Table 1.
Transitions and collision energies used in LC-MS/MS for the detection of meranzin hydrate, the probe substrates, metabolites and the internal standard.
Table 2.
IC50 and Ki values of MH against human CYP isoforms compared with that of specific inhibitors reported in literature.
Figure 1.
The concentration-velocity curve for meranzin hydrate metabolism after incubation with HLMs (A), recombinant CYP1A2 (B) and recombinant CYP2C19 (C).
Note: The incubation conditions are described in the materials and methods section. The curve was automatically fitted using nonlinear regression and Michaelis-Menten equation, the data were obtained in triplicates.
Figure 2.
Effect of specific inhibitors on CYP-mediated MH (10 µM) metabolism in HLMs.
Note: The concentrations of CHL was 5 µM and that for the other specific inhibitors was 1 µM. The incubation conditions are described in the materials and methods section. Each data point represents the average of triple determinations and error bars (n = 3). FUR, furafylline, the specific inhibitor for CYP1A2; TRA, trans-2-phenylcyclopropylamine, the specific inhibitor for CYP2A6; SUL, sulfaphenazole, the specific inhibitor for CYP2C9; QUI, quinidine, the specific inhibitor for CYP2D6; CLM, chlormethiazole, the specific inhibitor for CYP2E1; TIC, ticlopidine, the specific inhibitor for CYP2C19; and KET, ketoconazole, the specific inhibitor for CYP3A4. In the presence of FUR (1 µM) and TIC (1 µM), the metabolic clearance rate of MH decreased to 29.1% and 41.3% of that of the control, respectively, while the other inhibitors had no significant inhibitory effects on the metabolism of MH.
Figure 3.
Effect on the metabolic clearance rate (MCR) of meranzin hydrate (MH) under the inhibition of recombinant CYP1A2 and CYP2C19.
Note: 10 µM and 50 µM MH were incubated with the CYP recombinants and cofactors in the absence (control) or presence of the inhibitors by furafylline (1 µM) and ticlopidine (1 µM), respectively. Each point represents the average of triplicate incubations. The MCRs of MH were significantly decreased compared with that of the control for CYP1A2 and CYP2C19, for both concentrations of MH.
Figure 4.
Effect of meranzin hydrate on the metabolic reactions of the seven CYP specific substrates in HLMs.
Note: The incubation conditions are described in the materials and methods section. Each data point represents the average of triple determinations and error bars.
Figure 5.
Double reciprocal (Lineweaver-Burk) plots for direct inhibition of phenacetin O-deethylation (A) and Ki values.
Note: The inhibition of CYP1A2 activity by MH can be best described as a mixed full inhibition mechanism by different concentrations of MH (0–100 µM) in HLM incubations (0.5 mg·mL-1 protein). The data points are mean values of triplicate incubations. Non-linear regression analysis of the phenacetin O-deethylation versus substrate concentration was performed to obtain Ki values.
Figure 6.
Secondary plot of CYP1A2 activity using the slopes of the primary Lineweaver-Burk plots versus concentrations of MH.
Note: The effects of MH on the metabolism of phenacetin O-deethylation in human liver microsomes. Each point represents the mean of triplicate determinations.
Figure 7.
Double reciprocal (Lineweaver-Burk) plots for direct inhibition of S-Mephenytoin 4-hydroxylation (A) and Ki values (B).
Note: The inhibition of CYP1A2 activity by MH can be best described as a full competitive inhibition by different concentrations of MH (0–100 µM) in HLM incubations (0.5 mg·mL-1 protein). The data points are mean values of triplicate incubations. Non-linear regression analysis of the S-Mephenytoin 4-hydroxylation versus substrate concentration was performed to obtain Ki values.
Figure 8.
Secondary plot of CYP2C9 activity using the slopes of the primary Lineweaver-Burk plots versus concentrations of MH.
Note: illustrating the effects of MH on the metabolism of S-Mephenytoin via 4-hydroxylation in human liver microsomes. Each point represents the mean of triplicate determinations.