Characterization of Human Corneal Epithelial Cell Model As a Surrogate for Corneal Permeability Assessment: Metabolism and Transport

Abstract

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...

Potential pharmacokinetic role of organic cation transporters in modulating the transcorneal penetration of its substrates administered topically

“…5 Hence, cationic drugs may utilize some uptake transporters for their permeation across biological membranes. 19,20,28 Presence of transporters like OCT in the conjunctival and corneal epithelial cells to reabsorb various endogenous amines in the tear fluid has been well recognized. [6][7][8] Moreover, evidence from molecular biology studies from our laboratory and others showed the presence of various OCT in cornea.…”

“…In previous studies, transport processes due to OCT were showed using ex vivo models in excised cornea, 19 in vitro corneal cells, 19 and excised conjunctiva 20 for understanding the functional importance of OCT. However, from the literature it was also evident that cell line studies did not reveal (due to minimal expression of transporters in the cell lines) the active functional role of OCT in transporting the administered eye drop.…”

“…However, from the literature it was also evident that cell line studies did not reveal (due to minimal expression of transporters in the cell lines) the active functional role of OCT in transporting the administered eye drop. 19 Studying the functional importance of OCT in vivo can reveal their role in the transport of organic cations across the cornea, when administered topically. It is well known that tetraethylammonium chloride (TEA) and metformin are cation substrates of OCT, and atropine and quinidine can inhibit the uptake of cation substrates.…”

Interaction of Ocular Hypotensive Agents (PGF2α Analogs—Bimatoprost, Latanoprost, and Travoprost) With MDR Efflux Pumps on the Rabbit Cornea

“…Corneal permeability of these prostaglandin analogs are in agreement with previously reported values in the literature. 9,21 It was noticed that both latanoprost and travoprost (ester prodrugs) rapidly hydrolyze to its free acid form thus substantiating the fact that the free acid form is responsible for IOP reduction. But in case of bimatoprost (amide prodrug), hydrolysis was partial, though the free acid form was the predominant species.…”

“…[6][7][8] In addition, mRNA expression levels of other isoforms of MRP, MRP1 and MRP3, have been reported in the human cornea, though functional activity and localization still remain to be assessed. 9,10 With an array of effl ux transporters being identifi ed on the corneal epithelium and along with our earlier report that clearly demonstrates the role of effl ux pumps in restricting ocular bioavailability, it is imperative to screen potential ocular therapeutic agents for interaction with effl ux pumps.…”