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.