Characterization of Stable and Reactive Metabolites of the Anticancer Drug, Ensartinib, in Human Liver Microsomes Using LC-MS/MS: An in silico and Practical Bioactivation Approach

Abstract

Background

Ensartinib (ESB) is a novel anaplastic lymphoma kinase inhibitor (ALK) with additional activity against Abelson murine leukemia (ABL), met proto-oncogene (MET), receptor tyrosine kinase (AXL), and v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1) and is considered a safer alternative for other ALK inhibitors. ESB chemical structure contains a dichloro-fluorophenyl ring and cyclic tertiary amine rings (piperazine) that can be bioactivated generating reactive intermediates.

Methods

In vitro metabolic study of ESB with human liver microsomes (HLMs) was performed and the hypothesis of generating reactive intermediates during metabolism was tested utilizing trapping agents to capture and stabilize reactive intermediates to facilitate their LC-MS/MS detection. Reduced glutathione (GSH) and potassium cyanide (KCN) were utilized as trapping agents for quinone methide and iminium intermediates, respectively.

Results

Four in vitro ESB phase I metabolites were characterized. Three reactive intermediates including one epoxide and one iminium intermediates were characterized. ESB bioactivation is proposed to occur through unexpected metabolic pathways. The piperazine ring was bioactivated through iminium ions intermediates generation, while the dichloro-phenyl group was bioactivated through a special mechanism that was revealed by LC-MS/MS.

Conclusion

These findings lay the foundations for additional work on ESB toxicity. Substituents to the bioactive centers (piperazine ring), either for blocking or isosteric replacement, would likely block or interrupt hydroxylation reaction that will end the bioactivation sequence.

Keywords:

• in vitro phase-I metabolites
• iminium intermediates
• epoxide intermediates
• human liver microsomes

Acknowledgments

The authors extend their appreciation to the Deputyship for Research & Innovation, “Ministry of Education” in Saudi Arabia for funding this research work through the project number IFKSURG-1435-025

Abbreviations

ESB, ensartinib; ESI, electrospray ionization; HLMs, human liver microsomes; TKIs, tyrosine kinase inhibitors; LC-MS/MS, liquid chromatography tandem mass spectrometry; GSH, reduced glutathione; KCN, potassium cyanide; NSCLCs, Non-small cell lung cancers; ALK, Anaplastic lymphoma kinase; CSL, composite site lability; HPLC, high-performance liquid chromatography; ABL, Abelson murine leukemia; MET, met proto-oncogene; ROS1, v-ros UR2 sarcoma virus oncogene homolog 1; Axl, receptor tyrosine kinase.

Ethics

The study’s design (in vitro experiments using commercially available human liver microsomes) exempts it from the need of the Ethics Committees approval.

Author Contributions

All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest for this work.