In Vitro Acetylation of Seven Anti‐glaucoma Agents in Rat, Rabbit and Human Ocular and Liver S9 Fractions

Abstract

Ocular N‐acetyltransferase(NAT) research has been limited to probe substrate activity in specific tissues from rat or rabbit, but not for a model that represents the whole eye. The comparison of acetylation as a biotransformation pathway between preclinical species and human has not been well documented. We aimed to understand functional ocular NAT activity across rat, rabbit, and human ocular S9 fractions in comparison with liver S9 fractions for seven clinically approved anti‐glaucoma compounds. Specific probe substrates were examined in human ocular, liver, and kidney S9 fractions to asses human NAT1 and 2 activities. High resolution mass spectrometry was utilized to characterize the acetyl conjugate(s) for each anti‐glaucoma agent in ocular and liver S9 fractions. Direct acetyl conjugates were detected across all species in ocular and liver fractions for betaxolol, carteolol and levobunolol, indicating that these compounds are non‐specific NAT substrates. Oxidation and O‐acetylation of levobunolol was detected in rat and rabbit liver S9 only. Species‐ and organ‐specific metabolic differences were observed for metoprolol, metipranolol, propranolol, and timolol. Neither ocular nor liver fractions from rat or rabbit were useful to predict human ocular acetylation in vitro. Ocular and renal S9 fractions did not catalyze the acetylation of sulfamethazine, a NAT2 probe substrate, contrary to the observation in liver S9. The data suggests a strong role of NAT1 in human ocular S9 fractions. This is the first report of direct acetyl conjugates for the b‐adrenergic receptor antagonist class of anti‐glaucoma agents in rat, rabbit and human ocular S9s.