Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism.

The primary metabolite of nicotine in smokers is cotinine. Cotinine is further metabolized to trans-3'-hydroxycotinine, the major urinary metabolite of nicotine in tobacco users. It was recently reported that cytochrome P450 2A6 catalyzes the conversion of cotinine to trans-3'-hydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans-3'-hydroxycotinine but also to 5'-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products of baculovirus-expressed P450 2A6 [methyl-(3)H]cotinine metabolism were analyzed by radioflow HPLC. Three (3)H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5'-hydroxycotinine and trans-3'-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6-catalyzed cotinine metabolism was detected by LC/MS. It was identified by cochromatography with a standard and MS and MS/MS data to be norcotinine. An attempt was made to further characterize the unidentified (3)H-labeled metabolite by comparison to the cotinine metabolites generated by hamster liver microsomes. Hamster liver microsomes contain a P450, 2A8, which is closely related to P450 2A6, and have previously been shown to metabolize cotinine to three hydroxylated products, trans-3'-hydroxycotinine, 5'-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.     

N-glucuronidation of trans-3'-hydroxycotinine by human liver microsomes

trans-3'-Hydroxycotinine is the major nicotine metabolite excreted in the urine of smokers and other tobacco or nicotine users. On average, about 30% of the trans-3'-hydroxycotinine in urine is present as a glucuronide conjugate. The O-glucuronide of trans-3'-hydroxycotinine has been isolated from smokers urine and appears to be the major glucuronide conjugate of trans-3'-hydroxycotinine in urine. However, nicotine and cotinine are both glucuronidated at the nitrogen atom of the pyridine ring. We report here that human liver microsomes catalyze both the N-glucuronidation and the O-glucuronidation of trans-3'-hydroxycotinine. The N-glucuronide was purified by HPLC, and its structure was confirmed by NMR. Both N- and O-glucuronidation of trans-3'-hydroxycotinine were detected in 13 of 15 human liver microsome samples. The ratio of N-glucuronidation to O-glucuronidation was between 0.4 and 2.7. One sample only catalyzed N-glucuronidation, and one sample did not catalyze either reaction. The rates of N-glucuronidation varied more than 6-fold from 6 to 38.9 pmol/min/mg protein; rates of O-glucuronidation ranged from 2.8 to 23.4 pmol/min/mg protein. The rate of trans-3'-hydroxycotinine N-glucuronidation by human liver microsomes correlated well with both the rate of nicotine and the rate of cotinine N-glucuronidation. trans-3'-Hydroxycotinine O-glucuronidation correlated with neither nicotine nor cotinine N-glucuronidation. These results suggest that the same enzyme(s) that catalyzes the N-glucuronidation of nicotine and cotinine may also catalyze the N-glucuronidation of trans-3'-hydroxycotinine in the human liver but that a separate enzyme catalyzes trans-3'-hydroxycotinine O-glucuronidation.     

Metabolism of nicotine and cotinine by human cytochrome P450 2A13.

Nicotine, a major constituent of tobacco, plays a critical role in smoking addiction. In humans, nicotine is primarily metabolized to cotinine, which is further metabolized to trans-3'-hydroxycotinine. Recently, we have demonstrated that heterologously expressed human CYP2A13 is highly active in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived carcinogen. In the present study, CYP2A13-catalyzed NNK metabolism was found to be inhibited competitively by nicotine and N'-nitrosonornicotine (NNN), suggesting that both nicotine and NNN are also substrates of CYP2A13. We have further demonstrated that human CYP2A13 is indeed an efficient enzyme in catalyzing C-oxidation of nicotine to form cotinine, with the apparent K(m) and V(max) values of 20.2 microM and 8.7 pmol/min/pmol, respectively. CYP2A13 also catalyzes the 3'-hydroxylation of cotinine to form trans-3'-hydroxycotinine, with the apparent K(m) and V(max) values of 45.2 microM and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined.     

A convenient synthesis of trans-3'-hydroxycotinine, a major nicotine metabolite found in the urine of tobacco users

trans-3'-Hydroxycotinine is a major urinary metabolite of nicotine in smokers, but no straightforward method is available for its synthesis. A simple method was developed for preparation of trans-3'-hydroxycotinine from cotinine in two steps by using NaN[(CH3)3Si]2 and dibenzyl peroxydicarbonate, followed by base-catalyzed hydrolysis.     

Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.

Urinary nicotine metabolic output was profiled for 11 smokers who smoked their regular cigarette brands ad libitum. Thermospray liquid chromatography/mass spectrometry was used to monitor nicotine and eight metabolites, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine that were determined indirectly using enzyme hydrolysis. These results were used to estimate an average, steady-state concentration in a 24-hr urine sample during ad libitum smoking and to assess interindividual variability in the excretion of these metabolites. The variability in absolute amount among the nine analytes ranged from 35 to 70% for these smokers. The glucuronide conjugates constituted an average of 29% of all urinary metabolites monitored in this study. trans-3'-Hydroxycotinine in the free form constitutes the largest single metabolite in smokers' urine, with an average of 35% of the total. The sums of nicotine metabolites determined here are very close to the Federal Trade Commission yields of nicotine for the total number of cigarettes smoked by these subjects during the urine collection interval. These results indicate that a large proportion of the nicotine absorbed while smoking can be accounted for as urinary metabolites of nicotine, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine.     

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.     

Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.

In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine Delta(5'(1'))-iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-(3)H]nicotine and radioflow high-performance liquid chromatography analysis, we determined kinetic parameters for nicotine 5'-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4, and 2A5. The formation of both cotinine and nicotine Delta(5'(1'))-iminium ion was monitored. The K(m) and V(max) values for P450 2A6 were 144 +/- 15 muM and 1.30 +/- 0.05 pmol/min/pmol, respectively. Previously reported K(m) values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 muM. P450 2A13 was a somewhat better catalyst of nicotine Delta(5'(1'))-iminium formation, with 2-fold lower K(m) and 2-fold higher V(max) values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84% identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. Whereas 5'-oxidation was the major pathway of nicotine metabolism for all five P450 2A enzymes, these enzymes also catalyzed methyl oxidation. Nornicotine, the product of this reaction was detected as 5 to 15% of the total nicotine metabolites. Nornicotine is the amine precursor to the esophageal carcinogen N'-nitrosonornicotine. Therefore, methyl oxidation of nicotine by P450 2A6 or P450 2A13 followed by nitrosation of nornicotine are possible endogenous pathways of N'-nitrosonornicotine formation.     

Effects of whole deletion of CYP2A6 on nicotine metabolism in humans

We investigated the effects of CYP2A6 genotypes on nicotine metabolism, focused from nicotine to cotinine and its additional 3'-hydroxylating resulted in trans-3'-hydroxycotinine formation. In the subjects genotyped by PCR-RFLP method, one cigarette smoking experiment was performed and urine samples were collected for 24 h. In all subjects who smoked, we detected nicotine, cotinine and trans-3'-hydroxycotinine in urine by GC-MS analysis. In whole deletion of CYP2A6, urinary excretion amounts of cotinine and trans-3'-hydroxycotinine were significantly smaller than those in the wild-type of CYP2A6*1. A lack of CYP2A6 reduces the formation of cotinine and trans-3'-hydroxycotinine, but not entirely reduces the trans-3'-hydroxycotinine formation. Unknown cotinine 3'-hydroxylating activity except CYP2A6 are suspected in humans.     

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.     

Within-subject variation of the salivary 3HC/COT ratio in regular daily smokers: prospects for estimating CYP2A6 enzyme activity in large-scale surveys of nicotine metabolic rate

Nicotine is the major addictive compound in tobacco and is responsible for tobacco dependence. It is primarily metabolized to cotinine (COT) and trans-3'-hydroxycotinine (3HC) by the liver enzyme cytochrome P-450 2A6 (CYP2A6). The 3HC/COT ratio measured in the saliva of smokers is highly correlated with the intrinsic hepatic clearance of nicotine and, therefore, may be a useful non-invasive marker of CYP2A6 activity and metabolic rate of nicotine. This study assessed within-subject variation in salivary 3HC/COT ratios in six regular daily smokers. Our data provide evidence that 1. variation in the 3HC/COT ratio is not dependent on the time of sampling during the day (i.e., morning vs. night ) (P > 0.1) and 2. the average within-subject biological variation in the 3HC/COT ratio is approximately 26%. These findings should be useful for designing large-scale population surveys to assess the variation in the metabolic rate of nicotine (via CYP2A6) in smokers.     

Metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted

Generally, 70-80% of absorbed nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. There is genetic polymorphism in the human CYP2A6 gene. Among several mutated alleles, CYP2A6*4 allele is a whole deleted type. The purpose of the present study was to clarify the metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for nicotine and its nine metabolites. Excretion levels of nicotine and its metabolites in 24 h accumulated urine after the chewing of one piece of nicotine gum were evaluated in five Japanese subjects whose CYP2A6 genotype was determined. In three subjects with CYP2A6*1A/CYP2A6*1A, CYP2A6*1A/CYP2A6*1B, and CYP2A6*1A/CYP2A6*4 (group I), nicotine was mainly excreted as cotinine, trans-3'-hydroxycotinine, and their glucuronide (approximately 60%). In contrast, in two subjects with CYP2A6*4/CYP2A6*4 (group II), trace levels of cotinine, cotinine N-glucuronide, and cotinine 1'-N-oxide were detected. Trans-3'-hydroxycotinine and its O-glucuronide were not detected. The excretion levels of nicotine itself, nicotine N-glucuronide, and nicotine 1'-N-oxide were higher than those in the other three subjects. The total excretion levels of these three compounds were approximately 95% in group II versus 35% in group I. However, the sum of the excretion levels of nicotine and all metabolites was similar among these five subjects. This is the first report of the metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted.     

Assessment of prenatal smoke exposure by determining nicotine and its metabolites in maternal and neonatal urine

Urine specimens were collected from 75 pregnant women before childbirth and from their newborns within 48 postnatal hours. A high-performance liquid chromatography (HPLC) method was used to determine urinary nicotine and its metabolites, cotinine and trans-3'-hydroxycotinine (OH-cotinine) to objectivise prenatal smoke exposure. Using the sum of nicotine metabolites as a marker, 34 women were classed as not exposed to smoke ( < 15 nmol/l), 18 as passive smokers (15-400 nmol/l), and 23 as active smokers ( > 400 nmol/1). The newborns of active smokers exhibited significantly (P < 0.001) higher nicotine metabolite concentrations than did those of either non-exposed women or passive smokers. A close correlation was found to exist between maternal and neonatal nicotine and cotinine concentrations (r=0.8968 and r=0.9205, respectively). For OH-cotinine, this correlation was particularly close when maternal, but not neonatal, OH - cotinine was adjusted to creatinine (r=0.9792). The neonatal/maternal urine concentration ratios for cotinine and OH-cotinine were noted to not significantly depend on the time of postpartal urine collection. Within the first two postnatal days, the extent of current prenatal smoke exposure attributable to active smoking of the mother was best reflected by the urinary concentrations of cotinine plus OH-cotinine without adjustment to creatinine.     

Identification of cis-3'-hydroxycotinine as a urinary nicotine metabolite

1. cis-3'-Hydroxycotinine was detected as an S(-)-nicotine metabolite in the urine of smokers as well as in the urine of rats and hamsters dosed with nicotine. 2. The excreted amount of cis-3'-hydroxycotinine is lower than that of the trans-isomer.     

Oxidative metabolism of the alkaloid rutaecarpine by human cytochrome P450.

Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by alpha-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V(max) values of 1,388 to approximately 1,893 pmol/min/nmol P450, K(m) values of 4.1 to approximately 9.5 microM, and K(i) values of 45 to approximately 103 microM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC(50) values of 2.56 +/- 0.04, 2.57 +/- 0.11, and 0.09 +/- 0.01 microM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.     

Determination of the human cytochrome P450 isoforms involved in the metabolism of zolmitriptan

1. Zolmitriptan was extensively metabolized by freshly isolated human hepatocytes to a number of components including the three main metabolites observed in vivo (N-desmethyl-zolmitriptan, zolmitriptan N-oxide and the indole acetic acid derivative). In contrast, metabolism of zolmitriptan by human hepatic microsomes was extremely limited with only small amounts of the N-desmethyl and indole ethyl alcohol metabolites being produced. 2. Furafylline, a selective inhibitor of CYP1A2, almost completely abolished the hepatocellular metabolism of zolmitriptan and markedly inhibited formation of the N-desmethyl metabolite in microsomes. Chemical inhibitors, selective against other major human cytochrome P450 (CYP2C9, 2C19, 2D6 and 3A4), had no obvious effects. In addition, expressed human CYP1A2 was the only cytochrome P450 to form the N-desmethyl metabolite. 3. N-desmethyl-zolmitriptan was extensively metabolized by both human hepatocytes and microsomes. The indole acetic acid and ethyl alcohol derivatives were the major metabolites formed by hepatocytes, whereas only the indole ethyl alcohol derivative was produced by microsomes. Metabolism of N-desmethyl-zolmitriptan was not inhibited by cytochrome P450-selective chemical inhibitors nor was it observed following incubation with expressed human cytochrome P450. Clorgyline, a selective inhibitor of monoamine oxidase A (MAO-A), markedly inhibited the microsomal formation of the indole ethyl alcohol derivative. 4. Primary metabolism of zolmitriptan is dependent mainly on CYP1A2, whereas MAO-A is responsible for further metabolism of N-desmethyl-zolmitriptan, the active metabolite. Since the in vivo clearance of zolmitriptan is primarily dependent on metabolism, interactions with drugs that induce or inhibit CYP1A2 or MAO-A may be anticipated.     

Relationship between machine-derived smoke yields and biomarkers in cigarette smokers in Germany

In order to determine whether smokers of cigarettes in the contemporary yield ranges of the German market (0.1-1.0mg nicotine, 1-10mg tar) differ in their actual exposure to various smoke constituents, we performed a field study with 274 smokers and 100 non-smokers. The following biomarkers were determined: In 24-h urine: Nicotine equivalents (molar sum of nicotine, cotinine, trans-3'-hydroxycotinine and their respective glucuronides), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK), 3-hydroxypropylmercapturic acid (metabolite of acrolein), trans,trans-muconic acid, S-phenylmercapturic acid (metabolites of benzene), 1-hydroxypyrene (metabolite of pyrene); in saliva: Cotinine and trans-3'-hydroxycotinine; in exhaled air: Carbon monoxide; in blood: Methyl-, hydroxyethyl-, cyanoethyl- (biomarker of acrylonitrile) and carbamoylethylvaline (biomarker of acrylamide) hemoglobin adducts. All biomarkers were found to be significantly higher in smokers compared to non-smokers and showed strong correlations with the daily cigarette consumption. Biomarker levels and per cigarette increases in smokers were at most weakly related to the machine-derived smoke yields. It is concluded that machine-derived yields of cigarettes from the contemporary German cigarette market have little or no impact on the actual smoking-related exposure determined by suitable biomarkers.     

Determination of the human cytochrome P450 isoforms involved in the metabolism of zolmitriptan.

1. Zolmitriptan was extensively metabolized by freshly isolated human hepatocytes to a number of components including the three main metabolites observed in vivo (N-desmethyl-zolmitriptan, zolmitriptan N-oxide and the indole acetic acid derivative). In contrast, metabolism of zolmitriptan by human hepatic microsomes was extremely limited with only small amounts of the N-desmethyl and indole ethyl alcohol metabolites being produced. 2. Furafylline, a selective inhibitor of CYP1A2, almost completely abolished the hepatocellular metabolism of zolmitriptan and markedly inhibited formation of the N-desmethyl metabolite in microsomes. Chemical inhibitors, selective against other major human cytochrome P450 (CYP2C9, 2C19, 2D6 and 3A4), had no obvious effects. In addition, expressed human CYP1A2 was the only cytochrome P450 to form the N-desmethyl metabolite. 3. N-desmethyl-zolmitriptan was extensively metabolized by both human hepatocytes and microsomes. The indole acetic acid and ethyl alcohol derivatives were the major metabolites formed by hepatocytes, whereas only the indole ethyl alcohol derivative was produced by microsomes. Metabolism of N-desmethyl-zolmitriptan was not inhibited by cytochrome P450-selective chemical inhibitors nor was it observed following incubation with expressed human cytochrome P450. Clorgyline, a selective inhibitor of monoamine oxidase A (MAO-A), markedly inhibited the microsomal formation of the indole ethyl alcohol derivative. 4. Primary metabolism of zolmitriptan is dependent mainly on CYP1A2, whereas MAO-A is responsible for further metabolism of N-desmethyl-zolmitriptan, the active metabolite. Since the in vivo clearance of zolmitriptan is primarily dependent on metabolism, interactions with drugs that induce or inhibit CYP1A2 or MAO-A may be anticipated.     

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.     

Species variation and stereoselectivity in the metabolism of nicotine enantiomers

1. High performance liquid radiochromatographic systems have been developed for the identification and quantification of 7 urinary metabolites of both S-(-)-[3H-N'-CH3]nicotine and R-(+)-[3H-N'-CH3] nicotine in guinea pig, hamster, rat and rabbit. 2. 3'-Hydroxycotinine was a major urinary metabolite of both S-(-)-nicotine and R-(+)-nicotine in guinea pig, hamster and rabbit. Cotinine was not generally a significant urinary metabolite of either nicotine enantiomer, except in rat, where it constituted 14.6 and 10.4%, respectively, of the total radiolabel in the urine after administration of [3H]-S-(-)-nicotine or [3H]-R-(+)-nicotine. Nicotine N'-oxide was an important urinary metabolite of both nicotine isomers in guinea pig and rat, but in both cases, was not observable in hamster and rabbit. No N-methylated urinary metabolite of S-(-)-nicotine could be detected in any of the species examined. In R-(+)-nicotine experiments, only guinea pig afforded N-methylated metabolites. Significant amounts of 2 unidentified polar, non-basic urinary metabolites of both S-(-)- and R-(+)-nicotine-treated animals were observed. 3. Analysis of the comparative metabolism of the nicotine enantiomers in the four animals species studied, showed that stereoselective differences in the formation of oxidative metabolites existed, particularly in the formation of 3'-hydroxycotinine and nicotine-N'-oxide. A clear stereospecificity was observed in the guinea pig, in that only the R-(+)-nicotine enantiomer was N-methylated in this species. 4. Sex differences appear to exist in the metabolism of nicotine enantiomers in the rat. Female rats excreted more of the unidentified polar metabolite B than male rats, whereas the converse was true for nicotine-N'-oxide. In experiments with R-(+)-nicotine, urinary levels of 3'-hydroxycotinine and R-(+)-nicotine in female rats were higher than in male rats. Conversely, higher amounts of nicotine-N'-oxide were observed in the urine of male rats compared to those in female rats.     

Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.

Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K(m) of 10.0 +/- 0.8 mM and a V(max) of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K(m) = 1.6 +/- 0.1 mM and V(max) = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC(50) = 55 microM), androstanediol (a substrate of UGT2B15, IC(50) = 169 microM), and propofol (a substrate of UGT1A9, IC(50) = 296 microM). Interestingly, imipramine (IC(50) = 45 microM), androstanediol (IC(50) = 21 microM), and propofol (IC(50) = 41 microM) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.