Abstract
Furmonertinib (Alflutinib, AST2818), as a third-generation epidermal growth factor receptor inhibitor with an advanced efficacy and a relatively wide safety window, has been commercially launched in China recently. However, previous clinical studies demonstrated its time- and dose-dependent clearance in a multiple-dose regimen. In vitro drug metabolism and pharmacokinetic studies have suggested that furmonertinib is mainly metabolized by cytochrome P450 3A4 (CYP3A4) and can induce these enzymes via an increased mRNA expression. This study investigated two important evaluation criteria of CYP3A4 induction by furmonertinib through quantitative proteomics and probe metabolite formation: simultaneous (1) protein expression and (2) enzyme activity with sandwich-cultured primary human hepatocytes in the same well of cell culture plates. Results confirmed that furmonertinib was a potent CYP3A4 inducer comparable with rifampin and could be used as a positive model drug in in vitro studies to evaluate the induction potential of other drug candidates in preclinical studies. In addition, inconsistencies were observed between the protein expression and enzyme activities of CYP3A4 in cells induced by rifampin but not in groups treated with furmonertinib. As such, furmonertinib could be an ideal positive control in the evaluation of CYP3A4 induction. The cells treated with 10 µM rifampin expressed 20.16 ± 5.78 pmol/mg total protein, whereas the cells induced with 0.5 µM furmonertinib expressed 4.8 ± 0.66 pmol/mg protein compared with the vehicle (0.1% dimethyl sulfoxide), which contained 0.65 ± 0.45 pmol/mg protein. The fold change in the CYP3A4 enzyme activity in the cells treated with rifampin was 5.22 ± 1.13, which was similar to that of 0.5 µM furmonertinib (3.79 ± 0.52).
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Acknowledgements
The study was partially financially supported by a grant from the National Natural Science Foundation of China (No. 81903701). We would like to acknowledge Dr. Shou-yan Wu and Prof. Li-kun Gong for their help in cell transfection experiments. Also, we appreciate all members in our laboratory for their kind help in daily work.
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YLW and ZTG designed and performed the experiments. YLW, DFZ, and XXD wrote the manuscript. YLW and ZDC performed the sample analysis and LC-MS/MS maintenance. YLW and YRX performed the isolation of rat/mouse primary hepatocytes. YLW, YRX, and XYG analyzed the RNA and protein samples of mouse hepatocytes.
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Wu, Yl., Xue, Yr., Guo, Zt. et al. Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes. Acta Pharmacol Sin 43, 747–756 (2022). https://doi.org/10.1038/s41401-021-00692-7
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DOI: https://doi.org/10.1038/s41401-021-00692-7
