Effect of CYP2C9 antibodies on diclofenac and S-mephenytoin hydroxylation by human liver microsomes
| Subject | Diclofenac 4′-Hydroxylation | S-Mephenytoin 4′-Hydroxylation | ||
|---|---|---|---|---|
| Anti-CYP2C9-P | Anti-CYP2C9-M | Anti-CYP2C9-P | Anti-CYP2C9-M | |
| % Inhibition | % Inhibition | |||
| A | 971-a | 941-a | 1001-b | 0 (−22)1-b |
| B | 94 | 91 | 100 | 0 (−17) |
| C | 94 | 87 | 100 | 22 |
| H | 70 | 85 | 98 | 12 |
Diclofenac and S-mephenytoin hydroxylation were determined in incubation mixtures containing 100 mM KPO4 buffer (pH 7.4), human liver microsomes (25 pmol of P-450), 0.5 mM NADPH, 10 μM diclofenac, or 100 μM S-mephenytoin, and 125 μg of anti-CYP2C9-P, anti-CYP2C9-M, or preimmune (control) IgG. Microsomes were preincubated with IgG for 3 min at 37°C, then for 10 min at ambient temperature. The remaining components were added, reactions were initiated with NADPH, and terminated after 30 min at 37°C. Product formation was then measured by HPLC as described inExperimental Procedures. Control rates of diclofenac hydroxylation (determined in the presence of preimmune IgG) were 1.89 ± 0.33 nmol 4′-hydroxydiclofenac formed/min/nmol microsomal P-450 whereas those for S-mephenytoin hydroxylation were 0.22 ± 0.08 nmol 4′-hydroxymephenytoin formed/min/nmol microsomal P-450.