Supporting Information
The 34* Neuronal Nicotinic Acetylcholine Receptor Plays an
Important Role in Ethanol Consumption and Seeking
Susmita Chatterjee1, Pia Steensland2, Jeffrey A. Simms1, Joan Holgate1, Jotham W. Coe3,
Raymond S. Hurst3, Christopher L. Shaffer3, John Lowe3, Hans Rollema3* and Selena E. Bartlett1*
Supporting Information Methods
Alcohol self-administration and voluntary intake studies
Operant self-administration procedures
Operant self-administration testing was conducted in standard operant conditioning
chambers (Coulbourn Instruments, Allentown, PA) as described previously (Steensland et
al., 2007). In brief, rats were randomly divided into two groups each for CP-601932
(ethanol group: n=13; sucrose group: n=10) and PF-4575180 compound testing (ethanol
group: n=8; sucrose group: n= 14). Rats were trained to self-administer 10% ethanol (v/v)
or 5% sucrose, on a fixed ratio 3 (FR3; three active lever presses required for 0.1 ml
reward) To evaluate the acute effect of CP-601932 on ethanol and sucrose selfadministration, CP-601932 (5 and 10 mg/kg s.c.) or vehicle (saline) were administered 30
minutes before the operant session (mean body weight before treatment: ethanol group:
550 ± 11 g; sucrose group: 596 ± 12 g; 11 weeks; approximately 55 sessions). In a
separate groups of rats, PF-4575180 (1 and 10 mg/kg s.c.) or vehicle (saline) was
administered 2 h before the operant session (mean body weight before treatment: ethanol
group: 580 ± 16g; sucrose group: 534 ± 9 g; 10 weeks; approximately 50 sessions). Each
injection was given 7 days apart in a Latin square design, thus each animal served as its
own control. Between the injection days, the rats were exposed to their normal schedule
1
of reinforcement as described above with no injections for the remaining days of that
week.
Intermittent Access 20% Ethanol Two-Bottle-Choice Drinking Paradigm
The intermittent access 20% ethanol two-bottle-choice drinking paradigm induces high
ethanol consumption in standard laboratory rats and mimics the alcohol consumption
pattern seen among humans (Simms et al., 2008; Wise, 1973). In brief, rats were given
access to one bottle of 20% ethanol and one bottle of water for three 24-hour-sessions per
week (Mondays, Wednesdays and Fridays). The rats had unlimited access to two bottles
of water between the ethanol-access periods. Bottles were weighed at 30 minutes, 6 hours
and 24 hours after the fluids were presented and measurements were taken to the nearest
0.1 g. Water and total fluid intake are not measured at the 30-min time point because of
low baseline consumption of water (1.5 ± 0.1 ml/30 min). CP-601932 (1, 5 and 10 mg/kg
s.c.) or vehicle (saline) was administered to one group of rats (n = 12, 6 ± 1 g/kg/24 h, 11
weeks; approximately 33 drinking sessions). Also, 42* nAChR antagonist DHE (6
and 10 mg/kg s.c.) or vehicle (saline) and the 34 partial agonist PF-4575180 (1 and 10
mg/kg s.c.) or vehicle (saline) in two separate groups of rats (DHE: n = 12 , 5.1 ± 0.4
g/kg/24 h, 12 weeks; ~36 sessions); PF-4575180: n = 10, 6.1 ± 0.3 g/kg/24 h, 9 weeks; ~
27 session). All the treatments were administered 30 minutes before the presentation of
the ethanol bottles. The procedural descriptions are given in the materials and methods
section of the paper.
Intermittent Access 5% Sucrose Two-bottle-choice Drinking Paradigm
2
The intermittent access 5% sucrose two-bottle-choice drinking paradigm was performed
like the intermittent access paradigm for ethanol described above, with the only exception
that the rats were given access to 5% sucrose rather than 20% ethanol. Water and total
fluid intake are not measured at the 30-min time point because of low baseline
consumption of water (1.5 ± 0.1 ml/30 min).The preference for sucrose over water (the
ratio of sucrose to total fluid intake) was calculated. CP-601932 and PF-4575180
administration began once the animals had attained stable drinking levels of sucrose (CP601932 group: 25 ± 2 g/kg/24 h; PF-4575180 group: 27 ± 2 g/kg/24 h) for about 8 weeks;
24 sessions and 11 weeks; 33 sessions), respectively. CP-601932 (1, 5 and 10 mg/kg s.c.)
or vehicle (saline) were administered to one group of rats (n = 10) and PF-4575180 (1
and 10 mg/kg s.c.) or vehicle (saline) were given to a separate group of rats (n = 10), 30
minutes before the presentation of the sucrose bottles. Injection was given on the Monday
and the Friday of the 8th and the 9th week and the 11th and 12th week, respectively of the
first sucrose exposure using a Latin square design. The mean body weights of the rats at
the first CP-601932 and PF-4575180 treatment were 444 ± 16 g and 486 ± 20 g
respectively.
Blood Ethanol Concentration (BEC) measurements
To evaluate whether partial agonists at nAChRs affect blood ethanol clearance,
CP-601932 (10 mg/kg, s.c., n=8) or saline (n=7) was administered to rats, 30 minutes
before an acute ethanol injection (3.6 g/kg i.p). Blood samples were collected from the
lateral tail vein 30, 60, 120 and 240 minutes after ethanol injection. The BECs were
analyzed using the alcohol dehydrogenase assay (Zapata et al., 2006). In brief, 10 µl of
3
blood samples were precipitated in 40 µl of 3.5% perchloric acid on ice and centrifuged
at 4
o
C for 5 minutes at 2,000 rpm. Samples (7 µl) were then added to 343 µl of the
buffer (100 ml 0.5M Tris-HCl buffer (pH 8.8) containing 0.55 mg of alcohol
dehydrogenase and 100 mg -nicotinamide adenine dinucleotide (-NAD+) and
incubated for 40 minutes at room temperature. -NADH accumulation was measured by
reading the absorbance at 340 nm using Spectromax (Molecular Devices, Sunnyvale).
The BEC was estimated using a standard calibration curve and compared at each time
point between CP-601932 and saline pre-treatment.
Chemistry: synthesis of PF-4575180
A.
rac-exo-tert-Butyl-[6-(2-bromo,5-fluorophenoxy)]-3-azabicyclo[3.2.1]octane-3-
carboxylate
To
a
solution
of
2.0
g
(8.8
mmol)
rac-endo-tert-butyl-(6-hydroxy)-3-
azabicyclo[3.2.1]octane-3-carboxylate (prepared as outlined in US 2005020830) in 100
mL dry THF were added 1.85 g (9.68 mmol) 2-bromo-5-fluorophenol, 3.11 g (15.4
mmol) diisopropylazodicarboxylate, and 4.62 g (17.6 mmol) triphenylphosphine. The
reaction was refluxed 24 hours, cooled to room temperature, quenched with 1N aqueous
sodium hydroxide solution, and extracted into 3 X ethyl acetate. The organic phase was
washed with 2 X aqueous 1N NaOH solution, dried over magnesium sulfate, filtered, and
evaporated. The residue was chromatographed on silica gel using heptane and ethyl
acetate as eluants to afford 3.16 g (93 %) of an oil.
C-NMR (CDCl3, ): 28.66, 33.36, 33.84, 34.75, 37.55, 40.03, 47.66, 48.82, 49.96,
13
50.98, 79.96, 82.27, 102.55, 107.17, 108.455 (d, J=23), 133.76 (d, J=10), 155.225 (d,
4
J=9), 156.15, 162.71 (d, J=244) (some line doubling due to hindered rotation). LRMS:
parent+1 peak: 400/402 (Br79/Br81).
B. rac-exo-tert-Butyl-[6-(2-(pyridin-3-yl)-5-fluorophenoxy)]-3-azabicyclo[3.2.1]octane3-carboxylate
To a solution of 1.87 g (4.67 mmol) rac-exo-tert-butyl-[6-(2-bromo, 5-fluorophenoxy)]3-azabicyclo[3.2.1]octane-3-carboxylate in 18 mL ethanol, 2 mL water, and 2 mL toluene
were added 632 mg (5.14 mmol) pyridin-3-yl-boronic acid, 216 mg (0.187 mmol) tetrakis
triphenylphosphine, and 1.49 g (14.0 mmol) sodium carbonate.
The reaction was
refluxed overnight, cooled to room temperature, quenched with 1 N aqueous sodium
hydroxide solution, and extracted into 3 X ethyl acetate. The organic phase was dried
over magnesium sulfate, filtered, and evaporated. The residue was chromatographed on
silica gel using dichloromethane and ethyl acetate as eluants to afford 1.3 g (70 %) of an
oil.
C-NMR (CDCl3, ): 28.67, 33.95, 34.70, 37.69, 40.01, 47.69, 48.83, 49.94, 50.98,
13
80.02, 81.64, 101.82, 107.635 (d, J=21), 123.09, 123.77, 131.76 (d, J=10), 133.82,
136.81, 148.05, 150.44, 155.92, 163.55 (d, J=246) (some lines doubled due to hindered
rotation). LRMS: parent+1 peak: 399.
C.
rac-exo-[6-(2-(Pyridin-3-yl)-5-fluorophenoxy))]-3-azabicyclo[3.2.1]octane
dihydrochloride salt, PF-04575180-01
To a solution of 1.3 g (3.3 mmol)
rac-exo-tert-butyl-[6-(2-(pyridin-3-yl)-5-
fluorophenoxy)]-3-azabicyclo[3.2.1]octane-3-carboxylate in 3 mL ethyl acetate was
5
added 15 mL of ethyl acetate saturated with HCl, the reaction was stirred overnight at
room temperature, and the resulting white precipitate filtered to afford 1.15 g (94 %) of a
white solid, mp > 250C.
C-NMR (CD3OD, ): 32.67, 33.59, 36.28, 39.41, 46.32, 48.71, 80.19, 101.835 (d,
13
J=27), 108.385 (d, J=23), 119.605 (d, J=3), 127.12, 132.41 (d, J=10), 137.71, 139.39,
141.49, 147.14, 155.52 (d, J=10), 164.905 (d, J=249). LRMS: parent+1 peak: 299.
Purity: Single peak at 0.23 minutes by ultra-HPLC, 0.05% TFA 95/5 to 5/95
Water/CH3CN, 100% pure by mass trigger and 100% pure by UV @ 215 Å.
Pharmacodynamic studies
In vitro functional activity at 42nAChRs expressed in HEK cells by FLIPR
Agonist and antagonist activities were determined in HEK293 cell lines stably transfected
with human 42 nAChRs by measuring effects on calcium flux with a Fluorescence
Imaging Plate Reader (FLIPR, Molecular Devices, Silicon Valley, CA), as previously
described (Wishka et al., 2006). In brief, the agonist testing was done by a single addition
of test compound to the medium and compared with the effect of 100 M ACh;
antagonist testing was done by adding compounds before a 30 M ACh agonist
challenge. Each experiment was run in quadruplicate (n = 3-5) and EC50 and IC50 values
were estimated from the concentration-activation and concentration-inhibition curves.
Detailed procedures are further given in the materials and methods section of the paper.
Pharmacokinetic studies
Sprague-Dawley Rat Plasma and Brain Homogenate Nonspecific Binding.
6
Using an equilibrium dialysis apparatus fitted with a Regenerated Cellulose Membrane
(12,000–14,000 Dalton molecular weight cut-off), the unbound fractions (fu) of CP601932 (0.1 and 1.0 M) and PF-4575180 (1.0 M) were determined in Sprague-Dawley
rat plasma and brain homogenate (Kalvass and Maurer, 2002) .For all binding studies,
working solutions (standard curve and quality control) of test compounds were prepared
in methanol, and dialysis membranes were prepared prior to dialysis by soaking for 15
min in each of three respective solutions: H2O (deionized), 30% EtOH in H2O, and
Dulbecco’s phosphate buffered saline (PBS). Plasma and brain were obtained from inhouse sources and kept frozen until use. For both compounds, their stability in each rat
matrix and optimal dialysis time were determined separately prior to dialysis studies. For
plasma studies, frozen plasma was thawed and warmed to 37 °C, and adjusted to pH 7.4
with 0.1% phosphoric acid or 1 N NaOH. Plasma samples (15 L, N=4), spiked with
substrate, were dialyzed against an equal volume of PBS (pH 7.4) for 6 h at 37 °C on an
oscillating platform in a 5% CO2 atmosphere. For brain homogenate studies, frozen brain
was thawed, diluted with PBS (3 mL/g brain) and liquified with a probe-type
homogenizer.
Substrate-fortified brain homogenate samples (150 L, N=3–4) were
dialyzed identically as described for plasma. At the end of the dialysis period, the donor
(plasma or brain homogenate) and receiver (PBS) sides were removed and prepared for
concentration quantification (see Quantitative Analysis section).The unbound fractions
(fu) of CP-601932 (0.1 and 1.0 M) and PF-4575180 (1.0 M) were determined in
Sprague-Dawley rat plasma (N=4/concentration; fu,p) and brain homogenate (N=3–
4/concentration; fu,b) using equilibrium dialysis (76).
7
Sprague-Dawley rat pharmacokinetics studies
Male Sprague-Dawley rats were fasted overnight prior to compound administration, but
had unrestricted access to H2O before and throughout the studies. Individual animal
doses were calculated based on respective pre-treatment body weights and dose volumes
(1 mL/kg for subcutaneous (s.c.) doses). All blood samples were collected into EDTAcontaining tubes and processed immediately to obtain plasma. Control plasma and brains
were harvested from untreated animals that were sacrificed. All samples were stored
frozen at -20 °C until analysis.For CP-601932, single doses of 1 and 10 mg/kg, s.c.
(N=3/dose) were evaluated to afford plasma compound concentration-time curves from 0
to 24 h post-dose. A rat neuropharmacokinetics study with CP-601932 was conducted
previously, and provided an AUC0–24-derived unbound brain compound concentration-tounbound plasma compound concentration ratio (Cb,u:Cp,u) of 0.41; this value was used to
project CP-601932 Cb,u for a total plasma compound concentration (Cp) at any time point
using the following equation: Cb,u = Cp•fu,p•(Cb,u /Cp,u). Plasma concentrations (and
individual pharmacokinetic parameters) increased proportionally with dose from 1 to 10
mg/kg s.c., hence any compound concentrations from doses other than these were
predicted assuming linear pharmacokinetics for CP-601932.For PF-4575180, a single
dose of 1 mg/kg, SC (N=2/time point) was assessed to determine the compound
concentration-time curves for plasma and brain from 0- 24 h post-dose. PF-4575180 Cb,u
at any time point was determined by Cb fu,b. As with CP-601932, plasma and/or brain
concentrations from doses other than 1 mg/kg s.c. were projected assuming linear
pharmacokinetics for PF-4575180.
8
CP-601932 Pharmacokinetics study
A single dose (1 or 10 mg/kg s.c.) of CP-601932 in H2O (1 or 10 mg/mL) was
administered to each jugular vein-cannulated rat (N=3/dose). Serial blood samples (0.5
mL) were collected just prior to dosing and at 0.25, 0.5, 1, 2, 4, 5 and 24 h post-dose for
the pharmacokinetic evaluation of CP-601932.
PF-4575180 Pharmacokinetics study
A single dose (1 mg/kg s.c.) of PF-4575180 in H2O (1 mg/mL) was administered to each
rat. Blood and brain samples were collected at 0.25, 0.5, 1, 2, 6 and 24 h post-dose
following animal (N=2/time point) euthanasia using the same procedures as described for
CP-601932.
Quantitative analysis of CP-601932 and PF-4575180 in plasma and brain.
CP-601932: Plasma and brain concentrations of CP-601932 were quantified using an
acetonitrile-mediated protein precipitation sample preparation and a characterized LCMS/MS assay described elsewhere (Shaffer et al., 2009).
PF-4575180: Plasma and brain concentrations of PF-4575180 were also quantified using
a characterized LC-MS/MS assay. Thawed plasma samples were processed directly,
while thawed brains were weighed, diluted with 60% iPrOH in H2O (4 mL/g brain) and
homogenized with a probe-type homogenizer.
Standard curves were prepared in
respective matrices via serial dilution to concentrations of 0.5 to 1000 ng/mL. Aliquots
(50 L) of plasma or brain homogenate were combined with acetonitrile (300 L;
9
MeCN), vortex-mixed and centrifuged (1,811 rcf for 10 min).
The respective
supernatants (250 L) were transferred to a deep-well 96-well plate, concentrated under
N2 at 40 °C, and the resulting residues were reconstituted in 75% MeCN in H2O (100
L). Samples were analyzed by an LC-MS/MS composed of a Applied Biosystems
Sciex 4000 tandem quadrupole mass spectrometer (Foster City, CA), tertiary Shimadzu
LC20AD pumps (Shimadzu USA, Columbia, MD) and a CTC PAL autosampler (LEAP
Technologies, Carrboro, NC). Analytes within sample aliquots (10 L) were eluted
under gradient conditions, using MeCN and 0.1% acetic acid in 10 mM ammonium
acetate (pH 3.5), on a Synergi Max-RP analytical column (4 , 2.0  30 mm;
Phenomenex, Torrance, CA) over a 2.5-min period. Mass spectral data were collected in
positive ionization mode using multiple-reaction monitoring of the m/z 299.1→190.4
transition for PF-4575180. Instrument potentials and settings were adjusted to afford
optimal data. The assay dynamic range was 0.5 to 1000 ng/mL.
Supporting Information Results
CP-601932 with equal affinity at 34 and 42 nAChRs selectively decreases ethanol
self-administration and consumption
In the operant self- administration paradigm, while CP-601932 (10 mg/kg) significantly
decreased the number of presses on the active lever compared to vehicle (Figure 4A), it
had no overall main effect on the number of inactive lever presses [(F (2, 12 = 0.55, non
significant (n.s.); Table S1 ]. To determine if CP-601932’s effect in decreasing ethanol
self-administration behavior was specific, CP-601932 was given to a separate group of
rats trained to self-administer 5% sucrose .CP-601932 treatment did not have an overall
10
main effect on 5% sucrose self-administration [F (2, 9) = 0.19, n.s.; Figure 4B] and no
effect on the number of presses on the inactive lever was observed [F (2, 9) = 0.74, n.s.;
Table S1].Similarly, there was no overall main effect of CP-601932 on sucrose
consumption in the intermittent-access 5% sucrose paradigm [30 min: F(3, 10) = 1; n.s.;
Fig 3C; 6 h: F(3, 10) = 3; n.s.; Table S2; 24 hr: F(3, 10) = 1; n.s.; Fig 4D] or on the
preference of sucrose over water in the intermittent [30 min: F(3,10) = 0.5; n.s.; 6 hr:
F(3,10) = 0.5; n.s.; 24 hr: F(3,10) = 1; n.s., Table S2]. In addition, CP-601932 treatment
did not have an overall main effect on water consumption at any time point [30 min: F
(3,11) = 2.1; n.s,; 6 h: F(3,11) = 0.6; n.s.; 24 h: F(3,11) = 2.7; n.s.; Table S2].
CP-601932 does not affect blood ethanol concentrations
To examine the possibility that CP-601932 decreases ethanol intake by affecting blood
ethanol concentration (BEC), CP-601932 (10 mg/kg) or vehicle (saline) was given two
groups of ethanol naïve Wistar rats and BEC was measured at different time points. Twoway ANOVA of BECs at 30, 60, 120, and 240 minutes following the ethanol injection
showed a significant effect of time [F(3,52) = 5.1; P<0.001], but no significant effect of
treatment [F(1,52) = 0.001, n.s.] or interaction between treatment x time [F(3,52) = 0.6,
n.s.]. As there was no difference in the BECs between the two groups over time [Table
S3], these results indicate that CP-601932 is reducing ethanol consumption by directly
affecting the reinforcing properties of ethanol
PF-4575180 with high affinity at 34* nAChR and negligible affinity at 42* nAChR
selectively decreases ethanol self-administration and voluntary ethanol consumption
11
PF-4575180 treatment had an overall main effect on operant self-administration of 10%
ethanol [F(2, 7) = 4.6, P<0.05, Fig 5A]. However, PF-4575180 had no overall main effect
on the number of presses on the inactive lever [F(2, 7 = 3.5, n.s.; Table S1]. To evaluate
the specificity of PF-4575180 in decreasing ethanol administration, it was given to
another group of rats self-administering 5% sucrose .PF-4575180 treatment did not have
an overall main effect on 5% sucrose self-administration [F(2, 13) = 0.22, n.s.; Figure
5B] or have any effect on the number of presses on the inactive lever [F(2, 13) = 0.83,
n.s.; Table S1]. Similarly, there was no overall main effect of PF-4575180 on sucrose
consumption [30 min: F(2, 9) = 0.0009; n.s.; Figure 5D; 6 hr: F(2, 9) = 1.6; n.s.; Table
S4; 24 hr: F(2, 9) = 0.27; n.s.; Table S4 ] or the preference of sucrose over water [30 min:
F (2,9) = 0.06; n.s.; 6 hr: F(2,9) = 0.9; n.s.; 24 hr: F(2,9) = 2.6; n.s., Table S4] using the
5% sucrose intermittent access paradigm.
Dihydro--erythroidine (DHE) an 42*nAChR antagonist does not decrease heavy
ethanol consumption
Dihydro--erythroidine (DHE), previously shown (Le et al., 2000) to be have no effect
in other ethanol intake models, was re-examined in long-term drinking rats, using the
intermittent-access two bottle choice paradigm model. Vehicle or DHE (6 and 10 mg/kg
s.c.) was administered in a separate group of Wistar rats (n = 12) when the rats had
maintained a stable baseline for 12 weeks (~36 sessions). Vehicle (saline) or DHE (6
and 10 mg/kg s.c.) was administered when the rats had maintained stable drinking
baseline. The DHE treatment had no overall main effect on ethanol consumption [30
12
min: F(2, 11) = 1.2, n.s.; Table S5; 6 hr: F(2, 11) = 0.51, n.s.; Fig S2A ; 24 hr: F(2, 11) =
0.69, n.s.; Fig S2B], on preference of ethanol over water [30 min: F(2, 11) = 0.38, n.s.; 6
hr: F(2, 11) = 0.49 , n.s.; 24 hr: F(2, 11) = 0.58, n.s.; Table S5], or on water consumption
at any time point [30 min: F(2, 11) = 0.68, n.s.; 6 hr: F(2, 11) = 0.24, n.s.; 24 hr: F(2, 11)
= 0.03, n.s.; Table S5].
Supporting Information Figure Legends
Figure S1: Concentration dependent activation and inhibition curves of 42 nAChRs
expressed in HEK293 cells measured by FLIPR methodology. Activation data (filled
circles) are expressed as fraction of the response evoked by 100 M ACh. Inhibition data
(open squares) were generated by applying 30 M ACh in the presence of varying
concentrations of the test compound, and the data are normalized to the response evoked
by 30 M ACh in the absence of test compound. The activation and inhibition curves are
the curves of best fit through the data points. Vertical gray bars correspond to the
estimated unbound rat brain concentrations (in nM) measured at 30 min after 5 mg/kg
and 10 mg/kg of CP-601932 (A), 1 mg/kg and 10 mg/kg of PF-4575180 (B) and 1 mg/kg
and 2 mg/kg of varenicline (C).
Figure S2: DHE, an 42* antagonist did not decrease ethanol consumption in rats
using the intermittent access 20% ethanol two bottle choice drinking paradigm. DHE (6
and 10 mg/kg) did not affect ethanol consumption at 6 (A) and 24 hrs (B) after the onset
of drinking. The values are expressed as mean ethanol intake (g/kg) ± SEM. (repeated
measures ANOVA followed by Newman-Keuls post-hoc test, n.s.), n = 12
13
Supporting Information Table Legends
Table S1: CP-601932 and PF-4575180 treatment had no effect on inactive lever presses
in rats self-administrating ethanol or sucrose.
Table S2: CP-601932 treatment decreased ethanol consumption without any effect on
water and sucrose consumption using the intermittent–access two bottle choice drinking
paradigm
Table S3: CP-601932 treatment has no effect on Blood Ethanol Concentrations
Table S4: PF-4575180 treatment decreased ethanol consumption without any effect on
sucrose consumption, but increased water consumption using the intermittent–access two
bottle choice drinking paradigm
Table S5: DHE treatment had no effect on ethanol consumption in rats using the
intermittent–access two bottle choice drinking paradigm
Supporting Information References
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14
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15