Table 2.
Mean (±SD) kinetic parameters for flurbiprofen 4′-hydroxylation, naproxen O-demethylation, and diclofenac 4′-hydroxylation by WT and mutant CYP2C9 enzymes determined in the absence (−) and presence (+) of dapsone (100 μM)
| Substrate/Enzyme | (−Dapsone) |
(+Dapsone) |
||||
|---|---|---|---|---|---|---|
| Km (μM) | Vmax (pmol/min/pmol P450) | CLint (μL/min/pmol P450) | Km (μM) | Vmax (pmol/min/pmol P450) | CLint (μL/min/pmol P450) | |
| Flurbiprofen | ||||||
| WT | 12.4 ± 0.44 | 5.05 ± 0.08 | 0.407 ± 0.01 | 5.60 ± 0.31 | 5.30 ± 0.09 | 0.946 ± 0.033 |
| Arg108Ala | N/D | N/D | - | 27.9 ± 2.0 | 1.40 ± 0.04 | 0.050 ± 0.002 |
| Phe114Ala | N/D | N/D | - | 112 ± 5.4 | 2.50 ± 0.06 | 0.022 ± 0.001 |
| Lys206Ala | 16.2 ± 0.6 | 3.21 ± 0.14 | 0.20 ± 0.01 | 6.6 ± 0.3 | 4.27 ± 0.11 | 0.66 ± 0.03 |
| Naproxen | ||||||
| WT | 107 ± 11 | 2.50 ± 0.11 | 0.023 ± 0.00 | 33 ± 1.35 | 12.0 ± 0.18 | 0.38 ± 0.01 |
| Arg108Alaa | N/D | N/D | - | 155 ± 29 | 2.27 ± 0.28 | 0.015 ± 0.00 |
| Lys206Ala | 208 ± 20 | 1.80 ± 0.10 | 0.009 ± 0.00 | 64.4 ± 2.4 | 10.1 ± 0.29 | 0.157 ± 0.00 |
| Diclofenac | ||||||
| WT | 2.18 ± 0.16 | 5.20 ± 0.13 | 2.39 ± 0.16 | 1.60 ± 0.15 | 3.19 ± 0.09 | 1.99 ± 0.17 |
| Lys206Ala | 4.05 ± 0.35 | 6.53 ± 0.19 | 1.64 ± 0.10 | 2.45 ± 0.13 | 4.41 ± 0.12 | 1.81 ± 0.07 |
N/D, not determined. Insufficient 4′hydroxyflurbiprofen and desmethylnaproxen concentrations above the lower limits of quantification of the respective assays were available for kinetic characterization.
a
Kinetic data for naproxen demethylation by Arg108Ala in the presence of dapsone are from fitting with the substrate inhibition equation. The Ksi was 520 ± 12 μM.