Masayoshi Tani

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes.

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.

Development and preliminary application of a high-performance liquid chromatographic assay for omeprazole metabolism in human liver microsomes

A high-performance liquid chromatography assay was developed to measure the enzymatic activities of the 5-hydroxylation and sulphoxidation of omeprazole, a proton pump inhibitor, in human liver microsomes. The preliminary study was also undertaken to assess the assays applicability for the enzyme kinetic analysis of omeprazole metabolism by human liver microsomes. The recovery of 5-hydroxyomeprazole, omeprazole sulphone and phenacetin (internal standard) after the precipitation of microsomal protein by acetonitrile was nearly complete. The intra-assay relative standard deviations (n = 6) were 8.2 and 12.8% for quantitation of the 5-hydroxylation and sulphoxidation activities of omeprazole, respectively. Eadie-Hofstee plots for the formation of 5-hydroxyomeprazole and omeprazole sulphone gave a biphasic relationship for all the microsomal samples studied (n = 6). The respective mean (+/- SD) high- and low-affinity component kinetic parameters for the 5-hydroxylation and sulphoxidation of omeprazole estimated by a two-enzyme kinetic analysis were: Km1 = 6.3 +/- 1.7 and 10.4 +/- 6.3 microM, Km2 = 183.2 +/- 180.4 and 671.2 +/- 639.4 microM, Vmax1 = 109.8 +/- 75.4 and 77.5 +/- 46.1 pmol mg-1 min-1, and Vmax2 = 163.3 +/- 94.1 and 318.3 +/- 163.3 pmol mg-1 min-1. The results suggest that the assay is reproducible, accurate and applicable for studying the metabolism of omeprazole in human liver microsomes.