An in vitro approach to investigate ocular metabolism: Part I - Levobunolol

The number drugs on the market for topical ocular administration in treatment of ocular diseases have been steadily increasing in the recent past. Despite ocular transport and ocular delivery being very well documented, metabolism in the eye is not well-documented. We devised a novel yet simple in vitro approach coupled with high resolution mass spectrometry that may be readily applied to future ocular drug metabolism studies to measure rate and extent of metabolism. The current investigation documents in vitro ocular and liver metabolism of levobunolol, a clinically used, potent non-selective β adrenergic antagonist, in S9 fractions from rat, rabbit and human. In the present study, we identified sixteen metabolites of levobunolol, eight of which were not previously reported. A direct acetyl conjugate of levobunolol that was identified in all ocular and liver fractions was never reported in any prior in vitro or in vivo levobunolol metabolism studies. We identified two human ocular specific conjugative metabolites subsequent to oxidation. Overall, our results for levobunolol metabolism indicate that rat liver S9 and human ocular S9 incubations showed the presence of most metabolites, and that liver was a poor in vitro surrogate for eye across rat, rabbit and human for studying the rate and extent of metabolism. To the best of our knowledge, our investigation is the first documentation of comprehensive in vitro ocular metabolism of levobunolol and betaxolol (see companion paper, Bushee J.L. et. al.) in rat, rabbit and human ocular S9 and its comparison to liver S9 metabolic characterization