In vitro pharmacological characterization of AM1241: A protean agonist at the cannabinoid CB2 receptor?

Article abstract of DOI:10.1038/sj.bjp.0706838

Background and purpose: The CB₂ receptor has been proposed as a novel target for the treatment of pain, and CB₂ receptor agonists defined in in vitro assays have demonstrated analgesic activity in animal models. Based on its in vivo analgesic efficacy, AM1241 has been classified as a CB₂-selective agonist. However, in vitro characterization of AM1241 in functional assays has not been reported. Experimental approach: In this study, AM1241 was characterized across multiple in vitro assays employing heterologous recombinant receptor expression systems to assess its binding potencies at the human CB₂ and CB₁ receptors and its functional efficacies at the human CB₂ receptor. Key results: AM1241 exhibited distinct functional properties depending on the assay conditions employed, a unique profile in contrast to those of the agonist CP 55,940 and the inverse agonist SR144528. AM1241 displayed neutral antagonist activities in FLIPR and cyclase assays. However, when cyclase assays were performed using lower forskolin concentrations for stimulation, AM1241 exhibited partial agonist efficacy. In addition, it behaved as a partial agonist in ERK (or MAP) kinase assays. Conclusions and implications: The unusual phenomenon of inconsistent functional efficacies suggests that AM1241 is a protean agonist at the CB ₂ receptor. We postulate that functional efficacies displayed by protean agonists in various assay systems may depend on the levels of receptor constitutive activities exhibited in the assay systems, and therefore, efficacies observed in in vitro assays may not predict in vivo activities.

Full text of DOI:10.1038/sj.bjp.0706838

British Journal of Pharmacology (2006) 149, 145–154
2006 Nature Publishing Group All rights reserved 0007–1188/06
&
$30.00
www.brjpharmacol.org
RESEARCH PAPER
In vitro pharmacological characterization of
AM1241: a protean agonist at the cannabinoid
CB2 receptor?
BB Yao, S Mukherjee, Y Fan, TR Garrison, AV Daza, GK Grayson, BA Hooker, MJ Dart, JP Sullivan
and MD Meyer
Neuroscience Disease Research, Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, IL, USA
Background and purpose: The CB2 receptor has been proposed as a novel target for the treatment of pain, and CB2 receptor
agonists defined in in vitro assays have demonstrated analgesic activity in animal models. Based on its in vivo analgesic efficacy,
AM1241 has been classified as a CB2-selective agonist. However, in vitro characterization of AM1241 in functional assays has
not been reported.
Experimental approach: In this study, AM1241 was characterized across multiple in vitro assays employing heterologous
recombinant receptor expression systems to assess its binding potencies at the human CB2 and CB1 receptors and its functional
efficacies at the human CB2 receptor.
Key results: AM1241 exhibited distinct functional properties depending on the assay conditions employed, a unique profile in
contrast to those of the agonist CP 55,940 and the inverse agonist SR144528. AM1241 displayed neutral antagonist activities
in FLIPR and cyclase assays. However, when cyclase assays were performed using lower forskolin concentrations for stimulation,
AM1241 exhibited partial agonist efficacy. In addition, it behaved as a partial agonist in ERK (or MAP) kinase assays.
Conclusions and implications: The unusual phenomenon of inconsistent functional efficacies suggests that AM1241 is a
protean agonist at the CB2 receptor. We postulate that functional efficacies displayed by protean agonists in various assay
systems may depend on the levels of receptor constitutive activities exhibited in the assay systems, and therefore, efficacies
observed in in vitro assays may not predict in vivo activities.
British Journal of Pharmacology (2006) 149, 145–154. doi:10.1038/sj.bjp.0706838; published online 7 August 2006
Keywords: AM1241; CB2 receptor; cannabinoid; protean agonist; receptor constitutive activity; GPCR
Abbreviations: AM1241, (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone; CB1, cannabi-
noid receptor 1; CB2, cannabinoid receptor 2; CHO cells, Chinese hamster ovary cells; DMSO, dimethyl
sulfoxide; CP 55,940, 5-(1,1-dimethyl-heptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phe-
nol; DMEM, Dulbecco’s modified Eagle’s medium; D-PBS, Dulbecco’s phosphate-buffered saline; ERK (or MAP)
kinase, extracellular signal-regulated protein kinase; FLIPR, fluorometric image plate reader; FSK, forskolin;
G protein, guanine nucleotide binding protein; GPCR, G-protein coupled receptors; HEK cells, human
embryonic kidney cells; HuT 78 line, human T lymphoma line; PBS, phosphate-buffered saline; PTX, pertussis
toxin; SR141716A (SR1), 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid
piperidin-1-ylamide; SR144528 (SR2), 5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-car-
boxylic acid ((1S,2S,4R)-1,3,3-trimethyl-bicyclo[2.2.1]hept-2-yl)amide
Introduction
The analgesic properties of herbal and synthetic cannabinoid
agonists have been well established (Pertwee, 2001; Rice,
2001; Baker et al., 2003; Croxford, 2003; Howlett et al., 2004).
However, undesirable psychotropic effects produced by these
ligands have limited their therapeutic potential (Baker et al.,
2003). Cloning and functional characterization of cannabi-
noid receptors have led to a better understanding of the
underlining physiological function of this receptor class.
Two types of cannabinoid receptors, CB1 and CB2, have
been characterized to date (Wess, 1998; Karnik et al., 2003).
Both are G-protein coupled receptors (GPCRs) coupling to
Correspondence: Dr BB Yao, Neuroscience Disease Research, Global
Pharmaceutical Research
& Development, Abbott Laboratories, R47W,
AP9A, 100 Abbott Park Road, Abbott Park, IL 60064, USA.
E-mail: betty.yao@abbott.com
G
i/o, whose activation leads to the inhibition of the adenylyl
cyclase activity. Activation of CB1 and CB2 receptors has
been demonstrated to activate extracellular signal-regulated
Received 12 January 2006; revised 18 April 2006; accepted 19 June 2006;
published online 7 August 2006

AM1241: a protean agonist at the CB2 receptor
146
BB Yao et al
protein (ERK or MAP) kinase (Bouaboula et al., 1999a, b;
Powles et al., 2005). The CB1 receptor is predominantly
located in the CNS and the peripheral CB2 receptor is
abundant in the immune system (Matsuda et al., 1990;
Munro et al., 1993; Shire et al., 1996; Griffin et al.,
2000; Brown et al., 2002). The activation of the CB1 receptor
subtype has been clearly linked to the sedation, anxiety,
paranoia, increased appetite, mild euphoria and impaired
short-term memory associated with cannabis use. However,
activation of both the CB1 and CB2 receptor subtypes has
been shown to be involved in the analgesic activity of non-
selective cannabinoids (Fox et al., 2001; Malan et al., 2001;
Scott et al., 2004). An increasing body of evidence supports
the premise that the CB2 receptor may be the better target for
the treatment of inflammatory and neuropathic pain, since
CB2 receptor activation with receptor-selective agonists
has been shown in animals to be devoid of the centrally-
mediated side effects of hypothermia, catalepsy and loss of
motor coordination associated with the nonselective canna-
binoids (Jaggar et al., 1998; Malan et al., 2003; Walter et al.,
2003; Fox and Bevan, 2005). A possible third cannabinoid
receptor has recently been recognized in the orphan
receptor, GPR55, and is expressed in the smooth muscle
cells surrounding the blood vessels, as well as in the CNS
(Sawzdargo et al., 1999; Brown et al., 2005; Lauckner et al.,
2005; Reyes et al., 2005; Sjogren et al., 2005). Although some
herbal and synthetic cannabinoid ligands exhibit affinity for
GPR55, this receptor remains largely uncharacterized.
propyl)-cyclohexyl]-phenol) and SR144528 (5-(4-chloro-3-
methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-carbo-
xylic acid ((1S,2S,4R)-1,3,3-trimethyl-bicyclo[2.2.1]hept-2-
yl)amide), which exhibit consistent pharmacology across
various functional assays, AM1241 behaves as an apparent
agonist, an inverse agonist, or a neutral antagonist depend-
ing on the specific assay systems employed. This unique
property of inconsistent efficacies across functional assays
suggests that AM1241 behaves as a protean agonist (Kenakin,
2001) at the CB2 receptor.
Methods
Cell culture
Human embryonic kidney (HEK, American Type Culture
Collection, Rockville, MD, USA) cells stably expressing the
human CB2 receptor were grown in Dulbecco’s modified
Eagle’s medium (DMEM) containing high glucose supplemen-
ted with 10% fetal bovine serum and 25mg mlꢀ1 zeocin in a
371C incubator in the presence of 5% CO2. HEK cells stably
co-expressing the human CB2 receptor and the chimeric
Gaq/o5 protein were grown under similar conditions except
that in addition to the components described above, the
growth medium was further supplemented with 200 mg mlꢀ1
hygromycin. The Chinese hamster ovary (CHO) cell line
stably expressing the human CB1 receptor was purchased
from Euroscreen (Brussels, Belgium), and the cells were
grown under the conditions recommended by the vendor.
A body of evidence supporting the role of CB2 receptor
activation in pain is demonstrated by the efficacies of CB2
receptor-selective agonist ligands such as HU-308 (Hanus
et al., 1999), JWH-133 (Elmes et al., 2004), GW405833
(Valenzano et al., 2005) and AM1241 (2-iodo-5-nitro-
phenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-
methanone) (Ibrahim et al., 2003) in preclinical models of
inflammatory and neuropathic pain. Although HU-308,
JWH-133, GW405833 have been functionally characterized
as agonists at the CB2 receptor in in vitro assay systems,
in vitro characterization of AM1241 has not been reported.
Nevertheless, a large body of evidence in the literature has
established that AM1241 is efficacious in a variety of in vivo
animal pain models, a profile that is consistent with the
properties of an agonist at the CB2 receptor. For example,
AM1241 has been shown to produce antinociceptive effects
in an acute thermal pain model (Malan et al., 2003) and
antihyperalgesic effects in a spinal nerve ligation model of
neuropathic pain (Ibrahim et al., 2003). AM1241 is also
efficacious in the carrageenan-induced thermal hypersensi-
tivity model (Quartilho et al., 2003), as well as capsaicin-
induced tactile thermal hyperalgesia and tactile allodynia
(Hohmann et al., 2004). Further, using receptor-selective
antagonists, the in vivo analgesic effects of AM1241 have
been demonstrated to involve the CB2 receptor, with no
significant contribution from CB1 activation (Malan et al.,
2001; Ibrahim et al., 2003; Ibrahim et al., 2005).
Radioligand binding assay
Membrane samples were prepared from HEK cells stably
expressing the human CB2 receptors previously generated
(Mukherjee et al., 2004), or the CHO cell line that stably
expresses the human CB1 receptor. Radioligand binding assays
were performed as described previously (Mukherjee et al., 2004).
Briefly, the cells were harvested and homogenized using a
Polytron for 2 ꢁ 10 s bursts in a buffer containing 50 mM Tris-
HCl, pH 7.4, 1 mM MgCl2, and 1mM EDTA in the presence of
protease inhibitors followed by centrifugation at 45 000 g for
20min. The membrane pellets were washed and frozen at
ꢀ801C in aliquots until use. Saturation binding reactions were
performed at 301C for 90min using [3H]CP 55,940 (0.01–8 nM)
in an assay buffer containing 50 mM Tris-HCl, pH 7.4, 2.5mM
EDTA, 5 mM MgCl2, and 0.05% fatty acid free bovine serum
albumin (BSA) and the reactions were terminated by rapid
vacuum filtration through UniFilter-96 GF/C filter plates
(Perkin-Elmer Boston, MA, USA) and four washes with cold
assay buffer. Competition experiments were conducted using
0.5 nM [3H]CP 55,940 in the presence of test compounds
(0.1 nM–10 mM). Nonspecific binding was defined by 10 mM
unlabeled CP 55,940. KD values from saturation binding assays
and Ki values from competition binding assays were determined
with one site binding or one site competition curve fitting
using the Prism software (GraphPad, San Diego, CA, USA).
This study characterizes AM1241 in various in vitro assay
systems including radioligand binding assays and receptor
functional assays using a cell line expressing the recombinant
human CB2 receptor. Unlike the reference ligands, CP 55,940
(5-(1,1-dimethyl-heptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-
Fluorometric image plate reader (FLIPR) functional assay
FLIPR assays were performed using HEK cells stably expres-
sing the human CB2 receptor and chimeric Gaq/o5 protein
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
BB Yao et al
147
(Mukherjee et al., 2004) with modification. Briefly, cells were
seeded at 7.5 ꢁ 104 cells per well in Biocoat 96-well poly-L-
lysine coated clear-bottomed black wall plates (BD Bio-
sciences, San Jose, CA, USA) 1 day prior to the assay. The cells
were incubated with No-Wash Dye following vendor’s
instruction. For agonist assays, variable concentrations of
test compounds (0.3 nM–10 mM), CP 55,940 (at 10 mM final
concentration) positive control or vehicle negative control
were added to cells in the presence of assay buffer (10 mM
HEPES, pH 7.4, 130 mM NaCl, 1 mM MgCl2, 5 mM KCl, 2 mM
CaCl2, 0.05% BSA), and maximum fluorescence responses
were measured with a FLIPR machine immediately following
addition of compounds. Agonist responses were adjusted for
the fluorescence background with vehicle controls and the
activities were expressed as percentages of the CP 55,940
response. For antagonist assays, vehicle or variable concen-
tration of test compounds (0.3 nM–10 mM) were added to the
cells at the first addition and CP 55,940 (at 1 mM final
concentration) was added to all cells at the second addition.
The interval between the first and the second additions was
5 min. For antagonist assays, the maximum fluorescence
response was measured immediately after the addition of CP
55,940 at the second addition and the response was
compared with the control where vehicle instead of test
compounds was added at the first addition. EC50 and Kb
values were analyzed with sigmoidal dose response curve
fitting using Prism.
starved in DMEM plus 0.1% BSA and 1% penicillin–
streptomycin overnight with 0.1 ng mlꢀ1 pertussis toxin
(PTX) included where indicated. Following pretreatment
for 10 min with 10 mM SR141716A (5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid
piperidin-1-ylamide), SR144528 or vehicle where indicated,
the cells were treated for 5 min with 100 nM CP 55,940 or
1 mM AM1241. Cells were washed with cold PBS, solublized in
a lysis buffer (5 mM HEPES, pH 7.4, 0.5% NP-40, 250 mM
NaCl, 2 mM EDTA, 10% (v/v) glycerol 1 ꢁ complete protease
inhibitor Cocktail) and the lysate clarified by centrifugation
at 41C for 15 min at 14 000 r.p.m. Equal amounts of proteins
were separated on 4–12% Novex Bis-Tris gels (Invitrogen,
Carlsbad, CA, USA) and transferred onto nitrocellulose
membranes for immunoblotting. Phosphorylated p42/44
ERK and total ERK proteins were detected by immunoblot-
ting with polyclonal anti-phospho-p44/42 ERK antibodies at
1:2000 dilution, or anti-ERK antibodies at 1:10 000 dilution.
Chemiluminescent detection was performed using the
SuperSignal West Pico reagent.
Statistical analysis
Results are shown as means7s.e.m. or means with 95%
confidence interval (CI).
Compounds synthesized
Racemic AM1241 (Makriyannis and Deng, 2001), SR144528
(Barth et al., 1997) and SR141716A (Barth et al., 1995) were
synthesized at Abbott Laboratories according the methods
described, and their structures characterized by 1H NMR
spectroscopy, mass spectrometry and elemental analysis.
The synthesis of racemic AM1241 used in the current
study is shown in the scheme below, and its characterization
described here.
Cyclase functional assay
The cyclase functional assays were performed using the
HitHunter cAMP assay kit according to vendor’s protocols.
Briefly, HEK cells expressing the human CB2 receptor were
detached using cell dissociation buffer, dispersed and placed
in suspension at 104 cells per well in 96-well plates prior to
the assay. For agonist and inverse agonist assays, cells were
treated for 20 min with variable concentrations of test
ligands (6.4 pM–25 mM) and forskolin (8 or 37 mM) in Dulbec-
co’s phosphate-buffered saline (D-PBS) supplemented with
BSA (0.01% final concentration). In experiments assessing
the antagonist properties of AM1241, variable concentra-
tions of AM1241 (6.4 pM–25 mM) and forskolin (37 mM) were
added to the cells together with a fixed concentration of
either CP 55,940 (final concentration at 4 nM) or SR144528
(final concentration at 100 nM). The concentration of
forskolin used to stimulate the cAMP level in cyclase assays
was 37 mM unless indicated otherwise. Reactions were
incubated for 20 min at 371C and terminated by the addition
of lysis buffer and the luminescence was detected following
the procedure according to vendor’s instructions. The cyclase
activities were expressed as percent responses over the
forskolin-stimulated control levels, where cells received
vehicle instead of test compounds. EC50 values were
calculated using sigmoidal dose response curve fitting from
Prism (GraphPad).
I
EtMgBr
ZnCl2
I
O
O
NO2
N
H
Cl
NO2
N
H
I
OSO2Me
N
O
NO2
Me
N
NaH
N
Me
AM1241
Briefly, acylation of indole with 2-iodo-5-nitrobenzoyl
chloride followed by N-alkylation with the mesylate derived
from (1-methylpiperidin-2-yl)methanol afforded racemic
AM1241 as a yellow powder: 1H NMR (500 MHz, CD3OD)
d p.p.m. 1.10–1.29 (m, 3H), 1.45–1.71 (m, 3H), 2.20 (dt,
ERK activation assay
HEK cells stably expressing the human CB2 receptor were
seeded at 2 ꢁ 105 cells per well in six-well plates, serum-
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
148
BB Yao et al
J ¼ 11.2, 3.7 Hz, 1H), 2.43 (s, 3H), 2.51 (m, 1H), 2.89 (m, 1H),
4.00 (dd, J ¼ 13.9, 9.2 Hz, 1H), 4.67 (dd, J ¼ 13.9, 4.7 Hz, 1H),
7.37 (m, 2H), 7.57 (m, 1H), 7.69 (s, 1H), 8.07 (dd, J ¼ 8.8,
2.7 Hz, 1H), 8.22 (d, J ¼ 2.5 Hz, 1H), 8.26 (d, J ¼ 8.8 Hz, 1H),
Table 1 Summary of compiled KD in [3H]CP 55,940 saturation binding
assays
Receptor
n
KD (nM)
(mean with 95% CI)
Bmax (pmol (mg protein)ꢀ1
(mean7s.e.m.)
)
8.28 (m, 1H); MS (DCI) m/z 504 (M þ H)þ
.
CB2
CB1
11
7
0.75 (0.57–0.98)
2.4 (2.0–2.9)
9.474.9
4.972.2
Analysis: Calculated for C22H22IN3O3: C, 52.50; H, 4.41; N,
8.35. Found: C, 52.23; H, 4.59; N, 8.09.
All compounds used were dissolved in dimethyl sulfoxide
(DMSO) at a 10 mM concentration and stored at ꢀ201C until
use. The initial dilutions from 10 mM stock solution were
made in D-PBS supplemented with fatty acid free BSA at
0.01% final concentration at the assay for cyclase and ERK
activation assays, or in assay buffers for radioligand binding
and FLIPR assays at a greater than 1:100 fold dilution to
avoid compound precipitation. Subsequent serial dilutions
were performed in assay buffers according to the concentra-
tion range tested.
competition binding assays and these results are also
summarized in Table 2. As reported previously by Howlett
et al. (2002), SR144528 exhibited high selectivity at the
human CB2 receptor over the human CB1 receptor. In
contrast, CP 55,940 was essentially nonselective with high
potencies at both human CB1 and CB2 receptors.
AM1241 is an apparent antagonist at the human CB2 receptor
in FLIPR assays
In order to assess the functional efficacy of AM1241 at the
human CB2 receptor, a FLIPR functional assay was performed
using an HEK cell line as previously described (Mukherjee
et al., 2004), which co-expresses the human CB2 receptor and
the chimeric Gaq/o5 protein. The chimeric Gaq/o5 protein
facilitates the increase of intracellular Ca2 þ level upon
activation of Gai/o-coupled GPCRs, which can be readily
measured by a FLIPR machine (Conklin et al., 1993). The
stable HEK cell line used in FLIPR assays was developed
by introducing chimeric Gaq/o5 into the HEK cell line that
expresses the human CB2 receptor alone. Saturation binding
assays indicated that the resulting cell line co-expressing the
human CB2 receptor and chimeric Gaq/o5 exhibited [3H]CP
55,940 radioligand binding profiles comparable to that of
the parent cell line expressing the human CB2 receptor alone
with a similar KD value (0.60 nM) and slightly lower Bmax
value (3.5 pmol mgꢀ1 protein). In FLIPR assays, AM1241
exhibited antagonist activity, blocking the agonist CP
55,940-evoked Ca2 þ response (Figure 1a) in a concentration
dependent manner with a Kb value of 63 nM (Figure 1b).
Similarly, SR144528 exhibited antagonist activity at the
human CB2 receptor with a Kb value of 22 nM, whereas CP
55,940 was an agonist at the human CB2 receptor with an
EC50 of 55 nM (Figures 1a and b).
Other materials
CP 55,940 (5-(1,1-dimethyl-heptyl)-2-[(1R,2R,5R)-5-hydroxy-
2-(3-hydroxy-propyl)-cyclohexyl]-phenol) was purchased
from Tocris Inc. (Ellisville, MI, USA) and [3H]CP 55,940
(specific activity, 6.67 TBq/mmol) from Perkin Elmer Boston,
MA, USA.
D-PBS, cell dissociation buffer, penicillin–streptomycin
and other reagents for cell culture were purchased from
Invitrogen (Carlsbad, CA, USA). Fatty acid free BSA was from
Sigma (St Louis, MO, USA) and the No-Wash Dye for the
FLIPR Calcium Assay Kit was from Molecular Device,
(Sunnyvale, CA, USA). The HitHunter cAMP assay kit was
purchased from DiscoveRx (Fremont, CA, USA), PTX from
List Biological Laboratories, Inc. (Campbell, CA, USA) and
the complete protease Inhibitor Cocktail from Roche
Applied Sciences (Indianapolis, IN, USA). Polyclonal anti-
phospho-p44/42 ERK antibody was from Cell Signaling
Technology, Beverly, MA, USA and anti-ERK antibodies from
Upstate Group Inc., Lake Placid, NY, USA). The SuperSignal
West Pico reagent for chemiluminescent detection was
obtained from Pierce Biotechnology Inc. (Rockford, IL, USA).
Results
AM1241 is selective at the human CB2 receptor
The binding selectivity of AM1241 at the CB2 cannabinoid
receptor has been reported previously (Ibrahim et al., 2003)
and was confirmed in the current [3H]CP 55,940 radioligand
binding studies using membrane preparations from stable
HEK and CHO cell lines expressing the recombinant human
CB2 and CB1 receptors, respectively. In saturation binding
assays, [3H]CP 55,940 exhibited high potencies at these
cannabinoid receptors (Table 1). The host HEK and CHO
cells do not exhibit significant specific binding to the [3H]CP
55,940 ligand (data not shown).
In [3H]CP 55,940 competition binding assays, AM1241
displayed high affinity at the human CB2 receptor with a
Ki value of about 7 nM, whereas its affinity at the human
CB1 receptor was more than 80-fold weaker (Table 2). For
comparison, SR144528 and CP 55,940 were also tested in
AM1241 behaves as a neutral antagonist at the human CB2
receptor in cyclase assays
In order to further assess the efficacy of AM1241 at the
human CB2 receptor, cyclase functional assays were per-
formed and activation of the human CB2 receptor was
measured using the stable HEK cell line expressing the
human CB2 receptor. Forskolin induced a concentration-
dependent increase in cAMP levels in HEK cells expressing
the human CB2 receptor with an EC50 value of 15 mM (data
not shown). Forskolin, at BEC70 concentration (37 mM), was
used to stimulate cAMP production in cyclase assays, and the
abilities of test ligands to modulate cyclase activity were
measured and expressed as percent responses over the
forskolin-stimulated cAMP levels. AM1241 exhibited no
agonist or inverse agonist activities in the concentration
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
BB Yao et al
149
Table 2 Radioligand competition binding assays using [3H]CP 55,940
Ligand
Structure
Human CB2
95% CI
Human CB1
95% CI
Ki (nM)
n
Ki (nM)
n
5
AM1241
7.1
4.0–13
13
580
61–5600
I
O
NO2
N
N
OH
CP 55,940
SR144528
0.79
0.63–0.99
1.3–5.3
49
1.9
1.3–2.7
41
OH
HO
2.7
4
320
260–380
4
H
O
N
H
N
N
Cl
range tested at the human CB2 receptor in the cyclase assays
(Figure 2a and Table 3). In contrast, under similar assay
conditions, CP 55,940 displayed potent agonist activity with
an EC50 value of 0.36 nM reducing cyclase activity by 70% of
the forskolin-induced level, whereas SR144528 exhibited an
inverse agonist activity with EC50 value of 92 nM increasing
cyclase activity by 74% of the forskolin-induced level. The
lack of robust functional efficacy of AM1241 at the human
performed at a lower forskolin concentration (8 mM, BEC35).
In contrast to the neutral antagonist behavior observed with
a higher (37 mM) forskolin concentration, AM1241 now
exhibited partial agonist activity at the human CB2 receptor,
in the presence of 8 mM forskolin (Figure 3). Conversely, CP
55,940 and SR144528 still exhibited agonist and inverse
agonist properties, respectively, independent of the forskolin
concentration used (Figure 3).
CB2 receptor may indicate that AM1241 is
antagonist in this assay.
a neutral
AM1241 is an apparent agonist at the human CB2 receptor
in ERK activation assays
Subsequently, AM1241 was evaluated for its ability to
antagonize the effects of agonist CP 55,940 and the inverse
agonist SR144528. AM1241 dose-dependently blocked the
agonist activity of CP 55,940 (4 nM) and the inverse agonist
activity of SR144528 (100 nM) at the human CB2 receptor
with Kb values at 27 and 11 nM, respectively (Figure 2b).
Schild analysis was performed to ascertain the competitive
nature of AM1241 inhibition. Rightward shifts of the CP
55,940 concentration–response curve were observed in the
presence of increasing concentrations of AM1241 ranging
from 0.1 to 10 mM (Figure 2c). The Schild plot of these results
(Figure 2d) gave a pA2 value of 7.9, similar to the pKb value of
7.6 (Kb ¼ 27 nM) with the Hill slope approaching unity,
indicating that under the conditions tested, AM1241 func-
tions as a competitive antagonist of CP 55,940.
It has been reported previously that activation of the human
CB2 receptor enhances phosphorylation and activation of
p42/p44 ERK (Bouaboula et al., 1999b). Unlike the cyclase
and FLIPR assays described above, AM1241 demonstrated
partial agonist activity, inducing the phosphorylation of
p42/p44 ERK but to a lesser extent than that induced by CP
55,940 (Figure 4a). ERK phosphorylation induced by
AM1241 and CP 55,940 was blocked by SR144528 but not
by SR141716A, demonstrating that the ERK activation is
mediated through the CB2 receptor (Figure 4a). Further, PTX
abolished the ERK phosphorylation induced by AM1241 and
CP 55,940, indicating that the activation of ERK by AM1241
and CP 55,940 was mediated by the Gi/o protein (Figure 4b).
Discussion
At lower forskolin concentrations, AM1241 behaves as an agonist
at the human CB2 receptor in cyclase assays
An emerging model of a protean agonist describes a ligand
that changes its apparent behavior, and depending on the
assay systems, can operate as an agonist, antagonist or
To determine if the assay conditions affected the functional
properties of AM1241 at the CB2 receptor, cyclase assays were
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
150
BB Yao et al
standard for CB2 receptor activation in these assays. Multiple
structures have been published for AM1241 (Goutopoula
and Makriyannis, 2003; Ibrahim et al., 2003). The structure
of AM1241 used in the current studies is consistent with
that published by Makriyannis and Deng (2001) and
the compound is now available from Alexis Corporation
(Lausen, Switzerland).
a
Agonist
120
100
80
60
40
20
0
CP 55,940
SR144528
AM1241
Our present studies employing recombinant CB2 receptor
systems showed that AM1241 exhibited inconsistent func-
tional efficacies. In ERK activation assays, AM1241 exhibited
Gi/o-dependent partial agonist activity at the CB2 receptor,
stimulating ERK activation at a level lower than that of CP
55,940. In contrast, AM1241 was an apparent antagonist in
FLIPR assays, blocking the CP 55,940-evoked calcium influx
through the CB2 receptor, similar to the effect observed with
SR144528. In cyclase assays, AM1241 produced inconsistent
efficacies that were dependent upon the assay conditions
-10
-9
-8
-7
-6
-5
-4
Log Ligand [M]
used. When a higher forskolin concentration (37 mM, BEC75
)
was used to stimulate the adenylyl cyclase, AM1241 failed to
produce a change in efficacy. However, AM1241 reversed the
effects of agonist CP 55,940 and inverse agonist SR144528
in a concentration-dependent manner, demonstrating that
AM1241 behaved as a neutral antagonist. When assays were
performed using lower forskolin concentrations (8 mM,
BEC35), AM1241 consistently exhibited agonist efficacy,
reducing the cAMP level, as did CP 55,940. The divergence
among functional properties of AM1241 in various in vitro
assays and the lack of robust CB2 agonist efficacies may
suggest that AM1241 is a protean agonist at the CB2 receptor.
In contrast, the agonist CP 55,940 and inverse agonist
SR144528 exhibited consistent functional efficacies across
different assay systems.
Antagonist
b
120
100
80
60
40
20
0
AM1241
SR144528
In order to provide a direct comparison to preclinical
animal studies (Malan et al., 2001; Ibrahim et al., 2003;
Ibrahim et al., 2005), a racemic mixture of AM1241 has been
used in the current studies. In addition, it has been shown by
Uveges et al. (2005) that the individual enantiomers exhibit
similar potencies and efficacies at the human CB2 receptor in
cyclase assays compared with those of the racemic mixture,
indicating that neither enantiomer is likely to produce
confounding functional properties.
-11
-10
-9
-8
-7
-6
-5
-4
Log Ligand [M]
Figure 1 Characterization of AM1241 in FLIPR functional assays at
the human CB2 receptor. The FLIPR assays were performed using
stable HEK cell line co-expressing the human CB2 receptor and
chimeric Gaq/o5 protein. (a) In agonist assays, the maximum
fluorescence responses were expressed as the percent of positive
control, 10 mM CP 55,940. The results are the average of three
experiments performed in duplicates. (b) In antagonist assays,
compounds or vehicle were added at the first addition and 1 mM
CP 55,940 was added at the second addition. The maximum
fluorescence responses following the CP 55,940 addition were
measured and expressed as the percentage of the CP 55,940
response when vehicle instead of compounds was added in the first
addition. The results are the average of at least three experiments,
performed in duplicates.
CP 55,940 failed to show agonist activity in cyclase assays
in native cell lines such as HuT 78 (human T lymphoma)
that expresses the human CB2 receptor gene (data not
shown). In contrast, the recombinant HEK cell line used in
the current studies expresses the human CB2 receptor at a
high level, allowing readily detection of both agonists and
antagonists. According to the current receptor activation
theory (Chen et al., 2000; Seifert and Wenzel-Seifert, 2002;
Milligan, 2003; Prather, 2004), the increased receptor avail-
ability in recombinant systems increases the absolute
number of receptors activated by agonist ligands, leading
to significant augmentation of the signalling pathway as well
as the detection capability of the assay system, resulting in
significant amplification in maximal agonist efficacies
(Figure 5). Concomitantly, the higher level of receptor
expression in recombinant systems increases the total
number of spontaneously activated receptors regardless of
the presence of agonist ligands, and thus the assay system
displays higher levels of constitutive activity. The level of
inverse agonist at the same receptors (Kenakin, 2001, 2004;
Prather, 2004). Protean agonists have been reported for the
histamine H3 receptor (Gbahou et al., 2003; Baldi et al., 2005)
and a2A-adrenergic receptor (Jansson et al., 1998; Pauwels
et al., 2002). The present studies provide evidence that
AM1241 behaves as a protean agonist at the human CB2
receptor.
AM1241, a CB2-selective ligand, consistently demonstrates
broad-spectrum analgesic activities in various preclinical
animal pain models (Malan et al., 2001), and is a reference
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
BB Yao et al
151
a
b
100
50
SR144528
AM1241
CP 55,940
50
CP 55,940
SR144528
25
0
0
-25
-50
-50
-100
-11 -10 -9
-8
-7
-6
-5
-11 -10 -9
-8
-7
-6
-5 -4
Log Ligand [M]
Log AM1241 [M]
c
d
AM1241
pA2 = 7.89
Slope = 0.93
r2 = 0.86
0
-20
-40
-60
0
3
2
1
0
0.1 µM
0.3 µM
1 µM
10 µM
-8
-7
-6
-5
-11 -10 -9 -8 -7 -6 -5 -4
Log CP 55,940 [M]
Log AM1241 [M]
Figure 2 Characterization of AM1241 in cyclase functional assays at the human CB2 receptor. The cyclase functional assays were performed
using stable HEK cell lines expressing the human CB2 receptor. (a) In agonist/inverse agonist assays, compounds were added together with
37 mM forskolin. The luminescence responses in the presence of test compounds were compared with those when compounds were replaced
with the vehicle and expressed as percent responses over the forskolin-stimulated level. (b) AM1241 was tested as an antagonist to block the
agonist activity of CP 55,940 and the inverse agonist activity of SR144528. In antagonist assays, a range of AM1241 concentration was used in
conjunction with 4 nM CP 55,940 or 100 nM SR144528 in the presence of 37 mM forskolin. The luminescence responses in the presence of test
compounds were compared with those when compounds were replaced with the vehicle and expressed as percent responses over the
forskolin-stimulated level. (c) Rightward shift of concentration dependent curves of CP 55,940 with increased concentration of AM1241. (d)
Schild analysis of results shown in (c). Results from all above experiments are the average of at least four experiments performed in duplicate.
Table 3 Agonist and inverse agonist activities in human CB2 cyclase
assays
detection capabilities not only for agonists (Figure 5, B and
C), but also permit the characterization of inverse agonists.
It has been hypothesized that the ligand-induced receptor
conformation state is dependent upon the intrinsic char-
acteristics of the ligand, regardless of the assay system
employed (Kenakin, 2001, 2004; Perez and Karnik, 2005).
Therefore, the pharmacological definition of a ligand may
vary depending on the constitutive activity present in the
assay system. Due to its high intrinsic activity, the hypo-
thetical ligand C behaves as an agonist in native and
recombinant systems. Without any intrinsic activity, the
hypothetical ligand A is an antagonist in the native system,
and behaves as an inverse agonist in systems containing
constitutively active receptors. In contrast, the hypothetical
ligand B, possessing a low level of intrinsic activity, can
behave as a partial agonist in a system with a lower relative
level of constitutive activity (R1 state), but as an inverse
agonist when the receptor constitutive activity is higher (R2
state). By definition, ligand B is a protean agonist, as its
observed functional efficacy is dependent upon the relative
level of constitutive activity exhibited by the system. There-
fore, it is possible that inverse agonists defined in recombi-
nant systems in vitro may actually behave as antagonists or
even partial agonists in native systems that exhibit low levels
Ligand
EC50 (nM)
95% CI
of Ki (nM)
n
Efficacy over
FSK-stimulated
level (%)
AM1241
CP 55,940
SR144528
41600
9
88
NAa–NA
5.4–15
61–127
7
16
16
NA
ꢀ70
74
aNA: not available.
spontaneous activation of receptors is dependent upon
multiple factors – the thermodynamic nature of receptor
conformational changes from quiescent state to active state,
the cellular background, such as repertoires of G proteins,
GPCR binding and effector proteins, as well as the conditions
under which the cells were grown and the assays performed
(Chen et al., 2000; Kenakin, 2001; Seifert and Wenzel-Seifert,
2002; Milligan, 2003).
In contrast to native systems where the receptors are
quiescent in general and do not display significant consti-
tutive activity (R0 state, Figure 5), recombinant systems
often show variable levels of constitutive activity (R1 and
R2 states, Figure 5). Such constitutive systems increase the
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
152
BB Yao et al
C
C
150
100
50
R2
B
A
B
A
R1
C
B
A
R0
Quiescent System
Constitutive Systems
Figure 5 A model of receptor activation theory. R0 represents the
receptor resting state in quiescent system lacking receptor
a
constitutive activities, and R1 and R2 represent resting states in
systems with receptor constitutive activities. A, B and C represent
hypothetical ligands that possess different levels of intrinsic activities
and produce different levels of functional efficacies at the receptor.
0
-11
-10
-9
-8
-7
-6
-5
-4
Log Ligand [M]
receptor systems can produce confounding results for
compounds with low efficacies. In addition, the distinct
repertoire of G proteins and other proteins that interact with
target receptors in cell lines employed may also contribute to
the inconsistent pharmacology between in vitro and in vivo
systems. Therefore, physiologically relevant assay systems,
ideally derived from target tissues, should be employed
to evaluate the predictability of in vitro assay systems,
and, ultimately, in vivo assays are necessary for compound
selection for advancing through the drug discovery process.
In summary, although efficacious agonists and antago-
nists/inverse agonists can be identified using recombinant
systems, characterizing protean agonists may be more
complex and require multiple functional assay systems.
Further, physiologically relevant in vitro assay systems with
correlations to in vivo testing are essential for the accurate
prediction of compound efficacies in vivo. While both
agonists and inverse agonists have proven utility in regulat-
ing receptor activities, the therapeutic potential of protean
agonists is not clear. Perhaps their unique properties of
promoting a lower level of ligand-specific receptor activation
states may be advantageous over fully efficacious agonists
and inverse agonists, whose therapeutic utility may be
limited by the development of tolerance (Kenakin, 2001).
AM1241 - 8µM FSK
AM1241 - 37µM FSK
CP 55,940 - 37µM FSK
SR144528 - 8µM FSK
SR144528 - 37µM FSK
CP 55,940 - 8µM FSK
Figure 3 Efficacy of AM1241 in cyclase assays depends on level of
stimulus. All three compounds, AM1241, CP55,940 and SR144528,
were re-assessed in cyclase assays at a low (8 mM) concentration of
forskolin and compared with their effects at the higher concentration
of forskolin (37 mM; see Figure 2a).
a
vehicle
AM1241
SR1 SR2
CP 55,940
SR1 SR2
SR1 SR2
P-ERK Ab
b
vehicle
PTX
AM1241
PTX
CP 55,940
PTX
P-ERK Ab
Acknowledgements
Figure 4 AM1241 behaves as an agonist at the human CB2
receptor in ERK (MAPK) activation assays. (a) Stable HEK cell line
expressing the human CB2 receptor was treated with either 10 mM
SR141716A (SR1), 10 mM SR144528 (SR2) or vehicle followed by
5 min treatment of 1 mM AM1241 or 100 nM CP 55,940. (b) AM1241
or CP 55,940-induced ERK activation is blocked by treatment with
PTX (0.1 ng mlꢀ1).
We thank Dr Murali Gopalakrishnan, Dr John Malysz, Karen
Kage, Dr Monique Adams and Kristi Wyckoff for their
contributions that led to the current publication.
Conflict of interest
of constitutive receptor activity. The behavior of AM1241
as an agonist in vivo and as a weakly efficacious agonist
or antagonist/inverse agonist in recombinant systems is
consistent with this classification as a protean agonist.
In vitro recombinant systems have been shown to be
indispensable tools in drug discovery for high throughput
screening and functional characterization of compounds at
target receptors. While high levels of receptor expression in
in vitro systems make functional assays feasible, the increased
receptor tone in these systems compared with the native
All authors are employees of Abbott Laboratories who
funded the research.
References
Baker D, Pryce G, Giovannoni G, Thompson AJ (2003). The
therapeutic potential of cannabis. Lancet Neurol 2: 291–298.
Baldi E, Bucherelli C, Schunack W, Cenni G, Blandina P, Passani MB
(2005). The H3 receptor protean agonist proxyfan enhances the
expression of fear memory in the rat. Neuropharmacol 48: 246–251.
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
BB Yao et al
153
Barth F, Casellas P, Congy C, Martinez S, Rinaldi M, Anne-Archard G
(1995). Preparation of N-piperidino-5-(4-chlorophenyl)-1-(2,4-di-
chlorophenyl)-4-methylpyrazole-3-carboxamide as a cannabinoid
receptor antagonist. European Patent Appl. EP 656354.
Barth F, Casellas P, Millan J, Oustric D, Rinaldi M, Sarran M (1997).
3-Pyrazolecarboxamide derivatives having cannabinoid receptor.
PCT Patent Appl. WO 9721682.
Jaggar SI, Hasnie FS, Sellaturay S, Rice AS (1998). The anti-
hyperalgesic actions of the cannabinoid anandamide and the
putative CB2 receptor agonist palmitoylethanolamide in visceral
and somatic inflammatory pain. Pain 76: 189–199.
¨
Jansson CC, Kukkonen JP, Nasman J, Huifang G, Wurster S, Virtanen
R et al. (1998). Protean agonism at alpha2A-adrenoceptors. Mol
Pharmacol 53: 963–968.
Bouaboula M, Bianchini L, Mckenzie FR, Pouyssegur J, Casellas P
(1999a). Cannabinoid receptor CB1 activates the Naþ /Hþ ex-
changer NHE-1 isoform via Gi-mediated mitogen activated
protein kinase signaling transduction pathways. FEBS Lett 449:
61–65.
Bouaboula M, Desnoyer N, Carayon P, Combes T, Casellas P (1999b).
Gi protein modulation induced by a selective inverse agonist
for the peripheral cannabinoid receptor CB2: implication for
intracellular signalization cross-regulation. Mol Pharmacol 55:
473–480.
Karnik SS, Gogonea C, Patil S, Saad Y, Takezako T (2003). Activation
of G-protein-coupled receptors: a common molecular mechanism.
Trends Endocrinol Metab 14: 431–437.
Kenakin T (2001). Inverse, protean, and ligand-selective agonism:
matters of receptor conformation. FASEB J 15: 598–611.
Kenakin T (2004). Principles: receptor theory in pharmacology.
Trends Pharmacol Sci 25: 186–192.
Lauckner JE, Reyes F, Xu C, Hille B, Stella N, Mackie K (2005).
Distribution and functional characterization of a novel cannabi-
noid receptor. Society for Neuroscience 35th Annual Meeting
Washington, DC. pp 491.4.
Brown AE, Ueno S, Suen K, Dowell SJ, Wise A (2005). Molecular
identification of GPR55 as a third G protein-coupled receptor
responsive to cannabinoid ligands. ICRS 15th Symposium on the
Cannabinoids. Clearwater, Florida. pp 16.
Makriyannis
A,
Deng
H
(2001).
Receptor
selective
cannabimimetic aminoalkylindoles. PCT Patent Appl. WO 02/
060447 A1.
Malan Jr TP, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T et al.
(2001). CB2 cannabinoid receptor-mediated peripheral antinoci-
ception. Pain 93: 239–245.
Brown SM, Wager-Miller J, Mackie K (2002). Cloning and molecular
characterization of the rat CB2 cannabinoid receptor. Biochim
Biophys Acta 1576: 255–264.
Malan Jr TP, Ibrahim MM, Lai J, Vanderah TW, Makriyannis A,
Chen G, Way J, Armour S, Watson C, Queen K, Jayawickreme CK
Porreca
F
(2003). CB2 cannabinoid receptor agonists:
et al. (2000). Use of constitutive
G protein-coupled receptor
pain relief without psychoactive effects? Curr Opin Pharmacol 3:
62–67.
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI (1990).
Structure of a cannabinoid receptor and functional expression of
the cloned cDNA. Nature 346: 561–564.
Milligan G (2003). Constitutive activity and inverse agonists of G
protein-coupled receptors: a current perspective. Mol Pharmacol
64: 1271–1276.
Mukherjee S, Adams M, Whiteaker K, Daza A, Kage K, Cassar S et al.
(2004). Species comparison and pharmacological characterization
of rat and human CB2 cannabinoid receptors. Eur J Pharmacol 505:
1–9.
activity for drug discovery. Mol Pharmacol 57: 125–134.
Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR (1993).
Substitution of three amino acids switches receptor specificity of
G
qato that of Gia. Nature 363: 274–276.
Croxford JL (2003). Therapeutic potential of cannabinoids in CNS
disease. CNS Drugs 17: 179–202.
Elmes Jr S, Jhaveri MD, Smart D, Kendall DA, Chapman V (2004).
Cannabinoid CB2 receptor activation inhibits mechanically
evoked responses of wide dynamic range dorsal horn neurons in
¨
naıve rats and in rat models of inflammatory and neuropathic
pain. Eur J Neurosci 20: 2311–2320.
Fox A, Bevan
S (2005). Therapeutic potential of cannabinoid
receptor agonists as analgesic agents. Expert Opin Invest Drugs 14:
695–703.
Fox A, Kesingland A, Gentry C, Mcnair K, Patel S, Urban L et al.
(2001). The role of central and peripheral Cannabinoid1 receptors
in the antihyperalgesic activity of cannabinoids in a model of
neuropathic pain. Pain 92: 91–100.
Gbahou F, Rouleau A, Morisset S, Parmentier R, Crochet S, Lin JS et al.
(2003). Protean agonism at histamine H3 receptors in vitro and
in vivo. Proc Natl Acad Sci USA 100: 11086–11091.
Goutopoula A, Makriyannis A (2003). From cannabis to cannabiner-
gic: new therapeutic opportunities. Pharmacol Ther 95: 103–117.
Griffin G, Tao Q, Abood ME (2000). Cloning and pharmacological
characterization of the rat CB2 cannabinoid receptor. J Pharmacol
Exp Ther 292: 886–894.
Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M
et al. (1999). HU-308: a specific agonist for CB2, a peripheral
cannabinoid receptor. Proc Natl Acad Sci USA 96: 14228–14233.
Hohmann AG, Farthing JN, Zvonok AM, Makriyannis A (2004).
Selective activation of cannabinoid CB2 receptors suppresses
hyperalgesia evoked by intradermal capsaicin. J Pharmacol Exp
Ther 308: 446–453.
Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA et al.
(2002). International union of pharmacology XXVII classification
of cannabinoid receptors. Pharmacol Rev 54: 161–202.
Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE,
Porrino LJ (2004). Cannabinoid physiology and pharmacology: 30
years of progress. Neuropharmacology 47: 345–358.
Ibrahim MM, Deng H, Zvonok A, Cockayne DA, Kwan J, Mata HP
et al. (2003). Activation of CB2 cannabinoid receptors by AM1241
inhibits experimental neuropathic pain: pain inhibition by
receptors not present in the CNS. Proc Natl Acad Sci USA 100:
10529–10533.
Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A et al.
(2005). CB2 cannabinoid receptor activation produces antinoci-
ception by stimulating peripheral release of endogenous opioids.
Proc Natl Acad Sci USA 102: 3093–3098.
Munro S, Thomas KL, Abu-Shaar M (1993). Molecular chara-
cterization of a peripheral receptor for cannabinoids. Nature 365:
61–65.
Pauwels PJ, Rauly I, Wurch T, Colpaert FC (2002). Evidence for
protean agonism of RX 831003 at alpha 2A-adrenoceptors by co-
expression with different G alpha protein subunits. Neuropharma-
cology 42: 855–863.
Perez DM, Karnik SS (2005). Multiple signaling states of G-protein-
coupled receptors. Pharmacol Rev 57: 147–161.
Pertwee RG (2001). Cannabinoid receptors and pain. Prog Neurobiol
63: 569–611.
Powles T, te Poele R, Shamash J, Chaplin T, Propper D, Joel S et al.
(2005). Cannabis-induced cytotoxicity in leukemic cell lines: the
role of the cannabinoid receptors and the MAPK pathway. Blood
105: 1214–1221.
Prather PL (2004). Inverse agonists: tools to reveal ligand-specific
conformations of G protein-coupled receptors. Science’s STKE 215:
1–3.
Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F,
Makriyannis A et al. (2003). Inhibition of inflammatory hyper-
algesia by activation of peripheral CB2 cannabinoid receptors.
Anesthesiology 99: 955–960.
Reyes F, Lauckner JE, Xu C, Mackie K, Stella N (2005). Initial
characterization of a novel cannabinoid receptor in glial cells.
In Society for Neuroscience 35th Annual Meeting Washington, DC.
pp 491.9.
Rice AS (2001). Cannabinoids and pain. Curr Opin Invest Drugs 2:
399–414.
Sawzdargo M, Nguyen T, Lee DK, Lynch KR, Cheng R, Heng HH et al.
(1999). Identification and cloning of three novel human
G
protein-coupled receptor genes GPR52, CGPR53 and GPR55:
GPR55 is extensively expressed in human brain. Brain Res Mol
Brain Res 64: 193–198.
Scott DA, Wright CE, Angus JA (2004). Evidence that CB-1 and CB-2
cannabinoid receptors mediate antinociception in neuropathic
pain in the rat. Pain 109: 124–131.
British Journal of Pharmacology (2006) 149 145–154

AM1241: a protean agonist at the CB2 receptor
154
BB Yao et al
Seifert R, Wenzel-Seifert K (2002). Constitutive activity of G-protein-
coupled receptors: cause of disease and common property of wild-
type receptors. Naunyn Schmiedeberg’s Arch Pharmacol 366: 381–416.
CB2. In Society for Neuroscience 35th Annual Meeting Washington,
DC; pp 634.1.
Valenzano KJ, Tafesse L, Lee G, Harrison JE, Boulet JM, Gottshall SL
et al. (2005). Pharmacological and pharmacokinetic characteriza-
tion of the cannabinoid receptor 2 agonist, GW405833, utilizing
rodent models of acute and chronic pain, anxiety, ataxia and
catalepsy. Neuropharmacology 48: 658–672.
`
Shire D, Calandra B, Rinaldi CM, Oustric D, Pessegue B, Bonnin CO
et al. (1996). Molecular cloning, expression and function of the
murine CB2 peripheral cannabinoid receptor. Biochim Biophys Acta
1307: 132–136.
Sjogren S, Ryberg E, Lindblom A, Larsson N, Astrand A, Hjorth S et al.
(2005). A new receptor for cannabinoid ligands. In ICRS 15th
Symposium on the Cannabinoids. Clearwater: Florida, pp 106.
Uveges AJ, Sun S-C, Bingham B, Kotnis SA, Xu J, Kennedy JD et al.
(2005). Pharmacological comparison of human, mouse and rat
Walter L, Franklin A, Witting A, Wade C, Xie Y, Kunos G et al. (2003).
Nonpsychotropic cannabinoid receptors regulate microglial cell
migration. J Neurosci 23: 1398–1405.
Wess
J (1998). Molecular basis of receptor/G-protein-coupling
selectivity. Pharmacol Ther 80: 231–264.
British Journal of Pharmacology (2006) 149 145–154