Pharmacogenetic Influence on Stereoselective Steady-State Disposition of Bupropion.

Bupropion is used for treating depression, obesity, and seasonal affective disorder, and for smoking cessation. Bupropion is commonly prescribed, but has complex pharmacokinetics and interindividual variability in metabolism and bioactivation may influence therapeutic response, tolerability, and safety. Bupropion is extensively and stereoselectively metabolized, the metabolites are pharmacologically active, and allelic variation in cytochrome P450 (CYP) 2B6 affects clinical hydroxylation of single-dose bupropion. Genetic effects on stereoselective disposition of steady-state bupropion are not known. In this preplanned secondary analysis of a prospective, randomized, double-blinded, crossover study which compared brand and generic bupropion XL 300 mg drug products, we measured steady-state enantiomeric plasma and urine parent bupropion and primary and secondary metabolite concentrations. This investigation evaluated the influence of genetic polymorphisms in CYP2B6, CYP2C19, and P450 oxidoreductase on the disposition of Valeant Pharmaceuticals Wellbutrin brand bupropion in 67 participants with major depressive disorder. We found that hydroxylation of both bupropion enantiomers was lower in carriers of the CYP2B6*6 allele and in carriers of the CYP2B6 516G>T variant, with correspondingly greater bupropion and lesser hydroxybupropion plasma concentrations. Hydroxylation was 25-50% lower in CYP2B6*6 carriers and one-third to one-half less in 516T carriers. Hydroxylation of the bupropion enantiomers was comparably affected by CYP2B6 variants. CYP2C19 polymorphisms did not influence bupropion plasma concentrations or hydroxybupropion formation but did influence the minor pathway of 4'-hydroxylation of bupropion and primary metabolites. P450 oxidoreductase variants did not influence bupropion disposition. Results show that CYP2B6 genetic variants affect steady-state metabolism and bioactivation of Valeant brand bupropion, which may influence therapeutic outcomes. SIGNIFICANCE STATEMENT: Bupropion, used for depression, obesity, and smoking cessation, undergoes metabolic bioactivation, with incompletely elucidated interindividual variability. We evaluated cytochrome P450 (CYP) 2B6, CYP2C19 and P450 oxidoreductase genetic variants and steady-state bupropion and metabolite enantiomers disposition. Both enantiomers hydroxylation was lower in CYP2B6*6 and CYP2B6 516G>T carriers, with greater bupropion and lesser hydroxybupropion plasma concentrations. CYP2C19 polymorphisms did not affect bupropion or hydroxybupropion but did influence minor 4'-hydroxylation of bupropion and primary metabolites. CYP2B6 variants affect steady-state bupropion bioactivation, which may influence therapeutic outcomes.

Does sex alter the relationship between CYP2B6 variation, hydroxybupropion concentration and bupropion-aided smoking cessation in African Americans? A moderated mediation analysis.

BACKGROUND AND AIMS: CYP2B6, a genetically variable enzyme, converts bupropion to its active metabolite hydroxybupropion. CYP2B6 activity and bupropion-aided cessation differ between women and men. The aim of this study was to determine whether genetically normal (versus reduced) CYP2B6 activity increases bupropion-aided cessation in African American smokers via higher hydroxybupropion concentration, and whether this differs by sex. DESIGN AND SETTING: Secondary analysis of a smoking cessation clinical trial (NCT00666978). PARTICIPANTS/CASES: African American light smokers (≤ 10 cigarettes/day). INTERVENTIONS: Participants were treated with bupropion for 7 weeks. MEASUREMENTS: Participants with detectable bupropion and/or hydroxybupropion concentrations were divided into normal (n = 64) and reduced (n = 109) CYP2B6 activity groups based on the presence of decreased-function CYP2B6*6 and CYP2B6*18 alleles. Biochemically verified smoking cessation was assessed at week 3, end of treatment (7 weeks) and follow-up (26 weeks). FINDINGS: Normal (versus reduced) CYP2B6 activity was associated with increased cessation at week 7, which was mediated by higher hydroxybupropion concentration [odds ratio (OR) = 1.25, 95% confidence interval (CI) = 1.03, 1.78]; this mediation effect persisted at week 26 (OR = 1.23, 95% CI = 1.02, 1.70). The mediation effect was similar in women (n = 116; OR = 1.33, 95% CI = 1.01, 2.30) and men (n = 57; OR = 1.33, 95% CI = 0.92, 3.87). Moreover, sex did not appear to moderate the mediation effect, although this should be tested in a larger sample. CONCLUSIONS: In African American light smokers with verified early bupropion use, genetically normal CYP2B6 activity appears to be indirectly associated with greater smoking cessation success in a relationship mediated by higher hydroxybupropion concentration. The mediating effect of higher hydroxybupropion concentration on smoking cessation persists beyond the active treatment phase and does not appear to differ by sex.

Association of CYP2B6 genetic polymorphisms with bupropion and hydroxybupropion exposure: A systematic review and meta-analysis.

INTRODUCTION: Bupropion is metabolized to its active metabolite, hydroxybupropion (HB), by the genetically polymorphic cytochrome P450 2B6 (CYP2B6) enzyme. Despite its significant role in bupropion metabolism, the magnitude of the impact of CYP2B6 genotype on the exposure of bupropion has not been quantified. OBJECTIVES: A systematic review and meta-analysis was conducted to quantify the association of bupropion and HB exposure with CYP2B6 variant alleles and genotype-defined metabolizer phenotypes. METHODS: MEDLINE, EMBASE, Web of Science, Scifinder, PsycINFO, and CENTRAL were screened to identify studies that met the following inclusion criteria (search updated on February 2021): (1) area under the plasma drug concentration-time curve (AUC) of bupropion and/or HB in relation to CYP2B6 genotypes was studied, and (2) study participants were genotyped for common CYP2B6 variant alleles including at least CYP2B6*6. The Newcastle Ottawa Scale was used to assess risk of bias in each included study. The ratio of means (RoM) between CYP2B6 genotype or genotype-defined phenotype groups for bupropion exposure was calculated for each study and combined in a meta-analysis. RESULTS: Eleven studies met the inclusion criteria for this systematic review, and 10 (including N = 413 participants) were included in the meta-analysis. All 10 studies involved healthy adult volunteers, where other medications were not allowed. The AUCs of HB and the active moiety (bupropion + HB) were significantly reduced in CYP2B6*6 carriers compared with the non-carriers (HB: RoM 0.77, 95% CI 0.71-0.83; active moiety: RoM 0.81, 95% CI 0.75-0.88). Both CYP2B6 poor and intermediate metabolizers had significantly decreased exposures to HB and the active moiety than normal metabolizers. CONCLUSION: The CYP2B6*6 allele and genotype-determined CYP2B6 poor and intermediate metabolizer phenotypes are associated with significantly lower exposures to HB and the total active moiety. The findings of this study suggest opportunities to further study precision dosing strategies for bupropion therapy based on CYP2B6 genotype.

Characterization of the Effect of Upadacitinib on the Pharmacokinetics of Bupropion, a Sensitive Cytochrome P450 2B6 Probe Substrate.

This phase 1 study characterized the effect of multiple doses of upadacitinib, an oral Janus kinase 1 selective inhibitor, on the pharmacokinetics of the cytochrome P450 (CYP) 2B6 substrate bupropion. Healthy subjects (n = 22) received a single oral dose of bupropion 150 mg alone (study period 1) and on day 12 of a 16-day regimen of upadacitinib 30 mg once daily (study period 2). Serial blood samples for measurement of bupropion and hydroxybupropion plasma concentrations were collected in each study period. The central values (90% confidence intervals) for the ratios of change were 0.87 (0.79-0.96) for bupropion maximum plasma concentration (Cmax ), 0.92 (0.87-0.98) for bupropion area under the plasma-concentration time curve from time 0 to infinity (AUCinf ), 0.78 (0.72-0.85) for hydroxybupropion Cmax , and 0.72 (0.67-0.78) for hydroxybupropion AUCinf when administered with, relative to when administered without, upadacitinib. After multiple-dose administration of upadacitinib 30 mg once daily, upadacitinib mean ± SD AUC0-24 was 641 ± 177 ng·h/mL, and Cmax was 83.3 ± 30.7 ng/mL. These results confirm that upadacitinib has no relevant effect on pharmacokinetics of substrates metabolized by CYP2B6.

Subtherapeutic bupropion and hydroxybupropion serum concentrations in a patient with CYP2C19*1/*17 genotype suggesting a rapid metabolizer status.

Bupropion is hydroxylated to its primary active metabolite hydroxybupropion by cytochrome P450 enzyme CYP2B6. In vitro data suggest the existence of alternative hydroxylation pathways mediated by the highly polymorphic enzyme CYP2C19. However, the impact of its genetic variants on bupropion metabolism in vivo is still under investigation. We report the case of a 28-year-old male Caucasian outpatient suffering from major depressive disorder who did not respond to a treatment with bupropion. Therapeutic drug monitoring revealed very low serum concentrations of both bupropion and hydroxybupropion. Genotyping identified a heterozygous status for the gain-of-function allele with the genotype CYP2C19*1/*17 predicting enhanced enzymatic activity. The present case shows a reduced bupropion efficacy, which may be explained by a reduced active moiety of bupropion and its active metabolite hydroxybupropion, due to alternative hydroxylation pathways mediated by CYP2C19 in an individual with CYP2C19 rapid metabolizer status. The case report thus illustrates the clinical relevance of therapeutic drug monitoring in combination with pharmacogenetics diagnostics for a personalized treatment approach.

Determination of hydroxybupropion in human serum for routine therapeutic drug monitoring in psychiatry: A tool for dose-individualization in treatment with bupropion.

Therapeutic drug monitoring (TDM) has become a clinical routine in psychiatry. Nevertheless, for bupropion there is only one method available that is suitable for routine use. However, it involves a complex sample clean-up. Owing to the instability of bupropion in serum, the main and active metabolite hydroxybupropion was chosen as the target substance. Therefore, a simple and robust high-performance liquid chromatography method for the quantification of hydroxybupropion in serum was developed and validated. A volume of 30 µL serum was used for easy sample clean-up, based on protein precipitation with acetonitrile followed by online solid-phase extraction. As hydroxybupropion was present in high serum concentrations, UV detection was possible. Owing to the commonly available instrumentation, the method could easily be integrated in routine TDM. The newly developed method was validated following the guidelines for bioanalytical method validation of the European Medicines Agency and US Food and Drug Administration. The lower limit of quantification was 100 ng/mL (0.391 µm) and linearity was shown between 100 and 2500 ng/mL. Intraday and interday precision ranged from 1.17 to 6.79% and from 6.07 to 9.41%, respectively. Intraday and interday accuracy ranged from 89.97 to 110.86% and from 95.05 to 101.2%. The method was shown to be selective, accurate and precise. Additionally, the method was successfully implemented in the therapeutic drug monitoring laboratory of the Department of Psychiatry, Psychosomatics and Psychotherapy at the University Hospital of Würzburg, Germany. Six months of routine analysis showed a rather low correlation between applied dose and serum concentration and therefore the necessity of TDM for dose-individualization in the treatment with bupropion.

Effects of Genetic Polymorphisms of CYP2B6 on the Pharmacokinetics of Bupropion and Hydroxybupropion in Healthy Chinese Subjects.

BACKGROUND Bupropion (BUP) is an antidepressant and its pharmacological activity is mediated by its major metabolite, hydroxybupropion (HBUP). We investigated the effects of genetic polymorphisms of CYP2B6 on BUP and HBUP to provide certain evidence on the clinical rational administration of BUP. MATERIAL AND METHODS Plasma BUP and HBUP concentrations were assayed using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS A total of 23 healthy volunteers (eleven participants with CYP2B6*1/*1, 7 participants with CYP2B6*1/*6, 3 participants with CYP2B6*4/*6, and 2 participants with CYP2B6*1/*4) received orally administered 150 mg of BUP according to protocol. Blood samples were obtained up to 96 hours after administration. The whole blood was subject to genotyping by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The concentration-time curve (AUC(0→96)), maximum plasma concentration (Cmax), and terminal half-life (t1/2) values of BUP in CYP2B6*1/*4 were lower than those of CYP2B6*1/*1. By contrast, the time to Cmax (tmax) value of the former was higher than that of the latter. The HBUP AUC(0→96) values in CYP2B6*4/*6 and CYP2B6*1/*4 increased to values 1.12-fold and 1.98-fold, compared with CYP2B6*1/*1 carriers. However, the HBUP AUC(0→96) value in CYP2B6*1/*1 was 1.51-fold higher than that in CYP2B6*1/*6. Similarly, the HBUP Cmax values in CYP2B6*4/*6 and CYP2B6*1/*4 increased by 1.12-fold and 1.97-fold, whereas the HBUP Cmax value in CYP2B6*1/*6 decreased to a value 1.64-fold lower than that in CYP2B6*1/*1. CONCLUSIONS Genetic polymorphisms of CYP2B6 influence the pharmacokinetic parameters of BUP and HBUP and thus establish rational BUP administration for Chinese patients in clinical settings.

Chirality and neuropsychiatric drugs: an update on stereoselective disposition and clinical pharmacokinetics of bupropion.

1. Bupropion, an antidepressant drug has been approved as a racemate containing equal amounts of R- and S-enantiomers. Recently, the chirality of bupropion has received significant attention in the delineation of stereoselective pharmacokinetic (PK) and disposition data. Although the non-stereoselective metabolism of bupropion was well established, the emerging data suggest that bupropion exhibits complex stereoselective metabolism, leading to the formation of various stereoisomeric metabolites. Along with the chiral PKs of bupropion, hydroxybupropion, threohydrobupropion and erythrohydrobupropion, the metabolism data also provided insights into the roles of both CYP2B6 and CYP2C19 enzymes in the stereoselective disposition. Furthermore, the metabolism studies also suggested specific involvement of CYP2B6 pathway in the stereoselective hydroxylation of bupropion to R,R-hydroxybupropion, which was considered as a better marker for CYP2B6 activity. 2. Other significant learnings were: (1) understanding the in vivo CYP2D6 inhibitory potential of bupropion with respect to the chirality of parent drug and the metabolites; (2) the potential involvement of bupropion and metabolites towards significant down regulation of CYP2D6 mRNA; (3) significant in vivo CYP2D6 inhibitory activity (86%) exhibited by R,R-hydroxybupropion and threohydrobupropion. 3. The newly published data on chiral PKs and disposition of bupropion and its metabolites can be used to gauge the drug-drug interaction potential when bupropion is combined in clinical therapy. Moreover, such data would be useful to understand the consequences (if any), due to the combination of bupropion with other drugs both from a safety and efficacy perspective because of the prevalence of polypharmacy situations in many therapeutic areas including CNS indications.

Bupropion Overdose Resulted in a Pharmacobezoar in a Fatal Bupropion (Wellbutrin® ) Sustained-release Overdose: Postmortem Distribution of Bupropion and its Major Metabolites.

Bupropion (BUP) overdose commonly causes generalized seizures and central nervous system depression. The case of a 28-year-old woman who died from a massive lethal overdose with sustained-release bupropion (Wellbutrin® 300 mg) is herein presented. The autopsy revealed the presence of a pharmacobezoar consisting of at least 40 tablets in the stomach. Determination of bupropion and its active metabolites (hydroxybupropion, threobupropion, erythrobupropion) was achieved by a liquid chromatographic mass spectrometry (LC-MS/MS) method. Postmortem concentrations for bupropion, hydroxybupropion, threobupropion, and erythrobupropion were obtained in intracranial blood, urine, bile, liver, kidney, and vitreous humor. In this case, intracranial blood level of the parent drug was 1.9 mg/L. Threobupropion was the most abundant metabolite in both blood and urine, 59.3 and 890.6 mg/L. Tissue distribution showed the highest concentration in the liver, 12.3 mg/kg. The 0.8 bupropion concentration ratio vitreous/blood suggested that vitreous could be a valuable specimen for toxicological analysis should postmortem blood be unavailable.

Design of experiment assisted concurrent enantioseparation of bupropion and hydroxybupropion by high-performance thin-layer chromatography.

A simple and efficient high-performance thin-layer chromatographic method was developed for chiral separation of rac-bupropion (BUP) and its active metabolite rac-hydroxybupropion (HBUP). Design of experiment (DoE)-based optimization was adopted instead of a conventional trial-and-error approach. The Box-Behnken design surface response model was used and the operating variables were optimized based on 17 trials design. The optimized method involved impregnation of chiral reagent, L(+)-tartaric acid, in the stationary phase with simultaneous addition in the mobile phase, which consisted of acetonitrile : methanol : dichloromethane : 0.50% L-tartaric acid (6.75:1.0:1.0:0.25, v/v/v/v). Under the optimized conditions, the resolution factor between the enantiomers of BUP and HBUP was 6.30 and 9.26, respectively. The limit of detection and limit of quantitation for (R)-BUP, (S)-BUP, (R,R)-HBUP, and (S,S)-HBUP were 9.23 and 30.78 ng spot-1 , 10.32 and 34.40 ng spot-1 , 12.19 and 40.65 ng spot-1 , and 14.26 and 47.53 ng spot-1 , respectively. The interaction of L-tartaric acid with analytes and their retention behavior was thermodynamically investigated using van't Hoff's plots. The developed method was validated as per the International Conference on Harmonization guidelines. Finally, the method was successfully applied to resolve and quantify the enantiomeric content from marketed tablets as well as spiked plasma samples.

In vivo effect of borneol on rat hepatic CYP2B expression and activity.

CYP2B subfamily accounts for 2-10% of total hepatic CYP450 enzymes and participate in the metabolism of around 8% of clinical drugs. Borneol has been widely used in traditional Chinese medicine for thousands of years. There are many studies about borneol-induced promoting penetration role for a number of drugs through various physiologic barriers, whereas there is no report involved the effect of borneol on hepatic CYP2B. The present work studied the in vivo effect of borneol on the expression and activity of rat hepatic CYP2B. The results indicated that the oral administration of borneol (33, 100 and 300 mg/kg/d) to rats for consecutive 7 days increased the hepatic CYP2B1/2 activity by 1.4-, 1.7- and 2.8-fold, hepatic CYP2B1 mRNA expression by 6.3-, 8.7- and 18.1-fold, and hepatic CYP2B1/2 protein expression by 1.2-, 1.9- and 2.6-fold, respectively compared to the control. Additionally, in the borneol pre-dosing (300 mg/kg/d for consecutive 7 days) rats, the increased Clint and decreased AUC0-24 of bupropion were observed as compared to the control. Moreover, there were no obvious effects on CAR protein level in rat liver microsome and nucleus following the borneol treatment. Taken together, our observations indicate that borneol is an in vivo inducer of rat hepatic CYP2B with different regulatory mechanism from phenobarbital-like inducers which caused CYP2B induction with CAR activation.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors.

The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT3ARs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT3ARs were heterologously expressed in HEK-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT3ARs reversibly and dose-dependently with inhibitory potencies of 87 µM and 112 µM, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect.

Identification of non-reported bupropion metabolites in human plasma.

Bupropion and its three active metabolites exhibit clinical efficacy in the treatment of major depression, seasonal depression and smoking cessation. The pharmacokinetics of bupropion in humans is highly variable. It is not known if there are any non-reported metabolites formed in humans in addition to the three known active metabolites. This paper reports newly identified and non-reported metabolites of bupropion in human plasma samples. Human subjects were dosed with a single oral dose of 75 mg of an immediate release bupropion HCl tablet. Plasma samples were collected and analysed by LC-MS/MS at 0, 6 and 24 h. Two non-reported metabolites (M1 and M3) were identified with mass-to-charge (m/z) ratios of 276 (M1, hydration of bupropion) and 258 (M3, hydroxylation of threo/erythrohydrobupropion) from human plasma in addition to the known hydroxybupropion, threo/erythrohydrobupropion and the glucuronidation products of the major metabolites (M2 and M4-M7). These new metabolites may provide new insight and broaden the understanding of bupropion's variability in clinical pharmacokinetics. © 2016 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.

Bupropion and its photoreactive analog (±)-SADU-3-72 interact with luminal and non-luminal sites at human α4ß2 nicotinic acetylcholine receptors.

The interaction of (±)-bupropion [(±)-BP] with the human (h) α4ß2 nicotinic acetylcholine receptor (AChR) was compared to that for its photoreactive analog (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72]. Ca2+ influx results indicated that (±)-SADU-3-72 and (±)-BP inhibit hα4ß2 AChRs with practically the same potency. However, (±)-SADU-3-72 binds to the [3H]imipramine sites at resting and desensitized hα4ß2 AChRs with 3-fold higher affinity compared to that for (±)-BP, which is supported by molecular docking results. The docking results also indicate that each isomer of BP and SADU-3-72, in the protonated state, interacts with luminal and non-luminal sites. In the channel lumen, both ligands bind to two overlapping subsites, one that overlaps the imipramine site, and another much closer to the cytoplasmic side. The results suggest, for the first time, three differentiated non-luminal domains, including the transmembrane (TMD), extracellular (ECD), and ECD-TMD junction. In the ECD-TMD junction, BP and SADU-3-72 bind to overlapping sites. Interestingly, only SADU-3-72 binds to intrasubunit and intersubunit sites in the TMD, and to additional sites in the ECD. Our results are consistent with a model where BP and SADU-3-72 bind to overlapping sites in the luminal and ECD-TMD junctional domains of the hα4ß2, whereas only SADU-3-72 binds to additional non-luminal sites. The BP junctional site opens the door for additional inhibitory mechanisms. The pharmacological properties of (±)-SADU-3-72 showed in this work support further photolabeling studies to mapping the BP binding sites in the hα4ß2 AChR.

Stereoselective Metabolism of Bupropion to OH-bupropion, Threohydrobupropion, Erythrohydrobupropion, and 4'-OH-bupropion in vitro.

Bupropion is a widely used antidepressant, smoking cessation aid, and weight-loss therapy. It is administered as a racemic mixture, but the pharmacokinetics and activity of bupropion are stereoselective. The activity and side effects of bupropion are attributed to bupropion and its metabolites S,S- and R,R-OH-bupropion, threohydrobupropion, and erythrohydrobupropion. Yet the stereoselective metabolism in vitro and the enzymes contributing to the stereoselective disposition of bupropion have not been characterized. In humans, the fraction of bupropion metabolized (fm) to the CYP2B6 probe metabolite OH-bupropion is 5-16%, but ticlopidine increases bupropion exposure by 61%, suggesting a 40% CYP2B6 and/or CYP2C19 fm for bupropion. Yet, the CYP2C19 contribution to bupropion clearance has not been defined, and the enzymes contributing to overall bupropion metabolite formation have not been fully characterized. The aim of this study was to characterize the stereoselective metabolism of bupropion in vitro to explain the stereoselective pharmacokinetics and the effect of drug-drug interactions (DDIs) and CYP2C19 pharmacogenetics on bupropion exposure. The data predict that threohydrobupropion accounts for 50 and 82%, OH-bupropion for 34 and 12%, erythrohydrobupropion for 8 and 4%, and 4'-OH-bupropion for 8 and 2% of overall R- and S-bupropion clearance, respectively. The fm,CYP2B6 was predicted to be 21%, and the fm,CYP2C19, 6% for racemic bupropion. Importantly, ticlopidine was found to inhibit all metabolic pathways of bupropion in vitro, including threohydrobupropion, erythrohydrobupropion, and 4'OH-bupropion formation, explaining the in vivo DDI. The stereoselective pharmacokinetics of bupropion were quantitatively explained by the in vitro metabolic clearances and in vivo interconversion between bupropion stereoisomers.

Pharmacokinetics of Bupropion and Its Pharmacologically Active Metabolites in Pregnancy.

Bupropion sustained release is used to promote smoking cessation in males and nonpregnant females. However, its efficacy as a smoking cessation aid during pregnancy is not reported. The pregnancy-associated changes in maternal physiology may alter the pharmacokinetics and pharmacodynamics of bupropion and consequently its efficacy in pregnant smokers. Therefore, the aims of this study were to determine the steady-state pharmacokinetics of bupropion during pregnancy and the effect of functional genetic variants of CYP2B6 and CYP2C19 on bupropion pharmacokinetics in pregnant women. Plasma and urine concentrations of bupropion and its metabolites hydroxybupropion (OHBUP), threohydrobupropion, and erythrohydrobupropion were determined by liquid chromatography-mass spectrometry. Subjects were genotyped for five nonsynonymous single-nucleotide polymorphisms that result in seven CYP2B6 alleles, namely *2, *3, *4, *5, *6, *7, and *9, and for CYP2C19 variants *2, *3, and *17 The present study reports that the isoform-specific effect of pregnancy on bupropion-metabolizing enzymes along with the increase of renal elimination of the drug could collectively result in a slight decrease in exposure to bupropion in pregnancy. In contrast, pregnancy-induced increase in CYP2B6-catalyzed bupropion hydroxylation did not impact the plasma levels of OHBUP, probably due to a higher rate of OHBUP glucuronidation, and renal elimination associated with pregnancy. Therefore, exposure to OHBUP, a pharmacologically active metabolite of the bupropion, appears to be similar to that of the nonpregnant state. The predicted metabolic phenotypes of CYP2B6*6 and variant alleles of CYP2C19 in pregnancy are similar to those in the nonpregnant state.

The P450 oxidoreductase (POR) rs2868177 and cytochrome P450 (CYP) 2B6*6 polymorphisms contribute to the interindividual variability in human CYP2B6 activity.

AIM: To investigate whether single-nucleotide polymorphisms (SNPs) in the P450 oxidoreductase (POR) gene were correlated with interindividual variations in cytochrome P450 (CYP) 2B6 activity. METHODS: Thirty-six healthy volunteers who tested CYP2B6 and POR polymorphisms were enrolled in the study. CYP2B6 activity was measured by bupropion hydroxylation with LC/MS/MS. The ratio of hydroxybupropion versus bupropion (AUC_hyd/AUC_bup) in terms of area under the time-concentration curve (AUC) was used to represent the CYP2B6 activity. RESULTS: The volunteers carrying CYP2B6*1/*1 showed a significantly higher mean AUC_hyd/ AUC_bup than those CYP2B6*1/*6 and CYP2B6*6/*6 variants (15.66 ± 1.65 vs. 9.25 ± 1.92, P = 0.008 and 15.66 ± 1.65 vs. 8.21 ± 1.74, P = 0.006, respectively). POR rs2868177 (6593 A > G) AA homozygotes showed a significantly lower mean AUC_hyd/ AUC_bup than that of POR rs2868177 AG heterozygotes or GG homozygotes (8.13 ± 1.37 vs. 12.15 ± 2.97, P = 0.005 and 8.13 ± 1.37 vs. 17.59 ± 3.25, P = 0.001, respectively). Moreover, POR rs2868177 AG heterozygotes and GG homozygotes showed a significantly increased mean AUC_hyd/AUC_bup than AA homozygotes in the CYP2B6*1/*1 and CYP2B6*6 carriers (16.40 ± 2.01 vs. 12.40 ± 1.45, P = 0.006 and 10.65 ± 1.47 vs. 6.54 ± 1.25, P = 0.004, respectively). Meanwhile, a strong correlation between the genetic variations (POR rs2868177 and CYP2B6*6) and AUC_hyd/ AUC_bup was found (P = 0.009 and P = 0.001, respectively). There was no significant difference in the mean AUC_hyd/AUC_bup among different POR *28 genotypes (P > 0.05). CONCLUSION: POR rs2868177 and CYP2B6*6 variants contribute to the interindividual variability in human CYP2B6 activity, which may affect the disposition and interaction of other CYP2B6 substrate drugs.

Development, validation and application of a comprehensive stereoselective LC/MS-MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine.

A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion ß-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by ß-glucuronidase. Hydroxybupropion ß-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete ß-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion ß-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R) were 10, 50, 100, and 100ng/mL, respectively. The limits of quantification for (1R,2R)-threohydrobupropion ß-d-glucuronide, (1S,2S)-threohydrobupropion ß-d-glucuronide, and (1R,2R)-erythrohydrobupropion ß-d-glucuronide were each 50ng/mL. Due to the abundance of bupropion and metabolites in human urine, no efforts were made to optimize sensitivity. All analytes were stable following freeze thaw cycles at -80°C. This assay was applicable to clinical pharmacokinetic investigations of bupropion in patients and to in vitro metabolism of the primary bupropion metabolites to their glucuronides.

Bupropion therapy during pregnancy: the drug and its major metabolites in umbilical cord plasma and amniotic fluid.

BACKGROUND: Bupropion is used for treatment of depression during pregnancy. However, its use as a smoking cessation aid for pregnant women is currently under evaluation. OBJECTIVE: The aim of this opportunistic study was to investigate the transfer of bupropion and its major pharmacologically active metabolites, hydroxybupropion and threohydrobupropion, across the placenta in vivo. In addition, the concentrations of the drug and its metabolites were determined in the amniotic fluid. STUDY DESIGN: The following samples were collected at deliveries from 22 women taking bupropion: maternal blood (n = 22), umbilical cord venous blood (n = 22), and amniotic fluid (n = 9). The concentrations of the drug and its metabolites in blood plasma and amniotic fluid were determined by means of liquid chromatography-mass spectrometry. Placental passage was calculated as a ratio of umbilical cord venous plasma to maternal plasma concentrations. RESULTS: The levels of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were invariably lower than their corresponding concentrations in maternal plasma. The concentrations of bupropion in umbilical cord plasma were lower than in maternal plasma in the majority of the maternal-cord blood pairs. The median values of the umbilical cord venous plasma to maternal plasma ratios were: bupropion, 0.53 (interquartile range 0.35, n = 18), hydroxybupropion, 0.21 (interquartile range 0.12, n = 18), and threohydrobupropion, 0.61 (interquartile range 0.11, n = 21). In umbilical cord venous plasma, the median concentration of bupropion was 5.3 ng/mL; hydroxybupropion, 103.6 ng/mL; and threohydrobupropion, 59.6 ng/mL. Bupropion and its metabolites were detectable in the amniotic fluid but the concentrations of threohydrobupropion were higher than those in the corresponding umbilical cord venous plasma. CONCLUSION: Bupropion and its active metabolites cross the placenta to the fetal circulation. The concentrations of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were higher than bupropion concentrations suggesting a higher fetal exposure to the metabolites than the parent drug. The higher levels of threohydrobupropion in the amniotic fluid than those in umbilical cord venous plasma suggest that enzymes involved in the metabolism of bupropion to threohydrobupropion are most likely active in the fetus. The biological consequences of fetal exposure to maternally administered bupropion and/or its active metabolites via placental transfer and recirculation of the amniotic fluid are yet to be determined.

Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-min run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1ng/mL, respectively. All analytes were stable following freeze thaw cycles at -80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.