In vitro inhibitory effects of Friedelin on human liver cytochrome P450 enzymes

Figures & data

Table 1. Isoforms tested, marker reactions, incubation conditions, and K_m used in the inhibition study.

CYPs	Marker reactions	Substrate concentration (μM)	Protein concentration (mg/mL)	Incubation time (min)	Estimated Km (μM)
1A2	Phenacetin O-deethylation	40	0.2	30	48
3A4	Testosterone 6β-hydroxylation	50	0.5	10	53
2A6	Coumarin 7-hydroxylation	1.0	0.1	10	1.5
2E1	Chlorzoxazone 6-hydroxylation	120	0.4	30	126
2D6	Dextromethorphan O-demethylation	25	0.25	20	4.8
2C9	Diclofenac 4'-hydroxylation	10	0.3	10	13
2C19	S-Mephenytoin 4-hydroxylation	100	0.2	40	105
2C8	Paclitaxel 6α-hydroxylation	10	0.5	30	16

Figure 1. Effects of Friedelin (100 μM) on the activity of CYP450 enzymes in pooled HLMs. All data represent mean ± SD of the triplicate incubations. *p < 0.05, significantly different from the negative control. Negative control: incubation systems without Friedelin; Friedelin: incubation systems with Friedelin; positive control: incubation systems with their corresponding positive inhibitors.

Figure 2. Lineweaver–Burk plots (A) and the secondary plot for K_i (B) of effects of Friedelin on CYP3A4 catalyzed reactions (testosterone 6β-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with testosterone (20–100 μM) in the absence or presence of Friedelin (0–30 μM). All data represent mean ± SD of the triplicate incubations.

Figure 3. Lineweaver–Burk plots (A) and the secondary plot for K_i (B) of effects of Friedelin on CYP2E1 catalyzed reactions (chlorzoxazone 6-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with chlorzoxazone (25–200 μM) in the absence or presence of Friedelin (0–50 μM). All data represent mean ± SD of the triplicate incubations.

Figure 4. Time-dependent inhibition investigations of CYP3A4 catalyzed testosterone 6β-hydroxylation and CYP2E1 catalyzed chlorzoxazone 6-hydroxylation reactions by Friedelin (20 μM). All data represent mean ± SD of the triplicate incubations.

Figure 5. Time and concentration-inactivation of microsomal CYP3A4 catalyzed testosterone 6β-hydroxylation by Friedelin in the presence of NADPH. The initial rate constant of inactivation of CYP3A4 by each concentration (K_obs) was determined through linear regression analysis of the natural logarithm of the percentage of remaining activity versus preincubation time (A). The K_i and K_inact values were determined through nonlinear analysis of the K_obs versus the Friedelin concentration (B).