Metabolism of nicotine.

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DRUG METABOLISM REVIEWS, 23( 1&2), 3-41 (1991)

METABOLISM OF NICOTINE* GABRIEL A. KYEREMATEN and ELLIOT S. VESELLt Department of Pharmacology The Pennsylvania State University College of Medicine Hershey, Pennsylvania I7033

111.

....... . ..4 ABSORPTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 DISTRIBUTION.. . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . 5

IV.

EXCRETION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. 11.

V.

INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . .

6

METABOLISM.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 A. C-Oxidation.. . . . . . . . . . , . . . . . . . . . . . . . . . . . , . . . . . 7 B. N-Oxidation.. . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 10 C. N-Demethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 D. Conjugation.. . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . 13

*This paper was refereed by Daniel S. Sitar, Ph.D., Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada R3E OW3. 'Address correspondence to E. S. Vesell, M.D., Department of Pharmacology, The Pennsylvania State University, College of Medicine, l? 0. Box 850, Hershey, PA 17033. 3 Copyright 0 1991 by Marcel Dekker, Inc.


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VI.

KYEREMATEN AND VESELL E . Pyrrolidine Ring Cleavage . . . . . . . . . . . . . . . . . . . . . . . . F . Peroxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 17

DISPOSITION KINETICS. . . . . . . . . . . . . . . . . . . . . . . . . . .

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VII. HOST FACTORS THAT INFLUENCE NICOTINE PHARMA22 COKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Genetic and Developmental Factors. . . . . . . . . . . . . . . . . . 24 B. Smoking Habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 28 C. Drug Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 D. Dietary Habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Disease States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 31 F. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VIII. METHODOLOGICAL CONSIDERATIONS . . . . . . . . . . . . . . 3 I A. Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3I B. Models for Nicotine Metabolism. . . . . . . . . . . . . . . . . . . . 32

IX. CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . .

33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Nicotine, while not a prescribed drug, is widely consumed throughout the world as tobacco [ I ] . Recreational use of tobacco has persisted in virtually every culture exposed to it over the past few centuries [2]. As the principal pharmacologically active component of tobacco, nicotine is widely assumed to play a crucial role in establishing and maintaining this tobacco habit [3], which can become addictive (41. To attain better understanding and management of dependence problems, as well as numerous health hazards associated with tobacco consumption, it is desirable to establish more firmly nicotine’s exceedingly complex metabolic fate. This review examines the present state of knowledge concerning the metabolism and disposition of nicotine in man and experimental animals. Such a review seems warranted at this time because of several very recent advances, consisting of identification of new metabolites, elucidation of new pathways of nicotine biotransformation, and introduction of novel concepts regarding potential mechanisms. This progress stems from new applications of high-resolution analytical technology and in v i m experimental models of drug metabolism. Previous excellent reviews [5- I I ] comprehensively cover various aspects of nicotine disposition so that only brief summaries of nicotine absorption, distribution, and excretion are provided here.

METABOLISM OF NICOTINE

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11. ABSORPTION Perhaps the most salient aspect of nicotine absorption is its pH dependence [8, lo]. The main implication of this pH dependence, which follows predictions of the Henderson-Hasselbach equation, is that the extent of nicotine absorption varies markedly in different organs and regions of the body according to differences in the pH of their mucosal lining. As the undissociated base, nicotine is lipid soluble and readily permeates cell membranes [12]. Nicotine is thus absorbed not only through the lungs but also through the skin [ 13, 141, the gastrointestinal tract I IS], the buccal and nasal mucosae [l6-19], and also the renal tubule [7, 12, 201. Pulmonary absorption of nicotine is extremely rapid, occurring at a rate similar to that after intravenous administration [ 121. Nicotine is rapidly absorbed when cigarette smoke is inhaled, but negligibly absorbed when smoke is not inhaled, that is, retained in the mouth [16, 211. The actual amount of nicotine absorbed during the smoking process depends upon several factors. These include:

I . Type of tobacco: Since cigarette smoke is more acidic than cigar smoke, nicotine in cigar smoke is more nonionized and hence more absorbable at the higher pH of the mouth [22]. Thus, for absorption, cigar smoke does not require the inhalation needed for cigarette smoke. 2. Nicotine and moisture content of the tobacco. 3. Shape and configuration of smoke particles. 4. pH of body fluids and surfaces with which smoke comes in contact. 5 . Duration of contact between smoke and mucous membranes. Other factors, collectively referred to as a smoker’s “smoking profile,” encompass (i) degree and depth of smoke inhalation; (ii) number, duration, and volume of puffs; (iii) the force with which smoke is drawn; (iv) use of a holder or filter; (v) length of cigarette or cigar butt; and (vi) number of cigarettes smoked within a given time [6].

I I I. DISTRIBUTION Once absorbed, nicotine disappears rapidly from blood. This rapid plasma decay of nicotine has been attributed to its widespread uptake into tissues. as well as to its rapid metabolism [6]. Experiments conducted in vitro and in vivo have shown that nicotine, like many other alkaloids derived from plants, distributes extensively into virtually all tissues, a


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KYEREMATEN AND VESELL

phenomenon typical of many basic drugs 161. Nicotine, a weak base, has a pKa of 7.9. Thirty to 60 minutes after intravenous administration of nicotine to rats, concentrations 2 to 15 times higher than those of plasma occur in the adrenals, liver, brain, lung, heart, intestinal wall, spleen, thymus, skeletal muscle, and kidney (23-251. In dogs and humans, the nicotine concentration ratio between erythrocytes and plasma is 0.8 [8]. In human plasma, nicotine is bound to albumin and several serum lipoproteins. An initial estimate of 20% [26] was followed by later studies [27] indicating even less binding (about 5%). At a cellular level, nicotine accumulates in nonciliated epithelial cells of mouse bronchial tissue where it strongly binds to melanin [28, 291. SubcelMar distribution patterns show that nicotine is predominantly localized in the microsomal fraction in cats and in the cytosol in pigeons [30].

IV. EXCRETION In man and other mammals, the kidney is the main organ for excretion of nicotine [31]. As already indicated under Absorption, renal excretion of nicotine is also pH dependent 120, 32-34]. At urinary pH above 7, nicotine is readily reabsorbed through the renal tubule, and only 2% of a dose is recovered unchanged in urine. When urine is more acidic (pH less than 5 ) , as much as 23% of a dose can be recovered as the unmetabolized alkaloid. Thus, urinary acidification has been shown to result in increased cigarette consumption in man [35]. Other aspects of the renal elimination of nicotine metabolites are discussed later in Sec. V1. Saliva constitutes an important route for nicotine excretion. On exposure of humans and monkeys to nicotine, the ratio of nicotine concentration in saliva to plasma generally exceeds 10 [12, 251. Less important routes of nicotine elimination are sweat [36], breast milk [37-391, bile, gastric juice, and feces [31].

V. METABOLISM Recent applications of advances in separation technology and analytical spectrometry led to fresh insights into some of the complexities of nicotine metabolism. Specifically, new nicotine metabolites as well as pathways of biotransformation have been elucidated and proposed.



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Figure I summarizes several current concepts-some well established, others only postulated-for the pathways of nicotine metabolism in man and experimental animals. By combining data from man and experimental animals, this scheme of necessity depicts only a composite that probably differs in detail from one species to another. In most mammalian species, nicotine is rapidly and extensively metabolized primarily in the liver, but also to a small extent in the lung and kidney [9]. With respect to specific pathways and products, nicotine is converted directly to 6 primary metabolites: cotinine, nicotine- 1 '-N-oxide, nornicotine, N-methylnicotium, nicotine glucuronide, and ni~otine-A~'.~'-enamine (Fig. I). The contribution of each pathway to nicotine biotransformation differs markedly, not only between species, but also within a given species according to tissue as well as numerous host factors such as age, sex, smoking habits, dietary habits, drug pretreatment, and disease states. These factors are discussed in Sec. VII. Another important aspect of nicotine metabolism, emphasized recently in studies on nicotine biotransformation [40451,involves stereospecificity which is noted subsequently for each relevant reaction and pathway. For purposes of this review, nicotine metabolism is discussed under six general biotransformation pathways: C-oxidation, Noxidation, N-dernethylation, conjugation, ring (pyrrolidine) cleavage, and peroxidation. This format has the advantage of allowing inclusion of novel pathways into an overall scheme. A. C-Oxidation

Cotinine is the main primary product of the C-oxidation pathway of nicotine (Figs. 1 and 2). In almost every mammalian species studied, cotinine formation occurs both in v i m and in vivo [6, 91. Although considered a primary metabolite, cotinine has a complex genesis, involving an initial double-step oxidation of nicotine at the C-5' position. This oxidation results in formation of an iminium ion (Figs. 1 and 2) [46,471. Cytochrome P-450catalyzes essentially all production of the iminium ion [48].This stepwise reaction entails initial transfer of one electron from the lone pair on the pyrrolidine nitrogen atom of nicotine to a heme-bound electrondeficient oxygen atom of cytochrome P-450(Fig. 2). This oxidative step is stereoselective (471.Subsequent one-electron oxidation of the resulting radical cation causes a net loss of C-5' hydrogen atoms. This loss leads either directly to an iminium intermediate species, nic~tine-Al''~"-iminiurn ion, or indirectly to a carbinolamine intermediate, 5'-hydroxynicotine, via a carbon-centered free radical intermediate (Fig. 2). The two intermediate species, nicotine-A"'5''-iminium ion and 5'-hydroxynicotine, exist in equilibrium with each other. Further oxidation of the iminium ion intermediate


t

9


METABOLISM OF NlCOTINE

Nicotine

Nicotine -A'''')

r

/

4'-Hydroxycotinine (3-HydrOXyCOtinine)

Cotinine

1

\r

-iminium ion

It 5-Hydrox ynicotine

2'-Hydroxycotinine (Allohydroxycotinine)

FIG. 2. C-Oxidation pathways of nicotine metabolism. Brackets indicate hypothetical transient intermediates. Alternative names and designations are also included in brackets below some metabolites. to cotinine occurs through a reaction catalyzed by cytosolic aldehyde oxidases 149, 501. Secondary C-oxidation of cotinine involves hydroxylation of the pyrrolidine ring. Two separate reactions result in formation of 3-hydroxycotinine (4'-hydroxycotinine) and 2'-hydroxycotinine (allohydroxycotinine) (Fig. 2). Metabolic conversion of cot inine to 3-hydroxycotinine is highly stereospecific; in vivo, the trans isomer predominates (>98%) 1441. Recently truns3-hydroxycotinine has assumed a significant role in nicotine metabolism. It is now considered to be the major metabolite of nicotine in humans and

FIG. 1. Pathways of nicotine metabolism in mammals. The solid arrows show the established pathways; broken arrows denote hypothetical, as yet unconfirmed reactions. Brackets indicate transient intermediates. Alternative names and designations are also included in brackets below some metabolites. Asterisks indicate new metabolites described since the review by Nakayama II 1 1.


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guinea pigs [45, 51-54]. Cotinine was previously believed to be quantitatively the principal metabolite, but recently it was shown that in smokers, urinary concentrations of 3-hydroxycotinine exceed those of cotinine by 2- to 3-fold [40]. The other product of cotinine C-oxidation is 2’-hydroxycotinine (allohydroxycotinine). It has been identified in urine of dogs and monkeys after cotinine administration (Fig. 2) [ 5 5 , 56). It exists in equilibrium with its open-chain ketoamide form, y-3-(pyridyl)-y-oxo-n-methylbutyramide(Fig. I ) 1571. Detailed mechanisms for the metabolic formation of the hydroxy lactams 3- and 2’-hydroxycotinine remain to be reported.

B. N-Oxidation Although C-oxidation is the major pathway of nicotine metabolism, formation of N-oxide derivatives of nicotine and its metabolites, designated the N-oxidation pathway, constitutes an important route of nicotine biotransformation [ 5 8 ] . Nicotine- 1 ’-N-oxide, the main N-oxide metabolite of nicotine (Fig. 3), has been identified in urine of smokers, cats, and rabbits [31, 591, as well as in vitro in hepatic and lung preparations from several species [48, 60-621. Nicotine N-oxidation exhibits stereoselectivity in the formation of diastereomeric N’-oxides from nicotine enantiomers 143, 59, 621. N-Oxidation of nicotine and of most other tertiary amines occurs by a direct two-electron oxidation of the pyrrolidine nitrogen, an ionic mechanism not requiring intermediate radicals [63]. In mammals this reaction is catalyzed almost exclusively by the flavin-containing monooxygenase (FMO) system 111, 43, 631. Recent investigations 1481 suggest that formation of nicotine-l’-N-oxide in rabbit lung vesicles is mediated in part by rabbit lung P-45011, an isozyme of cytochrome P-450. Other oxides postulated to be N-oxidation products of nicotine metabolism include nicotineN-oxide and nicotine-1.1’-di-N-oxide(Fig. 3) [S]. The latter accounts for approximately 5% of the total N-oxidation pathway [SS]. N-Oxidation also occurs in the secondary metabolism of cotinine, to form cotinine-N-oxide (Fig. 3). Cotinine-N-oxide has been detected both in vitro [64-66) and in vivo in humans, dogs, rats, and monkeys 167-711. Unlike formation of nicotine- 1 ’-N-oxide, which involves the FMO system, production of cotinine-N-oxide is cytochrome P-450 dependent [66]. N-Oxidation of nicotine is of biological interest for at least two reasons. The first is the capacity for nicotine N’-oxides to be directly nitrosated to form several potent tobacco-specific carcinogens. These include N’-nitrosonornicotine, 4-(methylnitrosarnino)-I -(3-pyridyl)-1-butanone, 4(methylnitrosamino)-4-(3-pyridyl)butanol. and 4-(methylnitrosamino)-4-(3-



&ko Nicotine1‘-N-oxide

n

t

n

fl -N

t 0

0

Nicotine1, 1‘-di-N-oxide

“‘5’

CH3

NicotineN-oxide

f lI b H3C 0 CH3

N-Methyl-N’-oxonicotinium ion

Cotinine

Cotinlne-N-oxide

FIG. 3. N-Oxidation pathways of nicotine metabolism. pyridy1)-1-butanol [ 5 8 , 721. It has been claimed that since nitrite occurs in saliva and nitrogen oxides in inhaled mainstream tobacco smoke, additional amounts of tobacco-specific nitrosamines could be formed in vivo by reaction of these nitrosating agents with nicotine and other alkaloids [72]. While this concept is considered likely, as yet no nitrosated products have been isolated in vivo, and it thus appears as yet unjustified to include such pathways as separate routes of nicotine metabolism. The second reason is the potential for back-reduction of nicotine-1’-N-oxide and nicotine-1,l’di-N-oxide to nicotine. Such back-reduction can be performed both in vivo by gut flora and in virro by intestinal and hepatic microsomal enzymes [58, 62, 731. The latter type of back-conversion to nicotine can create a nicotine “reservoir” that may serve to reinforce the tobacco habit [58].

C. N-Demethylation N-Demethylation was established as a significant route of nicotine metabolism by identification of demethylcotinine in urine of cigarette smokers and of nicotine-injected rats, mice, rabbits, cats, and pigs (Fig. 4) [74-811.


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In rabbits, hepatic preparations were initially reported to convert nicotine to both nornicotinine and demethylcotinine 17 I]. Subsequently, however, nornicotine formation in vitro was questioned. Incubations of nicotine with supernatants of rabbit liver homogenates (9OOOXg), and later with microsomes or postmitochondrial fractions from hamsters, all failed to produce nornicotine [82, 83). Our recent demonstration of nornicotine formation from nicotine by hepatocyte preparations from rats, mice, guinea pigs, and hamsters 1841 suggests that “intact cell” experimental models may be required to demonstrate nornicotine formation in virro. Occurrence of demethylcotinine in urine of both smokers and subjects given nicotine intravenously, but not in the urine of volunteers after they receive cotinine [85, 861, provides evidence for the validity of the suggested pathway in Fig. 4 in which nicotine is converted initially to nornicotine and then to demethylcotinine. Recently, a capillary gas chromatographic method that involves derivitization to enhance sensitivity has confirmed the presence of nornicotine in urine of cigarette smokers [Sl]. Moreover, a sensitive radiometric HPLC assay capable of separating most nicotine metabolites recently disclosed nornicotine in urine from rats, as

Cotinine

Nicotine

r I

2-Hydroxycotinine

1

CH2 OH

Nicotine N’methyleneiminium ion

N-Hydroxymthyl nicotine

-

fi0 fy+l ----m

.N

Nornlcotine

H

H

Demthylcotlnine

2’-Hydroxydemthyicotinine

FIG. 4. N-Demethylation pathways of nicotine metabolism. Brackets indicate hypothetical transient intermediates.



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well as from cigarette smokers and nonsmokers given an intravenous dose of I4C-nicotine [70, 871. With respect to the mechanism of nicotine demethylation, the reaction has been postulated to proceed by initial two-electron-withdrawal oxidation at the a-carbon (N-methyl) position. This reaction yields the electrophilic iminium species, nicotine methylene-iminium ion, which exists in equilibrium with its a-carbinolamine analog (Fig. 4) [63, 881. Formation of the reactive iminium ion is cytochrome P-450 dependent and involves the same double-step, two-electron-withdrawal mechanism postulated for formation of the nic~tine-A“‘~”-iminium ion intermediate in cotinine production (Fig. 2) (88, 891. Nornicotine arises from spontaneous breakdown of the nicotine methylene-iminium ion (Fig. 4) [89]. We isolated from rat urine a novel metabolite, 2’-hydroxydemethylcotinine (allohydroxydemethylcotinine,ADC), with a half-life longer than cotinine [90]. ADC is the demethylated analog of 2’-hydroxycotinine (allohydroxycotinine). Figure 4 illustrates 2 pathways postulated for ADC formation. Mass spectral properties of another long-lived biotransformation product-which we term metabolite G (Figs. I and 4)-isolated from urine of human subjects given ‘‘C-nicotine suggest the structural assignment of demethylcotinine-A2’.”’-enamine[87]. Metabolite G is the demethylated analog of ~otinine-A*’*~’-enamine (Fig. 1). Cotinine-A2’*3‘-enamineis a C-oxidation metabolite of P-nicotyrine (Fig. 1 ), a naturally occurring tobacco alkaloid [91]. P-Nicotyrine has itself been suggested to be a metabolite of nicotine [92].

D. Conjugation Recent identification of glucuronidation as a major pathway of nicotine metabolism reemphasizes the significant role that conjugation mechanisms play in drug metabolism in general and nicotine metabolism in particular (Fig. 5 ) [87]. N-methylation and glucuronidation represent currently established conjugation pathways of nicotine metabolism. Glycine conjugation of the terminal nicotine metabolite, 3-pyridylacetic acid, has also been suggested 1931.

1. N-Methylation Identification in dogs of the N-methylnicotinium ion and the Nmethylcotinium ion as urinary metabolites of nicotine and cotinine, respectively, constitutes the first recognition of a methylation pathway in nicotine metabolism (941. More than 2 decades later, this N-methylation pathway



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” ‘ CH3 Nicotine glucuronlde

Colinine giucuronide

3-Hydroxycotinine glucuronlde

Cotinine

SHydroxycotlnlne

t Nlcotlne

CH 3

N-Melhylnornlcollniurn ion

CH 3

N-Methylnicotinium ion

CH3

N-Methyicotinlniumion

4 CH3 N-Methyl-N-oxonicotlniumion

FIG. 5. Conjugation pathways of nicotine metabolism. was shown to be stereospecific, involving only the R-( +)-enantiomer of nicotine 195, 961. Isolation of the N-methylnornicotiniurn ion (see Fig. 5) from urine and tissues of guinea pigs given either nicotine or the N-methylnicotinium ion provides a unique example of both N-demethylation and N-methylat ion occurring within the same molecule (97,981. The N-methylcotininium ion (see Fig. 5 ) was isolated from urine of guinea pigs given the N methylnicotinium ion [98] but was not excreted when guinea pigs received cotinine [54]. Both N-methylcotininium and N-methylnornicotinium ions are thus believed to be produced following formation of their precursor, the N-methlnicotinium ion (Fig. 5) 1981. Using the double-isotope technique, recent studies designed to determine the stability in vivo of the N-methyl and N’methyl groups of the N-methylnicotinium ion (NMN) indicate that N’-demethylation, but not N-demethylation. occurs [99]. These observations suggest that biotransformation of NMN involves either (i) initial N’demethylation to the methylnornicotinium ion followed by N'-methylat ion back to NMN or (ii) formation of an N’methylene-iminium species that could be reduced back to NMN [99].



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Formation of a new nicotine metabolite, the N-methyl-A”-oxonicotinium ion, has been reported and involves both N-methylation and ”oxidation (Fig. 5 ) (IOO]. This metabolite was identified in the urine of guinea pigs after administration of nicotine and N-methylnicotinium. N-Methyl-”oxonicotinium formation is stereospecific. This pathway most likely involves initial N-methylation followed by “oxidation (Fig. 5 ; also see Fig. 3). N-Methylation of nicotine has been demonstrated in human liver cytosolic preparations [ 1011. This group had shown earlier that cytosolic azaheterocycle-N-methyl transferases, purified from rabbit liver, were responsible for catalyzing the stereospecific formation of both N- and N’methylated metabolites of nicotine (96, 102).

2. Glucuronidation Previous reviews neglected the role of conjugation mechanisms, other than methylation, in the overall scheme of nicotine metabolism. The most plausible explanation for this omission is that only very recently has the excretion of glucuronides of nicotine and nicotine metabolites been demonstrated [87]. On incubation in v i m with P-glucuronidase, the material derived from the HPLC peak of a long-lived nicotine metabolite, detected in the urine of cigarette smokers and nonsmokers given a low intravenous dose of I4Cnicotine, was hydrolyzed almost completely (90%) to 3-hydroxycotinine [87j. The remainder was hydrolyzed to nicotine and cotinine. The kinetic disposition of this long-lived metabolite, identified as a 3-hydroxycotinine glucuronide, is unaltered by cigarette smoking [87]. This latter property, coupled to its longer half-life relative to that of cotinine, could render 3hydroxycotinine glucuronide an even more sensitive and reliable index than cotinine of passive exposure to tobacco smoke. Additional studies should be performed to clarify the role of glucuronidation in the overall scheme of nicotine metabolism. It would be useful, for example, to characterize the precise site or type of glucuronic acid binding to nicotine and its metabolites. It would also be desirable to validate in a larger population the use of 3-hydroxycotinine glucuronide as an index of exposure to tobacco.

E. Pyrrolidine Ring Cleavage The pioneering work by McKennis’ group in the 1960s contributed the main body of our present knowledge concerning mechanisms and biotransformation pathways involving cleavage of the pyrrolidine ring of nicotine


16


(Fig. 6). This chemical degradation has already been reviewed extensively 14, 5 , 7, 10). The only addition, recent identification of 3-pyridylcarbinol as a putative metabolite of nicotine, postulates that 3-pyridylcarbinol. rather than 3-pyridylacetic acid, may be the terminal degradation product of nicotine [ 103, 1041.

Cotinine

Dernethylcotinine

2’-Hydroxycotinine

I1

11

+

Y-(3-Pyridyl)-xoxobutyric acid

y-(3-Pyridyl)y-methylarninobutyric acid

+ flo S

O

O

Y-(3-Pyridyl)-Y-oxo-Nmethylbutyramide

H

N .

y-(3-Pyridy1)-tetrahydrofuran-2-one

-[QJ

Y-(3-Pyridyl)-Yhydroxybutyric acid

Y-(3-Pyridyl):3butenoic acid

dooH cfcH20H (----.

3-Pyridylcarbinol

+

3-Pyridylacetic acid

&COOH -N

Y-(3-Pyridyl)butyric acid

flH . IFI

COOH

N N-(3-Pyrldylacetyl) glycine

FIG. 6. Pyrrolidine ring cleavage of nicotine metabolism. The solid arrows indicate established pathways; broken arrows denote hypothetical reactions. Brackets indicate hypothetical transient intermediates.


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F. Peroxidation Recently, nicotine A4’.5’-enaminewas identified in rabbit lung and ram seminal vesicles as a peroxidatic metabolite of nicotine [105]. These in vifro studies involved incubation mixtures containing nicotine, arachidonic acid, and a source of prostaglandin H synthetase, that is. microsomes from lung or seminal vesicles. The hydroperoxidase component of prostaglandin H synthetase catalyzed this peroxidatic pathway of nicotine metabolism [ 1051. Nicotine A4””-enamine formation has been demonstrated in vivo both in rabbits after nicotine and from a cigarette smoker [ 1051. Catalysis involving the catalase-peroxidase system had earlier been implicated in cotinine formation in vivo [85, 1061. A recent communication indicates that nicotine A4’.”-enamine constitutes an artifact of the nicotine A”‘”)-iminium ion, the intermediate species in cotinine formation [ 1071. The enamine and the iminium ion are interconvertible, the tautomeric form that prevails depends on whether aqueous or anhydrous isolation procedures are used.

VI. DISPOSITION KINETICS In addition to elucidation of the chemical structure of its metabolites, characterization of the disposition of any xenobiotic should include the determination of its own pharmacokinetics and those of its biotransformation products. These requirements involve quantitative assessment of each metabolic pathway as well as determination of the urinary excretion profile, including the percentage of the dose accounted for by parent drug and each metabolite excreted in the urine 1451. Advances in understanding nicotine disposition have occurred rapidly over the past decade. This progress had to await development and application of highly sensitive analytical techniques required to determine accurately the rapidly changing, albeit small, concentrations of nicotine and its metabolites in biological matrices. Other factors that needed to be addressed to permit progress include (i) nicotine’s extreme toxicity, which severely limits the doses that can be administered to humans and animals under experimental conditions; and (ii) inherent difficulty in estimating accurately the actual amounts of nicotine absorbed systemically after tobacco use. Precise knowledge of this dose is required to calculate satisfactorily such critical pharmacokinetic parameters as nicotine clearance and apparent volume of distribution. Yet smoking itself consists of intermittent dosing of nicotine. Since nicotine is eliminated from the body very rapidly, large changes or oscillations can occur in nicotine concentrations during the smoking of even one cigarette. However, as Benowitz observes [41]: “Consistent with a



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half-life of two hours, nicotine accumulates over six to eight hours of regular smoking and nicotine levels persist overnight, even as the smoker sleeps. Thus, smoking results not in intermittent exposure, but in exposure to nicotine that lasts 24 hours of each day.” By contrast to the fluctuating nicotine blood concentrations that occur during smoking, Table 1 summarizes several reports over the past decade of pharmacokinetic parameters for nicotine obtained after only one intravenous injection or infusion. Variations in these values arise not only from differences among subjects with respect to critical host factors to be discussed in Sec. VII, but also from methodological differences between investigators in the dose and mode of nicotine administration, as well as in the analytical techniques employed [lo]. Disappearance of nicotine from plasma, after intravenous administration to humans and experimental animals, is biexponential; an initial rapid distributional phase is followed by a slower terminal phase representing metabolism and excretion 127, 90, 108, 1091. In rats, the biologic half-life, apparent volume of distribution, and total body clearance of nicotine following intravenous administration have been reported to be I .O k 0.2 h; 5 . 7 2 1.3 Lekg-’ and 4.0rt0.2 mL-min-’-kg-l, respectively [go, 1081. In man, the biologic half-life of nicotine (tl12P)ranges from I to 3 h (Table I). Differences in the protocols used for nicotine administration explain in part this variability in nicotine t,/2f3[lo]. On the basis of time- and dose-related changes in rates of nicotine elimination, Russell and Feyerabend (121 and Rosenberg et al. [34] suggested that nicotine exhibits an additional slower terminal elimination phase not readily detectable from plasma data. Urinary excretion profiles of nicotine in smokers and nonsmokers provide evidence for the existence of this “extra” phase. In smokers, the terminal half-life of this “extra” disposition phase is 4.720.4 h and can be as long as 10. I ? 1.3 h in nonsmokers [IOS]. Possibly with application of even more sensitive analytical methods, plasma nicotine profiles will show this latter, longer-lived elimination phase [I lo]. Wider adoption of this phase as truly representative of the terminal phase of plasma nicotine decay may yield more consistent values for nicotine tIl2P. The very large volume of distribution of nicotine (Table I ) reflects avid uptake and localization of this highly lipid soluble alkaloid by many tissues of the body [ I l l ] . Values for the apparent volume of distribution (aV,) of nicotine range from 2 to 3 L-kg-’ body weight. Variability in nicotine aV, cannot be explained on the basis of variable protein binding, as nicotine binding is only approximately 5%, too low to be biologically important ~71. The high metabolic clearance of nicotine, ranging from 1.3 to 2.5 L-min- , approaches normal hepatic blood flow. These values indicate high

’



19

hepatic extraction of nicotine (271, suggesting that it undergoes extensive first pass metabolism [Ill]. Drugs like nicotine that are very rapidly metabolized often exhibit marked interindividual variability in metabolic clearance secondary to changes in hepatic blood flow [27]. In addition to hepatic mechanisms, renal elimination of nicotine also occurs [ 10, 341. Under conditions of urinary alkalinization, renal clearance of nicotine in humans approximates 17 m l m i n - ' , whereas on urinary acidification its renal clearance increases to 245 mL-min-I, and total plasma clearance rises from 778 mL.min-' to 1027 m l m i n - ' . These observations show that nicotine is not only filtered at the glomerulus, but also secreted and reabsorbed by the tubule [lo, 341. As illustrated in Table 1, we observed higher nicotine clearance (87, 1091 than reported by others 127, 34, 1111. This difference may be explained by the fact that we administered the lowest dose of nicotine (2.7 p,g/kg). Svensson ( 101 described possible dose dependency of nicotine elimination and stressed the need for an appropriate dose-ranging study of nicotine elimination rates in human subjects. While studies in the rat indicate a tendency for nicotine clearance to be dose dependent (90, 1121, Benowitz et al. [27] and Feyerabend et al. [ I 1 11 found no evidence of dose dependency (over the range of 25 to 60 p,g/kg) in humans. However, at the lower dose of nicotine that we administered intravenously, 2.7 p,g/kg, which approximates the amount absorbed from inhaling a single puff of a regular cigarette, nicotine may be cleared faster. Another factor may be that by virtue of our administration of a tracer I4C-labeled dose, our data could be unaffected by, and therefore independent of, background levels developed from previous smoking and/or passive exposure to unlabeled nicotine in cigarette smoke. Following administration of tracer doses of I4C-nicotine to both humans and rats, only cotinine, of all the metabolites of nicotine, appears in plasma in sufficient concentrations to permit kinetic analysis by presently available methods. In rats, plasma cotinine tl/# ranges from 4.9 to 5.2 h [90, 1121, consistent with a report for cotinine tl12Pof 5.9 h after its bolus injection [113]. Plasma cotinine clearance and aV, are 0.2 mLmin-' and 0.4 Lekg-', respectively [ 1131. Increased total plasma clearance of cotinine observed after its prolonged infusion was attributed to increased renal clearance or to autoinduction [113], a phenomenon suggested earlier to account for time-dependent induction of drug metabolism by cotinine [ 1141. In man, cotinine tIl2P ranges from 10 to 17 h (Table I ) [87, 109, 1151201. Mean total plasma clearance and aVd of cotinine have recently been reported to be 0.9 mL.min-'.kg-' and 1.1 Lskg-', respectively (Table 1) [120]. Trans-3-Hydroxycotinine occurs in the plasma of cigarette smokers [5 153, 1211. However, in sera of cigarette smokers these concentrations are

20



TABLE I Plasma Nicotine and Cotinine Pharmacokinetic Parameters (mean f SE) in Humans After Intravenous Administration of Nicotine or Cotinine

Smokers (6) Smokers (4) Smokers (5) Smokers (6) Nonsmokers (6) Smokers (8) Smokers (5) Nonsmoker ( 1 ) Nonsmoker ( I ) Smokers (6) Smokers (4) Smokers (4) Smokers (4) Smokers (9) Smokers (9) Smokers (9) Smokers (6) Nonsmokers (6)

20.0

30.0 60.0 2.7 2.7

-

25.0

-

480

-

0 . 7 ~ 0 . 1 13.8 2 I .3 1.620.6 18.5 2 4 . 5 18.42 1 . 1 2.0 "0.1 - 0.8 k0.03 28.9 2 4.5 1.320.I 26.22 2.0 240.0 NA NA - 2.220.3 13.22 2.1 280 NA NA 14400 NA NA -

NA 2.8 k 0.4 3.02 0.4

-

NA NA NA NA NA NA NA NA NA NA NA NA NA

-

73 145 290 69 139 277

2.4 2.4

-

-

-

-

NA NA NA NA NA NA NA 0.9 2 0 . I 1.420.1

NA NA NA NA NA NA NA 31.123.1 21 - 8 2 3 . 0

2.7 2 0 . 9

3.32 1 . 1

1.020. I 2.5 k0.7 2.6k0.7 2 .O 2 0.3

3.020.3 NA 2.5 2 0 . 3 NA NA NA NA NA NA NA NA NA 2.3 2 0.4 2.9 2 0.5

Abbreviurions t,,,P = terminal half-life; CI, = total body clearance; CI, = renal clearance; V,P = apparent volume of distribution; NA = not available.

lower than those of cotinine I5 1, 1211. Plasma pharmacokinetic parameters for t,&. aV,, and total clearance of 3-hydroxycotinine following its unlabeled infusion in smokers are 5.9 h, 0.9 L-kg-', and 1.8 mL*min-'.kg-', respectively [121]. Traces of nicotine- I '-N-oxide, cotinine-N-oxide. and y-3-(pyridyl)methylaminobutyric acid have been identified in rat and human plasma after administration of 14C-nicotine[70, 87, 90, 1091. However, concentrations of these metabolites were too low or erratic to permit their pharmacokinetic analysis in plasma. Accordingly, urinary excretion data are required for this purpose.


21


TABLE 1 CONTINUED

Cotinine

NA NA NA 10.2 2 0.9 13.320.9 15.8k4.0 NA 10 II NA 12.7 2 I .6 11.9k2.1 12. I 2 2.3 14.9 2 2.5 15.8 k 3.2 16.224.9 9.8 5 0.6 14.3 2 1.2

NA NA NA NA NA 0.94 20.15 NA NA NA NA 0.85 +O. 13 0.91 20. 14 0.8820.16 0.9820.55 0.79 2 0.29 0.8520.29 NA NA

NA NA NA NA NA 0.16k 0.06 NA NA NA NA 0.1 I ? 0.01 0. I I ? 0.02 0.10 ? 0.01 0.09? 0.03 0.07 & 0.01 0.082 0.03 NA NA

NA NA NA NA NA 1.1 k0.2 NA NA NA NA 0.8 to 1 . 1 0.8 to 1 . 1 0.8 to 1.1 I .2-+0.4 I . 120.2 1.1 20.2 NA NA

To achieve this objective we used sigma-minus plots generated for the urinary excretion of nicotine and its metabolites. These data afford accurate pharmacokinetic interpretations of the disposition of many drugs and their metabolites in the body [ 122,1231. The sigma-minus method consists of semilogarithmic plots of Qdu,-Qdu, against time, where Qdu, is the absolute amount of nicotine or metabolite excreted in urine over the duration of the experiment and Qdu, represents the cumulative amount excreted up to any time t. Such sigma-minus plots are advantageous in that kinetic parameters generated from them are reliable because they are least influenced by fluctuations of urinary pH, urine volume, or by additional factors capable of affecting nicotine disposition.


22


A summary in rats and humans of values for t,,# and urinary recovery expressed as percent of the single intravenously administered dose excreted as either unchanged nicotine or of metabolite appears in Table 2 [87, 901. These urinary excretion data provide evidence that in both rats and humans two newly described metabolites are even longer-lived than cotinine. Previously cotinine was considered the most sensitive index of exposure to nicotine because it had been thought to be the longest-lived nicotine metabolite 1411. More recently, 3-hydroxycotinine has been advocated because in the urine of smokers it appears to be even more abundant than cotinine [45, 52, 1211. Our results shown in Table 2 confirm this impression since in the urine of smokers addition of 3-hydroxycotinine to its glucuronide yields a recovery value of 18.2%, compared to 15.6% for continine. In rats there occur 2 metabolites that are longer-lived than cotinine: In human cotinine-N-oxide and 2'-hydroxydemethylcotinine(Table 2) [W]. smokers, after intravenous administration of a tracer dose of 14C-nicotine. two other metabolites were identified that persisted longer than cotinine: 3-hydroxycotinine glucuronide and demethylcot inine-A2'3'-enamine [ 871. The latter metabolite appears to be a dehydrated metabolite of 2'hydroxydemethylcotinine. The implication is that these longer-lived metabolites of nicotine are potential candidates for tracing more sensitively than previously possible passive exposure of humans to tobacco smoke. Adoption of 3-hydroxycotinine glucuronide as a more sensitive and discriminating marker than cotinine for exposure to tobacco offers an additional advantage in that the kinetics of the glucuronidated metabolite seem to be unaltered by smoking [87]. By contrast, the rate of cotinine elimination is accelerated in cigarette smokers compared to nonsmokers [ 1031, thereby complicating the use of plasma or urinary cotinine concentrations to indicate exposure to cigarette smoke.

VII. HOST FACTORS THAT INFLUENCE NICOTINE PHARMACOKINETICS Smoking status is only one of numerous host factors that can alter, both individually and through dynamic interactions with each other, the disposition of xenobiotics, including nicotine [ 1241. These host factors are either genetic, developmental, or environmental in nature. Certain cytochrome P450s involved in nicotine metabolism are induced by drugs, carcinogens, and other xenobiotics in an age- and sex-dependent manner [ l l ] . Some are also inhibited. Flavin-containing monooxygenases that mediate N-oxidation

~~~

-a 5.320.9 5.620.9 6.4 2 0.5 2.2 2 0.3 5.5k0.4 9.9 k 1.5

1.020.1

4.5k0.6 1.8kO.l 4.4 20.4 2.0 k 0.1 2.2k0.3 3.1 20.4 ND ND

-

2.3k0.3 4.820.8 1.220.1 7.5k0.7 3.320.4

tl&

11.820.9 7.220.9 11.620.9 9.350.9 8.9k0.9

of dose

% recovery

~

29.124.4 16.322.1 0.920.2 4.920.9 2.420.3 5.823.4 1.1 k0.4 ND ND ND ND ND 11.92 1.8 8.4k1.6

of dose

% recovery

18.4? 1.8 11.2k0.7

-

-

-

-

-a

1.420.3 13.621.5 2.120.3 10.421.8 8.520.7 10.4k1.0

t,12fi

Nonsmokers

14.922.5 15.621.8 0.720.3 7.421.3 2.720.3 9.321.3 1.6k0.5 ND ND ND ND ND 16.620.7 11.32 1.7

of dose

% recovery

15.72 1.0 15.1 21.3

-

-

-

-

-

0.720.1 9.821.3 2.620.5 8.921.6 7.220.8 11.720.9

t,,2f3

Smokers

Source. From Refs. 87 and 90. Abbreviurions tl12P = terminal half life; ND = not detected. at,12P could not be determined accurately.

~

Nicotine Cotinine Nicotine-1 '-N-oxide Cot inine-N-oxide Nornicot ine Demet hylcot inine 3-H ydroxycotinine y-(3-Pyridyl)-y-oxobutyric acid y-(3-Pyridyl) methylaminobutyric acid y-(3-Pyridyl)-y-oxo-N-methylbutyramide 3-Pyridyl acetic acid Allohydroxydemethylcotinine 3-Hydroxycotinine glucuronide Demethylcotinine A2'.3'-enamine

Metabolite

Rats

Urinary Excretion of Nicotine and Metabolites 120 h After Administration of '*C-Nicdine in Rats and in SmokerdNonsmokers

Table 2


a

24

KY EREMATEN AND VESELL


pathways of nicotine biotransformation are also regulated by environmental, genetic, and developmental host factors, including age and sex 1631.

A. Genetic and Developmental Factors Although genetic studies using twins and families have not yet been performed in humans on causes of appreciable interindividual variability in nicotine pharmacokinetics and pharmacodynamics 1271. a recent study of serum cotinine concentrations in healthy Black and White smokers 18 to 30 years old described much higher mean values in the former (221 mg/mL) than the latter (I70 mg/mL) [125]. Multiple genetic and environmental causes were considered for this racial difference in nicotine metabolism. A genetic defect has been reported in the FMO system that converts nicotine to nicotine-N-oxide [ 1771. The metabolic lesion occurred in two sisters with trimethylaminuria, a rare autosomal recessive condition known as “fish-odor syndrome” [ 1781. This syndrome is characterized by a block in the metabolism of trimethylamine to its oxide. Affected subjects experience social problems because they exhibit markedly increased excretion in urine, sweat, and breath of a malodorous tertiary aliphatic amine, trimethylamine. Thus, these interesting sisters with an impaired FMO system were unable to metabolize at normal rates either of two substrates, nicotine and trimethylamine, to their respective oxides. Reports published several decades ago document age and sex differences in nicotine metabolism (126-1301. These reports are reviewed well by Nakayama [ I I], although two studies published in the 1980s on sex differences in nicotine disposition were unmentioned. The first made the important discovery that total plasma clearance of nicotine when normalized for body weight is lower in female than in male smokers [131]. This observation that men metabolize nicotine faster than women correlates with an earlier finding that male nonsmokers excreted more cotinine than female nonsmokers 11321. The second study provides detailed in virro and in vivo pharmacokinetic measurements on the sexual dimorphism of nicotine metabolism and distribution in rats [133]. In each of 4 rat strains, liver homogenates from males exhibited significantly higher rates than females for formation of both Cand N-oxidation metabolites of nicotine. Mature ( 100-day-old) Wistar and Long Evans rats showed the largest sex difference (>50%) for formation of both metabolites, whereas young (40-day-old) Fischer rats revealed least sex difference (Fig. 7). Thus, the sex difference is both strain and age dependent. Moreover, Sprague Dawley rats given ‘‘C-nicotine intravenously



25

FIG. 7. Sexual dimorphism of total nicotine-metabolizing activity of hepatic 10,OOOXg homogenates from four strains at two ages. Values represent female activity expressed as percentage of male activity. *p 5 O.O5;**p 5 0.01 [84].


26


exhibited sexual dimorphism in vivo in nicotine metabolism and distribution; plasma nicotine and cotinine half-lives were shorter in males than in females 11331. Nicotine aV, in female rats exceeded that in males; thus, nicotine plasma clearance showed no significant sex difference. Urinary excretion data for nicotine and its metabolites suggested more rapid metabolism of nicotine in males than in females [133]. Species differences in nicotine metabolism have been demonstrated in vivu [134], as well as in vitro using hepatocytes [84]. From the study conducted in vivo, 3-hydroxycotinine was the major urinary metabolite in hamsters, guinea pigs, and humans; but not in rats, where cotinine excretion became more significant. Nicotine- I '-N-oxide, an important metabolite in guinea pigs and rats, was not detected in hamsters and rabbits (1341. In the study performed in virro, hepatocytes isolated from guinea pigs, hamsters, mice, rats, and humans exhibited very large species differences in nicotine metabolism, ranging from extensive biotransformation in the guinea pig and hamster to much less in rats and humans (Fig. 8) [84]. A similar trend occurred in earlier studies in vitru utilizing hepatic homogenates 174, 1351.

B. Smoking Habits Cigarette smoking can alter the disposition of numerous drugs [136-1391. Better understanding of the relationship between chronic tobacco smoking and the disposition of tobacco's most important active principle, nicotine, is desirable. Such concepts could also serve as a basis for elucidating mechanisms that underlie other tobacco-drug interactions. Consistent with an inductive effect of smoking, an early study comparing the disposition kinetics of nicotine in age- and sex-matched smokers and nonsmokers disclosed shorter elimination half-lives of both nicotine and cotinine in smokers than in nonsmokers [ 1091. In this study cigarette smoking produced complex pharmacokinetic changes; a diminished nicotine aV, in smokers offset the shortened t,,# such that plasma clearance of nicotine was unaltered as a result of chronic smoking 1 1091. Other investigators confirmed that cigarette smoking accelerated nicotine metabolism [ 140, 14I ). However, still others [ 142,1431 questioned whether nicotine metabolism was induced in cigarette smokers. With the aid of an hepatocyte model unaffected by absorption and distribution factors, hepatocytes isolated from livers of chronic tobacco smokers metabolized nicotine faster than hepatocytes from nonsmokers 1841. Hepatocytes from ex-smokers exhibited a trend toward induction.



27

FIG. 8. Metabolism of nicotine in 5 species arranged in order from the species with most extensive metabolism of nicotine (guinea pig) at left to the species with least metabolism (rat and human) at right. Values indicated are percent contribution ? SE of remaining nicotine and of metabolites formed to the total starting radioactivity (771. A recent in vivo study of nicotine disposition in smokers and nonsmokers [87] using an HPLC procedure capable of separating most nicotine metabolites confirmed reduced t,,# of nicotine and cotinine in cigarette smokers. Smokers and nonsmokers did not, however, differ in their rates of urinary elimination of 3-hydroxycotinine glucuronide, cotinine-N-oxide, and demethylcotinine. Paradoxically, the elimination phase of metabolite G was shorter in nonsmokers compared to smokers. One explanation for selective modulation of individual pathways of nicotine metabolism in smokers may relate to the complex composition of


28


tobacco smoke [ 1441 with different constituents either enhancing or inhibiting different drug-metabolizing enzyme systems [ 871. Such selective effects of individual components of cigarette smoke on different pathways of caffeine metabolism have been documented [ 1451. Increased nicotine clearance following brief abstinence from smoking suggested that either some component of cigarette smoke itself or a nicotine metabolite inhibits nicotine metabolism in smokers 11461. Studies of theophylline disposition following I week of abstinence from smoking indicate partial restoration toward normal of an enzyme-inducing effect of smoking [146]. Smokeless tobacco [I141 or nicotine chewing gum [147, 1481 does not accelerate nicotine metabolism. Collectively these studies suggest that the polycyclic aromatic hydrocarbon fraction (PAHF) of tobacco smoke mediates inductive effects of smoking [139]. It is of interest that those who smoke both marijuana and cigarettes exhibit cotinine concentrations lower than those who only smoke tobacco [149]. The implication of this observation is that marijuana smoke may contain additional factors that enhance nicotine metabolism. In this connection, nicotine gum, which has become a popular method to assist smoking cessation, may lack an inductive effect on drug metabolism because in the absence of pyrolysis, no inductive PAHF arises. The gum consists of a cation exchange resin containing 2 to 4 mg of nicotine (101. Chewing the gum is required to release the nicotine, but nicotine absorption from gum across the buccal mucosa is slower than from a cigarette. Peak concentrations of nicotine, which depend on such host factors as vigor and duration of chewing, occur about 30 min after initiation of chewing, compared to only 5 to 10 min after smoking a cigarette (lo]. Plasma nicotine increases at an average rate of 12 pg.L-' after chewing a single piece of gum containing 4 mg nicotine compared to 28 p.g.L-' after smoking a cigarette containing 1.3 mg nicotine [ 121. These facts provide a possible pharmacokinetic basis for the relatively poor success rate of nicotine gum in promoting smoking cessation: The gum may not offer the cigarette smoker sufficient nicotine to satisfy his needs. Also the lower plasma nicotine concentrations produced by the gum may explain why the gum fails to maintain the effect needed by many smokers to overcome their psychological dependence on smoking.

C. Drug Pretreatments Inductive and inhibitory effects on nicotine metabolism exerted by nicotine itself and other chemicals, such as phenobarbital and P-naphthoflavone, have been extensively discussed [ 1 1 1. Therefore, we shall



29

review here effects of chemicals on nicotine disposition only with respect to developments since 1988. These developments include use of in v i m models of drug metabolism such as perfused isolated liver/lung preparations (150, 1511 and hepatocytes [84], as well as applications of molecular biology to the study of nicotine metabolism [152]. In studies using isolated perfused rat livers, phenobarbital-pretreated rats enhanced by 8-fold nicotine metabolism; by contrast, pretreatment with an inducer of cytochrome P-448, 5,6-benzoflavone, exerted only marginal influence [ 1501. After phenobarbital pretreatment, biliary excretion of radioactivity from ''C-nicotine accounted for 6-17%, representing a 2.7-fold increase over values obtained in untreated, control rats. Biliary metabolites excreted after phenobarbital induction are polar, possibly conjugated metabolites of nicotine. Nicotine clearance by isolated perfused lung systems almost doubled after pretreatment of rats with phenobarbital, whereas P-naphthoflavone pretreatment exerted no significant change 115 I]. A reconstituted enzyme system has been developed to investigate nicotine metabolism. This study used 6 cytochrome P-450 isozymes purified from rabbit liver. Two isozymes demonstrated highest rates for C-oxidation and N-demethylation pathways of nicotine metabolism; these activities were associated with a phenobarbital inducible isozyme, form 2, as well as a constitutive isozyme, form 3b 11521. In other in vitro studies using hepatocytes from obese humans, phenobarbital pretreatment of human subjects for 2 days prior to liver biopsy was associated with significant induction of nicotine biotransformation to cotinine, whereas nicotine-1 '-N-oxide production was unaffected 1841. Phenobarbital pretreatment significantly induced cotinine formation in hepatocytes not only from humans, but also from guinea pigs, rats, and mice [84]. In hepatocytes from hamsters, phenobarbital did not increase cot inine production, possibly because cotinine was further biotransformed to other secondary metabolites 1841. In hepatocytes from rats, phenobarbital pretreatment enhanced both cot inine and nornicotine formation, an inductive effect that was sex dependent, being more pronounced in males than females I841. Phenobarbital pretreatment did not alter formation of nicotine1'-N-oxide by rat and hamster hepatocytes. In hepatocytes from guinea pigs, formation of this N-oxide was paradoxically reduced, as previously described in rabbits using purified microsomes [ 1521. These large species- and sex-dependent variations in the induction of nicotine metabolism suggest that different isozymic forms of cytochrome P450 and FMO are involved in nicotine metabolism [153]. Differential induction and inhibition of these isozymes with various xenobiotics provide one approach to unraveling the species-dependent heterogeneity.


30


With respect to inhibitory effects of drug pretreatments on nicotine metabolism, three recent reports merit attention. An earlier pharmacokinetic study showed that chronic ethanol pretreatment in rats significantly induced cotinine formation in vivo [154]. A subsequent in virro study indicated that cotinine formation by microsomes isolated from ethanol pretreated hamster was unaltered, whereas nicotine-1 '-N-oxide production was induced [ 1531. Recent reports, however, demonstrate that ethanol administered acutely inhibits nicotine metabolism in vivo. Total nicotine clearance in rabbits after I4C-nicotine decreased by almost 50%; also the rate of CO, exhalation by rats after ''C-methyl-nicotine declined [ 1551. Moreover, in virro in isolated perfused rat liver and a liver homogenate, ethanol retarded not only nicotine clearance, but also its conversion to cotinine 11551. In a double-blind crossover study, cimetidine pretreatment significantly reduced nicotine and cotinine clearances [ 156). Interaction between cimetidine and nicotine was believed to occur at sites of renal tubular secretion of nicotine and cotinine. Since smokers regulate their smoke intake based in large part on their nicotine blood concentrations [ 1 I I , 1571, diminished clearance of nicotine in the presence of cimetidine could be important in assisting smoking reduction or cessation [ 1561.

D. Dietary Habits Several epidemiologic studies suggested a correlation between coffee consumption and cigarette smoking [ 158, 1591. Recent detailed studies [ 1601 observed no association between chronic caffeine consumption and nicotine disposition in cigarette smokers. Both nicotine and cotinine AUC, as well as 24-h urinary excretion of nicotine or cotinine, were similar during consumption of different doses of caffeine or noncaffeinated coffee [ 1601. Nevertheless, subjects tended to smoke more cigarettes while consuming coffee; also, plasma nicotine concentrations also tended to be high in smokers on a low-caffeine regimen [160]. These tendencies suggest that some undefined association exists between nicotine kinetics and caffeine consumption. This connection might emerge if measurements were made to determine total plasma nicotine clearance. Ingestion of a high-protein meal substantially increases hepatic blood flow [161]. For nicotine, whose hepatic clearance is flow limited, there exists a potential for such food-drug interactions to occur [lo]. Recently the effects of such a high-protein meal were investigated in 7 smokers [ 1621. Nicotine metabolism was accelerated and consuming this meal during a steady-state infusion of nicotine caused a small (18%) but consistent decline in blood nicotine concentrations [ 1621.


31


E. Disease States Effects of disease on nicotine disposition is undoubtedly one of the most unexplored and perhaps neglected aspects of nicotine disposition, Better elucidation is needed on the question of how various disease states, such as cirrhosis and nephrosis, influence nicotine metabolism and kinetics in smokers. Altered nicotine disposition that may occur in “sick” chronic tobacco smokers could contribute to poor management of their smoking dependence, which in turn could lead to an exacerbation of their smokingrelated complications. Since nicotine elimination depends on hepatic blood flow, any alteration in hepatic blood flow, as occurs in cases of acute viral hepatitis, cirrhosis, and congestive heart failure, might substantially influence nicotine clearance [lo]. Such changes may also exist in geriatric patients with compromised renal excretory function. It is known that in patients with urinary bladder cancer, oxidative pathways of nicotine metabolism shift [ 1631.

F. Pregnancy In pregnant smokers, exposure of the developing fetus to nicotine and its metabolites can be substantial [39] and in primates it has been shown that the fetus accumulates nicotine to higher levels than in the mother [ 1641. Under such conditions, smoking has been associated with an increased incidence of spontaneous abortion, a decreased birth weight, and an increased perinatal mortality [165]. Thus, it is of interest to determine how pregnancy affects the metabolism and disposition of nicotine in smokers. In pregnant smoking women, qualitative and quantitative changes in nicotine metabolism have been reported [166]. A reduction in the amount of oxidative nicotine metabolites occurred, as well as a change in the ratio of these metabolites. Similar changes occurred in rats [ 167).

VIII. METHODOLOGICAL CONSIDERATIONS

A. Analytical Methods Major advances in the understanding of nicotine metabolism have had to await development of highly sensitive analytical techniques for nicotine and its principal metabolites. There is no doubt that the imaginative coupling of mass spectrometry to gas chromatography (GC) [53, 8 1, 117, 168, 1691 and


32


high-performance liquid chromatography (HPLC) [ 1701 represents a most significant contribution and constitutes state-of-the-art technique for identification and quantitation of nicotine and its metabolites. Other significant advances include enhanced detector sensitivity through derivitization for both GC [ S l , 1711 and HPLC [103, 1041 analysis of nicotine and metabolites, as well as radiometric applications [70, 1091. Despite its theoretical appeal and high sensitivity, the radioimmunoassay technique has not become widely used for analysis of nicotine and metabolites, probably because of inherent technical complications arising from nicotine’s poor antigenicity and some potential for cross-reactivities to occur between antibiodies produced to nicotine and to its metabolites 1172, 1731.

B. Models for Nicotine Metabolism The application of hepatocyte preparations 1841 and of isolated perfused organs 1150, 1511 in studies of nicotine metabolism offers certain distinct advantages in establishing convenient in virro models for conditions that prevail in vivo. The main asset of hepatocytes resides in their potential for reflecting subtle metabolic changes that could be concealed in vivo, where such alterations can be offset by simultaneously occurring changes in rates of drug absorption, distribution and excretion 1841. When hepatocytes are investigated, each factor and environmental component can be examined individually, being systemically altered while all other variables are held constant. Thus, the role of each factor in affecting nicotine metabolism can be assessed independently of all others and dose-response relationships generated. Perfused organ systems are described as “semi vivo” experimental models because of their closeness to in vivo conditions in drug metabolism studies. For nicotine metabolism, the use of the perfused liver and lung systems provide valuable information especially on the kinetics of nicotine metabolism in virro [ISO, 1511. As intact cell systems, hepatocytes and perfused organs retain most metabolic capabilities of their tissue of origin. Thus, they allow under welldefined, carefully controlled conditions examination of effects produced by various biochemical and physiological manipulations. Finally, in vivo models of nicotine metabolism have been proposed in certain species for some aspects of nicotine metabolism in humans. For example, stumptailed macaques produce in their urine large quantities of metabolites A and G after intravenous injection of ‘‘C-nicotine [ 1741. This



33

suggests that stumptailed macaques can serve as models to study some aspects of the regulation in humans of these recently discovered metabolites ~371.

IX. CONCLUDING REMARKS Extensive information on nicotine metabolism reviewed in this paper forms the basis for much future research. This progress, founded on advances in analytical methology and development of appropriate drug metabolism models, will probably encompass at least some of the following directions, although we also anticipate entirely unpredictable discoveries: I . In addition to identification of new nicotine metabolites, isolation and characterization of more reactive intermediates. Nicotine, although itself highly toxic on acute administration, is generally not implicated as a major culprit in mediating many of the chronic deleterious health effects of smoking, such as cancer. However, recent mechanistic studies of nicotine metabolism identified certain chemically reactive intermediates capable of forming covalent bonds with tissue macromolecules 148, 89, 91, 105. 107, 1751. Formation of some of these reactive intermediates probably involves free radical generation [83]. Several as yet unidentified pathways of nicotine metabolism may produce even more chemically reactive intermediates. 2. Isolation of genes regulating those isozymes of cytochrome P-450 and flavin-dependent monooxygenases that mediate nicotine metabolism. Currently available state-of-the-art techniques in molecular biology favor a rapid expansion of research in this area [11, 152, 1761. 3. Identification of additional effects on nicotine disposition of disease states and numerous host factors, particularly smoking and diet. The potential of this subject, discussed in Sec. VII, is great. Studies of dynamic interactions between disease states and various host factors that influence nicotine disposition-such as dietary and smoking habits, drug use, age, and sex-could advance our understanding of how nicotine disposition is regulated. 4. Identification of the role of conjugation pathways in nicotine metabolism. Recent demonstration of the glucuronidation of nicotine and some of its metabolites suggests the potential for a role of extrarenal mechanisms, such as enterohepatic recycling and biliary excretion, in the elimination of nicotine and its metabolites. These extrarenal routes of nicotine excretion may be altered in a variety of conditions that influence and compromise hepatic and renal function.

34



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Metabolism of nicotine by hepatocytes.

Nicotine Dependence, Nicotine Metabolism, and the Extent of Compensation in Response to Reduced Nicotine Content Cigarettes.

Absorption and metabolism of nicotine from cigarettes.

[Nicotine metabolism in the pig (author's transl)].

Metabolism of nicotine by human liver microsomes: stereoselective formation of trans-nicotine N'-oxide.

Racial differences in the relationship between rate of nicotine metabolism and nicotine intake from cigarette smoking.

The Influence of Puff Characteristics, Nicotine Dependence, and Rate of Nicotine Metabolism on Daily Nicotine Exposure in African American Smokers.

Metabolism of nicotine by the isolated perfused dog lung.

Arachidonic acid metabolism in nicotine-treated rats and nicotine-incubated rabbit aortic smooth muscle cells.

Paternal nicotine exposure alters hepatic xenobiotic metabolism in offspring.

Cessation of alcohol consumption decreases rate of nicotine metabolism in male alcohol-dependent smokers.

Influence of route of administration on metabolism of [14C]nicotine in four species.

A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism.

Changes in the rate of nicotine metabolism across pregnancy: a longitudinal study.

Effects of maternal nicotine exposure on thyroid hormone metabolism and function in adult rat progeny.

The influence of maternal nicotine exposure on neonatal lung carbohydrate metabolism.

Brain Responses to Smoking Cues Differ Based on Nicotine Metabolism Rate.

Effects of nicotine on brain 1-phosphatidylinositol-4-phosphate and 1-phosphatidylinositol-3,4-bisphosphate synthesis and metabolism--possible relationship to nicotine-induced behaviors.

Drug Metabolizing Enzyme and Transporter Gene Variation, Nicotine Metabolism, Prospective Abstinence, and Cigarette Consumption.

Beneficial effects of nicotine.

Nicotine withdrawal.

Disposition kinetics and metabolism of nicotine and cotinine in African American smokers: impact of CYP2A6 genetic variation and enzymatic activity.

Plasma nicotine levels after cigarette smoking and chewing nicotine gum.

Nasal nicotine spray: a rapid nicotine delivery system.