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Division of Clinical Pharmacology and Experimental Therapeutics, Medical Service, San Francisco General Hospital Medical Center, and the Departments of Medicine and Psychiatry, University of California, San Francisco, California
Abstract
Abstract I. Introduction II. Nicotine and Related Alkaloids in Tobacco Products III. Absorption of Nicotine from Tobacco Smoke and Nicotine Medications IV. Distribution of Nicotine in Body Tissues V. Nicotine and Cotinine Blood Levels during Tobacco Use and Nicotine Replacement Therapy VI. Metabolism of Nicotine A. Primary Metabolites of Nicotine B. Cotinine Metabolism C. Quantitative Aspects of Nicotine Metabolism D. Liver Enzymes Responsible for Nicotine and Cotinine Metabolism 1. Cytochrome P450 Enzymes. 2. Aldehyde Oxidase. 3. Flavin-Containing Monooxygenase 3. 4. Amine N-Methyltransferase. 5. UDP-Glucuronosyltransferases. E. Genetic Variations in Nicotine Metabolizing Enzymes 1. CYP2A6 Polymorphisms. 2. Polymorphisms in Other Enzymes. F. Extrahepatic Nicotine Metabolism G. Factors Influencing Nicotine Metabolism 1. Physiological Influences. a. Diet and Meals. b. Age. c. Chronopharmacokinetics of Nicotine. d. Gender-Related Differences in Nicotine Metabolism. 2. Pathological Conditions. 3. Medications. a. Inducers. b. Inhibitors. 4. Smoking. a. Inhibiting Effect on Nicotine Clearance. b. Inducing Effect of Smoking on Glucuronidation. 5. Racial and Ethnic Differences. VII. Excretion A. Renal Excretion B. Excretion in Feces and Sweat VIII. Species Differences in Nicotine Metabolism IX. Metabolism of Minor Alkaloids of Tobacco X. Pharmacokinetics and Metabolism of Nicotine Analogs XI. Conclusions and Areas for Further Study
Nicotine is of importance as the addictive chemical in tobacco, pharmacotherapy for smoking cessation, a potential medication for several diseases, and a useful probe drug for phenotyping cytochrome P450 2A6 (CYP2A6). We review current knowledge about the metabolism and disposition kinetics of nicotine, some other naturally occurring tobacco alkaloids, and nicotine analogs that are under development as potential therapeutic agents. The focus is on studies in humans, but animal data are mentioned when relevant to the interpretation of human data. The pathways of nicotine metabolism are described in detail. Absorption, distribution, metabolism, and excretion of nicotine and related compounds are reviewed. Enzymes involved in nicotine metabolism including cytochrome P450 enzymes, aldehyde oxidase, flavin-containing monooxygenase 3, amine N-methyltransferase, and UDP-glucuronosyltransferases are represented, as well as factors affecting metabolism, such as genetic variations in metabolic enzymes, effects of diet, age, gender, pregnancy, liver and kidney diseases, and racial and ethnic differences. Also effects of smoking and various inhibitors and inducers, including oral contraceptives, on nicotine metabolism are discussed. Due to the significance of the CYP2A6 enzyme in nicotine clearance, special emphasis is given to the effects and population distributions of CYP2A6 alleles and the regulation of CYP2A6 enzyme.
Smoking has enormous negative health consequences worldwide, and the use of tobacco is still rising globally (Mackay and Eriksen, 2002
). Nicotine is not a direct cause of most tobacco-related diseases, but it is highly addictive (Benowitz, 1999; Balfour, 2002). The addictiveness of nicotine is the cause of the continuing use of tobacco products, which in turn results in exposure to the diverse array of carcinogens and other bioactive compounds in tobacco, making tobacco use the leading cause of premature deaths in developed countries (Peto et al., 1992; Hecht, 2003). Tobacco is the single greatest preventable cause of death due to cancer. The vast majority of smokers want to quit, but due to nicotine addiction only a few percent of smokers quit successfully each year (CDC, 1993; USDHHS, 2000).
Nicotine medications are widely used as nicotine replacement therapies to assist smoking cessation and more recently have been proposed for use concurrently with smoking as part of a risk reduction strategy. Nicotine has also been studied as an experimental therapy for Parkinson's disease, Alzheimer's disease, and ulcerative colitis (Jani and Regueiro, 2002; Quik and Kulak, 2002; Sabbagh et al., 2002). Besides having importance as an addictive substance and a pharmaceutical, nicotine is of consequence as a specific substrate for cytochrome P450 2A6 (CYP2A6), thus facilitating the study of this enzyme in humans (Raunio et al., 2001).
We review current knowledge about the pharmacokinetics and metabolism of nicotine, some other naturally occurring tobacco alkaloids, and nicotine analogs that are under development as potential therapeutic agents. The focus is on studies in humans, but animal data are mentioned when relevant to the interpretation of human data. The pharmacokinetics of tobacco-specific nitrosamines, which are formed from tobacco alkaloids, is beyond the scope of this review. Their metabolism has been extensively reviewed elsewhere (Hecht, 1998).
II. Nicotine and Related Alkaloids in Tobacco Products
Nicotine (Fig. 1) is a natural ingredient in tobacco leaves where it acts as a botanical insecticide (Soloway, 1976; Tomizawa and Casida, 2003). It is the principal tobacco alkaloid occurring to the extent of about 1.5% by weight in commercial cigarette tobacco and comprising about 95% of the total alkaloid content (Schmeltz and Hoffmann, 1977; Benowitz et al., 1983a). Oral snuff and pipe tobacco contain concentrations of nicotine similar to cigarette tobacco, whereas cigar and chewing tobacco have only about half of the nicotine concentration of the cigarette tobacco (Tilashalski et al., 1994; Lu and Ralapati, 1998; Jacob et al., 1999). An average tobacco rod contains 10 to 14 mg of nicotine (Kozlowski et al., 1998), and on average about 1 to 1.5 mg of nicotine is absorbed systemically during smoking (Benowitz and Jacob, 1984). The nicotine in tobacco is largely the levorotary (S)-isomer, only 0.1 to 0.6% of total nicotine content is (R)-nicotine (Armstrong et al., 1998). Chemical reagents and pharmaceutical formulations of (S)-nicotine have a similar content of (R)-nicotine (0.1–1.2%) as an impurity since plant-derived nicotine is used for their manufacture (Armstrong et al., 1998). The (R)-nicotine content of tobacco smoke is higher. Up to 10% of nicotine in smoke has been reported to be (R)-isomer, presumably resulting from racemization occurring during combustion (Klus and Kuhn, 1977; Pool et al., 1985).
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In most tobacco strains, nornicotine and anatabine are the most abundant of the minor alkaloids, followed by anabasine (Fig. 1) (Schmeltz and Hoffmann, 1977; Saitoh et al., 1985). This order of abundance is the same in cigarette tobacco and oral snuff, chewing, pipe, and cigar tobacco (Jacob et al., 1999). However, nornicotine levels are highest in cigar tobacco, anatabine levels are lowest in chewing tobacco and oral snuff, and anabasine levels are lowest in chewing tobacco (Jacob et al., 1999). Small amounts of the N'-methyl derivatives of anabasine and anatabine are found in tobacco and tobacco smoke. Several of the minor alkaloids are thought to arise by bacterial action or oxidation during tobacco processing rather than by biosynthetic processes in the living plant (Leete, 1983). These include myosmine, N'-methylmyosmine, cotinine, nicotyrine, nornicotyrine, nicotine N'-oxide, 2,3'-bipyridyl, and metanicotine (Fig. 1) (Schmeltz and Hoffmann, 1977). Myosmine has been thought to be tobacco-specific but recent studies show that myosmine is found in a variety of foods including nuts, cereals, milk, and potatoes (Zwickenpflug et al., 1998; Tyroller et al., 2002). Also nicotine is found in low levels in vegetables such as potatoes, tomatoes, and eggplants (Domino et al., 1993; Siegmund et al., 1999).
N-Nitroso derivatives of tobacco alkaloids arise by the action of nitrous acid on nicotine, nornicotine, anabasine, and anatabine. These nitroso compounds are important because some are carcinogenic (Hecht and Hoffmann, 1989). Eight tobacco-specific nitrosamines have been identified (Hecht, 2003). N'-Nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK1), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are the most carcinogenic. They all are derived from nicotine; N'-nitrosonornicotine is also derived from nornicotine (Hecht, 1998). Unlike nicotine, the minor alkaloids anatabine, nornicotine, and anabasine and their N-nitroso derivatives are present to a substantial degree as the (R)-isomer (about 16, 20, and 42%, respectively) in tobacco products (Armstrong et al., 1999; Carmella et al., 2000).
Of the minor alkaloids that have been studied, nornicotine, metanicotine, and anabasine have been shown to have significant pharmacologic activity (Clark et al., 1965). Qualitatively, their actions are similar to those of nicotine, but they are generally less potent, the relative potency depending upon the test system. Anabasine administered orally or sublingually has been reported to aid smoking cessation and to have cardiovascular effects (Nasirov et al., 1978). Ingestion of anabasine-containing Nicotiana glauca leaves has been reported to lead to nicotine-like poisoning and death by respiratory paralysis (Castorena et al., 1987; Mellick et al., 1999; Sims et al., 1999; Mizrachi et al., 2000; Steenkamp et al., 2002). Cotinine, the primary metabolite of nicotine in humans, has little or no effects on cognitive performance and no cardiovascular effects in humans, but has been reported to modify symptoms of nicotine withdrawal (Benowitz et al., 1983b; Keenan et al., 1994; Hatsukami et al., 1997, 1998a,b; Herzig et al., 1998; Zevin et al., 2000b). Trans-3'-hydroxycotinine, the main metabolite of cotinine, has no cardiovascular effects (Scherer et al., 1988; Benowitz and Jacob, 2001). To our knowledge, no studies of the pharmacologic effects of any of the other minor alkaloids in humans have been reported.
III. Absorption of Nicotine from Tobacco Smoke and Nicotine Medications
Nicotine is distilled from burning tobacco and is carried proximally on tar droplets (also called particulate matter) which are inhaled. Absorption of nicotine across biological membranes depends on pH. Nicotine is a weak base with a pKa of 8.0 (Fowler, 1954). In its ionized state, such as in acidic environments, nicotine does not rapidly cross membranes. The pH of smoke from flue-cured tobaccos, found in most cigarettes, is acidic (pH 5.5–6.0) (Sensabaugh and Cundiff, 1967; Brunnemann and Hoffmann, 1974). At this pH, nicotine is primarily ionized. As a consequence, there is little buccal absorption of nicotine from flue-cured tobacco smoke, even when it is held in the mouth (Gori et al., 1986). Smoke from aircured tobaccos, the predominant tobacco used in pipes, cigars, and some European cigarettes, is more alkaline (pH 6.5 or higher), and considerable nicotine is unionized (Sensabaugh and Cundiff, 1967). Smoke from these products is well absorbed through the mouth (Armitage et al., 1978). It has recently been proposed that the pH of cigarette smoke particulate matter is higher than previously thought, and thus, a larger portion of nicotine would be in the unionized form, facilitating rapid pulmonary absorption (Pankow, 2001). The effective pH values for particulate matter in various brands of cigarettes were measured to span a range of 6.0 to 7.8 (Pankow et al., 2003).
When tobacco smoke reaches the small airways and alveoli of the lung, the nicotine is rapidly absorbed. Blood concentrations of nicotine rise quickly during and peak at the completion of cigarette smoking (Fig. 2). The rapid absorption of nicotine from cigarette smoke through the lungs, presumably because of the huge surface area of the alveoli and small airways, and dissolution of nicotine in the fluid of pH 7.4 in the human lung facilitates transfer across membranes. On average, about 1 mg (range 0.3–2 mg) of nicotine is absorbed systemically during smoking (Benowitz and Jacob, 1984; Gori and Lynch, 1985). About 80 to 90% of inhaled nicotine is absorbed during smoking as assessed using 14C-nicotine (Armitage et al., 1975). The efficacy of absorption of nicotine from environmental smoke in nonsmoking women has been measured to be 60 to 80% (Iwase et al., 1991). After a puff, high levels of nicotine reach the brain in 10 to 20 s, faster than with intravenous administration, producing rapid behavioral reinforcement through the activation of the dopaminergic reward system (Benowitz, 1990, 1996b). The rapidity of rise in nicotine levels permits the smoker to titrate the level of nicotine and related effects during smoking and makes smoking the most reinforcing and dependence-producing form of nicotine administration (Benowitz, 1990; Henningfield and Keenan, 1993).
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The process of cigarette smoking is complex, and as mentioned above, the smoker can manipulate the dose of nicotine and nicotine brain levels on a puff by puff basis. The intake of nicotine during smoking depends on the puff volume, the depth of inhalation, the extent of dilution with room air, the rate of puffing, and the intensity of puffing. For this reason, the machine-determined nicotine yields of cigarettes (U.S. Federal Trade Commission, FTC yields) cannot be used to estimate the dose of nicotine by a smoker (Russell et al., 1980; Benowitz et al., 1983a; Jarvis et al., 2001). In general, cigarette smokers switching from a higher to a lower-yield cigarette will compensate, i.e., will change the smoking pattern to gain more nicotine (USDHHS, 2001).
Chewing tobacco and snuff are buffered to alkaline pH to facilitate absorption of nicotine through oral mucosa (Benowitz, 1999). Although the absorption through cell membranes is rapid for these more alkaline tobacco products, the rise in the brain nicotine level is slower than with smoking. Concentrations of nicotine in the blood rise gradually with the use of smokeless tobacco and plateau at about 30 min with levels persisting and declining only slowly over 2 h or more (Fig. 2) (Benowitz et al., 1988).
The various formulations of nicotine replacement therapy (NRT), such as nicotine gum, transdermal patch, nasal spray, inhaler, sublingual tablets, and lozenges, are buffered to alkaline pH to facilitate the absorption of nicotine through cell membranes. Absorption of nicotine from all NRTs is slower and the increase in nicotine blood levels more gradual than from smoking (Table 1). This slow increase in blood and especially brain levels results in low abuse liability of NRTs (Henningfield and Keenan, 1993; West et al., 2000). Only nasal spray provides a rapid delivery of nicotine that is closer to the rate of nicotine delivery achieved with smoking (Sutherland et al., 1992; Gourlay and Benowitz, 1997; Guthrie et al., 1999). The absolute dose of nicotine absorbed systemically from nicotine gum is much less than the nicotine content of the gum, in part, because considerable nicotine is swallowed with subsequent first-pass metabolism (Benowitz et al., 1987). Some nicotine is also retained in chewed gum. A portion of the nicotine dose is swallowed and subjected to first-pass metabolism when using other NRTs, inhaler, sublingual tablets, nasal spray, and lozenges (Johansson et al., 1991; Bergstrom et al., 1995; Lunell et al., 1996; Molander and Lunell, 2001; Choi et al., 2003). Bioavailability for these products with absorption mainly through the mucosa of the oral cavity and a considerable swallowed portion is about 50 to 80% (Table 1).
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Nicotine is poorly absorbed from the stomach because it is protonated (ionized) in the acidic gastric fluid, but is well absorbed in the small intestine, which has a more alkaline pH and a large surface area. Following the administration of nicotine capsules or nicotine in solution, peak concentrations are reached in about 1 h (Benowitz et al., 1991; Zins et al., 1997; Dempsey et al., 2004). The oral bioavailability of nicotine is about 20 to 45% (Benowitz et al., 1991; Compton et al., 1997; Zins et al., 1997). Oral bioavailability is incomplete because of the hepatic first-pass metabolism. Also the bioavailability after colonic (enema) administration of nicotine (examined as a potential therapy for ulcerative colitis) is low, around 15 to 25%, presumably due to hepatic first-pass metabolism (Zins et al., 1997). Cotinine is much more polar than nicotine, is metabolized more slowly, and undergoes little, if any, first-pass metabolism after oral dosing (Benowitz et al., 1983b; De Schepper et al., 1987; Zevin et al., 1997).
Nicotine base is well absorbed through the skin. That is the reason for the occupational risk of nicotine poisoning (green tobacco sickness) in tobacco harvesters who are exposed to wet tobacco leaves (McBride et al., 1998). That is also the basis for transdermal delivery technology (Benowitz, 1995). Currently in the United States several different nicotine transdermal systems are marketed. All are multilayer patches. The rate of release of nicotine into the skin is controlled by the permeability of the skin, rate of diffusion through a polymer matrix, and/or rate of passage through a membrane in the various patches. The rates of nicotine delivery and plasma nicotine concentrations vary among the different transdermal systems (Fig. 3) (Fant et al., 2000). In all cases, there is an initial lag time of about 1 h before nicotine appears in the bloodstream, and there is continued systemic absorption (about 10% of the total dose) after the patch is removed, the latter due to residual nicotine in the skin.
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IV. Distribution of Nicotine in Body Tissues
After absorption, nicotine enters the bloodstream where, at pH 7.4, it is about 69% ionized and 31% unionized. Binding to plasma proteins is less than 5% (Benowitz et al., 1982a). The drug is distributed extensively to body tissues with steady-state volume of distribution averaging 2.6 body weight (Table 2). Based on human autopsy samples from smokers, the highest affinity for nicotine is in the liver, kidney, spleen, and lung and the lowest affinity in adipose tissue (Urakawa et al., 1994). Cotinine concentrations are highest in the liver. In skeletal muscle the concentrations of nicotine and cotinine are close to that of whole blood. In a large postmortem case series of suicides and homicides with nicotine solution sold as a pesticide, brain and kidney nicotine levels were 75 to 80% of the level in liver, whereas blood levels were as high as in the liver (Grusz-Harday, 1967). In one fatal case of intentional overdose of nicotine patches, brain nicotine levels were about 2-fold higher than in peripheral blood and about 40% of the nicotine level in the liver (Kemp et al., 1997). Nicotine binds to brain tissues with high affinity, and the receptor binding capacity is increased in smokers compared with nonsmokers (Benwell et al., 1988; Breese et al., 1997; Court et al., 1998; Perry et al., 1999). The increase in the binding is caused by a higher number of nicotinic cholinergic receptors in the brain of the smokers. Nicotine accumulates markedly in gastric juice and saliva (Russell and Feyerabend, 1978; Lindell et al., 1993, 1996). Gastric juice/plasma and saliva/plasma concentration ratios are 61 and 11 with transdermal nicotine administration, and 53 and 87 with smoking, respectively (Lindell et al., 1993, 1996). The accumulation is caused by ion-trapping of nicotine in gastric juice and saliva. Nicotine also accumulates in breast milk (milk/plasma ratio 2.9) (Luck and Nau, 1984; Dahlstrom et al., 1990). Nicotine crosses the placental barrier easily, and there is evidence for the accumulation of nicotine in fetal serum and amnionic fluid in slightly higher concentrations than in maternal serum (Luck et al., 1985; Pastrakuljic et al., 1998; Dempsey and Benowitz, 2001).
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The time course of nicotine in the brain and in other body organs and resultant pharmacologic effects are highly dependent on the route and rate of dosing. Smoking a cigarette delivers nicotine rapidly to the pulmonary venous circulation, from which it moves quickly to the left ventricle of the heart and to the systemic arterial circulation and to the brain. The lag time between a puff of a cigarette and nicotine reaching the brain is 10 to 20 s. Although the delivery of nicotine to the brain is rapid, there is nevertheless significant pulmonary uptake and some delayed release of nicotine as evidenced by pulmonary positron emission tomography data and the slow decrease in the arterial concentrations of nicotine between puffs (Lunell et al., 1996; Rose et al., 1999). Nicotine concentrations in arterial blood after smoking a cigarette can be quite high, reaching up to 100 ng/ml, but usually ranging between 20 and 60 ng/ml (Armitage et al., 1975; Henningfield et al., 1993; Gourlay and Benowitz, 1997; Rose et al., 1999; Lunell et al., 2000). The usual peak arterial nicotine concentration after the first puff is lower, averaging 7 ng/ml (Rose et al., 1999). As high as 10-fold arterial/venous nicotine concentration ratios have been measured (Henningfield et al., 1993), but the mean ratio is typically around 2.3 to 2.8 (Gourlay and Benowitz, 1997; Rose et al., 1999). The rapid rate of delivery of nicotine by smoking (or intravenous injection, which presents similar distribution kinetics) results in high levels of nicotine in the central nervous system with little time for development of tolerance. The result is a more intense pharmacologic action (Porchet et al., 1987). The short time interval between puffing and nicotine entering the brain also allows the smoker to titrate the dose of nicotine to a desired pharmacologic effect, further reinforcing drug self-administration and facilitating the development of addiction.
In contrast, slow delivery of nicotine, such as by transdermal systems, results in little, if any, arterial-venous disequilibrium. The resultant brain levels of nicotine are much lower than after smoking, and the gradual rise in levels of nicotine in the central nervous system allows for the development of considerable tolerance to pharmacologic effects. Thus, the intensity of central nervous system effects is much less, and the addiction liability with the use of transdermal nicotine is virtually nil (Henningfield and Keenan, 1993). Routes of dosing that are associated with more rapid rates of delivery, such as nasal spray, are expected to result in higher intensity of effects and higher addiction liability when compared with products with slower absorption. Some indications of this were seen in a recent study comparing the abuse liability of the nicotine patch, gum, nasal spray, and inhaler in smoking cessation (West et al., 2000). Nasal spray had the highest rate of continuing use at the end of the study compared with the other NRTs; however, overall abuse liability was low for all products. These same considerations regarding rate of delivery and pharmacologic effects are expected to apply to nicotine-related compounds.
V. Nicotine and Cotinine Blood Levels during Tobacco Use and Nicotine Replacement Therapy
Blood or plasma nicotine concentrations sampled in the afternoon in smokers generally range from 10 to 50 ng/ml (Benowitz et al., 1990). Typical trough concentrations during daily smoking are 10 to 37 ng/ml, and typical peak concentrations range between 19 and 50 ng/ml (Schneider et al., 2001). The increment in venous blood nicotine concentration after smoking a single cigarette ranges from 5 to 30 ng/ml, depending on how a cigarette is smoked. In a recent study, the mean nicotine boost after smoking a cigarette was 10.9 ng/ml in smokers with no smoking abstinence on the study day (Patterson et al., 2003). Blood levels peak at the end of smoking a cigarette and decline rapidly over the next 20 min due to tissue distribution. The distribution half-life averages about 8 min.
Peak venous blood levels of nicotine are similar, although the rate of rise of nicotine is slower, for cigar smokers and users of snuff and chewing tobacco compared with cigarette smokers (Armitage et al., 1978; Benowitz et al., 1988). Pipe smokers, particularly those who have previously smoked cigarettes, may have blood and urine levels of nicotine as high as cigarette smokers (Turner et al., 1977; Wald et al., 1981; McCusker et al., 1982). Primary pipe smokers who have not previously smoked cigarettes tend to have lower nicotine levels. Likewise, cigar smokers who have previously smoked cigarettes may inhale more deeply and achieve higher blood levels of nicotine than primary cigar smokers (Armitage et al., 1978), although on average, based on urinary cotinine levels, daily nicotine intake appears to be less for cigar compared with cigarette or pipe smokers (Wald et al., 1984).
The plasma half-life of nicotine after intravenous infusion or cigarette smoking averages about 2 h (Table 2). However, when half-life is determined using the time course of urinary excretion of nicotine, which is more sensitive in detecting lower levels of nicotine in the body, the terminal half-life averages 11 h (Jacob et al., 1999). The longer half-life detected at lower concentrations of nicotine is most likely a consequence of slow release of nicotine from body tissues. Based on a half-life of 2 h for nicotine, one would predict accumulation over 6 to 8 h (3 to 4 half-lives) of regular smoking and persistence of significant levels for 6 to 8 h after cessation of smoking. If a smoker smokes until bedtime, significant levels should persist all night. Studies of blood levels in regular cigarette smokers confirm these predictions (Fig. 4) (Benowitz et al., 1982b). Peak and trough levels follow each cigarette, but as the day progresses, trough levels rise and the influence of peak levels become less important. Thus, nicotine is not a drug to which smokers are exposed intermittently and which is eliminated rapidly from the body. To the contrary, smoking represents a multiple dosing situation with considerable accumulation while smoking and persistent levels for 24 h of each day.
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Plasma levels of nicotine from nicotine replacement therapies tend to be in the range of low-level cigarette smokers. Thus, typical steady-state plasma nicotine concentrations with nicotine patches range from 10 to 20 ng/ml, for nicotine gum, inhaler, sublingual tablet, and nasal spray from 5 to 15 ng/ml (Benowitz et al., 1987, 1995; Schneider et al., 2001). Usually ad libitum use of NRTs results in one-third to two-thirds the concentration of nicotine that is achieved by cigarette smoking (Benowitz, 1993; Schneider et al., 2001). However, users of 4-mg nicotine gum may sometimes reach or even exceed the nicotine levels associated with smoking (McNabb et al., 1982, 1984). For the sake of comparison, systemic doses from various nicotine delivery systems are as follows: cigarette smoking, 1 to 2 mg per cigarette (Benowitz and Jacob, 1984; Jarvis et al., 2001); nicotine gum, 2 mg for a 4-mg gum (Benowitz et al., 1988; Stevens, 1994); transdermal nicotine, 5 to 21 mg per day, depending on the patch (Table 1); nicotine nasal spray, 0.7 mg per 1-mg dose of one spray in each nostril (Johansson et al., 1991; Gourlay and Benowitz, 1997); nicotine inhaler, 2 mg for a 4-mg dose released from the 10-mg inhaler (Molander et al., 1996); nicotine lozenge, 1 mg for a 2-mg lozenge (Choi et al., 2003); oral snuff, 3.6 mg for 2.5 g held in the mouth for 30 min (Benowitz et al., 1988); and chewing tobacco, 4.5 mg for 7.9 g chewed for 30 min (Benowitz et al., 1988).
Cotinine is present in the blood of smokers in much higher concentrations than those of nicotine. Cotinine blood concentrations average about 250 to 300 ng/ml in groups of cigarette smokers (Benowitz et al., 1983a; Gori and Lynch, 1985). We have seen levels in tobacco users ranging up to 900 ng/ml. After stopping smoking, levels decline in a log linear fashion with an average half-life of about 16 h (Table 2). The half-life of cotinine derived from nicotine is longer than the half-life of cotinine administered as cotinine (Zevin et al., 1997). This is caused by slow release of nicotine from tissues. Because of the long half-life there is much less fluctuation in cotinine concentrations throughout the day compared with nicotine concentrations. As expected, there is a gradual rise in cotinine levels throughout the day, peaking at the end of smoking and persisting at high concentrations overnight (Fig. 4). Because of the long half-life of cotinine, it has been used as a biomarker for daily intake, both in cigarette smokers and in those exposed to environmental tobacco smoke (Benowitz, 1996a). There is a high correlation among cotinine concentrations measured in plasma, saliva, and urine, and measurements in any one of these fluids can be used as a marker of nicotine intake. Cotinine levels produced by NRTs are usually 30 to 70% of the levels detected while smoking (Hurt et al., 1994; Schneider et al., 1995, 1996).
A. Primary Metabolites of Nicotine
Studies of nicotine metabolism have been conducted over several decades, and different authors have used different names for the same metabolite. The most frequently used alternative names for nicotine and many of its metabolites are listed in Table 3. Since Chemical Abstracts provides access to much of the world's literature on chemistry and pharmacology, Chemical Abstracts Registry numbers and index names are listed in this table.
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Nicotine is extensively metabolized to a number of metabolites (Fig. 5) by the liver. Six primary metabolites of nicotine have been identified. Quantitatively, the most important metabolite of nicotine in most mammalian species is the lactam derivative cotinine. In humans, about 70 to 80% of nicotine is converted to cotinine (Benowitz and Jacob, 1994). This transformation involves two steps. The first is mediated by a cytochrome P450 system to produce nicotine-
1'(5')-iminium ion, which is in equilibrium with 5'-hydroxynicotine (Murphy, 1973; Brandange and Lindblom, 1979b; Peterson et al., 1987). The second step is catalyzed by a cytoplasmic aldehyde oxidase (Brandange and Lindblom, 1979a; Gorrod and Hibberd, 1982). Nicotine iminium ion has received considerable interest since it is an alkylating agent and, as such, could play a role in the pharmacology of nicotine (Gorrod and Jenner, 1975; Hibberd and Gorrod, 1981; Shigenaga et al., 1988; Jacob et al., 1997).
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Nicotine N'-oxide is another primary metabolite (Fig. 6) of nicotine, although only about 4 to 7% of nicotine absorbed by smokers is metabolized via this route (Byrd et al., 1992; Benowitz et al., 1994). The conversion of nicotine to nicotine N'-oxide involves a flavin-containing monooxygenase 3 (FMO3), which results in formation of both possible diasteriomers, the 1'-(R)-2'-(S)-cis and 1'-(S)-2'-(S)-trans-isomers in animals (Cashman et al., 1992; Park et al., 1993). In humans, this pathway is highly selective for the trans-isomer (Cashman et al., 1992). Only the trans-isomer of nicotine N'-oxide was detected in urine after administration of nicotine by intravenous infusion, transdermal patch, or smoking (Park et al., 1993). It appears that nicotine N'-oxide is not further metabolized to any significant extent, except by reduction back to nicotine, which may lead to recycling of nicotine in the body (Dajani et al., 1975). A study by Beckett et al. (1970) indicated that reduction of nicotine N'-oxide to nicotine in humans is mediated by bacterial action in the large intestine. These investigators found that nicotine N'-oxide administered intravenously was excreted largely, if not entirely, in the urine unchanged, whereas administration rectally as an enema resulted in extensive conversion to nicotine and cotinine, which appeared in the urine. Oral administration of nicotine N'-oxide resulted in small but significant urinary excretion of nicotine and cotinine.
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In addition to oxidation of the pyrrolidine ring, nicotine is metabolized by two nonoxidative pathways, methylation of the pyridine nitrogen giving nicotine isomethonium ion (also called N-methylnicotinium ion) and glucuronidation (Fig. 6). The methylation pathway was first reported by McKennis, who found it in dogs dosed with (S)-nicotine (McKennis et al., 1963a). Studies of the N-methylation pathway using animal models and human liver homogenates show that S-adenosyl-L-methionine is the source of the methyl group in a reaction catalyzed by the amine N-methyltransferase (Crooks and Godin, 1988; Nwosu and Crooks, 1988). Human liver cytosol was capable of methylating both enantiomers of nicotine, but the (R)-isomer was methylated more rapidly than the (S)-isomer. Small amounts of nicotine isomethonium ion have been detected in smokers' urine (Neurath et al., 1987, 1988). In light of the reported pharmacologic activity of nicotine isomethonium ion (Dwoskin et al., 1992), further studies of this metabolite are warranted.
Nicotine glucuronidation results in an N-quaternary glucuronide in humans (Curvall et al., 1991; Byrd et al., 1992; Benowitz et al., 1994). This reaction is catalyzed by uridine diphosphate-glucuronosyltransferase (UGT) enzyme(s) producing (S)-nicotine-N-
-glucuronide (Seaton et al., 1993). About 3 to 5% of nicotine is converted to nicotine glucuronide and excreted in urine in humans.
Oxidative N-demethylation is frequently an important pathway in the metabolism of xenobiotics, but this route is, in most species, a minor pathway in the metabolism of nicotine. Conversion of nicotine to nornicotine in humans has been demonstrated. We found that small amounts of deuterium-labeled nornicotine are excreted in urine of smokers administered deuterium-labeled nicotine (Jacob and Benowitz, 1991). Metabolic formation of nornicotine from nicotine has also been reported by Neurath et al. (1991). Nornicotine is a constituent of tobacco leaves. However, the majority of urine nornicotine is derived from the metabolism of nicotine with less than 40% coming directly from tobacco, as estimated from the difference in nornicotine excretion in smokers during smoking and during transdermal nicotine treatment (0.65 and 0.41%, respectively) (Benowitz et al., 1994). The formation of nornicotine from nicotine has been shown to be mediated by cytochrome P450 system in rabbits (Williams et al., 1990b). Formation of an iminium ion as an intermediate in the demethylation of nicotine was reported by Castagnoli and coworkers, who characterized N'-cyanomethylnornicotine in extracts obtained following incubation of rabbit liver microsomes with nicotine and sodium cyanide (Nguyen et al., 1979). This observation implies the intermediacy of N'-methylene-iminium ion, which is captured by cyanide ion to form the stable cyano adduct. In the absence of cyanide, the iminium ion would be expected to hydrolyze to nornicotine and formaldehyde.
A new cytochrome P450 mediated metabolic pathway for nicotine metabolism was recently reported by Hecht et al. (2000). 2'-Hydroxylation of nicotine was shown to produce 4-(methylamino)-1-(3-pyridyl)-1-butanone with 2'-hydroxynicotine as an intermediate (Fig. 5). 2'-Hydroxynicotine also yields nicotine-1'(2')-iminium ion. 4-(Methylamino)-1-(3-pyridyl)-1-butanone is further metabolized to 4-oxo-4-(3-pyridyl)butanoic acid and 4-hydroxy-4-(3-pyridyl)butanoic acid. Previously these metabolites were thought to arise mainly through metabolism of cotinine (McKennis et al., 1964). The new pathway is potentially significant since 4-(methylamino)-1-(3-pyridyl)-1-butanone can be converted to carcinogenic NNK. However, endogenous production of NNK from nicotine has not been detected in humans or rats (Carmella et al., 1997; Hecht et al., 1999a).
About 10 to 15% of nicotine and metabolites is excreted as 4-oxo-4-(3-pyridyl)butanoic acid and 4-hydroxy-4-(3-pyridyl)butanoic acid in smokers' urine (Hecht et al., 1999b,c). Less than 0.5% of cotinine dosed to nonsmokers was recovered as these metabolites, demonstrating that the 2'-hydroxylation of nicotine is the primary, although not the only, pathway for their production (unpublished data reported in Hecht et al., 2000). 4-Hydroxy-4-(3-pyridyl)butanoic acid is further metabolized to 3-pyridylacetic acid, the so-called terminal metabolite of nicotine. It has been detected in human urine following oral administration of cotinine (McKennis et al., 1964). It is speculated (McKennis et al., 1964) that 3-pyridylacetic acid is formed via dehydration of the hydroxyacid 4-hydroxy-4-(3-pyridyl)butanoic acid to give 4-(3-pyridyl)-3-butenoic acid, reduction to 4-(3-pyridyl)-butanoic acid, -oxidation, and cleavage to 3-pyridylacetic acid. This metabolic scheme is analogous to the catabolism of fatty acids, although there is no experimental evidence for the intermediacy of 4-(3-pyridyl)-3-butenoic acid or of 4-(3-pyridyl)-butanoic acid.
Although on average about 70 to 80% of nicotine is metabolized via the cotinine pathway in humans, only 10 to 15% of the nicotine absorbed by smokers appears in the urine as unchanged cotinine (Benowitz et al., 1994). A number of cotinine metabolites have been structurally characterized (Fig. 5). Indeed, it appears that most of the reported urinary metabolites of nicotine are derived from cotinine. Six primary metabolites of cotinine have been reported in humans: 3'-hydroxycotinine (Bowman and McKennis, 1962; McKennis et al., 1963b; Neurath et al., 1987), 5'-hydroxycotinine (also called allohydroxycotinine) (Neurath, 1994), which exists in tautomeric equilibrium with the open chain derivative 4-oxo-4-(3-pyridyl)-N-methylbutanamide (Bowman and McKennis, 1962; McKennis et al., 1962), cotinine N-oxide (Shulgin et al., 1987; Kyerematen et al., 1990b), cotinine methonium ion (McKennis et al., 1963a), cotinine glucuronide (Curvall et al., 1991; Caldwell et al., 1992), and norcotinine (also called demethylcotinine) (Bowman et al., 1959; Kyerematen et al., 1990b).
3'-Hydroxycotinine is the main nicotine metabolite detected in smokers' urine. It is also excreted as a glucuronide conjugate (Fig. 5) (Curvall et al., 1991; Benowitz et al., 1994). 3'-Hydroxycotinine and its glucuronide conjugate account for 40 to 60% of the nicotine dose in urine (Byrd et al., 1992; Benowitz et al., 1994; Andersson et al., 1997; Hecht et al., 1999b). The conversion of cotinine to 3'-hydroxycotinine in humans is highly stereoselective for the trans-isomer, as less than 5% is detected as cis-3'-hydroxycotinine in urine (Jacob et al., 1990; Voncken et al., 1990). Although nicotine and cotinine conjugates are N-glucuronides, the only 3'-hydroxycotinine conjugate detected in urine is the O-glucuronide (Byrd et al., 1994). Recently, significant rates of N-glucuronidation of 3'-hydroxycotinine were detected in human liver microsomes, but this metabolite was not detected in urine (Kuehl and Murphy, 2003b). Thus, the N-glucuronide of 3'-hydroxycotinine might be unstable, or the concentration was too low to detect the N-glucuronide by the methodology employed.
As with nicotine N'-oxide, cotinine N-oxide can be reduced back to the parent amine in vivo as evidenced by a study in rabbits (Yi et al., 1977). Studies with P450 enzyme inhibitors in hamster and guinea pig liver microsomes show that, unlike nicotine N'-oxide, cotinine N-oxide is formed by P450 enzymes (Hibberd and Gorrod, 1985). Cotinine N-oxide accounts for 2 to 5% of the nicotine and metabolites in smokers' urine (Byrd et al., 1992; Benowitz et al., 1994; Meger et al., 2002). McKennis and coworkers showed that cotinine methonium ion is excreted in urine after cotinine administration to a single subject (McKennis et al., 1963a).
Norcotinine has been detected in smokers' urine (about 1% of total nicotine and metabolites) (Byrd et al., 1992, 1995b). Two pathways for its formation are possible, demethylation of cotinine or oxidative metabolism of nornicotine (Fig. 5). Animal studies have demonstrated the existence of both of these pathways. Wada et al. (1961) and Papadopoulos (1964) reported that norcotinine is a metabolite of nornicotine, and Harke et al. (1974) detected norcotinine in urine of pigs following cotinine administration. From studies in which deuterium-labeled nicotine and cotinine were administered, we have obtained evidence that both pathways occur in humans as well (P. Jacob III, L. Yu, and N. L. Benowitz, unpublished data). N'-Hydroxymethylnorcotinine has been detected in vitro in hamster hepatic microsomes after incubation with cotinine in some (Li and Gorrod, 1994) but not all (Murphy et al., 1999) studies. It is speculated to be an intermediate in the conversion of cotinine to norcotinine (Li and Gorrod, 1994). In rat liver microsomes, norcotinine is further metabolized to 4-oxo-4-(3-pyridyl)-butanamide (Eldirdiri et al., 1997). 4-Oxo-4-(3-pyridyl)-butanamide is metabolized further to 4-oxo-4-(3-pyridyl)butanoic acid. 4-Oxo-4-(3-pyridyl)-butanamide is in equilibrium with 5'-hydroxynorcotinine (allohydroxy-demethylcotinine), dehydration of which leads to the formation of norcotinine 2'(3')-enamine (Kyerematen et al., 1990b). Using liquid chromatography-tandem mass spectrometry, we have found that small amounts of 4-oxo-4-(3-pyridyl)-butanamide (5'-hydroxynorcotinine) are present in smokers' urine (P. Jacob III, L. Yu, and N. L. Benowitz, unpublished data). Recently, the human CYP2A6 enzyme was shown to catalyze formation of norcotinine and 5'-hydroxycotinine from cotinine (Murphy et al., 1999).
5'-Hydroxycotinine has been detected in smokers' urine in levels of less than 4% of those of the 3'-hydroxycotinine (Neurath, 1994). 5'-Hydroxycotinine N-oxide has been isolated from rat urine after nicotine administration, but was unmeasurable in urine samples from smokers (Schepers et al., 1999). The ketoamide 4-oxo-4-(3-pyridyl)-N-methylbutanamide derived from 5'-hydroxycotinine is presumably the precursor of a number of nicotine metabolites which result from degradation of the pyrrolidine ring. These include the keto acid 4-oxo-4-(3-pyridyl)butanoic acid, its reduction product 4-hydroxy-4-(3-pyridyl)butanoic acid, a hydroxyacid which can be in equilibrium with the lactone 5-(3-pyridyl)-tetrahydrofuran-2-one, and 3-pyridylacetic acid, the so-called terminal metabolite of nicotine. As mentioned earlier, these metabolites are probably formed mainly via nicotine 2'-hydroxylation (Hecht et al., 2000).
Cotinine is a lactam, and it is reasonable to expect that the open-chain form 
-(3-pyridyl)--methylaminobutyric acid [4-methylamino-4-(3-pyridyl)butanoic acid], an amino acid, might be an intermediate in cotinine formation or be formed via hydrolysis of cotinine. This open-chain derivative has indeed been reported to be present in urine of smokers (Bowman et al., 1959), but based on in vitro studies with liver homogenates, it appears that cotinine is formed directly by oxidation of the nicotine-1'(5')-iminium ion (Brandange and Lindblom, 1979a; Gorrod and Hibberd, 1982) rather than via the amino acid.
C. Quantitative Aspects of Nicotine Metabolism
Quantitative aspects of the pattern of nicotine metabolism have been elucidated fairly well in people (Fig. 7). About 90% of a systemic dose of nicotine can be accounted for as nicotine and metabolites in the urine (Benowitz et al., 1994). Based on studies with simultaneous infusion of labeled nicotine and cotinine, it has been determined that 70 to 80% of nicotine is converted to cotinine (Benowitz and Jacob, 1994). About 4 to 7% of nicotine is excreted as nicotine N'-oxide and 3 to 5% as nicotine glucuronide (Byrd et al., 1992; Benowitz et al., 1994). Cotinine is excreted unchanged in the urine to a small degree (10 to 15% of the nicotine and metabolites in urine). The remainder is converted to metabolites, primarily trans-3'-hydroxycotinine (33–40%), cotinine glucuronide (12–17%), and trans-3'-hydroxycotinine glucuronide (7–9%).
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The rate of metabolism of nicotine can be determined by measuring blood levels after administration of a known dose of nicotine. We have studied cigarette smokers and nonsmokers given intravenous infusions of nicotine for 30 to 60 min (Table 2). Total and renal clearance are computed directly and the nonrenal or metabolic clearance computed as the difference between the total and renal clearance. Total clearance of nicotine averages about 1200 ml/min. Nonrenal clearance represents about 70% of liver blood flow. Assuming most nicotine is metabolized by the liver, this means about 70% of the drug is extracted from the blood in each pass through the liver.
The metabolism of cotinine is much slower than that of nicotine. Cotinine clearance averages about 45 ml/min (Table 2). The clearance of (3'R,5'S)-trans-3'-hydroxycotinine is also quite slow, about 82 ml/min. Because these compounds are metabolized slowly, the rates of elimination of cotinine and 3'-hydroxycotinine are predicted to not be substantially influenced by changes in liver blood flow.
Recently, we evaluated the measurement of the 3'-hydroxycotinine/cotinine ratio in plasma and saliva as a noninvasive probe for CYP2A6 activity (Dempsey et al., 2004). The ratio was highly correlated with the oral clearance of nicotine and the oral clearance and half-life of cotinine. Correlation coefficients of oral nicotine and cotinine clearances with plasma 3'-hydroxycotinine/cotinine ratios were 0.78 and 0.63, respectively, at 6 h after oral nicotine dosing. The 3'-hydroxycotinine/cotinine ratio could be used to phenotype nicotine metabolism and CYP2A6 enzyme in nonsmokers after oral nicotine dosing and in smokers after smoking regular cigarettes. We have previously shown that this ratio is a predictor of cigarette consumption (Benowitz et al., 2003).
D. Liver Enzymes Responsible for Nicotine and Cotinine Metabolism
1. Cytochrome P450 Enzymes.
In vitro and in vivo studies show that CYP2A6 is the enzyme that is primarily responsible for the oxidation of nicotine and cotinine. The evidence for the role of CYP2A6 includes human liver microsome studies in which the rate and extent of nicotine oxidation to cotinine and cotinine oxidation to 3'-hydroxycotinine are highly correlated with coumarin 7-hydroxylase activity (known to be mediated by CYP2A6) and correlated with immunochemically determined hepatic CYP2A6 levels (Cashman et al., 1992; Berkman et al., 1995; Nakajima et al., 1996a,b). Nicotine oxidation to cotinine has also been shown to be inhibited by coincubation with coumarin (a competitive inhibitor of CYP2A6) and CYP2A6 antibodies (Nakajima et al., 1996b; Messina et al., 1997; Le Gal et al., 2003). CYP2A6 expression systems have high activity for metabolizing nicotine and cotinine (Nakajima et al., 1996a,b). Of note is that human liver specimens exhibit marked individual variability in levels of CYP2A6 mRNA and coumarin 7-hydroxylase activity, consistent with the known wide variability in the rate of nicotine metabolism in people (Pelkonen et al., 2000; Tyndale and Sellers, 2002). In addition to forming cotinine and 3'-hydroxycotinine, CYP2A6 is active in the 2'-hydroxylation of nicotine and in the formation of 5'-hydroxycotinine and norcotinine from cotinine (Murphy et al., 1999; Hecht et al., 2000).
In vivo studies support the significant role of CYP2A6 in nicotine metabolism. Methoxsalen, a CYP2A6 inhibitor, reduces the first-pass metabolism of oral nicotine, decreases clearance of subcutaneously administered nicotine, and decreases urinary levels of 3'-hydroxycotinine in smokers (Sellers et al., 2000, 2003a). Subjects homozygous for CYP2A6 deletion allele (CYP2A6*4) have very low plasma cotinine levels after smoking or nicotine administration (Nakajima et al., 2000, 2001; Kwon et al., 2001; Xu et al., 2002b; Dempsey et al., 2004). Urinary excretion of cotinine in homozygotes for the deletion allele is only 10 to 30% of the levels detected in subjects with functional genes, depending on the experimental design of the study (Kitagawa et al., 1999; Yang et al., 2001; Zhang et al., 2002). No 3'-hydroxycotinine is detected in plasma and saliva after oral nicotine administration in subjects homozygous for CYP2A6*4 (Dempsey et al., 2004). Also, urine 3'-hydroxycotinine excretion after smoking is markedly decreased in subjects with two CYP2A6*4 alleles (Zhang et al., 2002). Thus, the metabolism of nicotine can be used to phenotype CYP2A6 activity (Nakajima et al., 2002a).
Although the studies mentioned above demonstrate the significant role of CYP2A6 in nicotine metabolism, they illustrate that other enzymes must also be involved in formation of cotinine and 3'-hydroxycotinine, at least in subjects lacking the CYP2A6 enzyme. CYP2B6 is the second most active hepatic P450 enzyme in nicotine C-oxidation when investigated using hepatic tissues or expression systems in vitro, especially at high nicotine concentrations (Flammang et al., 1992; McCracken et al., 1992; Nakajima et al., 1996b; Yamazaki et al., 1999). Most of the studies with CYP2D6 expression systems show some activity toward nicotine metabolism (McCracken et al., 1992; Nakajima et al., 1996b; Yamazaki et al., 1999; Le Gal et al., 2003), but there are also studies showing no cotinine formation by expressed CYP2D6 (Flammang et al., 1992; Messina et al., 1997). In humans, CYP2D6 poor metabolizer and extensive metabolizer phenotypes have similar nicotine and cotinine pharmacokinetics (Benowitz et al., 1996), although ultrarapid metabolizer phenotype caused by amplification of CYP2D6 gene may be associated with accelerated nicotine metabolism (Saarikoski et al., 2000; Caporaso et al., 2001). CYP2E1 has some activity toward nicotine in in vitro systems at high nicotine concentrations (Flammang et al., 1992; Yamazaki et al., 1999; Le Gal et al., 2003). A preliminary report shows that heterologously expressed CYP2A13, a close relative of CYP2A6, has considerable activity toward nicotine and cotinine (Bao et al., 2000). CYP2A6 and CYP2A13 share several substrates, such as NNK and gasoline additive MTBE (methyl tertiary butyl ether) (Su et al., 2000). CYP2A13 is highly expressed in the human respiratory tract, especially in nasal mucosa (Getchell et al., 1993; Koskela et al., 1999; Su et al., 2000; Chen et al., 2003b). However, there is negligible expression of CYP2A13 in liver (Koskela et al., 1999; Su et al., 2000), indicating that it is probably of significance in nicotine clearance (if at all) only in subjects lacking functional CYP2A6 enzyme.
2. Aldehyde Oxidase.
Aldehyde oxidase is a cytosolic enzyme catalyzing the conversion of nicotine-1'(5')-iminium ion to cotinine (Brandange and Lindblom, 1979a). In in vitro studies with rabbit liver microsomes, nicotine-1'(5')-iminium ion accumulates in the absence of aldehyde oxidase (Obach and van Vunakis, 1990). Raloxifene, a selective estrogen receptor modulator and potent aldehyde oxidase inhibitor, inhibits the formation of cotinine in human liver cytosol with a Ki of 1.4 nM (Obach, 2004). In rat liver cytosol, aldehyde oxidase has been shown to catalyze the conversion of nicotine N'-oxide back to nicotine (Sugihara et al., 1996). There is no evidence for such a conversion in humans as nicotine N'-oxide administ
