Identification and characterization of in vitro, in vivo, and reactive metabolites of tandutinib using liquid chromatography ion trap mass spectrometry

Abstract

Tandutinib (TND) is a novel, oral small molecule designed for treating acute myeloid leukemia (AML) by inhibiting type III receptor tyrosine kinases. This study reports the use of in silico, in vivo, and in vitro methods to investigate the metabolism and possible metabolic bioactivation of TND. First, in silico metabolism of TND was assessed using the WhichP450™ module of the StarDrop® software to determine labile sites of metabolism in the TND chemical structure. Second, the XenoSite reactivity model, a web-based metabolism prediction software, was used to determine probable bioactive centers. Based on the in silico outcomes, a list of predicted metabolites and reactive intermediates were prepared. Third, in vitro and in vivo experiments were performed. In vitro TND metabolites were generated through incubation of TND with rat liver microsomes (RLMs). Another incubation of TND with RLMs was separately performed in the presence of GSH and KCN to check for the generation of reactive intermediates (soft and hard electrophiles). In vitro phase II metabolism was assessed by incubation of TND with isolated perfused rat hepatocytes. In vivo metabolism was investigated by oral gavage of TND (37 mg kg-1) in Sprague Dawley rats. Five in vitro phase I metabolites, one in vitro phase II and five reactive iminium intermediates (cyano adducts), six in vivo phase I, and one in vivo phase II metabolites of TND were characterized. The in vitro and in vivo metabolic pathways involved were O-dealkylation, α-hydroxylation, α-carbonyl formation, reduction, glucuronide, and sulfate conjugation. No GSH conjugate or its catabolic products were detected either in vitro or in vivo. Two cyclic tertiary rings of TND (piperazine and piperidine) were metabolically bioactivated to generate reactive iminium intermediates forming cyano adducts with KCN. The formed reactive intermediates may be the reason behind TND toxicity. In silico toxicological studies were performed for TND and its related (in vitro and in vivo) metabolites were evaluated using the DEREK software tool.