ABSTRACT:The recently introduced Clonetics human corneal epithelium (cHCE) cell line is considered a promising in vitro permeability model, replacing excised animal cornea to predict corneal permeability of topically administered compounds. The purpose of this study was to further characterize cHCE as a corneal permeability model from both drug metabolism and transport aspects. First, good correlation was found in the permeability values (P app ) obtained from cHCE and rabbit corneas for various ophthalmic drugs and permeability markers. Second, a previously established realtime quantitative polymerase chain reaction method was used to profile mRNA expression of drug-metabolizing enzymes (major cytochromes P450 and UDP glucuronosyltransferase 1A1) and transporters in cHCE in comparison with human cornea. Findings indicated that 1) the mRNA expression of most metabolizing enzymes tested was lower in cHCE than in excised human cornea, 2) the mRNA expression of efflux transporters [multidrug resistantassociated protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein], peptide transporters (PEPT1 and PEPT2), and organic cation transporters (OCTN1, OCTN2, OCT1, and OCT3) could be detected in cHCE as in human cornea. However, multidrug resistance (MDR) 1 and organic anion transporting polypeptide 2B1 was not detected in cHCE; 3) cHCE was demonstrated to possess both esterase and ketone reductase activities known to be present in human cornea; and 4) transport studies using probe substrates suggested that both active efflux and uptake transport may be limited in cHCE. As the first detailed report to delineate drug metabolism and transport characteristics of cHCE, this work shed light on the usefulness and potential limitations of cHCE in predicting the corneal permeability of ophthalmic drugs, including ester prodrugs, and transporter substrates.Topical instillation is the desired route of administration for ophthalmic drugs to treat diseases in the anterior segment of the eye including glaucoma, inflammations, infections, and dry eye. The primary pathway of drug permeation from the tear fluid to the anterior chamber of the eye is via the transcorneal route. The cornea has a multilayered structure constituted primarily of corneal epithelium, stroma, and endothelium. However, passage through the corneal epithelium is considered to be the rate-limiting step in the transcorneal penetration of most ophthalmic drugs (Maurice and Mishima, 1984).In the past decade, several in vitro corneal permeability models that can substitute for the isolated cornea to predict ocular absorption and facilitate ophthalmic drug discovery have been developed (Reichl and Becker, 2008). These models evolved from primary cultures using rabbit corneas and immortalized rabbit cell lines to the immortalized human corneal epithelial (HCE) cell lines, and even include complex systems such as human corneal constructs (summarized in Table 1). Given the pros and cons of each model, HCE cell lines seem to be most appealing owing to their human o...