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Vol. 27, Issue 10, 1117-1122, October 1999
Department of Pharmacokinetics and Drug Metabolism (C.M.M.,
T.B.A.), AstraZeneca Mölndal, Mölndal, Sweden; Department
of Molecular Biology (C.O., B.L., E.J., T.J., A.B., A.E.), AstraZeneca
Mölndal, Umeå, Sweden; and AstraZeneca Biotech Laboratory (M.B.,
M.J.), AstraZeneca, Södertälje, Sweden
Drug metabolism studies in the early phases of drug
discovery and development will improve the selection of new chemical
entities that will be successful in clinical trials. To meet the
expanding demands for these studies on the numerous chemicals generated through combinatorial chemistry, we have heterologously expressed nine
human drug-metabolizing cytochromes P-450 (CYPs) in
Saccharomyces cerevisiae. The enzymes were characterized
using known marker substrates CYP1A1/1A2 (ethoxyresorufin), 2C8
(paclitaxel), 2C9 (diclofenac), 2C19 (S-mephenytoin),
2D6 (bufuralol), 2E1 (chlorzoxazone), and 3A4/3A5 (testosterone). All
of the CYPs showed the expected substrate specificity except for
chlorzoxazone hydroxylation, which, in addition to CYP2E1 and 1A2, was
also catalyzed by CYP1A1 with a high turnover. The apparent
Michaelis-Menten parameters obtained for each CYP were within the
ranges of those reported in the literature using human liver microsomes
and/or recombinant CYPs. The Km for
CYP2E1-catalyzed chlorzoxazone hydroxylation was, however, much higher
(177 µM) than that obtained using liver microsomes (40 µM).
CYP-selective inhibitors, 
-naphthoflavone (CYP1A1/1A2), quercetin
(2C8), sulfaphenazole (2C9), quinidine (2D6), and ketoconazole
(3A4/3A5) showed significant isoform-selective inhibitory effects. We
have shown that ticlopidine is a potent inhibitor of CYP2C19
(IC50 = 4.5 µM) and CYP2D6 (IC50 = 3.5 µM) activities. We have therefore successfully set-up and
validated an "in-house" heterologous system for the production of
human recombinant CYPs for use in metabolism research.
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