Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: inhibition by sulfaphenazole, omeprazole, and ticlopidine

Abstract.

Objectives: To determine the relative contribution of cytochromes P ₄₅₀ (CYP) 2C9 and 2C19 to the formation of 5-(-4-hydroxyphenyl)-5-phenylhydantion (HPPH) from phenytoin (PPH). Design: Hydroxylation of PPH to form HPPH was studied in vitro using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. Results: Formation of HPPH from PPH in liver microsomes had a mean (±SEM) apparent Km [substrate concentration corresponding to 50% of maximal reaction velocity (V_max)] of 23.6±1.8 µmol/l. Coincubation with the CYP2C9 inhibitor, sulfaphenazole (SPA), at 5 µmol/l reduced reaction velocity to less than 15% of control values. The mean inhibitor concentration at which 50% inhibition is achieved (IC₅₀ value) for SPA versus PPH hydroxylation (0.49 µM) was similar to the SPA IC₅₀ versus flurbiprofen hydroxylation (0.46 µM) and tolbutamide hydroxylation (0.7–1.5 µM). In contrast, the CYP2C19 inhibitor omeprazole (OME) at 10 µmol/l produced only a small degree of inhibition. Incubation of PPH with microsomes from cDNA-transfected human lymphoblastoid cells containing CYP1A2, 2A6, 2B6, 2C8, 2D6, 2E1, or 3A4 yielded no detectable formation of HPPH. Only CYP2C9 and 2C19 had PPH hydroxylation activity, with apparent K_m values for the high-affinity component of 14.6 µmol/l and 24.1 µmol/l, respectively. Based on V_max values in liver microsomes, the V_max and K_m values in expressed CYPs and the relative abundance of the two isoforms in human liver, CYP2C9, and 2C19 were estimated to have relative contributions of 90% and 10%, respectively, to net intrinsic clearance. Conclusions: Formation of HPPH from PPH is mediated exclusively by CYP2C9 and 2C19, with CYP2C9 playing the major role.