Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues

As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tis-sues from human eyes were analyzed for the activity and expres-sion of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activi-ties. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and dil-tiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tis-sues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be de-tected. Inhibition studies showed that hydrolysis of fluorescein diace-tate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue -de-pendent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacoki-netic modeling of the eye.SIGNIFICANCE STATEMENT Novel and comprehensive data on the protein expression and ac-tivities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical spe-cies, this study will improve the understanding of interspecies dif-ferences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.

Automated summary

Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.