Composition or method including (t)ew-7197 for treating or preventing corneal endothelial diseases
A technology of EW-7197 and corneal endothelium, which is applied in the field of corneal endothelial cell preservation technology, can solve the problems of corneal endothelial malnutrition and insufficient supply of cornea
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Embodiment 1
[0164] (Example 1: Inhibition effect of EW-7197 on corneal endothelial cell disorder)
[0165] In this example, using immortalized human corneal endothelial cells (iFECD) from patients with Fuchs endothelial corneal dystrophy, the inhibitory effect of EW-7197 on corneal endothelial cell disorders was observed. The observation method of cells was performed based on the above-mentioned observation examples. As a subject, SB431542 (refer to International Publication No. 2015 / 064768 ), which is known to suppress corneal endothelial cell disorders, was used.
[0166] (Materials and methods)
[0167] Seed iFECD on 12-well plate, after culturing for 24 hours, remove the medium, add SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, S7530) to the final concentrations of 0.1, 0.3, 1, 3, 10 μM, respectively, Incubate for 24 hours. As a medium, DMEM+2%FBS+1%P / S was used.
[0168] After 24 hours, remove the medium, add SB431542 and EW-7197 to the final concentrations of 0.1, 0...
Embodiment 2
[0172] (Example 2: Discussion on the concentration of EW-7197)
[0173] In this example, the concentration of EW-7197 at which the effect of effectively suppressing corneal endothelial cell disturbance was observed was examined. The inhibitory effect of the corneal endothelial cell disorder was observed by the same method as in Example 1.
[0174] (Materials and methods)
[0175] at 7.0×10 4 Sow iFECD in a 12-well plate at a ratio of 12, culture for 24 hours, remove the medium, add EW-7197 (Selleck Chemicals, S7530) to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM Final concentration, cultured for 24 hours. As a medium, DMEM+2%FBS+1%P / S was used.
[0176] After 24 hours, remove the medium and add 10 ng / ml recombinant human transforming growth factor β2 (WAKO, 200-19911) and EW-7197 to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM The final concentration was incubated for 24 hours. As a medium, DMEM+2%FBS+1%P / S was used.
[0177] After 24 hours, cell morpholo...
Embodiment 3
[0180] (Example 3: cell viability and Caspase3 / 7 activity)
[0181] In this example, analysis of cell viability and Caspase 3 / 7 activity in the presence of EW-7197 was performed. SB431542 was used as a comparative control.
[0182] (Materials and methods)
[0183] (Analysis of cell viability)
[0184] Take 3 x 10 per hole 3 Seed iFECD in a 96-well plate at a ratio of 37 °C (5% CO 2 ) to confluence. As a medium, DMEM+10%FBS+1%P / S was used.
[0185] After 24 hours, the medium was removed, and SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, S7530) were added to final concentrations of 0.01, 0.1, 1, and 10 μM, respectively, and cultured for 48 hours. As a medium, DMEM+2%FBS+1%P / S was used.
[0186] After 24 hours, the cell morphology was observed under a phase-contrast microscope.
[0187] After the observation, the cell viability was analyzed by the Cell Titer-Glo luminescent cell viability assay (Luminescent Cell Viability Assay) in the following procedure. T...
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