Profiling of in vitro and reactive metabolites of Ponatinib using LC-MS/MS method: metabolic stability and bioactivation pathway elucidation | Abstract
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Journal of Computational Methods in Molecular Design


Profiling of in vitro & reactive metabolites of Ponatinib using LC-MS/MS method: metabolic stability and bioactivation pathway elucidation

Author(s): Sawsan Mohamed Amer

Ponatinib ( PNT) , marketed as (Iclusig™ tablets), is a drug taken orally for the management of some types of tumors including acute lymphoblastic leukemia  and chronic myeloid leukemia . The current work reports the identification and characterization of in vitro & reactive  metabolites of PNT. In vitro metabolites were generated by incubation with rat liver microsomes (RLMs). Extraction of ponetanib and its in vitro metabolites from the incubation mixtures were done by protein precipitation method. N-methyl piperazine ring of ponetanib, a cyclic tertiary amine ring , undergoes metabolism to form iminium intermediates that are reactive toward nucleophilic macromolecules. Incubation with RLMs in the presence of 1.0 mM KCN to check reactive metabolites as it is often responsible for observed toxicities including phototoxicity and prolongation of QT interval. Seven in vitro phase I metabolites, and four cyano conjugates of ponatinib were detected by LC-MS/MS. In vitro phase I metabolic pathways were N-demethylation, N-oxide formation, oxidation, reduction and hydroxylation . All metabolic reactions occurred in N-methyl piperazine ring of ponatinib which causes its instability and toxicity.
Validated LC–MS/MS method was established for the determination of PNT in RLMs . This method was applied in metabolic stability investigation of PNT. Resolution of PNT & VNT (IS) analytes were  performed using C18 column and isocratic mobile phase composed of binary system of 10 mM ammonium formate (pH 4.1) and acetonitrile in a ratio of 1:1. The flow rate was set at 0.25 mL/min and total run time was 4 min . Ions were generated by ESI source and analyzed by multiple reaction monitoring mode (MRM) in the Agilent 6410

QqQ analyzer. The linearity of the established method ranged from 5 to 400 ng/ ml . LOD and LOQ was 0.44  ng/mL, and 1.32ng/ml , respectivly . The intra-day and inter-day precision and accuracy , ranged from 0.97 to 2.31% and -1.65 to -0.3% respectivly with mean % recovery of 100.09 ± 2.31% . In vitro half-life was 6.26 min and intrinsic clearance was 15.18 ± 0.47 mL/min/kg, indecates low bioavailability.