Bupropion Hydroxylation Research Articles

Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction

The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug–drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5±0.3 and kinact value of 0.045±0.001min−1. Phenelzine inactivated CYP2B6 with KI and kinact values of 44.9±6.9μM and 0.085±0.003min−1 respectively. Inactivation of selegiline was characterized with KI and kinact values of 22.0±3.3 and 0.074±0.002min−1 respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index.

Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

In Vitro Inhibitory Effect of Licoricidin on Human Cytochrome P450s

Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori, and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/ MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human liver microsomes. When licoricidin was incubated at 0-25 μM with CYP probes for 60 min at 37oC, it showed potent inhibitory effects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4’-hydroxylation with half maximal inhibitory concentration (IC50) values of 3.4 and 4.0 μM, respectively. The inhibition mode of licoricidin was revealed as competitive, dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect of licoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with IC50 values of 4.5 and 0.73 μM, respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activity could be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B- and CYP2C9- mediated metabolism.

Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP) enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs) 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

Genetic Variants of Pregnane X Receptor (PXR) and CYP2B6 Affect the Induction of Bupropion Hydroxylation by Sodium Ferulate

This study investigated the effects of pregnane X receptor (PXR/NR1I2) and CYP2B6 genetic variants on sodium ferulate (SF)-mediated induction of bupropion hydroxylation. The pharmacokinetics of bupropion and hydroxybupropion were evaluated after an oral dose of bupropion (150 mg) administered with and without SF pretreatment for 14 days in 33 healthy subjects. The area under the time-concentration curve (AUC) ratio of AUC_hyd (AUC(0-∞) of hydroxybupropion)/AUC_bup (AUC(0-∞) of bupropion) represents the CYP2B6 hydroxylation activity, which was significantly lower in CYP2B6*6 carriers (NR1I2 TGT noncarriers or carriers) than in noncarriers in both the basal and SF-induced states (p-value<0.05). AUC ratio and AUC_hyd of NR1I2 -24113AA variant were markedly lower than GA and GG genotypes (7.5±2.1 versus 14.5±3.3 and 20.6±1.1, and 8873±1431 versus 14,504±2218 and 17,586±1046) in the induced states. However, -24020(-)/(-) variant didn't show significant difference in the induction of CYP2B6 hydroxylation activity by SF compared with other -24020[GAGAAG]/(-) genotypes. NR1I2 TGT haplotype (-25385T+g.7635G+g.8055T) carriers exhibited a significantly decreased AUC ratio, compared with TGT noncarriers, in the basal states (7.6±1.0 versus 9.7±1.0), while this result wasn't observed in CYP2B6*6 noncarriers. Moreover, individuals with complete mutation-type [CYP2B6*6/*6+NR1I2 TGT+ -24113AA+ -24020 (-)/(-)] showed even lower percent difference of AUC ratio (8.7±1.2 versus 39.5±8.2) than those with complete wild-type. In conclusion, it is suggested that NR1I2 variants decrease the bupropion hydroxylation induced by SF treatment, particularly in CYP2B6*6 carriers.Trial RegistrationChiCTR.org ChiCTR-TRC-11001285

Mechanism of Autoinduction of Methadone N-Demethylation in Human Hepatocytes

There is considerable interindividual and intraindividual variability in methadone metabolism and clearance. Methadone dosing is particularly challenging during initiation of therapy, because of time-dependent increases in hepatic clearance (autoinduction). Although methadone N-demethylation is catalyzed in vitro by cytochrome P4502B6 (CYP2B6) and CYP3A4, and clearance in vivo depends on CYP2B6, mechanism(s) of autoinduction are incompletely understood. In this investigation, we determined mechanism(s) of methadone autoinduction using human hepatocytes. Fresh human hepatocytes were exposed to 0.1 to 10 µM methadone for 72 hours. Cells were washed and methadone N-demethylation assessed. CYP2B6, CYP3A4, and CYP3A5 messenger RNA (mRNA), protein expression (by gel-free high-performance liquid chromatography mass spectrometry) and catalytic activity (bupropion hydroxylation and alfentanil dealkylation for CYP2B6 and CYP3A4/5, respectively) were measured. Mechanisms of CYP induction were characterized using pregnane X receptor and constitutive androstane receptor reporter gene assays. Methadone (10 µM) increased methadone N-demethylation 2-fold, CYP2B6 and CYP3A4 mRNA 3-fold, and protein expression 2-fold. CYP3A5 mRNA was unchanged. CYP2B6 and CYP3A4/5 activities increased 2-fold. Induction by methadone enantiomers (R-methadone versus S-methadone) did not differ. Induction was relatively weak compared with maximum induction by phenobarbital and rifampin. Lower methadone concentrations had smaller effects. Methadone was an agonist for the pregnane X receptor but not the constitutive androstane receptor. Methadone caused concentration-dependent autoinduction of methadone N-demethylation in human hepatocytes, related to induction of CYP2B6 and CYP3A4 mRNA expression, protein expression, and catalytic activity. Induction was related to pregnane X receptor but not constitutive androstane receptor activation. These in vitro findings provide mechanistic insights into clinical autoinduction of methadone metabolism and clearance.

Effects of Green Tea Catechins on Cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A Activities in Human Liver and Intestinal Microsomes

The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

Genetic Variants of Pregnane X Receptor (PXR) and CYP2B6 Affect the Induction of Bupropion Hydroxylation by Sodium Ferulate

Genetic Variants of Pregnane X Receptor (PXR) and CYP2B6 Affect the Induction of Bupropion Hydroxylation by Sodium Ferulate

Effect of TSHAC on Human Cytochrome P450 Activity, and Transport Mediated by P-Glycoprotein

TSAHC [4'-(p-toluenesulfonylamido)-4-hydroxychalcone] is a promising antitumorigenic chalcone compound, especially against TM4SF5 (four-transmembrane L6 family member 5)-mediated hepatocarcinoma. We evaluated the potential of TSAHC to inhibit the catalytic activities of nine cytochrome P450 isoforms and of P-glycoprotein (Pgp). The abilities of TSAHC to inhibit phenacetin Odeethylation (CYP1A2), coumarin 6-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine Ndeethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation (CYP3A) were tested using human liver microsomes. The P-gp inhibitory effect of TSAHC was assessed by [3H]digoxin accumulation in the LLCPK1-MDR1 cell system. TSAHC strongly inhibited CYP2C8, CYP2C9, and CYP2C19 isoform activities with Ki values of 0.81, 0.076, and 3.45 microM, respectively. It also enhanced digoxin accumulation in a dose-dependent manner in the LLCPK1-MDR1 cells. These findings indicate that TSAHC has the potential to inhibit CYP2C isoforms and P-gp activities in vitro. TSAHC might be used as a nonspecific inhibitor of CYP2C isoforms based on its negligible inhibitory effect on other P450 isoforms such as CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, and CYP3A.

Rapid clinical induction of bupropion hydroxylation by metamizole in healthy Chinese men

This study aimed to investigate the effect of metamizole on bupropion hydroxylation related to different CYP2B6 genotype groups in healthy volunteers. Sixteen healthy male volunteers (6 CYP2B6*1/*1, 6 CYP2B6*1/*6 and 4 CYP2B6*6/*6) received orally administered bupropion alone and during daily treatment with metamizole 1500 mg day(-1) (500 mg tablet taken three times daily) for 4 days. Serial blood samples were obtained up to 48 h after each bupropion dose. After metamizole treatment relative to bupropion alone, the geometric mean ratios (GMRs) and 90% confidence interval (CI) of the AUC(0,∞) ratio of 4-hydroxybupropion over bupropion were 1.99 (1.57, 2.55) for the CYP2B6*1/*1 group, 2.15 (1.53, 3.05) for the CYP2B6*1/*6 group and 1.86 (1.36, 2.57) for the CYP2B6*6/*6 group. The GMRs and 90% CI of bupropion were 0.695 (0.622, 0.774) for AUC(0,∞) and 0.400 (0.353, 0.449) for C(max) , respectively. The corresponding values for 4-hydroxybupropion were 1.43 (1.28, 1.53) and 2.63 (2.07, 2.92). The t(1/2) value was significantly increased for bupropion and decreased for 4-hydroxybupropion. The t(max) values of bupropion and 4-hydroxybupropion were both significantly decreased. The mean percentage changes in pharmacokinetic parameters among the CYP2B6 genotype groups were not significantly different. Oral administration of metamizole for 4 days significantly altered the pharmacokinetics of both bupropion and its active metabolite, 4-hydroxybupropion, and significantly increased the CYP2B6-catalyzed bupropion hydroxylation in all of the subjects. Cautions should be taken when metamizole is co-administered with CYP2B6 substrate drugs.

Quantitative Prediction of CYP2B6 Induction by Estradiol During Pregnancy: Potential Explanation for Increased Methadone Clearance During Pregnancy

There is considerable evidence that pregnancy changes the disposition of drugs in an enzyme- and gestational stage-specific manner. On the basis of probe drug studies, the activity of CYP3A4 and CYP2D6 increases and CYP1A2 decreases during human pregnancy. However, no studies of CYP2B6 activity during human pregnancy have been conducted. In rodent models and in HepG2 cells, CYP2B enzymes have been shown to be regulated by estradiol. Because estradiol concentrations increase by ∼50-fold during human pregnancy, it was hypothesized that the increasing estradiol concentrations during human pregnancy would result in induction of CYP2B6 activity. Hepatocytes from three female donors were treated with estradiol, and the EC(50) and E(max) were measured for CYP2B6 mRNA and bupropion hydroxylation activity. The measured values were used to predict the magnitude of CYP2B6 induction during human pregnancy. At 100 nM total estradiol, a concentration achievable during the third trimester of pregnancy, CYP2B6 activity was predicted to increase by 1.5-3-fold, based on increased CYP2B6 activity and mRNA. When the E(max) and EC(50) values were compared with those for carbamazepine and rifampin, estradiol was found to be as potent an inducer of CYP2B6 as rifampin and carbamazepine. These data suggest that, during human pregnancy, the increasing estradiol concentrations will result in increased clearance of drugs that have CYP2B6-mediated clearance pathways. This could in part explain the observed increase in methadone clearance during pregnancy.

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. The samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6β-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

Bupropion Hydroxylation as a Selective Marker of Rat CYP2B1 Catalytic Activity

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC(50) for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 μM, respectively) when using liver microsomes from β-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC(50) of 2.5 × 10(-3) ± 0.8 × 10(-3) μM for BROD and >100 μM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K(m) and V(max)) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 μM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the K(m) values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype-phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.

Meclizine is not an inverse agonist or antagonist of human constitutive androstane receptor

Certain tissues express both pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and various target genes are cross‐regulated by these receptors. Meclizine was reported to be an inverse agonist and antagonist of human CAR (hCAR) and an activator of human PXR (hPXR) in cell‐based reporter gene assays. Therefore, activation of hPXR by meclizine may attenuate its apparent hCAR inverse agonistic effects. To test this hypothesis, we investigated whether meclizine would still be capable of suppressing hCAR target gene expression (CYP2B6) in cultured human hepatocytes, which are known to express hCAR and hPXR. Meclizine (0.03–60 μM) did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist‐mediated increase in CYP2B6 mRNA and CYP2B6‐catalyzed bupropion hydroxylation levels. These findings reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a reporter gene assay and a mammalian two‐hybrid assay in transfected HepG2 cells. Control experiment indicated that PK11195, which is a hCAR inverse agonist and antagonist, decreased constitutive and hCAR agonist‐mediated hCAR activity. In conclusion, meclizine does not act as a hCAR inverse agonist or antagonist in human hepatocytes. Therefore, it is not appropriate to use this drug as a pharmacological tool to study hCAR function in this cell type.[Supported by CIHR and MSFHR]

Drug Interaction Study between Bupropion and Ticlopidine in Male CF-1 Mice

Bupropion is an atypical antidepressant that is biotransformed in humans to its major active metabolite hydroxybupropion by cytochrome P450 2B6 (CYP2B6). Co-administration of bupropion with an inhibitor of CYP2B6 can result in a serious drug interaction, leading to bupropion related adverse effects (e.g. seizures). The antiplatelet agent ticlopidine has been identified as a potent in vitro inhibitor of bupropion hydroxylation, however it is unknown if it interacts in vivo in rodents. In this study we investigated the potential pharmacokinetic (PK) drug interaction between bupropion and ticlopidine in mice. Using a destructive sampling design, male CF-1 mice were administered ticlopidine 1.0 mg/kg daily for 5 d, followed by single-dose bupropion 50 mg/kg. Bupropion and hydroxybupropion levels were measured by HPLC-UV in plasma and brain tissues at 30, 60, 90, 120 and 180 min post-dose, and compared between treatment groups. There was a strong trend in both plasma and brain data towards greater bupropion levels and smaller hydroxybupropion levels in ticlopidine treated mice. Analysis of variance indicated statistical differences (p<0.05) at many time points. The variance associated with the area under the curve was calculated using Bailer's method and significant differences were found between treatment groups. Taken together, the concentration time point statistical analysis followed by PK modeling demonstrate a significant PK drug interaction between bupropion and ticlopidine. To our knowledge, this is the first study to document an in vivo drug interaction between these drugs in mice. Our findings support future in vivo drug interaction studies in mice between bupropion and CYP2B6 inhibitors.

Differential Effect of Meclizine on the Activity of Human Pregnane X Receptor and Constitutive Androstane Receptor

Conflicting data exist as to whether meclizine is an activator of human pregnane X receptor (hPXR). Therefore, we conducted a detailed, systematic investigation to determine whether meclizine affects hPXR activity by performing a cell-based reporter gene assay, a time-resolved fluorescence resonance energy transfer competitive ligand-binding assay, a mammalian two-hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. In pregnane X receptor (PXR)-transfected HepG2 cells, meclizine activated hPXR to a greater extent than rat PXR. It bound to hPXR ligand-binding domain and recruited steroid receptor coactivator-1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene expression (CYP3A4) in human hepatocytes. However, it did not increase but decreased testosterone 6β-hydroxylation, suggesting inhibition of CYP3A catalytic activity. Meclizine has also been reported to be an inverse agonist and antagonist of human constitutive androstane receptor (hCAR). Therefore, given that certain tissues (e.g., liver) express both hPXR and hCAR and that various genes are cross-regulated by them, we quantified the expression of a hCAR- and hPXR-regulated gene (CYP2B6) in cultured human hepatocytes treated with meclizine. This drug did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist-mediated increase in CYP2B6 mRNA and CYP2B6-catalyzed bupropion hydroxylation levels. These observations reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a hCAR reporter gene assay and mammalian two-hybrid assay. In conclusion, meclizine is a hPXR agonist, and it does not act as a hCAR inverse agonist or antagonist in cultured human hepatocytes.

Effects of Pregnane X Receptor (NR1I2) and CYP2B6 Genetic Polymorphisms on the Induction of Bupropion Hydroxylation by Rifampin

We investigated genetic polymorphisms in the pregnane X receptor (NR1I2) in Korean individuals (n = 83) and the effects of NR1I2 genotypes on rifampin-mediated induction of bupropion hydroxylation. The pharmacokinetics of bupropion and hydroxybupropion were evaluated after an oral dose of bupropion (150 mg) administered before and after rifampin treatment for 7 days in 35 healthy subjects. The area under the time-concentration curve (AUC) ratio of hydroxybupropion to bupropion in CYP2B6*6 carriers was significantly lower than that in CYP2B6*6 noncarriers in both the basal and rifampin-induced states (p = 0.012). Among the CYP2B6*6 carriers (n = 13), the NR1I2 TGT (-25385T + g.7635G + g.8055T) carriers exhibited a significantly lower AUC ratio, representing the CYP2B6 hydroxylation activity, compared with the TGT noncarriers, in the induced state (11.9 versus 20.3, p = 0.045). The percent difference in the AUC ratio between the basal and induced states was also significantly different (212% versus 58.8%, p = 0.006). However, no significant difference was observed among the NR1I2 TGT genotypes for the CYP2B6*6 noncarriers (n = 22). In conclusion, it is suggested the NR1I2 TGT genotype decreases the bupropion hydroxylation induced by treatment with rifampin, particularly in CYP2B6*6 carriers.