Three concentrations of PTV (1, 10, 100 µM) were conducted to detect the luciferin PPXE activity against recombinant CYP3A4 and CYP3A5. A significant decrease in the luminescene activity in both recombinant CYP3A4 and CYP3A5 was observed in 10 µM and 100 µM of PTV (Fig 1). It seems that PTV is a candidate drug for metabolism against CYP3A4 and CYP3A5 enzymes.
Metabolite profiling and Identification
After incubating PTV with HLMs in the presence of a NADPH regenerating system, a metabolite (M2) of PTV was profiled, characterized, and identified by LC-MS/MS. A representative chromatograms are provided in Fig 2 (a) and (b). The LC-MS/MS analysis of the unchanged PTV and its metabolite produced informative and prominent product ions. The MS/MS spectrum of PTV, with a protonated molecular ion [M+H]+ at m/z …show more content…
The kinetic plots indicated that the Km values for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 23.40 ± 1.74 and 28.63 ± 2.38 µM, respectively. The Vmax for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 6.10 ± 1.04 pmol/mg protein/min and 6.93 ± 1.26 pmol/mg protein/min, respectively. The intrinsic clearance (Clint) was 0.26 ± 0.03 µl/mg protein/min for CYP3A5 expressers and 0.24 ± 0.04 µl/mg protein/min for CYP3A5 non-expressers. The results are demonstrated in Table …show more content…
Ketoconazole (1 µM), a potent CYP3A4/3A5 inhibitor, completely inhibited the formation of major metabolite. CYP3cide (0.5 µM) selectively inhibited the metabolic activity of CYP3A4