Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct.

The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea.

Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas.

mRNA expression of metabolic enzymes in human cornea, corneal cell lines, and hemicornea constructs.

PURPOSE: Drugs from ophthalmic formulations are mainly absorbed into the eye via the corneal route. However, little is known about drug metabolism during the transcorneal passage. The objective of this study was to determine the mRNA expression of phase I and II isoenzymes in human corneal epithelial tissue, corneal cell lines, and a tissue-engineered cornea equivalent (a hemicornea construct) as in vitro model for drug absorption studies. METHODS: The reverse transcription polymerase chain reaction was used to profile the mRNA expression of 10 cytochrome P450 enzymes (CYP) and seven phase II enzymes in the three human corneal cell lines and the hemicornea construct. The human corneal epithelial cell line (HCE-T), human corneal keratocyte cell line (HCK-Ca) and human corneal endothelial cell line (HENC) were used. Human liver tissue, human corneal epithelium from donor corneas, and the human colon adenocarcinoma cell line Caco-2 were also investigated. RESULTS: All the phase I and II mRNAs were expressed in the human liver tissue. The Caco-2 cell line showed an expression pattern similar to the liver tissue, although the signals for CYP1A2 and CYP3A4 were absent. In the case of the donor human corneal epithelium, all the detected phase I mRNAs had lower levels than did the liver tissue. By contrast, the phase II mRNA expression pattern was heterogeneous to the liver tissue. The expression patterns in the three human corneal cell lines were comparable, although the signals for a few phase I enzymes and N-acetyltransferase (NAT2) mRNAs were only detectable in the HCE-T. In the hemicornea construct, all the investigated phase I and II mRNA (except for CYP1A2, CYP2B6, CYP2C19, and NAT2) were expressed. CONCLUSIONS: Overall, the mRNA expressions of the tested phase I and phase II enzymes in the hemicornea construct and the three corneal cell lines correlated well with the expression patterns of the ex vivo human corneal epithelium.

In vitro cell culture models to study the corneal drug absorption.

INTRODUCTION: Many diseases of the anterior eye segment are treated using topically applied ophthalmic drugs. For these drugs, the cornea is the main barrier to reaching the interior of the eye. In vitro studies regarding transcorneal drug absorption are commonly performed using excised corneas from experimental animals. Due to several disadvantages and limitations of these animal experiments, establishing corneal cell culture models has been attempted as an alternative. AREAS COVERED: This review summarizes the development of in vitro models based on corneal cell cultures for permeation studies during the last 20 years, starting with simple epithelial models and moving toward complex organotypical 3D corneal equivalents. EXPERT OPINION: Current human 3D corneal cell culture models have the potential to replace excised animal corneas in drug absorption studies. However, for widespread use, the contemporary validation of existent systems is required.