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.

Influence of HIV antiretrovirals on methadone N-demethylation and transport.

Drug interactions involving methadone and/or HIV antiretrovirals can be problematic. Mechanisms whereby antiretrovirals induce clinical methadone clearance are poorly understood. Methadone is N-demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) by CYP2B6 and CYP3A4 in vitro, but by CYP2B6 in vivo. This investigation evaluated human hepatocytes as a model for methadone induction, and tested the hypothesis that methadone and EDDP are substrates for human drug transporters. Human hepatocyte induction by several antiretrovirals of methadone N-demethylation, and CYP2B6 and CYP3A4 transcription, protein expression and catalytic activity, and pregnane X receptor (PXR) activation were evaluated. Methadone and EDDP uptake and efflux by overexpressed transporters were also determined. Methadone N-demethylation was generally not significantly increased by the antiretrovirals. CYP2B6 mRNA and activity (bupropion N-demethylation) were induced by several antiretrovirals, as were CYP3A4 mRNA and protein expression, but only indinavir increased CYP3A activity (alfentanil dealkylation). CYP upregulation appeared related to PXR activation. Methadone was not a substrate for uptake (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1) or efflux (P-gp, BCRP) transporters. EDDP was a good substrate for P-gp, BCRP, OCT1, OCT3, OATP1A2, and OATP1B1. OATP1A2- and OCT3-mediated EDDP uptake, and BCRP-mediated EDDP efflux transport, was inhibited by several antiretrovirals. Results show that hepatocyte methadone N-demethylation resembles expressed and liver microsomal metabolism more than clinical metabolism. Compared with clinical studies, hepatocytes underreport induction of methadone metabolism by HIV drugs. Hepatocytes are not a good predictive model for clinical antiretroviral induction of methadone metabolism and not a substitute for clinical studies. EDDP is a transporter substrate, and is susceptible to transporter-mediated interactions.

Mechanism of autoinduction of methadone N-demethylation in human hepatocytes.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Methadone N-demethylation by the common CYP2B6 allelic variant CYP2B6.6.

The long-acting opioid methadone displays considerable unexplained interindividual pharmacokinetic variability. Methadone metabolism clinically occurs primarily by N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), catalyzed predominantly by CYP2B6. Retrospective studies suggest that the common allele variant CYP2B6*6 may influence methadone plasma concentrations. The catalytic activity of CYP2B6.6, encoded by CYP2B6*6, is highly substrate-dependent. This investigation compared methadone N-demethylation by CYP2B6.6 with that by wild-type CYP2B6.1. Methadone enantiomer and racemate N-demethylation by recombinant-expressed CYP2B6.6 and CYP2B6.1 was determined. At substrate concentrations (0.25-2 µM) approximating plasma concentrations occurring clinically, rates of methadone enantiomer N-demethylation by CYP2B6.6, incubated individually or as the racemate, were one-third to one-fourth those by CYP2B6.1. For methadone individual enantiomers and metabolism by CYP2B6.6 compared with CYP2B6.1, Vmax was diminished, Ks was greater and the in vitro intrinsic clearance was diminished 5- to 6-fold. The intrinsic clearance for R- and S-EDDP formation from racemic methadone was diminished approximately 6-fold and 3-fold for R- and S-methadone, respectively. Both CYP2B6.6 and CYP2B6.1 showed similar stereoselectivity (S>R-methadone). Human liver microsomes with diminished CYP2B6 content due to a CYP2B6*6 allele had lower rates of methadone N-demethylation. Results show that methadone N-demethylation catalyzed by CYP2B6.6, the CYP2B6 variant encoded by the CYP2B6*6 polymorphism, is catalytically deficient compared with wild-type CYP2B6.1. Diminished methadone N-demethylation by CYP2B6.6 may provide a mechanistic explanation for clinical observations of altered methadone disposition in individuals carrying the CYP2B6*6 polymorphism.

P-glycoprotein is a major determinant of norbuprenorphine brain exposure and antinociception.

Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of µ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(-/-) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(-/-) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(-/-) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine.