Structural characterization of in vivo and in vitro metabolites of bosutinib by LC-MS/MS, in combination with the in silico methodologies for toxicity and metabolism prediction

Bosutinib monohydrate, a second-generation tyrosine kinase inhibitor, is primarily used to treat Philadelphia chromosome-positive chronic myelogenous leukemia. Pharmacokinetic studies in humans identified 3 metabolites of bosutinib: oxidative dechlorinated bosutinib, N-desmethylated bosutinib, and bosutinib N-oxide. Although a few metabolites have been reported clinically, a comprehensive understanding of bosutinib's metabolic fate is essential for optimizing its therapeutic use and minimizing risks. Therefore, the present study aimed to investigate the detailed metabolism of bosutinib using a combination of in vitro, in vivo, and in silico methods. In vitro experiments were conducted using liver microsomes and S9 fractions in the presence of suitable cofactors, whereas in vivo studies employed Sprague-Dawley rats in which bosutinib was administered as an oral suspension, followed by the collection of blood, urine, and feces at respective time points. The biological samples were analyzed using liquid chromatography-quadrupole-Orbitrap mass spectrometer. A total of 10 metabolites were identified, including 8 novel ones. The diverse metabolic reactions included oxidative O-dealkylation (B-M1, B-M2, B-M4, and B-M7), N-oxidation (B-M5), oxidative dechlorination (B-M2 and B-M3), N-dealkylation (B-M8 and B-M9), hydroxylation (B-M8), and glycine conjugation (B-M10). Interestingly, no metabolites were detected in the plasma, and the major metabolites, B-M3 (13.91%) and B-M9 (10.58%), were found predominantly in the feces. In silico predictions using Meteor Nexus matched with 6 of the experimentally observed metabolites. Toxicity and mutagenicity were further assessed using Deductive Estimation of Risk from Existing Knowledge Nexus and Structure Activity Relationship Analysis using Hypotheses Nexus, which indicated a potential mutagenic concern for B-M7. The integration of experimental and computational approaches in this work contributes significantly to understanding bosutinib's metabolic profile and can guide future strategies for its safe and effective clinical application. SIGNIFICANCE STATEMENT: This study provides an in-depth exploration of bosutinib's metabolic pathways using in vitro models and in vivo analysis of plasma, urine, and fecal samples. Prominently, in silico toxicity assessments indicated that B-M7 may pose mutagenic risks, emphasizing the need for further investigation.