Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists

:
Correlation with desensitization of a4b2
Ã
receptors
Jiahui Zhang, Yun-De Xiao, Kristen G. Jordan, Phil S. Hammond, Katherine M. Van Dyke,
Anatoly A. Mazurov, Jason D. Speake, Patrick M. Lippiello, John W. James, Sharon R. Letchworth,
Merouane Bencherif, Terry A. Hauser

Targacept Inc., 200 East First Street, Winston-Salem, NC 27101, USA
a r t i c l e i n f o
Article history:
Received 13 February 2012
Received in revised form 17 August 2012
Accepted 14 September 2012
Available online 2 October 2012
Keywords:
a4b2
Ã
Neuronal nicotinic
acetylcholine receptors
Receptor desensitization
Analgesic effect
Formalin assay
a b s t r a c t
Nicotinic a4b2
Ã
agonists are knownto be effective ina variety of preclinical painmodels, but the underlying
mechanisms of analgesic action are not well-understood. In the present study, we characterized activation
and desensitization properties for a set of seventeen novel a4b2
Ã
-selective agonists that display druggable
physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies
observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to
be effective in the formalin assay, were included for comparison. The set of compounds displayed a range
of activities at human (a4b2)
2
b2 (HS-a4b2), (a4b2)
2
a5 (a4b2a5) and (a4b2)
2
a4 (LS-a4b2) receptors.
We report the novel finding that desensitization of a4b2
Ã
receptors may drive part of the antinociceptive
outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than
activation activities at a4b2
Ã
receptors are considered, there is a better correlation between analgesia
scores and combined in vitro properties.
Our results suggest that although all three a4b2 subtypes assessed are involved, it is desensitization of
a4b2a5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds.
For modulation of Phase I responses, correlations are significantly improved froman r
2
value of 0.53 to 0.67
and0.66 whenHS- and LS-a4b2 DC
50
values are considered, respectively. More profoundly, considering the
DC
50
at a4b2a5 takes the r
2
from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-a4b2 desen-
sitization potencies did not improve the correlations significantly. Considering the a4b2a5 DC
50
value
significantly increased the r
2
from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role
for a4b2a5 nAChRs in the modulation of pain in the formalin assay.
The present studies demonstrate that compounds which are more potent at desensitization of a4b2
Ã
receptors display better analgesia scores in the formalin test. Consideration of desensitization properties at
a4b2
Ã
receptors, especially at a4b2a5, in multiple linear regression analyses significantly improves corre-
lations with efficacies of analgesia. Thus, a4b2
Ã
nicotinic acetylcholine receptor desensitization may con-
tribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by
nicotinic agonists.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
Agonists selective for certain neuronal nicotinic acetylcholine
receptors (nAChRs) such as a4b2
Ã
(the asterisk indicates the
potential presence of additional subunits) are known to be analge-
sic in various pre-clinical pain models. One of the earliest demon-
strations of this effect was reported in 1932, when nicotine was
shown to be efficacious in a cat model of visceral pain (Flores,
2000). More recent studies have extended these findings to include
a number of additional nicotinic compounds. Specifically, epibati-
dine demonstrates powerful analgesic properties which are 200-
fold more potent than those of morphine (Yogeeswari et al.,
2006). Sazetidine-A is a potent analgesic in a rat model of forma-
lin-induced pain (Cucchiaro et al., 2008). In addition, ABT-594 is
not only potently effective in animal models of acute, inflamma-
tory and neuropathic pain (Bannon et al., 1998), but also showed
analgesic effects in patients with diabetic peripheral neuropathic
0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.09.014
Abbreviations: ACh, acetylcholine; a4b2a5, (a4b2)
2
a5; DC
50
, concentration
producing 50% of maximal desensitization response; D
max
, the maximal desensi-
tization response; EC
50
, concentration producing 50% of maximal activation
response; E
max
, the maximal activation response; HEK, human embryonic kidney;
HS-a4b2, high sensitivity (a4b2)
2
b2; LS-a4b2, low sensitivity (a4b2)
2
a4; MLR,
multiple linear regression; PSA, the molecular polar surface area; nAChR, neuronal
nicotinic acetylcholine receptor.

Corresponding author. Tel.: +1 336 480 2248; fax: +1 336 480 2113.
E-mail address: terry.hauser@targacept.com (T.A. Hauser).
European Journal of Pharmaceutical Sciences 47 (2012) 813–823
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
j our nal homepage: www. el sevi er. com/ l ocat e/ ej ps
pain (Rowbotham et al., 2009). These analgesic compounds are all
potent agonists at a4b2
Ã
receptors (Bannon et al., 1998; Cucchiaro
et al., 2008; Flores, 2000; Yogeeswari et al., 2006). Further, knock-
out mice lacking either a4 or b2 genes show reduced sensitivity to
the analgesic effects of nicotinic compounds in acute pain tests
(Marubio et al., 1999). Conversely, gain-of-function mice with
knock-in a4 subunits are more sensitive to nicotine-induced anal-
gesia (Damaj et al., 2007).
Although there is a general consensus about the involvement of
a4 and b2 subunits in analgesia, full understanding of the distinct
a4b2
Ã
subtypes and their required pharmacological manipulation
for the effect remain elusive. The existence of two subtypes of
a4b2
Ã
, high- and low-sensitivity (HS- and LS-) receptors, has been
demonstrated both in vitro and in vivo (Benwell et al., 1988;
Moroni et al., 2006; Nguyen et al., 2003; Zwart and Vijverberg,
1998). The HS- and LS-a4b2 receptors differ significantly not only
in their stoichiometries, with (a4b2)2b2 for HS- and (a4b2)2a4 for
LS-a4b2, but also in their sensitivity to activation by agonists,
desensitization kinetics and regulation by chronic exposure to
agonists (Moroni et al., 2006; Zwart and Vijverberg, 1998). Hence,
the separate expression of HS- and LS-a4b2 receptors and charac-
terization of the functional properties of these specific receptor
subtypes should be a logical approach for delineating a4b2
Ã
receptor-targeted compounds and their analgesic effects.
There has also been some controversy as to whether activation
of a4b2
Ã
receptors is sufficient to produce analgesia, in spite of a
wealth of supporting evidence. Recently, Gao and colleagues pro-
filed a series of nicotinic agonists, both selective and nonselective
for a4b2
Ã
receptors, in various in vitro and in vivo assessments.
They found that some highly selective a4b2
Ã
agonists did not ap-
pear to be analgesic, and concluded that activation of a4b2
Ã
recep-
tors was necessary but not sufficient to produce analgesia (Gao et
al., 2010). Additionally, the authors suggested that activities of
other receptor subtype(s) could produce an increase in the overall
level of inhibitory synaptic transmission in the spinal cord beyond
that produced by a4b2
Ã
agonism alone.
One potential target for optimization of nicotinic modulation of
analgesia is the activity at a5-containing nicotinic receptors (a5
Ã
).
The role for a5
Ã
nAChRs in analgesia is supported by a number of
studies using various experimental approaches. Specifically, spinal
expression of a5 was increased in response to spinal nerve ligation
(Vincler and Eisenach, 2004). Additionally, it was demonstrated
that antinociception induced by nicotinic agonists in wild-type
animals was lost when tested in a5 knockout mice (Jackson et
al., 2010). Interestingly, it has been shown that a5 subunits in
the brain are exclusively associated with a4b2
Ã
receptors (Mao et
al., 2008).
Taken together, these data raise questions regarding what as-
pect of functional activity at a4b2
Ã
nAChRs is relevant to analgesia,
and whether and how incorporation of a5 subunits into a4b2
Ã
may
affect activity in the mediation of pain. In the present studies, we
cotransfected concatenated human b2-a4 dimers with a4, b2 and
a5 monomers in order to express LS-, HS-a4b2, and (a4b2)2a5
(a4b2a5) nAChRs, respectively, in HEK293F cells. This approach
has been demonstrated previously to produce a homogeneous pop-
ulation of receptors, the stoichiometry of which was determined by
the monomer added Papke et al., 2010; Zhou et al., 2003. Using
these cells, we then conducted side-by-side examinations of acti-
vation and desensitization properties for a set of 17 a4b2
Ã
agonists
that demonstrated selectivity for a4b2
Ã
receptors, displayed a wide
range of agonist activities, and had adequate physical and pharma-
cokinetic profiles. In order to compare our results to those of oth-
ers, we examined two reference compounds in the same in vitro
and in vivo systems. These were ABT-894 and Sazetidine-A, both
known to be a4b2-selective (Xiao et al., 2006; Ji et al., 2007) and
effective in a variety of pre-clinical pain models including the
formalin assay (Cucchiaro et al., 2008; Jain, 2004). Functional activ-
ities observed at the three a4b2
Ã
receptor subtypes, along with var-
ious parameters that may play a role in the activity profile, were
then examined for correlations with analgesic efficacies observed
in a mouse formalin model. We further developed mathematical
models to quantitatively predict the analgesia efficacy as a function
of the in vitro pharmacological and physical profile of the
compounds.
2. Materials and methods
2.1. Chemicals
Acetylcholine (ACh) and atropine were obtained from Sigma (St.
Louis, MO). Novel nicotinic compounds, ABT-894 and Sazetidine-A
were synthesized at Targacept Inc. (Winston-Salem, NC). Dul-
becco’s modified Eagle’s medium(DMEM), fetal bovine serum, heat
inactivated horse serum, L-glutamine, sodium pyruvate, zeocin,
and Hank’s Balanced Salt Solution were obtained from Invitrogen
(Carlsbad, CA). Geneticin and hygromycin B were obtained from
Calbiochem (San Diego, CA).
2.2. Transient transfection
Human a4, b2 and a5 cDNA monomers and b2-a4 dimers were
provided by Dr. Isabel Bermudez (Oxford Brookes University, Ox-
ford, UK). Single a4 and b2 subunits were concatenated by syn-
thetic linkers into dimeric b2-a4 constructs using the T4 ligase
enzyme as specified by the manufacturer (NEB Biolabs, UK). Both
dimers and monomers of a4, a5 and b2 were subcloned into the
pCI plasmid vector (Promega, UK), with modifications of the vector
by oligo-hybridization to contain an AscI and EcoRV restriction site,
and a SwaI site downstream from the SV40 late region.
The FreeStyle 293 Expression System with HEK293F cells (Invit-
rogen, Carlsbad, CA) was used for the transient transfection accord-
ing to the manufacturer’s instructions. To control the expression
level, conditions for transfection were optimized by the number
of cells transfected and the amount and ratio of plasmid DNA used.
Receptor protein expression was optimized by harvesting cells
during a specific post-transfection period and plating cells at a spe-
cific density. Briefly, concatenated dimers of b2-a4 were co-trans-
fected with monomeric a4 for the expression of LS-a4b2, b2 for the
expression of HS-a4b2, or a5 for the expression of a4b2a5. The
cDNA ratio was 4:1 for b2-a4 dimer to a4, b2 or a5 monomer,
and the total amount of cDNA was 20 lg for the transfection of
4 Â 10
7
cells. Transfected cells were maintained in FreeStyle 293
Expression Medium for 48–72 h and then subcultured into poly-
D-lysine coated 96-well plates at a density of 0.8–1 Â 10
6
cells/
ml. Cells were further incubated at 29 °C for 48 h before functional
activities were examined.
2.3. Cell cultures
The SH-EP1 cells stably transfected with human (h) consensus
a4b2 nAChRs and SH-SY5Y human neuroblastoma cells were pro-
vided by Dr. Ronald J. Lukas (Barrow Neurological Institute, Phoe-
nix, AZ). HEK293 cells stably transfected with ha7/RIC3 were
provided by Dr. John Lindstrom (University of Pennsylvania, Phila-
delphia, PA). The rat pheochromocytoma PC-12 cell line, Shooter
subclone, was obtained from Dr. Eric Shooter (Stanford University,
Stanford, CA).
The SH-EP1ha4b2, SH-SY5Y and PC-12 cells were all main-
tained in DMEM with 10% horse serum, 5% fetal bovine serum,
1 mM sodium pyruvate and 4 mM L-Glutamine. Zeocin (0.25 mg/
ml) and hygromycin B (0.13 mg/ml) were used for a4 and b2
814 J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823
selection, respectively, for SH-EP1 ha4b2 cells. HEK ha7/RIC3 cells
were maintained in DMEM with 10% fetal bovine serum and 4 mM
L-glutamine, and zeocin (0.5 mg/ml) and geneticin (0.6 mg/ml)
were used for subunit expression selection. All cells were main-
tained in a humidified atmosphere containing 5% CO
2
in air at
37 °C.
2.4. Membrane potential assay
HEK293F cells transfected with ha4b2
Ã
receptors were incu-
bated at 29 °C with a fluorescent membrane potential dye (Molec-
ular Devices, Union City, CA) in the presence of 0.5 lM atropine in
Hank’s Balanced Salt Solution with 20 mM Tris-HEPES (pH 7.4).
Cells were then challenged with test compounds, and membrane
potential changes were detected using FLIPR
TETRA
(Molecular De-
vices, Union City, CA), with excitation at 510–545 nm and emis-
sion at 565–625 nm. The desensitization properties of
compounds were obtained by treating cells with various concen-
trations of compounds for 30 min (min), followed by challenging
cells with 10 lM ACh while measuring membrane potential
responses.
Under the conditions used, ACh activated HS-, LS-a4b2 and
a4b2a5 receptors with respective EC
50
values of 163 ± 1.2, 456 ±
1.4 and 297 ± 1.4 nM. The responses of ACh (10 lM) were always
measured in parallel in order to normalize the activation or desen-
sitization responses. Data were expressed as the Mean ± SEM from
independent experiments. Values for concentrations producing
50% of the maximal activation or desensitization response (EC
50
or
DC
50
), the maximal activation or desensitization response (E
max
or
D
max
) were generated by fitting concentration–response curves
with nonlinear regression using GraphPad Prism (La Jolla, CA).
2.5. Calcium flux assay
Two days before experiments, SH-EP1ha4b2 cells were cultured
in 96-well plates at a density of 6–8 Â 10
4
cells/well and incubated
at 37 °C overnight. The next day, cells were transferred to an incu-
bator set at 29 °C for 24 h. Cells were loaded with a calcium (Ca)-
sensitive fluorescent dye Calcium 4 (Molecular Devices, Union City,
CA) in modified Earle’s balanced salt solution composed of the fol-
lowing: 4 mM CaCl
2
, 0.8 mM MgSO
4
, 20 mM NaCl, 5 mM KCl, 5 mM
D-glucose, 20 mM Tris-HEPES, and 120 mM N-methyl-D-glucamine,
pH 7.4. The Ca mobilization was detected using FLIPR
TETRA
with
excitation of 470–495 nm and emission of 515–575 nm. The Ca
flux data were normalized as the percentage of the Ca response in-
duced by 10 lM nicotine, and expressed as mean ± SEM.
2.6. Competitive binding assays
All membrane preparation procedures were carried out at 4 °C,
and centrifugation was carried out at 40,000g for 20 min. Sources
for different nAChRs are: rat (r) cortex for ra4b2, SH-EP1ha4b2
cells for ha4b2, HEK ha7/RIC3 cells for ha7, SH-SY5Y for ha3b4
and PC-12 for ra3b4 nAChRs. Rat cortices (Analytical Biological
Services, Wilmington, Delaware) were isolated from adult Spra-
gue–Dawley rats. Cells or tissues were homogenized in ice-cold
phosphate buffered saline (PBS) and centrifuged. After centrifuga-
tion, pellets were then washed, suspended in PBS, aliquoted and
frozen at À20 °C if not immediately used.
The [
3
H]-epibatidine and [
3
H]-nicotine were obtained from
PerkinElmer (Waltham, MA). Protein concentrations were deter-
mined by a Pierce Coomassie Plus Assay Kit (Pierce Chemical Com-
pany, Rockford, IL). Membranes were incubated in PBS in the
presence of radioligands and various concentrations of compounds
for 2 h at 25 °C. Non-specific binding was defined in the presence
of 10 lM epibatidine. Incubation was terminated by rapid filtration
on a multi-manifold tissue harvester (Brandel, Gaithersburg, MD)
using GF/B filters presoaked in 0.33% polyethyleneimine. Radioac-
tivity was determined by liquid scintillation counting using a Per-
kin–Elmer Trilux Microbeta (Waltham, MA).
Binding data were expressed as the average percentage of total
specific binding, and plotted against the log concentration of the
compound. The IC
50
was determined by least squares non-linear
regression. Binding affinity Ki was calculated using the Cheng–
Prusoff equation of Ki = IC
50
/(1 + N/Kd), where N is the concentra-
tion of the radioligand and Kd is the affinity of [
3
H]-epibatidine
or [
3
H]-nicotine defined specifically for the assay under the condi-
tions used.
2.7. Animal care
Adult male CD-1 (ICR) mice weighing approximately 20–25 g
were supplied by Charles River Laboratories (Raleigh, NC). Animals
were housed and cared for in accordance with the ‘‘Guide for the
Care and Use of Laboratory Animals’’ as published by the National
Research Council (1996). All protocols were approved by the Insti-
tutional Animal Care and Use Committee at Targacept Inc.
2.8. Rotarod study
The effects of compounds on locomotor activity were quantita-
tively evaluated with the rotarod test in mice (Karl et al., 2003). For
this study, adult male mice were trained to perform on the accel-
erating rotarod (0–40 RPM over 120 s) apparatus. Training con-
sisted of three trials per day over the course of 3 days. On the
4th day animals (N = 6 per group) were administered test drug or
vehicle subcutaneously, placed on the 3.5 cm rotating rod and
the time/latency required for the mouse to fall from the rod (with
a maximum of 120 s) was recorded. Doses expressed as the base
compound that resulted in a significant decrease in latency com-
pared with vehicle treated animals were considered motor-impair-
ing, and the minimum effective dose (MED) was reported for each
compound.
2.9. Formalin study
The formalin test in mice was performed as described by Dub-
uisson and Dennis (1977). The doses of each compound tested
were chosen based on the rotarod study to exclude the possibility
of side effects affecting motor responses. Briefly, after an acclima-
tion period, test compound, morphine or saline were administered
subcutaneously 20 min before an intra-dermal injection of 25 ll of
2.5% formalin solution into the right hind paw. Once injected, mice
were immediately returned to a clear Plexiglas™ observation
chamber. Phase I of the pain response was defined as 0–9 min,
and Phase II was 10–40 min following formalin injection
(Malmberg and Bannon, 2007). Sessions were videotaped for
viewing and scoring at a later time by raters blinded to the exper-
imental conditions.
Each subject was observed for 1 min at 5-min intervals over a
40-min session, and the time spent licking the affected paw was re-
corded. Several doses of each compound were tested in different
groups of animals (N = 10 per treatment group) and relative anal-
gesia scores on a scale from 0 to 100 were obtained by normalizing
analgesic effects to that of saline and morphine. In every formalin
assay, the effect of 5 mg/kg morphine was tested in parallel as a
positive control with an assigned analgesia score of 100, and the ef-
fect of saline as a vehicle control with a score of 0. Analgesia scores
of each compound tested at a base dose of 3 mg/kg, a common dose
tested for all compounds, were reported and used for correlation
analyses. The analgesic effect of a compound was compared to
J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823 815
the saline control using one-way ANOVA followed by Dunn’s post
hoc, with P < 0.05 considered significant.
2.10. In vivo concentration evaluations
Plasma and brain concentration in mice coincident with tim-
ing of the behavioral evaluations in the formalin study were as-
sessed using a cassette dosing method similar to that described
by Berman and colleagues (Berman et al., 1997), where up to five
compounds were administered subcutaneously (N = 6 per group)
in combination at 1.5 lmol/kg each. All other parameters, includ-
ing timing of manipulations and administration of formalin, were
in line with the formalin test procedures described above. Follow-
ing drug administration, formalin was injected at 20 min, plasma
and brain samples were collected from half of the subjects at
25 min (corresponding to midpoint of Phase I) and from the
remaining subjects at 45 min (corresponding to midpoint of
Phase II). Quantitative analyses of the plasma and brain samples
were performed by HPLC-MS/MS with a sensitivity range up to
1000 nM. Results fell out of the sensitivity range are reported
as >1000 nM.
2.11. Physical properties of novel nicotinic compounds
Based on the compound structure, Pipeline Pilot (Accelerys, Inc.,
San Diego, CA) was used to determine the molecular properties for
each compound, including the number of hydrogen-bond (H-bond)
donors, the partition coefficient for n-octanol/water CLog P, the
distribution coefficient Log D at pH of 7.4, the molecular polar sur-
face area (PSA) and the molecular weight.
2.12. Principal components analysis
Principal component analysis (PCA) described by Jolliffe (1986)
was used to compare the differences of chemical structures of nic-
otinic compounds. PCAs were determined from 134 structural fea-
tures, with all calculations carried out by Molecular Operating
Environment (Chemical Computing Group Inc., Montreal, QC,
Canada). PCA reduces the dimensionality of molecular features into
a small number of uncorrelated variables (principal components),
with the first principal component accounting for as much variabil-
ity in the original data as possible, and each succeeding component
accounting for as much of the remaining variance as possible. Thus,
PCA can provide a lower-dimensional picture using two or three
dimensional plots, to show the relative distribution of compounds
in high dimensional chemical space.
2.13. Correlation analysis and statistics
In vitro pharmacological and physical parameters included
binding affinities at ra4b2
Ã
nAChRs, the number of H-bond donors
and receptor activation and desensitization potencies at ha4b2a5,
LS- and HS-a4b2 subtypes. In all cases, Ki, EC
50
and DC
50
values
were transformed by taking the log(10) of each value. Those num-
bers were then used for the correlation analyses. Correlations be-
tween analgesia scores and in vitro parameters were analyzed by
multiple linear regression (MLR), and coefficients of determination
(r
2
) were calculated using Pipeline Pilot (Accelerys Inc., San Diego,
CA). Significance tests of an individual coefficient in the regression
model, variance ratio (F) and probability factor related to F-ratio
(P-value) were performed or determined using Microsoft Excel
(Redmond, WA). Statistical qualities of MLR equations were judged
by parameters of coefficient of determination, variance ratio and
probability factor related to F-ratio.
3. Results
3.1. Structures of novel nicotinic compounds
The novel nicotinic compounds selected for this study were
chosen based on their chemical structural diversity, their relative
selectivity for a4b2
Ã
receptors, and their broad range of agonist
activities at a4b2
Ã
receptors. The structures of five compounds
used in the present studies are shown in Fig. 1A. Others have not
been revealed due to proprietary and patent-related issues. How-
ever, the structural differences of the remaining compounds as
compared to these five novel a4b2
Ã
-selective compounds, as well
as ABT-894 and Sazetidine-A, are graphically depicted in a three
dimensional picture using the method of principal component
analysis (Fig. 1B). As can be seen, a relatively diverse group of
chemotypes was included in our studies.
3.2. The selectivity for a4b2
Ã
receptors by novel nicotinic compounds
To investigate the selectivity for a4b2
Ã
receptors, competitive
binding assays were carried out with several nAChR subtypes.
The resulting Ki values are shown in Table 1. As demonstrated, Ki
values were similar between human and rat tissues, both for
a4b2
Ã
and a3b4 receptors. All compounds tested demonstrated
high affinity for a4b2
Ã
receptors, with Ki values less than 100 nM
at both human and rat a4b2
Ã
receptors with the exception of Com-
pound 15, which had a Ki value of 15 nM at ha4b2
Ã
but 330 nM at
ra4b2
Ã
receptors. Additionally, these compounds were more than
20-fold selective for ha4b2
Ã
over ha7 receptors, with the exception
of Compounds 9 and 11 which were only 7- and 3-fold selective,
respectively. Moreover, all of the novel nicotinic compounds were
more than 44-fold selective for ha4b2
Ã
over ha3b4 receptors.
For ABT-894 and Sazetidine-A, Ki values were all in the sub-
nanomolar range for both ha4b2
Ã
and ra4b2
Ã
receptors. Both com-
pounds were more than 100-fold selective for ha4b2
Ã
over ha7 and
ha3b4 receptors. Although an exact comparison is impossible due
to the differences of tissues and radioligands used, our results are
consistent with the previous reports that ABT-894 and Sazeti-
dine-A are potent and selective a4b2
Ã
agonists Xiao et al., 2006;
Ji et al., 2007.
3.3. Activation and desensitization of a4b2
Ã
receptors by novel
nicotinic compounds
Transient expression of ha4b2
Ã
nAChRs was achieved in
HEK293F cells by co-transfecting concatenated b2-a4 dimers with
monomeric b2, a5 or a4 for the expression of HS-a4b2, a4b2a5 or
LS-a4b2, respectively. To ensure that a nicotinic, a4b2
Ã
-mediated
mechanism of action is involved for each of these a4b2
Ã
receptor
types, the responses induced by 10 lM acetylcholine (ACh) in the
presence of various concentrations of dihydro-b-erythroidine and
mecamylamine, two different nicotinic receptor antagonists, were
determined. At each receptor subtype, full inhibition of ACh-
evoked responses was observed for both antagonists. The IC
50
val-
ues were 135, 88 and 161 nM for dihydro-b-erythroidine, and
1902, 249 and 236 nM for mecamylamine at HS-a4b2, a4b2a5
and LS-a4b2 subtypes, respectively (data not shown).
Subsequently the activation and desensitization activities of 17
novel nicotinic compounds were examined using these cells. The
activation and desensitization curves for Compounds 3, 4, 5, 7,
and 8 are shown in Fig. 2. The EC
50
and DC
50
values of all test com-
pounds at each ha4b2
Ã
receptor subtype are listed in Table 2. As
shown in Fig. 2A and Table 2, activation efficacies for these
compounds ranged from partial to full agonism, with some com-
pounds showing no detectable activity (EC
50
> 10,000 nM) over
816 J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823
Compound 7 Compound 8 Compound 5 Compound 3 Compound 4
(A)
(B)
Fig. 1. Structures and the structural differences of nicotinic compounds. (A) Structures of novel nicotinic compounds. (B) Principal component analysis of structural features
of compounds. The distribution of 19 compounds in the space of the first three principal components determined from 134 structural features is shown from two different
views of the 3-dimentional graph. The arrow indicates the rotation direction of the graph. The color of red, green, magenta, goldenrod, cyan, navy-blue and dark-gray
represent Compounds 3, 4, 5, 7, 8, ABT-894 and Sazetidine-A, respectively. Variances on the PCA1, PCA2 or PCA3 coordinate suggest the structural differences of compounds.
Table 1
The binding affinity of novel nicotinic compounds at different types of nAChRs.
Compound Ki ± SEM, nM
r_a4b2 h_a4b2 h_a7 r_a3b4 h_a3b4
ABT-894 0.9 ± 0.1 0.9 ± 0.2 750 ± 335 560 ± 114 180 ± 28
Sazetidine-A 0.05 ± 0.0 0.26 ± 0.2 3300 ± 745 3000 ± 630 30 ± 2
Compound 1 7 ± 1.4 2 ± 0.3 450 ± 60 330 ± 42 100 ± 19
Compound 2 6 ± 0.6 5 ± 0.7 420 ± 52 580 ± 144 310 ± 31
Compound 3 64 ± 11.5 16 ± 2.9 65,000 ± 13,622 9700 ± 1836 5100 ± 1086
Compound 4 8 ± 1.2 9 ± 3.4 990 ± 385 720 ± 127 420 ± 166
Compound 5 2 ± 1.1 1 ± 0.4 550 ± 224 1300 ± 367 800 ± 238
Compound 6 63 ± 11.5 6 ± 2.0 520 ± 96 320 ± 59 420 ± 96
Compound 7 1 ± 0.6 2 ± 0.4 76 ± 17 510 ± 159 790 ± 259
Compound 8 11 ± 9.7 3 ± 1.2 2000 ± 371 2200 ± 307 3400 ± 1671
Compound 9 12 ± 1.9 9 ± 2.1 70 ± 5 1100 ± 422 1800 ± 512
Compound 10 7 ± 2.3 2 ± 0.5 87 ± 26 300 ± 44 530 ± 73
Compound 11 110 ± 46 11 ± 2.9 29 ± 11 1700 ± 817 1300 ± 476
Compound 12 5 ± 3 5 ± 3.2 1000 ± 338 10,000 ± 8891 750 ± 75
Compound 13 20 ± 8.0 6 ± 2.4 59,000 ± 27,839 49,000 ± 21,222 9700 ± 1705
Compound 14 77 ± 32 33 ± 9.3 720 ± 307 260 ± 127 2000 ± 810
Compound 15 330 ± 98 15 ± 2.5 300 ± 54 1400 ± 174 2000 ± 890
Compound 16 25 ± 7.7 14 ± 2.8 790 ± 73 330 ± 113 1100 ± 159
Compound 17 38 ± 17 25 ± 13.1 1200 ± 464 670 ± 135 1100 ± 498
Competitive binding assays were carried out using [
3
H]-epibatidine and membranes made from rat cortical membranes, SH-EP1 ha4b2, HEK ha7/RIC3, PC-12 and SH-SY5Y
cells. The Ki values listed are mean ± SEM of 3–8 experiments. A majority of the compounds demonstrated high affinity and relative selectivity for a4b2
Ã
receptors over a7 or
a3b4 receptors.
r: rat.
h: human.
J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823 817
the concentration range tested (0.01–10,000 nM) and experimental
conditions used. Note that most of the compounds in this study
having no detectable agonist activity were not simply antagonists.
Nearly all of the compounds demonstrated partial agonist re-
sponses in a calcium (Ca) flux assay in SH-EP1 cells with mixed
subtypes of HS- and LS-a4b2 receptors, where sodium in the assay
buffer was replaced with N-methyl-D-glucamine to reveal any ago-
nist activity (Table 2). Compounds 1 and 6, two compounds that
did not show any agonist activity in the Ca flux assay, were equally
potent and efficacious at desensitization and antagonism of ha4b2
Ã
receptors in SH-EP1 cells (data not shown).
Regardless of the activation efficacies, almost all compounds
examinedproducedfull desensitizationat all three ha4b2
Ã
subtypes
(Fig. 2B and Table 2). We observed that for any given compound, the
DC
50
value was usually lower than the EC
50
value at the same recep-
tor subtype. While DC
50
values for a specific compound at the three
different a4b2
Ã
receptors might be similar, in several cases (includ-
ing Compounds 7 and 8), their EC
50
values differed greatly.
3.4. Activation and desensitization of a4b2
Ã
receptors by ABT-894 and
Sazetidine-A
We also examined the functional properties of ABT-894 and Saz-
etidine-A, using the same HEK293F cells expressing human HS-
a4b2, a4b2a5 and LS-a4b2 receptors. Concentration-dependent in-
creases in responses were observed for both agonists (Fig. 3). While
ABT-894 partially activated HS-a4b2 and a4b2a5, it fully activated
LS-a4b2. In contrast, Sazetidine-A fully activated HS-a4b2 but only
partially activated a4b2a5 and LS-a4b2 (Fig. 3A).
Unlike activation of a4b2
Ã
receptors by these two agonists, their
desensitization was more potent, and full inhibition of the ACh-in-
duced response was observed at all three receptor subtypes
(Fig. 3B). For each agonist, DC
50
values were comparable at all three
a4b2
Ã
receptors (Table 2). The Ca flux results for ABT-894 are sim-
ilar to those reported previously (Ji et al., 2007). The results for Saz-
etidine-A are in line with previous reports that it is a full agonist at
HS-a4b2, a poor partial agonist at LS-a4b2 (Carbone et al., 2009)
and it is more potent and efficacious at desensitization than at acti-
vation of the a4b2
Ã
receptors (Xiao et al., 2006).
3.5. Analgesic effects and rotarod study of novel nicotinic compounds
The formalin assay in mice was performed for the same set of 19
compounds. The doses for the assay were selected based on results
from a mouse rotarod study, in which latency to fall in the acceler-
ating rotarod was examined. The lowest doses which significantly
impaired coordination in the rotarod study (MED, Table 3) or high-
er were not included in the formalin assay, to exclude the possibil-
ity of a non-analgesic effect of compounds resulting in impairment
of the animal’s ability to elicit a motor response.
A normalized analgesia score was obtained by comparing the
analgesic effect of each dose of compounds to that of saline (the
vehicle control) and 5 mg/kg morphine (the positive control)
tested within the same experiment. The saline and morphine con-
trol in each phase of the assay were assigned a score of 0 and 100,
respectively. The analgesia scores during Phase I and Phase II for
each compound (3 mg/kg, s.c.) are reported in Table 3. In general,
an analgesia score of 40 or higher is reflective of a significant anal-
gesic effect of a compound compared to the saline control. It is of
note that both ABT-894 and Sazetidine-A demonstrated significant
analgesic effects in both Phases of the formalin test. These results
align with previously published data for Sazetidine-A in a rat for-
malin model (Cucchiaro et al., 2008).
Along with ABT-894 and Sazetidine-A, Compounds 2 and 7–10
displayed significant effects in Phase I of the formalin assay. The
compounds that were effective in Phase I of the assay also showed
significant analgesia in Phase II. As well, Compounds 4, 5, 12, 15
and 16 were effective in Phase II of the assay.
3.6. Physical properties and in vivo concentrations of novel nicotinic
compounds
Physical properties of a compound are known to correlate with
in vivo bioavailability and the ability of a compound to cross the
blood–brain barrier (Modi, 2003; Wang et al., 2007). As shown in
Table 3, the number of H-bond donors for all test compounds
was less than or equal to 2, while their CLogP values varied be-
tween À0.24 and 2.21 with most of them around 2. Whereas nearly
all of the compounds displayed values for Log D at pH 7.4 between
À1 and 1, their PSA values are between 17 and 62. The molecular
weight of these compounds range from 197.27 to 323.43 with a
median of 230.27. In addition, all 17 novel nicotinic compounds
as well as ABT-894 and Sazetidine-A showed no violations of Lipin-
ski’s rules.
In separate studies, plasma and brain samples were collected
from mice treated with compounds for examination of exposure
levels. Compound concentrations, at time points of 25 and
45 min (corresponding to midpoint of Phase I and Phase II of the
Fig. 2. Activation and desensitization of a4b2
Ã
receptors by novel nicotinic compounds. HEK293F cells separately expressing human a4b2a5, LS- and HS-a4b2 receptors were
treated with compounds along with (A) or 30 min before (B) 10 lM ACh challenge. Membrane potential data are mean ± SEM of more than three experiments. (A) Compounds
tested displayed a range of agonist activities at a4b2
Ã
receptors. (B) Each compound produced full desensitization of the ACh-induced response with comparable DC
50
values
at all three a4b2
Ã
subtypes.
818 J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823
formalin test, respectively) after compound administration, are
listed in Table 3. All compounds tested demonstrated brain pene-
tration, with detectable levels in the brain at both time points. A
majority of the compounds showed good in vivo plasma and brain
concentrations. Considering that doses used in this cassette dosing
study were approximately 10-fold lower than those in the formalin
study, animals in the formalin assay would have been exposed to
nanomolar to micromolar concentrations of each compound both
peripherally and in the CNS.
3.7. Correlation between analgesia scores and a4b2
Ã
receptor activities
of novel nicotinic compounds
To investigate the role that a4b2
Ã
nAChR activation or desensi-
tization plays in analgesia, multiple linear regression (MLR) was
used to analyze correlations between analgesic effects of the com-
pounds and their HS-, LS-a4b2 and a4b2a5 functional activities,
alone or in combination with other in vitro profiles (i.e. a4b2
Ã
binding affinities, physical properties including number of H-bond
donors, CLog P, PSA or Log D at pH 7.4), or in vivo plasma and brain
concentrations. In the MLR analyses, the Ki values obtained using
rat cortical membranes were used for the analyses. Previous stud-
ies have shown that binding affinity of competitive inhibitors of
nicotinic binding sites is very similar between rat and mouse brain
(Marks et al., 1986). The results of these MLR analyses along with
the variance ratio (F) and P-values are shown in Table 4. All F val-
ues shown in the table indicate that at least one of the coefficients
in each model was significant.
For all in vitro and in vivo parameters examined individually,
ra4b2
Ã
binding affinities showed the most highly significant corre-
lation with analgesia scores for both Phase I and Phase II. Although
inclusion of the number of H-bond donors did not achieve signifi-
cance for Phase I, it did result in an increase in correlation for Phase
II with an improved coefficient of determination (r
2
) from 0.41 to
0.70 (P = 0.001). Inclusion of in vivo concentration data neither
made a significant contribution nor improved the correlation (data
not shown).
We next explored the addition of a third term to the MLR
equations that include both the Ki and the number of H-bond do-
nors of the compound. For correlations with analgesic efficacies
of both Phase I and Phase II, adding activation potencies for all
three a4b2
Ã
subtypes maintained but did not significantly im-
prove the relationships. The r
2
values were 0.62, 0.62 or 0.53 as
compared to 0.53 for Phase I, and 0.72, 0.74 or 0.71 as compared
to 0.70 for Phase II after adding EC
50
values for HS-a4b2, a4b2a5
or LS-a4b2, respectively. No statistical significance was observed
for any of these changes in the r
2
values. In contrast, adding
desensitization potencies as a third term in the MLR equations
provided a significant improvement in correlations for Phase I
analgesia scores at all three subtypes of a4b2
Ã
receptors. The r
2
values were 0.67, 0.70 or 0.66 as compared to 0.53 for Phase I
after addition of DC
50
values at HS-a4b2, a4b2a5 or LS-a4b2,
respectively, with respective P-values of 0.02, 0.01 or 0.03. In
addition, the r
2
value significantly increased from 0.70 to 0.79
(P = 0.0001) for Phase II when the DC
50
values at a4b2a5 were
added. Interestingly, a significant effect on the r
2
for Phase II
was not seen when the HS- or LS-a4b2 DC
50
values were consid-
ered. Given the sample size of 19 for this study, MLR analyses
with more than three terms were not performed due to the po-
tential for over-fit.
Thus, MLR analysis demonstrated that better analgesia scores
were obtained for compounds showing higher affinities and great-
er desensitization potencies at a4b2
Ã
receptors, suggesting the
involvement of a4b2
Ã
desensitization, especially of a4b2a5, in pain
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J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823 819
Fig. 3. Activation and desensitization of a4b2
Ã
receptors by ABT-894 and Sazetidine-A. HEK293F cells separately expressing human a4b2a5, LS- and HS-a4b2 nAChRs were
treated with ABT-894 or Sazetidine-A along with (A) or 30 min before (B) 10 lM ACh challenge. Membrane potential data are mean ± SEM of 3 experiments. (A) ABT-894
partially activated HS-a4b2 and a4b2a5 but fully activated LS-a4b2 receptors. Sazetidine-A (Saz) was a potent full agonist at HS-a4b2 and a partial agonist at both a4b2a5
and LS-a4b2 subtypes. (B) Both agonists fully inhibited the ACh-induced response at all three a4b2
Ã
receptors, whereas Sazetidine-A was more potent compared to ABT-894.
Table 3
Analgesia scores, rotarod study, physical and pharmacokinetic properties of novel nicotinic compounds.
Compound Analgesia score RotaRod Physical Property In vivo concentrations
C
25
± SEM, nM C
45
± SEM, nM
Phase I Phase II MED, mg/kg H_Donor CLogP LogD pH 7.4 PSA MW Plasma Brain Plasma Brain
ABT-894 44
*
79
*
>30 1 1.98 À0.01 28 244.12 165 ± 12 426 ± 60 72 ± 53 446 ± 67
Sazetidine-A 125
*
100
*
NR 2 1.12 À0.79 54 260.33 58 ± 5 7 ± 0 20 ± 5 6 ± 0
Compound 1 5 22 56 1 2.21 À0.49 28 231.34 >1000 280 ± 6 >1000 290 ± 4
Compound 2 42
*
47
*
>30 1 0.36 À0.99 42 203.28 >1000 >1000 >1000 >1000
Compound 3 4 4 >100 1 2.13 À1.00 32 236.35 489 ± 171 41 ± 4 85 ± 40 36 ± 2
Compound 4 19 42
*
30 1 À0.24 À2.07 32 198.24 257 ± 85 8 ± 2 512 ± 238 6 ± 0
Compound 5 15 48
*
>100 1 0.58 À0.35 47 235.28 362 ± 94 102 ± 4 367 ± 182 109 ± 7
Compound 6 2 30 30 1 0.8 À0.92 28 229.32 >1000 405 ± 25 >1000 414 ± 33
Compound 7 40
*
61
*
>30 1 0.39 À0.12 37 223.25 277 ± 74 208 ± 10 279 ± 147 207 ± 16
Compound 8 54
*
44
*
>30 1 À0.21 À0.31 61 230.27 740 ± 224 64 ± 8 161 ± 71 54 ± 2
Compound 9 62
*
73
*
>100 1 1.86 À0.15 17 215.31 >1000 >1000 >1000 >1000
Compound 10 84
*
116
*
>100 1 1.86 À0.15 17 215.31 >1000 >1000 >1000 >1000
Compound 11 0 0 >100 1 1.69 0.88 25 212.29 481 ± 27 18 ± 1 269 ± 70 13 ± 0
Compound 12 19 57
*
100 1 1.69 0.88 25 212.29 505 ± 79 12 ± 0 308 ± 66 7 ± 0
Compound 13 7 15 >100 1 1.77 À1.29 62 323.43 177 ± 22 6 ± 0 84 ± 1 4 ± 0
Compound 14 0 0 >30 1 0.87 À1.37 48 300.40 >1000 166 ± 9 >1000 81 ± 4
Compound 15 7 48 10 0 2.01 0.87 30 197.27 >1000 4 ± 2 25 ± 15 6 ± 2
Compound 16 28 48
*
>10 1 1.22 À1.56 31 286.42 34 ± 16 12 ± 1 12 ± 4 10 ± 0
Compound 17 0 0 >30 1 1.77 À1.03 31 286.42 >1000 >1000 >1000 >1000
Analgesia scores, rotarod study, physical and pharmacokinetic properties of novel nicotinic compounds.
The analgesia scores of each compound were obtained in Phase I and Phase II of the mouse formalin assay (N = 10), and the minimum effective dose (MED) that impaired the
locomotor ability were from mouse rotarod study (N = 6). Physical properties were determined based on compound structure. The plasma and brain concentrations were
obtained at 25 and 45 min after injection of compounds in mice following a similar procedure of the formalin assay (N = 6). ABT-894 and Sazetidine-A both demonstrated
significant analgesic effects.
MED: the minimum effective dose.
H_Donor: number of H-bond donors.
PSA: the molecular polar surface area.
MW: molecular weight.
NR: not reported.
*
Significant analgesic activity compared to vehicle control.
820 J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823
3.8. Modeling of analgesic effects of novel nicotinic compounds using
desensitization potencies at a4b2a5 receptors
Considering ligand affinities at ra4b2
Ã
receptors, the number of
H-bond donors (H_Donor) and DC
50
values at ha4b2a5 receptors,
equations were generated to quantitatively predict analgesia
scores separately for Phase I and Phase II of the formalin assay
using MLR models. The model with the highest coefficient of deter-
mination value was chosen from all the models generated. The
mathematical equations to predict analgesic efficacy of Phase I
(Score I) and Phase II (Score II) were:
Score I ¼ 159:9 À58:8 ÂH Donor À26:6 ÂLogðKiÞ À17:8
ÂLogðDC50Þ
Score II ¼ 134:6 À40:2 ÂH Donor À19:5 ÂLogðKiÞ À25:6
ÂLogðDC50Þ
Subsequently, the predicted analgesia scores with the demon-
strated analgesic efficacies obtained in the formalin test for each
compound were compared. Plots of predicted vs. observed analge-
sic efficacy scores for novel nicotinic compounds are shown in
Fig. 4A and B for Phase I and Phase II, respectively. Correlation coef-
ficients of determination of 0.70 and 0.79 (F values of 11.4 and
18.6, respectively) were obtained for Phase I and Phase II between
predicted and observed analgesia scores for the set of compounds
tested, indicating that these models were suitable to predict anal-
gesic efficacy in the mouse formalin test. Additionally, these mod-
els suggest that the lower the number of H-bond donors, the Ki and
the DC
50
values are, the higher the analgesia scores will be for the
compound, again consistent with the observations.
4. Discussion
The novel and important finding of the current studies is the di-
rect correlation between desensitization of a4b2
Ã
nAChRs and the
observed analgesic efficacy in both acute (Phase I) and persistent
(Phase II) pain in a mouse formalin model, suggesting that desen-
sitization of a4b2
Ã
receptors may play a crucial role in the analge-
sic effects of nicotinic compounds. Although the role that nAChRs
play in antinociception has mainly focused on a4 and b2 subunits
and their activation (Damaj et al., 2007; Gao et al., 2010; Marubio
et al., 1999), the specific a4b2
Ã
subtypes involved, their functional
requirements and the underlying mechanisms have not been fully
elucidated. By determining the in vitro pharmacological profiles of
a group of novel nicotinic compounds at several prevalent sub-
types of a4b2
Ã
receptors and investigating their in vivo properties
in a controlled and normalized assessment of nociception, we be-
gin to get a clearer picture of how these receptors may be function-
ing in the mediation of pain.
In agreement with previous reports (Cucchiaro et al., 2008; Da-
maj et al., 2007; Gao et al., 2010; Marubio et al., 1999), the present
studies demonstrate that a4b2
Ã
nAChRs play a role in the media-
tion of analgesic effects, based on the observation that there is a
good correlation between the analgesic efficacy and the a4b2
Ã
affinity alone. These findings are consistent with our suggested
role for desensitization since the affinity of agonists for nAChRs
has been shown to influence both the onset of, and recovery from
desensitization (Wang and Sun, 2005). The additional positive cor-
relation between analgesic effects and the number of H-bond do-
nors may be the consequence of the greater blood–brain barrier
penetration (Modi, 2003).
As an extension of previous findings noting the involvement of
agonism at a4b2
Ã
receptors in analgesia, we report herein the no-
vel finding that desensitization of a4b2
Ã
receptors may drive part
of the antinociceptive outcome. Using a formalin assay in mice,
we observed that Sazetidine-A, which is known as a desensitizer
instead of activator of a4b2
Ã
receptors (Cucchiaro et al., 2008; Xiao
et al., 2006), displays robust analgesic effects both for Phase I and
Phase II. Likewise, Compound 10 demonstrates good analgesic ef-
fects, activates a4b2
Ã
receptors poorly, but desensitizes them po-
tently. Our molecular modeling approaches revealed that when
receptor desensitization rather than activation activities at a4b2
Ã
receptors are considered, there is a better correlation between
analgesia scores and combined in vitro properties. Exceptions to
this correlation are Compounds 11, 12, 13 and 17 which all po-
tently desensitize a4b2
Ã
receptors but are ineffective in the forma-
lin model. Table 3 shows that the brain levels of Compounds 11–13
are low at both time points examined. Due to poor brain perme-
ability, these compounds may not have reached critical levels nec-
essary for activity at the central site of action. The brain
concentration of Compound 17 is at the other end of the spectrum,
reaching micromolar levels. At the 3 mg/kg, s.c. dose tested in the
formalin model, the exposure levels of Compound 17 may have
been too high, exceeding the levels needed for activity in the assay.
Unfortunately the 3 mg/kg dose was the lowest tested in the model
for this compound, not allowing us to determine if a lower dose,
possibly one achieving nanomolar levels in the brain approximat-
ing its DC
50
, may have been efficacious.
Our results suggest that although all three a4b2a5, HS- and
LS-a4b2 subtypes assessed are involved, it is desensitization of
Table 4
Correlation of analgesia scores with a4b2
Ã
receptor activities and physical properties of novel nicotinic compounds.
In vitro parameters analyzed Coefficient of determination (r
2
) Variance ratio (F) P-value
Score_Phase I Score_Phase II Score_Phase I Score_Phase II Score_Phase I Score_Phase II
Ki_a4b2 0.47
*
0.41
*
15.0 11.7 0.001 0.003
Ki_a4b2, H_Donor 0.53 0.70
*
8.9 19.0 0.001, 0.18 <0.0001, 0.001
Ki_a4b2, H_Donor, EC
50
_HS-a4b2 0.62 0.72 8.1 12.6 0.002, 0.04, 0.08 <0.0001, 0.003, 0.44
Ki_a4b2, H_Donor, EC
50
_a4b2a5 0.62 0.74 8.2 14.4 0.02, 0.04, 0.07 0.001, 0.001, 0.16
Ki_a4b2, H_Donor, EC
50
_LS-a4b2 0.53 0.71 5.6 12.2 0.002, 0.36, 0.73 <0.0001, 0.01, 0.59
Ki_a4b2, H_Donor, DC
50
_HS-a4b2 0.67
*
0.76 10.4 15.5 0.02, 0.03, 0.02 0.001, 0.0003, 0.09
Ki_a4b2, H_Donor, DC
50
_a4b2a5 0.70
*
0.79
*
11.4 18.6 0.01, 0.01, 0.01 0.0003, 0.03, 0.0001
Ki_a4b2, H_Donor, DC
50
_LS-a4b2 0.66
*
0.73 9.6 13.4 0.01, 0.04, 0.03 0.0003, 0.001, 0.27
Correlation of analgesia scores with a4b2
Ã
receptor activities and physical properties of novel nicotinic compounds.
Parameters analyzed were obtained as described in the Methods. MLR was used to analyze the correlations. For analgesic scores in Phase I and Phase II of the mouse formalin
assay, the coefficient of determination (r
2
) and the variance ratio (F) for each set of parameters analyzed are listed separately, while the P-values for each individual parameter
analyzed are listed correspondingly under each conditions.
H_Donor: number of H-bond donors.
Bold numbers are with significance.
*
Significant contribution by the last term added.
J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823 821
a4b2a5 receptors that seems to play a more prominent role in the
antinociceptive action of nicotinic compounds. Correlations for
Phase I are significantly improved from an r
2
value of 0.53 to
0.67 and 0.66 when HS- and LS-a4b2 DC
50
values are considered,
respectively. More profoundly, considering the DC
50
at a4b2a5
takes the r
2
from 0.53 to 0.70, placing a focus on the involvement
of the a5-containing receptor. For Phase II analgesia scores, adding
HS- or LS-a4b2 desensitization potencies did not improve the cor-
relations significantly. Considering the a4b2a5 DC50 value signif-
icantly increased the r
2
from 0.70 to 0.79 for Phase II, and
strongly suggested a more prominent role for a4b2a5 nAChRs in
the modulation of pain in the formalin assay.
Phase I responses in the formalin assay are generally attributed
to activation of peripheral nociceptors and Phase II responses are
primarily due to a central loci of action (Alsharari et al., 2012).
With this understanding and the significant effects observed when
considering desensitization at each of the a4b2 subtypes, one may
suspect a role for all three receptor subtypes in the periphery in
relation to Phase I formalin responses, but a more prominent role
for a4b2a5 receptors centrally during Phase II responses.
The significance of the desensitization results with the a4b2a5
subtype are supported by previous reports showing that activation
of a4b2
Ã
receptors is necessary but not sufficient to produce analge-
sia (Gao et al., 2010). Additionally, a5
Ã
receptors have been shown
to be expressed in the spinal cord (Vincler and Eisenach, 2005), a
key site in pain pathways, and have been previously implicated in
analgesia (Jackson et al., 2010; Vincler and Eisenach, 2004, 2005).
How might desensitization be promoting analgesia? Is desensi-
tization the mechanism or is it a marker for other nicotinic recep-
tor activity? During prolonged exposure to a4b2
Ã
-selective
agonists, changes in receptor composition may be induced (Govind
et al., 2012; Lester et al., 2009; Fenster et al., 1999.). Altered incor-
poration of a5 subunits into a4b2-containing receptors, or modu-
lation of a4b2a5 receptor levels or ratios may be realized (Mao et
al., 2008). With such alteration of receptors, changes in function
may occur since a4b2a5 subtypes are more sensitive to desensiti-
zation than to activation (Brown et al., 2007; Gerzanich et al.,
1998; Kuryatov et al., 2008). Desensitization of a4b2
Ã
receptors
in return may lead to increased opioid release, decreased serotonin
release, changes in other neurotransmitters or second messengers
that can produce analgesic effects at the supraspinal, spinal or pri-
mary afferents level (Cucchiaro et al., 2008; Curzon et al., 1998;
Tassonyi et al., 2002). Recent reports have also described a co-ago-
nist character of nicotinic agonists (Prickaerts et al., 2012; Zwart
and Vijverberg, 2000). This property has been described at concen-
trations below those needed for intrinsic agonism; levels similar to
those at which desensitization is observed (Fenster et al., 1999;
Marks et al., 2010). Desensitization may be a surrogate measure
for compounds that may act as a co-agonist in vivo, enhancing
the activity of the endogenous ligand acetylcholine. At this point
in our understanding of nicotinic receptors in pain, one should
not discount the potential that nicotinic ligands may utilize multi-
ple mechanisms in analgesia, including desensitization, receptor
upregulation, co-agonism and intrinsic agonism.
We have described a role for desensitization in the modulation
of Phase I and Phase II responses in the formalin model. Future
work should extend the analyses to additional pain models to
understand the breadth of the role desensitization of nicotinic
receptors plays, especially that of a4b2a5. As ligands with different
nicotinic pharmacologic profiles may be effective in different pain
models, we may find that nicotinic receptor desensitization may
correlate with effectiveness against some types of pain and not
others.
In summary, we have demonstrated in the present studies that
compounds which are more potent at desensitization of a4b2
Ã
nAChRs display better analgesia scores in the formalin test. Consis-
tent with these observations, consideration of desensitization
properties at a4b2
Ã
nAChRs, especially at a4b2a5 receptors, in
MLR significantly improve correlations with efficacies of analgesia.
Thus, a4b2
Ã
nAChR desensitization may contribute to efficacy in
the mediation of pain, and represent an additional mechanism
for analgesic effects mediated by nicotinic agonists. Accordingly,
consideration of a4b2a5 receptor desensitization during the dis-
covery process should aid in the identification of nicotinic ligands
that have potential as pain therapeutics.
Acknowledgements
We thank M. Kiser and L.A. Guerra for generating binding and Ca
flux data, respectively; J.M. Stukes, S.P. Wene and RC Pritchard for
collecting in vivo concentration data; and Drs. J.P. Strachan, S. Akir-
eddy, B. Bhatti, S. Breining, M. Melvin and L. Miao, and R. Heemstra,
R. Whitaker and T. Showalter for their efforts to synthesize novel
nicotinic compounds. All of them are employees at Targacept Inc.
We also thank Dr. M.I. Damaj, Professor of Pharmacology at Virginia
Commonwealth University, for his constructive comments and
suggestions.
Score I = 159.9 − 58.8H_Donor–26.6
Log (Ki)–17.8 Log (DC50)
r
²
=0.79
0
20
40
60
80
100
0 20 40 60 80 100 120 140
Analgesia Score_Observed
Phase II
r
²
=0.70
-20
0
20
40
60
80
100
0 20 40 60 80 100 120 140
A
n
a
l
g
e
s
i
a

S
c
o
r
e
_
P
r
e
d
i
c
t
e
d
Analgesia Score_Observed
Phase I
Score II = 134.6 − 40.2 H_Donor–19.5
Log (Ki) –25.6 Log (DC50)
(A) (B)
Fig. 4. Modeling of analgesic effects of novel nicotinic compounds using desensitization potencies at a4b2a5 receptors. Considering the Ki obtained with rat brain tissue, the
number of H-bond donors (H_Donor) and the DC
50
at ha4b2a5 nAChRs, models were generated to quantitatively predict analgesia scores separately for Phase I (A) and Phase
II (B) of the mouse formalin test. Mathematical equations and coefficients of determination (r
2
) are listed for each Phase. Significant correlations were observed between
predicted and observed analgesia scores for both Phases of the formalin assay.
822 J. Zhang et al. / European Journal of Pharmaceutical Sciences 47 (2012) 813–823
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