Characterization of in vitro primary metabolic profile of SKLB-M8, a novel antitumor compound, using liquid chromatography coupled with triple quadrupole tandem mass spectrometry and quadrupole time-of-flight tandem mass spectrometry

• The structures of SKLB-M8 and its metabolites were identified by UPLC/Q-TOF-MS/MS.
• SKLB-M8 metabolic pathways were clarified by inhibitors of P450 at different concentrations.
• Drug–drug interactions of SKLB-M8 were further investigated.
• A UPLC–QqQ-MS/MS method was developed and applied to study SKLB-M8 enzymatic kinetics.

In early drug development, metabolism studies provide important parameters regarding efficacy and safety of drug candidates. Our previous pharmacological studies employing SKLB-M8 have demonstrated its potential as an anti-tumor drug candidate. The aim of this work was to evaluate its in vitro primary metabolism and determine the enzymatic kinetic parameters by UPLC–QqQ-MS/MS and UPLC/Q-TOF-MS/MS in human liver microsome samples (HLM), rat liver microsomes samples (RLM) and dog liver microsome samples (DLM). Our results showed that SKLB-M8 was metabolized to at least four major P450-mediated metabolites in HLM samples and respective two in RLM and DLM samples through the processes of hydroxylation, demethylation, ring opening and demethoxylation. The high in vitro intrinsic clearance of SKLB-M8 indicated that it is metabolized rapidly in vivo. Metabolism of SKLB-M8 was mainly catalyzed by CYP2E1, CYP2C19 and CYP3A4 while in RLM samples, Cyp1a2 and Cyp2a6 were also the main catalysts and a similar result was obtained for Cyp2a6 in DLM samples. Besides, in RLM samples, SKLB-M8 had weak inhibition to Cyp2d6, moderate inhibition to Cyp1a2, Cyp2c9, Cyp3a4 and strong inhibition to Cyp2c19. The results suggested that drug–drug interactions might significantly occur for SKLB-M8 when co-administrated with other drugs or compounds. The enzymatic kinetic parameters revealed a sigmoidal profile with S50 = 1.21 ± 0.07, 0.46 ± 0.045 and 610.97 ± 0.047 μM, Vmax = 247.19 ± 0.02, 44.50 ± 0.03 and 610.97 ± 0.047 μM/min/mg protein and h = 1.82 ± 0.17, 1.52 ± 0.21 and 1.24 ± 0.26 respectively. Results of this work provide critical data to give the first clues regarding SKLB-M8 metabolism.

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