Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry
Abstract
Aim: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes.
Methods: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system.
Results: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 μmol/L and 35 pmol·min−1·mg−1, respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin.
Conclusion: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.
Keywords:
Methods: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system.
Results: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 μmol/L and 35 pmol·min−1·mg−1, respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin.
Conclusion: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.