Download residence times and half-lives of nicotine metabolites in rat brain

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DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 12
Printed in U.S.A.
RESIDENCE TIMES AND HALF-LIVES OF NICOTINE METABOLITES IN RAT BRAIN
AFTER ACUTE PERIPHERAL ADMINISTRATION OF [2*-14C]NICOTINE
OMAR GHOSHEH, LINDA P. DWOSKIN, WEN-KUI LI,
AND
PETER A. CROOKS
College of Pharmacy, University of Kentucky, Lexington, Kentucky
(Received April 16, 1999; accepted September 8, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
70, and 7 nM for cotinine, nornicotine, and norcotinine, respectively. Even with potential accumulation of cotinine in brain after
chronic nicotine administration, it is likely that the brain concentration of cotinine will be insufficient to produce neuropharmacological effects resulting from activation of nicotinic receptors to
induce dopamine release. Conversely, the concentration of nornicotine in brain after acute nicotine approaches the range found to
be neuropharmacologically active. It is likely that nornicotine will
accumulate in brain on chronic nicotine administration based on
the brain half-life of this metabolite. Importantly, nornicotine is also
a major alkaloidal component of tobacco. Thus, as a consequence
of tobacco use, alkaloidal and metabolically formed nornicotine
may reach concentrations in brain sufficient to produce pharmacological effects.
Although peripheral metabolism of nicotine has been studied extensively (Gorrod and Jenner, 1975; Kyerematen and Vesell, 1991;
Crooks, 1993; Gorrod, 1993), nicotine metabolism in brain has been
given little attention until recently. The presence of nicotine metabolites in brain is an important factor to consider because nicotine
biotransformation products have been shown to be pharmacologically
active (Crooks and Dwoskin, 1997), and their presence in brain
constitutes a potential contribution to the neuropharmacological effects resulting from nicotine exposure. In earlier studies, only one
major metabolite, cotinine, was reported in mouse and cat brain after
i.v. injection of [14C-N-19-methyl]nicotine (Appelgren et al., 1962).
More recently, similar results were found in the rat (Deutsch et al.,
1992). Other studies have reported the presence of cotinine and four
other unidentified minor metabolites in the brain of [14C-N-19-methyl]nicotine-treated animals (Schmiterlow et al., 1967). The distribution and pharmacokinetics of radiolabeled nicotine in brain after
various routes of peripheral administration in several animal species
were reported (Saji et al., 1984; Nordberg et al., 1989; Plowchalk et
al., 1992; Yamada et al., 1992). Generally, these studies did not
determine the concentrations of nicotine biotransformation products.
In other studies, both [14C]cotinine and [14C-N-19-methyl]nicotine-N-
oxide have been detected by high pressure liquid radiochromatography (HPLRC)1 in mouse brain after peripheral administration of
[14C-N-19-methyl]nicotine (Stalhandske, 1970; Petersen et al., 1984).
The use of [14C]-methyl-labeled nicotine in these studies precludes
the detection of nicotine metabolites that have undergone oxidative
N-demethylation, e.g., metabolites such as nornicotine and norcotinine.
Previous HPLRC studies from our laboratory have shown that
when rats are injected s.c. with [3H-N-methyl]nicotine, a radiolabeled
metabolite coeluting with authentic cotinine standard was observed;
this had a long residence time in the central nervous system (CNS)
(Crooks et al., 1995). Also, a significant amount of tritium eluted in
the void volume of the brain supernatants, suggesting either dissociation of the tritium label, which is unlikely, or metabolism of the
tritiated methyl group through an oxidative process leading to loss of
tritium from the molecule. Thus, the tritium in the void volume was
attributed to a low molecular weight oxidized 1-carbon unit, such as
tritiated formaldehyde or tritiated formic acid. This led us to suspect
that other nicotine metabolites might be present in brain that would be
devoid of the 3H label and, therefore, not detectable by HPLRC. Such
metabolites would be nor-metabolites of which two are known to be
formed peripherally from nicotine, viz. nornicotine and norcotinine
(Bowman and McKennis, 1962; Zhang et al., 1990; Curvall and
Kazemi Valla, 1993; Gabrielsson and Gumbleton, 1993; Liu et al.,
1993).
This work was supported in part by National Institute of Drug Abuse Grants
DA08656 and DA00399, and by the Tobacco and Health Institute, Lexington,
Kentucky.
Send reprint requests to: Dr. Peter A. Crooks, College of Pharmacy, University of Kentucky, Rose Street, Lexington, Kentucky: 40536-0082; E-mail:
[email protected]
1
Abbreviations used are: HPLRC, high pressure liquid radiochromatography;
GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry;
CNS, central nervous system.
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The residence times of nicotine and its metabolites in rat brain
after acute peripheral nicotine administration were determined.
We hypothesize that nicotine metabolites will reach pharmacologically significant concentrations in brain. Cotinine, nornicotine, and
norcotinine were structurally identified by dual label radiochemical
and gas chromatography-mass spectrometric analysis as biotransformation products of nicotine present in rat brain after s.c.
injection of S(2)-nicotine. Two unidentified minor metabolites were
also detected in brain. The half-lives in brain of nicotine metabolites were determined after a single s.c. injection of [2*-14C]-(6)nicotine (0.8 mg/kg) and analysis of radiolabeled metabolites by high
pressure-liquid radiochromatography. The brain half-lives of nicotine, cotinine, and nornicotine were 52, 333, and 166 min, respectively. Peak brain concentrations of nicotine metabolites were 300,
NICOTINE METABOLITES RESIDENCE TIMES IN RAT BRAIN
Materials and Methods
3
Compounds. S(2)-[ H-N-Methyl]nicotine (specific activity: 81 Ci/mmol)
and (6)[29-14C]nicotine (specific activity: 55 mCi/mmol) were obtained from
New England Nuclear (Boston, MA) and Moravek Biochemicals (Brea, CA).
The radiochemical purities of each of the radiolabeled compounds were
.98%, as determined by HPLRC (Cundy and Crooks, 1983). Altima AP
scintillation cocktail (Packard BioSciences, Groningen, the Netherlands),
S(2)-nicotine tartarate (Research Biochemicals International, Natick, MA),
and S(2)-cotinine (Sigma, St. Louis, MO) were purchased. HPLC grade
triethylamine, acetonitrile, phosphoric acid, and sodium phosphate (dibasic,
anhydrous) were purchased from Fisher Scientific (Pittsburgh, PA). S(2)Nornicotine and S(2)-norcotinine were prepared in our laboratory as described
previously (McKennis et al., 1959; Jacob, 1982).
In Vivo Metabolic Experiments. Male Sprague-Dawley rats (250 –350 g)
were obtained from Harlan Laboratories (Indianapolis, IN) and were housed
two per cage with free access to food and water in the Division of Laboratory
Animal Resources at the College of Pharmacy, University of Kentucky.
Experimental protocols involving animals were approved by the Institutional
Animal Care and Use Committee at the University of Kentucky.
[2*-14C]Nicotine Experiments. Experiments were performed using groups
of four rats (n 5 4 per group for each time point analyzed). Rats were injected
s.c. with 0.8 mg/kg nicotine free-base equivalent containing 50 mCi of [2914
C]nicotine in saline (0.2 ml/100 g). This dose of nicotine is nontoxic and is
a commonly used dose in behavioral studies using rat as the model, and
provides the greatest probability of allowing quantification of nicotine metabolites in brain. In radiolabeled experiments, rats were sacrificed by rapid
decapitation at 5, 30, 60, 240, 360, 480, 720, 960, and 1440 min, and the trunk
blood and whole brain, including brain stem, were obtained within 1 to 2 min.
The presence of radiolabeled nicotine and its metabolites in brain was determined by homogenizing the brain in 3 volumes of ice-cold 1.15% w/v KCl
using a polytron homogenizer. The homogenate was then centrifuged at 3000g
for 30 min. The supernatant was separated and treated with 2% w/v ZnSO4 and
the mixture was maintained in a water bath for 1 h at 34°C. The precipitated
protein was then pelleted by centrifugation at 30,000g for 60 min. The
resulting supernatants were analyzed directly by HPLRC.
Dual-Labeled Nicotine Experiments. Experiments were performed using
a group of four rats, which were injected with 0.8 mg/kg nicotine free-base
equivalent containing 25 mCi of [29-14C]nicotine and 25 mCi of [3H-Nmethyl]nicotine in saline (0.2 ml/100 g). Rats were sacrificed at 240 min (a
time point at which significant amounts of all major metabolites are observed)
and trunk blood and brain were obtained. Tissue was treated as described
above, and supernatants were analyzed directly by HPLRC.
Unlabeled Nicotine Experiments. A group of five rats were each injected
s.c. with 0.8 mg/kg nicotine free-base equivalent, and sacrificed by rapid
decapitation 240 min postinjection. Brain supernatants were obtained as described previously, and pooled for analysis by GC-MS. The supernatants were
acidified with aqueous HCl to pH 3 to 4 and lyophilized. To the lyophilized
powder was added 0.5 ml of a 5 N aqueous NaOH solution, and the mixture
was extracted with two-10 ml volumes of methylene chloride. The methylene
chloride layers were combined and evaporated to dryness over a stream of
nitrogen gas. The resulting residue was reconstituted in 5 ml of methanol, and
this solution was injected onto the GC-MS analytical unit. Chromatography
was performed on a Hewlett Packard HP 6890 gas chromatograph (Avondale,
PA) equipped with an HP 7683 series automatic sampler with a HP 7683 series
injector, and interfaced to a HP 5973 mass selective detector operating in scan
mode. The GC was equipped with a fused silica capillary column (30 m 3 0.25
mm i.d.) containing DB-5 as stationary phase (1.0-mm film thickness) obtained
from J and W Scientific (Falson, CA). The total 5-ml brain supernatant extract
was injected in the pulsed splitless mode with the injection pulse (20.0 psi)
switched on for 0.6 min and the purge valve closed for 0.5 min. The temperatures of the injection port, interface, and electron impact source were 250,
280, and 230°C, respectively. The initial oven temperature was 110°C. After
holding for 1 min, the temperature was increased at a rate of 1°C/min to 190°C,
and then increased at a rate of 4°C/min to a final temperature of 270°C. The
total separation time was 120 min. Helium was used as a carrier gas at a flow
rate of 1 ml/min in the constant flow mode. Under these conditions, nicotine,
nornicotine, cotinine, and norcotinine eluted with retention times of 26.6, 32.8,
61.8, and 69.2 min, respectively. The electron multiplier voltage was set to
1700 mV. The nature of the peaks were identified and confirmed both by
retention time of the compounds and by ion extraction chromatograms at m/z:
162, 133, and 119 for nicotine; 148, 147, and 119 for nornicotine; 176, 145,
and 98 for cotinine; and 162, 134, and 80 for norcotinine.
HPLRC System. The HPLRC system comprised a Packard Series 1100b
Radiomatic flow-through detector (Packard Instruments, Meriden, CT) connected to a Spectroflow 783 UV detector (ABI Analytical, Ramsey, NJ)
operating at a fixed wavelength of 254 nm. A Beckman System Gold PSM 116
pump (Fullerton, CA) was used. Column effluent (mobile phase) was mixed in
a 1:3 ratio volume with Altima flow AP scintillation cocktail before entering
the radiomatic detector. Output of the two detectors was recorded simultaneously on the same chromatogram on different channels on an Epson LX 810
printer, and were corrected for lag time. Chromatographic analyses were
performed on a 25 3 0.46 cm Partisil 10 C8 column (Clifton, NJ) connected
to a Phenomenex 3.0 3 0.46 cm C8 guard column (Torrance, CA). The mobile
phase was 0.1 M sodium phosphate/acetonitrile (95:5 v/v%), and 30 ml/liter
triethylamine, and the pH was adjusted to 7.0 with phosphoric acid. The system
was run at a flow rate of 1.5 ml/min. Each supernatant sample was coinjected
with 100 ml of a standard solution containing 1.5 mg of authentic standards of
S(2)-nicotine, S(2)-cotinine, and S(2)-norcotinine. Radioactive metabolites
eluting from the HPLC column were identified by comparing their retention
times with those of the UV-active authentic standards. The retention times for
the analytes were 5, 13, 26, 28, 38, and 55 min for metabolite A, nornicotine,
norcotinine, metabolite B, nicotine, and cotinine, respectively. For dual label
analysis, the energy range settings for 3H detection were set at 0 keV for the
lower discriminator and 13 keV for the upper discriminator. For 14C detection,
energy range settings were 14 keV for the lower discriminator and 114 keV for
the upper discriminator. Energy spillover of 14C into the 3H spectrum was 70%
and the quenching efficiencies for 3H and 14C were 32.5 and 48.2%, respectively. The third channel of the analytical unit was used for UV detection at
254 nm.
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The presence of nornicotine and cotinine in brain after nicotine
administration may be of additional relevance to tobacco smoking,
because these compounds are also alkaloidal constituents of tobacco.
Thus, these compounds in smokers’ brain may arise from both nicotine metabolism and from self-administration of these alkaloids. The
presence of nornicotine and cotinine in brain may also have significance in understanding the neuropharmacological effects of tobacco
use, because both metabolites have been shown to be neuropharmacologically active (Risner et al., 1985; Goldberg et al., 1989; Dwoskin
et al., 1995, 1999a,b; Stolerman et al., 1995; Teng et al., 1997).
Therefore, we carried out similar metabolic experiments using nicotine with a 14C label at the 29-carbon to determine whether normetabolites of nicotine were indeed formed in the brain after peripheral nicotine administration to rats (Crooks et al., 1997). These
HPLRC experiments clearly showed that at 4 h postinjection of
peripheral nicotine, two metabolites were detected and quantified in
brain by coelution with authentic standards of nornicotine and norcotinine. These data represent the first comprehensive study on the
determination and quantitation of nicotine metabolites in brain after
acute nicotine administration. However, these brain metabolites have
been identified only tentatively by HPLRC. This tentative identification is based on their coelution with authentic UV absorbing metabolic standards, and on the observation that when [3H-N-methyl]nicotine is used instead of [29-14C]nicotine, none of the nor-metabolites
are detected, although cotinine is clearly observed. We now report the
definitive structural identity of cotinine, nornicotine, and norcotinine
in rat brain after peripheral nicotine administration using dual-label
tracers and gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, we have determined the residence time and half-life
in rat brain of each of these metabolites after a single peripheral dose
of [29-14C]nicotine.
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Top: cpm above background for the 14C channel; bottom: cpm above background
for the 3H channel. Radiolabeled bands were identified by comparing retention
times with those of coinjected UV-absorbing authentic metabolic standards. HPLRC
operating conditions are as described in Materials and Methods.
Results
Dual-labeled nicotine metabolism experiments were performed to
confirm the presence of nor-metabolites of nicotine in rat brain. One
label was 3H, which was placed on the N-methyl group of nicotine; the
second label was 14C, which was placed in the pyrrolidine ring
skeleton at C-29. The presence of nor-metabolites can be established
using this dual-labeled form of nicotine by dual-channel ratio radioactivity analysis. Nor-metabolites should only retain the 14C label, i.e.,
the 3H label is lost through demethylation, whereas N-methyl metabolites should retain both labels. Thus, rats were injected peripherally
with 14C-/3H-labeled nicotine, and 4 h later, brains were obtained and
supernatants analyzed by HPLRC dual channel analysis. Figure 1
clearly shows that the radiolabeled bands tentatively identified as
nornicotine and norcotinine have both lost their 3H label, demonstrating that they are nor-metabolites. The 14C radiochromatograms afforded radiolabeled bands that were much broader than the bands
observed in the 3H channel analysis. This is because the 14C-labeled
nicotine is racemic, whereas the [3H]nicotine is the pure S(2)-enantiomer, and the added mass of unlabeled nicotine is in the form of the
S(2)-enantiomer. Thus, a typical chirodiastaltic effect is observed
with the racemic 14C-labeled nicotine, causing significant peak broadening of both [14C]nicotine and its metabolites (Cundy and Crooks,
1983).
To prove conclusively that the metabolites present in brain after
acute peripheral nicotine administration are cotinine, nornicotine, and
norcotinine, GC-MS analysis was performed on rat brain supernatant
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FIG. 1. HPLRC Partisil-10 C8 chromatogram of supernatant from 4-h brain
homogenate from a representative rat administered a single s.c. dose of 0.8 mg/
kg nicotine containing 25 mCi each of [29-14C]nicotine and [3H-methyl]nicotine.
extracts. Because of the extremely small amounts of nicotine metabolites present in brain after acute administration, it was not possible to
quantify these metabolites in single animal experiments. Thus, brain
supernatants from five animals that had each been administered 0.8
mg/kg of nonradiolabeled S(2)-nicotine 4 h earlier were pooled and
processed, and the extract was injected onto the GC-MS. Figure 2
illustrates the GC chromatograph of this extract. Nicotine and the
three metabolites, nornicotine, cotinine, and norcotinine, were easily
detected by GC analysis, displaying retention times of 26.6, 32.8,
61.8, and 69.2 min, respectively. Cotinine was by far the most
abundant metabolite in brain. Nornicotine was the second most abundant, whereas norcotinine was only a minor metabolite in brain. The
unambiguous structural assignment of each of the above nicotine
metabolites was determined by ion extraction GC-MS. The retention
times and fragmentation patterns obtained for each metabolite were
consistent with their proposed structures. The fragmentation patterns
obtained for each of the metabolites and the selected ion chromatograms are shown in Fig. 3. Each metabolite spectrum was peak
matched with the spectrum of the authentic compound in the Tobacco
and Health Research Institute and Wiley library databases. These data
confirm unequivocally the presence of these metabolites in rat brain
4 h after acute peripheral nicotine exposure. The results are entirely
consistent with those obtained from radiochemical studies performed
at the 4-h time point (Crooks et al., 1997).
To determine the time course of appearance and half-life of CNS
metabolites, experiments were carried out with [29-14C]nicotine to
reliably quantitate the low levels of these metabolites in brain. Groups
of rats were administered 0.8 mg/kg of nicotine containing 50 mCi of
[29-14C]nicotine. At 5, 30, 60, 240, 360, 480, 720, 960, and 1440 min
after injection, brain supernatants from individual animals were obtained and analyzed by HPLRC. The half-lives of nicotine and its
metabolites were calculated from the terminal slope of log concentration versus time graphs of each component. The results of these
time course studies are shown in Figs. 4 and 5. Figure 4 illustrates
representative radiochromatographs obtained at the different time
points analyzed. At the 5-min time point, only nicotine was observed
in brain, whereas at 30 min, small amounts of both cotinine and
nornicotine began to appear. These two metabolites were relatively
greater in amount at the 60-min time point when compared with
nicotine, and reached their highest levels between 1 and 4 h. In
addition, the minor metabolite norcotinine was clearly apparent at 4 h.
At 8 h, nicotine was virtually nonquantifiable. At 8 h postinjection,
nicotine was completely cleared from the central compartment,
whereas only residual levels of nornicotine were present. The cyclic
lactam metabolites, cotinine and norcotinine, which have relatively
longer half-lives than the basic amino compounds (nicotine and nornicotine), were still present in brain 12 h postinjection. Two very
minor unidentified metabolites (A and B) appeared in brain at the 4-h
time point but were barely detectable at 16 h.
Figure 5 illustrates time-concentration profiles for nicotine, cotinine, nornicotine, norcotinine, and the two unidentified metabolites A
and B obtained from a group of four rats in which [29-14C]nicotine
was peripherally (s.c.) administered. Nicotine absorption from the s.c.
injection site was rapid, such that at the 5-min time point, the concentration of nicotine in brain had already peaked. The time-concentration profile was clearly biphasic, showing an initial rapid distribution phase followed by a slower elimination phase. By 6 h, nicotine
was almost completely cleared from brain. The nicotine T1/2 was 58
min. The time-concentration profile for cotinine indicated that it was
first detected at 30 min and peaked between 60 and 240 min, and was
still present in significant amounts at 8 and 16 h postinjection.
Cotinine was the major metabolite in brain over the entire time course
NICOTINE METABOLITES RESIDENCE TIMES IN RAT BRAIN
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Data are expressed as relative abundance in arbitrary units as a function of time (min). Separations were carried out on a fused silica capillary column containing DB-5
stationary phase (see Materials and Methods for operating conditions). Brain metabolites of nicotine were identified and structures confirmed by comparing the retention
times of the metabolites with those of authentic metabolic standards and by carrying out the analysis by ion extraction mass spectrometry (see Fig. 3 for details). NNIC,
nornicotine; COT, cotinine; NCOT, norcotinine.
studied and reached a peak concentration of 300 pmol/g brain tissue.
Thus, cotinine entered the CNS quickly, but was eliminated from the
CNS relatively slowly. The cotinine T1/2 was 5.5 h and was still
detectable at 24 h postinjection.
The next most abundant metabolite was nornicotine. Nornicotine
was not observed at 5 min, but peaked at 60 min with a maximal
concentration of 70 pmol/g brain, and was still detectable at 12 h. The
nornicotine T1/2 was 2.8 h, and its elimination rate was comparable
with that for nicotine.
Norcotinine was a very minor metabolite and was not observed
until 4 h, peaked at 7 pmol/g brain between 4 and 6 h, and was almost
nonquantifiable by 24 h. The two unidentified metabolites, A and B,
were also present in very minor amounts, similar to norcotinine. Thus,
several metabolites in brain were detected that had relatively longer
CNS residence times and half-lives than those of nicotine.
Discussion
S(2)-Cotinine is a major peripheral metabolite of nicotine and is
able to pass the blood-brain barrier from the periphery (Appelgren et
al., 1962; Schmiterlow et al., 1967; Deutsch et al., 1992; Crooks et al.,
1997; Crooks and Dwoskin, 1997). The origin of S(2)-cotinine in
brain has not been elucidated and could arise from oxidative formation from nicotine locally in the brain, or from oxidative formation in
the periphery followed by redistribution to the brain. It is generally
accepted that brain is a poor metabolizing organ compared with liver,
although the activity of certain metabolic enzymes have been detected in brain, including certain isozymes of cytochrome P-450 that
are induced on chronic administration of nicotine (S. Miksys, E.
Hoffman, and R. F. Tyndale, submitted). The relatively large amounts
of cotinine found in brain would suggest that cotinine is formed in the
periphery and then redistributed to the CNS. Nevertheless, one cannot
rule out the formation, at least in part, of cotinine from nicotine locally
in brain.
Compared with nicotine, the neuropharmacological effects of S(2)cotinine have not been widely investigated. In behavioral studies,
S(2)-cotinine has been reported to alter responding for food in rats,
Beagle dogs, and squirrel monkeys (Risner et al., 1985; Goldberg et
al., 1989). However, the rate-increasing effect of S(2)-cotinine during
fixed interval responding was not attenuated by the nicotinic receptor
antagonist, mecamylamine (Goldberg et al., 1989), suggesting that
this effect may not be nicotinic receptor mediated. Drug discrimination studies report generalization of S(2)-nicotine to S(2)-cotinine in
rats and squirrel monkeys, but large doses of S(2)-cotinine were
required (Goldberg et al., 1989; Tacada et al., 1989). Similar to
nicotine, cotinine releases dopamine from its presynaptic terminals in
striatum via a nicotinic receptor-mediated mechanism (Dwoskin et al.,
1999b). The EC50 for S(2)-cotinine to evoke [3H]dopamine overflow
from rat striatal slices was 30 mM. Furthermore, exposure to S(2)cotinine resulted in nicotinic receptor desensitization (Dwoskin et al.,
1999b). Thus, although the concentration of S(2)-cotinine in brain
(0.3 mM) after acute nicotine administration in the present study is not
within the concentration range found to be effective in the dopamine
release assay, cotinine may accumulate in brain during chronic smoking due to its long residence time in brain and due to the fact that
cotinine is also an alkaloidal constituent of tobacco (Crooks, 1999).
Nevertheless, taking into account potential accumulation with chronic
nicotine administration and the expected higher cotinine concentrations in brain from chronic smokers, it remains unlikely that the
concentration of cotinine in brain will reach the levels that were found
to be effective in the dopamine release assay. Thus, it appears unlikely
that cotinine will contribute to the dopamine-mediated neuropharmacological effects of nicotine exposure. In the current studies, cotinine
had a half-life of 333 min in rat brain after a single peripheral dose of
0.8 mg/kg nicotine. This is comparable with a plasma cotinine halflife of 294 to 318 min over a range of nicotine doses in the rat
(Kyerematen et al., 1988).
The next most abundant metabolite found in brain in the present
studies was nornicotine. It is important to note the concentration of
nornicotine (0.07 mM) observed in brain after acute peripheral nicotine administration. Nornicotine was found to have a half-life of 166
min in rat brain (present study); this compares to a half-life of 198 min
for this metabolite in plasma after i.v. administration of nicotine in the
rat (Kyerematen and Vesell, 1991). It has been reported that the
plasma half-life of nornicotine in smokers and nonsmokers after a
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FIG. 2. GC chromatograph of a sample of an extract of brain supernatant pooled from five rats 4 h after s.c. administration of 0.8 mg/kg of nonradiolabeled S(2)nicotine (NIC).
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FIG. 3. Structural identification of nicotine (A), cotinine (B), nornicotine (C), and norcotinine (D) in a pooled sample of an extract of brain supernatant from five rats
4 h after s.c. administration of 0.8 mg/kg of nonradiolabeled S(2)-nicotine by ion extraction chromatography and mass spectrometry.
A to D, ion extraction chromatograms (top) and electron impact mass fragmentation patterns (bottom) for nicotine (m/z 162, 133, 119), cotinine (m/z 176, 145, 98),
nornicotine (m/z 148, 147, 119), and norcotinine (m/z 162, 134, 80), respectively.
NICOTINE METABOLITES RESIDENCE TIMES IN RAT BRAIN
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FIG. 4. HPLRC chromatograms of brain supernatant from representative rats that have been injected s.c. with 0.8 mg/kg [29-14C]nicotine (NIC; 50 mCi).
Brains were removed at 5, 30, 60, 240, 360, 480, 720, 960, and 1440 min after injection. Brain supernatants were obtained and either 1-ml (for 5-, 30-, 60-, 240-, and
360-min time points), or 2-ml (for 480-, 720-, 960-, and 1440-min time points) samples were injected onto a Partisil-10 C8 column (for details, see Materials and Methods).
A and B, unidentified minor nicotine metabolites. NNIC, nornicotine; COT, cotinine; NCOT, norcotinine.
single i.v. dose of nicotine is 7.2 and 8.5 h, respectively; these values
are considerably higher than plasma half-lives for nornicotine observed in the rat (Kyerematen and Vesell, 1991). Nornicotine is a
minor biotransformation product (0.4% in humans and 8% in rats) of
nicotine in the periphery (Cundy and Crooks, 1984; Benowitz et al.,
1991; Curvall and Kazemi Valla, 1993). In guinea pigs, only 1.6% of
the total nicotine dose was detected as nornicotine in 24-h urine void
(Cundy and Crooks, 1984). After administration of a single arterial
dose of labeled nicotine to rats, it was found that nornicotine accounted for only 8% of total recovery of administered radioactivity
(Curvall and Kazemi Valla, 1993). Therefore, it is intriguing that such
significant amounts of this metabolite, relative to nicotine, are found
in rat brain after acute peripheral nicotine administration. Thus, redistribution of metabolically formed nornicotine from the periphery to
the brain appears to be an unlikely source of brain nornicotine,
considering the very small amount of nornicotine detected as a peripheral nicotine metabolite. A more likely origin of nornicotine in
brain is via local oxidative N-demethylation of nicotine.
Nornicotine is pharmacologically active, and similar to nicotine,
acts as an agonist at nicotinic receptors, which evoke the release of
dopamine from its presynaptic terminal stores (EC50 5 1.0 mM; Teng
et al., 1997). Nornicotine also causes nicotinic receptor desensitization
(EC50 5 0.095 mM; Dwoskin et al., 1999c). In behavioral studies,
nornicotine has been shown to have psychomotor effects that differ
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The half-lives for nicotine, cotinine, and nornicotine in brain are also shown.
qualitatively from those of S(2)-nicotine, in that behavioral sensitization was not apparent after chronic administration of nornicotine to
rats (Dwoskin et al., 1999a). Similar to nicotine, nornicotine has
reinforcing qualities, in that it maintains i.v. self-administration
(Bardo et al., 1999). The present study demonstrates that concentrations of nornicotine in rat brain after acute administration of nicotine
approach those that produce pharmacological effects. It is likely that
nornicotine will accumulate in brain on chronic nicotine administration, based on the longer residence time of nornicotine in comparison
to nicotine, and that accumulation will reach concentrations necessary
for pharmacological effects to be observed. This is particularly relevant in human tobacco smokers, in that not only is nornicotine a
biotransformation product of nicotine, but smokers are exposed to
significant quantities of nornicotine present in commercial tobacco
(15–20% total alkaloid content; Zhang et al., 1990; Curvall and
Kazemi Valla, 1993). Thus, a combination of “alkaloidal” nornicotine
and “metabolic” nornicotine may be sufficient to produce brain levels
of nornicotine that contribute to the neuropharmacological effects of
tobacco smoking.
The current study is the first to unequivocally identify and provide
detailed information on the residence times and half-lives of several
pharmacologically active nicotine metabolites in brain after acute
nicotine exposure. Nevertheless, correlation of the current results in
the rat with those in other species, including humans, may be limited
by factors such as species differences in nicotine metabolism and
distribution, as well as dose-related effects (i.e., only one dose of
nicotine was used in this study).
An important pharmacokinetic characteristic of nicotine metabolites appearing in brain is that their residence times are all longer than
that of nicotine. Therefore, the possibility exists that on repeated
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FIG. 5. Brain time-concentration profiles for nicotine, cotinine, nornicotine, norcotinine, and two unidentified minor metabolites (A and B) obtained from a group of
four rats (mean 6 S.E.) that have been injected s.c. with 0.8 mg/kg [29-14C]nicotine (50 mCi).
NICOTINE METABOLITES RESIDENCE TIMES IN RAT BRAIN
peripheral administration of nicotine, these metabolites have the potential to accumulate in brain. This is of particular significance with
regard to the chronicity of nicotine exposure during tobacco smoking.
Typically, a chronic smoker will smoke a cigarette every 20 min with
intermittent inhalation of main-stream smoke. Delivery of nicotine to
the CNS constitutes intermittent peaks, superimposed on a constant
plasma level (Benowitz et al., 1991; Jacob and Benowitz, 1993).
Because the residence time of nicotine in the CNS is short, its ability
to accumulate is less likely than that of its metabolites, which have
longer half-lives. Thus, it is possible that prolonged intermittent
administration of nicotine, as is the case with tobacco smoking, may
result in significantly higher levels of the slower effluxing metabolites. We hypothesize that slow effluxing metabolites that possess
pharmacological activity may well play a major role in the neuropharmacology of tobacco smoking. It will be important to determine in
future studies the ability of such metabolites to accumulate in brain
after chronic nicotine administration.
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