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Vol. 28, Issue 10, 1222-1230, October 2000
Divisions of Pharmacotherapy (S.R.F., R.L.H., S.S.S, C.M.L.) and
Drug Delivery and Disposition (E.L.L.), School of Pharmacy, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina;
Department of Biomedical Sciences (B.Y.), University of Rhode Island,
Kingston, Rhode Island; and Department of Bioanalysis and Drug
Metabolism (R.M.L.), Glaxo Wellcome, Inc., Research Triangle Park,
North Carolina
The purpose of this study was to establish bupropion (BUP)
hydroxylation as a selective in vitro marker of cytochrome P450 (CYP)
2B6 catalytic activity. Among a panel of 16 human liver microsomes
(HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 µM substrate and significantly correlated with CYP2B6 blotting
density (r2 = 0.99) and
S-mephenytoin N-demethylase activity
(r2 = 0.98). Kinetic analysis of BUP
hydroxylation was performed in a subset of seven HLMs representative of
the 80-fold range in activity. Sigmoidal kinetics suggestive of
allosteric activation was observed in five HLMs exhibiting low or high
activity; the mean apparent Km for BUP
hydroxylation in these HLMs (130 µM) was similar to the
Km for cDNA-expressed CYP2B6 (156 µM).
Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low
activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and
CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 µM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 µM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.
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