Substituted heteroaromatic compounds: Effect on nicotine self-administration in rats

Article abstract of DOI:10.1007/s00213-011-2608-6

Rationale: Certain compounds that nonselectively inhibit a prominent human nicotine-metabolizing enzyme (i.e., human cytochrome P-450 2A6, hCYP 2A6) showed inhibition of smoking in humans. However, a comprehensive examination of hCYP 2A6 inhibitors to dec

Full text of DOI:10.1007/s00213-011-2608-6

Psychopharmacology (2012) 221:637–648
DOI 10.1007/s00213-011-2608-6
ORIGINAL INVESTIGATION
Substituted heteroaromatic compounds: effect on nicotine
self-administration in rats
John R. Cashman & Karl Okolotowicz & Matt Cerny &
Robert Johnson & Aaron Janowsky & Marc R. Azar
Received: 1 August 2010 /Accepted: 2 December 2011 /Published online: 5 January 2012
#
Springer-Verlag 2012
Abstract
nicotine- vs. food-reinforced responding was evaluated for
compounds 10 and 11.
Rationale Certain compounds that nonselectively inhibit a
prominent human nicotine-metabolizing enzyme (i.e., hu-
man cytochrome P-450 2A6, hCYP 2A6) showed inhibition
of smoking in humans. However, a comprehensive exami-
nation of hCYP 2A6 inhibitors to decrease nicotine self-
administration in rats has not been reported.
Objectives We tested substituted heteroaromatic compounds
designed to selectively inhibit hCYP 2A6 in a model system
to (a) examine selective hCYP 2A6 inhibitors to decrease
cotinine formation in vivo in rats administered with nicotine
and (b) examine their efficacy to decrease nicotine self-
administration in rats.
Methods Rats were trained to IV self-administer nicotine in
1-h sessions. Nicotine self-administration was carried out at
a unit dose of 0.03 mg/kg/infusion in 0.1 ml/s. Pretreatment
with substituted heteroaromatic test compounds (0.5–
25 mg/kg, i.p., 30 min prior to nicotine self-administration
sessions) resulted in dose-dependent decreases of nicotine
self-administration. Using operant conditioning techniques,
Results Compounds 10 and 11 selectively decreased nico-
tine self-administration with estimated ED50 values 4 and
2.8 mg/kg, respectively. Of the test compounds examined,
none showed significant affinity for mammalian α4β2- or
α7-neuronal nicotinic acetylcholine (nAChR) receptors and
none were inhibitors of the human dopamine transporter
(hDAT); thus, neither the endogenous nAChRs nor DAT ap-
parently plays a role in decreasing nicotine self-administration
for this series of compounds.
Conclusion The results indicate that chemical analogs of
nicotine can play a role in nicotine self-administration harm
reduction but a non-nAChR and a non-hDAT mechanism are
likely involved.
.
Keywords Nicotine self-administration Nicotine
.
.
metabolism Cytochrome P-450 2A6 Neuronal nicotinic
.
acetylcholine receptors Dopamine transporter
Electronic supplementary material The online version of this article
(doi:10.1007/s00213-011-2608-6) contains supplementary material,
which is available to authorized users.
Introduction
In the USA, tobacco smoking is the leading cause of pre-
ventable death (Taylor and Bettcher 2000). Currently, al-
most 25% of Americans smoke some form of tobacco
product (Rock et al. 2007) despite convincing evidence that
smoking cigarettes is the primary cause of lung cancer (Doll
and Hill 1952) and that sidestream smoke is also unques-
tionably linked to lung cancer (Hirayama 1981) and heart
disease. Worldwide, death from tobacco smoking is pre-
dicted to increase to over six million individuals per year
by 2015 and account for 10% of all deaths (Mathews and
Loncar 2006).
:
:
J. R. Cashman (*) K. Okolotowicz M. Cerny
Human BioMolecular Research Institute,
5310 Eastgate Mall,
San Diego, CA 92121, USA
e-mail: JCashman@hbri.org
:
R. Johnson A. Janowsky
Veterans Administrative Hospital,
Portland, OR 97201, USA
M. R. Azar
Behavioral Pharma Inc.,
La Jolla, CA 92037, USA

638
Psychopharmacology (2012) 221:637–648
The pharmacological effects of smoking are mediated by
the investigation to 1-alkyl- or 1-benzyl imidazoles as inhib-
itors of human CYP 2A6 and studied the effects of these
agents on nicotine self-administration in rats. The goals of
the work described herein included the following: (1) examine
a role of selective CYP 2A6 inhibitors in nicotine self-
administration and clearance in rats and (2) examine a role
of nAChRs and the dopamine transporter (DAT) in mediating
this outcome. We determined that an association between
potency of inhibition of nicotine metabolism by rat CYP
2B1 or nicotine clearance in rats and efficacy of nicotine
self-administration in vivo was not observed. However, a
new class of potent nicotine self-administration blockers have
been discovered and characterized.
the alkaloid (S)-nicotine (Balfour 2002). Treatments for
tobacco smoking addiction have included nicotine replace-
ment therapy and use of the antidepressant bupropion
(Hughes 2007). However, these therapies only double the
placebo quit rate (West 2007) and the success rate for people
using these therapies is only about double that achieved with
no pharmacological treatment (Stead et al. 2008). Recently,
a smoking reduction agent, varenicline (Chantix, Pfizer),
based on partial agonism of the α4β2-nicotinic acetylcho-
line receptor (nAChR) was developed (Coe et al. 2005) and
has shown utility in humans (Cahill et al. 2009). Although
varenicline has promise as a therapeutic and has shown
clinical utility and a role for a nicotine receptor partial
agonist in smoking reduction, in view of the side effects,
additional drugs and approaches are needed.
Methods
In humans, the main way administered nicotine is cleared
is by metabolism via hepatic cytochrome P-450 2A6 (CYP
2A6)-mediated oxidation to cotinine via aldehyde oxidase
(Cashman et al. 1992). Nicotine is primarily eliminated by
metabolism that is quite variable among individuals (Beno-
witz and Jacob 1984; Benowitz 1988; Feyerabend et al.
1985). In rats, rCYP 2B1-mediated oxidation to cotinine
via aldehyde oxidase is the major way that nicotine is
metabolized and cleared (Miksys et al. 2000).
Various lines of evidence suggest that decreased CYP 2A6-
dependent metabolism of nicotine is associated with de-
creased risk for smoking and thus harm reduction. If humans
modulate their smoking to control nicotine consumption
(Benowitz and Jacob 1984), it is possible that individuals with
decreased nicotine metabolism will be predisposed to smoke-
less tobacco and have decreased addiction liability. In fact, the
CYP 2A6 inhibitor, methoxsalen (administered at 30 mg,
orally) attenuated nicotine clearance, increased nicotine bio-
availability, and decreased cigarette smoking by 24% in
humans compared with placebo (Sellers et al. 2000). Addi-
tional reports have suggested that individuals with defective
CYP 2A6 smoke fewer cigarettes (Pianezza et al. 1998).
While new, potent and selective CYP 2A6 inhibitors hold
promise as chemopreventative agents against N-nitrosamine-
induced lung tumorigenesis and possibly as nicotine harm
reduction agents (Shin et al. 2008; Takeuchi et al. 2003),
considerably more studies need to be done.
Chemicals
(−)-(S)-Nicotine hydrogen tartrate salt was purchased from
Aldrich Chemical Company (Milwaukee, WI) and dissolved
in isotonic saline solution, and the pH was adjusted to pH 7
before use in vivo. Mecamylamine hydrochloride salt was
obtained from Aldrich and dissolved in isotonic saline solu-
tion. Heparinized saline and Timentin® were purchased from
SavMart Pharmaceuticals, (San Diego, CA). Drug concentra-
tions refer to the salt form. The synthesis of compounds 1, 2,
and 4–6 and 8 was previously described (Denton et al. 2005;
Yano et al. 2006) and used as HCl salts.
Overview
Eleven compounds were tested and each compound was
referred to by a number between 1 and 11.
General procedure for the synthesis of 1-alky and 1-benzyl
imidazoles
The alkyl or benzyl halide (1.0 equiv) and imidazole (3.0
equiv) were placed together, heated to 90°C for 3 h, cooled
to room temperature and the resulting residue was parti-
tioned between 5 M NaOH and CH2Cl2 (Lu et al. 1997).
The organic portion was concentrated in vacuo and the
product was purified by silica gel chromatography and fully
characterized as described in the Appendix for each
compound.
Kinetic studies of CYP 2A6 inhibition (Denton et al. 2005)
and X-ray co-crystallographic studies with CYP 2A6 (Yano et
al. 2006) of numerous substituted 3-heteroaromatic pyridine
analogs of nicotine provided considerable insight into struc-
ture–activity relations (SAR) for interaction with CYP 2A6.
Certain pyridyl imidazoles were potent inhibitors of CYP 2A6
(Denton et al. 2005). Based on this information and previous
studies of the effect of inhibitors on human CYP 2A6 (Denton
et al. 2004, 2005; Berkman et al. 1995) and inhibition studies
of cigarette smoking (Sellers et al. 2000, 2003), we extended
Biological material
Wistar-derived male rats (250–300 g, Harlan (Livermore,
CA)) were housed in groups of two and maintained in a
temperature-controlled environment on a 12-h:12-h light/dark
cycle (0600 hours on–1800 hours off) upon arrival to

Psychopharmacology (2012) 221:637–648
639
the laboratory, given free access to food and water during a
1-week habituation period to the laboratory, and handled,
housed, and killed in accordance with current NIH guidelines.
Animals were handled daily for several days to desensitize
them to handling stress before experimental testing. The cell
sizes (n05–8) provided reliable estimates of drug effects. The
rats in this experiment were previously injected with a test
compound and, therefore, were not drug naive. However, an
appropriate washout period (14 days) was given before com-
pounds were tested, and no carry-over effect was apparent.
press) with a 1-s time-out (TO-1 s) after each reinforcement.
The schedule was gradually increased to achieve an FR1-
TO-20s schedule of reinforcement. Training sessions were
given two times per day, and each session lasted for 30 min.
Once the rats obtained steady baseline responding (defined
as less than 20% variability across three consecutive ses-
sions) at a FR1-TO-20s schedule of reinforcement, they
were returned to ad libitum food to allow for preparation
of studies using IV jugular catheter implant surgery.
Food training—food study
Test compound treatment
For food studies, two separate cohorts of rats (n08) were
used to test compounds 10 and 11. Lever pressing was
established as described above (Hyytia et al. 1996). Initially,
rats were restricted to 15 g of food daily (approximately
85% of their free-feeding body weight). After the second
day of food restriction, rats were trained to respond for food
under a FR1 schedule of reinforcement (one food pellet for
each lever press) with a 1-s time-out (TO-1 s) after each
reinforcement. The schedule was gradually increased to
achieve an FR1-TO-20s schedule of reinforcement. Training
sessions were given two times per day, and each session
lasted for 30 min. Once the rats obtained steady baseline
responding (defined as less than 20% variability across three
consecutive sessions) at an FR1-TO-20s schedule of rein-
forcement, dose response testing commenced. Rats were
baselined for 1–3 days (until stable) between each dose
probe.
In the study, separate groups of rats received compounds 1,
3, 4, 7, or 9 (vehicle, 15 mg/kg, i.p.), compounds 2, 5, and 6
(vehicle, 15 and 25 mg/kg, i.p.), compound 8 (vehicle, 15,
20 and 25 mg/kg, i.p.), compound 10 (vehicle, 0.5, 1.58, and
5 mg/kg, i.p.), compound 11 (vehicle, 1.6, 2.8, and 5 mg/kg,
i.p.), or a positive control compound, mecamylamine
(3.0 mg/kg, subcutaneously, s.c.). Each test compound and
mecamylamine as hydrochloride salts was dissolved in iso-
tonic saline, and administered to separate groups in a vol-
ume of 1 ml/kg. Test compounds were administered 30 min
prior to the start of self-administration (SA) sessions. The
experimental design was a within-subjects Latin square
design, and each rat in a full dose–response study received
all doses of test compound. Mecamylamine was tested after
the dose–response studies with the above test compounds
using a cross-over dose design.
Apparatus
Surgery
Food training and nicotine self-administration took place in
eight standard Coulbourn operant conditioning chambers
(standard “rat” chambers) housed in a sound-attenuated
box. Operant conditioning chambers were equipped with
two levers mounted 2 cm above the floor and a cue light
mounted 2 cm above the right lever (active lever) on the back
wall of the chamber. For food training, a food hopper was
located 2 cm to the left/right of either lever in the middle of the
front wall. Intravenous infusions were delivered in a volume
of 0.1 ml over a 1-s interval via an infusion pump (Razel, CT)
housed outside of the sound-attenuated operant conditioning
chamber.
Rats were anesthetized with an isoflurane–oxygen mixture
(1–3% isoflurane), and chronic silastic jugular catheters
were inserted into the external jugular and passed subcuta-
neously to a polyethylene assembly mounted on the ani-
mal’s back. The catheter was passed subcutaneously from
the back to the jugular vein of the rat where it was inserted
and secured with a nonabsorbable silk suture. Upon suc-
cessful completion of surgery, rats were given 5 days to
recover before baseline self-administration sessions started,
remained on ad libitum food access, and had their catheter
lines flushed daily with heparinized saline containing
66 mg/ml of Timentin® to prevent blood coagulation and
infection.
Food training—IVSA study
Nicotine self-administration
Lever pressing was established using a previously described
method (Hyytia et al. 1996). Initially, rats were restricted to
15 g of food daily (approximately 85% of their free-feeding
body weight). After the second day of food restriction, rats
were trained to respond for food under a fixed ratio 1 (FR1)
schedule of reinforcement (one food pellet for each lever
Following successful recovery from catheter implant sur-
gery, rats were again food deprived to 85% of their free-
feeding body weight. Once self-administration sessions
started, rats were trained to IV self-administer nicotine
(0.03 mg/kg/infusion, 0.1 ml in 1 s) in 1-h baseline sessions.

640
Psychopharmacology (2012) 221:637–648
Rats were studied 5–7 days per week, under an FR1-TO-20s
schedule of reinforcement, until stable responding was
achieved. A 20-s time-out (TO) period, concurrent with
the beginning of the infusion, was signaled by illumination
of the cue light above the active lever. Responding during
the TO period was not reinforced. After stable responding
for nicotine was achieved, various doses of test compound
were tested using a within-subjects Latin square design. Rats
were treated with one of several doses of test compound for
each test session and subsequently retested to establish that
baseline session values were obtained for 1–3 days before rats
were administered the next dose of test compound.
Newman–Keuls test, was conducted on dose–response curve
data, where appropriate.
In vitro pharmacology
Triplicate assays were done with each incubation containing
radioligand (1.5 nM [3H]cytisine for α4β2 and 1 nM [125I]
alpha-bungarotoxin for α7 nicotinic receptors), cell mem-
brane (10 μg rat cerebral cortex protein) and assay buffer
(pH 7.5, 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2), and
test compounds 9–11 as before (Sharples et al. 2000). Non-
specific binding was determined in the presence of nicotine
bitartrate salt (10 μM) for α4β2 and α-bungarotoxin (1 μM)
for α7 nicotinic receptors. Single determinations (1 μM) of
test agent binding were done at each nicotinic receptor and
were compared to those of the standard nicotinic reference
compounds examined at the same time. Membranes were
incubated with the test compounds for 75 min at 4°C (for the
α4β2 receptor) and 150 min at 37°C (for the α7 nicotinic
receptor) and centrifuged (1,500 rpm), and binding was
assessed using scintillation counting.
Effect of compounds 4–6 and 8 on pharmacokinetics
of nicotine clearance
The effect of pretreatment of compounds 4–6 and 8 (i.e.,
25 mg/kg, 30 mins prior, i.p.) on the clearance of nicotine
(3.0 mg/kg, IV) was determined by investigating the area
under the concentration–time profile (AUC) in male Wistar
rats (350–375 g, Harlan (Livermore, CA)). Briefly, blood
was obtained from an IV jugular catheter of rats at various
time points and centrifuged at 10,000×g at 4°C and the
plasma was extracted as previously described (Denton et
al. 2005); nicotine and cotinine were quantified by liquid
chromatography–mass spectrometry (LC–MS). The mean
and standard deviation of the time that the greatest or peak
plasma concentration (Cmax) of nicotine or cotinine occurred
defined as tmax, and AUC were calculated using the
WinNonlin-Pro program (Version 1.5, Pharsight, Inc.).
Enzyme inhibition
To measure human CYP 2A6 functional activity, coumarin
7-hydroxylation was determined as previously described
(Denton et al. 2005) using commercially available enzyme
(BD Biosciences, Woburn, MA). The IC50 value was con-
verted to an inhibition constant (Ki) value as described below.
To measure the effect of compounds 1–11 on rat CYP 2B1
functional activity, cotinine formation was determined by a
method previously described (MacDougall et al. 2003) using
commercially available rat CYP 2B1 (BD Biosciences). The
inhibitory effect of each test compound was determined at two
concentrations (i.e., 25 μM and 100 μM).
Data compilation, processing and analysis
Animal data were collected online simultaneously from
multiple operant conditioning chambers. Results of the (IV
self-administration) IVSA and food operant conditioning
procedures, using the Latin square design, were reported
as mean cumulative number of reinforced responses for
nicotine and food. In general, tests for homogeneity of
variance were first conducted on the data. If the scores did
not violate the assumption of homogeneity of variance,
appropriate analyses of variance (ANOVA) were conducted.
“Order effects” were examined by conducting repeated
measures ANOVA on the mean reinforcement values within
each dose day across the dose response testing. Order effects
were reported only if a significant finding was observed.
Test data were analyzed using the StatView statistical pack-
age on a PC-compatible computer. For the analysis of the
dose response testing and individual dose testing, a repeated
measures ANOVA was conducted, with number of reinforced
responses as the dependant variable and drug dose as the
independent variable. Post hoc analysis, using the Student–
Dopamine transporter binding
Dopamine transporter binding was determined as described
previously (Feng et al. 2003). Briefly, membranes from the
human dopamine transporter transfected into HEK cells
(HEK-hDAT) were incubated in the presence of [125I]-RTI-
55 and test compounds in HEPES with pH 7.4, and specific
binding was determined as the difference in radioactivity
observed in the presence and absence of mazindol using a
filtration assay. IC50 values for binding to the hDAT were
determined using GraphPad Prism (GraphPad Software, San
Diego, CA) and converted to Ki values as described before
(Cheng and Prusoff 1973). Ki values determined in the pres-
ence of the hDAT were listed in Table 7.
The specific ligand binding to the receptors was deter-
mined as the difference between the total binding and the
nonspecific binding determined in the presence of an excess

Psychopharmacology (2012) 221:637–648
641
of unlabelled ligand. The results were expressed as a percent
of control specific binding ((measured specific binding / con-
trol specific binding)×100) and as a percent inhibition of
control specific binding (100−((measured specific binding /
control specific binding)×100)) obtained in the presence of
each test compound. The IC50 values (concentration causing a
half-maximal inhibition of control specific binding) were
determined by non-linear regression analysis of the competi-
tion curves generated with mean replicate values using Hill
equation curve fitting (Y0D+[(A – D) / (1+(C / C50)nH)],
where Y0specific binding, D0minimum specific binding,
A0maximum specific binding, C0compound concentration,
C500IC50, and nH0slope factor). For both receptor-binding
studies and enzyme inhibition, the analysis was done using
SigmaPlot® 4.0 for Windows®. The Ki values were calculated
using the Cheng–Prusoff equation: (Ki 0 0 IC50 / (1+(L / KD)),
where L0concentration of radioligand in the assay, and KD0
affinity of the radioligand for the receptor). A Scatchard plot
was used to determine the KD (Cheng and Prusoff 1973).
Results
The synthesis of substituted 3-heteroaromatic pyridine or
1-alkyl- or 1-benzyl imidazole analogs of nicotine (com-
pounds 1–11, Tables 1 and 3) followed literature procedures
for related compounds (Lu et al. 1997), or alkylation of
imidazole was done using an alkyl or benzyl halide in the
Table 1 Effect of substituted heteroaromatic compounds on nicotine self-administration
Vehiclea Compounda % Decrease
(15 mg/kg)
Vehicle
Compounda % Change Nb
(25 mg/kg)
Compd
Structure
NH2
NH
13.6 2.6
11.6 + 2.9
14.7
19.2
NAc
NA
5
5
1
N
N
N
N
NH2
O
10.4 + 2.7
8.4 + 1.7
14.0 + 3.3
5.6 + 1.6d
60.
2
N
N
10.8 + 3.2
15.4 + 1.5
13.0 + 2.7
11.8 + 3.2
23.6
23.4
NA
NA
NA
NA
5
5
3
4
Cl
NH2
S
N
S
5
13.2 + 2.5
8.8 + 1.6
33.3
10.4 + 3.1
1.6 + 0.7d
84.6
64.2
5
CH3
N
NH2
O
23.2 + 1.8
10.5 + 1.3
16.8 + 2.3d
7.5 + 1.9d
27.6
28.6
23.2 + 1.8
8.3 + 2.1d
8
8
6
7
N
N
N
NA
NA
N
NA not available
a Number refers to the effects of the vehicle or compound (15.0 mg/kg or 25 mg/kg), i.p. 30 min before IV self-administration of 0.03 mg/kg/infusion of
nicotine under an FR1 schedule of reinforcement as described in the “Methods” section
b The number of animals used in the self-administration study test group
c Statistically different than vehicle-treated animals (p00.05)

642
Psychopharmacology (2012) 221:637–648
presence of excess imidazole (Baggaley et al. 1975; Kruse et
al. 1990). If only the benzyl alcohol was commercially avail-
able, it would have been converted to the benzyl chloride
using thionyl chloride, followed by alkylation of imidazole
as described above. This methodology resulted in efficient
preparation of a variety of analogs in good yield from readily
available precursors. In addition to CYP inhibition, selected 1-
alkyl- or 1-benzyl imidazoles were also studied for binding to
α4β2- and α7-nicotinic acetylcholine receptors (nAChRs)
and inhibition of binding to the hDAT. The effects of certain
test compounds (i.e., 4–6 and 8) on clearance of nicotine
administered to rats was also examined in an attempt to relate
in vitro CYP inhibition potency to in vivo nicotine clearance.
Finally, compounds 1–11 were examined in an animal model
of nicotine self-administration.
Effect of compounds 8–11 on nicotine self-administration
The dose-effect function of compound 8 on nicotine IVSA
is depicted in Fig. 1. At stable criteria, rats responded with
an average of 18.5 1.7 infusions (data not shown). That
value was not significantly different from the zero dose
within the Latin square design (i.e., the average vehicle dose
value was 23.5 3.1). An ANOVA of the treatment data (i.e.,
compound 8 dose response) failed to reveal a significant
effect of compound 8 on nicotine intake during the 1-h self-
administration sessions (F02.81, df 3,21, p00.0644), al-
though the analysis did reveal a “near-significant effect”.
As with all the compounds examined in this study, there was
no carry-over effect. That is, there was no carry-over effect
24 h after administration (i.e., during baseline sessions be-
tween drug test days) of compounds 1–11, indicating that
whatever effect that caused blockade of nicotine self-
administration was reversible. However, because the effica-
cy of compound 8 was low, it was not investigated further.
Pretreatment of a single dose (n08) with compound 9
(15 mg/kg, i.p., 30 min prior) significantly decreased nico-
tine self-administration (0.03 mg/kg/infusions of nicotine
under an FR1 schedule of reinforcement, F016.8, df 1,7,
p00.0046).
Effect of access to nicotine self-administration
Rats trained in the IVSA paradigm treated with test com-
pounds 1–7 at 15 mg/kg, i.p., showed decreases in nicotine
self-administration ranging from 14% to 33% (Table 1).
Certain compounds (i.e., 2, 5, and 6) tested in rats at
25 mg/kg showed decreases in nicotine self-administration
ranging from 60% to 85% (Table 1). However, compounds 8–
11 were more potent at decreasing nicotine self-administration
and, accordingly, were examined in more detail along with
mecamylamine (as a positive control) (Jones et al. 2010).
Analysis of the data revealed that 15 mg/kg of compound
9 produced a statistically significant decrease in IV nicotine
intake during a 1-h self-administration session (F016.8, df
1,7, p00.0046) when compared to control (i.e., vehicle-
treated) conditions. However, because the efficacy of
Effect of mecamylamine on nicotine self-administration
Mecamylamine (3.0 mg/kg, s.c., 30 min before the ses-
sion) decreased nicotine self-administration (IVSA of
0.03 mg/kg/infusion of nicotine under an FR1 schedule
of reinforcement). ANOVA of these data revealed a
significant decrease in the number of nicotine infusions
during 1-h self-administration sessions (F011.9, df 1,7,
p<0.02). At 3.0 mg/kg, responding for nicotine was de-
creased by 62% in rats when compared to control (i.e.,
vehicle-treated) conditions.
Effect of compounds 1–7 on nicotine self-administration
Initially, a select group of human CYP 2A6 inhibitors were
examined at one dose to examine a SAR on IVSA of
nicotine. Thus, pretreatment of 1–7 (15 mg/kg, i.p., 30 min
prior to 1-h IVSA sessions) showed that the test compounds
were effective in decreasing the number of nicotine infu-
sions compared to vehicle-treated rats. Compounds 2, 5, and
6 were tested at 25 mg/kg and showed a dose-dependent
decrease in the number of nicotine infusions compared to
vehicle-treated rats (Table 1).
Fig. 1 Effects of compound 8 on IV self-administration of 0.03 mg/kg/
infusions of nicotine under an FR1 schedule of reinforcement. The data
are expressed as mean ( S.E.M.) number of infusions per session (n08).
Analysis of dose response revealed a near-significant effect of compound
8 (p00.064)

Psychopharmacology (2012) 221:637–648
643
compound 9 was low, it was not investigated further. Pre-
treatment with compound 10 (Fig. 2) revealed a significant
dose effect on nicotine intake (F015.5, df 3,21, p<0.0001).
Post hoc Student–Newman–Keuls test of individual doses of
compound 10 revealed that a dose of 5 mg/kg produced a
statistically significant difference from control (vehicle-trea-
ted) conditions, with a corresponding p value of <0.0001. A
dose-effect function on nicotine self-administration for treat-
ment with compound 11 is shown in Fig. 3. As apparent
from Fig. 3, pretreatment with compound 11 produced a
significant effect of dose on nicotine self-administration
(F07.3, df 3,21, p00.0021). Post hoc Student–Newman–
Keuls test of individual doses of compound 11 revealed that
doses of 2.8 and 5 mg/kg were significantly different from
control (vehicle-treated) conditions, with corresponding p
values of 0.0017 and 0.009, respectively. There was a near-
significant effect of 1.6 mg/kg of compound 11 on nicotine
intake (p00.06).
Fig. 3 Effect of compound 11 on IV self-administration of 0.03 mg/kg/
infusions of nicotine under an FR1 schedule of reinforcement. The data
are expressed as mean ( S.E.M.) number of infusions per session (n07).
As indicated by a star, statistical significance from vehicle-treated rats
was p<0.05. There was a near-significant effect of 1.6 mg/kg of com-
pound 11 (#, p00.06)
Effect of compounds 10 and 11 on operant conditioning
food responding
The effect of the two most potent compounds was examined
for effects on food intake using operant conditioning tech-
niques. Table 4 shows the effects of compounds 10 and 11
on food intake during 30-min operant conditioning food
sessions. Results revealed that there was no effect of drug
dose order within the repeated measures of the dose design.
However, studies revealed that only the highest dose of com-
pound 11 (i.e., 5 mg/kg) produced a significant suppression on
food intake (p<0.05) in rats.
Pharmacodynamic profiles of selected test compounds
with CYPs, nicotinic receptors and the dopamine transporter
In the presence of compounds 3–6 and 10, significant inhibi-
tion of human CYP 2A6 was observed (Tables 2 and 3).
Compared to tranylcypromine, compounds 3–6 and 10 were
more potent human CYP 2A6 inhibitors. Compounds 1, 2, 7,
and 11 were less potent inhibitors of human CYP 2A6 com-
pared with tranylcypromine (Tables 2 and 3). The selectivity
of human CYP 2A6 inhibition was examined by studying the
effect of compounds 3, 10, and 11 on the selective functional
activity of human CYP 3A4, 2E1, 2 C9, 2 C19, 2D6, and 2B6
at a concentration of 10 μM (Table 4). As shown in Table 5,
compounds 3, 10, and 11 showed some inhibition of human
CYP 2E1. Compounds 3, 10, and 11 also inhibited human
CYP 2B6 to a certain extent. However, a 16- to 125-fold
greater concentration of compounds 3, 10, and 11 was
required to achieve the same degree of inhibition of human
CYP 2A6. The results indicated that compounds 3, 10, and 11
were relatively selective inhibitors of human CYP 2A6. How-
ever, even though compounds 3, 10, and 11 were selective
inhibitors of human CYP 2A6, the degree of blockade of
nicotine self-administration in rats for these compounds did
not exactly follow suit.
Fig. 2 Effect of compound 10 on IV self-administration of 0.03 mg/kg/
infusions of nicotine under an FR1 schedule of reinforcement. The data
are expressed as mean ( S.E.M.) number of infusions per session (n08).
As indicated by a star, statistical significance from vehicle-treated rats
was p<0.05

644
Psychopharmacology (2012) 221:637–648
Table 2 Inhibition of human CYP2A6 and rat CYP2B1 in the presence of substituted heteroaromatic compounds and physicochemical properties
of compounds 1–11
Compound
hCYP2A6
IC50 (μM)
hCYP2B6
IC50 (μM)
rCYP2B1
rCYP2B1
tPSA
CLogP
% inhibitiona
% inhibitionb
1
0.6
66.4
67.4
12
ND
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
53.8
55.3
35.5
42.7
47.9
46.6
43.3
NA
62.8
59.9
15.6
38.4
12.4
47.6
27.9
36.7
27.9
15.6
15.6
0
2
0.8
−0.2
2.5
1.4
2.6
0.9
0.3
0.7
0.6
2.6
2.1
3
0.16
0.16
0.2
4
52.2
3.9
5
6
0.2
191
ND
103
NA
11
7
>200
800
NA
170
618
8
9
53.5
56.9
67.7
10
11
>20
tPSA topological polar service area, CLogP log of the partition ratio, ND no detectable inhibition, NA not available
a Inhibitor present at 25 μM
b Inhibitor present at 100 μM
We examined the inhibition of rat CYP 2B1 by
compounds 1–7 and 9–11 because CYP 2B1 is the
prominent nicotine metabolizing enzyme in rat liver
and largely responsible for nicotine clearance in rat.
As shown in Table 3, compounds 1–7 and 9–11 were
ineffective at inhibiting rat CYP 2B1. With the possible ex-
ception of compound 11, all the compounds examined pos-
sessed an IC50 value estimated to be greater than 100 μM.
Accordingly, at the doses used in the in vivo experiments,
compounds 1–7 and 9–11 were unlikely to significantly in-
hibit rat CYP 2B1 and decrease nicotine metabolism in the rat
by this mechanism. To examine this point in greater detail, the
influence of selected test compounds on nicotine clearance
was studied in rats.
After pretreatment with compounds 4–6 and 8 (i.p. ad-
ministration 30 min before IV nicotine administration), the
pharmacokinetics of cotinine formation was examined as a
surrogate for nicotine clearance because the clearance of
nicotine was very rapid and quantitation of cotinine was
more reliable. Compounds 4–6 and 8 were studied because
they represented highly potent and non-potent inhibitors of
human CYP 2A6, respectively. LC–MS analysis of cotinine
Table 3 Interaction of N-alkyl-substituted imidazoles with CYP2A6 and nicotinic acetylcholine receptors
aNicotinic
receptors
µM
CYP2A6
Inhibition
IC50, nM
Compd
Structure
Tranylcypromine
233
-
N
NH
8
9
800
NA
N
N
N
NA
ND
N
N
N
10
11
170
618
ND
ND
N
N
NA not available, ND no detectable binding at 1 μM
a Mammalian α-7 and α4ß2 nicotinic receptors

Psychopharmacology (2012) 221:637–648
Table 4 Order and food effects
645
Compound 10
of compounds 10 and 11 on
operant responding for food in
rats
Order effects
Day 1
Day 2
78 1.7
Dose
Day 3
79 3.2
Dose
Day 4
75 3.3a
Doseb
80 1.9
Dose
Food effects
Vehicle
77 3.0
0.5
1.58
5.0
80 1.8
79 2.5
77 3.2
Compound 11
aAverage number of nicotine
self-administrations in
1 h SEM
bDose, mg/kg
cStatistically different than
Order effects
Day 1
Day 2
78 2.3
Dose
Day 3
76 3.7
Dose
Day 4
71 4.8
Dosec
5.0
76 4.1
Dosea
Food effects
Vehicle
79 1.0
1.6
2.8
81 1.2
79 1.3
63 5.6
vehicle-treated animals (p<0.05)
formation from organic extracts of plasma samples taken
after nicotine administration was examined in rats (Table 6).
Compared to vehicle-pretreated animals, rats treated with
test compounds 4–6 and 8 (25 mg/kg, i.p. route of admin-
istration) 30 min before nicotine administration (3 mg/kg,
IV route of administration) did not decrease cotinine AUC or
significantly change the tmax for cotinine formation (Table 6).
The data indicated that the AUC for cotinine formation was
increased possibly due to increased blood flow to the liver or
some other pharmacological mechanism.
To explain the pharmacological mechanism of action,
compounds 3, 10, and 11 were studied for their interaction
with the α4β2- and α7-nicotinic receptors and compounds
1–5, 7, and 9–11 were examined as inhibitors of the hDAT.
Compounds 3, 10, and 11 showed no apparent affinity for
α4β2- and α7-nicotinic receptors (Table 3). In contrast,
nicotine and α-bungarotoxin had KD values of 1.8 nM and
0.3 nM, respectively, for α4β4- and α7-nicotinic receptors.
The results indicated that compounds 3, 10, and 11 were not
agonists or antagonists of the nAChRs examined. As shown in
Table 7, compared with cocaine and mazindol, compounds 1–
5, 7, and 9–11 had no significant effect on inhibition of [125I]-
RTI-55 binding to the hDAT and therefore no detectable
affinity for the hDAT. We conclude that inhibition of dopa-
mine binding to the hDAT was not responsible for the phar-
macological activity of compounds 1–5, 7, and 9–11.
Discussion
1-Benzyl imidazole and 1-methylcyclohexylimidazole in-
hibit bacterial CYP (Verras et al. 2004) and pyridyl imidazoles
inhibit human CYP 2A6 (Denton et al. 2005). We extended
the investigation to 1-alkyl- and 1-benzyl imidazoles as
CYP 2A6 inhibitors and as antagonists of nicotine self-
administration. A survey of potent and inactive human CYP
2A6 inhibitors and in vivo efficacy (Table 1) did not reveal
any distinct SAR features that predicted in vivo efficacy for
the inhibition of nicotine self-administration. However, the
results showed that highly polarizable nucleophilic imidazoles
are associated with potent inhibition of human CYP 2A6
and, in some cases, efficacious decrease in nicotine self-
administration in animals. Based on the results (Tables 1
and 2), we conclude that structural features required for
human CYP 2A6 inhibition could be related to inhibition of
nicotine self-administration, but rat CYP 2B1 is not the main
pharmacological target responsible for inhibition of nicotine
self-administration in rats.
A comparison of Tables 1 and 2 showed no clear rela-
tionship between potency of human CYP 2A6 inhibition
determined in vitro on the basis of kinetic studies of selec-
tive functional substrates and percent decrease in nicotine
self-administration in vivo. This study shows that the use of
human CYP 2A6 inhibition SAR data is a useful guide, but
Table 5 Percent inhibition of CYP3A4, 2E1, 2 C9, 2 C19, or 2D6 by alkyl- or aryl-imidazolesa
Compound
CYP3A4
CYP2E1
CYP2C9
CYP2C19
CYP2D6
Percent inhibition
3
23
27
63
87
89
88
32
44
13
43
24
21
48
34
62
10
11
a 10 μM concentration of alkyl or aryl imidazole used. Values are the mean of triplicate determinations

646
Psychopharmacology (2012) 221:637–648
Table 6 Effect of heteroaromatic aryl compounds on nicotine distri-
Compounds 10 and 11 did not have a food effect, except
possibly at the largest dose of compound 11 (i.e., 5 mg/kg)
(Table 4).
bution in vivoa
Treatmenta
AUCb (ng×h/ml)
tmax (min)
Number of rats
Because compounds 1–11 structurally resemble nicotine,
we investigated possible molecular targets of action. Ac-
cordingly, we examined binding potency of certain com-
pounds to nicotinic receptors as a possible target to explain
the efficacy of inhibition of nicotine self-administration in
vivo. As shown in Table 3, some of the most potent inhib-
itors of nicotine self-administration examined did not have
any detectable direct binding affinity for mammalian α-7
and α4ß2-nAChRs. However, this does not rule out indirect
or allosteric mechanisms. Agents that agonize nAChRs have
shown utility in decreasing smoking. For example, the α4ß2
nAChR partial agonist varenicline blocks nicotine self-
administration with slightly greater efficacy (i.e., ED50 of
2–3 mg/kg; Rollema et al. 2007) than compounds 10 and 11.
Thus, despite the structural relatedness of the test com-
pounds described herein to nicotine, direct binding to the
nAChR apparently does not explain the complex interaction
responsible for the pharmacological efficacy of the test
compounds observed.
The data in Table 7 shows that compounds 1–5, 7, and 9–11
had no detectable affinity for the hDAT. Generally, small
molecules that show no affinity for the hDAT also do not
inhibit dopamine reuptake to the hDAT (Eshleman et al.
1999) and we conclude that the DAT is not the biological target
of compounds 1–5, 7, and 9–11.
It is likely that additional neurotransmitter and/or recep-
tor systems in addition to behavioral and environmental
secondary effectors contribute to the efficacy of the test
compounds. The mechanism by which 3-heteroaromatic
Vehicle
119.1 23.4
411.6 117.6c
287.6 84.2c
357.5 77.9c
180.0 45.8
90
230
93
4
3
4
4
4
4
5
6
8
89
112
a Preadministration of 25 mg/kg of compounds 4–6 and 8 was done by
the i.p. route of administration 30 min before nicotine administration.
Nicotine was administered at 3.0 mg/kg by the IV route of
administration
b Area under the concentration–time profile (AUC) was the mean SD
c Statistically different than vehicle-treated animals (p<0.05)
not an absolute indicator of the utility of a small molecule as
a blocker of nicotine self-administration.
While not as extensively studied herein, compared to
human CYP 2A6, rat CYP 2B1 possesses a distinct inhibitor
binding profile based on the lack of nicotine metabolism
inhibitory potency of compounds 1–7 and 9–11 (Table 2).
Moreover, at the dose of compounds 10 and 11 that are
effective at decreasing nicotine self-administration (i.e., IC50
values of 4 and 2.8 mg/kg, respectively), it is unlikely that any
significant degree of CYP 2B1 inhibition occurs in rats ad-
ministered compounds 10 or 11. Accordingly, inhibition of rat
CYP 2B1-mediated nicotine metabolism cannot explain the
pharmacological or behavioral response of compounds 10 and
11 observed in rats. This point is further reinforced because
pharmacokinetic studies of nicotine metabolism after the pre-
treatment of rats with compounds 4–6 or 8 did not show a
decrease on the AUC for cotinine formation compared to
vehicle administration (Table 6). Thus, relatively potent
CYP 2A6 inhibitors (i.e., compounds 4–6) afforded similar
effects on the PK of cotinine formation as a relatively non-
potent CYP 2A6 inhibitor (i.e., compound 8).
Table 7 Inhibition of radioligand binding in HEK-hDAT cells by
compounds 1–5, 7, and 9–11
Compound
Numbera
Reuptake inhibition
Ki (nM) SEM
CYP 2A6 inhibitors could have two additional effects in
humans in addition to its main mechanism of action: (a)
CYP 2A6 inhibition could decrease nicotine metabolism,
elevate nicotine levels and possibly provide an aversive
effect on nicotine self-administration, and (b) CYP 2A6
inhibition could decrease metabolism of the inhibitor and
increase the efficacy as an inhibitor of nicotine self-
administration. Notwithstanding the CYP inhibitory properties
of compounds 1–11, these compounds possess the appropriate
polar surface area and lipophilicity values to enable good
blood–brain barrier penetration and efficacy (Table 2). Further-
more, the mechanism of substances 1–11 does not involve
irreversible or covalent modification of a biomacromolecule
because there is no apparent carry-over effect in rats. For
compounds 10 and 11, no apparent order effect was apparent.
Cocaine
5
5
3
3
3
3
3
3
3
3
3
450 128b
38 15b
>10,000
>10,000
>9,525
Mazindol
1
2
3
4
>10,000
>10,000
>10,000
>10,000
>10,000
>9,874
5
7
9
10
11
a Number of independent determinations
b Hill slope and standard error of the mean (SEM) for cocaine and
mazindol was −1.02 0.08 and −0.89 0.13, respectively

Psychopharmacology (2012) 221:637–648
647
Feyerabend C, Ings RM, Russel MA (1985) Nicotine pharmacokinetics
and its application to intake from smoking. Br J Clin Pharmacol
19:239–247
Hirayama T (1981) Non-smoking wives of heavy smokers have a
higher risk of lung cancer: a study from Japan. Br Med J (Clin
Res Ed) 282:183–185
Hughes JR (2007) Antidepressants for smoking cessation. Cochrane Da-
tabase Syst Rev 3:CD000146. doi:10.1002/14651858CD000146.
pub2
Hyytia P, Schulteis G, Koob GF (1996) Intravenous heroin and
ethanol self-administration by alcohol-preferring AA and
alcohol-avoiding ANA rats. Psychopharmacol (Berl) 125:248–
254
pyridine or 1-alkyl- or 1-benzyl imidazole analogs of nico-
tine decrease nicotine self-administration remains unknown.
Because blockers of nicotine self-administration described
herein do not apparently have an irreversible effect (i.e.,
24 h after administration of 1–11, no apparent residual effect
was observed), we favor a direct effect on some specific
molecular target. However, substantial further research will
be required to determine this point.
Acknowledgments We thank Dr. Senait Ghirmai for the synthesis of
compound 2 and Dr. Travis Denton for the synthesis of compound 5 and
for the pharamacokinetic studies (Table 6). This work was supported by
funds from HBRI.
Jones J, Raiff BR, Dallery J (2010) Nicotine’s enhancing effects on
responding maintained by conditioned reinforcers are reduced by
pretreatment with mecamylamine, but not hexamethonium, in
rats. Exp Clin Psychophrmacol 18:350–358
Kruse LI, Kaiser C, DeWolf WE, Finkelstein JA, Frazee JS, Hilbert
EL, Ross ST, Flaim KE, Sawyer JL (1990) Some benzyl-
substituted imidazoles, triazoles, tetrazoles, pyridinethiones, and
structural relatives as multisubstrate inhibitors of dopamine beta-
hydroxylase. 4. Structure–activity relationships at the copper
binding site. J Med Chem 33:781–789
Lu P, Alterman MA, Chaurasia CS, Bambal RB, Hanzlik RP (1997)
Heme-coordinating analogs of lauric acid as inhibitors of fatty
acid omega-hydroxylation. Arch Biochem Biophys 337:1–7
MacDougall JM, Fandrick K, Zhang X, Serafin SV, Cashman JR
(2003) Inhibition of human liver microsomal (S)-nicotine oxida-
tion by (−)-menthol and analogues. Chem Res Toxicol 16:988–
993
References
Baggaley KH, Heald M, Hindley RM, Morgan B, Tee JL, Green J
(1975) Hypolipidemic imidazoles. J Med Chem 18:833–836
Balfour DJ (2002) The neurobiology of tobacco dependence: a com-
mentary. Respiration 69:7–11
Benowitz NL (1988) Drug therapy. Pharmacologic aspects of cigarette
smoking and nicotine addiction. N Engl J Med 319:1318–1330
Benowitz NL, Jacob P 3rd (1984) Daily intake of nicotine during
cigarette smoking. Clin Pharmacol Ther 35:499–504
Mathews CD, Loncar D (2006) Projections of global mortality and
burden of disease from 2002 to 2030. PloS Med 11:2011–
2030
Berkman CE, Park SB, Wrighton SA, Cashman JR (1995) In vitro–in
vivo correlations of human (S)-nicotine metabolism. Biochem
Pharmacol 50:565–570
Miksys S, Hoffmann E, Tyndale RF (2000) Regional and cellular
induction of nicotine-metabolizing CYP2B1 in rat brain by chron-
ic nicotine treatment. Biochem Pharmacol 59:1501–1511
Pianezza ML, Sellers EM, Tyndale RF (1998) Nicotine metabolism
defect reduces smoking. Nature 393:750
Rock VJ, Malarcher A, Kahende JW, Asman K, Husten C, Caraballo R
(2007) Cigarette smoking among adults—United States, 2006.
MMWR 56:1157–1161
Cahill K, Stead L, Lancaster T (2009) A preliminary benefit-risk
assessment of varenicline in smoking cessation. Drug Safety 32
(2):119–135. doi:10.2165/00002018-200932020-00005
Cashman JR, Park SB, Yang ZC, Wrighton SA, Jacob P 3rd, Benowitz
NL (1992) Metabolism of nicotine by human liver microsomes:
stereoselective formation of trans-nicotine N’-oxide. Chem Res
Toxicol 5:639–646
Cheng Y, Prusoff WH (1973) Relationship between the Ki value and
Rollema H, Coe JW, Chambers LK, Hurst RS, Stahl SM, Williams KE
(2007) Rationale, pharmacology and clinical efficacy of partial
agonists of α4ß2 nACh receptors for smoking cessation. Trends
Pharmacol Sci 28:316–325
Sellers EM, Kaplan HL, Tyndale RF (2000) Inhibition of cytochrome
P450 2A6 increases nicotine’s oral bioavailability and decreases
smoking. Clin Pharmacol Ther 68:35–43
Sellers EM, Tyndale RF, Fernandes LC (2003) Decreasing smoking
behavior and risk through CYP2A6 inhibition. Drug Discov To-
day 8:487–493
Sharples SC, Kaiser S, Soliakov L, Marks MJ, Collins AC, Washburn
M, Wright E, Spencer JA, Gallagher T, Whiteaker P, Wonnacutt S
(2000) UB-165: a novel nicotinic agonist with subtype selectivity
implicates the alpha4beta2* subtype in the modulation of dopa-
mine release from rat striatal synaptosomes. J Neurosci 20:2783–
2791
Shin VY, Jin HC, Ng EK, Yu J, Leung WK, Cho CH, Sung JJ (2008)
Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in-
duce cyclooxygenase-2 activity in human gastric cancer cells:
involvement of nicotinic acetylcholine receptor (nAChR) and
beta-adrenergic receptor signaling pathways. Toxicol Appl Phar-
macol 233:254–261
Stead LF, Perera R, Bullen C, Mant D, Lancaster T (2008) Nicotine
replacement therapy for smoking cessation. Cochrane Database
Syst Rev 1:CD000146
the concentration of inhibitor which causes 50% inhibition (IC50
of an enzymatic reaction. Biochem Pharmacol 22:3099–3108
)
Coe JW, Brooks PR, Vetelino MG, Wirtz MC, Arnold EP, Huang J,
Sands SB, Davis TI, Lebel LA, Fox CB, Shrikhande A, Heym JH,
Schaeffer E, Rollema H, Lu Y, Mansbach RS, Chambers LK,
Rovetti CC, Schulz DW, Tingley FD 3rd, O’Neill BT (2005)
Varenicline: an alpha4beta2 nicotinic receptor partial agonist for
smoking cessation. J Med Chem 48:3474–3477
Denton TT, Zhang X, Cashman JR (2004) Nicotine-related alkaloids
and metabolites as inhibitors of human cytochrome P-450 2A6.
Biochem Pharmacol 67:751–756
Denton TT, Zhang X, Cashman JR (2005) 5-Substituted, 6-substituted
and unsubstituted 3-heteroaromatic pyridine analogs of nicotine
as selective inhibitors of cytochrome P-450 2A6. J Med Chem
48:224–239
Doll R, Hill AB (1952) A study of the aetiology of carcinoma of the
lung. Br Med J 2(4797):1271–1286
Eshleman AJ, Carmolli M, Cumbay M, Neve KA, Janowsky A (1999)
Characteristics of drug interactions with recombinant biogenic
amine transporters expressed in the same cell type. J Pharmacol
Exper Ther 289:877–885
Feng X, Fandrick K, Johnson R, Janowsky A, Cashman JR (2003)
Synthesis of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in
vivo pharmacological evaluation: identification of inhibitors of the
human dopamine transporter. Bioorg Med Chem 11:775–780

648
Psychopharmacology (2012) 221:637–648
Takeuchi H, Saoo K, Yokohira M, Ikeda M, Maeta H, Miyazaki M,
Yamazaki H, Kamataki T, Imaida K (2003) Pretreatment with 8-
methoxypsoralen, a potent human CYP2A6 inhibitor, strongly
inhibits lung tumorigenesis induced by 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone in female A/J mice. Cancer Res 66:7581–
7583
Taylor AL, Bettcher DW (2000) WHO framework convention on
tobacco control: a global “good” for public health. Bull World
Health Organ 78:920–929
Verras A, Kuntz ID, Ortiz de Montellano PR (2004) Computer-assisted
design of selective imidazole inhibitors for cytochrome P450
Enzymes. J Med Chem 47:3572–3579
West R (2007) The clinical significance of “small” effects of smoking
cessation treatments. Addiction 102:506–509
Yano JK, Denton TT, Cerny MA, Zhang X, Johnson EF, Cashman JR
(2006) Synthetic inhibitors of cytochrome P-450 2A6: inhibitory
activity, difference spectra, mechanism of inhibition and protein
co-crystallization. J Med Chem 49:6987–7001