In vitro metabolites characterization of ponatinib in human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive‐Orbitrap tandem mass spectrometry

Abstract

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q‐Exactive‐Orbitrap‐MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N‐demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N‐demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450‐mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.

Use of Structural Alerts for Reactive Metabolites in the Application SpotRM

“…An older one is ponatinib (Entry 5) that was mentioned as an “outlier” with no clear SAs . However, GSH adducts on the imidazopyridazine heterocycle have been reported but reaction pathways have not been fully elucidated. , Besides, a major in vivo metabolite in human is the carboxylic acid cleavage product from amide hydrolysis, which means that an aniline product is concurrently formed, as captured by the indicated alert in Table .…”