Identification and characterization of in silico, in vivo, in vitro, and reactive metabolites of infigratinib using LC-ITMS: bioactivation pathway elucidation and in silico toxicity studies of its metabolites

Abstract

Infigratinib (INF) is a novel, small molecule that is orally administered to inhibit human fibroblast growth factor receptors (FGFRs), which are a family of receptor tyrosine kinases that may be upregulated in different tumor cell types. On 6 January 2020, the FDA granted fast track designation to INF for first-line treatment of cholangiocarcinoma. Prediction of susceptible sites of metabolism and reactivity pathways (cyanide and GSH) for INF was performed by the Xenosite web predictor tool. Then, we report the characterization and identification of in vitro, in vivo, and reactive intermediates of INF using liquid chromatography ion trap mass spectrometry (LC-ITMS). Finally, an in silico toxicity assessment of INF metabolites was carried out using the StarDrop DEREK module showing structural alerts. Rat liver microsomes (RLMs) and isolated perfused rat liver hepatocytes were incubated with INF in vitro and the generated metabolites were collected by protein precipitation. In vivo metabolism was evaluated by time-course urine sampling from Sprague-Dawley rats administered a single INF oral dose. A similar volume of acetonitrile was added to each collected urine sample and both organic and aqueous layers were analyzed by LC-ITMS to detect in vivo INF metabolites. N-Ethyl piperazine rings and benzene at part A of the INF structure are metabolized to form iminium and 1,4-benzoquinone, respectively, which are very reactive toward nucleophilic macromolecules. Incubation of INF with RLMs in the presence of 1.0 mM KCN and 1.0 mM glutathione was used to evaluate reactive metabolites potentially responsible for toxicities associated with INF. There were seven in vitro phase I metabolites, three in vitro phase II metabolites, three cyano adducts, and three GSH conjugate metabolites of INF detected by LC-ITMS. In vivo INF metabolites identified included four in vivo phase I and three in vivo phase II metabolites. In vitro and in vivo phase I metabolic pathways included N-dealkylation, N-demethylation, O-demethylation, hydroxylation, and dechlorination, while the in vivo phase II metabolic reaction was a direct conjugation of INF with glucuronic acid and sulphate.