Functional characterization of CYP3A4.16: Catalytic activities toward midazolam and carbamazepine

Abstract

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus–insect cell system.

2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them.

3. When midazolam was used as a substrate, K_m and V_max for 1′-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V_max/K_m) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K_m without a difference in V_max.

4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K_m2 and V_max2 of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance.

5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

Keywords::

• CYP3A4
• non-synonymous single nucleotide polymorphism (SNP)
• function
• substrate dependency

Acknowledgements

The authors thank Ms. Chie Sudo for her secretarial assistance.

Declaration of interest: This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences, and by the Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare.