Table 1

Effect of CYP2C9 antibodies on diclofenac and S-mephenytoin hydroxylation by human liver microsomes

SubjectDiclofenac 4′-HydroxylationS-Mephenytoin 4′-Hydroxylation
% Inhibition% Inhibition
A971-a 941-a 1001-b 0 (−22)1-b
B94911000 (−17)

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.

  • 1-a IgG/P-450 ratio = 5 mg/nmol.

  • 1-b IgG/P-450 ratio = 5 mg/nmol.