Stereoselective bupropion hydroxylation by cytochrome P450 CYP2B6 and cytochrome P450 oxidoreductase genetic variants

Article abstract of DOI:10.1124/DMD.119.090407

Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by CYP2B6. The metabolite hydroxybupropion derived from t-butylhydroxylation is considered to contribute to the antidepressant and smoking-cessation effect

Full text of DOI:10.1124/DMD.119.090407

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Stereoselective bupropion hydroxylation by cytochrome P450 CYP2B6 and
cytochrome P450 oxidoreductase genetic variants
DMD/2019/090407 Revised
Pan-Fen Wang, PhD Alicia Neiner, Evan D. Kharasch, MD PhD*
panfen.wang@duke.edu, neinera@wustl.edu, evan.kharasch@duke.edu
Department of Anesthesiology, Duke University School of Medicine, Durham, NC (P.-F.W., EDK)
Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO. (A.N.)

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Running Title: Bupropion enantiomers metabolism by CYP2B6 variants
*Address Correspondence to: Dr. Evan Kharasch, Department of Anesthesiology, Duke University
School of Medicine, Box 3094, 905 S. LaSalle St, GSRB1 Room 2031, Durham, NC 27710
Tel.: 919-613-1154 E-mail: evan.kharasch@duke.edu
Figures: 4
Tables: 3
References: 50
Word count: Abstract (197)
Word count: Introduction (506)
Word count: Discussion (1499)
No author has any competing or conflict of interest
Non-standard abbreviations: CYP, cytochrome P450; POR, NADPH cytochrome P450 oxidoreductase

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Abstract
Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by
cytochrome P4502B6 (CYP2B6). The metabolite hydroxybupropion derived from t-butylhydroxylation is
considered to contribute to the antidepressant and smoking-cessation effects of the parent drug.
Bupropion hydroxylation is the canonical in vitro and in vivo probe for CYP2B6 activity. P450 also
requires obligate partnership with P450 oxidoreductase (POR). Human CYP2B6 and POR genes are
highly polymorphic. Some CYP2B6 variants affect bupropion disposition. This investigation evaluated
the influence of several human CYP2B6 and POR genetic variants on stereoselective bupropion
metabolism, using an insect cell coexpression system containing CYP2B6, POR and cytochrome b5.
Based on intrinsic clearances, relative activities for S,S-hydroxybupropion formation were in the order
CYP2B6.4>CYP2B6.1>CYP2B6.17>CYP2B6.5>CYP2B6.6≈CYP2B6.26≈CYP2B6.19>CYP2B6.7>
CYP2B6.9>>CYP2B6.16 and CYP2B6.18; relative activities for R,R-hydroxybupropion formation were
in the order CYP2B6.17>CYP2B6.4>CYP2B6.1>CYP2B6.5≈CYP2B6.19≈CYP2B6.26>CYP2B6.6>
CYP2B6.7≈ CYP2B6.9>>CYP2B6.16 and CYP2B6.18. Bupropion hydroxylation was not influenced by
POR variants. CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Though Vmax and Km
varied widely among CYP2B6 variants, stereoselectivity was preserved, reflected by similar Clint(S,S-
hydroxybupropion)/Clint(R,R-hydroxybupropion) ratios (1.8 to 2.9), except CYP2B6.17, which was less
enantioselective. Established concordance between human bupropion hydroxylation in vitro and in vivo,
together with these new results, suggests additional CYP2B6 variants may influence human bupropion
disposition.

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Significance
Bupropion pharmacokinetics, metabolism and clinical effects are affected by the CYP2B6*6
polymorphism. Other expressed CYP2B6 polymorphisms had diminished (*5, *6, *7, *9, *19, *26) or
defective (*16, *18) in vitro bupropion hydroxylation. P450 oxidoreductase genetic variants had no
effect on metabolism, suggesting no clinical consequence of this polymorphism. These CYP2B6
polymorphisms may portend diminished in vivo bupropion hydroxylation and predict additional clinically
important variant alleles.

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Introduction
Bupropion is FDA-approved to treat depression (Dhillon et al., 2008) and for smoking cessation
therapy (Reid et al., 2016), and also used for obesity (Wilding, 2017), seasonal affective disorder (Cools
et al., 2018), attention deficit hyperactivity disorder (Verbeeck et al., 2017), drug abuse (Lee et al., 2018),
and chronic pain (Urits et al., 2019). Bupropion inhibits norepinephrine and dopamine reuptake
transporters, increasing extracellular norepinephrine and dopamine concentrations, and is a nicotinic
antagonist, blocking nicotinic acetylcholine receptors in the brain.
Bupropion is extensively metabolized by the liver, with less than 1% eliminated unchanged in
urine (Welch et al., 1987). The major primary metabolic pathway is t-butyl hydroxylation to
hydroxybupropion. Hydroxybupropion plasma exposure exceeds that of bupropion by an order of
magnitude. Bupropion is used clinically as a racemate, and metabolism is stereoselective (Xu et al., 2007;
Coles and Kharasch, 2008; Kharasch et al., 2008; Masters et al., 2016; Kharasch and Crafford, 2019).
Hydroxybupropion is pharmacologically active (Bondarev et al., 2003; Damaj et al., 2004), and is
considered to contribute to the antidepressant and smoking-cessation effects of bupropion (Lee et al.,
2007; Zhu et al., 2012; Laib et al., 2014; Malcolm et al., 2015). Hydroxylation of the t-butyl group and
subsequent cyclic hemiketal formation creates a second chiral center. Therefore, there is the potential for
four diastereomeric hydroxylated metabolites. However, only 2S,3S- and 2R,3R-hydroxybupropion have
been found in plasma in humans, presumably due to steric hindrance precluding formation of 2S,3R- and
2R,3S-hydroxybupropion (Suckow et al., 1997). Plasma concentrations of 2R,3R-hydroxybupropion are
approximately 20-fold greater than 2S,3S-hydroxybupropion. In contrast, R-bupropion concentrations are
only about twofold greater than S-bupropion (Kharasch et al., 2008; Masters et al., 2016; Kharasch and
Crafford, 2019). 2S,3S-hydroxybupropion was deemed responsible for antidepressant activity and
nicotine-cessation activities of bupropion, while 2R,3R-hydroxybupropion was inactive toward inhibition
of norepinephrine and dopamine reuptake (Lee et al., 2007; Zhu et al., 2012; Laib et al., 2014).
Human bupropion racemate and enantiomers hydroxylation is catalyzed exclusively by liver
CYP2B6 (Faucette et al., 2000; Coles and Kharasch, 2008). Bupropion hydroxylation is the standard in

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vitro and in vivo probe to assess CYP2B6 activity, polymorphisms, and drug interactions (Faucette et al.,
2000; Fahmi et al., 2016). The CYP2B6 gene is highly polymorphic, with at least 38 allelic variants have
been described (https://www.pharmvar.org/gene/CYP2B6), of which 25 are considered important, and 8
are common or unique in at least one racial or ethnic population (Zhou et al., 2017). Carriers of certain
CYP2B6 allelic variants have altered in vivo hydroxylation of bupropion racemate (Kirchheiner et al.,
2003; Chung et al., 2011; Zhu et al., 2012; Benowitz et al., 2013; Høiseth et al., 2015; Gao et al., 2016;
Lv et al., 2016; Ma et al., 2018) and enantiomers (Kharasch and Crafford, 2019). CYP2B6*4 and more so
CYP2B6*6 are the most studied common variants. CYP2B6*6 homozygotes had decreased bupropion
hydroxylation, while CYP2B6*4 carriers had somewhat increased hydroxylation. These polymorphisms
have been associated with clinical differences in bupropion-mediated smoking cessation rates (Tran et al.,
2019).
CYP2B6 metabolizes nearly 8% of marketed drugs, including for example methadone, ketamine,
efavirenz, cyclophosphamide, and artemisinin. Previous studies showed that the activity of CYP2B6
allelic variants is allele-, substrate-, and expression system-dependent, and cannot be extrapolated across
substrates, or expression systems, and requires substrate-specific evaluation (Coles and Kharasch, 2008;
Ariyoshi et al., 2011; Honda et al., 2011; Zhang et al., 2011; Xu et al., 2012; Radloff et al., 2013; Gadel et
al., 2015; Wang et al., 2018; Wang et al., 2019). Bupropion hydroxylation in vitro by CYP2B6 variants
has been evaluated, including CYP2B6.4, CYP2B6.5, CYP2B6.6, CYP2B6.7, and CYP2B6.9 (Zhang et
al., 2011; Xu et al., 2012; Radloff et al., 2013). Nevertheless, expression systems have variably used E.
coli, SF9 and COS-1 cells. Therefore the first purpose of this investigation was to characterize
stereoselective bupropion hydroxylation, and by a greater number of clinically relevant CYP2B6 variants,
and using a standard expression system for all.
CYP catalysis requires an obligate partnership with P450 oxidoreductase (POR), which transfers
electrons from NADPH to CYP. Cytochrome b5 can also function electively in this capacity. POR plays
important roles in metabolism of drugs and steroid hormones. Genetic variation in POR is associated
with altered metabolism of endogenous steroids (Burkhard et al., 2017) and xenobiotics (Riddick et al.,

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2013). Therefore the second purpose of the present investigation was to determine the influence of POR
variants on bupropion hydroxylation by wild-type CYP2B6.

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Materials and Methods
Materials
Racemic bupropion hydrochloride was purchased from Sigma Aldrich (St. Louis, MO). The
standards rac-hydroxybupropion was purchased from Cerilliant (Round Rock, Texas), and rac-
hydroxybupropion-d6 from Toronto Research Chemicals (TRC, Toronto, ON, Canada). Spodoptera
frugiperda (Sf9) cells and Sf-900 III SFM culture media were from ThermoFisher (Waltham, MA).
Trichoplusia ni (Tni) cells and ESF AF culture media were from Expression Systems (Davis, CA). All
other reagents were from Sigma Aldrich (St. Louis, MO).
Generation of baculovirus constructs and recombinant proteins expression
Production of recombinant proteins CYP2B6 variants, P450 reductase (POR) variants and
cytochrome b5 was carried out in insect cells by triple infection as described previously (Gadel et al.,
2015; Wang et al., 2018; Wang et al., 2019). All CYP2B6 variants were coexpressed with P450 reductase
(POR) and cytochrome b5. For each variant, protein contents of CYP2B6, POR and b5 were measured as
described previously (Wang et al., 2018; Wang et al., 2019).
Bupropion metabolism
All incubations were carried out in triplicate in 96-well PCR plates with raised wells in 100 mM
potassium phosphate buffer (pH 7.4), as adapted from published protocols with modifications (Coles and
Kharasch, 2008). RS-bupropion substrate (final racemate concentrations of 0, 2 ,5, 10, 25, 50, 100, 250,
500, and 1000 µM) was mixed with CYP2B6/POR/b5. Final CYP2B6 concentration was 2.5 pmol/ml and
total reaction volume is 200 µL. After preincubation for 5 min at 37°C, the reaction was initiated by
adding NADPH regenerating system (final concentrations: 10 mM glucose 6-phosphate, 1 mM ß-NADP,
1 U/ml glucose-6-phosphate dehydrogenase, and 5 mM magnesium chloride, preincubated at 37°C for 10
min). The reaction was terminated after 10 min by adding 40 µL 20% TCA containing 75 ng/mL internal
standard rac hydroxybupropion-d6. The plate was centrifuged at 2500 rpm for 5 min to remove
precipitated proteins, and the supernatant was transferred to a 96-well shallow well plate for LC/MSMS

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analysis. Preliminary experiments showed that under the assay conditions bupropion hydroxylation was
linear with time and CYP2B6 concentration.
Analysis of bupropion hydroxylation by HPLC/tandem mass spectrometry
Calibration samples were prepared using standard of rac hydroxybupropion, which is made up of
equal amount of the two enantiomers (R,R)- and (S,S)-hydroxybupropion. Aqueous working stock
solutions of rac hydroxybupropion were prepared at concentrations of 0.1, 1, and 100 µg/mL, by diluting
the certified methanolic stock solution (1 mg/mL) in deionized water. The calibration standards were
prepared at 2, 5, 10, 50, 100, 500, 1000 and 2000 ng/mL rac hydroxybupropion from the working stock
solutions. 200 µL calibration samples were treated with 40 µL 20% TCA containing 75 ng/mL internal
standard rac hydroxybupropion-d6, and followed by centrifugation at 2500 rpm for 5 min. The
supernatant was transferred to a 96-well shallow well plate for LC/MSMS analysis.
LC–MS/MS analysis was performed on a Shimadzu HPLC system composed of two LC-20AD
XR pumps, DGU20A5R degasser, CBM-20A system controller, CTO-20AC column oven, FCV-11AL
solvent selection valve, and a SIL-20AC XR temperature regulated autosampler. The LC system was
coupled to an API6500 triple quadrupole tandem mass spectrometer (Applied Biosystems/MDS Sciex,
Foster City, CA) operated with Analyst 1.6.2. MultiQuant 3.0.1(AB Sciex) was utilized for peak
integration, generation of calibration curves, and data analysis.
Bupropion metabolites were analyzed utilizing a Chiralpak α1-acid glycoprotein (AGP) analytical
column (100 x 2.0 mm, 5 µm, Chiral Technologies, Westchester, PA) equipped with a chiral AGP guard
cartridge (10 x 2.0 mm, Chiral Technologies). A 0.25 µm inline filter was placed prior to the sample
entering the column. The column oven was at ambient temperature and the autosampler was at 4 °C. The
flow rate was 0.22 mL/min with a mobile phase consisting of 20 mM aqueous ammonium formate, pH
5.7 (A) and methanol (B). The sample injection volume was 5 µL and total run time is 12 min. The time
program to achieve the separation was as follows: 10% B for 0.5 min, linear gradient to 20% B until 1
min, held at 20% B until 5 min, linear gradient to 50% B until 8 min, and then re-equilibrated to initial
conditions until 12 min. Flow was directed into the mass spectrometer at 2.5 min and diverted to waste at

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11.5 min. Under these conditions, the retention time was 6.7 min for (R,R)-hydroxybupropion, 4.4 min for
(S,S)-hydroxybupropion, 6.4 min for (R,R)-hydroxybupropion-d6, and 4.4 min for (S,S)-
hydroxybupropion-d6. The mass spectrometer was operated with a turbo spray ion source in the positive
mode (ESI+) with multiple reaction monitoring (MRM). Analytes were detected with the MRM
transition of m/z 256.2 > 238.2 for both (R,R)- and (S,S)-hydroxybupropion, and m/z 262.2 > 244.2 for
(R,R)- and (S,S)-hydroxybupropion-d6. Operating conditions for analysis on the mass spectrometer were
as previously described (27). Global parameters were optimized as follows: curtain gas 20 psig, ion spray
voltage 5000 V, source temperature 600 °C, Gas 1 40 psig, and Gas 2 40 psig. Dwell times were 500 ms.
Interday coefficients of variation for hydroxybupropion were 5% or less at all concentrations.
Data Analysis
Hydroxybupropion formation by enzyme variants was analyzed by ANOVA with post hoc
Dunnet’s test (SigmaPlot 12.5; Systat, USA). The results are the mean ± 95% confidence interval.
Statistical significance was assigned at P<0.05. Hydroxybupropion formation versus substrate
concentration data were analyzed by nonlinear regression analysis using Michaelis-Menten model. The
results are reported as the parameter estimate ± standard error of the estimate. In vitro intrinsic clearance
(Clint) was Vmax/Km.

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Results
Metabolism of bupropion by CYP2B6 was evaluated first at approximately peak therapeutic
concentrations. Based on previous publication (Coles and Kharasch, 2008), 2µM RS-bupropion (1 µM
enantiomers) was considered to be in the range of clinically relevant concentrations. As shown in Fig 1
for R,R- and S,S-hydroxybupropion formation at 2 µM RS-bupropion substrate, activities of CYP2B6
variants vary over a wide range. For S,S-hydroxybupropion formation, CYP2B6.4 showed similar
activity with the wild type, while all other variants had diminished activity compared with CYP2B6.1,
and CYP2B6.16 and CYP2B6.18 were essentially catalytically inactive. At clinically relevant
concentrations, hydroxylation rates were of the order CYP2B6.1 ≈ CYP2B6.4 > CYP2B6.17 ≈
CYP2B6.26 > CYP2B6.5 ≈ CYP2B6.6 ≈ CYP2B6.19 > CYP2B6.7 ≈ CYP2B6.9 >> CYP2B6.16 ≈
CYP2B6.18. For R,R-hydroxybupropion formation, the order is similar except that CYP2B6.17 was
more active than CYP2B6.1.
Racemic bupropion hydroxylation catalyzed by wild-type CYP2B6.1 and the catalytically active
CYP2B6 variants with co-expressed POR and b5 was determined as a function of bupropion enantiomer
concentrations (Fig 2). For CYP2B6.1 and all CYP2B6 variant proteins, S,S-hydroxyburopion formation
exceeded that of R,R-hydroxybupropion. Eadie-Hosfstee plots were generally linear (not shown).
Michaelis-Menten kinetic parameters were determined for all active enzymes (Table 1). There was a 3-
fold (S-bupropion) and 4-fold (R-bupropion) range in Vmax, and a 5-fold range in Km parameters for both
R- and S-bupropion. CYP2B6.19 had the highest Vmax, which was about 2-fold greater than that of
CYP2B6.1 (for both S- and R-bupropion), but the Km was also higher (4-fold), so the net effect was a
lower in vitro intrinsic clearance (Clint) compared with CYP2B6.1. Similarly, CYP2B6.6 Vmax was also
greater than that of CYP2B6.1 for S-bupropion, but the Km was also higher, so Clint was lower than that of
CYP2B6.1. All variants had Clint values lower than that of CYP2B6.1, except CYP2B6.4 (R- and S-
bupropion) and CYP2B6.17 (R-bupropion). Based on intrinsic clearances, relative activities for S,S-
hydroxybupropion
formation
were
in
the
order
CYP2B6.4≥CYP2B6.1>CYP2B6.17>

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CYP2B6.5>CYP2B6.6≈CYP2B6.26≈CYP2B6.19>CYP2B6.7>CYP2B6.9>>CYP2B6.16≈CYP2B6.18,
and relative activities for R,R-hydroxybupropion formation were in the order
CYP2B6.17≈CYP2B6.4≈CYP2B6.1>CYP2B6.5≈CYP2B6.19≈CYP2B6.26>CYP2B6.6>CYP2B6.7≈
CYP2B6.9>>CYP2B6.16≈CYP2B6.18.
Bupropion hydroxylation by CYP2B6.1 is stereoselective, with S,S-hydroxybupropion formation
greater than that of R,R-hydroxybupropion. The ratios of Vmax(S,S-hydroxybupropion)/Vmax(R,R-
hydroxybupropion) and Clint(S,S-hydroxybupropion)/Clint(R,R-hydroxybupropion) for CYP2B6.1 were
2.9 and 1.8, respectively, consistent with previous report.(Coles and Kharasch, 2008) All CYP2B6
variants showed similar stereoselectivity, with the Vmax(S,S-hydroxybupropion)/Vmax(R,R-
hydroxybupropion) ratio 1.9 to 4.2. The Clint(S,S-hydroxybupropion)/Clint(R,R-hydroxybupropion) ratio
for most 2B6 variants was also similar to the wild type, 1.8 to 2.9, except that CYP2B6.17 (1.2) was
lower.
The activity of POR variants was determined with co-expressed wild-type CYP2B6 and
cytochrome b5. In contrast to CYP, POR variants caused minor, albeit statistically significant changes to
bupropion (2 µM) hydroxylation compared with wild type POR (Fig 3). Bupropion hydroxylation by
POR variants over the full substrate concentration range is shown in Figure 4 and Table 2. The kinetic
parameters for the three POR variants POR.5, POR.28 and PORP228L are similar to the wild type.

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Discussion
The first major observation was that CYP2B6 allelic variants have altered and diverse activities
towards racemic bupropion hydroxylation compared with wild type CYP2B6, for both R- and S-
bupropion. CYP2B6 variants selection was based on allele frequency and consequence. The 516G>T
(Q172H) and 785G>T (K262R) polymorphisms are common, and variants with these mutations (2B6*4,
*6, *7, *9, *16, *19, *26) were studied, even though some have minor population frequency. The other
three variants (*5, *17, *18) have relatively high allele frequency. At clinically relevant concentrations,
CYP2B6.5, CYP2B6.6, CYP2B6.7, CYP2B6.9, CYP2B6.19 and CYP2B6.26 were less active than wild
type CYP2B1.1. CYP2B6.4 had activity similar to CYP2B1.1, while Clint was slightly higher than wild
type for both enantiomers. CYP2B6.7 and CYP2B6.9 had the lowest activities of the enzymatically
active variants, with 20-35% of wild type Clint. CYP2B6.16 and CYP2B6.18 were essentially inactive.
Bupropion hydroxylation by CYP2B6 is stereoselective. Although Vmax and Km vary over a wide
range among the variants, stereoselectivity was retained in most variants, reflected by similar ratios of
Vmax(S,S-hydroxybupropion)/Vmax(R,R-hydroxybupropion) and Clint(S,S-hydroxybupropion)/Clint(R,R-
hydroxybupropion). CYP2B6.17 was the only variant with a moderate difference in enantioselectivity
compared with CYP2B6.1.
Only limited kinetic data are available on bupropion hydroxylation by CYP2B6 variants, and none
report on enantiomer metabolism. Table 3 summarizes the previously reported activity of expressed
CYP2B6 variants towards RS-bupropion hydroxylation (Zhang et al., 2011; Xu et al., 2012; Radloff et al.,
2013), and the present results. For CYP2B6.6, our results are comparable with previous studies, and
lower Clint for CYP2B6.6 is consistently reported. CYP2B6.6 had both increased Vmax (1.5-fold for S,S-
hydroxybupropion and 1.2-fold for R,R- hydroxybupropion) and increased Km (2.9-fold for S,S-
hydroxybupropion and 3.4-fold for R,R-hydroxybupropion) compared with CYP2B6.1. Zhang et al
reported that E. coli-expressed CYP2B6.6 had 1.8-fold greater kcat versus wild type, and 4-fold greater
Km, although the protein expression system was different than used herein (Zhang et al., 2011). In

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contrast, the one previous investigation of CYP2B6.4 reported substantially lower Clint while we found
normal to slightly greater activity (Zhang et al., 2011).
CYP2B6 is an unusual P450 isoform, in that activity of variants is clearly substrate-dependent,
and generalizations about catalytic consequence or clinical implications of CYP2B6 polymorphisms are
not possible. CYP2B6.4 (785G>T, K262R) had greater activity in metabolism of methadone (Gadel et al.,
2015), efavirenz (Wang et al., 2019), artemether (Honda et al., 2011), and selegiline (Watanabe et al.,
2010), and decreased activity with cyclophosphamide (Ariyoshi et al., 2011; Raccor et al., 2012),
ifosfamide (Zanger and Klein, 2013; Calinski et al., 2015), ketamine (Wang et al., 2018), and nicotine
(Bloom et al., 2019). Bupropion metabolism in the present study, using the CYP2B6.4 expression system
used to study many of these other substrates, was slightly (10%) but not substantially greater than
CYP2B6.1. CYP2B6.5 (1459C>T, R487C) was less active than CYP2B6.1 in metabolizing bupropion, as
well as methadone (Gadel et al., 2015), ketamine (Wang et al., 2018), cyclophosphamide (Raccor et al.,
2012), artmether (Honda et al., 2011) and nicotine (Bloom et al., 2019). It had moderately decreased
activity in S-efavirenz hydroxylation and slightly increased activity in R-efavirenz hydroxylation (Wang
et al., 2019). CYP2B6.9 (516G>T, Q172H) was substantially less active than CYP2B6.1 in bupropion
metabolism, as well as methadone, ketamine, efavarenz, and nicotine, and moderately less (30%) with
artemether, but more active than CYP2B6.1 in ifosfamide bioactivation (Honda et al., 2011; Calinski et
al., 2015; Gadel et al., 2015; Wang et al., 2018; Bloom et al., 2019; Wang et al., 2019). CYP2B6.6
(516G>T-785A>G, Q172H-K262R) has been studied extensively in vitro and in vivo due to its high
frequency of occurrence and therapeutic significance. CYP2B6.6 was less active than wild-type in
metabolism of bupropion, as well as methadone, ketamine, efavirenz, bupropion, ifosfamide, and
nicotine, but more active in metabolizing cyclophosphamide and artemether (Ariyoshi et al., 2011; Honda
et al., 2011; Calinski et al., 2015; Gadel et al., 2015; Wang et al., 2018; Bloom et al., 2019; Wang et al.,
2019). CYP2B6.16 and CYP2B6.18, which were essentially inactive towards buproiopion, are also
inactive with methadone, ketamine, and efavirenz (Gadel et al., 2015; Wang et al., 2018; Wang et al.,
2019). These two variants are expressed as apoproteins only, without detectable cofactor heme bound.

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The mutation shared in the two variants, 983T>C (I328T), is located in the J-helix, and the structural
change caused by this mutation is relayed to C- and I-helices, which are directly involved in ligand/heme
recognition (Kobayashi et al., 2014). As a result heme binding is disrupted, leading to abolition of
catalytic activity. The common polymorphisms of CYP2B6 include 516G>T, 785G>T, 983T>C and
1459C>T. Based on in vitro studies with substrates evaluated so far, 516G>T, 983T>C and 1459C>T can
generally be considered loss of function variants.
The second major observation in this investigation was that the three POR polymorphisms
evaluated had very minor influence on bupropion metabolism. POR transfers electrons from NADPH to
the P450 heme, an essential step in P450-catalysed metabolism. Influence of POR polymorphism on drug
metabolism is substrate- and P450 isoform-dependent. POR.5 (859G>C) had minimal or no effect on
bupropion metabolism, and 25% less activity in a previous bupropion study (Chen et al., 2012). In
contrast, POR.5 had substantially lower activity with CYP3A4 and testosterone, and with CYP17A1 and
androgen biosynthesis (Chen et al., 2012; Pandey and Sproll, 2014). POR.28 had a small (R-bupropion)
or no (S-bupropion) influence on CYP2B6.1-catalyzed bupropion metabolism.
There is high concordance between POR and CYP2B6 genetic variant effects on bupropion
metabolism in vitro and in vivo. POR*28 had little effect on bupropion hydroxylation in vitro, or in vivo,
based on plasma hydroxybupropion/bupropion area under the curve (AUC) ratios and urine
hydroxybupropion formation clearance (Gao et al., 2016; Lv et al., 2016; Kharasch and Crafford, 2019).
Accordingly, the present results would also suggest little influence of POR*5 and P228L polymorphisms
on bupropion hydroxylation in vivo. The influence of CYP2B6*6, which consistently results in
diminished bupropion hydroxylation in vitro (Table 3), is highly concordant in vivo. Plasma racemic
hydroxybupropion/bupropion AUC ratios after single-dose bupropion were lower in CYP2B6*6 carriers
than noncarriers (Chung et al., 2011), lower in CYP2B6*6/*6 and CYP2B6*1/*6 versus CYP2B6*1/*1
genotypes (Gao et al., 2016; Lv et al., 2016), and hydroxybupropion concentrations were lower in
CYP2B6*6 heterozygotes than wild-types (Ma et al., 2018). Assessed stereoselectively, plasma AUC
ratios and urine hydroxybupropion formation clearances for both bupropion enantiomers, after a single

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bupropion dose, were lower in CYP2B6*6/*6 but not CYP2B6*1/*6 compared with CYP2B6*1/*1
genotypes (Kharasch and Crafford, 2019). At steady-state, plasma racemic hydroxybupropion/bupropion
AUC ratios in CYP2B6 slow (*6/*6, *18/*18 genotypes) and intermediate (*6 and *18 heterozygotes)
metabolizers were 34% and 54%, respectively, of those in normal metabolizers (*1/*1, *1/*4, *1/*5,
*1/*22, *22/*22), and hydroxybupropion plasma concentrations were 40% and 20% lower in slow and
intermediate than normal metabolizers (Zhu et al., 2012). Steady-state ratios were also lower in
CYP2B6*6 carriers than wild types (Benowitz et al., 2013). Steady-state ratios were also lower than wild-
types in CYP2B6*6/*6 and CYP2B6*1/*6 genotypes, and plasma hydroxybupropion concentrations were
less in CYP2B6*6/*6 but not CYP2B6*1/*6 genotypes (Høiseth et al., 2015). The influence of CYP2B6
polymorphisms on steady-state hydroxylation of bupropion enantiomers has not been reported.
CYP2B6*18 coded for markedly deficient activity in vitro, and at steady state,
hydroxybupropion/bupropion AUC ratios were lower in CYP2B6*18 carriers than wild types, and even
less than in CYP2B6*6 carriers (Benowitz et al., 2013). In vitro-in vivo concordance is less consistent for
CYP2B6*4 than for decreased metabolism polymorphisms, which may be influenced by the rarity of
CYP2B6*4. CYP2B6.4 had minimally increased bupropion hydroxylation in vitro, and numerically but
not significantly higher plasma hydroxybupropion/bupropion AUC ratios in CYP2B6*4 hererozygotes for
the racemate (Kirchheiner et al., 2003; Zhu et al., 2012; Ma et al., 2018; Kharasch and Crafford, 2019)
and enantiomers (Kharasch and Crafford, 2019). Based on these patterns, and the present in vitro results,
it could be expected that other CYP2B6 polymorphisms coding for diminished (*5, *6, *7, *9, *19, *26)
or defective (*16) in vitro bupropion hydroxylation might result in diminished in vivo bupropion
hydroxylation.
There are acknowledged potential limitations to this investigation. First, the purpose was only to
test singular CYP and POR variants, not combinations; hence CYP and POR variants coexpressed in
combination was not evaluated. Second, racemic bupropion was used as the substrate, rather than single
enantiomers, because bupropion is used clinically as a racemate and results were intended to more
accurately reflect and inform on clinical metabolism. Third, for the CYP variants, the CYP:POR ratio

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varied, ranging from 1:1.3 to 1:4.5. CYP and POR form a 1:1 complex, and if POR content equals or
exceeds CYP content all constructs should be fully catalytically competent and extra POR beyond 1:1
should not further increase activity. For the POR variants, the CYP:POR ratio exceeded 1, except for
POR*5 (1:0.6). While it is possible that POR.5 expression affected apparent activity, it was not different
than wild type for S-bupropion, and only marginally less with R-bupropion. In addition, there are several
types of CYP expression systems, including yeast, bacteria, mammalian, and insect cells, using transient
or stable transfection, with or without co-expressed POR and b5, and expression systems can influence
apparent catalytic activity (Wang et al., 2018). Yeast and bacteria are straightforward but require
exogenous POR and b5, and bacterial expression usually requires sequence modification of mammalian
CYPs. Mammalian systems (e.g. monkey kidney COS cells and human embryonic kidney HEK cells)
contain POR and b5, and allow easy CYP expression, protein expression and integrity can vary and thus
influence apparent catalytic activity. Baculovirus-mediated expression in insect cells is a mature
technology and allows simultaneous and flexible expression of individual genetic variants, using separate
or combination multi-protein virus cassettes.
The newly identified CYP2B6 genetic polymorphisms in bupropion hydroxylation may also
portend clinical consequence, because there are important relationships between CYP2B6 genetics and
clinical outcomes of bupropion pharmacotherapy. Bupropion hydroxylation is a bioactivation pathway,
and hydroxybupropion, specifically S,S-hydroxybupropion, is considered to contribute to bupropion anti-
smoking (Bondarev et al., 2003; Damaj et al., 2004; Lee et al., 2007; Zhu et al., 2012; Carroll et al., 2014;
Malcolm et al., 2015) and antidepressant effects (Carroll et al., 2014; Laib et al., 2014). Bupropion-
treated smokers who were CYP2B6 1459C>T carriers (*5, *7) reported greater cravings for cigarettes and
had higher relapse rates compared to non-carriers.(Lerman et al., 2002) Lower plasma racemic
hydroxybupropion concentrations, related to CYP2B6 slow and intermediate metabolizer status, were
associated with a lesser incidence of smoking abstinence (Zhu et al., 2012), although not all studies
agree.(Lee et al., 2007) Among depressed patients, lower plasma racemic hydroxybupropion
concentrations were associated with lesser improvement in depression (Laib et al., 2014). The incidence

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of other CYP2B6 polymorphisms coding for deficient (*5, *6, *7, *9, *19, *26) or defective (*16) in vitro
bupropion hydroxylation on clinical bupropion effectiveness is unknown.

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Acknowledgements
none

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Authorship Contributions
Participated in research design: Wang, Kharasch
Conducted experiments: Wang, Neiner
Contributed new reagents or analytic tools: none
Performed data analysis: Wang, Neiner, Kharasch
Wrote or contributed to the writing of the manuscript: Wang, Kharasch

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Footnotes
This work was supported by the National Institutes of Health [Grants R01-DA14211 and R01-
DA042985] and by the Washington University in St. Louis Department of Anesthesiology Russel B. and
Mary D. Shelden fund

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Figure Legends
Figure 1.
Metabolism of RS-bupropion catalyzed by coexpressed recombinant CYP2B6
variants/POR.1/b5 at concentration of 2 µM RS-bupropion (1 µM R- or S-bupropion). Results are the
mean ± 95% confidence interval of triplicate determinations. Asterisks denote rates significantly different
from the wild-type CYP2B6.1 (P<0.001). (A) R,R-hydroxybupropion formation from RS-bupropion. (B)
S,S-hydroxybupropion formation from RS-bupropion.
Figure 2. Hydroxybupropion formation from RS-bupropion catalyzed by co-expressed CYP2B6 variants,
wild-type P450 oxidoreductase and cytochrome b5. (●) S,S-hydroxybupropion from RS-bupropion, (○)
R,R-hydroxybupropion from RS-bupropion. Results are the mean ± 95% confidence interval of triplicate
determinations. Lines are predicted concentrations based on kinetic parameters obtained by nonlinear
regression analysis of measured concentrations
Figure 3. Metabolism of RS-bupropion catalyzed by coexpressed recombinant CYP2B6.1/POR
variants/b5 at concentration of 2 µM RS-bupropion (1 µM R- or S-bupropion). Results are the mean ±
95% confidence interval of triplicate determinations. Asterisks denote rates significantly different from
the wild-type POR (P<0.001 for R-bupropion and P=0.002 for S-bupropion). (A) R,R-hydroxybupropion
formation from RS-bupropion. (B) S,S-hydroxybupropion formation from RS-bupropion.
Figure 4. Hydroxybupropion formation from RS-bupropion catalyzed by co-expressed CYP2B6.1, P450
oxidoreductase variants and cytochrome b5. (●) S,S-hydroxybupropion from RS-bupropion, (○) R,R-
hydroxybupropion from R-bupropion. Results are the mean ± 95% confidence interval of triplicate
determinations. Lines are predicted concentrations based on kinetic parameters obtained by nonlinear
regression analysis of measured concentrations.

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Table 1. Kinetic parameters for (S,S)- and (R,R)-hydroxybupropion formation from RS-bupropion by CYP2B6
variants
CYP2B6
variant
(S,S)-hydroxybupropion formation from RS-
(R,R)-hydroxybupropion formation from RS-
bupropion
bupropion
a
a
Vmax
(pmol/min/pmol)
26.4 ± 0.5
23.5 ± 0.4
14.4 ± 0.2
39.0 ± 0.6
15.6 ± 0.2
20.7 ± 0.2
0.74 ± 0.03
22.3 ± 1.1
0.91 ± 0.03
49.2 ± 4.1
31.7 ± 2.1
Km
Clint
Vmax
Km
Clint
(µM)
(ml/min/nmol) (pmol/min/pmol)
(µM)
(ml/min/nmol)
0.79 (100)
0.86 (109)
0.42 (53)
CYP2B6.1
CYP2B6.4
CYP2B6.5
CYP2B6.6
CYP2B6.7
CYP2B6.9
CYP2B6.16b
CYP2B6.17
CYP2B6.18b
CYP2B6.19
CYP2B6.26
18.2 ± 1.5
14.5 ± 1.0
17.3 ± 0.8
53.0 ± 2.7
31.1 ± 1.5
53.3 ± 2.1
1.45 (100)
1.62 (112)
0.83 (57)
0.74 (51)
0.50 (34)
0.39 (27)
9.0 ± 0.1
8.0 ± 0.1
11.4 ± 0.8
9.3 ± 1.6
12.2 ± 0.7
39.3 ± 2.0
21.4 ± 1.5
37.7 ± 1.3
5.1 ± 0.1
10.8 ± 0.2
3.7 ± 0.1
0.27 (34)
0.17 (22)
6.0 ± 0.6
0.16 (20)
0.72 ± 0.02
11.8 ± 0.2
0.78 ± 0.01
16.2 ± 0.2
8.5 ± 0.1
21.3 ± 2.5
1.05 (72)
13.1 ± 0.8
0.90 (114)
75.6 ± 11.0
41.5 ± 5.3
0.65 (45)
0.76 (52)
45.4 ± 2.1
22.0 ± 1.4
0.36 (46)
0.39 (49)
aValues in brackets represent percentages of CYP2B6.1 Clint.
bFor CYP2B6.16 and CYP2B6.18, rates were measured at a fixed substrate concentration of 1000 µM RS-bupropion.

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Table 2. Kinetic parameters for (S,S)- and (R,R)-hydroxybupropion formation from RS-bupropion by POR
variants
POR variant
(S,S)-hydroxybupropion formation from RS-
(R,R)-hydroxybupropion formation from RS-
bupropion
bupropion
a
a
Vmax
(pmol/min/pmol)
26.4 ± 0.5
Km
Clint
Vmax
Km
Clint
(µM)
(ml/min/nmol) (pmol/min/pmol)
(µM)
(ml/min/nmol)
0.79 (100)
0.87 (110)
1.18 (149)
0.81 (103)
POR.1
POR.5
18.2 ± 1.5
15.5 ± 3.1
13.3 ± 1.1
18.8 ± 1.8
1.45 (100)
1.35 (93)
1.61 (111)
1.13 (78)
9.0 ± 0.1
9.1 ± 0.1
10.5 ± 0.1
10.5 ± 0.1
11.4 ± 0.8
10.5 ± 0.7
8.9 ± 0.5
13.0 ± 0.5
21.0 ± 1.6
POR.28
21.4 ± 0.7
POR P228L
21.3 ± 0.8
aValues in brackets represent percentages of CYP2B6.1 Clint.