stereoselective glucuronidation of bupropion metabolites in vitro and in...

DMD/2015/068908

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Stereoselective Glucuronidation of Bupropion Metabolites in vitro and in vivo.

Brandon T. Gufford, Jessica Bo Li Lu, Ingrid F. Metzger, David R. Jones, and

Zeruesenay Desta

Department of Medicine, Division of Clinical Pharmacology Indiana University School of

Medicine, Indianapolis, Indiana

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on January 22, 2016 as DOI: 10.1124/dmd.115.068908

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Running Title: Stereoselective bupropion glucuronidation

Corresponding Author: Zeruesenay Desta, PhD

Department of Medicine, Division of Clinical Pharmacology

Indiana University School of Medicine

950 W. Walnut Street, R2, Room 425

Indianapolis, IN 46202-5188

Phone: (317) 274-2823

Fax: (317) 278-2765

Email: [email protected]

Number of text pages: 20

Number of tables: 4

Number of figures: 7

Number of references: 51

Word Count

Total: 4,316

Abstract: 249

Introduction: 747

Discussion: 1,139

Abbreviations: CYP, cytochrome P450; HLM, human liver microsome; UDPGA, UDP-

glucuronic acid; UGT, UDP-glucuronosyl transferase; rUGT, recombinant UGT


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Abstract

Bupropion is a widely used antidepressant and smoking cessation aid in addition to

being one of two FDA recommended probe substrates for evaluation of cytochrome

P450 2B6 activity. Racemic bupropion undergoes oxidative and reductive metabolism

producing a complex profile of pharmacologically active metabolites with relatively little

known about the mechanisms underlying their elimination. An LC-MS/MS assay was

developed to simultaneously separate and detect glucuronide metabolites of (R,R)- and

(S,S)-hydroxybupropion, (R,R)- and (S,S)-hydrobupropion (threo), and (S,R)- and (R,S)-

hydrobupropion (erythro) in human urine and liver subcellular fractions to begin

exploring mechanisms underlying enantioselective metabolism and elimination of

bupropion metabolites. Human liver microsomal data revealed marked glucuronidation

stereoselectivity [Clint, 11.4 versus 4.3 µL/min/mg for the formation of (R,R)- and (S,S)-

hydroxybupropion glucuronide; and Clmax, 7.7 versus 1.1 µL/min/mg for the formation of

(R,R)- and (S,S)-hydrobupropion glucuronide] in concurrence with observed

enantioselective urinary elimination of bupropion glucuronide conjugates. Approximately

10% of the administered bupropion dose was recovered in the urine as metabolites with

glucuronide metabolites accounting for approximately 40, 15, and 7% of total excreted

hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively.

Elimination pathways were further characterized using an expressed UGT panel with

bupropion enantiomers (both individual and racemic) as substrates. UGT2B7 catalyzed

the stereoselective formation of glucuronides of hydroxybupropion, (S,S)-

hydrobupropion, (S,R)- and (R,S)-hydrobupropion while UGT1A9 catalyzed the

formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the


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metabolic pathways underlying bupropion metabolite disposition and significantly

expand our knowledge of potential contributors to the inter- and intra-individual

variability in therapeutic and toxic effects of bupropion in humans.


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Introduction

Bupropion [(±)-1-(3-chlorophenyl)-2-[(1,1-dimethylethyl) amino]-1-propanone] is

widely used for the treatment of depression, seasonal affective disorder, as a smoking

cessation aid (Dwoskin et al., 2006; Dhillon et al., 2008), and recently co-formulated

with naltrexone for weight management in obese patients (Yanovski and Yanovski,

2015). However, bupropion antidepressant (Thase et al., 2005) and smoking cessation

(Hurt et al., 1997; Jorenby et al., 1999; Dale et al., 2001) treatment outcomes vary

widely among patients. The use of immediate release bupropion is also associated with

dose-dependent adverse effects, including seizures (Davidson, 1989).

Mechanisms contributing to the variable clinical response and adverse effects of

bupropion are not fully known. Alterations in metabolism of bupropion and its primary

metabolites have been posited to contribute to this variability. Early animal studies

suggested that bupropion is a more effective antidepressant in mice than in rats (Soroko

et al., 1977; Ferris et al., 1983) and hydroxybupropion plasma exposure was much

higher in mice than in rats (Welch et al., 1987). Thus, differences in the formation of

potentially active metabolites may explain species differences in bupropion effect. In

humans, bupropion undergoes extensive hepatic metabolism via oxidation of the t-butyl

moiety to hydroxybupropion and reduction of the aminoketone group to two amino

alcohols (erythro- and threo-hydrobupropion), with <1% of the administered dose

excreted unchanged in urine (Findlay et al., 1981; Lai and Schroeder, 1983; Schroeder,

1983; Laizure et al., 1985; Posner et al., 1985; Welch et al., 1987). Several preclinical

studies in addition to human data provided compelling evidence that bupropion

metabolites are pharmacologically active (Perumal et al., 1986; Golden et al., 1988;


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Bondarev et al., 2003; Damaj et al., 2004; Silverstone et al., 2008; Zhu et al., 2012).

Bupropion has also been reported as a clinically relevant inhibitor of CYP2D6 with in

vitro evidence suggesting that bupropion metabolites (rather than parent drug) mediated

this interaction (Reese et al., 2008). Consideration of metabolite pharmacokinetics

further reinforces their potential contribution to overall bupropion effect. Steady-state

plasma exposures of hydroxybupropion and threo-hydrobupropion following bupropion

administration are 17- and 7-fold higher, respectively, than the parent drug (Laizure et

al., 1985; Posner et al., 1985; Benowitz et al., 2013) and metabolites accumulate

substantially in plasma and CSF during repeated administration. Therefore, it is

important to understand factors that contribute to differential tissue and plasma

exposure of active bupropion metabolites.

Of the bupropion metabolites so far described, hydroxybupropion has been

studied in detail. Bupropion has one chiral center and is administered clinically as a

50:50 racemic mixture. Hydroxylation of racemic bupropion to hydroxybupropion is

exclusively catalyzed by cytochrome P450 2B6 (CYP2B6) (Faucette et al., 2000; Hesse

et al., 2000). This reaction is one of two FDA recommended in vitro and in vivo probes

of CYP2B6 (FDA CDER, 2012). Elevated hydroxybupropion plasma exposure is

associated with improved bupropion treatment outcomes in depression (Laib et al.,

2014) and smoking cessation (Zhu et al., 2012). However, hydroxybupropion plasma

exposure varied widely among patients and is only partially explained by CYP2B6

genetic variation (Benowitz et al., 2013). Bupropion hydroxylation creates an additional

chiral center, and two diastereomers [(2R,3R)- and (2S,3S)-hydroxybupropion] have

been quantified in human plasma (Suckow et al., 1997; Coles and Kharasch, 2007; Xu


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et al., 2007). While plasma exposure of (2R,3R)-hydroxybupropion is >20-fold higher

than (2S,3S)-hydroxybupropion (Kharasch et al., 2008), some pharmacological activity

of bupropion may reside in (2S,3S)-hydroxybupropion (Bondarev et al., 2003; Damaj et

al., 2004; Hansard et al., 2011). The marked stereoselectivity in hydroxybupropion

disposition observed in vivo is not fully explained by CYP2B6 as the rate of (S)-

bupropion hydroxylation in expressed CYP2B6 and human liver microsomes is only 3-

and 1.5-fold higher, respectively, than (R)-bupropion (Coles and Kharasch, 2008).

Mechanisms other than CYP2B6-mediated formation (e.g. further elimination

processes) may account for observed stereoselective disposition.

In vitro (Skarydova et al., 2014; Connarn et al., 2015) and in vivo (Benowitz et al.,

2013) studies suggest that bupropion reduction to form two hydrobupropion metabolites

is the major mechanism of bupropion clearance. Reduction of the keto group by 11β-

hydroxysteroid dehydrogenase type 1 and other carbonyl reductases (Molnari and

Myers, 2012; Meyer et al., 2013; Skarydova et al., 2014; Connarn et al., 2015) appears

to favor formation of threo-hydrobupropion (Benowitz et al., 2013). Although, this

reduction creates an additional chiral center, potentially generating two distinct threo-

hydrobupropion and two erythro-hydrobupropion diastereomers, data describing

stereoselective elimination pathways and effect are lacking.

The main objective of the present study was to explore the mechanisms

underlying enantioselective metabolism of bupropion metabolites by investigating the

UGT-mediated metabolism of hydroxybupropion and threo- and erythro-

hydrobupropion.


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Materials and Methods

Chemicals

All chemicals and solvents were HPLC grade or higher. Acetonitrile (ACN),

methanol (MeOH) and acetic acid were purchased from Fisher Scientific Company

LLC.(Hanover Park, IL). Lab water was prepared for LC-MS/MS applications using a

Nanopure Infinity® UV system (Barnsteas/Thermolyne, Dubuque, IA). UDPGA,

alamethicin, Trizma® base and magnesium chloride (MgCl2) were purchased from

Sigma-Aldrich (St. Louis, MO). Internal standard, nevirapine, was supplied through the

National Institutes of Health AIDS Research and Reference Reagent Program

(Germantown, MD). Bupropion, R-Bupropion, S-bupropion, (2R,3R)-hydroxybupropion

(R,R-hydroxybupropion), (2S,3S)-hydroxybupropion (S,S-hydroxybupropion), racemic

erythro-hydrobupropion, racemic threo-hydrobupropion, (S,S)- and (R,R)-

hydrobupropion β-D-glucuronides (threo-hydrobupropion glucuronides) and racemic

erythro-hydrobupropion β-D-glucuronide (EGLUC1 and EGLUC2) were purchased

from Toronto Research Chemicals (Toronto, Ontario).

Microsomal preparations

Mixed gender pooled human liver microsomes (HLM) (20mg/ml) were purchased

from Corning (Woburn, MA) and recombinant UGTs (rUGT) (UGT1A1, UGT1A3,

UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7,

UGT2B10, UGT2B15, and UGT2B17) (5mg/ml) were purchased from Discovery

Labware, Inc. (Woburn, MA). All microsomal preparations were stored at -80°C until

analysis.

LC-MS/MS method development


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LC-MS/MS analytical method development to separate and detect bupropion,

hydroxybupropion, and threo- and erythro-hydrobupropion was performed using an API

3200 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA)

equipped with an electrospray ionization (ESI) source and coupled to an HPLC system

consisting of two LC-20AD pumps, SIL-20AHT UFLC autosampler, DGU-20A3

degasser and a CBM-20A system controller (Shimadzu, Columbia, MD). Data

acquisition and processing were performed using Analyst® software (version 1.5.1, AB

SCIEX™, Ontario, Canada). Analyte concentrations were quantified using Analyst®

software by interpolation from matrix-matched calibration curves and quality controls

with dynamic assay ranges of 0.1-1000 ng/mL for all analytes. The calibration standards

and quality controls were judged for batch quality based on the 2013 FDA guidance for

industry regarding bioanalytical method validation (Food and Drug Administration

Center for Drug Evaluation and Research, 2013). Chromatographic separation was

achieved using a Luna C18-2 column (150 x 4.6 mm i.d.; 5 µm particle size;

Phenomenex, Torrance, CA) and mobile phase consisting of methanol (mobile phase B)

and water containing 0.1% acetic acid (mobile phase A) using the following gradient:

initial conditions of 50% mobile phase B followed by a linear gradient to 90% mobile

phase B between 0.01 and 16 min, then re-equilibrated to initial conditions between

16.01 min and 20 min using a total flow rate of 0.8 mL/min.

Initially, no glucuronide standards were commercially available so urine collected

from healthy volunteer(s) following the administration of a single 100 mg oral tablet of

racemic bupropion hydrochloride (Sandoz Inc., Princeton, NJ) was used for FIA (flow

injection analysis) to obtain optimal instrument and compound dependent parameters


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for the assay. Healthy volunteers were enrolled into a trial at the Indiana University

School of Medicine Clinical Research Center. The Indiana University School of

Medicine Institutional Review Board (IRB) approved the study protocol and all

participants signed an IRB-approved informed consent prior to enrollment. The trial is

registered at the ClinicalTrials.gov (Identified # NCT02401256). The obtained urine

sample was centrifuged at 12,000 rpm for 10 mins prior to injection of resulting

supernatant. Initial parameters for FIA were set according to parent drug FIA results. All

data were collected in positive ion mode. As some authentic standards [(S,S)- and

(R,R)-hydrobupropion (threo-hydrobupropion) β-D-glucuronides and racemic erythro-

hydrobupropion β-D-glucuronide] were later commercially available, FIA was

repeated for these glucuronides following acquisition. Matrix-matched calibration curves

were generated to directly quantify the glucuronide metabolites of threo- and erythro-

hydrobupropion using the available glucuronide standards with a dynamic assay range

of 0.5-1000 ng/mL. (R,R)- and (S,S)-hydroxybupropion glucuronides were quantified

using the standard curves of threo-hydrobupropion glucuronides with the same isomeric

configuration as glucuronide standards were not commercially available for these

metabolites. The possibility that ionization efficiency and other system parameters may

yield differing LC-MS/MS response between these structurally related metabolites could

not be excluded. As such, the reported nmol formation and excretion rates of (R,R)- and

(S,S)-hydroxybupropion glucuronides could be viewed more appropriately as nmol

equivalents relative to threo-hydrobupropion glucuronides with the same isomeric

configuration.

Urine sample analysis


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Urine samples were collected during 4 intervals (0-12, 12-24, 24-36, and 36-48

hours) over a 48 hour period following bupropion administration to healthy volunteers

(n=10). An aliquot of each urine sample (50 μL) was diluted with water:methanol (1:1) to

100 µL, 25 µL of 250 ng/ml nevirapine was added as internal standard, the sample was

centrifuged (12,000 rpm x 10 mins), and the resulting supernatant (10μl) was injected

into the LC-MS/MS system.

Optimization of microsomal incubation conditions

Pilot incubation experiments were performed using HLMs to identify potential

glucuronide metabolites of hydroxybupropion and to optimize incubation and LC-MS/MS

analysis conditions. Racemic hydroxybupropion was dissolved and serially diluted in

methanol to the required concentration. Methanol was removed by drying in a speed

vacuum before reconstituting with HLMs. HLMs were diluted with Tris HCl buffer (pH

7.4, 100 mM) containing MgCl2 (5 mM) and activated by addition of alamethicin (50

µg/mg protein) on ice for 15 min with gentle agitation every five minutes. For the pilot

studies, hydroxybupropion (100µM) and 25 µL of activated HLMs (desired

concentrations) were diluted in 125 µL Tris HCl buffer and pre-incubated for 5 min at

37°C. The reaction was initiated by adding 100 µL of UDPGA (5 mM dissolved in buffer)

to yield a final reaction volume of 250µL. The reaction was terminated by addition of 100

µL of ACN at pre-determined time intervals. Nevirapine (25 µl of 500ng/mL in methanol)

was added as an internal standard to the incubation samples prior to analysis. The

samples were vortexed vigorously for 20s and left on ice for 15 min. Samples were then

evaporated for 10 min followed by centrifugation at 12,000 rpm for 5 min in an

Eppendorf model 5415C centrifuge (Brinkman Instruments, Westbury, NY). The


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resulting supernatant (10 µL) was injected onto the LC-MS/MS system.

Chromatographic separation of the bupropion glucuronide metabolites was achieved on

a Luna C18-2 column (150 × 4.6 mm i.d.; 5µm particle size; Phenomenex, Torrance,

CA) using a mobile phase consisting of methanol (mobile phase B) and water

containing 0.1% acetic acid (mobile phase A) using the following gradient: initial

conditions of 2% mobile phase B followed by a linear gradient to 90% mobile phase B

between 0.01 and 10 min, then re-equilibrated to initial conditions between 10.01 min

and 14 min using a total flow rate of 0.8 mL/min.

Using the incubation and LC-MS/MS assay conditions described above, linearity

in the formation rate of the observed hydroxybupropion glucuronides was established

with regard to microsomal protein concentration and incubation time. hydroxybupropion

(100uM) was incubated in HLMs (0.0-1.0mg protein/mL) in the presence of the UDPGA

(5 mM) at 37°C across a range of incubation times (0-90min). Optimal incubation

conditions were determined to be 60 min incubation time using 1 mg/mL microsomal

protein.

Kinetic Analysis.

An abbreviated substrate concentration range was used to obtain initial kinetic

parameter estimates using Phoenix® WinNonlin® with simulations to guide final

concentration selection. Rates of hydroxybupropion, threo-hydrobupropion and erythro-

hydrobupropion glucuronide formation were determined by incubating a range of

substrate concentrations (25-1000 μM) encompassing the predicted Km or S50.

Incubation conditions were optimized for linearity with respect to time and protein

concentration for each substrate as described above (data not shown).


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Reaction phenotyping using a recombinant UGT enzyme panel

Hydroxybupropion, (R,R) and (S,S)-hydroxyburpopion, threo-hydrobupropion or

erythro-hydrobupropion (50 µM) were incubated with HLMs (1 mg/mL) or each

individual recombinant UGT enzyme (0.5mg/ml, UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8,

1A9, 1A10, 2B4, 2B7, 2B15, 2B17 and vehicle/vector control). Reactions were

terminated at 60 minutes by the addition of acetonitrile and analyzed via LC-MS/MS as

described above.

Data Analysis

Urinary excretion kinetics were analyzed both as absolute (ng) and molar (nmol)

amounts (based on individual urine collection volumes) of each metabolite in relation to

bupropion using Phoenix® WinNonlin® (version 6.4, Pharsight Corp., Cary, NC).

Apparent in vitro enzyme kinetic parameters, maximum formation rate (Vmax) and

substrate concentration resulting in 50% of Vmax (Km or S50), were obtained via nonlinear

regression by fitting the single site Michaelis-Menten (V=Vmax x [S]/Km+[S]) or Hill

(V=Vmax x [S]h/S50h+[S]h) equation (where h=hill coeffficient) to substrate concentration

versus apparent metabolite formation velocity data using Phoenix® WinNonlin®. Clint

(Vmax/Km) or Clmax (Vmax x (h-1)/Km + h(h-1)1/h) (Houston and Kenworthy, 2000) was

calculated for substrates described by the simple Michaelis-Menten or Hill equation,

respectively. Unless noted, data are presented as mean of duplicate incubations, with

error bars showing data variability for N = 2.


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Results

LC-MS/MS method development. The optimized instrument parameters were:

interface temperature at 500°C, nebulizer gas (nitrogen) (GS1) 60.0 psi, heater gas

(GS2) 55.0 psi, curtain gas (CUR) 25.0 psi, IonSpray Voltage (IS) 3500 V and collision

activated dissociation (CAD) 6.0 psi. The optimized compound dependent MS/MS

parameters are outlined in Table 1.

Chromatographic separation, detection and identification of bupropion

metabolites. Retention times and mass transitions of bupropion substrates and

metabolites are outlined in Table 2. Baseline chromatographic separation of individual

enantiomers of hydroxybupropion and threo- and erythro-hydrobupropion was not

achieved using the outlined chromatographic conditions (Fig 1, A-C). In contrast,

baseline separation and detection of the enantiomeric bupropion glucuronide

metabolites was achieved for the first time (Fig 1, D and E). The glucuronides of

hydroxybupropion (racemic or enantiomers) were not available commercially. Thus,

identification of the two urinary glucuronides of hydroxybupropion were carried out by

monitoring metabolites formed during incubation of racemic-, (S,S)-, and (R,R)-

hydroxybupropion with HLMs supplemented with UDPGA. In microsomal incubations

containing racemic hydroxybupropion, two glucuronide metabolites (Figure 2A) that had

the same retention times with those observed in urine (Figure 1D) were generated. The

glucuronide metabolite eluting first (RT 7.79 min) was exclusively formed in incubations

utilizing (S,S)-hydroxybupropion as the substrate (Figure 2C) while the later eluting

glucuronide (RT 8.09 min) was exclusively formed in incubations utilizing (R,R)-

hydroxybupropion as the substrate (Figure 2B). Peaks corresponding to these


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glucuronide metabolites were not observed in the negative control incubations (no

cofactor, no substrate, or no incubation). Although no hydroxybupropion glucuronides

(racemic or enantiomers) were available to us commercially to confirm the specific

identity of the metabolites, it is reasonable to suggest that (S,S)- and (R,R)-

hydroxybupropion undergo UGT-mediated conjugation to form (S,S)- and (R,R)-

hydroxybupropion glucuronides, respectively. Similarly, as shown in Figure 3, two

putative glucuronide peaks for each substrate were identified when racemic threo- (RTs

7.89, 8.63 min) (Figure 3A) or erythro-hydrobupropion (RTs, 8.06 and 8.97 min) (Figure

3B) was incubated with HLMs supplemented with UDPGA. Based on the retention times

of authentic standards, the two glucuronide peaks formed from racemic threo-

hydrobupropion were consistent with (1R,2R)-hydrobupropion glucuronide (RT, 7.89

min) and (1S,2S)-hydrobupropion glucuronide (RT, 8.63 min) (Figure 3A). Our data from

hydroxybupropion suggest that (1R,2R)-hydrobupropion and (1S,2S)-hydrobupropion

undergo conjugation by UGTs to form (1R,2R)-hydrobupropion glucuronide (RT, 7.89

min) and (1S,2S)-hydrobupropion glucuronide (RT, 8,63 min), respectively.

Confirmation of the identity of the two glucuronides formed from incubations of racemic

erythro-hydrobupropion, erythro-hydrobupropion glucuronide (EGLUC) 1 and EGLUC2,

remains to be determined as neither individual enantiomers of the potential substrates

(erythro-hydrobupropion) nor enantiomers of the products (putative glucuronide

metabolites) are commercially available. However, injection of an authentic

glucuronide standard obtained from TRC (labeled: racemic erythro-hydro

bupropion β-D-glucuronide) into the LC-MS/MS system yielded only a single peak

that was consistent with the second glucuronide peak (RT, 8.97 min). This


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standard was confirmed to be a mislabelled single enantiomer of erythro-hydro

bupropion β-D-glucuronide (data available from TRC), which suggests that the

metabolite at RT 8.97 min is (1R, 2S)-hydrobupropion glucuronide (EGLUC2) and the

glucuronide peak at RT, 8.06 min is (1S, 2R)-hydrobupropion (EGLUC1).

Urinary excretion of bupropion glucuronides. Less than 1% of the bupropion dose

was excreted unchanged in the urine (Table 3). Approximately 10% of the administered

bupropion dose was recovered in the urine as bupropion and metabolites by 48 hours.

Threo-hydrobupropion was the predominant unconjugated bupropion metabolite

detected in urine followed by erythro-hydrobupropion and hydroxybupropion (Fig 4A,

Table 3). Threo-hydrobupropion accounted for approximately 50% of the total urinary

bupropion metabolites. The predominant glucuronide metabolite excreted in the urine

was (R,R)-hydroxybupropion glucuronide, followed (in order of magnitude) by (1R,2R)-

hydrobupropion glucuronide, EGLUC1, (S,S)-hydroxybupropion glucuronide, (1S,2S)-

hydrobupropion glucuronide, and EGLUC2 (Fig 4B, Table 3). Glucuronide metabolites

accounted for approximately 40, 15, and 7% of total excreted hydroxybupropion,

erythro-hydrobupropion, and threo-hydrobupropion, respectively.

Bupropion metabolite glucuronidation kinetics. Glucuronide formation kinetics in

HLMs demonstrated stereoselective glucuronidation of hydroxybupropion (Fig 5A),

threo-hydrobupropion (Fig 5B) and erythro-hydrobupropion (Fig 5C). Glucuronidation

kinetics of hydroxybupropion were best described by the simple Michaelis-Menten

equation while threo- and erythro-hydrobupropion kinetics were best described by the

Hill equation. Glucuronidation efficiency (Clint or Clmax) varied nearly 10-fold across the


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enantiomers evaluated. Kinetic parameters for glucuronide formation are displayed in

table 4.

Isoform selective bupropion glucuronidation. UGT2B7 was identified as the most

active isoform catalyzing in vitro hydroxybupropion glucuronidation with UGT2B4

forming the glucuronides to a lesser extent. UGT2B7 and UGT2B4 catalyzed the

glucuronidation of (R,R)-hydroxybupropion, while only UGT2B7 catalyzed

glucuronidation of (S,S)-hydroxybupropion. Recombinant UGT1A4 and UGT1A9

catalyzed threo-hydrobupropion glucuronidation while UGT2B7 was the most active

isoform catalyzing erythro-hydrobupropion glucuronidation with UGT1A9 and UGT2B4

also forming glucuronides of erythro-hydrobupropion. These data expand our

knowledge of the proposed human metabolic pathways involved in the stereoselective

glucuronidation of bupropion and its metabolites (Figure 7).


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Discussion

Racemic bupropion undergoes complex metabolism catalyzed by multiple

enzymes to yield numerous metabolites in humans. These metabolites are important

contributors to the therapeutic and toxic effects of the drug, but relatively little is known

about the mechanisms governing their metabolism and elimination from the body. This

is the first report describing: the stereoselective separation and detection of

glucuronides of bupropion enantiomers excreted in the urine of healthy human

volunteers; UGT-mediated elimination pathways using HLMs and bupropion

enantiomers (both individual and racemic) as substrates to infer the glucuronide

stereochemistry and in vitro to in vivo extrapolation; and the predominant UGT isoforms

responsible for catalyzing glucuronidation of bupropion metabolites using a panel of

recombinant UGTs. These data provide a deeper understanding of mechanisms

underlying the disposition of pharmacologically active bupropion metabolites.

Introduction of a hydroxy functional group occurs during both the oxidative and

reductive metabolism of bupropion to yield hydroxybupropion as well as erythro- and

threo-hydrobupropion. These metabolites would be expected to undergo further

metabolism via conjugation reactions (glucuronidation and/or sulfation) to enhance

hydrophilicity and promote renal elimination. Indeed, glucuronide metabolites of

hydroxybupropion and threo- and erythro-hydrobupropion have been detected in human

urine following the therapeutic doses of racemic bupropion (Welch et al., 1987; Petsalo

et al., 2007; Benowitz et al., 2013). The estimated amount of free and total bupropion

metabolites excreted in the urine from healthy volunteers administered bupropion to

steady-state indicates that glucuronide metabolites account for approximately 75, 25,


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and 10% of total excreted hydroxybupropion, erythro- and threo-hydrobupropion,

respectively (Benowitz et al., 2013). These percentages mirror the observed rank order

in our study but differ slightly in absolute amount. However, the previously reported

indirect method (subtractive using deconjugation by β-glucuronidase) (Benowitz et al.,

2013) does not specifically provide information on glucuronide and sulfate conjugates,

as nonselective cleavage of sulfate conjugates (qualitatively identified in urine of healthy

volunteers administered bupropion, (Petsalo et al., 2007)) can occur during extended

enzymatic incubations, and only the sum of racemic mixtures were reported. This

important difference in analytical methodology likely explains the discrepancy between

our work and previous report. Nonetheless, these findings provide further confirmation

that bupropion primary metabolites undergo metabolism via conjugation prior to urinary

excretion.

Herein, is the first report describing stereoselective glucuronidation of bupropion

metabolites. We have directly identified two glucuronides each of hydroxybupropion,

erythro-hydrobupropion, and threo-hydrobupropion in the urine of healthy volunteers

administered a single 100 mg oral dose of bupropion. A previous study reported two

glucuronide metabolites of hydroxybupropion and four glucuronides of threo-/erythro-

hydrobupropion mixtures in human urine (Petsalo et al., 2007), which could suggest

stereoselective glucuronidation. However, this study did not elucidate isomeric

structures nor identify specific enantiomeric substrates. In addition, no quantitative

information was provided to describe glucuronide disposition. Our data provide for the

first time structural description of these glucuronides and, using in vitro experiments,

show enantiomers from which these glucuronide metabolites originate. Of note, these


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glucuronides were chromatographically separated using achiral column chemistry while

enantiomers of their proximate substrates are not [present data and (Petsalo et al.,

2007)], suggesting substantially unique configuration of the glucuronide metabolites

compared to their corresponding substrates. Following a single oral dose of racemic

bupropion, the glucuronides of hydroxybupropion were the predominant conjugates

detected in urine, in agreement with a previous report conducted at steady state

(Benowitz et al., 2013). Of the glucuronides detected in urine, (R,R)-hydroxybupropion

was the most prevalent species, providing further evidence for enantioselective urinary

elimination of bupropion metabolites and conjugates. We confirm that threo-

hydrobupropion is the primary unconjugated urinary bupropion metabolite. Overall,

direct description of stereoselective urinary excretion of bupropion and metabolites at 48

hours agreed well with a previous (indirect, non-stereoselective) report of urinary

recovery at 24 hours (Benowitz et al., 2013) and with findings following administration of

C14 labeled bupropion (Johnston et al., 2002).

The enzymes responsible for the oxidative and reductive metabolism of

bupropion to form hydroxybupropion, erythro-hydrobupropion, and threo-

hydrobupropion have been investigated extensively (Faucette et al., 2000; Hesse et al.,

2000; Faucette et al., 2001; Bondarev et al., 2003; Damaj et al., 2004; Molnari and

Myers, 2012; Zhu et al., 2012; Meyer et al., 2013; Skarydova et al., 2014). Metabolite

exposure is presumably dependent upon metabolic pathways leading to their formation

and elimination. However, the enzymes responsible for glucuronidation and subsequent

renal elimination of the pharmacologically important bupropion metabolites have not

been fully elucidated. Here, we quantitatively describe the stereoselective hepatic


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glucuronidation of bupropion. The most efficiently formed bupropion glucuronide in vitro,

(R,R)-hydroxybupropion glucuronide, was also the predominant urinary conjugate

observed in vivo. Similarly, the next most efficiently formed glucuronide in vitro, threo-

hydrobupropion glucuronide 1, was also the second most abundant glucuronide

excreted in the urine. Interestingly, erythro-hydrobupropion glucuronide formation

demonstrated similar in vitro efficiency but nearly 4-fold differences in mean urinary

excretion, suggesting that additional mechanisms may underlie differential urinary

excretion of these metabolites.

Characterization of the UGTs responsible for the conjugation of active bupropion

metabolites is essential to understanding factors that may influence the inter- and intra-

individual variability in bupropion metabolite exposure, including the evaluation of

potential drug-drug interactions and pharmacogenetic implications. UGT2B7 and

UGT1A9, both being prominently expressed in the liver (Margaillan et al., 2015b), are

likely the dominant isoforms responsible for hepatic glucuronidation of bupropion

metabolites. These isoforms may also play an important role in the extrahepatic

glucuronidation of bupropion, particularly in the kidney where UGT1A9 is the

predominant isoform expressed (Margaillan et al., 2015a) and in the gut where UGT2B7

and UGT1A9 are present (Harbourt et al., 2012; Fallon et al., 2013). Further exploration

of extrahepatic bupropion metabolism could more comprehensively describe bupropion

metabolite disposition and facilitate quantitative in vitro-in vivo extrapolation of

bupropion metabolite kinetics (Gill et al., 2012; Gill et al., 2013).

In summary, racemic bupropion undergoes complex metabolism catalyzed by

multiple enzymes to yield numerous metabolites in humans. These metabolites are


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important contributors to the therapeutic and toxic effects of the drug. Despite their

pharmacological importance, relatively little has been known about the mechanisms

governing their metabolism and elimination from the body. This is the first LC-MS/MS

assay that allowed simultaneous separation and quantification of enantiomers of

bupropion glucuronide metabolites in human urine and liver subcellular fractions. We

elucidated for the first time isomeric structures of the glucuronides and identified specific

enantiomeric substrates (Figure 7). Furthermore, we provided quantitative information

regarding the amount of each enantiomer excreted in urine and show enantioselective

urinary elimination of bupropion glucuronide conjugates that is in concurrence with

observed in vitro glucuronidation efficiency. Our data suggest that plasma exposure of

active bupropion metabolites may be influenced by differences not only in their

formation but also in their elimination via UGTs, notably UGT2B7 and UGT1A9. These

novel findings significantly expand our knowledge of the metabolic pathways underlying

bupropion disposition in humans (Fig 7) and should form the basis for future studies

designed to link bupropion metabolism and its clinical effect.


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Author Contributions

Participated in research design: JBL, BTG, ZD

Conducted experiments: JBL, IFM

Contributed new reagents or analytical tools: JBL, DRJ

Performed data analysis: BTG, ZD

Wrote or contributed to writing of the manuscript: BTG, JBL, DRJ, ZD


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Footnotes

a. This work was supported by the National Institutes of Health National Institute of

General Medical Sciences [Grant R01 GM078501]. B.T.G. is supported by the

National Institute of General Medical Sciences [Grant T32 GM008425].

b. Reprint requests: Zeruesenay Desta, PhD

Division of Clinical Pharmacology

Indiana University School of Medicine

950 W. Walnut Street, R2, Room 425

Indianapolis, IN 46202-5188

Phone: (317) 274-2823

Fax: (317) 278-2765

Email: [email protected]


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Figure Legends

Figure 1. Representative chromatographic traces for bupropion and its

metabolites detected in urine collected from a healthy volunteer administered a

single 100 mg oral dose of bupropion. Urine (200 µL) was centrifuged and the

resulting supernatant (50 µL) was injected into the LC-MS/MS system after filtration.

Left: traces of bupropion (A), racemic hydroxybupropion (B) and racemic erythro-/threo-

hydrobupropion (C) at the retention times (RT) of 6.97, 6.72, 7.08, 7.21 min,

respectively, corresponding to Q1/Q3 m/z transitions of 240/184, 256/238, 242.1/168

and 242.1/168. Right: D) traces of (SS)-hydroxybupropion glucuronide (RT, 7.79 min)

and (RR)-hydroxybupropion glucuronide (RT, 8.09 min) corresponds to Q1/Q3 m/z

transitions of 432/184 for both metabolites. Right: E) traces of (1R,2R)-hydrobupropion

glucuronide (RT, 7.86 min) and (1S,2S)-hydrobupropion glucuronide (RT, 8.62 min),

and erythro-hydrobupropion glucuronide (EGLUC) 1 (RT, 8.06 min) and EGLUC 2 (RT,

8.97 min), corresponding to Q1/Q3 m/z transitions of 418/168.

Figure 2. Representative chromatographic traces for glucuronides of racemic

hydroxybupropion (A), (RR)-hydroxybupropion (B), and (SS)-hydroxybupropion

(C) in human liver microsomal incubates. Racemic hydroxy-, (S,S)-hydroxy- or

(R,R)-hydroxy-bupropion (10 µM) was incubated in duplicate for 60 min at 37°C with

alamethicin (50 µg/mg protein final concentration) activated HLMs (1 mg/ml protein) and

UDPGA (5 mM). Reactions were terminated by the addition of acetonitrile (100 µL).

After the addition of the internal standard (nevirapine, 25 µl of 500ng/mL in methanol),

sample was vortex mixed, centrifuged and dried for 10 min and the resulting

supernatant (50µL) was injected into the LC-MS/MS system. A) traces of (S,S)-


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hydroxybupropion glucuronide (RT, 7.79 min) and (R,R)-hydroxybupropion glucuronide

(RT, 8.09 min) obtained from incubation containing racemic hydroxybupropion. B)

traces of (S,S)-hydroxybupropion glucuronide (RT, 7.79 min) and (R,R)-

hydroxybupropion glucuronide (RT, 8.10 min) obtained from incubation containing

(R,R)-hydroxybupropion. C) traces of (S,S)-hydroxybupropion glucuronide (RT, 7.79

min) and (R,R)-hydroxybupropion glucuronide (RT, 8.09 min) obtained from incubation

containing (S,S)-hydroxybupropion. All peaks correspond to Q1/Q3 m/z transitions of

432/184.

Figure 3. Representative chromatographic traces for glucuronides of threo- (A)

and erythro-hydrobupropion (B) in human liver microsomal incubates. Erythro-

hydrobupropion (25 µM) or threo-hydrobupropion (25 µM) was incubated for 60 min at

37°C with alamethicin (50 µg/mg protein final concentration) activated HLMs (1 mg/ml

protein) and UDPGA (5 mM). Reaction was terminated by the addition of 100 µL of

acetonitrile. After the addition of the internal standard (nevirapine, 25 µl of 500ng/mL in

methanol), sample was vortex mixed, centrifuged and dried for 10 min and the resulting

supernatant (50µL) was injected into LC-MS/MS system. A) (1R,2R)-hydrobupropion

glucuronide (RT, 7.86 min) and (1S,2S)-hydrobupropion glucuronide (RT, 8.62 min)

obtained from incubations containing threo-hydrobupropion; and B) erythro-

hydrobupropion glucuronide (EGLUC) 1 (RT, 8.06 min) and EGLUC 2 (RT, 8.97 min)

obtained from incubation containing erythro-hydrobupropion; all peaks correspond to

Q1/Q3 m/z transitions of 418/168.

Figure 4. Urinary excretion kinetics of bupropion and metabolites. Urine was

collected (0-48 hr) from healthy volunteers (n=10) following oral administration of a


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single 100 mg dose of racemic bupropion. Cumulative urinary excretion was determined

by LC-MS/MS monitoring of A) bupropion (blue), hydroxybupropion (red), threo-

hydrobupropion (black), erythro-hydrobupropion (gray) and B) (R,R)-hydroxybupropion

glucuronide (solid red line and symbols), (S,S)-hydroxybupropion glucuronide (dashed

red line, open red symbols), (1R,2R)-hydrobupropion glucuronide (solid black line and

symbols), (1S,2S)-hydrobupropion glucuronide (dashed black line, open black symbols),

erythro-hydrobupropion glucuronide (EGLUC) 1 (solid gray line and symbols), and

EGLUC2 (dashed gray line, open gray symbols) over time. Glucuronide 1 and

glucuronide 2 assigned based upon retention times in Figures 1-3 and outlined in Table

2. Symbols and error bars denote the mean and 90% confidence interval of the

observed cumulative urinary excretion (nmol) at the end of the collection interval,

respectively.

Figure 5. Kinetics for the formation of hydroxybupropion (A), threo-

hydrobupropion (B), and erythro-hydrobupropion (C) glucuronides in HLMs.

Racemic hydroxybupropion, threo-hydrobupropion, or erythro-hydrobupropion (25 µM to

1000 µM) was incubated for 60 min at 37°C in duplicate with HLMs (1 mg/ml protein)

and 5 mM UDPGA. The two putative glucuronide metabolites in each panel are

consistent with (R,R)-hydroxybupropion glucuronide (black) and (S,S)-

hydroxybupropion glucuronide (gray) (A), (1R,2R)-hydrobupropion glucuronide (black)

and (1S,2S)- hydrobupropion glucuronide (gray) (B), erythro-hydrobupropion

glucuronide (EGLUC) 1 (black) and EGLUC2 (gray) (C). Glucuronide 1 and glucuronide

2 assigned based upon retention times in Figures 1-3 and outlined in Table 2. Curves

denote model-generated values obtained from nonlinear regression performed by fitting


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the Michaelis-Menten or Hill equation to substrate concentration versus apparent

metabolite formation velocity using Phoenix® WinNonlin® (version 6.4). Symbols and

error bars denote means and S.D.’s, respectively, of duplicate incubations.

Figure 6. Bupropion glucuronide formation using HLMs and a panel of thirteen

rUGT isoforms. HLMs (1 mg/mL) or rUGT (0.5 mg/mL) were incubated with

hydroxybupropion, (R,R)-hydroxybupropion (50 µM), (S,S)-hydroxybupropion (50 µM),

threo-hydrobupropion (100 µM), or erythro-hydrobupropion (100 µM) and UDPGA (5

mM) (250 µL final reaction volume) at 37°C for 60 minutes. Velocities correspond to the

formation of (S,S)-hydroxybupropoin glucuronide (A), (R,R)-hydroxybupropion

glucuronide (B), (1R,2R)-hydrobupropion glucuronide (C), (1S,2S)-hydrobupropion

glucuronide (D), erythro-hydrobupropion glucuronide 1 (EGLUC1) (E), or erythro-

hydrobupropion glucuronide 2 (EGLUC2) (F). Glucuronide 1 and glucuronide 2

assigned based upon retention times in Figures 1-3 and outlined in Table 2. Bars and

error bars denote the mean and S.D., respectively, of duplicate incubations.

Figure 7. Proposed human metabolic pathways involved in the stereoselective

oxidation, reduction, and glucuronidation of bupropion and its metabolites.

aConfirmation of the identity of the two glucuronides formed from incubations of racemic

erythro-hydrobupropion, erythro-hydrobupropion glucuronide (EGLUC) 1 and EGLUC2,

remains to be definitively determined as neither individual enantiomers of the potential

substrates (erythro-hydrobupropion) nor unambiguously identified enantiomers of the

products (putative glucuronide metabolites) are commercially available.


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Tables

Table 1. Compound dependent parameters optimized to hydroxybupropion glucuronide.

Analytes Q1/Q3

(m/z)

DP

(v)

EP

(v)

CEP

(v)

CE

(v)

CXP

(v)

Erythro-hydrobupropion 242.10/168.00 36 5.5 12 24 3

EGLUC 1 418.00/168.00 45 8.5 30 35 5

EGLUC 2 418.00/168.00 45 8.5 30 35 5

Threo-hydrobupropion 242.10/168.00 36 5.5 12 24 3

(1R,2R)-hydrobupropion

glucuronide 418.00/168.00 45 8.5 30 35 5

(1S,2S)-hydrobupropion

glucuronide 418.00/168.00 45 8.5 30 35 5

hydroxybupropion 256.00/238.00 30 5.5 25 24 4

(S,S)-hydroxybupropion

glucuronide 432.00/184.00 56 8.5 16 24 3

(R,R)-hydroxybupropion

glucuronide 432.00/184.00 56 8.5 16 24 3

Declustering Potential (DP); Entrance Potential (EP); Collision cell entrance potential

(CEP); Collision Energy (CE); Collision Cell Exit Potential (CXP), Erythro-

hydrobupropion glucuronide (ELGUC).


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Table 2. Retention times and mass transitions of bupropion metabolites.

Substrates/Metabolites RT

(min)

Observed Mass

Q1[DA]

Fragment

Quantifier

Q3[DA]

Erythro-hydrobupropion 6.92 242.10 168.00

EGLUC 1 8.03 418.00 168.00

EGLUC 2 8.93 418.00 168.00

Threo-hydrobupropion 7.05 242.10 168.00

(1R,2R)-hydrobupropion

glucuronide

7.84 418.00 168.00

(1S,2S)-hydrobupropion

glucuronide

8.59 418.00 168.00

hydroxybupropion 6.75 256.00 238.00


glucuronide

7.80 432.00 184.00


glucuronide

8.10 432.00 184.00

(R,R)-hydroxybupropion 6.59 256.00 131.00


glucuronide

7.78 432.00 184.00


glucuronide

8.08 432.00 184.00

(S,S)-hydroxybupropion 6.60 256.00 131.00


glucuronide

7.75 432.00 184.00


glucuronide

8.05 432.00 184.00

Erythro-hydrobupropion glucuronide (EGLUC)


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Table 3. 48 hour bupropion and metabolite urinary excretion.

Analyte 48-h recovery, nmola Mean (90% CI)

% of bupropion dose Mean (90% CI)

Bupropion 2172 (1218-3125) 0.60 (0.34-0.86) hydroxybupropion 2266 (722-3811) 0.67 (0.21-1.12) Threo-hydrobupropion 9463 (5384-13542) 5.24 (2.98-7.51) Erythro-hydrobupropion 1953 (894-3011) 1.08 (0.50-1.67) (R,R)-hydroxybupropion glucuronide

1275 (732-1818) 1.27 (0.73-1.81)

(S,S)-hydroxybupropion glucuronide 205 (135-275) 0.20 (0.13-0.27)

EGLUC 1 275 (179-372) 0.26 (0.17-0.36)

EGLUC 2 67 (42-92) 0.06 (0.04-0.09) (1R,2R)-hydrobupropion glucuronide

591 (325-857) 0.57 (0.31-0.82)

(1S,2S)-hydrobupropion glucuronide 172 (119-225) 0.17 (0.11-0.22)

Erythro-hydrobupropion glucuronide (EGLUC). Values denote means (n=10 subjects)

and 90% confidence intervals (CI).

a(R,R)- and (S,S)-hydroxybupropion glucuronides represent nmol equivalents relative to

threo-hydrobupropion glucuronides with the same isomeric configuration.


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Table 4. Bupropion glucuronidation kinetics.a

Substrate Km or S50 (µM) Vmax

(pmol/min/mg) Clint

c or Clmaxd

(µL/min/mg) (S,S)-hydroxybupropion glucuronidea 172 ± 38.9 739 ± 59.6 4.31

(R,R)-hydroxybupropion glucuronidea 488 ± 98.3 5550 ± 507 11.4

(1R,2R)-hydrobupropion glucuronide

343 ± 37.5 3290 ± 269 7.7

(1S,2S)-hydrobupropion glucuronide 248 ± 27.1 358 ± 25.5 1.1

EGLUC1b 373 ± 63.0 1740 ± 212 3.1

EGLUC2b 360 ± 55.2 2280 ± 241 3.8 Values represent the parameter estimate ± S.E. obtained by fitting the Michaelis-Mentena (hydroxybupropion) or Hillb (threo- and erythro-hydrobupropion) equation to [substrate] versus metabolite formation velocity using Phoenix® WinNonlin® (version 6.4). b Glucuronide 1 and glucuronide 2 denoted based upon retention times in Figures 1-3 and outlined in Table 2. Erythro-hydrobupropion glucuronide (EGLUC). c Intrinsic clearance (Clint), calculated as the ratio of Vmax to Km. d Maximal clearance (Clmax), calculated as the ratio of Vmax x (h-1) to S50 + h(h-1)1/h.


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