Liquid chromatography-tandem mass spectrometry metabolic profiling of nazartinib reveals the formation of unexpected reactive metabolites.

Ali S Abdelhameed,Mohamed W Attwa,Adnan A Kadi

doi:10.1098/rsos.190852

Ali S Abdelhameed, Mohamed W Attwa + Show 1 more

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https://doi.org/10.1098/rsos.190852

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Abstract

Nazartinib (EGF816, NZB) is a promising third-generation human epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. This novel irreversible mutant-selective EGFR inhibitor targets EGFR containing both the resistance mutation (T790M) and the activating mutations (L858R and Del19), while it does not affect wild-type EGFR. However, the metabolic pathway and bioactivation mechanisms of NZB are still unexplored. Thus, using liquid chromatography–tandem mass spectrometry, we screened for products of NZB metabolism formed in vitro by human liver microsomal preparations and investigated the formation of reactive intermediates using potassium cyanide as a nucleophile trap. Unexpectedly, the azepane ring was not bioactivated. Instead, the carbon atom between the aliphatic linear tertiary amine and electron-withdrawing system (butenoyl amide group) was bioactivated, generating iminium intermediates as reactive species. Six NZB phase I metabolites, formed by hydroxylation, oxidation and N-demethylation, were characterized. Moreover, two reactive iminium ions were characterized and their corresponding bioactivation mechanisms were proposed. Based on our results, we speculate that bioactivation of NZB can be blocked by small sterically hindering groups, isosteric replacement or a spacer. This approach might reduce the toxicity of NZB by avoiding the generation of reactive species.

Highlights

Non-small-cell lung cancer (NSCLC) encompasses a heterogeneous group of lung cancer subtypes [1,2,3,4,5], which affects 90% of patients with lung cancer [6]
Tyrosine kinase inhibitors (TKIs) regulate the activity of human epidermal growth factor receptor (EGFR) and have become the standard treatment for patients suffering from advanced EGFR-mutant NSCLC
The first-generation EGFR TKIs bind reversibly and competitively to the ATP-binding site of the EGFR tyrosine kinase (TK) domain, which improves the outcome of NSCLC patients bearing EGFR-activating mutations (L858R and Del19) [7,8]

Summary

Introduction

Non-small-cell lung cancer (NSCLC) encompasses a heterogeneous group of lung cancer subtypes [1,2,3,4,5], which affects 90% of patients with lung cancer [6]. Second-generation EGFR TKIs (e.g. avitinib and dacomitinib) were designed to target tumours with T790M mutation and EGFR-activating mutations These compounds showed promising anti-T790M activity in laboratory experiments. Reactive metabolites are usually generated by phase I metabolic pathways and their identification is hindered by their transient nature To overcome this limitation, a nucleophile can be used to capture reactive intermediates, and the resulting adducts can be characterized and identified by mass spectrometry technique [26,27]. We used a scavenging molecule ( potassium cyanide) to trap reactive intermediates of NZB metabolism This approach was used because when reactive metabolites form in vivo, they bind to DNA and proteins via covalent bonds and cannot be detected [24,27,32]

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