Profiling of in vivo, in vitro and reactive zorifertinib metabolites using liquid chromatography ion trap mass spectrometry

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.

Fig. 1. Predicted atomic sites of metabolism for zorifertinib by XenoSite web predictor.

Fig. 2. Predicted bioactive sites of zorifertinib by XenoSite web predictor including GSH and cyano bioactive centers (A), outcomes of in silico toxicity studies of zorifertinib using DEREK toxicity module of StarDrop software. Red color indicates structural alerts (B).

Fig. 3. MS² mass spectrum of zorifertinib (A). Proposed zorifertinib structure and its corresponding fragment ions (B).

Fig. 4. MS² mass spectrum of M1 (A). Proposed M1 structure and its corresponding fragment ions (B).

Fig. 5. MS² mass spectrum of M7 (A). Constant neutral loss scan of M7 (B). Proposed M7 structure and its corresponding fragment ions (C).

Fig. 6. MS² mass spectrum of M8 (A). Constant neutral loss scan of M8 (B). Proposed M8 structure and its corresponding fragment ions (C).

Fig. 7. MS² mass spectrum of M15 (A). Constant neutral loss scan of M15 (B). Proposed M15 structure and its corresponding fragment ions (C).

Fig. 8. MS² mass spectrum of AZDCN485 (A). Proposed AZDCN485 structure and its corresponding fragment ions (B).

Fig. 9. MS² mass spectrum of AZDGSH715 (A). Constant neutral loss scan of AZDGSH715 (B). Proposed AZDGSH715 structure and its corresponding fragment ions (C).

Fig. 10. Proposed pathways for zorifertinib bioactivation for cyano adducts and GSH conjugates.

Fig. 11. Chemical structure of zorifertinib showing bioactivation pathways including iminium, and electro deficient conjugated system.