Li_2025_Drug.Metab.Dispos_53_100145

Hydrolases in the eye play an important role in the metabolism of ophthalmic drugs, especially those administered locally to the eyes. With the growing interest in peptide-based therapeutics for treating eye disease, it has become increasingly important to characterize the enzymatic activities of ocular tissues against both small molecules and peptides to better understand their ocular metabolism. In this study, we characterized the activities of hydrolases, including carboxylesterase 1 and 2, arylacetamide deacetylase, paraoxonases, cytidine deaminase, fatty-acid amide hydrolase, and peptidases by incubating probe substrates in whole eye homogenates and vitreous humors from human donors and 3 preclinical species, including New Zealand White rabbits, Gottingen minipigs, and Cynomolgus monkeys. The enzyme activities were also tested in human liver S9 fractions as a comparison. The activities of all the investigated enzymes were detected and characterized in eye homogenates of humans and the 3 preclinical species, except that arylacetamide deacetylase activity was only observed in rabbits. To our knowledge, the activities of paraoxonases, cytidine deaminase, and fatty-acid amide hydrolase were identified for the first time in the eyes. In addition to eye homogenates, the activities of carboxylesterase 1 and 2 and peptidases were also confirmed in vitreous humor samples from humans and the 3 preclinical species. In general, the extent of in vitro hydrolytic activities of the tested hydrolases in monkeys better mimics those in humans after 24 hours of incubation. The results from this study will offer a better insight for understanding the pharmacokinetic principles of ophthalmic administration in the eyes. SIGNIFICANCE STATEMENT: This study investigated the activities of key hydrolases in the eye homogenates and vitreous humor from humans and preclinical species. The findings of the study provide an understanding of ocular drug metabolism and potentially contributes to ocular drug design and delivery.