High resolution mass spectrometry‐driven metabolite profiling of baricitinib to report its unknown metabolites and step‐by‐step reaction mechanism of metabolism

Abstract

Rationale Metabolite profiling is an integral part of the drug development process for selecting candidates with high therapeutic efficacy and low risk. Baricitinib (BARI) was approved in 2018 by the US Food and Drug Administration to treat rheumatoid arthritis. According to the available literature, no systematic study has been reported on the metabolite profiling of BARI. The biotransformation pathway of the drug has also not been established until recently. This study aims to identify BARI metabolites generated in in vitro matrices. Methods The in vitro metabolism study was carried out using rat liver microsome, human liver microsomes, and human S9 fraction. Ultra high‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (U‐HPLC‐Q/TOF) and ultra‐high‐performance liquid chromatography/linear ion trap‐Orbitrap mass spectrometry (U‐HPLC/LTQ‐Orbitrap‐MS/MS) were used to identify and characterize the metabolites of BARI. The in silico toxicity of BARI and its metabolite was studied using ProTox‐II toxicity predictor software. Results A total of five new metabolites have been identified amongst which two (M1 and M2) were detected on both U‐HPLC/LTQ‐Orbitrap‐MS/MS and U‐HPLC‐Q/TOF and two additional metabolites (M4 and M5) were detected on U‐HPLC/LTQ‐Orbitrap‐MS/MS. Moreover, one metabolite (M3) was only detected on LC‐QTOF. Conclusions The major metabolic changes were found to be N‐dealkylation, demethylation, hydroxylation, and hydrolysis. Metabolites M3 and M4 were found to have the potential for carcinogenicity. The novelty of the study can be justified by the unavailability of any previous research on in vitro metabolite profiling of BARI. Furthermore, this is the first time the biotransformation pathway of BARI and the toxicity potential of its metabolites have been reported.

Comprehensive characterization and preclinical assessment of an imidazopyridine‐based anticancer lead molecule

“…Pro-Tox-II, an in silico software was employed to predict different levels of toxicities (Rachmale et al, 2022;Sahu & Sengupta, 2020).…”

Forced degradation study of baricitinib and structural characterization of its degradation impurities by high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy

“…The metabolic changes observed in this study were N ‐dealkylation, demethylation, hydroxylation, and hydrolysis. Furthermore, no structural similarities were found between the metabolites and the degradation impurities (DIs) 11 . Due to the limitations in the previously reported studies, it was considered necessary to conduct systematic degradation experiments on BARI.…”

Ultra‐high‐performance liquid chromatography–quadrupole time of flight tandem mass spectrometry based in vitro metabolite profiling of DK‐GV‐04P, a novel anticancer molecule under drug discovery

“…The quinazolinone class of compounds shows anticancer activity by inhibition of the PI3K/Akt signalling pathway, protein lysine methyltransferases, topoisomerase I, protein kinase and tubulin polymerization (Wani et al, 2016). An investigational molecule, in recent times owing to the rising concern about metabolite-induced toxicity (Park et al, 2005, Rachmale, Rajput, Jadav, Sahu, Sharma, & Sengupta, 2022, Rachmale, Rajput, Jadav, Sahu, Tekade, & Sengupta, 2022. Generally, estimation of metabolites during the early drug discovery stage is carried out employing in vitro systems like rat liver microsomes/human liver microsomes (HLM), hepatocytes, liver slices or human S9 fractions (HS9) (Li et al, 2020, Liu et al, 2023, Panday et al, 2021.…”

“…Metabolite profiling studies predict if there is any possibility of forming toxic metabolites during biotransformation and help to deduce the possible pathway of formation of different metabolites. To identify and characterize the metabolites, quadrupole time of flight tandem mass spectrometry (Q‐TOF‐MS/MS) serves a vital role in the assignment of putative structure to the metabolites and identification of functional moiety responsible for metabolism (Rachmale, Rajput, Jadav, Sahu, Sharma, & Sengupta, 2022; Sahu & Sengupta, 2020). Comparative mass fragmentation analysis along with the accurate mass measurement of the fragment ions of the analyte and metabolites provides necessary information on the structural changes during biotransformation.…”

“…In a drug discovery program, in vitro metabolism studies are crucial and are carried out in the early lead optimization phase to understand the metabolic fate of the molecule inside the human body. Moreover, the importance of carrying out early metabolism studies has been felt in recent times owing to the rising concern about metabolite‐induced toxicity (Park et al, 2005, Rachmale, Rajput, Jadav, Sahu, Sharma, & Sengupta, 2022, Rachmale, Rajput, Jadav, Sahu, Tekade, & Sengupta, 2022). Generally, estimation of metabolites during the early drug discovery stage is carried out employing in vitro systems like rat liver microsomes/human liver microsomes (HLM), hepatocytes, liver slices or human S9 fractions (HS9) (Li et al, 2020, Liu et al, 2023, Panday et al, 2021, Wang et al, 2020).…”