Identification and Structural Characterization of Three New Metabolites of Bupropion in Humans

Article abstract of DOI:10.1021/acsmedchemlett.6b00189

Bupropion is a widely used antidepressant and the recommended CYP2B6 probe drug. However, current understanding of bupropion elimination pathways is limited. Bupropion has three active circulating metabolites, OH-bupropion, threohydrobupropion, and erythrohydrobupropion, but together with bupropion these metabolites and their conjugates in urine represent only 23% of the dose, and the majority of the elimination pathways of bupropion result in uncharacterized metabolites. The aim of this study was to determine the structures of the uncharacterized bupropion metabolites using human clinical samples and in vitro incubations. Three new metabolites, 4′-OH-bupropion, erythro-4′-OH-hydrobupropion, and threo-4′-OH-hydrobupropion, were detected in human liver microsome incubations and were isolated from human urine. The structures of the metabolites were confirmed via comparison of UV absorbance, NMR spectra, and mass spectral data to those of the synthesized standards. In total, these metabolites represented 24% of the drug related material excreted in urine.

Full text of DOI:10.1021/acsmedchemlett.6b00189

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Letter
Identification and Structural Characterization of
Three New Metabolites of Bupropion in Humans
Jennifer E Sager, John R. Choiniere, Justine Chang, Alyssa
Stephenson-Famy, Wendel L. Nelson, and Nina Isoherranen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00189 • Publication Date (Web): 17 Jun 2016
Downloaded from http://pubs.acs.org on June 20, 2016
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Identification and Structural Characterization of Three New Metab-
olites of Bupropion in Humans
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Jennifer E. Sager, John R. Choiniere, Justine Chang , Alyssa StephensonꢀFamy , Wendel L. Nelson,
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*
Nina Isoherranen
1
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Department of Pharmaceutics , Obstetrics and Gynecology and Medicinal Chemistry , University of Washington, Seattle,
Washington, USA
KEYWORDS: Bupropion, structural characterization, metabolites, aromatic hydroxylation.
ABSTRACT: Bupropion is a widely used antidepressant and the recommended CYP2B6 probe drug. However, current underꢀ
standing of bupropion elimination pathways is limited. Bupropion has three active circulating metabolites, OHꢀbupropion, threohyꢀ
drobupropion and erythrohydrobupropion, but together with bupropion these metabolites and their conjugates in urine represent
only 23% of the dose, and the majority of the elimination pathways of bupropion result in uncharacterized metabolites. The aim of
this study was to determine the structures of the uncharacterized bupropion metabolites using human clinical samples and in vitro
incubations. Three new metabolites, 4’ꢀOHꢀbupropion, erythroꢀ4’ꢀOHꢀhydrobupropion and threoꢀ4’ꢀOHꢀhydrobupropion were
detected in human liver microsome incubations and were isolated from human urine. The structures of the metabolites were
confirmed via comparison of UV absorbance, NMR spectra and mass spectral data to those of the synthesized standards. In total,
these metabolites represented 24% of the drug related material excreted in urine.
Bupropion is a norepinephrine and dopamine reuptake inꢀ
additional, yet unidentified metabolites of bupropion may also
have pharmacological activity and contribute to the effects of
bupropion in vivo. Such metabolites and novel metabolic
pathways may also contribute to the interindividual variability
in bupropion disposition, efficacy and toxicity. Thus better
characterization of the elimination pathways of bupropion is
necessary.
1
hibitor approved as an antidepressant and for smoking cessaꢀ
2
3
tion and weight loss therapy. It is also the preferred sensitive
4
in vivo CYP2B6 probe substrate recommended by the FDA.
Yet, despite its numerous clinical and research applications,
the factors contributing to the observed interꢀ and intraindividꢀ
ual variability in bupropion disposition and in its safety and
5–8
efficacy are poorly understood.
Current knowledge of bupropion metabolism is incomplete.
Following a single oral radiolabeled dose, only 23 % of the
dose was recovered in urine as bupropion, OHꢀbupropion,
erythrohydrobupropion, threohydrobupropion 17,1a8 nd their
conjugates (Nꢀ and Oꢀglucuronides and sulfates ) while 22
% of the dose was excreted in urine as mꢀchlorohippuric acid
1
1
and 36 % as uncharacterized polar metabolites. Possible
structures of some of the uncharacterized metabolites such as
aromatic hydroylation products or products of hydroxylation
of the methyl group (indicated by red arrows in Figure 1) have
been proposed based on mass spectral data from in vitro
Figure 1. Bupropion and its active metabolites. Red arrows indiꢀ
cate potential additional hydroxylation sites
18
incubations and urine samples from clinical subjects.
Furthermore, analysis of human urine samples revealed the
1
8,19,20
presence of a reduced and hydroxylated metabolite.
In vivo, bupropion undergoes extensive metabolism, with <1%
of the dose excreted unchanged in urine after a single oral
However, the structures of these metabolites and their relative
importance in circulation or elimination of bupropion are
unknown. The goal of this study was to characterize the major
unknown metabolites of bupropion and generate standards of
these metabolites for quantitative characterization of bupropiꢀ
on elimination pathways. These metabolites can be evaluated
for their potential pharmacological and toxicological activity.
9–11
dose.
Much of the pharmacological activity and toxicity of
bupropion is attributed to its three known primary metabolites,
OHꢀbupropion,
hydrobupropion (Figure 1),
concentrations higher than bupropion. OHꢀbupropion appears
to be the main contributor to the efficacy of bupropion in
smoking cessation, while both OHꢀbupropion and
threohydrobupropion
and
erythroꢀ
1
2,13
which circulate at
1
In order to identify the primary bupropion metabolites
formed in vitro, bupropion was incubated with human liver
microsomes (HLM) and metabolite formation was measured
using multiple reaction monitoring (MRM) (See Supporting
Information for experimental procedures). Upon incubation of
threohydrobupropion are suggested to be primarily responsible
12,14,15
for the antiꢀdepressant activity.
All three metabolites
11
also have lower LD50 values than bupropion and cause
1
6
increased convulsion risk in mice. Thus, it is possible that
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bupropion with HLM, NADPH dependent formation of six To determine whether M1ꢀM3 are present in vivo, urine and
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metabolites was observed (Figure 2). In addition to OHꢀ
bupropion (Figure 2a), erythrohydrobupropion and threohyꢀ
drobupropion (Figure 2b), three additional monohydroxylation
products (M1ꢀM3) were detected with a parent (M+H) mass of
m/z 256 and a main MRM transition of m/z 256→ 182 (Figure
plasma were obtained from 5 subjects on bupropion therapy
and analyzed for M1ꢀM3 using MRM of m/z 256→ 182.
Urine was analyzed before and after acid deconjugation since
sulfate and glucuronide conjugates of bupropion metabolites
have been reported. Metabolite M1 was detectable in plasma
and urine from all 5 subjects while M2 was not detected in any
of the samples (Supplemental Figure S2). Metabolite M3 was
detected in the urine of 3 subjects prior to acid deconjugation,
and in the acid deconjugated urine of all 5 subjects, but at
much lower abundance than M1. Acid deconjugation markedꢀ
ly increased M1 and M3 ion abundance (Figure 3a), suggestꢀ
ing an important role for conjugation in the elimination of
these metabolites.
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2
c). On the basis of ion abundance, M1 and M2 appeared to
have similar importance in bupropion metabolism, while M3
formation appeared minor. All three metabolites had similar
fragmentation pathways (Figures 2c and d). The main fragꢀ
mentation of loss of 56 Da indicates a loss of the t-butyl group
as isobutylene corresponding to hydroxylation outside of the tꢀ
butyl group. However, the fragmentation patterns did not
allow for the determination of the exact hydroxylation site.
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Figure 3. (a) A representative MRM chromatogram of m/z
transition 256→182 from urine before or after acid deconjugaꢀ
tion. (b) A representative MRM chromatogram of m/z transiꢀ
tion 256→182 with an MS/MS spectrum of m/z 256 and (c)
and NMR spectrum of M1 isolated from urine. (d) Structure of
M1 (4’ꢀOHꢀbupropion) and (e) UV spectrum for M1 isolated
from acid deconjugated urine.
Figure 2. Characterization of metabolites formed from buꢀ
propion in HLM incubations. (Representative MRM chromaꢀ
tograms of m/z 256→238 (a), m/z 242→168 (b) and m/z
256→182 (c) depicting NADPHꢀdependent formation of OHꢀ
bupropion (a), erythroꢀ (E) and threoꢀ (T) hydrobupropion (b)
and M1, M2 and M3 (c) and the MS/MS spectra of m/z 256 (a
and c) and m/z 242 (b) for the detected metabolites. (d) Proꢀ
posed fragmentation pathway of M1ꢀM3.
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Figure 4. Characterization of metabolites formed in erythrohydrobupropion and threohydrobupropion incubations. Representative
MRM chromatogram of m/z transition 258→184 depicting NADPHꢀdependent formation of M4 and M5 from erythrohydrobuꢀ
propion (a) and M6 and M7 from threohydrobupropion (b) in HLM. The insets show the MS/MS spectra of m/z 258 for M4 and M5
(a) and M6 and M7 (b). (c) A representative MRM chromatogram of m/z transition 267→184 depicting NADPHꢀdependent forꢀ
mation of M6ꢀd and M7ꢀd following threohydrobupropionꢀd incubation in HLM and MS/MS spectra of m/z 267 for M6ꢀd and
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M7ꢀd . (d) Structures of threohydrobupropionꢀd and its potential hydroxylation products. (e) Proposed fragmentation pathways of
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M4ꢀM7.
Based on ion abundance, M1 appeared to be the predominant
unknown hydroxylation product of bupropion in vitro and in
vivo. To determine the structure of M1, it was extracted from
acid deconjugated urine and purified using preparative chroꢀ
matography. A sufficient quantity of M1 was isolated to allow
structural characterization via NMR, UVꢀVis and MS/MS
spectroscopy (Figure 3bꢀe). The presence of only 3 protons in
the aromatic region of the NMR spectrum suggested that M1
was a product of aromatic hydroxylation (Figure 3c). The
hydroxylation site was assigned to the 4’ position based on
NMR chemical shifts and coupling constants (Figure 3c and
supporting information). Reference standard of 4’ꢀOHꢀ
bupropion was then synthesized and the structure of M1 conꢀ
firmed by comparison of the LC retention time (6.42 min), and
MS/MS, NMR and UV (λmax 350 nm) spectra between the
isolated metabolite and the synthesized compound (Figure 3
and Supplemental Figures S3 and S4).
ylated metabolites from both erythrohydrobupropion (M4 and
M5) and threohydrobupropion (M6 and M7) was detected with
an M+1 ion of m/z 258 and MRM transition of 258→184,
following incubation of erythroꢀ and threohydrobupropion in
HLM (Figure 4). No NADPH dependent formation of metabꢀ
olites with m/z transitions of 258→184 was observed in incuꢀ
bations of threohydrobupropion, erythrohydrobupropion, OHꢀ
bupropion or 4’ꢀOHꢀbupropion in cytosol or following OHꢀ
bupropion incubation in HLM (Supplemental figure S5).
Based on ion abundance, metabolites M4 and M6 were the
major hydroxylation products of erythroꢀ and threohydrobuꢀ
propion, respectively, while M5 and M7 appeared to be minor
metabolites. Chromatograms and representative spectra for
M4ꢀM7 are shown in Figure 4. While the [M+H] m/z 258 ion
of all four metabolites (M4ꢀM8) suggested that these are hyꢀ
droxylated metabolites of threoꢀ and erythrohydrobupropion
([M+H] 242), the MS/MS fragmentation patterns (Figure 4a
and b) did not allow determination of hydroxylation sites.
Due to the hydroxylation, these metabolites lack the loss of 56
Da alone and loose H O or C H + H O, as shown in the proꢀ
Previous studies of bupropion metabolism have reported the
presence of reduced and hydroxylated bupropion metabolites,
yet the precursor(s) for the metabolite(s) have not been identiꢀ
fied. To characterize the sequential metabolism, OHꢀ
bupropion, threohydrobupropion erythrohydrobupropion and
4’ꢀOHꢀbupropion were incubated in HLM or human liver cyꢀ
tosol to determine which substrate(s) and subcellular fractions
are involved in the formation of the reduced and hydroxylated
metabolite(s). NADPH dependent formation of two hydroxꢀ
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posed fragmentation pathway in Figure 4e. To identify the site
of hydroxylation in these compounds, threohydrobupropionꢀ
d9, containing deuteriums in the tꢀbutyl group, was incubated
in HLM to determine whether M6 and M7 were hydroxylated
18
in the tꢀbutyl group, as was previously proposed. Depending
on the hydroxylation site, the products were expected to differ
by one Da in the M+H ion, as shown in Figure 4d, allowing
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for detection of tꢀbutyl hydroxylation. Parent masses of m/z ucts of aromatic ring hydroxylation. The hydroxylation site
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267 for both M6 and M7 suggested no loss of tꢀbutyl deuteriꢀ
ums (Figure 4c), confirming that the hydroxylation was either
in the aromatic ring or in the methyl group (Figure 4d).
was proposed to be at the 4’ꢀposition based on NMR chemical
shifts and coupling contants. A reference standard was synꢀ
thesized (Supplemental information) and the structures of M4
(erythroꢀ4’ꢀOHꢀhydrobupropion) and M6 (threoꢀ4’ꢀOHꢀ
hydrobupropion) were confirmed by comparison of LC retenꢀ
tion times, NMR, UV and MS/MS spectra between the isolatꢀ
ed and synthesized compounds (Figure 5, Supplemental Figꢀ
ures S7 and S8).
To establish the relative importance of the new metabolites
in vivo, their concentrations were quantified in plasma and
urine. The doseꢀnormalized concentrations of 4’ꢀOHꢀ
bupropion (M1), threoꢀ4’ꢀOHꢀhydrobupropion (M4) and
erythroꢀ4’ꢀOHꢀhydrobupropion (M6) in plasma were low, 12
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6 nM for 4’ꢀOHꢀbupropion, 7 ± 1 nM for threoꢀ4’ꢀOHꢀ
hydrobupropion (M4) and 33 ± 20 nM for erythroꢀ4’ꢀOHꢀ
hydrobupropion (M6). Concentrations of all three compounds
were below the lower limit of quantification (3.3 nM) in one
subject. On average, 4’ꢀOHꢀbupropion (M1) concentrations
were less than 0.01 % of those of bupropion. Circulating conꢀ
centrations of threoꢀ4’ꢀOHꢀhydrobupropion (M4) and erythroꢀ
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’ꢀOHꢀhydrobupropion (M6) were 1.8 and 2.2 % of the averꢀ
age concentration of bupropion at steady state. Metabolite
safety is typically considered to be a concern when steady
state concentrations are greater than 10% of the total drug
21
related material.
Thus, it is likely unnecessary to screen
these metabolites for toxicity. However, despite the low circuꢀ
lating concentrations of these compounds, they were abundant
in urine. The overall recovery of bupropion metabolites is
shown in Table 1. On average, 39 % of the total oral dose was
recovered in urine over the dosing interval. The amount of
threohydrobupropion and its metabolites represented 26 % of
the dose, while only 5 % and 6 % of the dose was excreted as
OHꢀbupropion and erythrohydrobupropion or their metaboꢀ
lites.
On
average,
4’ꢀOHꢀbupropion,
threoꢀ4’ꢀOHꢀ
hydrobupropion and erythroꢀ4’ꢀOHꢀhydrobupropion and their
conjugates accounted for 0.4 %, 14 % and 11 % of the total
oral bupropion dose excreted in urine, respectively (Table 1).
Based on the amounts of threoꢀ and erythrohydrobupropion
excreted unchanged, as conjugates and as 4’ꢀhydroxylation
products, 4’hydroxylation accounts for about 70 % and 20 %
of the elimination of erythroꢀ and threohydrobupropion, reꢀ
spectively, and changes in the formation of the 4’OHꢀ metaboꢀ
lites could have a substantial impact on the circulating levels
of threoꢀ and erythrohydrobupropion. To identify the enzymes
responsible for the 4’ꢀhydroxylation of bupropion, threohyꢀ
drobupropion and erythrohydrobupropion each of the three
compounds were incubated with a panel of recombinant CYP
enzymes (see supplemental information for experimental deꢀ
tails). Of the recombinant CYP enzymes only CYP2C19
formed 4’ꢀOHꢀbupropion from bupropion and threoꢀ4’ꢀOHꢀ
hydrobupropion and erythroꢀ4’ꢀOHꢀhydrobupropion from
threo and erythrohydrobupropion, respectively.
Figure 5. (a) A representative MRM chromatogram of m/z
transition 258→184 from urine before or after acid deconjugaꢀ
tion. (b) A representative MRM chromatogram of m/z transiꢀ
tion 258→184 with the MS/MS spectra of M4 and M6 for m/z
2
58 and (c) NMR spectrum of M4+M6 isolated from urine. (d)
Structure of threo/erythroꢀ4’ꢀOHꢀhydrobupropion (M4 and
M6) and (e) UV spectrum for M4+M6 isolated from acid deꢀ
conjugated urine.
Metabolites M4, M6 and M7 were all detectable in plasma and
urine from 5 subjects (Figure 5 and Supplemental Figure S6).
Acid deconjugation resulted in substantially greater ion abunꢀ
dance for M4, M6 and M7 than in untreated urine (Figure 5).
Metabolite M5 was only detectable in urine following acid
deconjugation (Figure 5a, Supplemental Figure S6). Based on
the ion abundances from incubations and deconjugated urine,
M4 and M6 were considered to be major contributors to the
elimination of threo and erythrohydrobupropion. The metaboꢀ
lites M4 and M6 were isolated and purified from deconjugated
urine as a mixture of the erythro and threo products (Figure
5
b) to allow for characterization via NMR, UVꢀVis, and
MS/MS. The presence of three protons in the aromatic region
of the NMR spectrum indicated that M4 and M6 were prodꢀ
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Supplemental information (pdf)
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Table 1. The percent of the oral bupropion dose recovered in
urine as bupropion and its metabolites over a steady state dosꢀ
ing interval
AUTHOR INFORMATION
Corresponding Author
*
Nina Isoherranen, Eꢀmail: ni2@uw.edu, Phone: 206ꢀ543ꢀ3204.
Total as free
+
conjugated
Free
(µmole)
Conjugated
(µmole)
Author Contributions
*
*
Compound
Bupropion
(% of dose)
1.4 ± 1.1
5.0± 3.2
The manuscript was written through contributions of all authors.
All authors participated in research design. J.E.S, J.R.C. and N.I.
performed experiments, J.E.S, J.R.C., N.I. and W.L.N. performed
data analysis and wrote or contributed to the writing of the manuꢀ
script.
59 ± 71
77 ± 69
6.5 ± 7.3
110 ± 63
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OHꢀbupropion
Threoꢀ
hydrobupropion
Erythroꢀ
hydrobupropion
21±14
1.9±1
610 ± 550
63 ± 65
250 ± 140
22 ±12
Funding Sources
This work was funded by the National Institutes of Health grants
P01 DA032507 and T32 GM007750 (J.E.S).
4
’ꢀOHꢀ
bupropion
Threoꢀ4’ꢀOHꢀ
hydrobupropion
Erythroꢀ4’ꢀOHꢀ
hydrobupropion
mꢀchloroꢀ
0.8±1.0
5.3±3.5
4.1±2.7
0.3 ± 0.2
0.9 ± 0.9
3.3 ± 3.2
29 ± 36
ACKNOWLEDGMENT
The authors would like to thank Dr. Libin Xu for generously
providing access to the speed vac in his laboratory. Additionally,
the authors thank Dr. Venkata Narayana Vidala for his assistance
collecting NMR spectra.
200 ± 140
160 ± 100
hippuric acid
3.0 ±0.7
39±15
167 ±130
980 ± 850
ꢀ
ABBREVIATIONS
Total
770 ± 220
*
The molar amounts are normalized to the administered dose
in each subject.
HLM, Human liver microsomes. MRM, multiple reaction monitorꢀ
ing.
In conclusion, we report the identification and characterization of
three new metabolites of bupropion, the 4’OHꢀbupropion, erythroꢀ
REFERENCES
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4
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2
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS
Publications website. The supporting information includes full deꢀ
scriptions of all experimental procedures, analytical assays, comꢀ
pound synthesis and characterization, and supporting results and
NMR spectra.
1
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HO
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4’-hydroxybupropion; M1
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Bupropion
Hydroxybupropion
Cl
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OH
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Threo/erythrohydrobupropion 4’-hydroxythreo/erythrohydrobupropion; M4, M6
Cl