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Ayumi Mori, chiho Yabuta, Akira Ohtori, Mitsuyoshi Azuma; Intracellular uptake of drugs in cultured human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):263. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The fist site of action for ophthalmic drugs is at the corneal epithelial cell. The concentrations of drugs and metabolites in these cells are needed to predict pharmacological effects and toxicities. Only limited information is available concerning the intracellular uptake of drugs into ocular cells. Thus, the purposes of the present study were to 1) determine the uptake of drugs with different properties into cultured corneal epithelial cells, and 2) establish a model for prediction of pharmacological effects and toxicities. We tested three E-64 inhibitors, which inhibit the non-lysosomal, cysteine proteases called calpains. E-64 and the stronger inhibitor E-64c do not permeate membranes well. E-64d, the ethyl ester of E-64c is membrane-permeable and has been designed as a pro-drug.
Immortalized human corneal epithelial cells (HCE-T) were suspended in growth medium or were mono-layered onto culture plates. After incubation with E-64 inhibitors, the medium was rapidly layered onto silicone oil, and the cells were pelleted by centrifugation. Intracellular concentrations of E-64 inhibitors were measured by LC/MS/MS.
Incubation of suspended cell cultures with 10 mM E-64 or E-64c from 5 to 180 min caused only minor uptake. In contrast, after 5 min incubation with 10 mM E-64d, a large uptake of E-64c (de-esterified E-64d) was observed. Intracellular E-64c peaked at 60 min and gradually decreased until 180 min. A small amount of un-hydrolyzed E-64d co-existed with E-64c at each time point. These data suggested that a large amount of E-64d rapidly penetrated into cells and was hydrolyzed to E-64c. Uptake of E-64d and conversion to E-64c occurred in a dose-dependent manner. Similar intracellular uptake of E-64d was observed in the monolayer culture.
Our model is useful for understanding the relationship between the physicochemical properties of drugs and their permeability across corneal epithelial cell membranes.
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