Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer, [3H]AZ11637326: Target localization, biodistribution and ligand occupancy in the rat brain

Article abstract of DOI:10.1016/j.neuropharm.2011.03.024

The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation of the biological actions of small molecul

Full text of DOI:10.1016/j.neuropharm.2011.03.024

Neuropharmacology 61 (2011) 161e171
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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer,
[3H]AZ11637326: Target localization, biodistribution and ligand
occupancy in the rat brain
,
a
a
a
a
a
Donna L. Maier a , Geraldine Hill , Min Ding , David Tuke , Emily Einstein , David Gurley ,
John C. Gordon d, Mary J. Bock b, Jeff S. Smith a, Russell Bialecki a, Mark Eisman b,
Charles S. Elmore c, Jennifer L. Werkheiser a
*
a Department of Neuroscience, CNS Discovery, AstraZeneca Pharmaceuticals, Wilmington, DE 19850, USA
b Department of DMPK, CNS Discovery, AstraZeneca Pharmaceuticals, Wilmington, DE 19850, USA
c Department of Chemistry, CNS Discovery, AstraZeneca Pharmaceuticals, Wilmington, DE 19850, USA
d Department of Lead Generation, CNS Discovery, AstraZeneca Pharmaceuticals, Wilmington, DE 19850, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive
disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation
of the biological actions of small molecules at a target receptor. We discovered a high affinity nicotinic
alpha-7 subtype-selective ligand, AZ11637326, with physicalechemical and pharmacokinetic properties
suitable for an in vivo radioligand tool. [3H]AZ11637326 synthesis by tritiodehalogenation of the cor-
responding tribromide precursor yielded a high specific activity radiotracer with high affinity alpha-7
receptor binding in the rat hippocampus determined by autoradiography (Kd ¼ 0.2 nM). When [3H]
AZ11637326 was administered to rats by intravenous bolus, rapid uptake was measured in the brain
followed by a 3e4 fold greater specific binding in regions containing the alpha-7 receptor (frontal cortex,
hippocampus, hypothalamus and midbrain) when compared to non-target regions (striatum and cere-
bellum). Systemic administration of the high affinity alpha-7 receptor antagonist, methyllycaconitine
(MLA), or pretreatment with alpha-7 selective agonists (AR-R17779, PyrQTC, DBCO-4-POM, and DBCO-3-
POM) significantly blocked the alpha-7 specific binding of [3H]AZ11637326 in the rat brain. The rank
order of ligand ED50 values for in vivo alpha-7 receptor occupancy in rat hippocampus was: DBCO-4-
POM > DBCO-3-POM w MLA > PyrQTC > AR-R17779. The occupancy affinity shift was consistent with in
vitro binding affinity in autoradiography. Our studies established the optimal conditions for [3H]
AZ11637326 in vivo specific binding in the rat brain and support the use of [3H]AZ11637326 as a pre-
clinical tool for assessment of novel alpha-7 compounds in drug discovery.
Received 9 September 2010
Received in revised form
3 March 2011
Accepted 30 March 2011
Keywords:
[3H]AZ11637326
AR-R17779
Alpha-7 neuronal nicotinic receptor
In vivo receptor occupancy
Autoradiography
Rat brain
Ligand binding
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
synaptic and perisynaptic localization on neurons primarily in the
hippocampus, neocortex and limbic regions. Presynaptic alpha-7
The alpha-7 receptor is an abundant neuronal nicotinic receptor
(NNR) in the mammalian brain. The alpha-7 NNR subtype is
a ligand gated ion channel distinguished from other pentameric
NNRs by subunit composition, functional activity, pharmacological
ligands, and its low affinity for acetylcholine and high affinity for
receptors play a key role in neurotransmitter release, while post-
synaptic receptors facilitate depolarization through cation entry
and perisynaptic receptors modulate glutamergic and GABA-ergic
neurotransmission directly and dopaminergic activity indirectly.
(Albuquerque et al., 2009; Gotti et al., 2009).
a
-Bungarotoxin (
a
-Bgtx). Distributed in mesocorticolimbic and
Alpha-7 receptor agonists have generated interest as promising
treatments for cognitive disorders in schizophrenia (Buccafusco and
Terry, 2009; Buchanan et al., 2007; Olincy et al., 2006), inflammation
(Conejero-Goldberga et al., 2008; de Jonge and Ulloa, 2007; Wang
et al., 2003) and Alzheimer’s Disease (Carson et al., 2008; Kem,
2000; Parri and Dineley, 2010). Drugs targeting alpha-7 receptors,
such as AR-R17779 (Levin, 2002) and AZD0328 (Sydserff et al., 2009;
mesostriatal circuitry, the alpha-7 NNR has presynaptic, post-
* Corresponding author. Present address: BrainCells Inc., 3565 General Atomics
Court, Suite 200, San Diego, CA 92121, USA. Tel.: þ1 858 812 7666.
E-mail address: dmaier5285@aol.com (D.L. Maier).
0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2011.03.024

162
D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
Castner et al., 2011) have shown enhancement of cognition and
attention in animal models (reviewed by Leiser et al., 2009) and in
humans (Olincy et al., 2006). Improvements seen in animal models
have clear implications for the therapeutic potential in humans
(Martin et al., 2004; Olincy and Stevens, 2007; Thomsen et al., 2010),
providing scientists with both models and NNR tools with rich
translational value.
In CNS drug discovery, selective radiotracer tools help us to
interpret the biological actions of small molecules at a target
receptor. Generally, radiotracers suitable for in vivo binding require
high affinity and selectivity at the target receptor. Moderate values
for radiotracer lipophilicity have been shown to improve brain
penetration and reduce non-specific binding. Radiotracers target-
ing targets in the CNS require low efflux ratio and are not substrates
for efflux pumps (Passchier et al., 2002). While NNR radiotracers for
development of novel therapeutics. AZ11637326, a spirofuropyr-
idine, is a NNR alpha-7 subtype-selective compound shown to
compete with
[ a
125I] -BTX at sub-nanomolar affinity. [3H]
AZ11637326 high affinity binding in rodent hippocampal
membranes (Kd ¼ 0.40 nM) and in HEK-cells transfected with the
human alpha-7 receptor (Kd ¼ 0.18 nM) is blocked competitively by
compounds with high selectivity for the alpha-7 receptor (Gordon
et al., 2010). Here, we add to these and other previous findings
(Ding et al., 2007; Elmore et al., 2008) to describe the in vitro auto-
radiography, the in vivo biodistribution and the pharmacological
properties of [3H]AZ11637326 to further demonstrate the usefulness
of this pre-clinical radiotracer for comparing novel compounds using
alpha-7 receptor occupancy.
2. Methods
the a4b2 receptor are available for in vivo binding and PET imaging
2.1. Subjects
(Ding et al., 2004; Ogawa et al., 2009), fewer high affinity radio-
tracers selective for the alpha-7 receptor have been successfully
developed. Radioligands that bind to the alpha-7 receptor with
high affinity, such as the potent and selective antagonist [3H]MLA
(Davies et al., 1999; Whiteaker et al., 1999) or the large polypeptide
Male Sprague Dawley adult rats (150e170 g) were purchased from Charles
River, USA. Rats were group housed in the vivarium on a 50:50 light:dark cycle and
received ad libitum food and water. All procedures were approved by the Institu-
tional Animal Care and Use Committee in accordance with The Guide for the Care
and Use of Laboratory Animals. All procedures were conducted in a manner to
minimize animal suffering, to reduce the number of animals used and to utilize
alternative techniques where available. The rats used for in vitro binding autora-
diography and slice electrophysiology were not cannulated prior to euthanasia.
Rats used for in vivo binding were anesthetized with isoflurane and a 2e3 French
catheter was inserted into the jugular vein and terminated at the right atrium. The
exterior end of the cannula was fed subcutaneously, anchored to the skin dorsal to
the shoulder blades and flushed with 0.9% saline with 100 units/mL heparin to test
patency. The jugular cannula was inserted one-two days prior to intravenous
injection of the radioligand.
[
125I]
a-BTX have been useful for in vitro binding in rat (Clarke et al.,
1985), monkey (Han et al., 2003) and human brain tissue (Breese
et al., 1997), yet lack the physical and chemical properties to
demonstrate in vivo occupancy. The in vitro binding profile and
biodistribution of [125I]iodo-MLA in the rat brain (Navarro et al.,
2000, 2002) suggests it may provide a selective tool for alpha-7
receptor imaging, while recent autoradiography data showing
high density [125I]iodo-MLA localization in the monkey thalamus
(Kulak et al., 2006) challenges the cross-species translational value
of this tool. One promising radiotracer is [3H]A-58553 (Anderson
et al., 2008) which shows high affinity and low non-specific
binding in rat and in human brain tissue using in vitro binding
techniques.
2.2. Materials
Methyllycaconitine citrate (MLA) was purchased from Tocris Bioscience (Ellis-
ville, MO). [3H]MLA was purchased from Tocris Bioscience (Bristol, UK). AZ11637326
(Dorff et al., 2005), PyrQTC (Phillips and Schmiesing, 2001), DBCO-3-POM (Ernst
et al., 2005), DBCO-4-POM (Ernst et al., 2005), and AR-R17779 (Balestra et al.,
1996; Mullen et al., 2000) were synthesized at AstraZeneca (Wilmington, DE).
Several novel in vivo imaging agents have been developed for the
alpha-7 receptor subtype NNRs. For example, [11C](R)-MeQAA,
a moderate affinity (41 nM) alpha-7 ligand, showed high brain
uptake that was reduced by pretreatment with MLA in bio-
distribution studies in mice, yet PET imaging studies in the monkey
showed only modest selectivity for the alpha-7 receptor (Ogawa
et al., 2009, 2010). The radiotracer [125I]I-TSA, showed high
affinity (0.54 nM) binding at alpha-7 receptors in the brain,
however, it showed high non-specific binding in bioavailability
studies in the mouse (Ogawa et al., 2006). Other groups have
examined bioactive NNR compounds such as analogs of SSR108711
(Hashimoto et al., 2008; Toyohara et al., 2009; Tanibuchi et al., 2010)
and quinuclidine derivatives (Pomper et al., 2005) with moderate to
high affinity for the alpha-7 receptor in order to develop a tool for
PET or SPECT imaging. 11C-Chiba-1001 (11C-Methyl-SSR 180711)
uptake and pharmacological selectivity studies conducted in the
conscious monkey concluded that this was a alpha-7 selective
radiotracer (Hashimoto et al., 2008). Other findings suggest that
[3H]Chiba-1001 has low affinity for the rat and human alpha-7
receptor in vitro (Tanibuchi et al., 2010) and poor in vivo regional
and pharmacological selectivity in the rodent brain (personal
communication, Min Ding), presenting challenges for using this tool
for translational studies. Specific quinuclidine radiotracers provided
an acceptable radiotracer yield, yet, in rodent biodistribution
studies these compounds showed only modest selectivity in the
hippocampus, the target region for alpha-7 binding (Pomper et al.,
2005).
2.3. Preparation of [3H]AZ11637326
[3H]AZ11637326 (Fig. 1) was synthesized at the radiochemistry laboratory at
AstraZeneca (Wilmington, DE). A slurry of 1 mg (1.83
of 5% Pd/C and 50 L of NEt3 in 0.50 mL of EtOH was attached to a tritec manifold and
degassed twice and the reaction vessel was charged with 38 mBar (666 mCi,
11
mol) of 3H2 and stirred for 1 h. The unreacted 3H2 was recovered and the
m
mol) Tribromoarene #, 1.5 mg
m
m
volatiles transferred from the reaction mixture to a waste flask. The residue was
taken up in 3 mCi of EtOH to give 37 mCi (90% radiochemical purity) and the sample
was purified by RP-semi-preparative HPLC (10e60% MeCNe0.1% TFA over 20 min,
Phenomenex Luna C-18(2), 3 mL/min) to give 18.6 mCi (69 Ci/mmol, 99.6 radio-
chemical purity).
2.4. In vitro autoradiography
Saturation and in vitro competition binding using autoradiography was deter-
mined for [3H]AZ11637326 and compared to [3H]MLA. Untreated rats (N ¼ 3 per
treatment condition) were euthanized by decapitation. The whole brain was
removed, rapidly frozen and stored at ꢀ70 ꢁC prior to cryosectioning. Coronal
sections (20 microns thick) were thaw mounted onto microscopy subbed-plus slides
and stored at ꢀ70 ꢁC. On the day of the experiment, slide boxes were removed from
the freezer and allowed to reach room temperature prior to opening. Autoradiog-
raphy was performed as reported by Mugnaini et al. (2002), Davies et al. (1999) and
Whiteaker et al. (1999), with slight modifications. For all experiments, slides were
O
N
O
N
F
F
3H2, Pd/C,
N
N
Br
3H
3H
NEt3, EtOH
In light of these findings, a radiotracer selective for the alpha-7
receptor with high affinity for the receptor, and that shows brain
uptake and target receptor localization after systemic administration
would provide an essential in vivo translational tool for the
Br
Br
3H
1
Fig. 1. [3H]AZ11637326 synthesis.

D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
163
preincubated in Krebs’ ringers’ HEPES (KRH) buffer containing 1 M HEPES, 1 M NaCl,
1 M KCl, 0.5 M CaCl2, and 1 M MgSO4, pH adjusted to 7.5 with 1 M NaOH (30 min,
ꢁC). Then, incubated in KRH buffer containing [3H]AZ11637326 (90 min at 4 ꢁC) or
2.7. In vivo receptor occupancy of novel alpha-7 ligands
4
Selective alpha-7 ligands PyrQTC (0.3, 1, 3, 10 mg/kg, s.c.), DBCO-3-POM (0.001,
0.01, 0.1, 0.3, 3, 10, 17, 25 mg/kg, s.c.), DBCO-4-POM (0.001, 0.01, 0.1, 0.3, 3, 10, 17,
25 mg/kg, s.c.) or vehicle treatment was administered 30 min prior to [3H]
AZ11637326 (0.15 nmol/kg, i.v.). MLA (3.0 mg/kg, i.v.) was administered 15 min prior
to [3H]AZ11637326. MLA served as the positive control for these studies. Rats
(N ¼ 6e12 per treatment group) were euthanized 90 min after radioligand injection
and brain regions were dissected and counted. Brain regions included the hippo-
campus (HPC) and cerebellum (CRB). [3H]AZ11637326 binding was determined for
all pretreatment groups and represented as fmol/mg tissue. In vivo receptor occu-
pancy (% occupancy) for unlabeled pretreatment compounds was indicated by
a reduction in specific binding of [3H]AZ11637326 and compared to vehicle controls.
Whole brain and trunk blood samples were collected for determination of
compound concentration in satellite rats following pretreatment for each
compound. For PyrQTC (3.0 or 10 mg/kg sc), DBCO-3-POM or DBCO-4-POM (0.01, 0.1,
0.3, 1, 3, 10, 17, 25 mg/kg) whole brain and trunk blood samples were collected at the
time of radiotracer dosing (30 min). For PyrQTC, samples were also collected at study
termination (120 min) following drug treatment. Methods were according to Bock
et al. (2007), with the following slight modifications to the ion monitored and
gradient conditions. Brain samples were weighed and homogenized with 4 volumes
Saline (w/v). Samples and matrix standards were prepared by protein precipitation
with four volumes of MeCNe0.1% TFA. The supernatant extracts were dried down
under nitrogen and re-solubilized in H2Oe0.1% TFA. Extracts were chromatographed
on a Zorbax SB-Phenyl analytical reverse phase column using gradient elution.
Quantification was performed on an Agilent MSD (LC/MS) instrument (ESI source e
positive mode) set up such that the standard concentrations bracketed the calcu-
lated concentrations of the samples.
[3H]MLA (120 min at 4 ꢁC) alone or with competitor compound for competition
binding studies. The reaction was stopped by removing slides from the radioactive
bath, draining off the liquid and washing (5 s at 4 ꢁC) in 2ꢂ KRH buffer and in 2ꢂ
20 mM HEPES (pH 7.5) followed by a rapid dip in deionized water. All slides were
dried under the hood using a cool air fan and stored in a desiccated slide box prior to
imaging. Surface radioactivity was measured in brain images generated using
a
b-imager (Biospace, France).
Optimization experiments of various types were performed to increase the
robustness of data comparisons. First, saturation binding of [3H]AZ11637326
(0.03e1 nM) or [3H]MLA (0.03e3 nM) was determined by bath incubation alone or
in competition with 10 mM MLA. The ventral hippocampus was used to determine
the radioligand affinity (Kd) for both [3H]AZ11637326 and [3H]MLA. Second, incu-
bation time (30, 60, 90, or 120 min) was tested for each radiotracer and the optimal
time was chosen for the competition binding experiments. Third, optimal protocol
conditions were used for the competition binding assays for AR-R17779, PyrQTC and
MLA (10
R17779 (12 concentrations from 0.03 nM to 100.0
tions from 0.1 nM to 1.0
M) were tested to determine the inhibition (IC50) of [3H]
m
M) to demonstrate inhibition of [3H]MLA (2.0 nM) binding. Finally, AR-
m
M) or PyrQTC (8 concentra-
m
AZ11637326 (0.15 nM) in vitro binding.
2.5. Ex vivo autoradiography
Brain penetration and regional localization of [3H]AZ11637326 in vivo binding
was demonstrated using autoradiography. [3H]AZ11637326 (0.15 nmol/kg) was
administered by intravenous (i.v.) bolus and rats were euthanized by decapitation
90 min following injection. The whole brain was removed, rapidly frozen and stored
at ꢀ70 ꢁC prior to cryosectioning. Coronal sections (20
m thick) were thaw mounted
onto microscope plus slides, dried on a warming plate for 1e2 h and stored in
2.8. Slice electrophysiology
Standard techniques were used to prepare prefrontal cortex slices from juvenile
rats and to record spontaneous inhibitory post-synaptic currents (sIPSCs) as in
(Smith et al., 2004; Christian et al., 2005; Biton et al., 2007). sIPSCs were measured in
whole cell patch clamp mode at 0 mV holding potential with an Axon 200B patch
clamp and recorded with PClamp ver. 9 software (MDS-Axon). Analysis of sIPSCs and
statistics were performed using MiniAnalysis ver. 6 software from Synaptosoft Inc.
(Chapel Hill, NC).
a desiccated slide box prior to imaging. Surface radioactivity was measured in brain
images generated using a b-imager (Biospace, France).
2.6. Biodistribution and selectivity using in vivo binding
On the day of the experiment, cannulated rats were brought to the laboratory,
weighed, and individually housed in clean clear plastic cages. To establish regional
uptake and dose, [3H]AZ11637326 (0.05, 0.10, 0.15 or 0.20 nmol/kg) was adminis-
tered to freely moving rats by jugular cannula as an intravenous (i.v.) bolus followed
by 0.5e1.0 cc sterile saline (0.9%). Dosing solutions were prepared from [3H]
AZ11637326 stock (specific activity ¼ 69 Ci/mmol) diluted with 0.9% saline. Each rat
was administered the radiotracer at a volume of 1 ml/kg body weight followed by
saline flush of the cannula and euthanized 15 min following injection. To establish
the time course for in vivo binding, [3H]AZ11637326 (0.15 nmol/kg) was adminis-
tered as an i.v. bolus and rats were euthanized 0.5, 1, 5, 15, 30, 60, 90, 120 or 240 min
following radioligand injection.
For all studies, the whole brain was rapidly removed and placed on a chilled
plate for free-hand dissection into brain regions. Brain regions included the frontal
cortex (FC), hippocampus (HPC), striatum (STR), hypothalamus (HT), midbrain
including the colliculi and substantia nigra (MID) and cerebellum (CRB). The bio-
distribution vs. time was also determined for peripheral organs: heart, liver, kidney
and testis. Each tissue region was frozen on dry ice-chilled foil, weighed and
transferred to scintillation vials containing tissue solubilizer (Soluene 350, Perkin
Elmer). Tissue was solubilized overnight, Ultima-Gold scintillation fluid (Perki-
nElmer) was added and total radioactivity was measured by scintillation count (Tri-
Carb, Perkin Elmer). Trunk blood samples were processed for scintillation count of
radioactivity in the plasma. An aliquot of serum was added to the scintillation fluid
and counted. To obtain data for calculating the injected dose, an aliquot of the
radioligand dosing solution was counted (2 min count protocol). Assay conditions
for [3H]AZ11637326 in vivo receptor occupancy was established based on the dose
response and time course experiments.
2.9. Data analysis and statistics
2.9.1. In vitro autoradiography
Radioactivity was measured and brain images were generated from well
desiccated sections on microscope slides using a b-imager (Biospace, France). The
Biospace Measures BetaVisionPlus acquisition and analysis program (Biospace,
France) was used analyze images. Surface radioactivity (cpm/mm2) in the prefrontal
cortex (PFC), striatum (STR), hippocampus (HPC), hypothalamus (HT) and cere-
bellum (CRB) was measured by outlining the anatomical region of interest. The data
for tissue in each treatment group from three rats is represented as the mean ꢃ SEM
surface radioactivity. IC50 values were determined for concentration response curves
using Prism GraphPad (GraphPad, San Diego CA).
2.9.2. Data analysis for in vivo receptor occupancy
Radioactivity count (cpm), counter efficiency and brain region weight were used
to calculate fmol/mg tissue. Specific binding was calculated from total binding for
each target region by subtracting the cerebellum (the non-specific binding region).
Between treatment group comparisons were analyzed using a one-way analysis of
variance (ANOVA) with Tukey post-hoc test (SigmaStat). Percent receptor occupancy
of the unlabeled compounds was calculated from specific binding values for each
brain region and occupancy of the vehicle was set at zero. Occupancy curves were fit
and non-linear regression analysis was used to calculate the ED50 values using Prism
GraphPad (GraphPad, San Diego CA).
Pharmacological selectivity of [3H]AZ11637326 was determined by pretreat-
ment with MLA, AR-R17779 and ondansetron. MLA (0.1, 0.3, 1, 3 mg/kg, i.v.) or AR-
R17779 (1, 3, 10, 30 mg/kg, i.v.) was administered 15 min prior to [3H]AZ11637326.
Ondansetron (1 mg/kg, sc) and vehicle treatment (saline; volume 1 cc/kg, s.c.) were
administered 30 min prior to [3H]AZ11637326. All animals (N ¼ 6 per treatment
group) were euthanized 90 min after the [3H]AZ11637326 (0.15 nmol/kg, i.v.)
injection. The brain regions included the frontal cortex (FC), hippocampus (HPC),
striatum (STR), hypothalamus (HT), midbrain including the colliculi and substantia
nigra (MID) and cerebellum (CRB) were dissected and counted. Inhibition of [3H]
AZ11637326 binding by pretreatment was demonstrated by the change in specific
radioactivity in each brain region and represented as fmol/mg tissue. Whole brain
and trunk blood samples were collected for determination of compound concen-
tration in satellite rats following AR-R17779 pretreatment. For AR-R17779 (10 or
30 mg/kg, iv), whole brain and trunk blood samples were collected at the time of
radiotracer dosing (15 min) and at study termination (105 min) following drug
treatment.
3. Results
3.1. Properties of AZ11637326
AZ11637326 has a molecular weight of 310.37. The LogD of 2.1 at
pH 7.4 is a lipophilicity value that suggests low potential for non-
specific binding. The ClogP of 3.3, clearance measured in human
microsomes (hClint ¼ 7
ml/min/mg) and Efflux ratio of 0.9
(permeability of 46.4), indicate adequate brain penetration. The
functional potency of AZ11637326 at the human (hu) alpha-7
receptor was determined to be 0.2
physiology, see Fig. 13. For chemical structures see Figs. 1 and 2. For
mM in Xenopus oocyte electro-

164
D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
incubation time was determined to be 120 min and specific to non-
specific binding ratio was 9 fold at the 2.0 nM concentration.
In vitro competition binding for AR-R17779, PyrQTC and MLA
indicated that all alpha-7 ligands inhibited [3H]MLA in vitro
binding in alpha-7 receptor regions (Fig. 4). In vitro competition
response data for AR-R17779 and PyrQTC was tested using [3H]
AZ11637326 (Fig. 5). Both compounds inhibited binding in specific
regions compared to non-specific regions (data shown for PFC,
ventral HPC and STR). There was a 10 fold concentration shift in the
inhibition curve for AR-R17779 to the right compared to PyrQTC in
the HPC. The IC50 values for inhibition in the HPC:PFC for AR-
R17779 are 537.5 nM: 351.3 nM compared to the IC50 values in
the HPC:PFC for PyrQTC (44.15 nM:141.9 nM).
3.3. Ex vivo binding autoradiography
Fig. 2. Chemical structures for AR-R17779, PYRQTC, DBCO-3-POM, DBCO-4-POM.
[3H]AZ11637326 in vivo accumulation and regional distribution
in the rat brain was determined using autoradiography (Fig. 6). The
data is represented as cpm/mm2. The localization pattern of [3H]
AZ11637326 ex vivo total binding is consistent with the uptake
measured by scintillation count (fmol/mg tissue) and with our in
vitro autoradiography data. Regions of highest uptake include the
HPC and HT followed by moderate uptake in the CTX, then lowest
uptake in the STR and CRB.
pharmacology and DMPK results for AZ11637326, AR-R17779,
PyrQTC, DBCO-3-POM, DBCO-4-POM see Table 1.
3.2. In vitro binding autoradiography
Saturation binding assay showed the typical localization profile
for the alpha-7 receptor in the rat brain. [3H]MLA and [3H]
A11637326 showed high receptor density in the hippocampus
(HPC), frontal cortex (FC) and hypothalamus (HT), moderate
receptor density in the cerebral cortex (CTX, containing parietal,
temporal and occupital cortex) and midbrain (MID, including the
colliculi and substantia nigra) and low receptor density in the
striatum (STR) and cerebellum (CRB). The radioligand affinity (Kd),
determined in the ventral hippocampus (Fig. 3), was sub-
nanomolar for both [3H]AZ11637326 (Kd ¼ 0.2 nM) and [3H]MLA
(Kd ¼ 0.5 nM). Incubation time was tested at four time points (30,
60, 90, or 120 min) for each radiotracer to determine the in vitro
binding kinetics. The kinetic analysis for [3H]AZ11637326 indicated
that incubation for 90 min produced a specific to non-specific
binding ratio of 3 fold at 0.15 nM. For [3H]MLA, the optimal
3.4. In vivo biodistribution and selectivity of [3H]AZ11637326
[3H]AZ11637326 accumulated in the rat brain following intra-
venous administration. Regional uptake of [3H]AZ11637326 was
consistent with the regional distribution of alpha-7 receptors seen
in our in vitro and ex vivo studies. Uptake in the hippocampus,
hypothalamus, midbrain, cerebral cortex and prefrontal cortex
(brain regions with alpha-7 receptors) was significantly higher than
in the striatum and cerebellum (brain regions with low alpha-7
receptor density).
The accumulation of [3H]AZ11637326 in selected brain regions
was determined 15 min following the intravenous administration
Table 1
Pharmacology of alpha-7 ligands.
AZ11637326
PyrQTC
DBCO-3-POM
DBCO-4-POM
AR-R17779
Patent
WO 2005/030778
0.109
16.2, >100 fold
WO 2001/060821 WO 2005/061510
WO 2005/061510
3.03
WO 1996/006098
40.1
8300 nM, >200 fold
Binding Affinitya (Ki at hu alpha-7 receptor, nM)
Bindinga alpha-7 vs. 5-HT3 (nM and fold difference)
0.381
4.5
>17,000 nM,
>10,000 fold
58.97, >10,000 fold >17,000, 5610 fold
Bindinga alpha-7 vs.
Functional potencyb
a
4
b
2 (nM and fold difference)
3730, >10,000 fold NV
31.92, 7093 fold
12,000, 3960 fold
5.4
>60300, 1503 fold
0.2
mM
0.7
m
M
4.4
m
M
mM
11.0
mM
(hu alpha-7 receptor, Xenopus oocyte)
Functional potencyc (rat PFC slice electrophysiology) 10 nM
NV
10
m
M
>30
m
M
50
m
M
Molecular weight
Solubility pH 7.4 (
310.40
>5000
7.154
313.42
>5000
NV
4.4
24 (likely)
84 ꢃ 6, 3 mg/kg,
sc, 1 h for
this cmpd, all
parameters
559 ꢃ 97
0.154 ꢃ 0.033
36 ꢃ 4
284.32
>500
<6.2
10.17
0.66 (unlikely)
739 ꢃ 131, 3.3
mg/kg, s.c.
1 hr for this cmpd
all parameters
622 ꢃ 170
1.3 ꢃ 0.4
NV
284.32
>500
41.67
21.1
1.8 (unlikely)
182.22
1500
<12
NV
m
M)
hCLint (
m
l/min/ml)d
Permeability (A:B)e
7
Effluxe
0.9 (unlikely)
2080
NV
Brain concentration (nmol/kg ꢃ S.D.) where
858 ꢃ 125, 3.3 mg/kg, 163 ꢃ 25, 3 mg/kg, sc,
available for this plasma and ratios
sc, 1 h for this cmpd,
all parameters
1 h for this cmpd,
all parameters
Plasma concentration (nM ꢃ S.D.)
Brain:plasma ratio
CSF (nM)
253
8.42
125
615 ꢃ 119
1.4 ꢃ 0.2
NV
3867 ꢃ 329
0.042 ꢃ 0.005
154 ꢃ 24
NV e No value determined.
a
Binding at hu alpha-7 receptor in recombinant HEK-cell membrane using [125I]BTX, method according to Gordon et al. (2010).
Method according to Sydserff et al. (2009).
Method outlined above.
hCLint determined with isolated human microsomes using AstraZeneca’s internally validated automated assay.
Permeability and efflux determined using MDR1eMDCK cell lines according to AstraZeneca’s internally validated automated assay and Polli et al., 2001.
b
c
d
e

D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
165
Fig. 3. In vitro autoradiography of [3H]MLA or [3H]AZ11637326 in the rat brain. Saturation binding of (A) [3H]MLA (0.1e10 nM) or (B) [3H]AZ11637326 (0.03e10 nM) was determined
by bath incubation alone or in competition with 10
(Kd ¼ 0.5 nM).
m
M MLA. The ventral hippocampus was used to determine the radioligand affinity: [3H]AZ11637326 (Kd ¼ 0.2 nM) and [3H]MLA
of acute and ascending doses (0.05, 0.1, 0.15 or 0.2 nmol/kg). [3H]
AZ11637326 accumulation was dose dependent. Consistent with
alpha-7 receptor localization seen in our autoradiography studies,
uptake in the hippocampus and hypothalamus was greater than
midbrain and frontal cortex (Fig. 7). Uptake in the cerebellum was
low (<0.1 fmol/mg tissue) at the 0.05, 0.1 and 0.15 nmol/kg dose
and increased to 0.23 fmol/mg tissue at the 0.2 nmol/kg dose,
defining the alpha-7 specific dose range for [3H]AZ11637326. All
subsequent experiments utilized the 0.15 nmol/kg dose which
provided a 3e4 fold specific to non-specific binding ratio.
[3H]AZ11637326 radioactivity measured (dpm) in the plasma
from trunk blood increased in a dose dependent manner. At
radiotracer doses of 0.1, 0.15 or 0.2 nmol/kg, the plasma radioac-
tivity was 112.8 ꢃ 9.3, 133.0 ꢃ 12 and 273.0 ꢃ 14.2 respectively.
The in vivo kinetics of [3H]AZ11637326 (0.15 nmol/kg, iv) accu-
mulation in the brain was determined at nine terminal time points:
0.5,1, 5,15, 30, 60, 90,120, 240 min (Fig. 8). [3H]AZ11637326 entered
the brain rapidly with peak distribution throughout all regions
5 min after i.v. injection. The specific to non-specific binding ratio
was 1.0 at 5 min. By 15 min, [3H]AZ11637326 binding was target
Fig. 4. In vitro autoradiography of [3H]MLA was blocked by alpha-7 ligands in the rat brain. [3H]MLA total binding (2.0 nM) in the frontal cortex, hippocampus and hypothalamus
was blocked by 10
M of MLA, PyrQTC or AR-R17779. [3H]MLA total binding is minimal in the striatum and cerebellum. The stereotaxic drawings on the left represent the frontal
cortex, striatum, dorsal hippocampus, ventral hippocampus and thalamus and the cerebellum and brain stem (top to bottom) and provide an anatomical guide for the autora-
diographic images. High resolution images were captured at 12 h acquisition time using the -imager.
m
b

166
D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
Fig. 5. [3H]AZ11637326 binding (0.15 nM) in the frontal cortex (PFC) and hippocampus (HPC) was blocked by co-incubation with (A) AR-R17779 (HPC, IC50 ¼ 537.5 nM and PFC,
IC50 ¼ 351.3 nM) or (B) PyrQTC (HPC, IC50 ¼ 44.15 nM and PFC, IC50 ¼ 141.9 nM). Binding in the striatum (STR) and cerebellum (not shown) was not inhibited by the competing
alpha-7 agonists. Surface radioactivity was measured and represented as cpm/mm2.
specific in the hippocampus (0.71 ꢃ0.04 fmol/mg tissue) and frontal
cortex (0.62 ꢃ 0.05 fmol/mg tissue), both alpha-7 receptor target
regions. Uptake in the cerebellum (0.11 ꢃ 0.01 fmol/mg tissue), the
non-specific binding region, was significantly reduced. Binding in
target regions (FC, HPC, HTand MID) was maintained from 15 min to
time points greater than 90 min. The specific to non-specific binding
ratio was 4.2 at 90 min. Binding in non-target region (CRB) fell from
1.09 fmol/mg tissue at 5 min to 0.11 fmol/mg tissue at 15 min and
remained low (0.10e0.13 fmol/mg) over the remaining time
periods. [3H]AZ11637326 accumulation in the HPC was not signifi-
cantly different from 60 to 240 min following injection, indicating
that the binding had reached equilibrium in vivo.
radioactivity was rapidly excreted and <0.5 fmol/mg tissue by
240 min (Fig. 9).
3.5. In vivo receptor occupancy in the rat brain
The in vivo pharmacological selectivity of [3H]AZ11637326 was
demonstrated using MLA pretreatment (Fig. 10A). MLA (0.1, 0.3,
1, 3 mg/kg, iv) significantly blocked [3H]AZ11637326 binding in the
HPC (p < 0.05) and PFC (p < 0.05) in a dose dependent manner
compared to saline pretreatment. MLA dosed at 0.1 mg/kg i.v. was
found to be significantly greater than 0.3, 1 and 3 mg/kg doses in
both regions (p > 0.05). MLA pretreatment significantly blocked
[3H]AZ11637326 binding (p ꢄ 0.05) in the HT (MLA 0.3, 1 and
3 mg/kg vs. saline) and in the MID (MLA 1 and 3 mg/kg vs. saline). In
the STR, a low density alpha-7 receptor region, no significant effect
of MLA pretreatment was found.
In all specific brain regions, pretreatment with ondansetron
(1 mg/kg, sc), a 5-HT3 ligand did not inhibit [3H]AZ11637326
binding and was not significantly different from saline treatment,
figure not shown. We tested the 5-HT3 ligand because the 5-HT3
receptor has similar sequence homology to the alpha-7 nicotinic
The % injected dose (ID) per gram tissue for [3H]AZ11637326
kinetic study was determined for each brain region. At the optimal
dose, 0.15 nmol/kg, the %ID was greatest at 15 min following
radiotracer injection in PFC (1.54%), HPC (1.38%), while non-specific
binding in the CRB (0.21%) remained low. At 90 min following
radiotracer injection, the %ID in PFC (0.59%) and HPC (0.73%) was
greater than the 0.5% target %ID, while the %ID in the CRB remained
consistently low (0.18%).
[3H]AZ11637326 was measured in the kidney, heart, spleen,
liver, testis, lung & thigh muscle. In all peripheral tissues sampled,
Fig. 6. [3H]AZ11637326 regional uptake after i.v. injection was demonstrated in slide mounted brain sections analyzed by -Imager. Dense [3H]AZ11637326 binding was observed in
b
the medial frontal cortex (PFC), hippocampus (HPC) from the dorsal (dHPC) to ventral/posterior (vHPC), hypothalamus (ventral medial region in the dHPC and HPC autoradiograms)
and midbrain (seen in the vHPC autoradiogram) in comparison to the lower surface radioactivity detected in the striatum (STR) and cerebellum (CRB).

D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
167
FC
HPC
HT
MID
CRB
0.6
0.4
0.2
0.0
0.05
0.10
nmol/kg
0.15
0.20
Fig. 7. [3H]AZ11637326 (0.05e0.20 nmol/kg) accumulated in alpha-7 receptor-rich
regions of the rat brain. Binding (fmol/mg tissue) in the hippocampus, hypothalamus,
midbrain and frontal cortex was significantly greater than in the cerebellum. Optimal
working dose and 3e4 fold specific to non-specific binding ratio was determined by
the 0.15 nmol/kg dose. At higher doses (ꢅ0.20 nmol/kg, i.v.) [3H]AZ11637326 non-
specific binding begins to increase. Frontal cortex, FC, hippocampus, HPC, hypothal-
amus, HT, midbrain, MID, and cerebellum, CRB.
Fig. 9. Accumulation of [3H]AZ11637326 in peripheral organs following 0.5e240 min
exposure time. Kidney exposure peaked at 1 min and is reduced by 5 min. In all
peripheral tissues sampled, radioactivity was rapidly excreted and <0.5 fmol/mg tissue
by 240 min, indicating that [3H]AZ11637326 does not selectively bind to peripheral
organs, but is distributed then eliminated at an acceptable rate for a radiotracer.
One-way ANOVA for [3H]AZ11637326 binding showed a signifi-
cant main effect of AR-R17779 treatment (p < 0.05). Pairwise
comparisons indicated that AR-R17779 significantly blocked [3H]
AZ11637326 binding in the HPC at 10 and 30 mg/kg (p < 0.05)
receptor. These data further substantiate [3H]AZ11637326 binding
to the alpha-7 receptor site.
[3H]AZ11637326 binding in target regions of the rat brain was
dose dependently reduced following pretreatment with all alpha-7
agonist compounds tested. See Table 1 for information about each
compound. Generally, all compounds tested had high affinity for
the human alpha-7 receptor with greater than 100 fold difference
in binding at the human 5-HT3 and
a4b2 receptors. Functional
potency at the human alpha-7 receptor in Xenopus oocytes was
sub-micromolar with similar potency in native rat tissue for DBCO-
3-POM, DBCO-4-POM and AR-R1779. AZ11637326 showed 1000
fold higher affinity using the native hippocampus compared to
oocyte electrophysiology. In Vitro permeability and efflux ratios
calculated for AZ11637326, DBCO-3-POM and DBCO-4-POM are
compatible with the measured in vivo brain, plasma and brain to
plasma ratios, indicating good brain penetration for these
compounds. Modest brain penetration was expected based on the
moderate efflux ratio for PyrQTC. While efflux ratio was not tested
for AR-R1779, we measured modest but adequate brain exposure
for this compound. Taken together, all compounds tested for in vivo
receptor occupancy showed receptor selectivity, functional potency
and adequate exposure when administered systemically.
Fig. 10. [3H]AZ11637326 specific in vivo binding was blocked by pretreatment with
MLA (A) or AR-R17779 (B) in a dose dependent manner. (A) MLA significantly blocked
[3H]AZ11637326 in the PFC and HPC at all doses administered (*p < 0.05). All target
regions were significantly blocked at the two highest doses (1 and 3 mg/kg, i.v.). No
inhibition was measured in the striatum. (B) AR-R17779 significantly (*p < 0.05)
blocked [3H]AZ11637326 in the HT, HPC and MID at the highest dose (30 mg/kg iv).
MLA (3 mg/kg iv) was the positive control compound in this study. Frontal cortex, PFC,
hippocampus, HPC, hypothalamus, HT, midbrain, MID, and striatum, STR.
Fig. 8. [3H]AZ11637326 binding time course (0.5e240 min) indicates that the radio-
tracer enters the brain rapidly and distributes to specific receptor sites by 15 min. Total
binding was measured in the frontal cortex, FC, hippocampus, HPC and cerebellum,
CRB.

168
D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
compared to saline pretreatment (Fig. 10B). AR-R17779 (3, 10 and
30 mg/kg) significantly blocked [3H]AZ11637326 in the HT
(p < 0.05), while AR-R17779 (30 mg/kg) significantly blocked [3H]
AZ11637326 in the MID (p < 0.05). AR-R17779 showed a dose
dependent trend toward significant block of [3H]AZ11637326
binding in the PFC, yet the effect did not reach significance. In the
striatum, a region with few alpha-7 receptors, [3H]AZ11637326
binding was not significantly different across treatment conditions.
Plasma and whole brain exposure for AR-R17779 was collected at
15 and 105 min following administration. Exposure in the brain at
AZ11637326 binding in the HPC at all doses administered
(0.001e25 mg/kg, p < 0.05) compared to saline pretreatment.
Plasma and whole brain samples were collected 30 min following
administration of DBCO-4-POM (0.3, 3, 10, 17, 25 mg/kg ¼ 58,
739 nmol/kg, 2.6, 6.5, 11.71 mmol/kg) and reflect a dose dependent
concentration of DBCO-4-POM when [3H]AZ11637326 is injected.
Brain to plasma ratio was consistently 1.3 across doses.
Receptor occupancy of PyrQTC, AR-R17779, DBCO-3-POM or
DBCO-4-POM was calculated from specific binding of [3H]
AZ11637326 and represented as a percent of the vehicle occupancy
(Fig. 12). In the hippocampus, the % receptor occupancy for DBCO-
4-POM > DBCO-3-POM w MLA > PyrQTC > AR-R17779. For all
compounds, % receptor occupancy curves for all target regions were
aligned, with variations across the dose range according to affinity.
The affinity shift reflects the difference in ED50 values for each
compound tested: ED50 value for DBCO-4-POM ¼ 0.0195, DBCO-3-
15 min after administration (10 mg/kg ¼ 1.29
mmol/kg and 30 mg/
mmol/kg) reflects the concentration of AR-R17779 when
kg ¼ 3.58
[3H]AZ11637326 is injected. The brain to plasma ratio is 0.1 at both
doses. The concentration of AR-R17779 in the brain 105 min after
drug administration is 10 mg/kg ¼ 0.470
mmol/kg and 30 mg/
kg ¼ 1.90 mol/kg. The brain to plasma ratio was 0.9.
m
Pretreatment with PyrQTC, DBCO-3-POM and DBCO-4-POM
dose dependently inhibited [3H]AZ11637326 binding in the rat
HPC (Fig.11). One-way ANOVA for [3H]AZ11637326 binding showed
a significant main effect of PyrQTC treatment (p < 0.05). Pairwise
comparisons indicated that PyrQTC significantly blocked [3H]
AZ11637326 binding at 3 and 10 mg/kg in the HPC (p < 0.05)
compared to saline pretreatment. Plasma and whole brain expo-
sure for PyrQTC was collected at 30 and 120 min following
administration. Exposure in the brain at 30 min after administra-
tion (3 mg/kg ¼ 87 nmol/kg and 10 mg/kg ¼ 483 nmol/kg) reflects
the concentration of PyrQTC when [3H]AZ11637326 is injected. The
brain to plasma ratio is 0.3 at both doses. The concentration of
PyrQTC in the brain 120 min after drug administration for 3 mg/kg
is 52 nmol/kg and for 10 mg/kg is 104 nmol/kg. Brain to plasma
ratio is 0.81 and 1.5, respectively.
POM
¼
0.1695, MLA
¼
0.1766, PyrQTC
¼
4.232 and AR-
R17779 ¼ 24.47 mg/kg. The ED50 values represent 50% receptor
occupancy in the rat hippocampus.
3.6. Slice electrophysiology
Several groups including ourselves have demonstrated the
modulation of gaba-ergic and glutamatergic synaptic activity by
alpha-7 agonists in acute brain slice preparations (Biton et al.,
2007). In order to evaluate the functional efficacy of AZ11637326
on native alpha-7 receptors, we recorded spontaneous inhibitory
post-synaptic currents (sIPSCs) from a rat prefrontal cortex slice
preparation. AZ11637326 was found to increase the frequency of
sIPSCs at a bath concentration of 10 nM (Table 1). This activity was
blocked by a 5 min pre-application of 10 nM MLA to the bath (not
shown). We show that other alpha-7 agonist compounds (DBCO-4-
POM, DBCO-3-POM, PyrQTC and AR-R17779) showed similar
activity albeit at different bath concentrations (see Table 1).
One-way ANOVA for [3H]AZ11637326 binding showed a signifi-
cant main effect of DBCO-3-POM treatment (p < 0.05). Pairwise
comparisons indicated that DBCO-3-POM significantly blocked [3H]
AZ11637326 binding in the HPC at doses of 0.01, 0.1, 0.3, 3,10,17 and
25 mg/kg (p < 0.05) compared to saline pretreatment. Plasma and
whole brain samples were collected 30 min following administra-
tion of DBCO-3-POM (0.1, 0.3, 3, 10, 17, 25 mg/kg ¼ 34, 108,
4. Discussion
The high affinity alpha-7 radiotracer, [3H]AZ11637326, shows
high specific binding to the alpha-7 receptor in the rodent brain,
using both in vitro and in vivo binding techniques. In vitro binding
studies in tissue sections prepared for autoradiography support the
properties of high affinity, target localization and pharmacological
selectivity for [3H]AZ11637326, making it a likely candidate for in
vivo characterization. When administered to rats by bolus intra-
venous injection, [3H]AZ11637326 rapidly entered the rat brain and
858 nmol/kg, 3.4, 7.6, 13.7 mmol/kg) and reflect the dose dependent
concentration of DBCO-3-POM when [3H]AZ11637326 is injected.
One-way ANOVA for [3H]AZ11637326 binding showed a signifi-
cant main effect of DBCO-4-POM treatment (p < 0.05). Pairwise
comparisons indicated that DBCO-4-POM significantly blocked [3H]
Fig. 11. [3H]AZ11637326 specific in vivo binding in the hippocampus was blocked by
pretreatment with PyrQTC, DBCO-3-POM or DBCO-4-POM. DBCO-3-POM or DBCO-4-
Fig. 12. In vivo receptor occupancy for alpha-7 agonist compounds, AR-R17779, MLA,
PyrQTC, DBCO-3-POM or DBCO-4-POM was determined in the rat hippocampus using
[3H]AZ11637326. The affinity relationship between compounds indicates that DBCO-4-
POM > DBCO-3-POM w MLA > PyrQTC > AR-R17779. This relative rank order rela-
tionship is seen in both binding and functional measures.
POM significantly blocked [3H]AZ11637326 at 0.01e10 mg/kg, sc (*p
< 0.05)
compared to saline pretreatment. PyrQTC blocked [3H]AZ11637326 at 3 and 10 mg/kg,
sc (*p < 0.05) compared to saline pretreatment. MLA (3 mg/kg i.v.) was the positive
control compound in these studies.

D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
169
5-HT3 receptor selective ligand odansetron and our in vivo occu-
pancy data for odansetron, suggest that the 5-HT3 receptor is an
unlikely binding site for [3H]AZ11637326.
Functional selectivity was demonstrated using electrophysi-
ology. In an electrophysiological functional assay AZ11637326 is
a potent partial agonist (EC50 ¼ 64 ꢃ17 nM) when tested in voltage-
clamped Xenopus oocytes expressing the recombinant human
alpha-7 receptor, while it was not active when combinations of
100
75
50
25
0
Frog donor #1
Frog donor #2
cRNA from
a and b NNR subtypes were tested (Gordon et al., 2010).
Functional activity of AZ11637326 was confirmed using native
alpha-7 receptors by recording spontaneous inhibitory post-
synaptic currents (sIPSCs) from a rat prefrontal cortex slice prepa-
ration (see Table 1).
-9
-8
-7
-6
-5
-4
log[AZ11637326],(M)
Binding kinetics for [3H]AZ11637326 using autoradiography
were slowed by incubation at 4 ꢁC and allowed us to utilize the
enthalpic role on binding affinity for agonist ligand binding. Incu-
bation required 90 min exposure to the radioligand alone or in
competition with MLA to produce a high affinity selective alpha-7
localization profile. The dissociation kinetics reported by Gordon
(Gordon et al., 2010) reflect a rapid association and rapid dissoci-
ation profile which did not interfere with the regional differentia-
tion of [3H]AZ11637326. This is also reflected in our in vivo kinetic
data were rapid and uniform distribution of [3H]AZ11637326 was
measured throughout the brain at 5 min. At 15 min following
administration, [3H]AZ11637326 was distributed in specific binding
regions such as the frontal cortex, hippocampus and midbrain
selectively over low density regions, such as the striatum and
cerebellum. This pattern of regional distribution in dissected
regions of the rat brain by scintillation count is also shown in our ex
vivo autoradiography sections. In addition, the selectivity profile in
vivo was sustained for greater than 60 min following administra-
tion, making it possible to test compound occupancy at equilibrium
in the in vivo kinetic binding curve for [3H]AZ11637326.
Fig. 13. Functional potency of AZ11637326 is 0.2
Xenopus oocyte expressing the human alpha-7 receptor. Data for two frog donors are
shown with close alignment between donors.
m
M using in vitro electrophysiology in
distributed selectively to alpha-7 receptor-rich regions. In vivo
pharmacological selectivity was supported by inhibition of [3H]
AZ11637326 by alpha-7 agonists. The in vivo occupancy for DBCO-
4-POM, DBCO-3-POM, MLA, PyrQTC and AR-R17779 mirrors the
affinity relationship and compound differentiation determined
using in vitro competition binding experiments in recombinant
human alpha-7 HEK-cell membranes (see Table 1 and Gordon et al.,
2010). These findings together suggest that [3H]AZ11637326 is
a suitable tool for profiling novel compounds with alpha-7 receptor
pharmacology.
Saturation binding studies conducted in recombinant human
alpha-7 receptors indicated that [3H]AZ11637326 bound with high
affinity to a single receptor site. In membranes prepared from the
rat hippocampus, [3H]AZ11637326 bound to a high affinity site
(Kd ¼ 0.510 nM) with Bmax ¼ 55 fmol/mg, values comparable to
those measured for [125I]BTX (Gordon et al., 2010). The affinity of
[3H]AZ11637326 determined by saturation binding studies using
autoradiography (Kd ¼ 0.15 nM in the ventral hippocampus) was
comparable to values determined using membrane preparations.
[3H]MLA affinity (Kd ¼ 0.2 nM) in the ventral hippocampus was
approximately 10 fold higher affinity than what has been reported
for rat (Davies et al., 1999) and mouse (Whiteaker et al., 1999) brain
membranes, due to measurement of surface radioactivity in
a region of high receptor density.
High affinity binding was required to selectively label the alpha-
7 receptor population. Selectivity in the autoradiography prepara-
tion was diminished with higher concentrations of [3H]AZ11637326
in the incubation bath suggesting that the in vitro working
concentration for [3H]AZ11637326 in autoradiography studies was
optimal at less than 3 nM. The narrow margin for high affinity and
selectivity was also reflected in the in vivo receptor occupancy dose
response data for [3H]AZ11637326. We were able to utilize an in
vitro concentration and in vivo dose of [3H]AZ11637326 that fully
defined the alpha-7 specific range. Concentrations higher than this
defined range resulted in an increase in non-specific binding. In the
native membrane preparation, Gordon found a nicotine-insensitive
binding site at Kd w4.9 nM for [3H]AZ11637326, approximately 10
fold lower than the alpha-7 selective high affinity site, lending
support to our findings. However, based on the selectivity profile
for AZ11637326, determined by MDS SpectrumScreen Assay, the
serotoninergic receptor 5-HT3 was the only target that elicited
The alpha-7 binding pattern for [3H]AZ11637326 was consistent
with the mRNA localization in the rat and receptor distribution
reported for [3H]MLA (Mugnaini et al., 2002) using autoradiog-
raphy. [3H]AZ11637326 showed highest binding pattern in the
hippocampus, hypothalamus, midbrain superior colliculus and
frontal cortex, representing the functional role of the alpha-7
receptor in the brain. The localization pattern of [3H]AZ11637326
in the rodent is representative of the alpha-7 receptor pattern
reported for the human brain (Breese et al., 1997), further sup-
porting the value of [3H]AZ11637326 as a possible translational
neuroscience tool.
AR-R17779 has been widely cited and reported to be efficacious
in rodent behavioral models of cognition, including learning and
memory (Levin et al., 1999), social recognition test (Van Kampen
et al., 2004) and associative learning (Li et al., 2008). AR-R17779
improved working memory in fimbria fornix lesioned rats in the
radial arm maze and improved short-term memory following
scopolamine treatment in rats, respectively. Much like other NNR
selective agonists (Werkheiser et al., 2007), AR-R17779 has been
shown to upregulate alpha-7 receptors in the frontal cortex and
hippocampus of the rabbit brain (Li et al., 2008). AR-R17779 was
recognized as the first full alpha-7 agonist to show high affinity,
subtype selectivity and potent efficacy for the alpha-7 subtype
when tested across alpha and beta NNR subtypes in Xenopus
oocytes (Astles et al., 2002; Papke et al., 2004). These findings
further support our use of AR-R17779 for validation of [3H]
AZ11637326.
a >50% inhibition response at 0.2 mM, which is more than 1000 fold
greater than the Kd value for [3H]AZ11637326 in autoradiography.
When compared to inhibition measured at the alpha-7 receptor,
AZ11637326 showed 80e150 fold selectivity for serotoninergic
receptor 5-HT3 (Gordon et al., 2010). This evidence along with
Gordon’s findings that specific binding was not inhibited by the
Based on our in vitro findings, the functional potency in slice
electrophysiology assays and the receptor occupancy data pre-
sented here, DBCO-4-POM, DBCO-3-POM and PyrQTC would show
efficacy in behavioral models. For DBCO-4-POM and DBCO-3-POM,
the brain and plasma exposure values following systemic

170
D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
administration, the low efflux ratio and the moderate permeability
value all suggest that adequate brain exposure was achieved. The
relationship between the in vitro inhibition values, the functional
potency and the plasma concentration indicate that an in vivo
efficacious dose can be achieved within the ED50 range for receptor
occupancy in the rat brain. For PyrQTC, the in vitro potency is
approximately 10 fold better than the other two compounds, yet
the efflux ratio is 26, indicating that it is a likely substrate for efflux
which would compromise brain penetration, providing an expla-
nation for the right shift for in vivo occupancy of this compound.
Receptor occupancy studies in rodents using a high affinity
radioligand can demonstrate that a compound has entered the
brain and reached the target receptor. It can also indicate the
relationship between drug dose, plasma concentration and target
occupancy. Information about compound in vivo occupancy in
rodents can be used to establish our understanding of the rela-
tionship between receptor binding, behavioral efficacy and
compound exposure. This approach to elucidating novel compound
pharmacology has been used to improve our ability to predict the
dose range for human studies and to increase the translational
science value for our compounds. Our occupancy studies in rodents
indicate that [3H]AZ11637326 is a suitable in vivo radioligand tool
for the alpha-7 receptor. Comparisons between the four alpha-7
compounds tested suggest that the relative difference between
compound occupancy match in vitro findings and exposure rela-
tionships. The Ki value and potency in the PFC slice preparation
show that AZ11637326 is more potent than DBCO-4-POM which is
greater than AR-R17779. The exposure relationship, as indicated by
the brain to plasma ratio, show the same pattern such that
AZ11637326 > DBCO-4-POM > AR-R17779. Compound exposure in
the brain and plasma measured following administration of all four
compounds indicated that they were systemically available and had
entered the brain at the time point when the radioligand was
administered to the animals. Pharmacological selectivity was
demonstrated in vitro with competition binding for AR-R17779 and
PyrQTC using [3H]AZ11637326. IC50 values indicated that the rela-
tive affinity of PyrQTC was 10 fold greater than AR-R17779.
Compound receptor occupancy demonstrated in vivo using [3H]
AZ11637326 indicated that the relationship between compounds is
DBCO-4-POM > DBCO-3-POM w MLA > PyrQTC > AR-R17779. The
rank order potency we observed reflects the in vitro affinity in
membrane preparations and the functional potency in Xenopus
oocytes.
References
Albuquerque, E.X., Pereira, E.F.R., Alkondon, M., Rogers, S.W., 2009. Mammalian
nicotinic acetylcholine receptors: from structure to function. Physiol. Rev. 89,
73e120.
Anderson, D.J., Bunnelle, W., Surber, B., Du, J., Surowy, C., Tribollet, E., Marguerat, A.,
Bertrand, D., Gopalakrishan, M., 2008. [3H]A-585539 [(1S,4S)-2,2-dimethyl-5-
(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane],
a novel high
affinity 7 neuronal nicotinic receptor agonist; Radioligand binding character-
a
ization to rat and human brain. J. Pharmacol. Exp. Ther. 324, 179e187.
Astles, P.C., Baker, S.R., Boot, J.R., Broad, L.M., Dell, C.P., Keenan, M., 2002. Recent
progress in the development of subtype selective nicotinic acetylcholine receptor
ligands. Current Drug Targets e CNS & Neurological Disorders 1, 337e348.
Balestra, M., Gordon, J.C., Griffith, R.C., Murray, R.J., 1996. Preparation of spi-
roazabicyclic compounds for treatment of psychosis, anxiety, and intellectual
impairment. WO/1996/006098, 1e21.
Biton, B., Bergis, O.E., Galli, F., Nedelec, A., Lochead, A.W., Jegham, S., Godet, D.,
Lanneau, C., Santamaria, R.I., Chesney, F., Le’onardon, J., Granger, P.,
Debono, M.W., Bohme, G.A., Sgard, F., Besnard, F., Graham, D., Coste, A., Oblin, A.,
Curet, O., Vige, X., Voltz, C., Rouquier, L., Souilhac, J., Santucci, V., Gueudet, C.,
Francon, D., Steinberg, R., Griebel, G., Oury-Donat, F., George, P., Avenet, P.,
Scatton, B., 2007. SSR180711, a novel selective
agonist: (1) binding and functional profile. Neuropsychopharmacology 32,
1e16.
a7 nicotinic receptor partial
Bock, M.J., Neilson, K.L., Dudley, A., 2007. Use of trifluoroacetic acid to quantify
small, polar compounds in rat plasma during discovery-phase pharmacokinetic
lunationion. J. Chromb. 856 (1e2), 165e170.
Breese, C.R., Adams, C., Logel, J., Drebing, C., Rollins, Y., Barnhart, M., Sullivan, B.,
Demasters, B.K., Freedman, R., Leonard, S., 1997. Comparison of the regional
expression of nicotine acetylcholine receptor a7 mRNA and [125I]-a-bungar-
otoxin binding in the human postmortem brain. J. Comp. Neurol. 387, 385e398.
Buccafusco, J.J., Terry, A.V., 2009. A reversible model of the cognitive impairment
associated with schizophrenia in monkeys: potential therapeutic effects of two
nicotinic acetylcholine receptor agonists. Biochem. Pharmacol. 78, 852e862.
Buchanan, R.W., Freedman, R., Javitt, D.C., Abi-Dargham, A., Lieberman, J.A., 2007.
Recent advances in the development of novel pharmacological agents for the
treatment of cognitive impairments in schizophrenia. Schizophr. Bull. 33 (5),
1120e1130.
Carson, R., Craig, D., McGuinness, B., Johnston, J.A., O’Neill, F.A., Passmore, A.P.,
Ritchie, C.W., 2008. a7 Nicotinic acetylcholine receptor gene and reduced risk of
Alzheimer’s disease. J. Med. Genet. 45, 244e248.
Castner, S.A., Smagin, G.N., Piser, T.M., Wang, Y., Smith, J.S., Christian, E.P., Mrzljak, L.,
Williams, G.V., 2011. Immediate and sustained improvements in working
memory after selective stimulation of a7 nicotinic acetylcholine receptors. Biol.
Psychiatry 69 (1), 12e18.
Christian, E.P., Doherty, J.J., Gadient, R., Paschetto, K., Smith, J., 2005. Functional
Consequences of 7 nACh Agonists and Positive Modulators on Neurotrans-
a
mitter Release: Divergent Therapeutic Implications. Soc. for Neurosci. 558.2.
Clarke, P.B.S., Schwartz, R.D., Paul, S.M., Pert, C.B., Pert, A., 1985. Nicotine binding in
the rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine,
and [125I]-
a-bungarotoxin. J. Neurosci. 5 (5), 1307e1315.
Conejero-Goldberga, C., Daviesa, P., Ulloa, L., 2008. Alpha7 nicotinic acetylcholine
receptor: a link between inflammation and neurodegeneration. Neurosci. Bio-
behav. Rev. 32, 693e706.
Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J.,
Wonnacott, S., 1999. Characterization of the binding of [3H]methyllycaconitine:
a
new radioligand for labeling a7-type neuronal nicotinic acetylcholine
Our in vitro and in vivo characterization of a novel alpha-7
radioligand tool indicates that [3H]AZ11637326 is a high affinity
pre-clinical tool for investigating novel compound binding to the
alpha-7 receptor in the rat brain using in vivo occupancy tech-
niques. [3H]AZ11637326 may be most effective as an imaging agent
in regions of high alpha-7 receptor density. At the optimal dose for
in vivo studies [3H]AZ11637326 was found to produce high specific
binding while maintaining a strong, reproducible assay. Further
investigation will be required to support AZ11637326 as a possible
molecular imaging tool for translational medicine in drug
discovery.
receptors. Neuropsychopharmacology 38, 679e690.
de Jonge, W.J., Ulloa, L., 2007. The alpha7 nicotinic acetylcholine receptor as
a pharmacological target for inflammation. Br. J. Pharmacol. 151, 915e929.
Ding, Y.S., Fowler, J.S., Logan, J., Wang, G.-J., Telang, F., Garza, V., Biegon, A.,
Pareto, D., Rooney, W., Shea, C., Alexoff, D., Volkow, N., Vocci, F., 2004. 6-[18F]
Fluoro-A-85830, anew PET tracer for the nicotinic acetylcholine receptor:
studies in the human brain and in vivo demonstration of specific binding in
white matter. Synapse 53, 184e189.
Ding, M., Werkheiser, J.L., Heys, J.R., Elmore, C.S., Mrzljak, L., Maier, D.L., 2007. In
Vivo Receptor Occupancy using [3H]AZ11637326, an Alpha-7 Nicotinic Receptor
Radiotracer. Soc. for Neurosci.. 574.13.
Dorff, P., Gordon, J., Heys, J.R., Keith, R.A., McCarthy, D.J., Phillips, E., Smith, M.A.,
2005. A preparation of isotope-labeled spiro(azabicyclooctane-furopyridine)
derivatives, useful as ligands for nicotinic acetylcholine receptor. WO/2005/
030778, 1e25.
Elmore, C.S., Werkheiser, J.L., Heys, J.R., Mrzljak, L., Maier, D.L., 2008. In vivo receptor
occupancy of alpha-7 nicotinic receptor agonist in rat brain using a high affinity
tritium labeled radioligand. J. Labeled Compd. Radiopharm. 51, 257.
Ernst, G., Frietze, W., Jacobs, R., Phillips, E., 2005. Preparation of 1,3-ethanopiper-
azines as nicotinic acetylcholine receptor ligands. WO/2005/061510, 1e40.
Gordon, J.C., Phillips, E., Gurley, D.A., Heys, J.R., Lazor, L.A., Barthlow, H.,
Mallamaci, M.A., Keith, R.A., 2010. In vitro binding characteristics of [3H]
AZ11637326, a novel a7-selective neuronal nicotinic receptor agonist radio-
ligand. Eur. J. Pharmacol. 645, 63e69.
Acknowledgments
The authors would like to acknowledge the following individ-
uals for their contribution to this work: Amy Hehman & Lynn India
Neveras for jugular cannulation surgery, Eifion Phillips for synthesis
of AZ11637326, J. Richard Heys for the original radiochemical
synthesis of [3H]AZ11637326, Ladislav Mrzljak, Kaoru Kondo for the
generous and rigorous experimental support she provided to the in
vitro autoradiography work, Mark Pietras for mining the data base
and Kathy Neilson for her tireless DMPK support.
Gotti, C., Clementi, F., Fornari, A., Gaimarri, A., Guiducci, S., Manfredi, I., Moretti, M.,
Pedrazzi, P., Pucci, L., Zoli, M., 2009. Structural and functional diversity of native
brain neuronal nicotinic receptors. Biochem. Pharmacol. 78, 703e711.

D.L. Maier et al. / Neuropharmacology 61 (2011) 161e171
171
Han, Z.-Y., Zoli, M., Cardona, A., Bourgeois, J.-P., Changeux, J.-P., Le Novere, N., 2003.
Localization of [3H]nicotine, [3H]cytosine, [3H]epibatidine, and [125I]a-bungar-
otoxin binding sites in the brain of Macaca mulatta. J. Comp. Neurol. 461, 49e60.
Hashimoto, K., Nishiyama, S., Ohba, H., Matsuo, M., Kobashi, T., et al., 2008. [11C]
CHIBA-1001 as a novel PET ligand for a7 nicotinic receptors in the brain: a PET
study in conscious monkeys. PLoS ONE 3 (9), e3231.
Olincy, A., Harris, J.G., Johnson, L.L., Pender, V., Kongs, S., Allensworth, D., Ellis, J.,
Zerbe, G.O., Leonard, S., Stevens, K.E., Stevens, J.O., Martin, L., Adler, L.E., Soti, F.,
Kem, W.R., Freedman, R., 2006. Proof-of-concept trial of an alpha-7 nicotinic
agonist in schizophrenia. Arch. Gen. Psychiatry 63, 630e638.
Papke, R.L., Porter Papke, J.K., Rose, G.M., 2004. Activity of a-7-selective agonists at
nicotinic and serotonin 5HT3 receptors expressed in Xenopus oocytes. Bioorg.
Med. Chem. Lett. 14, 1849e1853.
Parri, H.R., Dineley, K.T., 2010. Nicotinic acetylcholine receptor interaction with b-
amyloid: molecular, cellular, and physiological consequences. Curr. Alzheimer
Res. 7, 27e39.
Passchier, J., Gee, A., Willemsen, A., Vaalberg, W., Waarde, A.V., 2002. Measuring
drug-related receptor occupancy with positron emission tomography. Methods
27, 278e286.
Kem, W.R., 2000. The brain a7 nicotinic receptor may be an important therapeutic
target for the treatment of Alzheimer’s disease: studies with DMXBA (GTS-21).
Beh. Brain Res. 113, 169e181.
Kulak, J.M., Carroll, F.I., Schneider, J.S., 2006. [125I]iodomethyllycaconitine binds to
a7 nicotinic acetylcholine receptors in monkey brain. Eur. J. Neurosci. 23,
2604e2610.
Leiser, S.C., Bowlby, M.R., Comery, T.A., Dunlop, J., 2009. A cog in cognition: how the
a
7
nicotinic acetylcholine receptor is geared towards improving cognitive
Phillips, E., Schmiesing, R., 2001. Preparation and formulation of biarylcarbox-
amides as nicotinic acetylcholine receptor agonists for therapeutic use in the
treatment or prophylaxis of psychotic and intellectual impairment disorders.
WO/2001/060821, 1e84.
Polli, J.W., Wring, S.A., Humphreys, J.E., Huang, L., Morgan, J.B., Webster, L.O., 2001.
Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol.
Exp. Ther. 299, 620e628.
deficits. Pharmacol. Ther. 122, 302e311.
Levin, E.D., Bettegowda, C., Blosser, J., Gordon, J., 1999. AR-R17779, and alpha7
nicotinic agonist, improves learning and memory in rats. Behav. Pharmacol. 10,
675e680.
Levin, E.D., 2002. Nicotinic receptor subtypes and cognitive function. J. Neurobiol.
53, 633e640.
Li, J.G., Lehr, M., Liu-Chen, L.Y., Woodruff-Pak, D.S., 2008. Nicotinic acetylcholine
receptors and modulation of learning in 4- and 27-month-old rabbits. Neuro-
psychopharmacology 33, 2820e2830.
Pomper, M.G., Phillips, E., Fan, H., McCarthy, D.J., Keith, R., Gordon, J.C., Scheffel, U.,
Dannals, R.F., Musachio, J.L., 2005. Synthesis and biodistribution of radiolabeled
a
7 nicotinic acetylcholine receptor ligands. J. Nucl. Med. 46, 326e334.
Martin, L.F., Kem, W.R., Freedman, R., 2004. Alpha-7 nicotinic receptor agonists:
potential new candidates for the treatment of schizophrenia. Psychopharma-
cology 174, 54e64.
Smith, J., Li, C., Doherty, J.J., 2004. Alpha 7 Agonists and Positive Modulators
Increase Gaba and Glutamate Function in Prefrontal Cortex. Soc. for Neurosci..
248.5.
Mugnaini, M., Tessari, M., Tarter, G., Pich, E.M., Chiamulera, C., Bunnemann, B., 2002.
Sydserff, S., Sutton, E.J., Song, D., Quirk, M.C., Maciag, C., Li, C., Jonak, G., Gurley, D.,
Gordon, J.C., Christian, E.P., Doherty, J.J., Hudzik, T., Johnson, E., Mrzljak, L., Piser, T.,
Upregulation of [3H]methyllycaconitine binding sites following continuous
infusion of nicotine, without changes of
a
7 or
a
6 subunit mRNA: an auto-
Smagin, G.N., Wang, Y., Widzowski, D., Smith, J.S., 2009. Selective a7 nicotinic
radioraphy and in situ hybridization study in rat brain. Eur. J. Neurosci. 16,
1633e1646.
receptor activation by AZD0328 enhances cortical dopamine release and improves
learning and attentional processes. Biochem. Pharmacol. 78, 880e888.
Mullen, G., Napier, J., Balestra, M., DeCory, T., Hale, G., Macor, J., Mack, R., Loch III, J.,
Wu, E., Kover, A., Verhoest, P., Sampognaro, A., Phillips, E., Zhu, Y., Murray, R.,
Griffith, R., Blosser, J., Gurley, D., Machulskis, A., Zongrone, J., Rosen, A.,
Gordon, J., 2000. (-)-Spiro[1-azabicyclo[2.2.2]octane-3,50-oxazolidin-20-one],
a conformationally restricted analogue of acetylcholine, is a highly selective full
Tanibuchi, Y., Wu, J., Toyohara, T., Fujita, Y., Iyo, M., Hashimoto, K., 2010. Charac-
terization of [3H]China-1001 binding to
a nicotinic acetylcholine receptors in
the brain from rat, monkey and human. Brain Res. 1348, 200e208.
Thomsen, M.S., Hansen, H.H., Timmerman, D.B., Mikkelsen, J.D., 2010. Cognitive
improvement by activation of a7 nicotinic acetylcholine receptors: from animal
agonist at the
a
7 nicotinic acetylcholine receptor. J. Med. Chem. 43, 4045e4050.
models to human pathophysiology. Curr. Pharm. Des. 16, 323e343.
Toyohara, J., Sakata, M., Wu, J., Ishikawa, M., Oda, K., Ishii, K., Iyo, M., Hashimoto, K.,
Ishiwata, K., 2009. Preclinical and the first clinical studies on [11C]CHIBA-1001
for mapping alpha7 nicotinic receptors by positron emission tomography. Ann.
Nucl. Med. 23 (3), 301e309.
Navarro, H.A., Zhong, D., Abraham, P., Xu, H., Carroll, F.I., 2000. Synthesis and
pharmacological characterization of [(125)I]iodomethyllycaconitine ([(125)I]
iodo-MLA). A new ligand for the alpha (7) nicotinic acetylcholine receptor.
J. Med. Chem. 43, 142e145.
Navarro, H.A., Xu, H., Zhong, D., Abraham, P., Xu, H., Carroll, F.I., 2002. In vitro and in
vivo characterization of [125I]iodomethyllycaconitine in the rat. Synapse 44,
117e123.
Van Kampen, M., Selbach, K., Schneider, R., Schiegel, E., Boess, F., Schreiber, R., 2004.
AR-R 17779 improves social recognition in rats by activation of nicotinic
a7
receptors. Psychopharmacology (Berl) 172, 375e383.
Ogawa, M., Tatsumi, R., Fujio, M., Katayama, J., Magata, Y., 2006. Synthesis and
evaluation of [125I]I-TSA as a brain nicotinic acetylcholine receptor alpha7
subtype imaging agent. Nucl. Med. Biol. 33 (3), 311e316.
Wang, H., Yu, M., Ochani, M., Amella, C.A., Tanovic, M., Susarla, S., Li, J.H., Wang, H.,
Yang, H., Ulloa, L., Al-Abed, Y., Czura, C.J., Tracey, K.J., 2003. Nicotinic acetyl-
choline receptor a7 subunit is an essential regulator of inflammation. Nature
Ogawa, M., Tsukada, H., Hatano, K., Ouchi, Y., Saji, H., Magata, Y., 2009. Central in
vivo nicotinic acetylcholine receptor imaging agents for positron emission
tomography (PET) and single photon emission computed tomography (SPECT).
Biol. Pharm. Bull. 32 (3), 337e340.
Ogawa, M., Nishiwana, S., Tsukada, H., Hatano, K., Fuchigami, T., Yamaguchi, H.,
Matsushima, Y., Ito, K., Magata, Y., 2010. Synthesis and evaluation of new
imaging agents for central nicotinic acetylcholine receptor alpha 7 subtype.
Nucl. Med. Biol. 37 (3), 347e355.
421, 384e388.
Werkheiser, J.L., Hubbs, S., Ding, M., Peng, T., Tuke, D., Smith, J.S., Mrzljak, L.,
Maier, D.L., 2007. AZD0328, A Selective Alpha-7 Receptor Agonist, Induces
Long-lasting Potentiation of Alpha-7 Receptor Binding in Rat Brain after Acute
Administration, vol. 37. Soc. for Neurosci.. 906.24.
Whiteaker, P., Davis, A.R.L., Marks, M.J., Blagbrough, I.S., Potter, B.V.L.,
Wolstenholme, A.J., Collins, A.C., Wonnacott, S., 1999. An autoradiographic
study of the distribution of binding sites for the novel a7-selective nicotinic
Olincy, A., Stevens, K.E., 2007. Treating schizophrenia symptoms with an a7 nico-
tinic agonist, from mice to men. Biochem. Pharmacol. 74, 1192e1201.
radioligand [3H]methyllycaconitine in the mouse brain. Eur. J. Neurosci. 11,
2689e2696.