Carbonyl Reduction of Bupropion in Human Liver Cytosol
Jillissa C. Molnari and Alan L. Myers
Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences, Des Moines, IA
RESULTS:
BACKGROUND:
 Human carbonyl reductases (CBRs) are primarily cytosolic enzymes
that catalyze the reduction of many clinically relevant drugs.1
SUMMARY:
Bupropion is reduced to TBUP and to a lesser extent EBUP in h
liver cytosol (Figure 1).
(a)
 Pharmacogenetic variations in CBRs have led to serious adverse
consequences with drugs metabolized by CBRs, such as anthracycline
chemotherapeutics.2
Bupropion reduction was linear with increases in cytosolic prote
concentration and incubation time, and was nullified in the presen
heat-inactivated cytosol (data not shown).
 Phenotypic probe substrates (e.g. dextromethorphan) are used to
identify pharmacogenetic variations in drug metabolizing enzymes (e.g.
CYP2D6).3
Metabolism of bupropion to EBUP and TBUP is NADPH depen
(Figure 2).
Apparent Michaelis-Menten constants (Km) for EBUP and TBUP
formation were 150 µM and 100 µM, respectively. The maximal v
(Vmax) for EBUP and TBUP formation were 43 pmol/min/mg and 2
pmol/min/mg, respectively.
 However, such substrates for CBRs are poorly studied.
 Bupropion may be a novel probe substrate since it is reduced to two
major metabolites: erythrohydrobupropion (EBUP) and
H
H
threohydrobupropion (TBUP):
CH
EBUP
When tested at their reported literature IC50 values1, dicumarol,
flufenamic acid and 4-methylpyrazole did not significantly inhibit
metabolite formation (Figure 3).
3
OH
N H C (C H 3 ) 3
(b)
O
H
Cl
CH3
Menadione (100 µM) significantly inhibited EBUP and TBUP fo
(p< 0.001) (Figure 3).
OH
N H C (C H 3 ) 3
H
CH3
Cl
H
Figure 3: Percent inhibition (of control) of EBUP and TBUP formation
(by selective CBR inhibitors: dicumarol (0.2 µM), F.A. (5 µM), 4-MP
(400 µM), and menadione (100 µM). F.A. (flufenamic acid); 4-MP (4methylpyrazole); ## p<0.001
TBUP
N H C (C H 3 ) 3
Bupropion
Cl
CONCLUSIONS:
 Although no studies have been published to date, bupropion reduction
is likely catalyzed by human cytosolic CBRs in the liver.
Ketone reduction of bupropion in human liver cytosol appears t
preferentially catalyzed by carbonyl reductase (CR), as evidence
selective inhibition by menadione.
OBJECTIVE:
The main purpose of this study was to identify a major human CBR
catalyzing bupropion reduction in human liver cytosol.
EXPERIMENTAL METHODS:
In Vitro Incubations: General incubation conditions were adapted from a
literature method.4 Briefly, bupropion (100-700 µM), NADPH
regeneration system (BD Gentest), potassium phosphate buffer (pH 7.4)
and human liver cytosol (200 µg) were combined and incubated at 37ºC
in a shaking water bath incubator. Control reactions contained heatinactivated cytosol. Incubation conditions were optimized for the
bupropion concentration, cytosolic protein and incubation time that
resulted in linear EBUP and TBUP formation. NADPH dependence was
tested in the presence of NADPH regeneration system, NADPH, NADH
or no co-factor. All reactions were terminated with a solution of 90%
acetonitrile/0.10 M HCl, centrifuged and analyzed by HPLC for the
formation of EBUP and TBUP.
In Vitro Inhibition Studies: Dicumarol (0.2 µM), flufenamic acid (5 µM),
4-methylpyrazole (400 µM), and menadione (100 µM), specific inhibitors
of quinone reductase, aldo-keto reductase, alcohol dehydrogenase, and
carbonyl reductase enzymes, respectively1, were tested for inhibition of
bupropion reduction.
Data Analysis: Enzyme kinetic data was fitted to a Michaelis-Menten
model using Enzyme Kinetics v1.1 software (SPSS, Inc.). IC50 plots and
data analysis were performed by GraphPad Prism Software (v 5.0).
IC50 values for inhibition of this reaction by menadione were 27
88 µM, respectively (Figure 4).
The extent of this reaction in human liver microsomes (which e
the 11-HSD enzyme) will be studied in the near future.
Figure 1: Bupropion concentration (100 µM to 700 µM) vs. Rate of
metabolite (a) EBUP and (b) TBUP formation (pmol/min/mg).
To conclude, bupropion shows promise as a novel in vivo probe
substrate for identifying pharmacogenetic variations in CR, which
ultimately benefit patients prescribed drugs that are substrates fo
REFERENCES:
Figure 2: Reaction Co-factor vs. Rate of EBUP and TBUP formation
(pmol/min/mg) plot showing the NADPH dependence of the reaction.
R.S. (NADPH regeneration system, BD Gentest)
Figure 4: Percent inhibition (of control) of EBUP and TBUP formation by
the selective carbonyl reductase (CR) inhibitor menadione (50 µM to
400 µM). IC50 values for inhibition of EBUP and TBUP formation were
calculated as 27 µM and 88 µM, respectively.
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and a strategy for their study in vitro. Drug Metab Rev 2004;
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2) Lakhman SS, Ghosh D and Blanco JG. Functional significanc
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CYP2D6 phenotyping with dextromethorphan. Pharmacol Rep
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ACKNOWLEDGEMENTS:
Research funding was graciously provided by the Drake Universi
College of Pharmacy and Health Sciences. We thank Dr. Brian G
(Drake University) for assisting in the IC50 analyses.