Phenobarbital induces monkey brain CYP2E1 protein but not hepatic CYP2E1, in vitro or in vivo chlorzoxazone metabolism

Cytochrome P450 2E1 (CYP2E1) is expressed in the brain and liver, and can metabolize clinical drugs and activate toxins. The effect of phenobarbital on hepatic and brain CYP2E1 is unclear. We investigated the effect of chronic phenobarbital treatment on in vivo chlorzoxazone disposition (a CYP2E1 probe drug), in vitro chlorzoxazone metabolism, and hepatic and brain CYP2E1 protein levels in African Green monkeys (Cercopithecus aethiops). Monkeys were given oral saccharine or saccharine supplemented with 20 mg/kg phenobarbital (N = 6/group) for 22 days. Phenobarbital did not induce in vivo chlorzoxazone disposition, in vitro chlorzoxazone metabolism or hepatic CYP2E1 protein levels (all P > 0.05). However, phenobarbital induced brain CYP2E1 protein levels, using immunoblotting, by 1.26-fold in the cerebellum (P = 0.01) and 1.46-fold in the putamen (P = 0.04). Phenobarbital also increased cell-specific CYP2E1 expression, for example in the frontal cortical pyramidal neurons and cerebellar Purkinje cells. This data indicates that phenobarbital does not alter hepatic metabolism, but may alter metabolism of CYP2E1 substrates within the brain.     

CYP2B6 is expressed in African Green monkey brain and is induced by chronic nicotine treatment

CYP2B6 is a drug-metabolizing enzyme expressed in human tissues that can activate bupropion (a smoking cessation drug) and tobacco smoke nitrosamines and can inactivate drugs such as nicotine. Smokers have higher brain CYP2B6 protein levels compared to non-smokers but the cause of this elevation is unknown. We investigated the basal expression and the effect of chronic nicotine treatment on CYP2B6 protein in African Green monkey (Cercopithecus aethiops) brain. Basal expression of brain CYP2B6 was strong in specific cells such as the frontal cortical pyramidal cells, the cerebellar Purkinje cells and the neurons in the substantia nigra. Basal CYP2B6 protein levels varied 2.7-fold (non-significant) among 12 brain regions. All monkeys were given a subcutaneous 0.1 mg/kg nicotine test dose prior to treatment and the maximum plasma concentration achieved was 87 +/- 69 ng/ml and the half-life was 2.6 +/- 1.5 h. Monkeys were treated subcutaneously twice daily with nicotine at 0.05 mg/kg for 2 days, 0.15 mg/kg for 2 days followed by 0.3 mg/kg for 18 days (n = 6) or saline (n = 6). Chronic nicotine treatment induced CYP2B6 expression in specific cells such as astrocytes and neurons in the frontal cortex, caudate, thalamus and hippocampus. CYP2B6 protein levels were induced 1.5-fold in the frontal cortex (p < 0.01). Hepatic CYP2B6 expression was not altered by nicotine. In conclusion, CYP2B6 protein is expressed in specific cells in monkey brain and is induced by chronic nicotine treatment which may impact central metabolism of CYP2B6 substrates such as bupropion and nicotine.     

Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K _m values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V _max values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes. Received: 27 October 1998 / Accepted: 11 January 1999     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.     

Induction of CYP2B1/2 and nicotine metabolism by ethanol in rat liver but not rat brain

A higher proportion of alcoholics than non-alcoholics smoke (>80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4-6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.     

Induction of the drug metabolizing enzyme CYP2D in monkey brain by chronic nicotine treatment

Cytochrome P450 (CYP) 2D6, an enzyme found in the liver and the brain, is involved in the metabolism of numerous centrally acting drugs (e.g. antidepressants, neuroleptics, opiates), endogenous neurochemicals (e.g. catecholamines) and in the inactivation of neurotoxins (e.g. pesticides, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)). Although CYP2D6 is essentially an uninducible enzyme in the liver, we show that smokers have higher CYP2D6 in the brain, especially in the basal ganglia. In order to determine whether nicotine, a component of cigarette smoke, could increase brain CYP2D, African Green monkeys were treated chronically with nicotine (0.05 mg/kg for 2 days, then 0.15 mg/kg for 2 days followed by 0.3 mg/kg for 18 days s.c., b.i.d.). Monkeys treated with nicotine showed significant induction of CYP2D in brain when compared to saline-treated animals as detected by western blotting and immunocytochemistry. No changes in liver CYP2D were observed in nicotine-treated monkeys. Induction was observed in various brain regions including those affected in Parkinson's disease (PD) such as substantia nigra (3-fold, p = 0.01), putamen (2.1-fold, p = 0.001) and brainstem (2.4-fold, p = 0.001), with the caudate nucleus approaching significance (1.6-fold, p = 0.07). Immunocytochemistry revealed that the expression of CYP2D in both saline- and nicotine-treated monkeys is cell-specific particularly in the cerebellum, frontal cortex and hippocampus. These results suggest that monkey brain expresses CYP2D, which is induced in specific cells and brain regions upon chronic nicotine treatment. Smokers, or those using nicotine treatment, may have higher levels of brain CYP2D6 that may result in altered localized CNS drug metabolism and inactivation of neurotoxins.     

Metabolism of (+)-fenchone by CYP2A6 and CYP2B6 in human liver microsomes

The in vitro metabolism of (+)-fenchone was examined in human liver microsomes and recombinant enzymes. Biotransformation of (+)-fenchone was investigated by gas chromatography-mass spectrometry. (+)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolite of (+)-fenchone was determined by relative abundance of mass fragments and retention time with GC. CYP2A6 and CYP2B6 in human liver microsomes were major enzymes involved in the hydroxylation of (+)-fenchone, based on the following lines of evidence. First, of eleven recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalyzed oxidation of (+)-fenchone. Second, oxidation of (+)-fenchone was inhibited by thioTEPA, (+)-menthofuran anti-CYP2A6 and anti-CYP2B6 antibodies. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (+)-fenchone hydroxylation activities in liver microsomes of 8 human samples.     

Regional and cellular distribution of CYP2E1 in monkey brain and its induction by chronic nicotine

CYP2E1 is expressed in liver and extrahepatic tissues, including brain. It metabolizes ethanol and other drugs and toxins, such as acetaminophen, chlorzoxazone and tobacco-derived nitrosamines. Hepatic CYP2E1 is inducible by nicotine, and cigarette smoke accelerates chlorzoxazone metabolism. Smokers have higher levels of brain CYP2E1 expression than non-smokers, but the specific regions and cell types which have the higher expression differ from nicotine-induced rat brain. We therefore investigated the expression and distribution of brain CYP2E1 in a non-human primate, the African green monkey, and determined the effect of nicotine treatment. CYP2E1 levels varied among saline-treated monkey brain regions (P < 0.01) and expression was cell-type specific. Chronic nicotine treatment induced CYP2E1 expression in the frontal cortex (1.5-fold, P < 0.05) and cerebellum (1.5-fold, P < 0.01), specifically in cortical pyramidal neurons and cerebellar Purkinje cells but no change was seen in temporal cortex (P = 0.20), hippocampus (P = 0.29), putamen (P = 0.26) and thalamus (P = 0.08). CYP2E1 expression pattern in monkey brain following chronic nicotine treatment is similar to that in smokers, suggesting that nicotine may be the primary component in cigarette smoke that induces CYP2E1. Increased CYP2E1 in brain may contribute to oxidative stress and alter localized metabolism, and resulting pharmacology, of centrally acting drugs metabolized by CYP2E1.     

In vivo effects of protein kinase and phosphatase inhibitors on CYP2B induction in rat liver

Effects of inhibiting protein kinases and phosphatases on induction of CYP2B by triphenyldioxane (TPD) and phenobarbital (PB) were investigated. Male Wistar rats were treated with test inhibitors before TPD or PB administration. Inhibitors of phosphatidylinositol-3-kinase (Wortmannin) and protein kinase C (bisindolylmaleimide I) did not have appreciable effects on TPD- or PB-induced pentoxyresorufin O-dealkylase (PROD) activity specific for CYP2B, although bisindolylmaleimide I did give substantial induction alone. W-7, an inhibitor of Ca2+/calmodulin-dependent kinase II, produced a 6-fold increase in the TPD-induced PROD activity and did not lead to a significant increase in basal PROD activity. Treatment of rats with okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, caused considerable decreases in PROD activity during the induction by TPD and PB (8- and 2.5-fold, respectively). Results of multiplex RT-PCR showed that the increase in enzymatic activity from W7 and OA treatment reflected at least in part increased mRNA levels. CYP2B mRNA level in the liver of rats treated with W-7 and TPD was 1.5 times higher than in the liver of TPD-treated rats. This effect was not observed for PB-induction. OA treatment caused a decrease of the CYP2B mRNA levels of 44% and 33% respectively, for TPD- and PB-induction. Thus, our results are consistent with the hypothesis that phosphorylation/dephosphorylation signaling pathways are involved in regulation of CYP2B induction in rat liver.     

Styrene monomer primarily induces CYP2B1 mRNA in rat liver

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600 mg kg(-1) styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.     

Styrene monomer primarily induces CYP2B1 mRNA in rat liver

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600?mg?kg^?1 styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.     

CYP2A6 genetic variation and dexmedetomidine disposition

Purpose

There is a large interindividual variability in dexmedetomidine dose requirements for sedation of patients in intensive care units (ICU). Cytochrome P450 2A6 (CYP2A6) mediates an important route of dexmedetomidine metabolism, and genetic variation in CYP2A6 affects the clearance of other substrate drugs. We examined whether CYP2A6 genotypes affect dexmedetomidine disposition.

Methods

In 43 critically ill ICU patients receiving dexmedetomidine infusions adjusted to achieve the desired level of sedation, we determined a median of five plasma dexmedetomidine concentrations each. Forty subjects were genotyped for five common CYP2A6 alleles and grouped into normal (n?=?33), intermediate (n?=?5), and slow metabolizers (n?=?2).

Results

Using a Bayesian hierarchical nonlinear mixture model, estimated dexmedetomidine clearance was 49.1?L/h (posterior mean; 95% credible interval 41.4–57.6?L/h). There were no significant differences in dexmedetomidine clearance among normal, intermediate, and slow CYP2A6 metabolizer groups.

Conclusion

Genetic variation in CYP2A6 does not appear to be an important determinant of dexmedetomidine clearance in ICU patients.     

Metabolism of (-)-fenchone by CYP2A6 and CYP2B6 in human liver microsomes

The in vitro metabolism of (-)-fenchone was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-fenchone was investigated by gas chromatography-mass spectrometry. (-)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on gas chromatography (GC). CYP2A6 and CYP2B6 were major enzymes involved in the hydroxylation of (-)-fenchone by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalysed the oxidation of (-)-fenchone. Second, oxidation of (-)-fenchone was inhibited by thioTEPA and (+)-menthofuran. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (-)-fenchone hydroxylation activities in liver microsomes of 11 human samples. CYP2A6 may be more important than CYP2B6 in human liver microsomes. Kinetic analysis showed that the Vmax/Km values for (-)-fenchone 6-endo-, 6-exo- and 10-hydroxylation catalysed by liver microsomes of human sample HG-03 were 24.3, 44.0 and 1.3nM(-1)min(-1) , respectively. Human recombinant CYP2A6 and CYP2B6 catalysed (-)-fenchone 6-exo-hydroxylation with Vmax values of 2.7 and 12.9 nmol min(-1) nmol(-1) P450 and apparent Km values of 0.18 and 0.15 mM and (-)-fenchone 6-endo-hydroxylation with Vmax values of 1.26 and 5.33nmolmin(-l) nmol(-1) P450 with apparent Km values of 0.29 and 0.26mM. (-)-Fenchone 10-hydroxylation was catalysed by CYP2B6 with Km and Vmax values of 0.2 mM and 10.66 nmol min(-1) nmol(-1) P450, respectively.     

Characterization of the novel CYP2A6*21 allele using in vivo nicotine kinetics

Objective The impact of CYP2A6*21 (K476R) on in vivo nicotine metabolism and disposition was investigated.Methods A two-step allele-specific PCR assay was developed to detect the 6573A>G single nucleotide polymorphism (SNP) in CYP2A6*21. Nicotine metabolism phenotypes from a previously described intravenous labeled nicotine and cotinine infusion study [1] was used to assess the impact of CYP2A6*21. Genomic DNA samples from 222 (111 monozygotic and dizygotic twin pairs) Caucasian subjects were genotyped for CYP2A6 alleles (CYP2A6*1X2, -*1B, -*2, -*4, -*7, -*9, -*10, -*12, and -*21). The pharmacokinetic parameters were compared between individuals with no detected CYP2A6 variants (CYP2A6*1/*1, n=163) and individuals heterozygous for the CYP2A6*21 allele (CYP2A6*1/*21, n=9).Results The frequency of the CYP2A6*21 allele was found to be 2.3% in Caucasians (n=5/222 alleles, evaluated in one twin from each twin pair). In vivo pharmacokinetic parameters, such as nicotine clearance (1.32±0.37 vs. 1.18±0.20 L/min), fractional clearance of nicotine to cotinine (1.02±0.36 vs. 0.99±0.23 L/min), nicotine half-life (111±37 vs. 116±29 min), and the trans-3′-hydroxycotinine to cotinine ratio (1.92±1.0 vs. 1.55±0.58) indicated no substantial differences in nicotine metabolism between those without the variant (CYP2A6*1/*1, n=163) and those with the variant (CYP2A6*1/*21, n=9), respectively.Conclusions CYP2A6*21 does not have a detectable impact on nicotine metabolism in vivo. Our data suggest that CYP2A6*21 may not be important for future studies of nicotine metabolism and the resulting impacts on smoking behaviors.Nael Al Koudsi and Jill C. Mwenifumbo contributed equally to this work.     

CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.     

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 µM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 µM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 µM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 µM and 10 µM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.     

Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro: implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo

The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V(max) and K(m) values for 8-hydroxyefavirenz formation and ～2-fold lower intrinsic clearance (Cl(int)) were noted in expressed CYP2B6.6 protein (-b5) compared with that of CYP2B6.1 protein (-b5); this effect was abolished by Cyt b5. The V(max) and Cl(int) values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K(m), whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower V(max) (and moderate increase in K(m)) and thus lower Cl(int) values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (K(i) = 1.6 ± 0.8 μM) than HLMs with CYP2B6*1/*1 genotype (K(i) = 3.0 ± 1.1 μM). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate- and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.     

Olanzapine disposition in humans is unrelated to CYP1A2 and CYP2D6 phenotypes

OBJECTIVE: Limited data suggest that CYP1A2 and CYP2D6 are involved in the metabolism of olanzapine. The purpose of this study was to further elucidate the role of these enzymes in the disposition of olanzapine in vivo. METHODS: Seventeen healthy non-smoking male volunteers were included in the study. Five subjects were CYP2D6 poor metabolisers (PMs), and 12 were CYP2D6 extensive metabolisers (EMs). All subjects received a single oral dose of 7.5 mg olanzapine, and serum concentrations were measured for 96 h using gas chromatography. A cross-over study was undertaken in the 12 CYP2D6 EMs who at least 2 weeks before or after the olanzapine dose received a single oral dose of 200 mg caffeine. The concentrations of caffeine and paraxanthine were measured in saliva 10 h after caffeine intake, and the paraxanthine/caffeine ratio was calculated as a measure of CYPIA2 activity. RESULTS: A threefold inter-individual variability in oral clearance (CLoral) and maximum serum concentration (Cmax) of olanzapine was observed and a 2.3-fold inter-individual variability in CYPIA2 activity. There was no significant correlation between CYP1A2 activity and oral clearance of olanzapine (r=-0.19, P=0.56). Moreover, there were no significant differences in any of the olanzapine pharmacokinetic parameters between the CYP2D6 PMs and EMs (CLoral=0.246 l h(-1) kg(-1) and 0.203 l h(-1) kg(-1), respectively, P=0.30). CONCLUSION: Neither CYP1A2 nor CYP2D6 seem to have a dominating role in olanzapine biotransformation after intake of a single dose.