Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/09/19/dmd.115.068908.DC1

1521-009X/44/4/544–553$25.00 http://dx.doi.org/10.1124/dmd.115.068908 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:544–553, April 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics 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

Received December 16, 2015; accepted January 20, 2016

ABSTRACT Bupropion is a widely used antidepressant and smoking cessa- (S,S)-hydrobupropion glucuronide], in concurrence with ob- tion aid in addition to being one of two US Food and Drug served enantioselective urinary elimination of bupropion glucu- Administration–recommended probe substrates for evaluation ronide conjugates. Approximately 10% of the administered of cytochrome P450 2B6 activity. Racemic bupropion undergoes bupropion dose was recovered in the urine as metabolites with Downloaded from oxidative and reductive metabolism, producing a complex profile glucuronide metabolites, accounting for approximately 40%, 15%, of pharmacologically active metabolites with relatively little and 7% of the total excreted hydroxybupropion, erythro- known about the mechanisms underlying their elimination. A hydrobupropion, and threo-hydrobupropion, respectively. Elimina- liquid chromatography-tandem mass spectrometry assay was tion pathways were further characterized using an expressed developed to simultaneously separate and detect glucuronide UDP-glucuronosyl transferase (UGT) panel with bupropion

metabolites of (R,R)- and (S,S)-hydroxybupropion, (R,R)- and enantiomers (both individual and racemic) as substrates. dmd.aspetjournals.org (S,S)-hydrobupropion (threo) and (S,R)- and (R,S)-hydrobupropion UGT2B7 catalyzed the stereoselective formation of glucuronides (erythro), in human urine and liver subcellular fractions to begin of hydroxybupropion, (S,S)-hydrobupropion, (S,R)- and (R,S)- exploring mechanisms underlying enantioselective metabolism hydrobupropion; UGT1A9 catalyzed the formation of (R,R)- and elimination of bupropion metabolites. Human liver micro- hydrobupropion glucuronide. These data systematically describe somal data revealed marked glucuronidation stereoselectivity the metabolic pathways underlying bupropion metabolite disposi-

[Clint, 11.4 versus 4.3 ml/min per milligram for the formation of tion and significantly expand our knowledge of potential contribu-

(R,R)- and (S,S)-hydroxybupropion glucuronide; and Clmax,7.7 tors to the interindividual and intraindividual variability in therapeutic at ASPET Journals on September 27, 2021 versus 1.1 ml/min per milligram for the formation of (R,R)- and and toxic effects of bupropion in humans.

Introduction exposure was much higher in mice than in rats (Welch et al., 1987). Bupropion [(6)-1-(3-chlorophenyl)-2-[(1,1-dimethylethyl) amino]- Thus, differences in the formation of potentially active metabolites may 1-propanone] is widely used for the treatment of depression, for explain species differences in bupropion effect. In humans, bupropion seasonal affective disorder, as a smoking cessation aid (Dwoskin undergoes extensive hepatic metabolism via oxidation of the t-butyl et al., 2006; Dhillon et al., 2008), and recently has been coformulated moiety to hydroxybupropion and reduction of the aminoketone group to , with naltrexone for weight management in obese patients (Yanovski two amino alcohols (erythro- and threo-hydrobupropion), with 1% of and Yanovski, 2015); however, bupropion antidepressant (Thase et al., the administered dose excreted unchanged in urine (Findlay et al., 1981; 2005) and smoking cessation (Hurt et al., 1997; Jorenby et al., 1999; Lai and Schroeder, 1983; Schroeder, 1983; Laizure et al., 1985; Posner Dale et al., 2001) treatment outcomes vary widely among patients. The et al., 1985; Welch et al., 1987). Several preclinical studies in addition use of immediate release bupropion is also associated with dose- to human data provide compelling evidence that bupropion metabolites dependent adverse effects, including seizures (Davidson, 1989). are pharmacologically active (Perumal et al., 1986; Golden et al., 1988; The mechanisms contributing to the variable clinical response and Bondarev et al., 2003; Damaj et al., 2004; Silverstone et al., 2008; Zhu adverse effects of bupropion are not fully known. Alterations in the et al., 2012). Bupropion has also been reported as a clinically relevant metabolism of bupropion and its primary metabolites have been posited inhibitor of CYP2D6, with in vitro evidence suggesting that bupropion to contribute to this variability. Early animal studies suggest that metabolites (rather than the parent drug) mediated this interaction bupropion is a more effective antidepressant in mice than in rats (Reese et al., 2008). Consideration of metabolite pharmacokinetics (Soroko et al., 1977; Ferris et al., 1983), and hydroxybupropion plasma further reinforces their potential contribution to overall bupropion effect. Steady-state plasma exposures of hydroxybupropion and threo- hydrobupropion after bupropion administration are 17- and 7-fold This work was supported by the National Institutes of Health (NIH) National higher, respectively, than the parent drug (Laizure et al., 1985; Posner Institute of General Medical Sciences [Grant R01 GM078501]. B.T.G. is supported et al., 1985; Benowitz et al., 2013), and metabolites accumulate by the NIH General Medical Sciences [Grant T32 GM008425]. substantially in plasma and cerebrospinal fluid during repeated dx.doi.org/10.1124/dmd.115.068908. administration. Therefore, it is important to understand the factors that

ABBREVIATIONS: FDA, US Food and Drug Administration; FIA, flow injection analysis; HLM, human liver microsome; LC-MS/MS, liquid chromatography-tandem mass spectrometry; P450, cytochrome P450; RT, retention time; rUGT, recombinant UGT; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyl transferase.

544 Stereoselective Bupropion Glucuronidation 545

TABLE 1 Compound dependent parameters optimized to hydroxybupropion glucuronide

Analytes Q1/Q3 DP EP CEP CE CXP

m/z vv v v 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

CE, collision energy; CEP, cell entrance potential; CXP, collision cell exit potential; DP, declustering potential; ELGUC, erythro- hydrobupropion glucuronide; EP, entrance potential. contribute to differential tissue and plasma exposure of active CYP2B6-mediated formation (e.g., further elimination processes) may bupropion metabolites. account for observed stereoselective disposition. Downloaded from Of the bupropion metabolites so far described, hydroxybupropion In vitro (Skarydova et al., 2014; Connarn et al., 2015) and in vivo has been studied in detail. Bupropion has one chiral center and is (Benowitz et al., 2013) studies suggest that bupropion reduction to form administered clinically as a 50:50 racemic mixture. Hydroxylation of two hydrobupropion metabolites is the major mechanism of bupropion racemic bupropion to hydroxybupropion is exclusively catalyzed by clearance. Reduction of the keto group by 11b-hydroxysteroid de- cytochrome P450 2B6 (CYP2B6) (Faucette et al., 2000; Hesse et al., hydrogenase type 1 and other carbonyl reductases (Molnari and Myers, 2000). This reaction is one of two US Food and Drug Administration 2012; Meyer et al., 2013; Skarydova et al., 2014; Connarn et al., 2015) dmd.aspetjournals.org (FDA)-recommended in vitro and in vivo probes of CYP2B6 (FDA appears to favor formation of threo-hydrobupropion (Benowitz et al., CDER, 2012). Elevated hydroxybupropion plasma exposure is associ- 2013). Although this reduction creates an additional chiral center, ated with improved bupropion treatment outcomes in depression (Laib potentially generating two distinct threo-hydrobupropion and two et al., 2014) and smoking cessation (Zhu et al., 2012); however, erythro-hydrobupropion diastereomers, data describing stereoselective hydroxybupropion plasma exposure varied widely among patients and elimination pathways and effect are lacking. is only partially explained by CYP2B6 genetic variation (Benowitz et al., The main objective of the present study was to explore the

2013). Bupropion hydroxylation creates an additional chiral center, and mechanisms underlying enantioselective metabolism of bupropion at ASPET Journals on September 27, 2021 two diastereomers [(2R,3R)- and (2S,3S)-hydroxybupropion] have been metabolites by investigating the UGT-mediated metabolism of hydroxy- quantified in human plasma (Suckow et al., 1997; Coles and Kharasch, bupropion and threo- and erythro-hydrobupropion. 2007; Xu et al., 2007). Wheres plasma exposure of (2R,3R)- . hydroxybupropion is 20-fold higher than (2S,3S)-hydroxybupropion Materials and Methods (Kharasch et al., 2008), some pharmacologic activity of bupropion may Chemicals. All chemicals and solvents were high-performance liquid reside in (2S,3S)-hydroxybupropion (Bondarev et al., 2003; Damaj et al., chromatogrphy grade or higher. Acetonitrile, methanol, and acetic acid were 2004; Hansard et al., 2011). The marked stereoselectivity in hydroxybu- purchased from Fisher Scientific Company LLC (Hanover Park, IL). Laboratory propion disposition observed in vivo is not fully explained by CYP2B6 as water was prepared for liquid chromatography-tandem mass spectrometry the rate of (S)-bupropion hydroxylation in expressed CYP2B6 and human (LC-MS/MS) applications using a Nanopure Infinity UV system (Barnsteas/ liver microsomes is only 3- and 1.5-fold higher, respectively, than Thermolyne, Dubuque, IA). UDPGA, alamethicin, Trizma base, and magnesium

(R)-bupropion (Coles and Kharasch, 2008). Mechanisms other than chloride (MgCl2) were purchased from Sigma-Aldrich (St. Louis, MO). Internal

TABLE 2 RTs and mass transitions of bupropion metabolites

Substrates/Metabolites RT Observed Mass Q1 Fragment Quantifier Q3

min Da 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 (S,S)-hydroxybupropion glucuronide 7.80 432.00 184.00 (R,R)-hydroxybupropion glucuronide 8.10 432.00 184.00 (R,R)-hydroxybupropion 6.59 256.00 131.00 (S,S)-hydroxybupropion glucuronide 7.78 432.00 184.00 (R,R)-hydroxybupropion glucuronide 8.08 432.00 184.00 (S,S)-hydroxybupropion 6.60 256.00 131.00 (S,S)-hydroxybupropion glucuronide 7.75 432.00 184.00 (R,R)-hydroxybupropion glucuronide 8.05 432.00 184.00

EGLUC, Erythro-hydrobupropion glucuronide. 546 Gufford et al. standard, nevirapine, was supplied through the National Institutes of Health 10 minutes before injection of resulting supernatant. Initial parameters for FIA AIDS Research and Reference Reagent Program (Germantown, MD). Bupropion, were set according to parent drug FIA results. All data were collected in positive R-bupropion, S-bupropion, (2R,3R)-hydroxybupropion (R,R-hydroxybupropion), ion mode. As some authentic standards [(S,S)- and (R,R)-hydrobupropion (2S,3S)-hydroxybupropion (S,S-hydroxybupropion), racemic erythro-hydrobupropion, (threo-hydrobupropion) b-D-glucuronides and racemic erythro-hydrobupropion racemic threo-hydrobupropion, (S,S)- and (R,R)-hydrobupropion b-D-glucuronides b-D-glucuronide] were later commercially available, FIA was repeated for these (threo-hydrobupropion glucuronides) and racemic erythro-hydrobupropion b-D- glucuronides fter acquisition. Matrix-matched calibration curves were generated glucuronide (EGLUC1 and EGLUC2) were purchased from Toronto Research to directly quantify the glucuronide metabolites of threo- and erythro- Chemicals (Toronto, ON). hydrobupropion using the available glucuronide standards with a dynamic assay Microsomal Preparations. Mixed-gender pooled human liver microsomes range of 0.5-1000 ng/ml. (R,R)- and (S,S)-hydroxybupropion glucuronides were (HLMs) (20 mg/ml) were purchased from Corning (Woburn, MA), and quantified using the standard curves of threo-hydrobupropion glucuronides with recombinant UGTs (rUGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, the same isomeric configuration as glucuronide standards were not commercially UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, available for these metabolites. The possibility that ionization efficiency and UGT2B15, and UGT2B17) (5 mg/ml) were purchased from Discovery Labware, other system parameters may yield differing LC-MS/MS response between these Inc. (Woburn, MA). All microsomal preparations were stored at 280C until structurally related metabolites could not be excluded. As such, the reported analysis. nmol formation and excretion rates of (R,R)- and (S,S)-hydroxybupropion LC-MS/MS Method Development. LC-MS/MS analytical method devel- glucuronides could be viewed more appropriately as nmol equivalents relative to opment to separate and detect bupropion, hydroxybupropion, and threo- and threo-hydrobupropion glucuronides with the same isomeric configuration. erythro-hydrobupropion was performed using an API 3200 triple quadrupole Urine Sample Analysis. Urine samples were collected during four intervals – – – – mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an (0 12, 12 24, 24 36, and 36 48 hours) over a 48-hour period after bupropion Downloaded from electrospray ionization (ESI) source and coupled to a high-performance liquid administration to healthy volunteers (n = 10). An aliquot of each urine sample chromatography system consisting of two LC-20AD pumps, SIL-20AHT UFLC (50 ml) was diluted with water-methanol (1:1) to 100 ml, 25 ml of 250 ng/ml autosampler, DGU-20A3 degasser, and a CBM-20A system controller (Shi- nevirapine was added as internal standard, the sample was centrifuged (12,000 madzu, Columbia, MD). Data acquisition and processing were performed using rpm  10 minutes), and the resulting supernatant (10ml) was injected into the Analyst software (version 1.5.1, AB SCIEX). Analyte concentrations were LC-MS/MS system. quantified using Analyst software by interpolation from matrix-matched Optimization of Microsomal Incubation Conditions. Pilot incubation calibration curves and quality controls with dynamic assay ranges of 0.1–1000 experiments were performed using HLMs to identify potential glucuronide dmd.aspetjournals.org ng/ml for all analytes. The calibration standards and quality controls were judged metabolites of hydroxybupropion and to optimize incubation and LC-MS/MS for batch quality based on the 2013 FDA guidance for industry regarding analysis conditions. Racemic hydroxybupropion was dissolved and serially bioanalytical method validation (FDA CDER, 2013). Chromatographic separa- diluted in methanol to the required concentration. Methanol was removed by tion was achieved using a Luna C18-2 column (150  4.6 mm i.d.; 5-mm particle drying in a speed vacuum before reconstituting with HLMs. HLMs were diluted size; Phenomenex, Torrance, CA) and mobile phase consisting of methanol with Tris HCl buffer (pH 7.4, 100 mM) containing MgCl2 (5 mM) and activated (mobile phase B) and water containing 0.1% acetic acid (mobile phase A) using by addition of alamethicin (50 mg/mg protein) on ice for 15 minutes with gentle the following gradient: initial conditions of 50% mobile phase B followed by a agitation every 5 minutes. For the pilot studies, hydroxybupropion (100 mM) and linear gradient to 90% mobile phase B between 0.01 and 16 minutes, then re- 25 ml of activated HLMs (desired concentrations) were diluted in 125 ml Tris at ASPET Journals on September 27, 2021 equilibrated to initial conditions between 16.01 minutes and 20 minutes using a HCl buffer and preincubated for 5 minutes at 37C. The reaction was initiated by total flow rate of 0.8 ml/min. adding 100 ml of UDPGA (5 mM dissolved in buffer) to yield a final reaction Initially, no glucuronide standards were commercially available, so urine volume of 250 ml. The reaction was terminated by the addition of 100 mlof collected from healthy volunteer(s) fter the administration of a single 100-mg acetonitrile at predetermined time intervals. Nevirapine (25 ml of 500 ng/ml in oral tablet of racemic bupropion hydrochloride (Sandoz Inc., Princeton, NJ) was methanol) was added as an internal standard to the incubation samples before used for flow injection analysis (FIA) to obtain optimal instrument and analysis. The samples were vortexed vigorously for 20 seconds and left on ice for compound-dependent parameters for the assay. Healthy volunteers were enrolled 15 minutes. Samples were then evaporated for 10 minutes, followed by into a trial at the Indiana University School of Medicine Clinical Research centrifugation at 12,000 rpm for 5 minutes in an Eppendorf model 5415C Center. The Indiana University School of Medicine Institutional Review Board centrifuge (Brinkman Instruments, Westbury, NY). The resulting supernatant approved the study protocol, and all participants signed an approved, informed (10 ml) was injected onto the LC-MS/MS system. Chromatographic separation consent before enrollment. The trial is registered at the ClinicalTrials.gov of the bupropion glucuronide metabolites was achieved on a Luna C18-2 column (NCT02401256). The obtained urine sample was centrifuged at 12,000 rpm for (150  4.6 mm i.d.; 5-mm particle size; Phenomenex, Torrance, CA) using a

Fig. 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 ml) was centrifuged, and the resulting supernatant (50 ml) 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 RTs of 6.97, 6.72, 7.08, 7.21 minutes, 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 minutes) and (RR)-hydroxybupropion glucuronide (RT, 8.09 minutes) correspond to Q1/Q3 m/z transitions of 432/184 for both metabolites. Right: (E) Traces of (1R,2R)-hydrobupropion glucu- ronide (RT, 7.86 minute) and (1S,2S)-hydrobupropion glucuronide (RT, 8.62 minutes), and erythro-hydrobupropion glucuronide (EGLUC) 1 (RT, 8.06 minutes), and EGLUC 2 (RT, 8.97 minutes), corresponding to Q1/Q3 m/z transitions of 418/168. Stereoselective Bupropion Glucuronidation 547 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 minutes and then reequilibrated to initial conditions between 10.01 and 14 minutes using a total flow rate of 0.8 ml/min. Using the incubation and LC-MS/MS assay conditions already described, linearity in the formation rate of the observed hydroxybupropion glucuronides was established with regard to microsomal protein concentration and incubation time. Hydroxybupropion (100 mM) was incubated in HLMs (0.0–1.0mgprotein/ml) in the presence of the UDPGA (5 mM) at 37C across a range of incubation times (0–90 minutes). Optimal incubation conditions were determined to be 60 minutes of 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 (Pharsight Corp., Cary, NC) with simulations to guide final concentration selection. Rates of hydroxybupropion, threo-hydrobupropion, and erythro-hydrobupropion glu- curonide formation were determined by incubating a range of substrate concentrations (25–1000 mM) encompassing the predicted Km or S50. Incubation

conditions were optimized for linearity with respect to time and protein Downloaded from concentration for each substrate as described here (data not shown). Reaction Phenotyping Using a Recombinant UGT Enzyme Panel. Hydroxybupropion, (R,R) and (S,S)-hydroxyburpopion, threo-hydrobupropion, or erythro-hydrobupropion (50 mM) were incubated with HLMs (1 mg/ml) or each individual recombinant UGT enzyme (0.5 mg/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 dmd.aspetjournals.org analyzed via LC-MS/MS as described herein. 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.). 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 h h Michaelis-Menten (V = Vmax  [S]/Km +[S]) or Hill (V = Vmax  [S] /S50 + Fig. 2. Representative chromatographic traces for glucuronides of racemic at ASPET Journals on September 27, 2021 [S]h) equation (where h = Hill coeffficient) to substrate concentration versus hydroxybupropion (A), (RR)-hydroxybupropion (B), and (SS)-hydroxybupropion apparent metabolite formation velocity data using Phoenix WinNonlin. Cl (C) in HLM incubates. Racemic hydroxy-, (S,S)-hydroxy-, or (R,R)-hydroxy- int bupropion (10 mM) was incubated in duplicate for 60 minutes at 37C with (V /K )orCl (V  (h 2 1)/K + h(h 2 1)1/h) (Houston and Kenworthy, max m max max m alamethicin (50 mg/mg protein final concentration) activated HLMs (1 mg/ml 2000) was calculated for substrates described by the simple Michaelis-Menten or protein) and UDPGA (5 mM). Reactions were terminated by the addition of Hill equation, respectively. Unless noted, data are presented as mean of duplicate acetonitrile (100 ml). After the addition of the internal standard (nevirapine, 25 mlof incubations, with error bars showing data variability for n =2. 500 ng/ml in methanol), sample was vortex-mixed, centrifuged, and dried for 10 minutes, and the resulting supernatant (50ml) was injected into the LC-MS/MS system. (A) Traces of (S,S)-hydroxybupropion glucuronide (RT, 7.79 minute)s and Results (R,R)-hydroxybupropion glucuronide (RT, 8.09 minutes) obtained from incubation containing racemic hydroxybupropion. (B) Traces of (S,S)-hydroxybupropion LC-MS/MS Method Development. The following were the glucuronide (RT, 7.79 minutes) and (R,R)-hydroxybupropion glucuronide (RT, optimized instrument parameters: interface temperature at 500C, 8.10 minutes) obtained from incubation containing (R,R)-hydroxybupropion. C) nebulizer gas (nitrogen) (GS1) 60.0 psi, heater gas (GS2) 55.0 psi, Traces of (S,S)-hydroxybupropion glucuronide (RT, 7.79 minutes) and (R,R)- hydroxybupropion glucuronide (RT, 8.09 minutes) obtained from incubation curtain gas (CUR) 25.0 psi, IonSpray Voltage 3500 V and collision containing (S,S)-hydroxybupropion. All peaks correspond to Q1/Q3 m/z transitions activated dissociation 6.0 psi. The optimized compound-dependent of 432/184. MS/MS parameters are outlined in Table 1. Chromatographic Separation, Detection, and Identification of Bupropion Metabolites. Retention times (RTs) and mass transitions of incubations using (S,S)-hydroxybupropion as the substrate (Fig. 2C); bupropion substrates and metabolites are outlined in Table 2. Baseline the later eluting glucuronide (RT 8.09 minutes) was exclusively formed chromatographic separation of individual enantiomers of hydroxybu- in incubations utilizing (R,R)-hydroxybupropion as the substrate (Fig. propion and threo- and erythro-hydrobupropion was not achieved using 2B). Peaks corresponding to these glucuronide metabolites were not the outlined chromatographic conditions (Fig. 1, A–C). In contrast, observed in the negative control incubations (no cofactor, no substrate, baseline separation and detection of the enantiomeric bupropion or no incubation). Although no hydroxybupropion glucuronides glucuronide metabolites were achieved for the first time (Fig. 1, D (racemic or enantiomers) were available to us commercially to confirm and E). The glucuronides of hydroxybupropion (racemic or enantio- the specific identity of the metabolites, it is reasonable to suggest that mers) were not available commercially. Thus, identification of the two (S,S)- and (R,R)-hydroxybupropion undergo UGT-mediated conjuga- urinary glucuronides of hydroxybupropion were carried out by tion to form (S,S)- and (R,R)-hydroxybupropion glucuronides, re- monitoring metabolites formed during incubation of racemic-, (S,S)-, spectively. Similarly, as shown in Fig. 3, two putative glucuronide and (R,R)-hydroxybupropion with HLMs supplemented with UDPGA. peaks for each substrate were identified when racemic threo- (RTs 7.89, In microsomal incubations containing racemic hydroxybupropion, two 8.63 minutes) (Fig. 3A) or erythro-hydrobupropion (RTs, 8.06 and 8.97 glucuronide metabolites (Fig. 2A) that had the same retention times as minutes) (Fig. 3B) was incubated with HLMs supplemented with those observed in urine (Fig. 1D) were generated. The glucuronide UDPGA. Based on the RTs of authentic standards, the two glucuronide metabolite eluting first (RT 7.79 minutes) was exclusively formed in peaks formed from racemic threo-hydrobupropion were consistent with 548 Gufford et al.

erythro-hydro bupropion b-D-glucuronide) into the LC-MS/MS system yielded only a single peak that was consistent with the second glucuronide peak (RT, 8.97 minutes). This standard was confirmed to be a mislabeled single enantiomer of erythro-hydrobupropion b-D-glucuronide (data available from TRC), which suggests that the metabolite at RT, 8.97 minutes, is (1R, 2S)-hydrobupropion glucuro- nide (EGLUC2) and the glucuronide peak at RT, 8.06 minutes, 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 metab- olite 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 Downloaded from urine was (R,R)-hydroxybupropion glucuronide, followed (in order of magnitude) by (1R,2R)-hydrobupropion glucuronide, EGLUC1, (S,S)-hydroxybupropion glucuronide, (1S,2S)- hydrobupropion glucu- Fig. 3. Representative chromatographic traces for glucuronides of threo- (A) and erythro-hydrobupropion (B) in HLM incubates. Erythro-hydrobupropion (25 mM) or ronide, and EGLUC2 (Fig. 4B; Table 3). Glucuronide metabolites threo-hydrobupropion (25 mM) was incubated for 60 minutes at 37C with accounted for approximately 40%, 15%, and 7% of total excreted alamethicin (50 mg/mg protein final concentration) activated HLMs (1 mg/ml hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, protein) and UDPGA (5 mM). Reaction was terminated by the addition of 100 mlof dmd.aspetjournals.org acetonitrile. After the addition of the internal standard (nevirapine, 25 ml of 500 ng/ respectively. ml in methanol), sample was vortex-mixed, centrifuged and dried for 10 minutes, Bupropion Metabolite Glucuronidation Kinetics. Glucuronide and the resulting supernatant (5 ml) was injected into LC-MS/MS system. (A) formation kinetics in HLMs demonstrated stereoselective glucuronida- (1R,2R)-hydrobupropion glucuronide (RT, 7.86 minutes) and (1S,2S)-hydrobupro- tion of hydroxybupropion (Fig. 5A), threo-hydrobupropion (Fig. 5B), pion glucuronide (RT, 8.62 minutes) obtained from incubations containing threo- hydrobupropion and (B) erythro-hydrobupropion glucuronide (EGLUC) 1 (RT, 8.06 and erythro-hydrobupropion (Fig. 5C). Glucuronidation kinetics of minutes) and EGLUC 2 (RT, 8.97 minutes) obtained from incubation containing hydroxybupropion is best described by the simple Michaelis-Menten erythro-hydrobupropion; all peaks correspond to Q1/Q3 m/z transitions of 418/168. equation, whereas threo- and erythro-hydrobupropion kinetics is best

described by the Hill equation. Glucuronidation efficiency (Clint or at ASPET Journals on September 27, 2021 Clmax) varied nearly 10-fold across the enantiomers evaluated. Kinetic (1R,2R)-hydrobupropion glucuronide (RT, 7.89 minutes) and (1S,2S)- parameters for glucuronide formation are displayed in Table 4. hydrobupropion glucuronide (RT, 8.63 minutes) (Fig. 3A). Our data Isoform Selective Bupropion Glucuronidation. UGT2B7 was from hydroxybupropion suggest that (1R,2R)-hydrobupropion and identified as the most active isoform catalyzing in vitro hydroxybupro- (1S,2S)-hydrobupropion undergo conjugation by UGTs to form pion glucuronidation, with UGT2B4 forming the glucuronides to a (1R,2R)-hydrobupropion glucuronide (RT, 7.89 minutes) and lesser extent. UGT2B7 and UGT2B4 catalyzed the glucuronidation (1S,2S)-hydrobupropion glucuronide (RT, 8,63 minutes), respectively. of (R,R)-hydroxybupropion, whereas only UGT2B7 catalyzed glucur- Confirmation of the identity of the two glucuronides formed from onidation of (S,S)-hydroxybupropion. Recombinant UGT1A4 incubations of racemic erythro-hydrobupropion, erythro-hydrobupropion and UGT1A9 catalyzed threo-hydrobupropion glucuronidation, glucuronide (EGLUC) 1, and EGLUC2 remains to be determined as wheres UGT2B7 was the most active isoform catalyzing erythro- neither individual enantiomers of the potential substrates (erythro- hydrobupropion glucuronidation with UGT1A9 and UGT2B4 also hydrobupropion) nor enantiomers of the products (putative glucuronide forming glucuronides of erythro-hydrobupropion. These data expand our metabolites) are commercially available; however, injection of an knowledge of the proposed human metabolic pathways involved in the authentic glucuronide standard obtained from TRC (labeled: racemic stereoselective glucuronidation of bupropion and its metabolites (Fig. 6).

TABLE 3 48-Hour bupropion and metabolite urinary excretion Values denote means (n = 10 subjects) and 90% confidence intervals (CI).

Analyte 48-h Recovery, nmola Mean (90% CI) % of Bupropion Dose Mean (90% CI) Bupropion 2172 (1218–3125) 0.60 (0.3–0.86) Hydroxybupropion 2266 (722–3811) 0.67 (0.21–1.12) Threo-hydrobupropion 9463 (5384–13,542) 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). a(R,R)- and (S,S)-hydroxybupropion glucuronides represent nmol equivalents relative to threo-hydrobupropion glucuronides with the same isomeric configuration. Stereoselective Bupropion Glucuronidation 549 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 4. Urinary excretion kinetics of bupropion and metabolites. Urine was collected (0–48 hours) from healthy volunteers (n = 10) after oral administration of a 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)-hydroxybu- propion 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 Fig. 5. Kinetics for the formation of hydroxybupropion (A), threo-hydrobupropion (EGLUC) 1 (solid gray line and symbols), and EGLUC2 (dashed gray line, open (B), and erythro-hydrobupropion (C) glucuronides in HLMs. Racemic hydroxybu- gray symbols) over time. Glucuronide 1 and glucuronide 2 were assigned based on propion, threo-hydrobupropion, or erythro-hydrobupropion (25 mM–1000 mM) was RTs in Figs. 1–3 and are outlined in Table 2. Symbols and error bars denote the incubated for 60 minutes at 37C in duplicate with HLMs (1 mg/ml protein) and mean and 90% confidence interval of the observed cumulative urinary excretion 5 mM UDPGA. The two putative glucuronide metabolites in each panel are consistent (nmol) at the end of the collection interval, respectively. 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 Discussion (EGLUC) 1 (black) and EGLUC2 (gray) (C). Glucuronide 1 and glucuronide 2 were assigned based on RTs in Figs. 1–3 and re outlined in Table 2. Curves denote Racemic bupropion undergoes complex metabolism catalyzed by model-generated values obtained from nonlinear regression performed by fitting the multiple enzymes to yield numerous metabolites in humans. These Michaelis-Menten or Hill equation to substrate concentration versus apparent metabolites are important contributors to the therapeutic and toxic effects metabolite formation velocity using Phoenix WinNonlin (version 6.4). Symbols and error bars denote mean and S.D., respectively, of duplicate incubations. of the drug, but relatively little is known about the mechanisms governing their metabolism and elimination from the body. This report is the first to describe the stereoselective separation and detection of glucuronides of wouldbeexpectedtoundergofurther metabolism via conjugation bupropion enantiomers excreted in the urine of healthy human volunteers, reactions (glucuronidation and/or sulfation) to enhance hydrophilicity UGT-mediated elimination pathways using HLMs and bupropion enan- and promote renal elimination. Indeed, glucuronide metabolites of tiomers (both individual and racemic) as substrates to infer the glucuronide hydroxybupropion and threo- and erythro-hydrobupropion have been stereochemistry and in vitro to in vivo extrapolation, and the predominant detected in human urine after therapeutic doses of racemic bupropion UGT isoforms responsible for catalyzing glucuronidation of bupropion (Welch et al., 1987; Petsalo et al., 2007; Benowitz et al., 2013). The metabolites using a panel of recombinant UGTs. These data provide a estimated amount of free and total bupropion metabolites excreted in the deeper understanding of mechanisms underlying the disposition of urine from healthy volunteers administered bupropion to steady-state pharmacologically active bupropion metabolites. indicates that glucuronide metabolites account for approximately 75%, Introduction of a hydroxy functional group occurs during both the 25%, and 10% of total excreted hydroxybupropion, erythro- oxidative and reductive metabolism of bupropion to yield hydroxybupro- hydrobupropion, and threo-hydrobupropion, respectively (Benowitz pion as well as erythro- and threo-hydrobupropion. These metabolites et al., 2013). These percentages mirror the observed rank order in our 550 Gufford et al.

TABLE 4 Bupropion glucuronidation kineticsa

d e Substrate Km or S50 (mM) Vmax Clint or Clmax

mM pmol/min/mg ml/min/mg (S,S)-hydroxybupropion glucuronidea 172 6 38.9 739 6 59.6 4.31 (R,R)-hydroxybupropion glucuronidea 488 6 98.3 5550 6 507 11.4 (1R,2R)-hydrobupropion glucuronideb 343 6 37.5 3290 6 269 7.7 (1S,2S)-hydrobupropion glucuronideb 248 6 27.1 358 6 25.5 1.1 EGLUC1c 373 6 63.0 1740 6 212 3.1 EGLUC2c 360 6 55.2 2280 6 241 3.8

Values represent the parameter estimate 6 S.E. obtained by fitting the Michaelis-Mentena (hydroxybupropion) or Hill. bGlucuronide 1 and glucuronide 2 denoted based on retention times in Figs. 1–3 and outlined in Table 2. Erythro-hydrobupropion glucuronide (EGLUC). c(threo- and erythro-hydrobupropion) equation to [substrate] versus metabolite formation velocity using Phoenix WinNonlin (version 6.4). d Intrinsic clearance (Clint), calculated as the ratio of Vmax to Km. e 1/h Maximal clearance (Clmax), calculated as the ratio of Vmax  (h 2 1) to S50 +h(h2 1) . Downloaded from study but differ slightly in absolute amount; however, the previously healthy volunteers administered bupropion) (Petsalo et al., 2007) can reported indirect method (subtractive using deconjugation by occur during extended enzymatic incubations, and only the sum b-glucuronidase) (Benowitz et al., 2013) does not specifically provide of racemic mixtures were reported. This important difference in information on glucuronide and sulfate conjugates, as nonselective analytical methods likely explains the discrepancy between our work cleavage of sulfate conjugates (qualitatively identified in urine of and previous reports. Nonetheless, these findings provide further dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 6. Bupropion glucuronide formation using HLMs and a panel of 13 rUGT isoforms. HLMs (1 mg/ml) or rUGT (0.5 mg/ml) were incubated with hydroxybupropion, (R,R)-hydroxybu- propion (50 mM), (S,S)-hydroxybupropion (50 mM), threo- hydrobupropion (100 mM), or erythro-hydrobupropion (100 mM) and UDPGA (5 mM) (250 ml final reaction volume) at 37C for 60 minutes. Velocities correspond to the formation of (S,S)-hydroxybupropoin glucuronide (A), (R,R)-hydroxybupro- pion glucuronide (B), (1R,2R)-hydrobupropion glucuronide (C), (1S,2S)-hydrobupropion glucuronide (D), erythro-hydro- bupropion glucuronide 1 (EGLUC1) (E), or erythro-hydro- bupropion glucuronide 2 (EGLUC2) (F). Glucuronide 1 and glucuronide 2 were assigned based on RT in Figs. 1–3 and are outlined in Table 2. Bars and error bars denote the mean and S.D., respectively, of duplicate incubations. Stereoselective Bupropion Glucuronidation 551 confirmation that bupropion primary metabolites undergo metabo- metabolite. Overall, direct description of stereoselective urinary excretion lism via conjugation before urinary excretion. of bupropion and metabolites at 48 hours agreed well with a previous Herein is the first report describing stereoselective glucuronidation of (indirect, nonstereoselective) report of urinary recovery at 24 hours bupropion metabolites. We have directly identified two glucuronides (Benowitz et al., 2013) and with findings after administration of each of hydroxybupropion, erythro-hydrobupropion, and threo- C14-labeled bupropion (Johnston et al., 2002). hydrobupropion in the urine of healthy volunteers administered a The enzymes responsible for the oxidative and reductive metabolism single 100-mg oral dose of bupropion. A previous study reported two of bupropion to form hydroxybupropion, erythro-hydrobupropion, and glucuronide metabolites of hydroxybupropion and four glucuronides of threo-hydrobupropion have been investigated extensively (Faucette threo-/erythro-hydrobupropion mixtures in human urine (Petsalo et al., et al., 2000, 2001; Hesse et al., 2000; Bondarev et al., 2003; Damaj 2007), which could suggest stereoselective glucuronidation; however, et al., 2004; Molnari and Myers, 2012; Zhu et al., 2012; Meyer et al., this study did not elucidate isomeric structures nor identify specific 2013; Skarydova et al., 2014). Metabolite exposure is presumably enantiomeric substrates. In addition, no quantitative information was dependent on metabolic pathways leading to their formation and provided to describe glucuronide disposition. Our data provide for the first elimination; however, the enzymes responsible for glucuronidation time structural description of these glucuronides and, using in vitro and subsequent renal elimination of the pharmacologically important experiments, show enantiomers from which these glucuronide metabolites bupropion metabolites have not been fully elucidated. Here we originate. Of note, these glucuronides were chromatographically separated quantitatively describe the stereoselective hepatic glucuronidation of using achiral column chemistry, whereas enantiomers of their proximate bupropion. The most efficiently formed bupropion glucuronide in vitro, Downloaded from substrates are not (present data and Petsalo et al., 2007), suggesting (R,R)-hydroxybupropion glucuronide was also the predominant urinary substantially unique configuration of the glucuronide metabolites com- conjugate observed in vivo. Similarly, the next most efficiently formed pared with their corresponding substrates. After a single oral dose of glucuronide in vitro, threo-hydrobupropion glucuronide 1, was also the racemic bupropion, the glucuronides of hydroxybupropion were the second most abundant glucuronide excreted in the urine. Interestingly, predominant conjugates detected in urine, in agreement with a previous erythro-hydrobupropion glucuronide formation demonstrated similar in report conducted at steady state (Benowitz et al., 2013). Of the vitro efficiency but nearly 4-fold differences in mean urinary excretion, glucuronides detected in urine, (R,R)-hydroxybupropion was the most suggesting that additional mechanisms may underlie differential dmd.aspetjournals.org prevalent species, providing further evidence for enantioselective urinary urinary excretion of these metabolites. elimination of bupropion metabolites and conjugates. We confirm that Characterization of the UGTs responsible for the conjugation of threo-hydrobupropion is the primary unconjugated urinary bupropion active bupropion metabolites is essential to understanding factors that at ASPET Journals on September 27, 2021

Fig. 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. 552 Gufford et al. may influence the interindividual and intraindividual variability in Fallon JK, Neubert H, Goosen TC, and Smith PC (2013) Targeted precise quantification of 12 human recombinant uridine-diphosphate glucuronosyl transferase 1A and 2B isoforms using bupropion metabolite exposure, including the evaluation of potential nano-ultra-high-performance liquid chromatography/tandem mass spectrometry with selected drug-drug interactions and pharmacogenetic implications. UGT2B7 reaction monitoring. Drug Metab Dispos 41:2076–2080. Faucette SR, Hawke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM, and Lindley CM (2000) and UGT1A9, both being prominently expressed in the liver (Margaillan Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 et al., 2015b), are likely the dominant isoforms responsible for hepatic catalytic activity. Drug Metab Dispos 28:1222–1230. glucuronidation of bupropion metabolites. These isoforms may also Faucette SR, Hawke RL, Shord SS, Lecluyse EL, and Lindley CM (2001) Evaluation of the contribution of cytochrome P450 3A4 to human liver microsomal bupropion hydroxylation. play an important role in the extrahepatic glucuronidation of bupropion, Drug Metab Dispos 29:1123–1129. particularly in the kidney where UGT1A9 is the predominant isoform Ferris RM, Cooper BR, and Maxwell RA (1983) Studies of bupropion’s mechanism of antide- – expressed (Margaillan et al., 2015a) and in the gut where UGT2B7 and pressant activity. J Clin Psychiatry 44:74 78. Findlay JW, Van Wyck Fleet J, Smith PG, Butz RF, Hinton ML, Blum MR, and Schroeder DH UGT1A9 are present (Harbourt et al., 2012; Fallon et al., 2013). Further (1981) Pharmacokinetics of bupropion, a novel antidepressant agent, following oral adminis- exploration of extrahepatic bupropion metabolism could more com- tration to healthy subjects. Eur J Clin Pharmacol 21:127–135. Food and Drug Administration Center for Drug Evaluation and Research (FDA CDER) (2012) prehensively describe bupropion metabolite disposition and facilitate Drug interaction studies: study design, data analysis, implications for dosing, and labeling quantitative in vitro-in vivo extrapolation of bupropion metabolite recommendations (draft guidance). Food and Drug Administration Center for Drug Evaluation and Research (FDA CDER) (2013) kinetics (Gill et al., 2012, 2013). Bioanalytical Method Validation (draft guidance). In summary, racemic bupropion undergoes complex metabolism Gill KL, Gertz M, Houston JB, and Galetin A (2013) Application of a physiologically based catalyzed by multiple enzymes to yield numerous metabolites in pharmacokinetic model to assess propofol hepatic and renal glucuronidation in isolation: utility of in vitro and in vivo data. Drug Metab Dispos 41:744–753. humans. These metabolites are important contributors to the therapeutic

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