Identification of reactive intermediate formation and bioactivation pathways in Abemaciclib metabolism by LC-MS/MS: in vitro metabolic investigation

Abstract

Abemaciclib (Verzenio^®) is approved as a tyrosine kinase inhibitor (TKI) for breast cancer treatment. In this study, in vitro phase I metabolic profiling of Abemaciclib (ABC) was done using rat liver microsomes (RLMs). We checked the formation of reactive intermediates in ABC metabolism using RLMs in the presence of potassium cyanide (KCN) that was used as a capturing agent for iminium reactive intermediates forming a stable complex that can be characterized by LC-MS/MS. Nine in vitro phase I metabolites and three cyano adducts were identified. The metabolic reactions involved in the formation of these metabolites and adducts are reduction, oxidation, hydroxylation and cyanide addition. The bioactivation pathway was also proposed. Knowing the electrodeficient bioactive centre in ABC structure helped in making targeted modifications to improve its safety and retain its efficacy. Blocking or isosteric replacement of α-carbon to the tertiary nitrogen atoms of piperazine ring can aid in reducing toxic side effects of ABC. No previous articles were found about in vitro metabolic profiling for ABC or structural identification of the formed reactive metabolites for ABC.

Keywords: Abemaciclib; iminium intermediates; reactive metabolites; side effects.

Figure 1.

ABC chemical structure.

Figure 2.

ABC PI chromatogram (a) and ABC PI MS spectrum (b).

Scheme 1.

PIs of ABC.

Figure 3.

ABC493 PI chromatogram (a), ABC493a PI MS spectrum (b) and ABC493b PI MS spectrum (c).

Scheme 2.

PIs of ABC493a and ABC493b.

Figure 4.

ABC509 PI chromatogram (a) and ABC509 PI MS spectrum (b).

Scheme 3.

PIs of ABC509.

Figure 5.

ABC521 PI chromatogram (a) and ABC521 PI MS spectrum (b).

Scheme 4.

PIs of ABC521.

Figure 6.

ABC523 PI chromatogram (a), ABC523a PI MS spectrum (b) and ABC523b PI MS spectrum (c).

Scheme 5.

PIs of ABC523a and ABC523b.

Figure 7.

ABC525 PI chromatogram (a), ABC525a PI MS spectrum (b) and ABC525b PI MS spectrum (c).

Scheme 6.

PIs of ABC525a and ABC525b.

Figure 8.

ABC537 PI chromatogram (a) and ABC537 PI MS spectrum (b).

Scheme 7.

PIs of ABC537.

Figure 9.

ABC532 PI chromatogram (a), ABC532a PI MS spectrum (b) and ABC532b PI MS spectrum (c).

Scheme 8.

PIs of ABC532a and ABC532b.

Figure 10.

ABC548 PI chromatogram (a) and ABC548 PI MS spectrum (b).

Scheme 9.

PIs of ABC548.

Scheme 10.

Proposed pathways for the iminium intermediate generation and trapping strategy.

Figure 11.

In vitro metabolic pathways and bioactivation centres of ABC.