In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes

Figures & data

Figure 1. The chemical structure of peucedanol.

Table 1. CYP450 isoforms tested, maker 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
2A6	Coumarin 7-hydroxylation	1.0	0.1	10	1.5
3A4	Testosterone 6β-hydroxylation	50	0.5	10	53
2C8	Paclitaxel 6α-hydroxylation	10	0.5	30	16
2C9	Diclofenac 4′-hydroxylation	10	0.3	10	13
2C19	S-Mephenytoin 4-hydroxylation	100	0.2	40	105
2D6	Dextromethorphan O-demethylation	25	0.25	20	4.8
2E1	Chlorzoxazone 6-hydroxylation	120	0.4	30	126

Table 2. HPLC conditions for the analyses of CYP450s corresponding metabolics.

CYP450s	Conditions
1A2	methanol : phosphate buffer = 30: 70; UV 245 nm
2A6	acetonitrile : Acetic acid = 35: 65; fluo Ex/Em 340/456 nm
3A4	methanol : water = 60: 40; UV 254 nm
2C8	methanol : water = 65: 35; UV 230 nm
2C9	acetonitrile : phosphate buffer = 35: 65; UV 280 nm
2C19	methanol : potassium phosphate = 32: 68
2D6	acetonitrile : phosphate buffer = 25: 75; fluo Ex/Em 235/310 nm
2E1	acetonitrile : Acetic acid = 22: 78; UV 287 nm

Figure 2. Effect of peucedanol on the activity of eight CYP isoforms, including CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1. *p < 0.05. Negative control: without any treatment; Peucedanol: treated with 100 μM peucedanol; Positive control: treated with corresponding inhibitors.

Figure 3. The inhibition of CYP1A2 (A), 3A4 (B), and 2D6 (C) by peucedanol was in a dose-dependent manner, with the IC₅₀ values of 6.03, 7.58, and 13.57 μM, respectively.

Figure 4. The inhibition model fitting and corresponding parameters calculation of CYP1A2 catalysed reactions (phenacetin O-deethylation). (A) Lineweaver-Burk plots of inhibition of peucedanol on CYP1A2 with phenacetin (20–100 μM) in the absence or presence of peucedanol (0–30 μM). The inhibition of CYP1A2 was best fitting with competitive manner. (B) Secondary plot with the values of K_m/V_max in Figure 4(A). All data represent the mean of the incubations (performed in triplicate).

Figure 5. The inhibition model fitting and corresponding parameters calculation of CYP2D6 catalysed reactions (diclofenac 4′-hydroxylation) in pooled HLM. (A) Lineweaver-Burk plots of inhibition of peucedanol on CYP2D6 with dextromethorphan (10–50 μM) in the absence or presence of peucedanol (0–50 μM). The inhibition of CYP2D6 was best fitting with competitive manner. (B) Secondary plot with the values of K_m/V_max in Figure 5(A). All data represent the mean of the incubations (performed in triplicate).

Figure 6. The inhibition model fitting and corresponding parameters calculation of CYP3A4 catalysed reactions (testosterone 6 b-hydroxylation) in pooled HLM. (A) Lineweaver-Burk plots of the of inhibition of peucedanol on CYP3A4 with testosterone (20–100 μM) in the absence or presence of peucedanol (0–30 μM). The inhibition of CYP3A4 was best fitting with non-competitive manner. (B) Secondary plot with the value of K_m/V_max in Figure 6(A). All data represent the mean of the incubations (performed in triplicate).

Figure 7. Time and concentration-inactivation of microsomal CYP3A4 activity by peucedanol in the presence of NADPH. (A) 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. (B) The K_i and K_inact values were determined through non-linear analysis of the K_obs versus the peucedanol concentration.