Abstract
Zorifertinib (AZD-3759; ZFB) is a potent, novel, oral, small molecule used for the treatment of non-small cell lung cancer (NSCLC). ZFB is Epidermal Growth Factor Receptor (EGFR) inhibitor that is characterized by good permeability of the blood–brain barrier for (NSCLC) patients with EGFR mutations. The present research reports the profiling of
in vitro
,
in vivo
and reactive metabolites of ZFB. Prediction of vulnerable metabolic sites and reactivity pathways (cyanide and GSH) of ZFB were performed by WhichP450™ module (StarDrop software package) and XenoSite reactivity model (XenoSite Web Predictor-Home), respectively. ZFB
in vitro
metabolites were done by incubation with isolated perfused rat liver hepatocytes and rat liver microsomes (RLMs). Extraction of ZFB and its related metabolites from the incubation matrix was done by protein precipitation.
In vivo
metabolism was performed by giving ZFB (10 mg kg
−1
) through oral gavage to Sprague Dawley rats that were housed in metabolic cages. Urine was collected at specific time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120 h) from ZFB dosing. The collected urine samples were filtered then stored at −70 °C.
N
-Methyl piperazine ring of ZFB undergoes phase I metabolism forming iminium intermediates that were stabilized using potassium cyanide as a trapping agent. Incubation of ZFB with RLMs were performed in the presence of 1.0 mM KCN and 1.0 mM glutathione to check reactive intermediates as it is may be responsible for toxicities associated with ZFB usage. For
in vitro
metabolites there were six
in vitro
phase I metabolites, three
in vitro
phase II metabolites, seven reactive intermediates (four GSH conjugates and three cyano adducts) of ZFB were detected by LC-IT-MS. For
in vivo
metabolites there were six
in vivo
phase I and three
in vivo
phase II metabolites of ZFB were detected by LC-IT-MS.
In vitro
and
in vivo
phase I metabolic pathways were
N
-demethylation,
O
-demethylation, hydroxylation, reduction, defluorination and dechlorination.
In vivo
phase II metabolic reaction was direct sulphate and glucuronic acid conjugation with ZFB.