Characterization of the metabolites of tirabrutinib generated from rat, dog and human liver microsomes using ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry

Rationale: Tirabrutinib is an orally administered Bruton's tyrosine kinase (Btk) inhibitor developed for the treatment of autoimmune disorders and haematological malignancies. The goals of this study were to identify the metabolites of tirabrutinib and to propose the metabolic pathways.

Methods: Tirabrutinib was individually incubated with rat, dog and human liver microsomes at 37°C for 1 h. To trap the potential reactive metabolites, glutathione (GSH) was incorporated into the incubation samples. The incubation samples were analysed using ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS). The metabolites were identified and characterized by exact masses, product ions and retention times.

Results: A total of 18 metabolites, including four GSH conjugates, were identified and characterized in terms of elemental compositions and product ions. The metabolic pathways of tirabrutinib included amide hydrolysis, O-dealkylation, mono-oxygenation, di-oxygenation and GSH conjugation. Among these metabolites, M10 was the most abundant metabolite. Compared with dog, rat has the closer metabolic profiles to humans, and thus it would be more suitable for toxicity study.

Conclusions: This study provides valuable data regarding the in vitro metabolism of tirabrutinib, which may be helpful for further safety assessment of this drug.