(251) In Vitro Drug Metabolism and Pharmacokinetic Evaluation of Ent-Verticilide

Objective : The goal of this project was to evaluate the drug metabolism and pharmacokinetic properties of ent-verticilide in vitro.

Methods: To estimate intrinsic clearance, we measured the in vitro half-life of ent-verticilide using liver S9 fractions from human, mouse, rat, and dog. Subsequently, the intrinsic clearance values were incorporated into the well-stirred model of hepatic clearance to estimate the hepatic metabolic clearance in vivo. To identify major metabolites of ent-verticilide, we incubated ent-verticilide with microsomal fractions of human, mouse, rat, and dog. We then subjected extracts from these incubations to LC/MS analysis for structural interpretation of resulting metabolites. Finally, we determined the enzymes responsible for ent-verticilide metabolism by incubating ent-verticilide with human liver microsomes in the presence and absence of various cytochrome P450 inhibitors.

Results: Predicted hepatic clearance for mouse, rat, dog, and human was 76, 41, 29, and 18 mL/min/kg, respectively. The metabolism was determined to be NADPH-dependent, indicating cytochrome P450 metabolism. Metabolites formed via mono-hydroxylation and di-hydroxylation of the hydrocarbon side chains are the major metabolites. Metabolites formation by oxidation of the hydrocarbon side chains were also observed. In addition, CYP3A4 was the major CYP450 implicated in the biotransformation of ent-verticilide.

Conclusions: These data indicate that ent-verticilide is extensively metabolized by hepatic enzymes (high hepatic extraction ratio), and that the metabolism is predominantly mediated by CYP3A4. The major elimination pathway was determined to be via hydroxylation of the hydrocarbon chains.