. The cell morphology was observed using a phase contrast microscope after 28 hours under the above conditions. Thereafter, caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay (Promega, # G8091).
- DMEM + 2% FBS + 1% P / S was used as the medium.
- the caspase 3/7 activity and cell viability were measured using a Veritas TM Microplate Luminometer. With such an apparatus, it is possible to measure a large number of samples simultaneously.
- DMSO Dimethylsulfoxide, Sterile-filtered
- nacalai tesque, 13408-64 a solvent of each reagent was added.
- a caspase inhibitor Z-VD-FMK isomer mixture
- Caspase activity was measured after incubation in Dulbecco's modified Eagle medium (DMEM) + 2% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S) for 28 hours.
- DMEM Dulbecco's modified Eagle medium
- FBS fetal bovine serum
- P / S penicillin / streptomycin
- FIG. 3 shows representative results of caspase 3/7 activity as measured by Caspase-Glo® 3/7 Assay in the primary screen.
- the horizontal axis of the graph in FIG. 3 is, from the left, the control group, the TGF- ⁇ 2 (5 ng / mL) added group, the TGF- ⁇ 2 (5 ng / mL) + Z-VD-FMK added group (10 ⁇ M), and the TGF- ⁇ 2 (5 ng).
- CV value coefficient of variation
- CV (%) standard deviation (SD) / average (Av) is an index indicating how much variation is present. Screening generally requires a CV value within 10%.
- the S / B ratio is preferably 2 or more.
- FIG. 4 shows the case where the control is 0%, the TGF- ⁇ 2 reaction is 100%, the TGF- ⁇ 2 reaction is 100%, and the positive control TGF- ⁇ 2 + Z-VD-FMK reaction is 0%.
- the result of calculating each index is shown. As shown, it satisfies the values generally required for screening systems.
- Cell viability was measured after incubation in Dulbecco's modified Eagle medium (DMEM) + 2% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S) for 28 hours.
- DMEM Dulbecco's modified Eagle medium
- FBS 2% fetal bovine serum
- P / S penicillin / streptomycin
- FIG. 7 and FIG. 8 show the results of secondary screening of the drug name of each drug in SCREEN-WELL® FDA Applied Drug Library V2, Japan version, caspase 3/7 activity in TGF- ⁇ 2 stimulation, and cell survival.
- the result of the rate and the value obtained by dividing caspase 3/7 activity by cell viability ((caspase 3/7 activity) / (cell viability) (%)) are shown.
- the value of caspase 3/7 activity in the secondary screening indicates that the lower the cell viability value ((caspase 3/7 activity) / (cell viability) (%)), the more useful the drug.
- FIG. 7 and 8 have caspase 3/7 activity / cell viability (%) of 80% or less, have inhibitory activity against TGF- ⁇ 2, have low toxicity, and are corneal endothelium. It is expected to be effective for diseases and the like.
- FIG. 7 shows drugs with caspase 3/7 activity / cell viability (%) values ranked 1-25
- FIG. 8 shows caspase 3/7 activity / cell viability (%). Indicates a drug ranked in the 26th to 47th position.
- Example 3 Screening using immortalized human corneal endothelial cells
- immortalized human corneal endothelial cells prepared in Example 1 were used to screen for compounds effective for diseases in the corneal endothelium.
- MG-132 known as a substance that induces endoplasmic reticulum (ER) -related stress was used as a substance that induces cell damage.
- Immortalized human corneal endothelial cells were seeded at 7 ⁇ 10 3 in a 96-well plate and cultured at 37 ° C. under 5% CO 2 for 24 hours.
- the medium used was Dulbecco's Modified Eagle Medium (DMEM) (nacalai tesque, 26252-94) + 10% FBS (Biological Industries / 04-001-1A) + 1% penicillin-streptomycin (nacalai tesque, 26252-94).
- MG-132 SIGMA, M7449
- MG-132 0.1 ⁇ M
- Example 2 had a value of (caspase 3/7 activity) / (cell viability) (%)
- Both the top 40 types of drugs (10 ⁇ M) showing 80% or less were added, and cell morphology was observed using a phase contrast microscope after 18 hours at 37 ° C. under 5% CO 2 conditions.
- caspase 3/7 activity was measured using Caspase-Glo (registered trademark) 3/7 Assay (Promega, # G8091).
- MG-132 has the effect of increasing the accumulation of unfolded protein in cells, similar to that observed in patients with Fuchs corneal endothelial dystrophy, Induces death.
- DMEM + 2% FBS + 1% P / S was used as the medium.
- DMSO Dimethylsulfoxide, Sterile-filtered
- nacalai tests, 13408-64 as a solvent of each reagent was added to the control group.
- a caspase inhibitor Z-VD-FMK isomer mixture
- DMEM Dulbecco's Modified Eagle Medium
- FBS fetal calf serum
- P / S penicillin / streptomycin
- FIG. 10 shows the results of screening for each drug name, caspase 3/7 activity upon stimulation with MG-132 (0.1 ⁇ M), results of cell viability upon stimulation with MG-132 (0.1 ⁇ M), and caspase 3 / A value obtained by dividing 7 activity by cell viability ((caspase 3/7 activity) / (cell viability) (%)) is shown.
- the drug shown in FIG. 10 has a caspase 3/7 activity / cell survival rate (%) of 80% or less, has an inhibitory activity against TGF- ⁇ 2, has low toxicity, and causes diseases in the corneal endothelium. Is expected to be effective.
- a method for screening a compound effective for a disease in the corneal endothelium using a model system of Fuchs corneal endothelial dystrophy is provided, and a technique that can be used in industries related to the treatment of corneal endothelial diseases (pharmaceuticals, etc.) is provided.
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Abstract
Description
(項目1)
角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)トランスフォーミング増殖因子(TGF)-βの存在下で候補化合物とフックス角膜内皮ジストロフィの不死化細胞とを接触させ、該候補化合物の該TGF-βに対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、
(c)必要に応じて、該工程(a)において選択された該化合物の該TGF-βに対する阻害活性を評価する工程と、
(d)該工程(b)において該細胞に対する毒性が低いと評価され、かつ、該工程(a)または(c)において該TGF-βに対する阻害活性を有すると評価された化合物を選択する工程と
を含む、方法。
(項目2)
前記TGF-βに対する阻害活性は、前記TGF-βシグナルに起因する前記細胞のアポトーシスを阻害する活性である、項目1に記載の方法。
(項目3)
前記工程(a)および(c)は、カスパーゼ活性を測定することを含み、前記TGF-βに対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、項目1または2に記載の方法。
(項目4)
前記工程(c)における前記TGF-βに対する阻害活性の評価の試行回数は、前記工程(a)における前記TGF-βに対する阻害活性の評価の試行回数よりも多い、項目1~3のいずれか一項に記載の方法。
(項目5)
前記工程(c)における前記TGF-βに対する阻害活性の評価の試行回数は、n=4~6であり、前記工程(a)における前記TGF-βに対する阻害活性の評価の試行回数は、n=1~3である、項目1~4のいずれか一項に記載の方法。
(項目6)
前記カスパーゼ活性は、カスパーゼ3/7活性である、項目3~5のいずれか一項に記載の方法。
(項目7)
前記工程(a)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記TGF-βに対する阻害活性を有する化合物として選択される、項目3~6のいずれか一項に記載の方法。
(項目8)
前記工程(b)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、項目1~7のいずれか一項に記載の方法。
(項目9)
前記工程(d)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、項目1~8のいずれか一項に記載の方法。
(項目10)
前記工程(d)において、前記細胞生存率が約90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、項目1~9のいずれか一項に記載の方法。
(項目11)
前記角膜内皮における症状、障害または疾患が、フックス角膜内皮ジストロフィである、項目1~10のいずれか一項に記載の方法。
(項目12)
(e)小胞体関連ストレス誘導物質の存在下で前記工程(d)において選択された化合物と不死化ヒト角膜内皮細胞とを接触させて、前記工程(d)において選択された化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価する工程と、
(f)前記工程(d)において選択された化合物の該不死化ヒト角膜内皮細胞に対する毒性を評価する工程と、
(g)工程(f)において該不死化ヒト角膜内皮細胞に対する毒性が低いと評価され、かつ、工程(e)において該小胞体関連ストレス誘導物質に対する阻害活性を有すると評価された化合物を選択する工程と
をさらに含む、項目1~11のいずれか一項に記載の方法。
(項目13)
前記小胞体関連ストレス誘導物質に対する阻害活性は、前記小胞体関連ストレス誘導物質に起因する前記不死化ヒト角膜内皮細胞のアポトーシスを阻害する活性である、項目12に記載の方法。
(項目14)
前記工程(e)は、カスパーゼ活性を測定することを含み、前記小胞体関連ストレス誘導物質に対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、項目12または13に記載の方法。
(項目15)
前記カスパーゼ活性は、カスパーゼ3/7活性である、項目14に記載の方法。
(項目16)
前記工程(g)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記小胞体関連ストレス誘導物質に対する阻害活性を有する化合物として選択される項目12~15のいずれか一項に記載の方法。
(項目17)
前記工程(f)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、項目12~16のいずれか一項に記載の方法。
(項目18)
前記工程(g)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、項目12~17のいずれか一項に記載の方法。
(項目19)
前記工程(g)において、前記細胞生存率が約90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、項目12~18のいずれか一項に記載の方法。
(項目20)
前記角膜内皮における症状、障害または疾患が、フックス角膜内皮ジストロフィである、項目12~19のいずれか一項に記載の方法。
(項目21)
前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、項目12~20のいずれか一項に記載の方法。
(項目22)
前記小胞体関連ストレス誘導物質は、MG-132である、項目12~21のいずれか一項に記載の方法。
(項目23)
角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)トランスフォーミング増殖因子(TGF)-βの存在下で候補化合物とフックス角膜内皮ジストロフィの不死化細胞とを接触させ、該候補化合物のカスパーゼ活性を評価し、候補化合物非接触群に対して実質的に低下したカスパーゼ活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該フックス角膜内皮ジストロフィの不死化細胞に対する毒性を評価する工程と、
(c)該工程(a)において選択された該化合物のカスパーゼ活性を評価する工程と、
(d)該工程(b)において該フックス角膜内皮ジストロフィの不死化細胞に対する毒性が低いと評価され、かつ、該工程(c)において実質的に低下したカスパーゼ活性を有すると評価された化合物を選択する工程と
(e)MG-132の存在下で該工程(d)において選択された化合物と不死化ヒト角膜内皮細胞とを接触させて、該工程(d)において選択された化合物の該候補化合物のカスパーゼ活性を評価する工程と、
(f)該工程(e)において選択された化合物の該不死化ヒト角膜内皮細胞に対する毒性を評価する工程と、
(g)該工程(f)において該不死化ヒト角膜内皮細胞に対する毒性が低いと評価され、かつ、該工程(e)において実質的に低下したカスパーゼ活性を有すると評価された化合物を選択する工程と
を含む、方法。
(項目24)
項目1~11のいずれか一項に記載の方法を使用して、角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングするためのキットであって、該キットは、
TGF-βと、
フックス角膜内皮ジストロフィの不死化細胞と、
該TGF-βに対する阻害活性を測定するための試薬または手段と、
細胞毒性を測定するための試薬または手段と
を備える、キット。
(項目25)
前記TGF-βは、TGF-β2である、項目24に記載のキット。
(項目26)
前記フックス角膜内皮ジストロフィの不死化細胞は、SV40ラージT抗原およびhTERTが導入されたフックス角膜内皮ジストロフィ患者の角膜内皮細胞に由来する、項目24または25に記載のキット。
(項目27)
前記フックス角膜内皮ジストロフィの不死化細胞は、4-[4-(1,3-ベンゾジオキソール-5-イル)-5-(2-ピリジニル)-1H-イミダゾール-2-イル]ベンズアミドおよび4-[4-(4-フルオロフェニル)-2-(4-メチルスルフィニルフェニル)-1H-イミダゾール-5-イル]ピリジン)を含む培地中で培養されたフックス角膜内皮ジストロフィ患者の角膜内皮細胞に由来する、項目24~26のいずれか一項に記載のキット。
(項目28)
前記TGF-βに対する阻害活性を測定するための試薬または手段は、カスパーゼ活性を測定するための試薬または手段である、項目24~27のいずれか一項に記載のキット。
(項目29)
前記カスパーゼ活性は、カスパーゼ3/7活性である、項目24~28のいずれか一項に記載のキット。
(項目30)
前記細胞毒性を測定するための試薬または手段は、細胞生存率を測定するための試薬または手段である、項目24~29のいずれか一項のいずれか一項に記載のキット。
(項目31)
前記TGF-βに対する阻害活性を測定するための試薬または手段は発光試薬を含む、項目24~30のいずれか一項のいずれか一項に記載のキット。
(項目32)
前記細胞毒性を測定するための試薬または手段は発光試薬を含む、項目24~31のいずれか一項に記載のキット。
(項目33)
角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)小胞体関連ストレス誘導物質の存在下で候補化合物と不死化ヒト角膜内皮細胞とを接触させ、該候補化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、
(c)必要に応じて、該工程(a)において選択された該化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価する工程と、
(d)該工程(b)において該細胞に対する毒性が低いと評価され、かつ、該工程(a)または(c)において該小胞体関連ストレス誘導物質に対する阻害活性を有すると評価された化合物を選択する工程と
を含む、方法。
(項目34)
前記小胞体関連ストレス誘導物質に対する阻害活性は、前記小胞体関連ストレス誘導物質に起因する前記細胞のアポトーシスを阻害する活性である、項目33に記載の方法。
(項目35)
前記工程(a)および(c)は、カスパーゼ活性を測定することを含み、前記小胞体関連ストレス誘導物質に対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、項目33または34に記載の方法。
(項目36)
前記工程(c)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、前記工程(a)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数よりも多い、項目33~35のいずれか一項に記載の方法。
(項目37)
前記工程(c)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、n=4~6であり、前記工程(a)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、n=1~3である、項目33~36のいずれか一項に記載の方法。
(項目38)
前記カスパーゼ活性は、カスパーゼ3/7活性である、項目33~37のいずれか一項に記載の方法。
(項目39)
前記工程(a)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記小胞体関連ストレス誘導物質に対する阻害活性を有する化合物として選択される、項目33~38のいずれか一項に記載の方法。
(項目40)
前記工程(b)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、項目33~39のいずれか一項に記載の方法。
(項目41)
前記工程(d)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、項目33~40のいずれか一項に記載の方法。
(項目42)
前記工程(d)において、前記細胞生存率が90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、項目33~41のいずれか一項に記載の方法。
(項目43)
前記角膜内皮における疾患が、フックス角膜内皮ジストロフィである、項目33~42のいずれか一項に記載の方法。
(項目44)
前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、項目33~43のいずれか一項のいずれか一項に記載の方法。
(項目45)
前記小胞体関連ストレス誘導物質は、MG-132である、項目33~44のいずれか一項に記載の方法。
(項目46)
項目33~45のいずれか一項に記載の方法を使用して、角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングするためのキットであって、該キットは、
小胞体関連ストレス誘導物質と、
不死化ヒト角膜内皮細胞と、
該小胞体関連ストレス誘導物質に対する阻害活性を測定するための試薬または手段と、
細胞毒性を測定するための試薬または手段と
を備える、キット。
(項目47)
前記不死化ヒト角膜内皮細胞は、SV40ラージT抗原およびhTERTが導入された正常角膜内皮細胞に由来する、項目46に記載のキット。
(項目48)
前記不死化ヒト角膜内皮細胞は、4-[4-(1,3-ベンゾジオキソール-5-イル)-5-(2-ピリジニル)-1H-イミダゾール-2-イル]ベンズアミドおよび4-[4-(4-フルオロフェニル)-2-(4-メチルスルフィニルフェニル)-1H-イミダゾール-5-イル]ピリジン)を含む培地中で培養された正常角膜内皮細胞に由来する、項目46または47に記載のキット。
(項目49)
前記TGF-βに対する阻害活性を測定するための試薬は、カスパーゼ活性を測定するための試薬である、項目46~48のいずれか一項に記載のキット。
(項目50)
前記カスパーゼ活性は、カスパーゼ3/7活性である、項目46~49のいずれか一項に記載のキット。
(項目51)
前記細胞毒性を測定するための試薬は、細胞生存率を測定するための試薬である、項目46~50のいずれか一項に記載のキット。
(項目52)
前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、項目46~51のいずれか一項に記載のキット。
(項目53)
前記小胞体関連ストレス誘導物質は、MG-132である、項目46~52のいずれか一項に記載のキット。
(項目54)
前記小胞体関連ストレス誘導物質に対する阻害活性を測定するための試薬または手段は発光試薬を含む、項目46~53のいずれか一項に記載のキット。
(項目55)
前記細胞毒性を測定するための試薬または手段は発光試薬を含む、項目46~54のいずれか一項に記載のキット。
本明細書において、数値の前の「約」とは、後に続く数値の±10%を意味する。
以下に好ましい実施形態の説明を記載するが、この実施形態は本発明の例示であり、本発明の範囲はそのような好ましい実施形態に限定されないことが理解されるべきである。当業者はまた、以下のような好ましい実施例を参考にして、本発明の範囲内にある改変、変更などを容易に行うことができることが理解されるべきである。
本発明のスクリーニング法では、フックス角膜内皮ジストロフィの不死化細胞を使用する。このフックス角膜内皮ジストロフィの不死化細胞は、特許文献1、2などに概説されており、必要に応じて本明細書において参考として援用される。
(1)細胞外マトリクス層の肥厚
正常細胞(通常2~4μm)より厚くなっていることを確認する。好ましくは正常細胞の少なくとも1.5倍、より好ましくは少なくとも2倍の厚さであることを観察することにより対象となる細胞がフックス角膜内皮ジストロフィの不死化細胞であるかどうか判断することができる。
(2)コラーゲンI、コラーゲンIV、コラーゲンVIIIおよびフィブロネクチンからなる群より選択される少なくとも1つの細胞外マトリクスタンパク質の過剰産生
正常細胞の発現量より多くなっていることを確認する。好ましくは正常細胞の少なくとも1.5倍、より好ましくは少なくとも2倍の発現量であることを観察することにより対象となる細胞がフックス角膜内皮ジストロフィの不死化細胞であるかどうか判断することができる。あるいは、TGF-βに対して応答して細胞外マトリクスタンパク質の発現が亢進しているかどうかをも判断の基礎とすることができる。
(3)ZEB1およびSnail1からなる群より選択される少なくとも1つの上皮間葉移行(EMT)関連マーカー発現亢進
正常細胞の発現量より多くなっていることを確認する。好ましくは正常細胞の少なくとも1.5倍、より好ましくは少なくとも2倍の発現量であることを観察することにより対象となる細胞がフックス角膜内皮ジストロフィの不死化細胞であるかどうか判断することができる。あるいは、TGF-βに対して応答してEMT関連マーカーの発現が亢進しているかどうかをも判断の基礎とすることができる。
(4)非線維芽形態
細胞の形状から判断する。正常細胞同様に多角形の形態を維持して、著しい重層化を生じず一層の上皮細胞様形態を示すことを確認することにより判断することができる。
(5)正常細胞機能
Na+/K+-ATPaseおよびZO-1が正常細胞と同様に免疫染色などにより発現が確認でき、正常細胞と同程度の発現により判断することができる。
本発明において使用されるフックス角膜内皮ジストロフィの不死化細胞は、SV40ラージT抗原およびhTERTをフックス角膜内皮ジストロフィの患者由来の角膜内皮細胞に導入することにより生産される。このフックス角膜内皮ジストロフィの不死化細胞の調製方法は、特許文献1、2などに概説されており、必要に応じて本明細書において参考として援用される。
1つの局面において、本発明は、フックス角膜内皮ジストロフィの不死化細胞(iFECD)を使用した、角膜内皮における疾患に有効な化合物をスクリーニングする方法を提供する。本発明の方法は、TGF-βに対する阻害活性を有する化合物を選択する1次スクリーニングと、細胞に対する毒性が低く、かつ、TGF-βに対する阻害活性を有する化合物を選択する2次スクリーニングを含む。本発明は、1次スクリーニングおよび2次スクリーニングの両方を行うことにより、信頼性の高いスクリーニング方法を提供する。特定の実施形態では、本発明の方法は、(a)トランスフォーミング増殖因子(TGF)-βの存在下で候補化合物とフックス角膜内皮ジストロフィの不死化細胞とを接触させ、該候補化合物の該TGF-βに対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、(b)工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、必要に応じて(c)工程(a)において選択された該化合物の該TGF-βに対する阻害活性を評価する工程と、(d)工程(b)において該細胞に対する毒性が低いと評価され、かつ、工程(c)において該TGF-βに対する阻害活性を有すると評価された化合物を選択する工程とを含み得る。
いくつかの実施形態では、工程(a)におけるTGF-βに対する阻害活性の評価の試行回数は、1回、2回、3回、4回、5回、またはそれ以上の回数であり得、工程(c)におけるTGF-βに対する阻害活性の評価の試行回数は、工程(a)における試行回数よりも多くなるように、2回、3回、4回、5回、6回、7回、8回、9回、10回、またはそれ以上の回数であり得る。特定の実施形態では、工程(c)におけるTGF-βに対する阻害活性の評価の試行回数は、n=4~6であり得、工程(a)におけるTGF-βに対する阻害活性の評価の試行回数は、n=1~3であり得る。好ましい実施形態では、工程(c)におけるTGF-βに対する阻害活性の評価の試行回数は、n=5であり得、工程(a)におけるTGF-βに対する阻害活性の評価の試行回数は、n=2であり得る。当業者であれば、候補化合物の数を考慮して、適宜試行回数を設定することが可能である。
2次スクリーニングでは、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択され得る。そのような化合物は、細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.9以下、約0.8以下、約0.75以下、約0.7以下、約0.65以下、約0.6以下、約0.55以下、約0.5以下、約0.45以下、約0.4以下、約0.35以下、約0.3以下、約0.25以下、または約0.2以下を示す化合物であり得る。特定の実施形態では、工程(d)において、細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択され得る。
1つの局面において、本発明は、不死化ヒト角膜内皮細胞(iHCEC)を使用した、角膜内皮における疾患に有効な化合物をスクリーニングする方法を提供する。本発明の方法は、小胞体(ER)関連ストレス誘導物質に対する阻害活性を有する化合物を選択する1次スクリーニングと、細胞に対する毒性が低く、かつ、小胞体(ER)関連ストレス誘導物質に対する阻害活性を有する化合物を選択する2次スクリーニングを含む。小胞体(ER)関連ストレスを誘導した不死化ヒト角膜内皮細胞(iHCEC)を使用することによって、iFECDを使用するスクリーニング方法とは異なり、小胞体(ER)関連ストレスという観点で角膜内皮における疾患に有効な化合物をスクリーニングすることが可能である。本発明は、1次スクリーニングおよび2次スクリーニングの両方を行うことにより、信頼性の高いスクリーニング方法を提供する。特定の実施形態では、本発明の方法は、(a)小胞体(ER)関連ストレス誘導物質の存在下で候補化合物と不死化ヒト角膜内皮細胞とを接触させ、該候補化合物の該小胞体(ER)関連ストレス誘導物質に対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、(b)工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、(c)工程(a)において選択された該化合物の該小胞体(ER)関連ストレス誘導物質に対する阻害活性を評価する工程と、(d)工程(b)において該細胞に対する毒性が低いと評価され、かつ、工程(c)において該小胞体(ER)関連ストレス誘導物質に対する阻害活性を有すると評価された化合物を選択する工程とを含み得る。
別の局面において、本発明は、上記スクリーニング方法を使用して、角膜内皮における疾患に有効な化合物をスクリーニングするためのキットを提供する。
本実施例では、フックス角膜内皮ジストロフィ患者由来および健常者由来の角膜内皮細胞から不死化角膜内皮細胞株(iFECDおよびiHCEC)を作製した。
シアトルアイバンクから購入した研究用角膜より角膜内皮細胞を基底膜とともに機械的に剥離し、コラゲナーゼを用いて基底膜よりはがして回収後、初代培養を行った。培地はOpti-MEM I Reduced-Serum Medium, Liquid(INVITROGEN カタログ番号:31985-070)に、8%FBS(BIOWEST、カタログ番号:S1820-500)、200mg/ml CaCl2・2H2O(SIGMA カタログ番号:C7902-500G)、0.08% コンドロイチン硫酸(SIGMA カタログ番号:C9819-5G)、20μg/mlアスコルビン酸(SIGMA カタログ番号:A4544-25G)、50μg/mlゲンタマイシン(INVITROGEN カタログ番号:15710-064)および5ng/ml EGF(INVITROGEN カタログ番号:PHG0311)を加えた3T3フィーダー細胞用の馴化させたものを基本培地として用いた。また、基本培地にSB431542(1μmol/l)およびSB203580(4-(4-フルオロフェニル)-2-(4-メチルスルホニルフェニル)-5(4-ピリジル)イミダゾール<4-[4-(4-フルオロフェニル)-2-(4-メチルスルフィニルフェニル)-1H-イミダゾール-5-イル]ピリジン)(1μmol/l)を添加したもの(「SB203580+SB431542+3T3馴化培地」という)で培養した。
フックス角膜内皮ジストロフィの臨床診断により水疱性角膜症に至り、角膜内皮移植(デスメ膜内皮角膜移植=DMEK)を実施されたヒト患者3名より文書による同意および倫理員会の承認のもと角膜内皮細胞を得た。DMEKの際に機械的に病的な角膜内細胞と基底膜であるデスメ膜とともに剥離し、角膜保存液であるOptisol-GS(ボシュロム社)に浸漬した。その後、コラゲナーゼ処理を行い酵素的に角膜内皮細胞を回収して、SB203580+SB431542+3T3馴化培地により培養した。培養したフックス角膜内皮ジストロフィ患者由来の角膜内皮細胞はSV40ラージT抗原およびhTERT遺伝子をPCRにより増幅して、レンチウイルスベクター(pLenti6.3_V5-TOPO; Life Technologies Inc)に導入した。その後、レンチウイルスベクターを3種類のヘルパープラスミド(pLP1、pLP2、pLP/VSVG; Life Technologies Inc.)とともにトランスフェクション試薬(Fugene HD; Promega Corp., Madison, WI)を用いて293T細胞 (RCB2202; Riken Bioresource Center, Ibaraki, Japan)に感染させた。48時間の感染後にウイルスを含む培養上清を回収して、5 μg/mlのポリブレンを用いて、培養したフックス角膜内皮ジストロフィ患者由来の角膜内皮細胞の培養液に添加して、SV40ラージT抗原およびhTERT遺伝子を導入した。フックス角膜内皮ジストロフィ患者由来の不死化角膜内皮細胞株(iFECD)の位相差顕微鏡像を確認した。シアトルアイバンクから輸入した研究用角膜より培養した角膜内皮細胞を同様の方法で不死化し、正常角膜内皮細胞の不死化細胞株を作製した(iHCEC)。健常ドナー由来の不死化角膜内皮細胞株(iHCEC)および不死化角膜内皮細胞株(iFECD)の位相差顕微鏡像をみると、iHCECおよびiFECDはいずれも正常の角膜内皮細胞同様に一層の多角形の形態を有する。iHCECおよびiFECDはダルベッコ改変イーグル培地(DMEM)+10%ウシ胎仔血清(FBS)により維持培養を行った。
本実施例では、実施例1で作製したフックス角膜内皮ジストロフィの不死化細胞を用いて、角膜内皮における疾患に有効な化合物のスクリーニングを行った。本実施例では、角膜内皮細胞の細胞障害を誘導する物質としてTGF-β2を使用した。
フックス角膜内皮ジストロフィの不死化細胞を96ウェルプレートに7×103個播種し、37℃で5%CO2の条件下にて24時間培養した。培地はダルベッコ改変イーグル培地(DMEM)(nacalai tesque,26252-94)+10% FBS(Biological Industries/04-001-1A)+1%ペニシリン-ストレプトマイシン(nacalai tesque、26252-94)を使用した。次に、TGF-β2(製造元:R&D Systems, Inc.、販売元:和光純薬工業株式会社/製造元コード302-B2-002,302-B2-010、販売元コード:553-62881,559-62883)(5ng/mL)のみまたはTGF-β2(5ng/mL)とSCREEN-WELL(登録商標) FDA Approved Drug Library V2, Japan versionの各薬剤(10μM)両方を添加し、37℃で5%CO2の条件下にて28時間後に位相差顕微鏡を用いて細胞形態を観察した。その後Caspase-Glo 3/7 Assay(Promega,#G8091)を用いてカスパーゼ3/7活性の測定を行った。培地は、DMEM+2% FBS+1% P/Sを使用した。本実施例におけるカスパーゼ3/7活性および細胞生存率の測定は、VeritasTM Microplate Luminometerを使用した。このような装置であれば、多数の試料について同時に測定することが可能である。
(1次スクリーニング)
図3は、1次スクリーニングにおけるCaspase-Glo(登録商標) 3/7 Assayにより測定されたカスパーゼ3/7活性の代表的な結果を示す。第1次スクリーニングとしてn=2で測定し、TGF-β2(5ng/mL)添加群を100%とした。図3におけるグラフの横軸は、左から、コントロール群、TGF-β2(5ng/mL)添加群、TGF-β2(5ng/mL)+Z-VD-FMK添加群(10μM)、TGF-β2(5ng/mL)+SCREEN-WELL(登録商標) FDA Approved Drug Library V2, Japan version各薬剤添加群(10μM)を示す。図3に示されるコントロール群等の(1)(2)は、96ウェルプレートの左端、右端に置いた結果をそれぞれ示しており、コントロール群、TGF-β2(5ng/mL)添加群、TGF-β2(5ng/mL)+Z-VD-FMK添加群(10μM)は(1)(2)合わせてn=5である。
一般的にスクリーニングで使われる代表的な指標を以下に示す。
・CV値(変動係数、coefficient of variation);CV(%)=標準偏差(SD)/平均(Av)は、どれ位ばらつきがあるのかを示す指標である。スクリーニングでは概ね、CV値10%以内が求められる。
・S/B比(Signal/Background ratio);S/B=Av100%/Av0%は、反応前後におけるシグナル強度の比である。S/B比が大きいほど読み取り幅が大きくなるので活性有無を判定しやすくなる。S/B比は2以上が好ましい。
・S/N比(Signal/Noise ratio);S/N=(Av100%-Av0%)/SD0%は、ベースのばらつきに対するシグナルの大きさの比である。
・Z’-factor;Z’-factor=1-(3×SD100%+3×SD0%)/(Av100%-Av0%)は、データのばらつきやシグナル強度から計算される、アッセイ系の質の目安となる数字である。アッセイ系の精度を表すもっとも重要な指標で、一般的に、Z’値が0.5以上であればスクリーニングの系として合格とみなされる。
CellTiter-Glo(登録商標) Luminescent Cell Viability Assay(Promega カタログ番号:G7570)で、n=2で毒性の指標として細胞生存率を算出した。細胞生存率は、ダルベッコ改変イーグル培地(DMEM)+2% ウシ胎仔血清(FBS)+1%ペニシリン/ストレプトマイシン(P/S)中28時間培養後に測定した。細胞生存率の測定はTGF-β非存在下で行った。2次スクリーニングにおける細胞生存率の測定の代表的な結果を図5に示す。細胞生存率が90%を切った場合、ランキングから除外した。
2次スクリーニングにおいてCaspase3/7活性の評価をn=5で行った。代表的な結果を図6に示す。
図7および図8は、SCREEN-WELL(登録商標) FDA Approved Drug Library V2, Japan versionでの各薬剤の薬剤名、TGF-β2刺激におけるカスパーゼ3/7活性の2次スクリーニングの結果、細胞の生存率の結果、及びカスパーゼ3/7活性を細胞生存率で割った値((カスパーゼ3/7活性)/(細胞生存率)(%))を示す。2次スクリーニングにおけるカスパーゼ3/7活性の値を細胞生存率の値((カスパーゼ3/7活性)/(細胞生存率)(%))が低いほど有用な薬剤であることを示している。図7および図8に示される薬剤は、カスパーゼ3/7活性/細胞生存率(%)が80%以下を示しており、TGF-β2に対する阻害活性を有し、かつ、毒性が低く、角膜内皮における疾患等に有効であることが予測される。図7は、カスパーゼ3/7活性/細胞生存率(%)の値が1~25位にランク付けられた薬剤を示しており、図8は、カスパーゼ3/7活性/細胞生存率(%)の値が26~47位にランク付けられた薬剤を示している。
本実施例では、実施例1で作製した不死化ヒト角膜内皮細胞を用いて、角膜内皮における疾患に有効な化合物のスクリーニングを行った。本実施例では、細胞障害を誘導する物質として小胞体(ER)関連ストレスを誘導する物質として知られるMG-132を使用した。
不死化ヒト角膜内皮細胞(iHCEC)を96ウェルプレートに7×103個播種し、37℃で5%CO2の条件下にて24時間培養した。培地はダルベッコ改変イーグル培地(DMEM)(nacalai tesque,26252-94)+10% FBS(Biological Industries/04-001-1A)+1%ペニシリン-ストレプトマイシン(nacalai tesque、26252-94)を使用した。次に、MG-132(SIGMA、M7449)(0.1μM)のみまたはMG-132(0.1μM)と実施例2において(カスパーゼ3/7活性)/(細胞生存率)(%)の値が80%以下を示した上位40種類の各薬剤(10μM)両方を添加し、37℃で5%CO2の条件下にて18時間後に位相差顕微鏡を用いて細胞形態を観察した。その後Caspase-Glo(登録商標) 3/7 Assay(Promega,#G8091)を用いてカスパーゼ3/7活性の測定を行った。MG-132は、フックス角膜内皮ジストロフィ患者で認められるのと同様に、折りたたまれなかったタンパク質(unfolded protein)の細胞内への蓄積を増加させる作用を有しており、小胞体ストレスを介した細胞死を誘導する。培地は、DMEM+2% FBS+1% P/Sを使用した。なお、コントロール群には各試薬の溶媒であるDMSO(Dimethyl Sulfoxide、Sterile-filtered)(nacalai tesque、13408-64)を添加した。また、陽性対照としてカスパーゼ阻害剤であるZ-VD-FMK(異性体混合物)(和光純薬工業株式会社/262-02061)(10μM)を使用した。本実施例におけるスクリーニングの概要を図9に示す。実施例2と同様の手順で、カスパーゼ3/7活性および細胞生存率の測定を行った(それぞれn=2)。カスパーゼ活性および細胞生存率は、ダルベッコ改変イーグル培地(DMEM)+2% ウシ胎仔血清(FBS)+1%ペニシリン/ストレプトマイシン(P/S)中18時間培養後に測定した。
図10は、各薬剤名、MG-132(0.1μM)刺激におけるカスパーゼ3/7活性のスクリーニングの結果、MG-132(0.1μM)刺激での細胞の生存率の結果、及びカスパーゼ3/7活性を細胞生存率で割った値((カスパーゼ3/7活性)/(細胞生存率)(%))を示す。図10に示される薬剤は、カスパーゼ3/7活性/細胞生存率(%)が80%以下を示しており、TGF-β2に対する阻害活性を有し、かつ、毒性が低く、角膜内皮における疾患等に有効であることが予測される。
Claims (55)
- 角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)トランスフォーミング増殖因子(TGF)-βの存在下で候補化合物とフックス角膜内皮ジストロフィの不死化細胞とを接触させ、該候補化合物の該TGF-βに対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、
(c)必要に応じて、該工程(a)において選択された該化合物の該TGF-βに対する阻害活性を評価する工程と、
(d)該工程(b)において該細胞に対する毒性が低いと評価され、かつ、該工程(a)または(c)において該TGF-βに対する阻害活性を有すると評価された化合物を選択する工程と
を含む、方法。 - 前記TGF-βに対する阻害活性は、前記TGF-βシグナルに起因する前記細胞のアポトーシスを阻害する活性である、請求項1に記載の方法。
- 前記工程(a)および(c)は、カスパーゼ活性を測定することを含み、前記TGF-βに対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、請求項1または2に記載の方法。
- 前記工程(c)における前記TGF-βに対する阻害活性の評価の試行回数は、前記工程(a)における前記TGF-βに対する阻害活性の評価の試行回数よりも多い、請求項1~3のいずれか一項に記載の方法。
- 前記工程(c)における前記TGF-βに対する阻害活性の評価の試行回数は、n=4~6であり、前記工程(a)における前記TGF-βに対する阻害活性の評価の試行回数は、n=1~3である、請求項4に記載の方法。
- 前記カスパーゼ活性は、カスパーゼ3/7活性である、請求項3~5のいずれか一項に記載の方法。
- 前記工程(a)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記TGF-βに対する阻害活性を有する化合物として選択される、請求項6に記載の方法。
- 前記工程(b)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、請求項1~7のいずれか一項に記載の方法。
- 前記工程(d)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、請求項8に記載の方法。
- 前記工程(d)において、前記細胞生存率が約90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、請求項9に記載の方法。
- 前記角膜内皮における症状、障害または疾患が、フックス角膜内皮ジストロフィである、請求項1~10のいずれか一項に記載の方法。
- (e)小胞体関連ストレス誘導物質の存在下で前記工程(d)において選択された化合物と不死化ヒト角膜内皮細胞とを接触させて、前記工程(d)において選択された化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価する工程と、
(f)前記工程(d)において選択された化合物の該不死化ヒト角膜内皮細胞に対する毒性を評価する工程と、
(g)工程(f)において該不死化ヒト角膜内皮細胞に対する毒性が低いと評価され、かつ、工程(e)において該小胞体関連ストレス誘導物質に対する阻害活性を有すると評価された化合物を選択する工程と
をさらに含む、請求項1~11のいずれか一項に記載の方法。 - 前記小胞体関連ストレス誘導物質に対する阻害活性は、前記小胞体関連ストレス誘導物質に起因する前記不死化ヒト角膜内皮細胞のアポトーシスを阻害する活性である、請求項12に記載の方法。
- 前記工程(e)は、カスパーゼ活性を測定することを含み、前記小胞体関連ストレス誘導物質に対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、請求項12または13に記載の方法。
- 前記カスパーゼ活性は、カスパーゼ3/7活性である、請求項14に記載の方法。
- 前記工程(g)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記小胞体関連ストレス誘導物質に対する阻害活性を有する化合物として選択される請求項15に記載の方法。
- 前記工程(f)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、請求項12~16のいずれか一項に記載の方法。
- 前記工程(g)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、請求項17に記載の方法。
- 前記工程(g)において、前記細胞生存率が約90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、請求項18に記載の方法。
- 前記角膜内皮における症状、障害または疾患が、フックス角膜内皮ジストロフィである、請求項12~19のいずれか一項に記載の方法。
- 前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、請求項12~20のいずれか一項に記載の方法。
- 前記小胞体関連ストレス誘導物質は、MG-132である、請求項21に記載の方法。
- 角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)トランスフォーミング増殖因子(TGF)-βの存在下で候補化合物とフックス角膜内皮ジストロフィの不死化細胞とを接触させ、該候補化合物のカスパーゼ活性を評価し、候補化合物非接触群に対して実質的に低下したカスパーゼ活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該フックス角膜内皮ジストロフィの不死化細胞に対する毒性を評価する工程と、
(c)該工程(a)において選択された該化合物のカスパーゼ活性を評価する工程と、
(d)該工程(b)において該フックス角膜内皮ジストロフィの不死化細胞に対する毒性が低いと評価され、かつ、該工程(c)において実質的に低下したカスパーゼ活性を有すると評価された化合物を選択する工程と
(e)MG-132の存在下で該工程(d)において選択された化合物と不死化ヒト角膜内皮細胞とを接触させて、該工程(d)において選択された化合物の該候補化合物のカスパーゼ活性を評価する工程と、
(f)該工程(e)において選択された化合物の該不死化ヒト角膜内皮細胞に対する毒性を評価する工程と、
(g)該工程(f)において該不死化ヒト角膜内皮細胞に対する毒性が低いと評価され、かつ、該工程(e)において該実質的に低下したカスパーゼ活性を有すると評価された化合物を選択する工程と
を含む、方法。 - 請求項1~11のいずれか一項に記載の方法を使用して、角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングするためのキットであって、該キットは、
TGF-βと、
フックス角膜内皮ジストロフィの不死化細胞と、
該TGF-βに対する阻害活性を測定するための試薬または手段と、
細胞毒性を測定するための試薬または手段と
を備える、キット。 - 前記TGF-βは、TGF-β2である、請求項24に記載のキット。
- 前記フックス角膜内皮ジストロフィの不死化細胞は、SV40ラージT抗原およびhTERTが導入されたフックス角膜内皮ジストロフィ患者の角膜内皮細胞に由来する、請求項24または25に記載のキット。
- 前記フックス角膜内皮ジストロフィの不死化細胞は、4-[4-(1,3-ベンゾジオキソール-5-イル)-5-(2-ピリジニル)-1H-イミダゾール-2-イル]ベンズアミドおよび4-[4-(4-フルオロフェニル)-2-(4-メチルスルフィニルフェニル)-1H-イミダゾール-5-イル]ピリジン)を含む培地中で培養されたフックス角膜内皮ジストロフィ患者の角膜内皮細胞に由来する、請求項26に記載のキット。
- 前記TGF-βに対する阻害活性を測定するための試薬または手段は、カスパーゼ活性を測定するための試薬または手段である、請求項24~27のいずれか一項に記載のキット。
- 前記カスパーゼ活性は、カスパーゼ3/7活性である、請求項24~28のいずれか一項に記載のキット。
- 前記細胞毒性を測定するための試薬または手段は、細胞生存率を測定するための試薬または手段である、請求項24~29のいずれか一項に記載のキット。
- 前記TGF-βに対する阻害活性を測定するための試薬または手段は発光試薬を含む、請求項24~30のいずれか一項に記載のキット。
- 前記細胞毒性を測定するための試薬または手段は発光試薬を含む、請求項24~31のいずれか一項に記載のキット。
- 角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングする方法であって、該方法は、
(a)小胞体関連ストレス誘導物質の存在下で候補化合物と不死化ヒト角膜内皮細胞とを接触させ、該候補化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価し、該阻害活性を有する化合物を選択する工程と、
(b)該工程(a)において選択された該化合物の該細胞に対する毒性を評価する工程と、
(c)必要に応じて、該工程(a)において選択された該化合物の該小胞体関連ストレス誘導物質に対する阻害活性を評価する工程と、
(d)該工程(b)において該細胞に対する毒性が低いと評価され、かつ、該工程(a)または(c)において該小胞体関連ストレス誘導物質に対する阻害活性を有すると評価された化合物を選択する工程と
を含む、方法。 - 前記小胞体関連ストレス誘導物質に対する阻害活性は、前記小胞体関連ストレス誘導物質に起因する前記細胞のアポトーシスを阻害する活性である、請求項33に記載の方法。
- 前記工程(a)および(c)は、カスパーゼ活性を測定することを含み、前記小胞体関連ストレス誘導物質に対する阻害活性が、候補化合物非接触群に対して実質的に低下したカスパーゼ活性である、請求項33または34に記載の方法。
- 前記工程(c)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、前記工程(a)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数よりも多い、請求項33~35のいずれか一項に記載の方法。
- 前記工程(c)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、n=4~6であり、前記工程(a)における前記小胞体関連ストレス誘導物質に対する阻害活性の評価の試行回数は、n=1~3である、請求項36に記載の方法。
- 前記カスパーゼ活性は、カスパーゼ3/7活性である、請求項35~37のいずれか一項に記載の方法。
- 前記工程(a)において、候補化合物非接触群に対して約75%以下のカスパーゼ3/7活性を示す化合物が、前記小胞体関連ストレス誘導物質に対する阻害活性を有する化合物として選択される、請求項38に記載の方法。
- 前記工程(b)は、細胞生存率を測定することを含み、該細胞生存率が実質的に減少していない場合、前記毒性が低いと評価される、請求項33~39のいずれか一項に記載の方法。
- 前記工程(d)において、実質的に減少していない細胞生存率、および候補化合物非接触群に対して実質的に低下したカスパーゼ3/7活性を示す化合物が選択される、請求項40に記載の方法。
- 前記工程(d)において、前記細胞生存率が90%以上を示し、かつ、該細胞生存率(%)に対するカスパーゼ3/7活性(%)の比が約0.8以下を示す化合物が選択される、請求項41に記載の方法。
- 前記角膜内皮における疾患が、フックス角膜内皮ジストロフィである、請求項33~42のいずれか一項に記載の方法。
- 前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、請求項33~43のいずれか一項に記載の方法。
- 前記小胞体関連ストレス誘導物質は、MG-132である、請求項44に記載の方法。
- 請求項33~45のいずれか一項に記載の方法を使用して、角膜内皮における症状、障害または疾患に有効な化合物をスクリーニングするためのキットであって、該キットは、
小胞体関連ストレス誘導物質と、
不死化ヒト角膜内皮細胞と、
該小胞体関連ストレス誘導物質に対する阻害活性を測定するための試薬または手段と、
細胞毒性を測定するための試薬または手段と
を備える、キット。 - 前記不死化ヒト角膜内皮細胞は、SV40ラージT抗原およびhTERTが導入された正常角膜内皮細胞に由来する、請求項46に記載のキット。
- 前記不死化ヒト角膜内皮細胞は、4-[4-(1,3-ベンゾジオキソール-5-イル)-5-(2-ピリジニル)-1H-イミダゾール-2-イル]ベンズアミドおよび4-[4-(4-フルオロフェニル)-2-(4-メチルスルフィニルフェニル)-1H-イミダゾール-5-イル]ピリジン)を含む培地中で培養された正常角膜内皮細胞に由来する、請求項46または47に記載のキット。
- 前記TGF-βに対する阻害活性を測定するための試薬は、カスパーゼ活性を測定するための試薬である、請求項46~48のいずれか一項に記載のキット。
- 前記カスパーゼ活性は、カスパーゼ3/7活性である、請求項46~49のいずれか一項に記載のキット。
- 前記細胞毒性を測定するための試薬は、細胞生存率を測定するための試薬である、請求項46~50のいずれか一項に記載のキット。
- 前記小胞体関連ストレス誘導物質は、MG-132、タプシガルギンおよびツニカマイシンからなる群から選択される、請求項46~51のいずれか一項に記載のキット。
- 前記小胞体関連ストレス誘導物質は、MG-132である、請求項46~52のいずれか一項に記載のキット。
- 前記小胞体関連ストレス誘導物質に対する阻害活性を測定するための試薬または手段は発光試薬を含む、請求項46~53のいずれか一項に記載のキット。
- 前記細胞毒性を測定するための試薬または手段は発光試薬を含む、請求項46~54のいずれか一項に記載のキット。
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