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

Figures & data

Figure 1. The chemical structure of CEP.

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 2. Inhibition of CEP on CYP 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 CEP; CEP: incubation systems with CEP (100 μM); Positive control: incubation systems with their corresponding positive inhibitors.

Figure 3. Lineweaver–Burk plots (A) and the secondary plot for Ki (B) of inhibition of CEP on CYP3A4 catalysed 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 CEP (0–30 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 4. Lineweaver-Burk plots (A) and the secondary plot for Ki (B) of inhibition of CEP on CYP2E1 catalysed reactions (chlorzoxazone 6-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with chlorzoxazone (25–250 μM) in the absence or presence of CEP (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 5. Lineweaver–Burk plots (A) and the secondary plot for Ki (B) of inhibition of CEP on CYP1A2 catalysed reactions (diclofenac 4′-hydroxylation) in pooled HLM. Data are obtained from a 30 min incubation with phenacetin (2–20 μM) in the absence or presence of CEP (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 6. Time-dependent inhibition investigations of CYP3A4, CYP2E1, or CYP1A2 catalysed reactions by CEP (20 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 7. Time and concentration-inactivation of microsomal CYP3A4 activity by CEP 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 pre-incubation time (A). The K_I and K_inact values were determined through non-linear analysis of the K_obs versus the CEP concentration (B).