No abstract

Although ocular transport and delivery have been well studied, metabolism in the eye is not well documented, even for clinically available medications such as levobunolol, a potent and nonselective b-adrenergic receptor antagonist. Recently, we reported an in vitro methodology that could be used to evaluate ocular metabolism across preclinical species and humans. The current investigation provides detailed in vitro ocular and liver metabolism of levobunolol in rat, rabbit, and human S9 fractions, including the formation of equipotent active metabolite, dihydrolevobunolol, with the help of high-resolution mass spectrometry. 11 of the 16 metabolites of levobunolol identified herein, including a direct acetyl conjugate of levobunolol observed in all ocular and liver fractions, have not been reported in the literature. The study documents the identification of six human ocular metabolites that have never been reported. The current investigation presents evidence for ocular and hepatic metabolism of levobunolol via non-cytochrome P450 pathways, which have not been comprehensively investigated to date. Our results indicated that rat liver S9 and human ocular S9 fractions formed the most metabolites. Furthermore, liver was a poor in vitro surrogate for eye, and rat and rabbit were poor surrogates for human in terms of the rate and extent of levobunolol metabolism.

While the compounds tested from "withdrawn and warning category" all formed the glutathione adduct in buffer, none from "safe" category formed the glutathione adduct. In contrast, none of the compounds from any category formed methoxylamine conjugate, which reacts with putative aldehyde moiety formed via acyl migration. These results, highly favorite the nucleophilic displacement as a cause of the reactivity rather than the acyl migration via aldehyde formation. The workflow herein could also be applied in the discovery setting to triage new chemical entities of interest.

ABSTRACT:In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.

The liver is known to be the principal site of drug metabolism. Depending on the route of administration, especially in cases of topical and local delivery, evaluation of local drug metabolism in extrahepatic tissues is vital to assess fraction of the drug metabolized. This parameter becomes important from the point of view of drug availability or the contribution to overall clearance. Examples include fraction metabolized in the gut for oral drugs and contribution of pulmonary or renal clearance to total clearance of a drug. Diseases of the eye represent a rising unmet medical need and a number of therapeutics are currently being developed in the form of small molecules and biologics. Treatment of ocular diseases has expanded to explore various topical formulations and local short- and long-term therapies by ocular routes of administration. Until recently, metabolism in the eye for any species, including human, was not well documented, but this topic is gaining wide interest. Many in vitro-ex vivo models, each with separate pros and cons, are being used for studying ocular metabolism. This review is aimed at providing a perspective on the relevance and application of ocular metabolism, melanin binding, and the use of tissue- and cell-derived ocular models in discovery and preclinical development.