Inhibition of Human CYP2B6-Catalyzed Bupropion Hydroxylation
by Ginkgo biloba Extract: Effect of Terpene Trilactones
Aik Jiang Lau and Thomas K. H. Chang
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
Received April 18, 2009; accepted May 28, 2009
Cytochrome P450 2B6 (CYP2B6) is expressed predominantly in
human liver. It catalyzes the oxidative biotransformation of various
drugs, including bupropion, which is an antidepressant and a to-
bacco use cessation agent. Serious adverse effects of high dos-
ages of bupropion have been reported, including the onset of
seizure. As Ginkgo biloba extract may be consumed with bupro-
pion or another CYP2B6 drug substrate, potential exists for an
herb-drug interaction. Therefore, we investigated the effect of G.
biloba extract and some of its chemical constituents (terpene
trilactones and flavonols) on the in vitro catalytic activity of
CYP2B6 as assessed by the bupropion hydroxylation assay with
recombinant enzyme and hepatic microsomes. The amount of
hydroxybupropion was quantified by a novel and validated ultra-
performance liquid chromatography/mass spectrometry method.
Enzyme kinetic analysis indicated that G. biloba extract competi-
tively inhibited hepatic microsomal CYP2B6-catalyzed bupropion
hydroxylation (apparent Kiwas 162 ? 14 ?g/ml). Bilobalide and
ginkgolides A, B, C, and J were not responsible for the inhibition of
CYP2B6 catalytic activity by the extract. Whereas the 3-O-glu-
coside and 3-O-rutinoside of quercetin, kaempferol, and isorham-
netin had no effect, the corresponding aglycones (10 and 50 ?g/ml)
decreased hepatic microsomal bupropion hydroxylation. The inhi-
bition of CYP2B6 by kaempferol was competitive (apparent Kiwas
10 ? 1 ?g/ml). In summary, G. biloba extract and its flavonol
aglycones are naturally occurring inhibitors of in vitro CYP2B6
catalytic activity and bupropion hydroxylation. Future studies are
needed to investigate whether G. biloba extract interacts in vivo
with bupropion or other clinically important CYP2B6 drug sub-
Cytochrome P450 2B6 (CYP2B6) is expressed mainly in human
liver, although this enzyme has also been detected in various extra-
hepatic tissues (Gervot et al., 1999). Considerable variability exists
not only in hepatic expression of CYP2B6 mRNA (280-fold) (Chang
et al., 2003) and protein (?288-fold) (Hesse et al., 2004) but also
CYP2B6 enzyme activity (80-fold) (Faucette et al., 2000). The basis
for the interindividual variability may relate to pharmacogenetics
(Hofmann et al., 2008) and the fact that this enzyme is subject to
induction by various drugs and other chemicals in a mechanism that
involves transcription factors such as the constitutive androstane
receptor (Sueyoshi et al., 1999), which also exhibits large interindi-
vidual differences (240-fold) in hepatic expression (Chang et al.,
2003). The magnitude of CYP2B6 catalytic activity may also be
altered as a result of enzyme inhibition by various synthetic drugs
(Turpeinen et al., 2004; Walsky et al., 2006); naturally occurring
compounds, including phenethyl isothiocyanate (Nakajima et al.,
2001), ?-viniferin (Piver et al., 2003), and citral (Kim et al., 2008);
and herbal supplements, such as Woohwangcheongsimwon (Kim et
al., 2008), Andrographis paniculata extract (Pekthong et al., 2008),
and curcuminoid extract (Volak et al., 2008). Important CYP2B6 drug
substrates include the alkylating anticancer prodrug cyclophospha-
mide (Chang et al., 1993) and the tobacco use cessation agent bupro-
pion (Faucette et al., 2000; Hesse et al., 2000). The biotransformation
of bupropion to hydroxybupropion is catalyzed predominantly by
CYP2B6 in human liver (Faucette et al., 2000; Hesse et al., 2000). As
a result, hepatic microsomal bupropion hydroxylation is used as an
enzyme-selective catalytic marker for CYP2B6 in human liver (Tur-
peinen et al., 2004; Walsky et al., 2006).
Bupropion inhibits dopamine and noradrenaline reuptake, and it
acts as an antagonist of neuronal nicotinic acetylcholine receptor
(Dwoskin et al., 2006). This drug was available initially as an anti-
depressant. It is now widely used as a non-nicotine drug for smoking
cessation. However, the use of high dosages of bupropion is associ-
ated with serious adverse effects (e.g., seizure), particularly among
susceptible individuals (Beyens et al., 2008). In humans, bupropion
undergoes extensive hepatic biotransformation to form hydroxybu-
propion, threohydrobupropion, and erythrohydrobupropion, which are
pharmacologically active metabolites (Dwoskin et al., 2006). Given
that inhibition of bupropion biotransformation leads to greater plasma
drug concentrations and the potential for the onset of serious adverse
effects, it is therefore important to identify factors (e.g., concomitant
This work was supported by the Canadian Institutes of Health Research [Grant
informationcan be foundat
ABBREVIATIONS: UPLC, ultraperformance liquid chromatography; MS, mass spectrometry; DMSO, dimethylsulfoxide; LLOQ, lower limit of
quantification; QC, quality control.
DRUG METABOLISM AND DISPOSITION
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
DMD 37:1931–1937, 2009
Vol. 37, No. 9
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at ASPET Journals on October 27, 2015
data from those in vitro studies may help in predicting the potential of
G. biloba and flavonols to inhibit CYP2B6-mediated drug biotrans-
formation in vivo. The use of in vitro data to predict in vivo inhibitory
effects in a given individual is complicated by many factors, including
genetic variation (Wienkers and Heath, 2005). For example, when
compared with the wild-type CYP2B6, the CYP2B6*4 and
CYP2B6*6 variants appear to be less prone to inhibition (Talakad et
In summary, our major findings indicate that 1) G. biloba extract
competitively inhibited human hepatic microsomal CYP2B6-cata-
lyzed bupropion hydroxylation, with an apparent Kiof 162 ?g/ml; 2)
bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide
J were not responsible for the inhibitory effect of the extract; and 3)
whereas a monoglycoside and a diglycoside of kaempferol, quercetin,
and isorhamnetin had no effect, the corresponding aglycones inhibited
hepatic microsomal CYP2B6-mediated enzyme activity. The discov-
ery of G. biloba and flavonols as in vitro inhibitors of CYP2B6
provides an impetus for future investigations to expand our under-
standing of the pharmacological and toxicological consequences of
CYP2B6 inhibition by these natural products. Interestingly, it has
been suggested that CYP2B6 inhibitors may be beneficial in prevent-
ing tamoxifen-mediated endometrial cancer (Stiborova ´ et al., 2002).
Acknowledgments. We thank Indena S. A. (Milan, Italy) for the G.
biloba extract and bilobalide. We also thank Andras Szeitz for tech-
nical assistance with the UPLC/MS instrumentation. T.K.H.C. re-
ceived a Senior Scholar Award from the Michael Smith Foundation
for Health Research.
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Address correspondence to: Thomas K. H. Chang, Faculty of Pharmaceuti-
cal Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC,
V6T 1Z3, Canada. E-mail: email@example.com
G. biloba AND CYP2B6 CATALYTIC ACTIVITY
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