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

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.

HWD, 0000-0002-3437-2748; MWA, 0000-0002-1147-4960 Abemaciclib (Verzenio w ) 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 a-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.

Chromatographic conditions
An Agilent 6410 QQQ equipped with an ESI coupled to an Agilent 1200 HPLC was used. The liquid and mass chromatographic parameters were adjusted for each drug. ABC and its metabolites were produced in the collision cell by CID. The optimized conditions for chromatographic resolution of incubation mixture extract are given in table 2.

RLM incubations
ABC was incubated at 20 mM with 1 mg ml 21 RLMs, 1 mM NADPH, 1 mM KCN and 50 mM Na/K phosphate buffer ( pH 7.4) containing 3.3 mM MgCl 2 . The mixtures were incubated at 378C in a shaking water bath for 60 min before the metabolic reactions were terminated using protein precipitation by adding 2 ml of ice-cold ACN followed by centrifugation at 9000 g for 10 min at 48C. The supernatants were removed to clean vials then evaporated to dryness, reconstituted in the mobile phase and analysed by the LC/MS system [8,12,17,20]. Two controls were done in the absence of NADPH or RLMs to confirm that ABC phase I metabolites were metabolically formed.

Characterization of ABC reactive intermediates in in vitro metabolic reactions
The same RLM metabolic incubation with ABC, previously described in §2.3, was repeated but in addition to 1.0 mM KCN to trap reactive iminium intermediates. This experiment was repeated three   times to confirm the results and support our conclusions. Two controls were done in the absence of NADPH or KCN to confirm that cyano adducts are formed due to metabolic bioactivation.

Identification of ABC reactive metabolites
MS scan and extracted ion chromatogram (EIC) detection modes were used to characterize and locate metabolites in the incubation mixtures, while product ion (PI) was used to identify ABC in vitro metabolites and adducts of reactive intermediates formed in ABC metabolism. Locating metabolites in metabolic mixture extract chromatogram was performed by EIC of m/z of the supposed ABC metabolites.

Results and discussion
3.1. PI study of ABC

Identification of ABC521 phase I metabolite of ABC
ABC521 chromatographic peak appears at 28.7 min in PI chromatogram (figure 5a). CID of ABC521 generates two FIs at m/z 407 and m/z 365 (figure 5b). Compared with PIs of ABC, an increase of 14 m/z units was identified, which indicates that oxidation metabolic reaction occurred in benzimidazole ring (scheme 4).

Conclusion
Three potential iminium reactive metabolites were detected and the bioactivation pathways were proposed ( figure 11). Nine in vitro phase I metabolites were identified. The findings of potentially reactive intermediates of these drugs may give a deeper understanding of their adverse effects. Further drug discovery studies in ABC structure can shed more light on the possibility of blocking or reducing the formation of reactive intermediates by introducing alkyl substituents or isosteric replacement to the alpha position of the piperazine partial moiety which would probably block or interrupt enzymatic oxidation/hydroxylation on a-carbon atoms. This study opens the way for new drug development with more safety profile.
Ethics. Male Sprague-Dawley rats were maintained according to the Animal Care Center instructions at KSU that were accepted by the Local Animal Care and Use Committee of KSU. The animal experimental design used in