Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues

Abstract

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC ( d -luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Mechanistic Model for the Prediction of Small-Molecule Vitreal Clearance Combining Diffusion-Limited and Permeability-Limited Clearance

“…Esterase and peptidase activity in the vitreous have been frequently reported in literature [10][11][12]32 and carboxyesterase 1 was meanwhile also quantified in the rabbit vitreous. 12 As our investigated compound set is unfortunately not suitable to gain deeper insights into the relevance of esterase activities for vitreal CL, we recommend reviewing the predictivity of the model when working with potential substrates for the above-mentioned metabolic pathways and adapting the model if needed. The compound set is, on the other hand, rich in substrates for various phase I and II metabolic reactions including cytochrome P450s, aldehyde oxidase, glucuronidation, and sulfation.…”

“…Some metabolizing enzymes have been detected in ocular tissues of different preclinical species. Esterase and peptidase activity in the vitreous have been frequently reported in literature − , and carboxyesterase 1 was meanwhile also quantified in the rabbit vitreous . As our investigated compound set is unfortunately not suitable to gain deeper insights into the relevance of esterase activities for vitreal CL, we recommend reviewing the predictivity of the model when working with potential substrates for the above-mentioned metabolic pathways and adapting the model if needed.…”

“…The barriers before RPE or retinal endothelium, such as the inner limiting membrane, are not expected to pose a permeability barrier for small molecules. The elimination via the anterior segment is driven by diffusion of the compounds through the opening between the vitreous and the posterior chamber to the aqueous humor followed by further elimination through the anterior segment tissues or aqueous humor outflow. , As the limited data available argue for a low expression of metabolizing enzymes in the vitreous itself, metabolism is likely a minor pathway of vitreal elimination and only relevant for certain molecules susceptible to esterase or peptidase activity. − Therefore, most of the CL is expected to occur through the anterior and posterior routes and depends essentially on two mechanisms, diffusion in the vitreous and permeation through the posterior segment tissues. While the diffusion rate depends mainly on MW, permeability in the narrow MW range of small molecules is affected more by the molecular structures which define permeability-relevant properties such as lipophilicity or PSA.…”

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

“…Similarly, the highest rates of NPA hydrolysis are observed in rabbit cornea and iris-ciliary body (Heikkinen et al, 2018). We published recently, for the first time, significant species and tissue differences in protein expression and activities of esterases (Hammid et al, 2021).…”

Topical ophthalmic administration: Can a drug instilled onto the ocular surface exert an effect at the back of the eye?

“…Rabbit conjunctiva allows permeation of molecules of 20 kDa (~3.2 nm) through the paracellular pore (Huang et al, 1989), with a surface area ~nine times the corneal counterpart (Watsky et al, 1988). Moreover, metabolism may take place for drugs that are substrates of esterases (as observed in rabbit and porcine conjunctiva) (Heikkinen et al, 2018;Hammid et al, 2021). Most of the drugs absorbed in the conjunctiva will reach the conjunctival vessels and be effectively cleared into the systemic circulation.…”