Abstract
Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.
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Acknowledgements
We thank Hannele Jaatinen for technical assistance during the incubation experiments and Miia Reponen for technical assistance during the LC/MS analyses. We further thank Ivan Vokřál, Ph.D. for valuable comments and Daniel Paul Sampey for the English revision.
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This work was funded by the Charles University Grant Agency (Project GAUK No. 1302120), the Czech Science Foundation (Grant no. 18-09946S), and Charles University (Research Project SVV 260 550). The School of Pharmacy mass spectrometry laboratory is supported by Biocenter Finland and Biocenter Kuopio.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by MŠ, ROJ and SA. The first draft of the manuscript was written by MŠ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Human liver tissue was obtained from the University Hospital of Oulu (Oulu, Finland) as a surplus from cadaveric kidney transplantation donors. The procedure was approved by the Ethics Committee of the Medical Faculty of the University of Oulu (January 21, 1986). This study was performed in line with the principles of the Declaration of Helsinki. The Wistar rats used in this study were maintained according to European guidelines on the protection of animals used for scientific purposes (Directive 2010/63/EU). The animal experiments were approved by the Ethics Committee for Animal Experiments, University of Kuopio (Document 01-38, June 1, 2000).
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Šadibolová, M., Juvonen, R.O., Auriola, S. et al. In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity. Arch Toxicol 96, 793–808 (2022). https://doi.org/10.1007/s00204-021-03218-6
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DOI: https://doi.org/10.1007/s00204-021-03218-6