Inhibitory effects of neurotransmitters and steroids on human CYP2A6.

Human CYP2A6 catalyzes the metabolism of nicotine, cotinine, and coumarin as well as some pharmaceutical drugs. CYP2A6 is highly expressed in liver and, also, in brain and steroid-related tissues. In this study, we investigated the inhibitory effects of neurotransmitters and steroid hormones on CYP2A6 activity. We found that coumarin 7-hydroxylation and cotinine 3'-hydroxylation by recombinant CYP2A6 expressed in baculovirus-infected insect cells were competitively inhibited by tryptamine (both K(i) = 0.2 microM), serotonin (K(i) = 252 microM and 167 microM), dopamine (K(i) = 49 microM and 22 microM), and histamine (K(i) = 428 microM and 359 microM). Cotinine formation from nicotine was inhibited by tryptamine (K(i) = 0.7 microM, competitive), serotonin (K(i) = 272 microM, noncompetitive), dopamine, noradrenaline, and adrenaline (K(i) = 11 microM, 54 microM, and 81 microM, uncompetitive). Estrogens (K(i) = 0.6-3.8 microM), androgens (K(i) = 60-149 microM), and corticosterone (K(i) = 36 microM) also inhibited cotinine formation, but coumarin 7-hydroxylation and cotinine 3'-hydroxylation did not. Nicotine-Delta(5'(1'))-iminium ion formation from nicotine was not affected by these steroid hormones, indicating that the inhibition of cotinine formation was due to the inhibitory effects on aldehyde oxidase. The nicotine-Delta(5'(1'))-iminium ion formation was competitively inhibited by tryptamine (K(i) = 0.3 microM), serotonin (K(i) = 316 microM), dopamine (K(i) = 66 microM), and histamine (K(i) = 209 microM). Thus, we found that some neurotransmitters inhibit CYP2A6 activity, being related with inter- and intraindividual differences in CYP2A6-dependent metabolism. The inhibitory effects of steroid hormones on aldehyde oxidase may also contribute to interindividual differences in nicotine metabolism.     

Isoflavones inhibit nicotine C-oxidation catalyzed by human CYP2A6

The inhibitory effects of isoflavones (daidzein, genistein, and glycitein) on human cytochrome P450 (CYP) 2A6 activities were investigated. Daidzein, genistein, and glycitein uncompetitively inhibited nicotine C-oxidation catalyzed by recombinant CYP2A6 expressed in baculovirus-infected insect cells with Ki values of 1.3 +/- 0.3 microM, 0.7 +/- 0.2 microM, and 5.2 +/- 0.8 microM, respectively, but not coumarin 7-hydroxylation. Effects of the intake of soy isoflavones on in vivo nicotine metabolism were investigated with 7 healthy Japanese homozygotes of CYP2A6*1. The cotinine/nicotine ratio of the plasma concentrations 2 hours after chewing 1 piece of nicotine gum under the basal condition (after abstaining from soy foods for 1 week) was 8.8 +/- 2.6 (4.4-11.4). The ratio was significantly (P < .05) reduced to 6.7 +/- 1.6 (4.0-8.2) after consumption of a soy isoflavone supplement (60 mg of total isoflavones/d) for 5 days. The authors found that isoflavone contained in soy products significantly decreased nicotine metabolism.     

Functional characterization of CYP2A13 polymorphisms

CYP2A13 is an efficient catalyst of metabolic activation of the human carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This study investigated the functional consequences of CYP2A13 polymorphisms that result in single amino acid substitutions. Five CYP2A13 variants, namely CYP2A13*2 (R257C), CYP2A13*5 (F453Y), CYP2A13*6 (R494C), CYP2A13*8 (D158E), and CYP2A13*9 (V323L), were expressed and evaluated for coumarin binding affinity, coumarin 7-hydroxylation, and -hydroxylation of (S)-NNN and NNK. In addition, the 133_134 Thr deletion variant, coded for by CYP2A13*3, was expressed but was not stable to the protein purification procedure. A 30-42% decrease in coumarin 7-hydroxylation catalytic efficiency was determined for R257C and D158E. No effect on coumarin binding or (S)-NNN metabolism was observed. Three variants, R257C, D158E, and V323L, had two- to threefold decreased catalytic efficiency for NNK -hydroxylation. CYP2A13 polymorphisms resulted in modest changes in coumarin 7-hydroxylation and NNK -hydroxylation activities in vitro. Although these changes are not likely to impact in vivo metabolism, these data should aid in the interpretation and design of future epidemiology studies.     

Characterization of novel CYP2A6 polymorphic alleles (CYP2A6*18 and CYP2A6*19) that affect enzymatic activity.

Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6(*)18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6(*)18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6(*)19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3'-untranslated region, in which the latter two changes correspond to CYP2A6(*)7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6(*)18, CYP2A6(*)19, and CYP2A6(*)7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the K(m) values were increased, and V(max) values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The K(m) and V(max) values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased K(m) and decreased V(max)) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the K(m) and V(max) values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n = 56) and Koreans (n = 40). Although the CYP2A6(*)18 and CYP2A6(*)19 alleles were found only heterozygously, a subject with CYP2A6(*)7/CYP2A6(*)19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6(*)1A, supporting the in vitro results that the CYP2A6(*)19 allele leads to decreased enzymatic activity.     

Interindividual variability in nicotine metabolism: C-oxidation and glucuronidation

Nicotine has roles in the addiction to smoking, replacement therapy for smoking cessation, as a potential medication for several diseases such as Parkinson's disease, Alzheimer's disease, and ulcerative colitis. The absorbed nicotine is rapidly and extensively metabolized and eliminated to urine. A major pathway of nicotine metabolism is C-oxidation to cotinine, which is catalyzed by CYP2A6 in human livers. Cotinine is subsequently metabolized to trans-3'-hydroxycotinine by CYP2A6. Nicotine and cotinine are glucuronidated to N-glucuronides mainly by UGT1A4 and partly by UGT1A9. Trans-3'-hydroxycotinine is glucuronidated to O-glucuronide mainly by UGT2B7 and partly by UGT1A9. Approximately 90% of the total nicotine uptake is eliminated as these metabolites and nicotine itself. The nicotine metabolism is an important determinant of the clearance of nicotine. Recently, advances in the understanding of the interindividual variability in nicotine metabolism have been made. There are substantial data suggesting that the large interindividual differences in cotinine formation are associated with genetic polymorphisms of the CYP2A6 gene. Interethnic differences have also been observed in the cotinine formation and the allele frequencies of the CYP2A6 alleles. Since the genetic polymorphisms of the CYP2A6 gene have a major impact on nicotine clearance, its relationships with smoking behavior or the risk of lung cancer have been suggested. The metabolic pathways of the glucuronidation of nicotine, cotinine, and trans-3'-hydroxycotinine in humans would be one of the causal factors for the interindividual differences in nicotine metabolism. This review mainly summarizes recent results from our studies.     

Competitive inhibition of coumarin 7-hydroxylation by pilocarpine and its interaction with mouse CYP 2A5 and human CYP 2A6.

1. We have shown earlier that pilocarpine strongly inhibits mouse and human liver coumarin 7-hydroxylase activity of CYP 2A and pentoxyresorufin O-deethylase activity of CYP 2B in vitro. Since pilocarpine, like coumarin, contains a lactone structure we have studied in more detail its inhibitory potency on mouse and human liver coumarin 7-hydroxylation. 2. Pilocarpine was a competitive inhibitor of coumarin 7-hydroxylase in vitro both in mouse and human liver microsomes although it was not a substrate for CYP 2A5. Ki values were similar, 0.52 +/- 0.22 microM in mice and 1.21 +/- 0.51 microM in human liver microsomes. 3. Pilocarpine induced a type II difference spectrum in mouse, human and recombinant CYP 2A5 yeast cell microsomes, with Ka values of 3.7 +/- 1.6, 1.6 +/- 1.1 and 1.5 +/- 0.1 microM, respectively. 4. Increase in pH of the incubation medium from pH 6 to 7.5 increased the potency of inhibition of coumarin 7-hydroxylation by pilocarpine. 5. Superimposition of pilocarpine and coumarin in such a way that their carbonyls, ring oxygens and the H-7' of coumarin and N-3 of pilocarpine overlap yielded a common molecular volume of 82%. 6. The results indicate that pilocarpine is a competitive inhibitor and has a high affinity for mouse CYP 2A5 and human CYP 2A6. In addition the immunotype nitrogen of pilocarpine is coordinated towards the haem iron in these P450s.     

In vivo evaluation of coumarin and nicotine as probe drugs to predict the metabolic capacity of CYP2A6 due to genetic polymorphism in Thais

The association between the distribution characteristics of CYP2A6 catalytic activities toward nicotine and coumarin, and the frequency distribution of CYP2A6 variant alleles reported was estimated in 120 healthy Thais. The distributions of the subjects as classified by the amounts of 7-hydroxycoumarin (7-OHC) excreted in the urine and by cotinine/nicotine ratio in the plasma were clearly bimodal. However, the numbers of apparently poor metabolizers for coumarin and nicotine were different. The inter-individual variability in the in vivo dispositions of coumarin and nicotine closely related to the CYP2A6 genetic polymorphism. There was a close correlation between the rate of 7-OHC excretion in the urine and cotinine/nicotine ratio in the plasma among subjects (R=0.92, p<0.001). The frequency of CYP2A6 allele found in the present study was: CYP2A6*1A=32% (95% CI, 22.1-39.4%), CYP2A6*1B=27% (95% CI, 19.4-33.5%), CYP2A6*9=20% (95% CI, 17.6-23.3%), CYP2A6*4=14% (95% CI, 9.6-17.8%), CYP2A6*7=5% (95% CI, 3.7-9.4%), CYP2A6*10=2% (95% CI, 0.8-5.1%). Subjects having CYP2A6*1A/*1B were found to have a higher rate of 7-OHC excretion, as well as a higher cotinine/nicotine ratio in the plasma compared with those of the other genotypes. In contrast, subjects with CYP2A6*4/*7 and CYP2A6*7/*7 almost lacked any cotinine formation, whereas urinary 7-OHC was still detectable. CYP2A6*9 allele clearly resulted in reduced enzyme activities. Despite the absence of the homozygote for CYP2A6*10 allele, the presence of CYP2A6*10 allele significantly decreased the enzyme activities. The results of the present study demonstrate that in vivo phenotyping of CYP2A6 using nicotine and coumarin are not metabolically equivalent. Nicotine is a better probe according to its specificity, while coumarin is still valuable to be used for a routine CYP2A6 phenotyping since the test employs a non-invasive method.     

Pronounced differences in inhibition potency of lactone and non-lactone compounds for mouse and human coumarin 7-hydroxylases (CYP2A5 and CYP2A6)

1. The structural requirements for a compound to be a potent inhibitor for mouse CYP2A5 and human CYP2A6 enzymes catalysing coumarin 7-hydroxylase activity have been studied. 2. The IC50 of 28 compounds for the pyrazole-treated male DBA/2 mouse and human liver microsomal coumarin 7-hydroxylation were determined at 10 microm coumarin concentration 15 times over Km of coumarin. 3. The three most potent inhibitors for CYP2A5 were gamma-nonanoic lactone, gamma-decanolactone and gamma-phenyl-gamma-butyrolactone with an IC50 = 1.9+/-0.4, 2.1+/-0.2 and 2.4+/-0.3 microM and for CYP2A67-methylcoumarin, butylcyclohexane and indan with an IC50. = 30+/-3.2, 43+/-9 and 50+/-11 microM. 4. Among the 28 compounds studied, only 2-benzoxazolinone, 2-indanone and gamma-valerolactone showed similar inhibitory activity in both species. Indan had a lower IC50 for human than for mouse coumarin 7-hydroxylation, whereas the IC50 of 24 other compounds was higher for human than for mouse coumarin 7-hydroxylation. 5. The largest difference in IC50 between mouse and human activity was observed with 5-substituted phenyl, pentyl, hexyl, heptyl or octyl gamma-lactones or 6-substituted delta-lactones. IC50 of gamma-undecanolactone and gamma-decanolactone was 500 times lower for mouse than human coumarin 7-hydroxylation. 6. The difference in the IC50 between human and mouse coumarin 7-hydroxylation decreased substantially with the corresponding compounds without the lactone ring. 7. It is concluded that certain 5- or 6-position substituted gamma- and delta-lactones are potent inhibitors for mouse CYP2A5 but not for the orthologous human CYP2A6 and that the active site of CYP2A6 could be smaller than the active site of CYP2A5.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro.

CYP2A6 is the principle enzyme metabolizing nicotine to its inactive metabolite cotinine. In this study, the selective probe reactions for each major cytochrome P450 (P450) were used to evaluate the specificity and selectivity of the CYP2A6 inhibitors methoxsalen, tranylcypromine, and tryptamine in cDNA-expressing and human liver microsomes. Phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), diclofenac 4'-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), 7-ethoxy-4-trifluoromethylcoumarin deethylation (CYP2B6), p-nitrophenol hydroxylation (CYP2E1), and omeprazole sulfonation (CYP3A4) were used as index reactions. Apparent K(i) values for inhibition of P450s' (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) activities showed that tranylcypromine, methoxsalen, and tryptamine have high specificity and relative selectivity for CYP2A6. In cDNA-expressing microsomes, tranylcypromine inhibited CYP2A6 (K(i) = 0.08 microM) with about 60- to 5000-fold greater potency relative to other P450s. Methoxsalen inhibited CYP2A6 (K(i) = 0.8 microM) with about 3.5- 94-fold greater potency than other P450s, except for CYP1A2 (K(i) = 0.2 microM). Tryptamine inhibited CYP2A6 (K(i) = 1.7 microM) with about 6.5- 213-fold greater potency relative to other P450s, except for CYP1A2 (K(i) = 1.7 microM). Similar results were also obtained with methoxsalen and tranylcypromine in human liver microsomes. R-(+)-Tranylcypromine, (+/-)-tranylcypromine, and S-(-)-tranylcypromine competitively inhibited CYP2A6-mediated metabolism of nicotine with apparent K(i) values of 0.05, 0.08, and 2.0 microM, respectively. Tranylcypromine [particularly R-(+) isomer], tryptamine, and methoxsalen are specific and relatively selective for CYP2A6 and may be useful in vivo to decrease smoking by inhibiting nicotine metabolism with a low risk of metabolic drug interactions.     

Determination of coumarin metabolism in Turkish population

Cytochrome P450 2A6 is an important human hepatic P450 which activates precarcinogens and oxidizes some drug constituents such as coumarin, halothane, and the major nicotine C-oxidase. Genetic polymorphism exists in the CYP2A6 gene. CYP2A6*1 (wild type) is responsible for the 7-hydroxylation of coumarin. The point mutation (T to A) in codon 160 leads to a single amino acid substitution (Leu to His) and the resulting protein, CYP2A*2 is unable to 7-hydroxylate coumarin. Gene conversion in exons 3, 6, and 8 between the CYP2A6 and the CYP2A7 genes creates another variant, CYP2A6*3. In this study, healthy male and female Turkish volunteers (n = 50) were administered 2 mg coumarin, and urine samples were analyzed for their content of the coumarin metabolite, 7-hydroxycoumarin (7OHC), by high-performance liquid chromatography (HPLC). Genetic polymorphism for CYP2A6 was detected by using two-step polymerase chain reaction (PCR) to identify CYP2A6*1, CYP2A6*2, and CYP2A6*3 in 13 of these subjects. The percentage of the dose excreted of total 7OHC in relation to CYP2A6 genotype and excretion of nicotine/cotinine was also evaluated to demonstrate the role of CYP2A6 in nicotine metabolism. The majority of Turkish subjects (68%) excreted less than 60% of the 2-mg dose as coumarin metabolite. The allelic frequencies were detected as 0.88 for CYP2A6*1 allele; 0.12 for CYP2A6*3 allele in 13 individuals. No heterozygous and homozygous individuals were identified for the CYP2A6*2 allelic variant. Phenotyping and genotyping for drug metabolizing enzymes are of great importance in studies correlating precarcinogen activation or drug metabolism to the CYP2A6 genotype in smoking behavior when populations are investigated.     

Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6 (CYP2A6*5, *7, *8, *18, *19, and *35)

Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F-G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased K(m) values whereas CYP2A6*18 and *19 showed decreased k(cat) values, which resulted in lower catalytic efficiencies (k(cat)/K(m)). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased K(m) value, whereas CYP2A6*18 and *35 showed much greater decreases in k(cat) values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.     

Functional characterization of three human cytochrome p450 2E1 variants with amino acid substitutions

1. Cytochrome p450 (p450) 2E1 is a hepatic enzyme of importance for the metabolism of xenobiotics such as drugs and environmental toxicants. Genetic polymorphisms of CYP2E1 in 5'-flanking and coding regions have been found previously in Caucasian and Chinese populations. 2. In order to investigate the effects of amino acid substitutions on the function of CYP2E1, the enzymes of all known CYP2E1 variants in the coding region (CYP2E1.2, CYP2E1.3 and CYP2E1.4) with Arg76His, Val389Ile and Val179Ile substitutions, respectively, as well as the wild-type CYP2E1 (CYP2E1.1) were expressed in COS-1 cells, and their chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation activities were determined. 3. The protein level of CYP2E1.2 was reduced to 29% compared with that of CYP2E1.1. The profiles of the level of activity relative to CYP2E1.1 for chlorzoxazone 6-hydroxylation (300 microM substrate) and 4-nitrophenol 2-hydroxylation (150 microM substrate) were very similar. 4. Although the K(m) values were not significantly different among wild-type and variant CYP2E1s in any oxidation metabolism, the V(max) and V(max)/K(m) of CYP2E1.2 on the basis of the CYP2E1 protein level were 2.7-3.0-fold higher than those of CYP2E1.1. In contrast, the levels of CYP2E1 protein and catalytic activity of CYP2E1.3 and CYP2E1.4 were not affected by the corresponding amino acid substitutions. 5. The findings suggest that Arg76 is closely associated with the function of CYP2E1, and that the genetic polymorphism of CYP2E1 is one cause of interindividual differences in the toxicity of xenobiotics.     

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.     

Interindividual differences in nicotine metabolism and genetic polymorphisms of human CYP2A6

Nicotine is widely consumed throughout the world, and exerts a number of physiological effects. After nicotine is absorbed through the lungs by cigarette smoking, it undergoes extensive metabolism in humans. Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. CYP2A6 can metabolize some pharmaceutical agents such as halothane, valproic acid, and fadrozole, and activate tobacco-specific nitrosamines. There are large interindividual differences in nicotine metabolism, and it has been found that the interindividual differences are attributed to the genetic polymorphisms of CYP2A6 gene. This review describes the techniques for determination of in vivo nicotine metabolism, characteristics of each human CYP2A6 alleles, and ethnic differences. The relationship between CYP2A6 genetic polymorphism and potency of nicotine metabolism, smoking behavior, and cancer risk are extensively reviewed. Finally, the usefulness of nicotine metabolism for phenotyping of CYP2A6 in individuals and implication of the significance of CYP2A6 genetic polymorphism in a clinical perspective are discussed.     

Interaction of buprenorphine and its metabolite norbuprenorphine with cytochromes p450 in vitro.

Buprenorphine is a thebaine derivative used in the treatment of heroin and other opiate addictions. In this study, the selective probe reactions for each of the major hepatic cytochromes P450 (P450s) were used to evaluate the effect of buprenorphine and its main metabolite norbuprenorphine on the activity of these P450s. The index reactions used were CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2C19 (omeprazole 5-hydrxoylation), CYP2D6 (dextromethorphan O-demethylation), CYP2B6 (7-ethoxy-4-trifluoromethyl-coumarin 7-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (omeprazole sulfoxidation). Buprenorphine exhibited potent, competitive inhibition of CYP2D6 (Ki 10 +/- 2 microM and 1.8 +/- 0.2 microM) and CYP3A4 (Ki 40 +/- 1.6 microM and 19 +/- 1.2 microM) in microsomes from human liver and cDNA-expressing lymphoblasts, respectively. Compared with buprenorphine, norbuprenorphine demonstrated a lower inhibitory potency with CYP2D6 (22.4% inhibition at 20 microM norbuprenorphine) and CYP3A4 (13.6% inhibition at 20 microM) in microsomes from human cDNA-expressing lymphoblast cells. Furthermore, buprenorphine was shown to be a substrate of CYP2D6 (Km = 600 microM; Vmax = 0.40 nmol/min/mg protein) and CYP3A4 (Km = 36 microM; Vmax = 0.19 nmol/min/mg protein). The present in vitro study suggests that buprenorphine and its major metabolite norbuprenorphine are inhibitors of CYP2D6 and CYP3A4; however, at therapeutic concentrations they are not predicted to cause potentially clinically important drug interactions with other drugs metabolized by major hepatic P450s.     

Nicotine metabolism,human drug metabolism polymorphisms,and smoking behaviour

Large interindividual differences occur in human nicotine disposition, and it has been proposed that genetic polymorphisms in nicotine metabolism may be a major determinant of an individual's smoking behaviour. Hepatic cytochrome P4502A6 (CYP2A6) catalyses the major route of nicotine metabolism: C-oxidation to cotinine, followed by hydroxylation to trans-3'-hydroxycotinine. Nicotine and cotinine both undergo N-oxidation and pyridine N-glucuronidation. Nicotine N-1-oxide formation is catalysed by hepatic flavin-containing monooxygenase form 3 (FMO3), but the enzyme(s) required for cotinine N-1'-oxide formation has not been identified. trans-3'-Hydroxycotinine is conjugated by O-glucuronidation. The uridine diphosphate-glucuronosyltransferase (UGT) enzyme(s) required for N- and O-glucuronidation have not been identified. CYP2A6 is highly polymorphic resulting in functional differences in nicotine C-oxidation both in vitro and in vivo; however, population studies fail to consistently and conclusively demonstrate any associations between variant CYP2A6 alleles encoding for either reduced or enhanced enzyme activity with self-reported smoking behaviour. The functional consequences of FMO3 and UGT polymorphisms on nicotine disposition have not been investigated, but are unlikely to significantly affect smoking behaviour. Therefore, current evidence does not support the hypothesis that genetic polymorphisms associated with nicotine metabolism are a major determinant of an individual's smoking behaviour and exposure to tobacco smoke.