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Cytochrome P450 2D6 (CYP2D6) exhibits rich genetic polymorphism, and functional changes caused by variations are the key reasons for differences in substrate drug systemic exposure. Discovering novel variants and defining their enzymatic kinetic characteristics can contribute to the personalized application of drugs. In this study, a data chain of variant-function-structure was established through population-based sequencing, baculovirus insect cell expression, in vitroenzymatic incubation, and ultrahigh performance liquid chromatography tandem mass spectrometry. Results revealed nine novel missense mutations in the exonic regions. After the corresponding microsomes were obtained, the kinetics of the variants were investigated using dextromethorphan as a probe substrate. It was found that the activities of CYP2D6.2, 10, 17, 35, 65, R28G, T76M, and E215K were significantly reduced, while D301V almost led to loss of enzyme function. Additionally, the relative clearance rate of R25Q was significantly increased. From the molecular structure perspective, the mutation sites are distributed outside the dextromethorphan binding pocket, suggesting that they primarily influence CYP2D6 activity via allosteric modulation. These research findings provide fundamental data for the precise application of CYP2D6 substrate drugs.

Purpose: To investigate the effects of hepatic enzyme activity variations and CYP2B6 gene polymorphisms on the in vivoand in vitrometabolism of efavirenz. Main methods: In vitro enzyme systems using rat and human liver microsomes (RLM/HLM) were established, with in vivo studies conducted on Sprague–Dawley rats. Metabolite detection was performed via LC-MS/MS. Human recombinant CYP2B6 microsomes were prepared using a baculovirus-insect cell system and ultracentrifugation, with efavirenz serving as the substrate to study enzyme kinetics. Results: Isavuconazole exhibited an IC50of 21.14 ± 0.57 μM in RLM, indicating a mixed competitive and noncompetitive mechanism, and an IC50of 40.44 ± 4.23 μM in HLM, suggesting an anticompetitive mechanism. In rats, coadministration of efavirenz and isavuconazole significantly increased the AUC, Tmax, and Cmaxof efavirenz. Co-administration of efavirenz and rifampicin significantly elevated the AUC, Tmax, and Cmaxof 8-OH-efavirenz. The activity of CYP2B6.4, 6, and 7 increased significantly compared to CYP2B6.1, with relative clearance ranging from 158.34% to 212.72%. Conversely, the activity of CYP2B6.3, 8, 10, 11, 13–15, 18–21, 23–27, 31–33, and 37 was markedly reduced, ranging from 4.30% to 79.89%. Conclusion: Variations in liver enzyme activity and CYP2B6 genetic polymorphisms can significantly alter the metabolism of efavirenz. It provides laboratory-based data for the precise application of efavirenz and other CYP2B6 substrate drugs.