Identification and characterization of in vivo, in vitro and reactive metabolites of vandetanib using LC-ESI-MS/MS

Abstract

Vandetanib (Caprelsa tablets, VNT) is an orally inhibitor of vascular endothelial growth factor receptor 2. The current research reports the characterization and identification of in vitro, in vivo and reactive intermediates of VNT. In vitro metabolites of VNT were performed by incubation with rat liver microsomes (RLMs). Extraction of vandetanib and its in vitro metabolites from the incubation mixtures were done by protein precipitation. In vivo metabolism was done by giving one oral dose of vandetanib (30.8mg/kg) to Sprague Dawley rats in metabolic cages by using oral gavage. Urine was gathered then filtered at certain time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120h) from vandetanib dosing. A similar volume of ACN was added to each collected urine sample. Both layers (organic and aqueous) were injected into liquid chromatography electro spray ionization tandem mass spectrometry (LC-ESI-MS/MS) to detect in vivo vandetanib metabolites. N-methyl piperidine ring of vandetanib is considered a cyclic tertiary amine that undergoes metabolism forming iminium intermediates that are very reactive toward nucleophilic macromolecules. Incubation of vandetanib with RLMs in the presence of 1.0mM KCN was made to check reactive metabolites as it is usually responsible for noticeable idiosyncratic toxicities including phototoxicity and QT interval prolongation. Four in vivo phase I, one in vivo phase II metabolites, six in vitro phase I metabolites and four cyano conjugates of vandetanib were detected by LC-MS/MS. In vitro and in vivo phase I metabolic reactions were N-oxide formation, N-demethylation, α-carbonyl formation and α-hydroxylation. In vivo phase II metabolic reaction was direct conjugation of vandetanib with glucuronic acid. All metabolic reactions occurred in N-methyl piperidine of vandetanib which causes toxicity and instability of vandetanib.[Figure not available: see fulltext.]