Bupropion Enantiomers Research Articles

Characterization of the Stereoselective Disposition of Bupropion and Its Metabolites in Rat Plasma and Brain.

Bupropion is an atypical antidepressant and smoking cessation aid; its use is associated with wide intersubject variability in efficacy and safety. Knowledge of the brain pharmacokinetics of bupropion and its pharmacologically active metabolites is considered important for understanding the cause-effect relationships driving this variability. Brain concentrations from rats administered a 10 mg/kg subcutaneous dose of racemic bupropion were analyzed using a stereoselective LC/MS-MS method. A 2 mg/kg dose of (S,S)-hydroxybupropion, which has comparable pharmacologic potency to bupropion, was administered to a separate group of rats. Plasma exposure and unbound concentrations in both matrices from companion equilibrium dialysis experiments were determined to assess potential carrier-mediated transport at the blood-brain barrier. Exposures to unbound forms of bupropion enantiomers were similar in plasma; this was also true in brain. This trend held for reductive diastereomer metabolite pairs in the two matrices. Unbound (R,R)-hydroxybupropion exposure was 1.5-fold higher than (S,S)-hydroxybupropion exposure in plasma and brain following bupropion administration. Unbound concentration ratios (Kp,uu) of a given molecular form decreased over time: between 4 and 6 h, these were < 1 for the two bupropion enantiomers, and they were ~ 1 for metabolites that formed. Administration of preformed (S,S)-hydroxybupropion also demonstrated a declining Kp,uu. The temporal shift in Kp,uu among the different molecular forms provides evidence regarding the operation of carrier-mediated transport and/or within-brain metabolism of bupropion, and, thereby, fresh insight regarding the causes of intersubject variability in the safety and efficacy of bupropion therapy.

Enantiomeric separation of bupropion by liquid chromatography on derivatized cyclofructan chiral stationary phase.

High-performance liquid chromatography (HPLC) is an ideal tool for enantiomeric separations of different drugs. In this study, the direct enantioseparation of bupropion, an atypical antidepressant structurally related to cathinone, was explored by using five chiral columns, including three based on derivatized cyclofructans, macrocyclic glycopeptide teicoplanin, and an immobilized amylose derivative under multimodal elution conditions. Baseline enantioseparation was obtained on the LarihcShell CF6-RN column, with derivatized cyclofructan 6, in the polar organic mode. The effects of the mobile-phase composition, type and content of major components, the nature and the amount of mobile-phase additives, and the column temperature on the retention, selectivity, and resolution were investigated to optimize enantioseparation. The calibration curve was linear in the range of 10-125 μg/ml for each enantiomer. The limits of detection and quantification were 0.1 and 0.3μg/ml for both enantiomers of bupropion. The chiral recognition was controlled especially by H bonds, π-π, dipole-dipole interactions, and steric effects. Finally, the developed method was applied to the determination of bupropion in the commercially available drug.

Population model analysis of chiral inversion and degradation of bupropion enantiomers, and application to enantiomer specific fraction unbound determination in rat plasma and brain

Pharmacologic effects elicited by drugs most directly relate to their unbound concentrations. Measurement of binding in blood, plasma and target tissues are used to estimate these concentrations by determining the fraction of total concentration in a biological matrix that is not bound. In the case of attempting to estimate R- and S-bupropion concentrations in plasma and brain following racemic bupropion administration, reversible chiral inversion and irreversible degradation of the enantiomers were hypothesized to confound attempts at unbound fraction estimation. To address this possibility, a kinetic modeling approach was used to quantify inversion and degradation specific processes for each enantiomer from separate incubations of each enantiomer in the two matrices, and in pH 7.4 buffer, which is also used in binding experiments based on equilibrium dialysis. Modeling analyses indicated that chiral inversion kinetics were two to four-fold faster in plasma and brain than degradation, with only inversion observed in buffer. Inversion rate was faster for S-bupropion in the three media; whereas, degradation rates were similar for the two enantiomers in plasma and brain, with overall degradation in plasma approximately 2-fold higher than in brain homogenate. Incorporation of degradation and chiral inversion kinetic terms into a model to predict enantiomer-specific binding in plasma and brain revealed that, despite existence of these two processes, empirically derived estimates of fraction unbound were similar to model-derived values, leading to a firm conclusion that observed extent of plasma and brain binding are accurate largely because binding kinetics are faster than parallel degradation and chiral inversion processes.

Common Polymorphisms of CYP2B6 Influence Stereoselective Bupropion Disposition

Bupropion hydroxylation is a bioactivation and metabolic pathway, and the standard clinical CYP2B6 probe. This investigation determined the influence of CYP2B6 allelic variants on clinical concentrations and metabolism of bupropion enantiomers. Secondary objectives evaluated the influence of CYP2C19 and P450 oxidoreductase variants. Healthy volunteers in specific cohorts (CYP2B6*1/*1, CYP2B6*1/*6, CYP2B6*6/*6, and also CYP2B6*4 carriers) received single-dose oral bupropion. Plasma and urine bupropion and hydroxybupropion was quantified. Subjects were also genotyped for CYP2C19 and P450 oxidoreductase variants. Hydroxylation of both bupropion enantiomers, assessed by plasma hydroxybupropion/bupropion AUC ratios and urine hydroxybupropion formation clearances, was lower in CYP2B6*6/*6 but not CYP2B6*1/*6 compared with CYP2B6*1/*1 genotypes, and numerically greater in CYP2B6*4 carriers. CYP2C19 and P450 oxidoreductase variants did not influence bupropion enantiomers hydroxylation or plasma concentrations. The results show that clinical hydroxylation of both bupropion enantiomers was equivalently influenced by CYP2B6 allelic variation. CYP2B6 polymorphisms affect S-bupropion bioactivation, which may affect therapeutic outcomes.

Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS.

Bupropion metabolites formed via oxidation and reduction exhibit pharmacological activity, but little is known regarding their stereoselective disposition. A novel stereoselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify enantiomers of bupropion, 4-hydroxybupropion, and erythro- and threo-dihydrobupropion. Liquid-liquid extraction was implemented to extract all analytes from 50 μL human plasma. Acetaminophen (APAP) was used as an internal standard. The analytes were separated on a Lux 3 μ Cellulose-3 250×4.6 mm column by methanol: acetonitrile: ammonium bicarbonate: ammonium hydroxide gradient elution and monitored using an ABSciex 5500 QTRAP triple-quadrupole mass spectrometer equipped with electrospray ionization probe in positive mode. Extraction efficiency for all analytes was ≥70%. The stability at a single non-extracted concentration for over 48 h at ambient temperature resulted in less than 9.8% variability for all analytes. The limit of quantification (LOQ) for enantiomers of bupropion and 4-hydroxybupropion was 0.3 ng/mL, while the LOQ for enantiomers of erythro- and threo-hydrobupropion was 0.15 ng/mL. The intra-day precision and accuracy estimates for enantiomers of bupropion and its metabolites ranged from 3.4% to 15.4% and from 80.6% to 97.8%, respectively, while the inter-day precision and accuracy ranged from 6.1% to 19.9% and from 88.5% to 99.9%, respectively. The current method was successfully implemented to determine the stereoselective pharmacokinetics of bupropion and its metabolites in 3 healthy volunteers administered a single 100mg oral dose of racemic bupropion. This novel, accurate, and precise HPLC-MS/MS method should enhance further research into bupropion stereoselective metabolism and drug interactions.

Resolution of enantiomers of bupropion and its metabolites by liquid chromatography.

Bupropion, a tricyclic aminoketone, is used primarily in the treatment of depression, the management of patients with bipolar and schizo-affective disorder, and the treatment of Parkinson's disease. Bupropion is marketed as a racemate, but the racemic mixture is known to have several disadvantages while the two isomers of bupropion and its metabolite differ significantly in their pharmacological activities. Therefore, the stereoselective determination of the drug enantiomers in pharamaceutical dosages, plasma or urine is of potential clinical and analytical importance. Different chromatographic methods have been employed for the separation of the two enantiomers. This is the first attempt to review the methods of enantiosepartion of bupropion using both direct and indirect approaches in both HPLC and TLC. The review presents a detailed discussion on the use of chiral stationary phase (based on polysaccharide, α1 acid glycoprotein and ovomucoid column) and chiral derivatizing reagents (based on isothiocyanate and cyanuric chloride) along with TLC separation of bupropion enantiomers using ligand exchange and impregnation methods. The focus is also on the separation mechanism for enantioresolution using the various methods described herein.

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.

Use of Enantiomeric Bupropion and Hydroxybupropion to Assess CYP2B6 Activity in Glomerular Kidney Diseases

Chronic kidney disease affects more than 50 million people in the U.S.1. The top three etiologies for end-stage kidney disease are diabetes mellitus (45% of patients), hypertension (29% of patients), and glomerulonephritis (19% of patients).2 While it is well established that chronic kidney diseases can result in decreased elimination of drugs via the kidneys, the effects of kidney disease on non-renal clearance processes, especially specific metabolic routes, are less well described. Experimental models of chronic kidney disease have reported reductions in hepatic cytochrome P450 (CYP) enzymes including 3A1, 3A2, 2C11, and N-acetyltransferases.3–5 Reductions in nonrenal clearance ranging from 30% to 67% have been reported for substrates of the CYP3A4, CYP2D6, CYP2B6, and CYP2C9 enzymes in patients with kidney disease.6 In order to fully understand the clinical significance of altered metabolic routes associated with kidney disease, it will be necessary to evaluate the effects that specific forms of kidney diseases have on these pathways and whether these processes effect drug disposition. Numerous studies have reported various probe drugs or cocktail approaches to evaluate the in vivo function of various drug metabolizing enzymes and transporters in patients.7, 8 However, only a few studies9–12 were actually conducted in chronic kidney disease patients, and hence applicability of most of the published studies beyond the healthy control population remains to be established. As chronic kidney disease patients are commonly prescribed ~10–12 different daily medications13, studies that evaluate metabolic pathway alterations should address requirements for modifications in drug regimens as well as drug interaction potential. While studies designed to assess and report the pharmacokinetics of bupropion exist14, 15, studies designed specifically to assess the influence of chronic kidney diseases on CYP2B6 activity are currently nonexistent. CYP2B6 is responsible for the metabolism of 3–8% of the currently marketed drugs16, 17 including bupropion, cyclophosphamide, efavirenz, selegiline, methadone, and sertraline18–22 and several drugs, including clopidrogrel, ticlopidine, clotrimazole, itraconazole, sertraline, and raloxifene have been purported to inhibit CYP2B6.23 Data from healthy subjects demonstrate that both the R and S enantiomers of bupropion and its hydroxy-metabolite are present in plasma, but only stereoselective S bupropion and (S,S) hydroxybupropion formation clearance have been shown to be a phenotypic probe for CYP2B624, complicating the assessment of in vivo activity. The purpose of the current study was to evaluate the pharmacokinetics of enantiomeric bupropion and its hydroxybupropion metabolite in patients with kidney diseases affecting the glomerulus in order to provide an assessment of CYP2B6 activity in this disease state.

Enantiomeric separation of bupropion enantiomers by electrokinetic chromatography: Quantitative analysis in pharmaceutical formulations

The first CE method enabling the quantitation of the two enantiomers of bupropion was developed in this work. Electrokinetic chromatography (EKC) mode using cyclodextrins as chiral selectors was employed. A study on the enantiomeric separation ability of different neutral and anionic CDs was carried out. Sulfated-β-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, temperature, and separation voltage on the enantiomeric separation of bupropion was also studied. The use of 10 mM sulfated-β-CD in 50 mM borate buffer (pH 9.0) with an applied voltage of 30 kV and a temperature of 30 °C enabled the separation of the enantiomers of bupropion with high resolution ( R s > 7) and short analysis time (∼3.5 min). Finally, the method was successfully applied to the quantitation of bupropion in two pharmaceutical formulations.

Stereoselective Metabolism of Bupropion by Cytochrome P4502B6 (CYP2B6) and Human Liver Microsomes

Hydroxylation of the antidepressant and smoking deterrent drug bupropion is a clinically important bioactivation and elimination pathway. Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6). CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions. Bupropion is chiral, used clinically as a racemate, and disposition is stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Hydroxylation of racemic bupropion by recombinant CYP2B6 and human liver microsomes was evaluated using a stereoselective assay. At therapeutic concentrations, hydroxylation of (S)-bupropion was threefold and 1.5-greater than (R)-bupropion, respectively, by recombinant CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise different for bupropion enantiomers. Stereoselective bupropion hydroxylation may have implications for the therapeutic efficacy of bupropion as an antidepressant or smoking cessation therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6 activity.

Bupropion hydrochloride: the development of a chiral separation using an ovomucoid column

The separation of bupropion enantiomers on an ovomucoid stationary phase was investigated. The mobile- and stationary-phase parameters that may influence the separation were identified. The parameters that were studied include: type and concentration of organic modifier, mobile phase pH, ionic strength, type of buffer, and column temperature, as well as the effect that the amount of sample injected had on the separation. The optimized chiral separation baseline-resolved the enantiomers in less than 10 min. Calibration curves for a standard were linear over a range of 0.27–53.0 μg/g (ppm) with a correlation coefficient of 0.999 for both enantiomers. A detection limit of 0.13 μg/g and a quantitation limit of 0.27 μg/g were also found. The system precision of the method was 0.2%.

Royal malady.

Bupropion is a widely used antidepressant and smoking cessation aid in addition to being one of two US Food and Drug Administration–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. A liquid chromatography-tandem mass spectrometry 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 [<i>Cl</i>_int, 11.4 versus 4.3 <i>µ</i>l/min per milligram for the formation of (R,R)- and (S,S)-hydroxybupropion glucuronide; and <i>Cl</i>_max, 7.7 versus 1.1 <i>µ</i>l/min per milligram 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 the total excreted hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively. Elimination pathways were further characterized using an expressed UDP-glucuronosyl transferase (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; UGT1A9 catalyzed the formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the metabolic pathways underlying bupropion metabolite disposition and significantly expand our knowledge of potential contributors to the interindividual and intraindividual variability in therapeutic and toxic effects of bupropion in humans.