Evaluation of EE as an inhibitor of human P450s in vitro
P450 | Incubationa | Substrate (Activity) | IC50(total) | IC50(free) No Preincubationc | ||
|---|---|---|---|---|---|---|
| No Preincubation | Preincubation (IC50(t))b | |||||
| μM | μM | μM | ||||
| 1A1 | Recombinant | Phenacetin (O-deethylase) | 2.7 ± 0.3 | 2.1 ± 0.5 | 2.7 ± 0.3 | |
| 3A4 | Recombinant | Midazolam (1′-hydroxylase) | 8.5 ± 0.7 | 1.5 ± 0.3 (0.031)d | 8.5 ± 0.7 | |
| 3A5 | Recombinant | Midazolam (1′-hydroxylase) | 8.9 ± 2.8 | 3.3 ± 0.3 (0.014) | 8.9 ± 2.8 | |
| 1B1 | Recombinant | Melatonin (6-hydroxylase) | 9.2 ± 2.2 | 11 ± 0.5 | 9.2 ± 2.2 | |
| 1A2 | Recombinant | Phenacetin (O-deethylase) | 14 ± 0.4 | 41 ± 0.7 | 14 ± 0.4 | |
| 2J2 | Recombinant | Terfenadine (hydroxylase) | 31 ± 5.0 | 15 ± 4.0e | 31 ± 5.0 | |
| 2B6 | Recombinant | Bupropion (hydroxylase) | 41 ± 2.4 | 38 ± 4.4 | 41 ± 2.4 | |
| 2C19 | HLM | Mephenytoin (4′-hydroxylase) | 4.4 ± 1.6 | 9.7 ± 1.4 | 2.8 ± 1.0 | |
| 2C9 | HLM | Diclofenac (4′-hydroxylase) | 13 ± 3.2 | 9.2 ± 0.3 | 11 ± 0.3 | |
| 2C8 | HLM | Paclitaxel (6α-hydroxylase) | 16 ± 2.0 | 8.3 ± 0.5e | 13 ± 1.7 | |
| 2B6 | HLM | Bupropion (hydroxylase) | 23 ± 1.5 | 23 ± 3.5 | 19 ± 1.3 | |
| 2D6 | HLM | Dextromethorphan (O-demethylase) | 28 ± 2.0 | 32 ± 12 | 24 ± 1.7 | |
| 3A4 | HLM | Testosterone (6β-hydroxylase) | 39 ± 9.0 | 8.9 ± 0.8 (0.005) | 34 ± 7.8 | |
| 3A4 | HLM | Midazolam (1′-Hydroxylase) | 42 ± 1.3 | 8.0 ± 0.7 (0.006) | 37 ± 1.1 | |
| 1A2 | HLM | Phenacetin (O-deethylase) | >45 (∼40%)f | >45 (∼40%)f | >39 | |
↵ a Incubations were performed in 0.1 M potassium phosphate buffer, pH 7.4, containing EDTA (1 mM), HLM protein (0.05-0.15 mg/ml), or rP450 (2-5 pmol/ml). EE was dissolved in DMSO (final concentration range of 10 nM to 45 μM). The final concentration of DMSO was <0.2% (v/v). Incubations and preincubations were initiated with NADPH (1.0 mM). Data represent mean ± S.D. of three determinations.
↵ b Furafylline (CYP1A1, IC50 = 2.8 μM, IC50(t) = 0.46 μM; CYP1A2, IC50 = 1.8 μM, IC50(t) = 0.11 μM), phencyclidine (CYP2B6, IC50 = 44 μM, IC50(t) = 0.67 μM), paroxetine (CYP2D6, IC50 = 1.0 μM, IC50(t) = 0.09 μM), phenelzine (CYP2C8, IC50 = 308 μM, IC50(t) = 114 μM), tienilic acid (CYP2C9, IC50 = 2.1 μM, IC50(t) = 0.21 μM), and ticlopidine (CYP2C19, IC50 = 2.1 μM, IC50(t) = 0.91 μM) served as positive controls (kinact/KI = 0.10, 0.42, 0.07, 0.54, 0.0004, 0.22, and 0.051 min-1 μM-1, respectively).
↵ c Total IC50 corrected for EE free fraction in the incubation (fu,inc), where IC50 free = IC50 total · fu,inc. For rP450s, binding of EE to total protein was negligible (fu,inc ∼ 1.0). For HLM, fu,inc was 0.63 (0.25 mg/ml), 0.87 (0.1 mg/ml), and 0.84 (0.05 mg/ml).
↵ d Data in parentheses represent the kinact/KI ratio calculated from the IC50 after 30 min of preincubation (IC50(t)), where kinact/KI = [0.693 · (1 + [S]/Km)]/[IC50(t) · t] (Maurer et al., 2000; Berry and Zhao, 2008). There was no dilution of incubate. For each P450 assay, the substrate concentration approximated the Km ([S]/Km ∼1.0), and the preincubation time was 30 min (t = 30). Troleandomycin was used as a positive control for recombinant CYP3A4 (IC50 = 2.5 μM, IC50(t) = 0.20 μM) and CYP3A5 (IC50 = 11 μM, IC50(t) = 2.4 μM), and kinact/KI ratios of 0.23 and 0.019 min-1μM-1 were calculated, respectively. The kinact/KI ratio for troleandomycin in HLM (IC50 = 19 μM, IC50(t) = 0.55 μM) was 0.084 min-1μM-1.
↵ e IC50 > IC50(t) (∼2-fold) for CYP2C9 activity (HLM) and recombinant CYP2J2, but no attempt was made to calculate a kinact/KI ratio.
↵ f Percentage inhibition at the highest concentration of EE tested (45 μM).