Diquafosol tetrasodium (INS365)

Cell experiment:

The viabilities of human corneal epithelial cells (HCECs) are determined using a MTT assay. Cells are subconfluent Diquafosol (100 mL diluted 10%, 20%, or 30%) or DMEM (100 mL) is added to controls. After 1, 6, and 24 h, plates are washed three times with PBS to remove the drugs. Cell viabilities are evaluated after incubating for 24 h. MTT is then added to each well. Samples are incubated in the dark for 4 h at 37oC, and media are then removed. Precipitates are resuspended in DMSO. Absorbances are measured on a plate reader at 570 nm[1].

Animal experiment:

Rats: An SD rat dry eye model is used in which exorbital lacrimal gland extirpation decreased the Schirmer test score by at least 50%. After 8 weeks, when significant increases occurred in corneal epithelial permeability, INS365-containing eye drops are applied six times daily for the next 4 weeks at concentrations from 0.03% to 3.0%. Corneal barrier function is evaluated based on measurements with a modified anterior fluorometer of fluorescein penetrance at 1, 2, and 4 weeks after initial application. After INS365 application, the periodic acid–Schiff reagent (PAS)–stained area is evaluated in histologic sections of the tarsal and bulbar conjunctiva[2].

References:

[1]. Lee JH, et al. Comparison of cytotoxicities and wound healing effects of diquafosol tetrasodium and hyaluronic acid on human corneal epithelial cells. Korean J Physiol Pharmacol. 2017 Mar;21(2):189-195.
[2]. Fujihara T, et al. Improvement of corneal barrier function by the P2Y(2) agonist INS365 in a rat dry eye model. Invest Ophthalmol Vis Sci. 2001 Jan;42(1):96-100.
[3]. Fujihara T, et al. INS365 suppresses loss of corneal epithelial integrity by secretion of mucin-like glycoprotein in a rabbit short-term dry eye model. J Ocul Pharmacol Ther. 2002 Aug;18(4):363-70.