Assessment of Active Metabolites of Levobunolol and Betaxolol in Rat, Rabbit and Human Ocular and Liver S9 Fractions

Abstract

Little is known about ocular metabolism and formation of pharmacologically active metabolites for drugs dosed topically to the eye. We devised a simple in vitro approach coupled with high resolution mass spectrometry to measure the rate and extent of ocular metabolism for comparison with liver. Levobunolol and betaxolol are antagonists of β‐adrenergic receptors and have been clinically utilized for treatment of primary open‐angle glaucoma and/or ocular hypertension. Levobunolol has been reported to be metabolized to an equipotent active metabolite, dihydrolevobunolol, in plasma and urine of mice, rat, dog, and human. Hydroxybetaxolol, an active metabolite of betaxolol, has been reported in the liver. We aimed to understand whether the eye has functional metabolic activity and to understand the extent of formation of two historically reported in vivo active metabolites, one each for levobunolol and betaxolol, across rat (R), rabbit (Rbt) and human (H) ocular S9 (OS9) fractions and liver S9 (LS9) fractions. Based on high resolution mass spectrometry M11 was identified as the active dihydroxylevobunolol while M2 was identified as the active hydroxybetaxolol. The in vitro data shows that betaxolol's M2 was formed in the eye across preclinical species and human. M2 pharmacological activity for antagonism toward β1‐ or β2‐receptors has not been assessed to date. This is the first report of hydroxybetaxolol being formed in the eye. Results from levobunolol and betaxolol metabolism studies point toward species differences in conversion of levobunolol and betaxolol to their respective active metabolites and strongly indicate that LS9 fractions are a poor surrogate for assessment of active metabolites formed in the eye.