IDENTIFICATION OF HUMAN CYTOCHROME-P450 ISOFORMS INVOLVED IN THE 3-HYDROXYLATION OF QUININE BY HUMAN LIVER-MICROSOMES AND 9 RECOMBINANT HUMAN CYTOCHROMES P450

Studies using human liver microsomes and nine recombinant human cytoch rome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 a nd 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie- Hofstee plots for the formation of 3-hydroxyquinine exhibited apparent ly monophasic behavior for all of the 10 different microsomal samples studied. There was interindividual variability in the kinetic paramete rs, as follows: 1.8-, 3.2- and 3.5-fold for K-m, V-max and V-max/K-m, respectively. The mean +/- S.D. values for K-m, V-max and V-max/K-m we re 106.1 +/- 19.3 mu M, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 mu l/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine a nd the metabolic activities for substrates of the respective CYP isofo rms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testos terone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4' -hydroxylase activities was also observed. However, no significant cor relation existed between the 3-hydroxylation of quinine and the oxidat ive activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac ), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and trolean domycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinin e by human liver microsomes with respective mean IC50 values of 0.026 mu M and 28.9 mu M. Anti-CYP3A antibodies strongly inhibited quinine 3 -hydroxylation, whereas weak inhibition was observed in the presence o f S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant h uman CYP isoforms, CYP3A4 exhibited the highest catalytic activity wit h respect to the 3-hydroxylation of quinine, compared with the minor a ctivity of CYP2C19 and little discernible or no effect of other CYP is oforms. Collectively, these data suggest that the 3-hydroxylation of q uinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance i n this major pathway of quinine in humans.