Identification of Stereoisomeric Metabolites of Meisoindigo in Rat Liver Microsomes by Achiral and Chiral Liquid Chromatography/Tandem Mass Spectrometry

Abstract

ABSTRACT:N-Methylisoindigotin, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vitro metabolism of meisoindigo is limited. In this study, in vitro stereoisomeric metabolites of meisoindigo in rat liver microsomes were identified for the first time by achiral and chiral liquid chromatography/tandem mass spectrometry, together with proton NMR spectroscopy and synchrotron infrared spectroscopy. The major in vitro phase I metabolites of meisoindigo were tentatively identified as stereoselective-reduced meisoindigo, which comprised a pair of (3-R, 3-R) and (3-S, 3-S) enantiomers with lower abundance, as well as another pair of (3-R, 3-S) and (3-S, 3-R) enantiomers with higher abundance. One type of minor in vitro metabolites was tentatively identified as stereoselective N-demethyl-reduced meisoindigo including a pair of (3-R, 3-R) and (3-S, 3-S) enantiomers, as well as one meso compound. Another type of minor in vitro metabolites was tentatively identified as both stereoselective and regioselective monohydroxyl-reduced meisoindigo. Based on the metabolite profiling, three parallel metabolic pathways of meisoindigo in rat liver microsomes were proposed.

Characterization of metabolites of meisoindigo in male and female rat kidney microsomes by high‐performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

“…6). These two metabolic pathways were partially consistent with those metabolic pathways in rat liver microsomes reported previously 12. Rat kidney microsomes did not form any metabolites that were not produced by liver microsomes.…”

“…2). A similar phenomenon was also observed from an incubation sample of meisoindigo in male rat liver microsomes 12. Comparison of the retention times of these two potential metabolites from liver and kidney microsomes showed that they could be the same compounds (data not shown).…”

“…However, the MRM in combination with EPI scanning was unable to detect and identify unexpected metabolites as compared to the conventional full MS scan (EMS) in combination with MS/MS (EPI), if the MRM transitions only depended on the generation through the script in Analyst™ software to cover the unknown metabolites. This disadvantage was improved by addition of possible MRM transitions or removal of unlikely MRM transitions based on the knowledge of the metabolic predictions and the MS/MS fragmentation pattern prediction, as well as experimental results obtained by EMS in combination with EPI scanning reported previously 12. Therefore, integration of MRM with conventional metabolic profiling methodology enables a wide range of potential transitions to be targeted for metabolite detection and could be a powerful tool to provide more comprehensive metabolism information of the drug.…”

“…A list of MRM transitions, including 279.1 → 147.1 for hydrogenation, 265.1 → 133.1 for hydrogenation followed by demethylation, 295.1 → 163.1 and 295.1 → 147.1 for hydrogenation followed by oxidation, 293.1 → 249.1 for oxidation, 309.1 → 265.1 for dioxidation, 263.1 → 235.1 for demethylation, 311.1 → 178.1 for hydrogenation followed by dioxidation, was used to cover the most common phase I biotransformation reactions of meisoindigo. The Q1 and Q3 values were based on the phase I biotransformation prediction of meisoindigo by MetaboliteID 1.4 software (Applied Biosystems/MDS Sciex) as well as the previous results of meisoindigo and its phase I metabolites in rat liver microsomes 12. The ionization parameters were the same as described above except declustering potential and collision energy were adjusted to 60.0 V and 30.0 eV, respectively.…”

“…[10][11][12] Three parallel metabolic pathways of meisoindigo in male rat liver microsomes were proposed as direct 3,3 0 double-bond reduction, reduction followed by N-demethylation, as well as reduction followed by phenyl monooxidation. 11,12 It was also reported that, after oral administration of 3 H-meisoindigo, the excretion of radioactivity in the 48 h feces was about 17.3%, whereas about 34.3% was present in the 48 h urine. 2 It is not clear, in addition to liver, whether kidney is also involved in the metabolism of meisoindigo.…”