Metabolism of bupropion by baboon hepatic and placental microsomes

Article abstract of DOI:10.1016/j.bcp.2011.04.014

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein^-1 min^-1), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.

Full text of DOI:10.1016/j.bcp.2011.04.014

Biochemical Pharmacology 82 (2011) 295–303
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Metabolism of bupropion by baboon hepatic and placental microsomes§
Xiaoming Wang, Doaa R. Abdelrahman, Valentina M. Fokina, Gary D.V. Hankins,
*
Mahmoud S. Ahmed, Tatiana N. Nanovskaya
Department of Obstetrics & Gynecology, University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555-0587, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic
and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics.
Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-
BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by
hepatic microsomes (Km 36 Æ 6 mM, Vmax 258 Æ 32 pmol mg proteinÀ1 minÀ1), however the formation of
OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion
for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of
CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme
caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-
keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11b-hydroxysteroid dehydro-
genases (18b-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental
microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of
bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser
extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.
Received 1 March 2011
Accepted 27 April 2011
Available online 5 May 2011
Keywords:
Bupropion
Metabolism
Baboon
Liver
Placenta
ß 2011 Elsevier Inc. All rights reserved.
metabolized in vivo and less than 10% of the drug is excreted
unchanged in urine and feces [5,6]. Furthermore, recent preclinical
data obtained from in vitro studies revealed that bupropion is also
1. Introduction
Smoking is the largest modifiable risk factor for pregnancy-
related morbidity and mortality in the U.S. [1]. Approximately 5–
10% of prenatal deaths, 20–35% of low-birth-weight infants, and 8–
15% of preterm deliveries have been attributed to smoking [2,3].
Despite the substantial risks to the fetus, most pregnant smokers
do not quit smoking during pregnancy because of the highly
addictive nature of nicotine.
Bupropionisanantidepressantthathas beensuccessfullyusedas
an alternative to nicotine replacement therapy to aid in smoking
cession in non-pregnant patients. However, due to limited data on
its safety and efficacy in pregnant women, its use in this patient
population is restricted. Additionally, the onset of pregnancy is
accompanied by changes in maternal physiology that affect the
absorption, distribution, metabolism, and elimination of adminis-
tered medications [4]. In humans, bupropion is extensively
metabolized by human placenta [7]. Consequently, if pregnancy-
induced changes alter the activity of enzymes metabolizing
bupropion, the pharmacokinetics of bupropion reported for non-
pregnantpatientscannotbeextrapolatedtothosewhoarepregnant.
There are several challenges associated with drug development
for the pregnant patient. First, ethical and safety concerns for the
mother and fetus. Second, the anatomical and functional
differences between the human placenta and the placenta of
other mammals limit availability of a recognized pregnant animal
model that approximates drugs disposition in the pregnant
human. In order to elucidate the effect of pregnancy on the
metabolism of bupropion in vivo and due to the above mentioned
concerns, the use of an animal model that best simulates drug
metabolism and placentation in humans is required. Previously,
interspecies differences in the biotransformation of bupropion
between laboratory animals have been reported [8,9] and it was
concluded that the metabolic fate of bupropion in humans more
closely resembles that of guinea pig than either rats or mice [9].
Although the use of a non-primate animal model to study drug
disposition in vivo has its advantages (e.g. short gestation and
lower expense), the distinct differences in placental development,
structure, and functions limit its validity in extrapolating data to
humans.
§
This work was supported by the National Institute of Drug Abuse RO1DA024094
to TN.
Abbreviations: Vmax, maximum reaction velocity; Km, substrate concentration
causing 50% of the reaction velocity; P450, cytochrome P450; IC50, concentration
of compound causing 50% inhibition of the reaction; OH-BUP, hydroxybupropion;
TB, threohydrobupropion; EB, erythrohydrobupropion.
* Corresponding author. Tel.: +1 409 772 3908; fax: +1 409 772 5297.
E-mail address: tnnanovs@utmb.edu (T.N. Nanovskaya).
0006-2952/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2011.04.014

296
X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
During the last 5 years, data obtained from our laboratory
revealed similarities between baboon (Papio cynocephalus) and
2.4. Identification of the enzyme(s) catalyzing the hydroxylation of
bupropion by baboon hepatic microsomes
human hepatic and placental microsomes in their biotransforma-
tion of the 17
a
-hydroxyprogesterone caproate [10] and hypogly-
2.4.1. Effect of chemical inhibitors on the formation of
cemic drug glyburide [11]. The baboon was chosen as a non-human
primate animal model because of its similarities with humans in
placental structure, functions [12] as well as in fetal development
[13].
Therefore, the aim of this investigation is to determine the
biotransformation of bupropion by baboon hepatic and placental
microsomes, identify the enzyme(s) catalyzing the reaction(s) and
determine its kinetics.
hydroxybupropion
The effect of chemical inhibitors selective for CYP isoforms [14]
on the biotransformation of bupropion to OH-BUP by baboon’s
hepatic microsomes was determined. The final concentration used
for each inhibitor was approximately 10-fold its reported Ki value
to maintain selectivity for its specific CYP isozyme and to obtain at
least ꢀ80% inhibition of the reaction. These inhibitors were
dissolved in several solvents: (1) in 0.1 M potassium phosphate
buffer:quinidine, CYP2D6 (4
ride, CYP 2E1 (120 M) [16], (+)-nootkatone, CYP2C19 (5
[17], phencyclidine hydrochloride, CYP2B6 (100 M) and ticlopi-
dine hydrochloride, CYP2B6 (2 M) [18,19]; (2) in 0.5% (v/v)
ethanol:aminoglutethimide, CYP19 (7 M) [20], sulfaphenazole,
CYP2C9 (3 M) [15], -naphthoflavone, CYP1A1 (0.1 M) [15],
ketonconazole, CYP 3A4 (1.8 M) [15] and furafylline, CYP1A2
(8 M) [15]; (3) in 0.3% (v/v) DMSO:trimethoprim, CYP2C8
(320 M) [21] and trans-2-phenylcyclopropyl-amine hydrochlo-
ride, CYP2A6 (0.4 M) [22]. Furafylline (the mechanism-based
m
M) [15], chlomethiazole hydrochlo-
2. Materials and methods
m
m
M)
m
2.1. Chemicals and biological reagents
m
m
Hydroxybupropion (OH-BUP), erythro- (EB) and threohydro-
bupropion (TB) were purchased from Toronto Research Chemicals
Inc. (North York, Canada). HPLC-grade methanol, acetic acid, 4-
methylpyrazole, dicumarol, and menadione were purchased from
Fisher Scientific (Fair Lawn, NJ). Phencyclidine hydrochloride was a
gift from the National Institute on Drug Abuse drug supply unit. All
other chemicals were purchased from Sigma Chemical Co. (St.
Louis, MO). Polyclonal antibodies raised against CYPs 2C19, 2D6
and 3A4 were obtained from AbD Serotec (Oxford, UK). Monoclonal
and polyclonal antibodies against CYPs 2B6, 2C9, 2E1, 2A6, 2C8,
1A2 and 1A1 were purchased from XenoTech, LLC (Lenexa, KS).
Rabbit antiserum to human placental aromatase (CYP19) was
purchased from Hauptman-Woodward Institute (Buffalo, NY).
m
a
m
m
m
m
m
inhibitor) was pre-incubated with hepatic microsomes (0.25 mg)
and the NADPH-regeneration system at 37 8C for 10 min, and then
50
reaction. Other inhibitors were pre-incubated with hepatic
microsomes (0.25 mg) and 50 M of bupropion for 5 min at
m
M (ꢁKm value) of bupropion was added to initiate the
m
37 8C. The reaction was initiated by the addition of NADPH-
regenerating system, incubated for 30 min at 37 8C, and then
terminated by the addition of TCA. The control reactions included
all the above-mentioned components in presence of either 0.5%
ethanol (v/v) or 0.3% (v/v) DMSO instead of inhibitors.
2.2. Preparation of microsomes from baboon hepatic and placental
tissues
2.4.2. The effect of antibodies against CYP isozymes on the formation
of hydroxybupropion
Adult baboon livers (n = 4) were obtained from female animals
that were sacrificed for herd reduction. Placentas of baboons
(n = 3) were obtained by cesarean section. All tissues were
obtained according to a protocol approved by the Institution
Animal Care and Use Committee of the Southwest Foundation for
Biomedical Research, San Antonio, TX. The hepatic and placental
microsomal fractions were prepared by differential centrifugation
as previously described [7,11]. The microsomes were re-suspended
in 0.1 M potassium phosphate buffer (pH 7.4) and stored at À80 8C
until used. Protein content of microsomes was determined by a
commercially available kit (BioRad Laboratories, Hercules, CA)
using bovine serum albumin as a standard.
Monoclonal and polyclonal antibodies raised against human
hepatic CYP isoforms 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1,
3A4, and rabbit antiserum to human CYP19 were used to identify
the CYP isozymes responsible for the biotransformation of
bupropion to OH-BUP. The reaction volume was 250
mL of 0.1 M
potassium phosphate buffer (pH 7.4). Each monoclonal or
polyclonal antibody was added to the reaction components at
the concentration causing 80% inhibition of the human CYP
isoform it was raised against. Baboon hepatic microsomal proteins
(13
followed by the addition of bupropion at a final concentration of
300 M. The reaction was initiated by the addition of the NADPH-
mg) were pre-incubated with the antibody at 25 8C for 10 min
m
2.3. Biotransformation of bupropion by baboon hepatic and placental
microsomes
regenerating system, incubated for 45 min at 37 8C, and terminat-
ed by the addition of TCA. Mouse IgG replaced the antibodies in the
control reaction.
The activity of baboon hepatic and placental microsomes in the
biotransformation of bupropion was determined in the reaction
2.5. Identification of the enzyme(s) catalyzing the reduction of the
carbonyl group in bupropion by baboon hepatic and placental
microsomes
solution (250
mL) of 0.1 M potassium phosphate buffer (pH 7.4).
Each reaction solution contained 0.25 mg protein of the hepatic or
placental microsomes and bupropion at a final concentration of
250
m
M was pre-incubated for 5 min at 37 8C. The reaction was
Identification of hepatic and placental microsomal enzyme(s)
catalyzing the reduction of the carbonyl group in bupropion was
performed as previously reported [7]. The concentration used for
each chemical inhibitor was based on their IC50 or Ki values
reported for a specific carbonyl reductase. The following are the
carbonyl reducing enzymes, their selective inhibitors and the
concentrations used: alcoholdehydrogenase (ADH), 4-methylpyr-
initiated by the addition of NADPH-regenerating system, made of
0.4 mM NADP+, 4 mM glucose 6-phosthate, 1 U/mL glucose-6-
phosphate dehyrogenase, and 2 mM MgCl2 followed by incubation
at 37 8C for 30 min or 40 min in presence of either hepatic or
placental microsomes, respectively. The reaction was terminated
by the addition of 25
Phenacetin (10 L of 2.4
standard. The effect of bupropion (10–400
m
m
L of 40% trichloroacetic acid (TCA).
g/mL) was then added as an internal
M) on the reaction
m
azole (500
(500 M) [24], aldo-ketoreductase (AKR), flufenamic acid
(5 M) [25], carbonyl reductase (CR) menadione (100 M) [25],
quinone oxidoreductase, dicumarol (500 M) [26], 11 -hydro-
mM) [23], aldehyde/aldose reductase, barbital
m
m
velocity was used to construct the saturation curve and to calculate
the Vmax and apparent Km values.
m
m
m
b

X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
297
xysteroid dehydrogenase (11
b
-HSD), 18
b
-glycyrrhetinic acid
lystTM 1.5 Software (MDS Inc. and Applera Corporation, USA). The
separation of metabolites was achieved by using a Waters
(18 -GA) (0.1 M) [27]. Stock solutions of inhibitors were
b
m
prepared in 0.5% ethanol (v/v) and an aliquot of each was used
to attain the final concentrations specified above. Each inhibitor
was pre-incubated in presence of hepatic or placental microsomes
(1 mg/mL) for 10 min at 37 8C. The final concentration of
Symmetry C18 column (150 mm  4.6 mm, 5
mm) connected to
a Phenomenex C18 guard column (4 mm  3.0 mm). The aqueous
mobile phase consisted of 10 mM ammonium acetate buffer
(pH = 6.0, adjusted with 0.1 M acetic acid). Samples were eluted at
flow rate of 1.0 mL/min using a linear gradient from 5 to 90%
bupropion in the reaction solution was 30
m
M (ꢁKm value). The
reaction was initiated by the addition of NADPH-regenerating
system, incubated for 40 min at 37 8C, and terminated by the
addition of TCA. The control reaction included all the above-
mentioned components in presence of 0.5% ethanol (v/v) instead of
inhibitor.
methanol for 60 min. A volume of 250
to the mass spectrometer.
mL of effluent was directed
The API 4000 triple quadrupole mass spectrometer equipped
with a Turbo (V) ion source (ESI) was operated in positive mode.
Optimal MS parameters were as follows: ionspray voltage, 2000 V;
curtain gas, 30 L/h; ion source gas1, 25 L/h; ion source gas2, 50 L/h;
temperature, 300 8C; collision gas 5 L/h; declustering potential,
20 V; entrance potential, 5 V; collision energy, 25 V; collision cell
exitpotential,14 V.Themassscanrangewassetupfromm/z80tom/
z 450 for Q1 full scan, and the quasi molecular ion of each metabolite
was transferred into Q3 to generate the product ion spectrum.
The formation of chromatographic peaks in the presence of
NADPH-regenerating system that were not observed in its absence
were considered as metabolites of bupropion. Further characteri-
zation of these peaks as corresponding to metabolites of bupropion
was confirmed by MS/MS analysis.
The IC50 for menadione, flufenamic acid, and 18b-GA for their
inhibition of bupropion reduction by hepatic and placental
microsomes was determined. The concentration of bupropion in
the reaction solution was 30
added in the following range of concentrations: menadione (5–
250 M), flufenamic acid (1–100 M), and 18 -GA (1–300 nM).
m
M (ꢁKm), and each inhibitor was
m
m
b
Each IC50 value was calculated from plots of the remaining activity
as percent of control (absence of inhibitor) versus the log of its
concentration.
2.6. Identification of bupropion metabolites formed by baboon hepatic
and placental microsomes
2.7. Quantitative determination of bupropion metabolites in baboon
hepatic and placental microsomes
The analysis of bupropion metabolites was achieved by using an
Agilent HPLC 1200 series system coupled with an API 4000 triple
quadrupole mass spectrometer (Applied Biosystems, Foster City,
CA). The HPLC system consisted of a degasser, binary pump
delivery system and Hip-ALS auto-sampler controlled by Ana-
Bupropion and its metabolites were extracted from reaction
solution as previously reported by our laboratory [7]. The HPLC and
MS instruments used consisted of Waters 600E multisolvent
TIC of +Q1: from Sample 3 (Blank of...
Max. 3.3e8 cps.
TIC of +Q1: from Sample 2 (BLM Q1...
3.0e8
Max. 3.1e8 cps.
3.3e8
3.0e8
B
A
Bupropion
2.5e8
2.5e8
2.0e8
1.5e8
1.0e8
5.0e7
0.0
M4&M5
M2
M6
2.0e8
1.5e8
M3
1.0e8
M1
5.0e7
0.0
10
20
30
40
50
60
10
20
30
40
50
60
Time, min
Time, min
TIC of +Q1: from Sample 4 (Blank of...
3.3e8
Max. 3.3e8 cps.
TIC of +Q1: from Sample 4 (BPM Q1...
Max. 3.2e8 cps.
3.2e8
3.0e8
D
C
3.0e8
2.5e8
2.0e8
1.5e8
1.0e8
5.0e7
0.0
Bupropion
2.5e8
2.0e8
1.5e8
1.0e8
5.0e7
0.0
M4
M3
M2
M1
10
20
30
40
50
60
10
20
30
40
50
60
Time, min
Time, min
Fig. 1. Representative total ion chromatograms of bupropion metabolites formed in vitro by baboon hepatic (A and B) and placental (C and D) microsomes in the absence (A
and C) and presence (B and D) of NADPH-regenerating system.

298
X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
delivery system, 717 autosampler controlled by EmpowerTM
2
Waters Symmetry C18 column (150 mm  4.6 mm, 5
mm) con-
chromatography Data Software (Waters, Milford, MA). The mobile
phase consisted of A: 40% methanol and B: 60% 10 mM ammonium
acetate buffer (pH = 6.0, adjusted with 0.1 M acetic acid). The
separation of bupropion and metabolites was performed using
nected to a Phenomenex C18 guard column (4 mm  3.0 mm).
Isocratic elution of metabolites was achieved at a rate of 1.0 mL/min
and mobile phase was transferred into mass spectrometer with flow
rate of 100
mL/min.
Fig. 2. Proposed fragmentation pathway of bupropion and its metabolites (M1–M6) formed by baboon microsomes.

X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
299
The mass spectrometer (Waters EMD 1000 single-quadrupole;
Milford, MA) equipped with an electrospray ion source (ESI) was
operated in positive mode. MS parameters were as follows: capillary
voltage, 2.2 kV; cone voltage, 40 V; source temperature, 95 8C;
desolvation temperature, 350 8C; desolvation gas flow rate, 450 L/h;
cone gas flow rate, 100 L/h. The metabolites and internal standard
were monitored by selective ion monitoring (SIM) at m/z 180 for
phenacetin (IS), m/z 238 for OH-BUP and m/z 168 for TB and EB.
The MS2 spectrum of M5 (Fig. 3E) shows the fragment ion at m/z
238, indicating the hydroxylation of the chain of the molecule. The
fragment at m/z 200 indicates the loss of intact butyl group.
The MS2 spectrum of M6 (Fig. 3G) shows the same fragment
ions at m/z 200, 182 and 147 with those of M1 and M2. The ion at
m/z 139 is derived from fragment ion at m/z 182 by losing C2H5N,
indicating that hydroxylation is most likely located at the methine
group in the side chain. The fragment ions at m/z 164 and 154 is not
readily identifiable and may arise via a rearrangement process.
Metabolites M3 and M4 show the protonated molecular ion at
m/z 242, 2 Da higher than quasi molecular ion of bupropion (m/z
240), suggesting that these metabolites were hydrogenated
derivatives of the parent bupropion. The MS2 spectrum of M3
and M4 (Fig. 3C and D) have the similar fragmentation patterns
indicating that M3 and M4 are the stereo isomers. The identifica-
tion of M3 as EB and M4 as TB was confirmed by comparing their
MS2 spectrum with the authentic compounds.
2.8. Data analysis
For each inhibition experiment the control was set at 100% and
the amount of metabolite formed in the presence of an inhibitor
(antibody or chemical) was expressed as percent of control. All
data are presented as mean Æ S.D. The variance (one way-ANOVA)
was used for all multiple comparative analyses.
The Vmax and apparent Km values were determined using
nonlinear regression analysis of the saturation equation (GraphPad
Prism 5, Vision 5.01, Graph Pad Software Inc.).
3.3. Quantification of bupropion metabolites formed by baboon
hepatic and placental microsomes
3. Results
The individual baboon hepatic microsomes (n = 4) and placen-
tal microsomes (n = 3) were utilized to investigate the metabolism
of bupropion. Metabolites M1, M5, and M6 formed by baboon
microsomes were not quantified in the present study due to the
lack of commercially available standard compounds of these
metabolites. The metabolites OH-BUP (M2), EB (M3), and TB (M4)
were quantitatively determined by using LC/MS-ESI method. The
quantitative determination of these metabolites was validated for
specificity, linearity, sensitivity, precision and accuracy as previ-
ously described from our laboratory [7].
3.1. Biotransformation of bupropion by baboon hepatic and placental
microsomes
The incubation of bupropion in the presence of baboon hepatic
microsomes and NADPH-regenerating system resulted in the
formation of 6 metabolites (Fig. 1A and B). The chromatographic
peaks of the formed metabolites were eluted at retention times of
18.5 (M1), 25.8 (M2), 28.6 (M3), 29.5 (M4), 29.9 (M5), and 50.4
(M6) minutes, respectively.
The total ion chromatograms of the reaction solutions revealed
4 peaks corresponding to metabolites formed by baboon placental
microsomes. The formation of bupropion metabolites by placental
microsomes also required the presence of NADPH-regenerating
system (Fig. 1C and D). The retention times of the metabolites M1–
M4 formed by placental microsomes were identical to the
retention times of the metabolites M1–M4 formed by hepatic
microsomes.
The major metabolite formed by baboon hepatic microsomes
was OH-BUP (6992 Æ 982 pmol/mg protein) which accounted for
62% of the sum of the three metabolites formed. The amounts of TB
(3241 Æ 577 pmol/mg protein) and EB (1222 Æ 233 pmol/mg pro-
tein) comprised 28% and 10%, respectively.
The amounts of bupropion metabolites formed by placental
microsomes were significantly lower than those by hepatic
microsomes. The major metabolite formed by placental micro-
somes was TB (1042 Æ 880 pmol/mg protein) representing 82% of
the sum of the three metabolites formed, while the formation of EB
(243 Æ 238 pmol/mg protein) accounted for 18%. The formation of
OH-BUP was below the lower limit of quantification (LLOQ) of the
analytical method used.
Subsequent identification of the metabolites formed by baboon
hepatic and placental microsomes was performed by MS/MS
analysis.
3.2. Identification of bupropion metabolites formed by baboon hepatic
and placental microsomes
3.4. Kinetic parameters of bupropion metabolism by baboon hepatic
microsomes
The structural identification of metabolites was performed by
comparing the fragment ion of metabolites with that of bupropion,
and the proposed fragmentation pattern of metabolites is shown in
Fig. 2.
Metabolites M1, M2, M5 and M6 show the protonated
molecular ion at m/z 256, which is 16 Da higher than quasi
molecular ion of bupropion (m/z 240), suggesting that these
metabolites were hydroxylated derivatives of the parent bupro-
pion.
The MS2 spectrum of M1 (Fig. 3A) shows the fragment at m/z
200, which indicates the loss of the intact butyl group (m/z 56). The
m/z 182 is the fragment ion derived from the ion at m/z 200 by
losing water. The fragment ions at m/z 147 and 155 of M1 are 16 Da
greater than the fragment ions at m/z 131 and 139 of bupropion
(Fig. 3F), indicating the hydroxylation of aromatic ring of M1 [28].
Metabolite M2 has the fragment ion at m/z 131 indicating that
the hydroxylation located at the methyl group in the side chain
[28]. The MS2 analysis of M2 confirmed the identification of OH-
BUP by comparing the MS2 fragmentation (Fig. 3B) with the
authentic bupropion and OH-BUP [29].
The rate of bupropion biotransformation to its three metabolites
OH-BUP, TB and EB was dependent on its concentration and
exhibited saturation kinetics. The hydroxylation of bupropion by
hepaticmicrosomesrevealedanapparentKm valueof36 Æ 6 mMand
Vmax value of 258 Æ 32 pmol mg proteinÀ1 minÀ1 (Fig. 4). The reduc-
tion of carbonyl group of bupropion to TB and EB revealed apparent Km
values of 28 Æ 2 mM and 32 Æ 3 mM, respectively. The rates of TB and
EB formation were lower than that for OH-BUP with mean Vmax values
of 116 Æ 19 and 46 Æ 8 pmol mg proteinÀ1 minÀ1, respectively.
3.5. Identification of the hepatic enzymes responsible for
hydroxylation and reduction of bupropion
3.5.1. Identification of baboon hepatic enzyme(s) catalyzing the
hydroxylation of bupropion
Chemical inhibitors selective for the various CYP 450 isoforms
were utilized to identify the enzyme(s) catalyzing the hydroxyl-
ation of bupropion by baboon hepatic microsomes. Phencyclidine

300
X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
Fig. 3. MS2 spectra of bupropion and its metabolites formed by baboon hepatic (M1–M6) and placental (M1–M4) microsomes. (A) M1 (aromatic hydroxylated metabolite); (B)
M2 (hydroxybupropion); (C) M3 (erythrohydrobupropion); (D) M4 (threohydrobupropion); (E) M5 (methyl hydroxylated metabolite); (F) Bupropion; (G) M6 (methine
hydroxylated metabolite).
(64%, p < 0.01) and ticlopidine hydrochloride (77%, p < 0.01)
selective for CYP2B6 caused the maximum inhibition of OH-BUP
formation (Fig. 5A). Chemicals selective for CYP2E1 (chlomethia-
zole hydrochloride) and CYP2C19 (+)-nootkatone inhibited the
formation of OH-BUP by 10–15%, whereas other compounds did
not affect its formation.
Monoclonal and polyclonal antibodies raised against human
hepatic CYP isoforms 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1,
3A4, and rabbit antiserum to human CYP19 were used to confirm
the identity of the microsomal enzyme(s) catalyzing the biotrans-
formation of bupropion to OH-BUP. Antibodies against human
CYP2B6 were the most potent and inhibited the formation of OH-
BUP by 82% (p < 0.01). The remaining antibodies had null effect on
the hydroxylation of bupropion.
3.5.2. Identification of baboon hepatic enzyme(s) catalyzing the
reduction of bupropion
Chemical inhibitors known as selective for carbonyl-reducing
isoforms were used to identify baboon hepatic enzyme(s)
catalyzing the reduction of bupropion to TB and EB (Fig. 5B).
The formation of TB by baboon hepatic microsomes was inhibited
by menadione (80%, p< 0.01), 18b-glycyrrhetinic acid (80%,
p < 0.01), while flufenamic acid caused 40% of inhibition
(p < 0.01). On the other hand, these three compounds inhibited

X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
301
250
200
150
100
50
formation of EB cannot be determined in the concentration range
tested.
3.6. Kinetic parameters of bupropion metabolism by baboon placental
microsomes
Km=44 μM
The rates of TB and EB formation were dependent on bupropion
concentrations and exhibited saturation kinetics (Fig. 7A). The
reduction of bupropion to TB and EB by placental microsomes
Vmax=254pmol/mg protein/min
revealed
a
mean apparent Km values of 20 Æ 14 mM and
0
32 Æ 14 mM, respectively. The Vmax value for formation of TB was
of 28 Æ 24 pmol mg proteinÀ1 minÀ1, while the rate for formation of
EB (7 Æ 7 pmol mg proteinÀ1 minÀ1) was approximately 25% that of
TB formation.
0
100
200
300
400
Bupropion (μM)
Fig. 4. A representative saturation curve of OH-BUP formation by baboon hepatic
microsomes. The rate of bupropion hydroxylation to OH-BUP was dependent on
bupropion concentration and exhibited typical saturation kinetics.
3.7. Identification of placental enzymes responsible for reduction of
bupropion
The chemical inhibitors known as selective for carbonyl-
reducing isoforms were used to identify baboon enzyme(s)
catalyzing the reduction of bupropion to TB and EB by placental
the formation of EB by approximately 40%, p < 0.01. Barbital, 4
methylpyrazole and dicumarol did not affect TB and EB formation.
The IC50 values calculated for the effect of 18
acid, flufenamic acid, and menadione on the TB formation were
15 nM, 11.4 M, and 47 M, respectively (Fig. 6). The IC50 value of
flufenamic acid for inhibition of EB formation was 7.7 M, whereas
IC50 values of 18 -glycyrrhetinic acid and menadione on the
b
-glycyrrhetinic
microsomes (Fig. 7B). Menadione and 18b-GA inhibited (80%,
p < 0.01) the formation of TB and EB, while flufenamic acid caused
40% (p < 0.01) inhibition. On the other hand, 4-methylpyrazole,
barbital, and dicumarol did not affect the formation of TB and EB.
m
m
m
b
The IC50 for the inhibition of the TB and EB by 18b-GA was 25 and
Fig. 5. Effect of chemical inhibitors selective for CYP isoforms on the formation of OH-BUP (A) and effect of chemical inhibitors selective for carbonyl reductases on the
formation of threo- and erythrohydrobupropion (B) by baboon hepatic microsomes. The inhibitors of CYP450 isoforms (A) are: sulfaphenazole (SUL, 3 M), chlomethiazole
(CHL, 120 M), quinidine (QUI, 4 M), trimethoprim (TRI, 320 M), -naphthoflavone (NAP, 0.4 M), (+)-nootkatone (NOK, 5 M), phencyclidine (PHE, 8 M), ticlopidine
hydrochloride (TIC, 2 M), trans-2-henmylcyclopropyl-anmie (TRN, 0.4 M), furafylline (FUR, 8 M), ketonconazole (KET, 1.8 M) and aminoglutethimide (AMG, 7 M).
The inhibitors on carbonyl reductases (B) are: 4-methylpyrazole (4-MP, 500 M), barbital (BAR, 500 M), flufenamic acid (FLU, 5 M), dicumarol (DIC, 500 M), menadione
(MEN, 100 M), and 18 -glycyrrhetinic acid (18 -GA, 0.1 M). The rates of hydroxybupropion (OH-BUP), threo-(TB) and erythrohydrobupropion (EB) are expressed as
m
m
m
m
a
m
m
m
m
m
m
m
m
m
m
m
m
m
b
b
m
percent of control (absence of inhibitor) and represent the mean Æ S.D. of triplicate experiments. ** Statistical significance of p < 0.01 as determined by one-way ANOVA with
Tukey’s comparison.
120
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
EB
TB
A
B
C
EB
TB
EB
TB
1
10
100
1000
1
10
100
1
10
100
1000
18β-Glycyrrhetinic acid concentration (nM)
Flufenamic acid concentration (μM)
Menadione concentration (μ M)
Fig. 6. The effect of increasing concentrations of (A) 18b-glycyrrhetinic acid, (B) flufenamic acid and (C) menadione on erythro- and threohydrobupropion formation by
baboon hepatic microsomes. The rates of erythro- and threohydrobupropion formation are expressed as a percent of their formation in the absence of inhibitors (control)
which was set at 100%. Each data point represents the mean Æ S.D. of triplicate experiments.

302
X. Wang et al. / Biochemical Pharmacology 82 (2011) 295–303
120
B
TB
EB
16
12
8
A
100
80
**
**
60
Km=34 μM
max=16 pmol/mg protein/min
V
40
20
0
4
**
**
**
**
0
0
100
200
300
400
4-MP
BAR
MEN
FLU
18β-GA
DIC
Chemical inhibitors against carbonyl reductases
Bupropion (μM)
Fig. 7. The representative saturation curve of threohydrobupropion formation by baboon placental microsomes (A) and the effect of chemical inhibitors selective to carbonyl
reductases on the formation of threo- and erythrohydrobupropion by baboon placental microsomes (B). The inhibitors of carbonyl reductase are: 4-methylpyrazole (4-MP,
500
mM), barbital (BAR, 500 mM), flufenamic acid (FLU, 5 mM), dicumarol (DIC, 500 mM), menadione (MEN, 100 mM), and 18b-glycyrrhetinic acid (18b-GA, 0.1 mM). The
rates of threo-(TB) and erythrohydrobupropion (EB) are expressed as percent of control (absence of inhibitor) and represent the mean Æ S.D. of triplicate experiments. **
Statistical significance of p < 0.01 as determined by one-way ANOVA with Tukey’s comparison.
22 nM, by menadione 27.9 and 30.32
9.9 and 5.6 M, respectively.
m
M, and by flufenamic acid
metabolites of bupropion in human urine samples by using
time-of-flight mass spectrometry [29].
m
The second part of this investigation focused on the characteri-
zation of the metabolites of bupropion (OH-BUP, TB and EB)
formed by baboon hepatic and placental microsomes as well as the
identification of the enzymes involved in their formation.
The data obtained in this investigation revealed that hydro-
xybupropion (62%) is the major hepatic metabolite while
threohydrobupropion (82%) is the major placental metabolite of
bupropion formed in vitro by baboons. Accordingly, in baboons, the
primary metabolic pathway for the biotransformation of bupro-
pion in the liver is by hydroxylation and in the placenta by
reduction. Similar tissue specific differences in the in vitro
biotransformation of bupropion were reported earlier for human
liver and placenta [7,31–33].
The major human hepatic enzyme responsible for the
hydroxylation of bupropion to hydroxybupropion was identified
as CYP2B6 [33]. In this investigation, two experimental approaches
were utilized to identify the enzyme(s) responsible for hydroxyl-
ation of bupropion by baboon hepatic microsomes, namely,
inhibition of the reaction by compounds that are known as
selective inhibitors of specific CYP isoforms as well as by antibodies
raised against each isozyme. It was assumed that an antibody
raised against a human CYP isoform will cross-react with the same
CYP subfamily of baboon isozymes.
Phencyclidine and ticlopidine hydrochloride (Fig. 5A), at their
concentrations selective for CYP2B6, were the most potent
inhibitors of OH-BUP formation by baboons. In addition, the
formation of OH-BUP was inhibited (82%) by monoclonal
antibodies raised against human CYP2B6. Taken together these
data indicate that the main enzyme responsible for the hydroxyl-
ation of bupropion to OH-BUP by baboon hepatic microsomes is
very similar or identical to human CYP2B6 and most likely belongs
to the CYP2B subfamily.
The enzyme(s) catalyzing the reduction of bupropion to threo-
and erythrohydrobupropion by baboon hepatic and placental
microsomes were identified using chemical inhibitors selective for
human carbonyl-reducing enzymes. However, it is plausible that
members of the same subfamily of enzymes from different species
may exhibit different substrate selectivity. Inhibition of aldo-keto
reductases (by flufenamic acid), carbonyl reductases (by menadi-
4. Discussion
The aim of this investigation was to determine the biotransfor-
mation of bupropion by baboon hepatic and placental microsomes,
identify the enzyme(s) catalyzing the reaction(s) and determine its
kinetics.
Data obtained in this investigation revealed that the biotrans-
formation of bupropion by baboon hepatic and placental micro-
somes resulted in the formation of six and four metabolites,
respectively. These metabolites, depending on the order of their
elution on LC/MS chromatograms, were named M1–M6 (Fig. 1).
Based on the similar retention times and MS2 spectrum as
compared to the standards compounds, the metabolites M2–M4
formed by baboon hepatic and placental microsomes were
identified as hydroxybupropion, erythro- and threohydrobupro-
pion. Metabolite M1 formed by both hepatic and placental
microsomes was identified by its fragmentation pattern as an
aromatic hydroxylated metabolite of bupropion. The two meta-
bolites of bupropion (M5 and M6) were formed only by hepatic
microsomes and based on their fragmentation patterns were
identified as methyl and methine hydroxylated metabolites,
respectively. The retention times and the fragmentation pattern
of the six metabolites formed by baboon hepatic microsomes (M1–
M6) and the four metabolites formed by placental microsomes
(M1–M4) were identical to those formed by human hepatic and
placental microsomes under the same experimental (in the
presence of NADPH) and chromatographic conditions (Figs. 8
and 9, see supplementary notes). Therefore, the data obtained in
this investigation revealed similarities between baboon and
human hepatic and placental microsomes for their in vitro
biotransformation of bupropion. However, it is plausible that
the hydroxylated metabolites of bupropion identified in vitro can
undergo further metabolism by phase II enzymes in vivo.
The formation of three hydroxylated metabolites of bupropion
identified in the current investigation (M1, M5, and M6) in addition
to previously described OH-BUP (M2) [30] is in agreement with the
recently published study by Chen et al. [28]. Using QTRAP-mass
spectrometry, the authors reported a total of nine metabolites of
bupropion formed by cDNA-expressed P450s and human hepatic
microsomes [28]. Furthermore, metabolites M1–M4 described in
this investigation were also identified among 20 reported
one), and 11b-hydroxysteroid dehydrogenase (by 18b-GA)
significantly decreased the formation of threo- and erythrohy-
drobupropion by baboon hepatic and placental microsomes

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The authors sincerely appreciate the support of the physicians
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Appendix A. Supplementary data
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