In vitro and in vivo characterization of A-796260: A selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in rodent pain models

Article abstract of DOI:10.1038/sj.bjp.0707568

Background and purpose: Selective cannabinoid CB₂ receptor agonists have demonstrated analgesic activity across multiple preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB₂ binding site and broad spectrum analgesic activity in rodent models, but is not an antagonist of CB₂ in vitro functional assays. Additionally, its analgesic effects are μ-opioid receptor-dependent. Herein, we describe the in vitro and in vivo pharmacological properties of A-796260, a novel CB₂ agonist. Experimental approach: A-796260 was characterized in radioligand binding and in vitro functional assays at rat and human CB₁ and CB₂ receptors. The behavioural profile of A-796260 was assessed in models of inflammatory, post-operative, neuropathic, and osteoarthritic (OA) pain, as well as its effects on motor activity. The receptor specificity was confirmed using selective CB₁, CB₂ and μ-opioid receptor antagonists. Key results: A-796260 exhibited high affinity and agonist efficacy at human and rat CB₂ receptors, and was selective for the CB₂ vs CB₁ subtype. Efficacy in models of inflammatory, post-operative, neuropathic and OA pain was demonstrated, and these activities were selectively blocked by CB₂, but not CB₁ or μ-opioid receptor-selective antagonists. Efficacy was achieved at doses that had no significant effects on motor activity. Conclusions and implications: These results further confirm the therapeutic potential of CB₂ receptor-selective agonists for the treatment of pain. In addition, they demonstrate that A-796260 may be a useful new pharmacological compound for further studying CB₂ receptor pharmacology and for evaluating its role in the modulation of pain.

Full text of DOI:10.1038/sj.bjp.0707568

British Journal of Pharmacology (2008) 153, 390–401
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RESEARCH PAPER
In vitro and in vivo characterization of A-796260: a
selective cannabinoid CB2 receptor agonist
exhibiting analgesic activity in rodent pain models
BB Yao1, GC Hsieh1, JM Frost, Y Fan, TR Garrison, AV Daza, GK Grayson, CZ Zhu, M Pai,
P Chandran, AK Salyers, EJ Wensink, P Honore, JP Sullivan, MJ Dart1 and MD Meyer
Neurological Diseases Research, Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, IL, USA
Background and purpose: Selective cannabinoid CB2 receptor agonists have demonstrated analgesic activity across multiple
preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB2 binding site and
broad spectrum analgesic activity in rodent models, but is not an antagonist of CB2 in vitro functional assays. Additionally, its
analgesic effects are m-opioid receptor-dependent. Herein, we describe the in vitro and in vivo pharmacological properties of
A-796260, a novel CB2 agonist.
Experimental approach: A-796260 was characterized in radioligand binding and in vitro functional assays at rat and human
CB1 and CB2 receptors. The behavioural profile of A-796260 was assessed in models of inflammatory, post-operative,
neuropathic, and osteoarthritic (OA) pain, as well as its effects on motor activity. The receptor specificity was confirmed using
selective CB1, CB2 and m-opioid receptor antagonists.
Key results: A-796260 exhibited high affinity and agonist efficacy at human and rat CB2 receptors, and was selective for the
CB2 vs CB1 subtype. Efficacy in models of inflammatory, post-operative, neuropathic and OA pain was demonstrated, and
these activities were selectively blocked by CB2, but not CB1 or m-opioid receptor-selective antagonists. Efficacy was achieved at
doses that had no significant effects on motor activity.
Conclusions and implications: These results further confirm the therapeutic potential of CB2 receptor-selective agonists for
the treatment of pain. In addition, they demonstrate that A-796260 may be a useful new pharmacological compound for
further studying CB2 receptor pharmacology and for evaluating its role in the modulation of pain.
British Journal of Pharmacology (2008) 153, 390–401; doi:10.1038/sj.bjp.0707568; published online 12 November 2007
Keywords: cannabinoid; CB2; A-796260; inflammatory pain; neuropathic pain
Abbreviations: A-796260, [1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)-methanone; AM1241,
(2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl-methanone; CP 55,940, 5-(1,1-dimethyl-
heptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol; GW-842166X, 2-(2,4-dichloro-phenyl-
amino)-4-trifluoromethyl-pyrimidine-5-carboxylic acid (tetrahydro-pyran-4-ylmethyl)-amide; L-768,242
(GW405833), (2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone;
SR141716A (SR1), 5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piper-
idin-1-ylamide; SR144528 (SR2), 5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-carboxylic
acid ((1S,2S,4R)-1,3,3-trimethyl-bicyclo[2.2.1]hept-2-yl)-amide; WIN 55,212-2, (R)-( þ )-[2,3-Dihydro-5-
methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone
Introduction
Although the analgesic properties of cannabinoid ligands
have been recognized for many years, the therapeutic
potential of cannabis-based medicines is severely restricted
by the undesired psychotropic side-effects associated with
this class of drugs (Pertwee, 2001; Fox and Bevan, 2005;
Ibrahim et al., 2006; Mackie, 2006; Whiteside et al., 2007).
The pharmacology of natural and synthetic cannabinoid
ligands is derived from their interaction with two cannabi-
noid receptor subtypes, CB1 and CB2. It is well established
that the psychotropic effects of cannabis are derived largely,
if not exclusively, by activity at the CB1 subtype; and based
on rodent studies, it is becoming apparent that the CB2
receptor plays an important role in the mediation of pain
Correspondence: Dr MD Meyer, Neurological Diseases Research, Global
Pharmaceutical Research & Development, Abbott Laboratories, R47W, AP9A-3,
100 Abbott Park Road, Abbott Park, IL 60064, USA.
E-mail: mike.d.meyer@abbott.com
1These authors contributed equally to this work.
Received 2 July 2007; revised 24 September 2007; accepted 16 October 2007;
published online 12 November 2007

A-796260: a selective CB2 receptor agonist
BB Yao et al
391
processing (Malan et al., 2002, 2003; Ibrahim et al., 2005; Fox
and Bevan, 2005). There is an emerging body of evidence to
suggest that expression of the CB2 receptor is upregulated as
(Valenzano et al., 2005), but unlike AM1241, the effects of
L-768,242 are not antagonized by
a m-opioid receptor
antagonist (Whiteside et al., 2005).
a
consequence of tissue or nerve injury, supporting a
Finally, GW-842166X, a pyrimidine derivative structurally
distinct from other known CB2 agonists, has been shown to
exhibit agonist activity at human and rat CB2 receptors in a
yeast reporter gene assay, and is effective in a complete
Freund’s adjuvant (CFA) model of chronic inflammatory
pain (Giblin et al., 2007). This compound has been selected
as a clinical candidate for the treatment of pain associated
with osteoarthritis and rheumatoid arthritis.
potential role for CB2-selective ligands in the treatment of
inflammatory, postoperative and neuropathic pain (Zhang
et al., 2003; Wotherspoon et al., 2005; Romero-Sandoval and
Eisenach, 2007).
Over the years, a number of CB2-selective ligands have
emerged and have been shown to exhibit analgesic,
antihyperalgesic and antiallodynic activity in various rodent
pain models. JWH-133 and HU-308 are structurally related to
D9-tetrahydrocannabinol, and display high radioligand-
binding affinity and selectivity towards the human CB2
receptor (Hanus et al., 1999; Huffman et al., 1999). JWH-133
has been characterized as a full agonist in a GTPgS-binding
assay at the human CB2 receptor (Huffman et al., 2004), and
HU-308 has been characterized as a selective full agonist in
human CB2 cyclic GMP assays (Hanus et al., 1999). However,
their activities at rodent CB1 and CB2 receptors have not
been studied. In preclinical studies, JWH-133 has demon-
strated efficacy in models of both inflammatory and
neuropathic pain, and antagonist blockade studies have
indicated that these activities are mediated through activa-
tion of the CB2 subtype (Elmes et al., 2004, 2005; Sagar et al.,
2005). HU-308 exhibited activity in a formalin-induced
peripheral pain model (Hanus et al., 1999) and a skin-
incision model of postoperative pain (LaBuda et al., 2005). In
both these models, the effects of HU-308 were blocked by the
CB2 antagonist SR144528.
Despite supporting evidence provided by multiple CB2-
selective ligands for the therapeutic potential of this
pharmacological class as novel analgesic agents, inconsis-
tencies exist with respect to both their in vitro and in vivo
profiles. The compounds described above either fail to
exhibit potent or robust agonist efficacy or have not been
fully characterized in functional assays at rodent CB2
receptors. Differences exist with respect to potential interac-
tions with the opioid system, suggesting that different
mechanisms may be contributing to the in vivo properties
of these compounds. These shortcomings and inconsisten-
cies highlight the need for additional compounds to help
assess the potential that this pharmacological class may hold
for the development of new drugs for the treatment of pain.
Methods
Compounds
Several structurally related indole derivatives, including
AM1241 and L-768,242 (GW405833) have been identified as
CB2-selective ligands exhibiting activity in several rodent
pain models. AM1241 has been found to exhibit high
affinity at human, rat and mouse CB2 receptors (Makriyannis
and Deng, 2002; Ibrahim et al., 2003a; Nackley et al., 2003a;
Yao et al., 2006; Bingham et al., 2007). However, the agonist
efficacy profile of AM1241 (as the racemate) is complex and
condition-dependent, ranging from inverse agonist to partial
agonist at the human CB2 receptor (Yao et al., 2006; Bingham
et al., 2007) and is an apparent inverse agonist at the rat CB2
receptor (Bingham et al., 2007). Nevertheless, the behavioural
profile of AM1241 across multiple pain models is consistent
with CB2 agonist activity. The efficacy of AM1241 in models
of inflammatory (Malan et al., 2001), neuropathic (Ibrahim
et al., 2003a) and postoperative pain (LaBuda et al., 2005) has
been shown to be mediated by the CB2 receptor, as the
effects are blocked by CB2- but not CB1-selective antagonists.
Despite an apparent lack of affinity for the m-opioid receptor,
the in vivo analgesic activity of AM1241 in a model of
acute thermal pain is antagonized by both naloxone and
b-endorphin antiserum, and AM1241 is inactive in the
m-opioid receptor knockout mouse (Ibrahim et al., 2005). L-
768,242 is characterized by high affinity for human and
rat CB2 receptors and high selectivity vs the CB1-binding
sites (Gallant et al., 1996). Functionally, it displays partial
agonist activity in human CB2 receptor-mediated inhibition
of forskolin-stimulated cAMP release assays (Valenzano et al.,
2005). Like AM1241, L-768,242 is effective in models
of inflammatory, postoperative and neuropathic pain
Racemic AM1241, SR141716A and SR144528 were prepared
at Abbott Laboratories as previously described (Yao et al.,
2006). CP 55,940, WIN 55,212-2 and AM630 were purchased
from Tocris (Ellisville, MO, USA). L-768,242 was synthesized
as described by Valenzano et al. (2005). GW-842166X was
synthesized as described by Giblin et al. (2007). The para-
toluenesulphonate salt of A-796260 was synthesized as
depicted in the scheme and described herein. Briefly,
acylation of indole with 2,2,3,3-tetramethylcyclopropane-
carbonyl chloride afforded a 1.6:1 mixture of the desired
C(3)- and undesired N-acylated regioisomers, which were
separated by silica gel chromatography. Alkylation of the
indole with the methanesulphonate ester of 2-morpholi-
noethanol gave the free base of A-796260 that was subse-
quently transformed into its toluenesulphonate salt, which
was utilized in all biological experiments in the current
study. Doses of A-796260 are presented as mg kgÀ1 of the free
base (mol wt ¼ 354.50). Characterization of the free base
and toluenesulphonate salt of A-796260 are as described
below: Free base of A-796260, 1H NMR (CD3OD, 300 MHz)
d p.p.m. 1.33 (s, 12H), 2.13 (s, 1H), 2.46–2.54 (m, 4H),
2.79 (t, J ¼ 6.4 Hz, 2H), 3.61–3.71 (m, 4H), 4.37 (t, J ¼ 6.4 Hz,
2H), 7.16–7.30 (m, 2H), 7.45–7.53 (m, 1H), 8.11 (s, 1H), 8.20–
8.30 (m, 1H), MS (DCI/NH3) m/z 355 (M þ H)þ ; Tosylate salt
of A-796260: [1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl]-
(2,2,3,3-tetramethylcyclopropyl)-methanone p-toluenesul-
phonic acid, 1H NMR (MeOH-d4, 300 MHz) d 1.33 (s, 6H),
1.34 (s, 6H), 2.15 (s, 1H), 2.36 (s, 3H), 3.40 (m, 4H), 3.68
(dd, J ¼ 7.1, 7.1 Hz, 2H), 3.90 (m, 4H), 4.73 (dd, J ¼ 7.1,
7.1 Hz, 2H), 7.23 (br d, J ¼ 7.8 Hz, 2H), 7.26 (ddd, J ¼ 8.1, 8.1,
British Journal of Pharmacology (2008) 153 390–401

A-796260: a selective CB2 receptor agonist
392
BB Yao et al
1.4 Hz, 1H), 7.33 (ddd, J ¼ 7.1, 7.1, 1.0 Hz, 1H), 7.56 (br d,
J ¼ 8.1 Hz, 1H), 7.72 (br d, J ¼ 8.5 Hz, 2H), 8.15 (s, 1H), 8.29
assay buffer. Nonspecific binding was defined by 10 mM
unlabelled CP 55,940. Ki values from competition binding
assays were determined with one-site competition curve
fitting using the Prism software (GraphPad, San Diego, CA,
USA).
(dt, J ¼ 7.8, 1.0 Hz, 1H); MS (DCI/NH3) m/z 355 (M þ H)þ
anal (C22H30N2O2 Á C7H8O3S) C,H,N.
;
Me
Me
O
O
Cyclase functional assays
EtMgBr
ZnCl2
Me
Me
Me
Me
Me
The cyclase functional assays were performed using the
HitHunter cAMP assay kit from DiscoveRx (Fremont, CA,
USA) in suspension forms according to the manufacturer’s
protocol and as described previously (Yao et al., 2006).
Briefly, cell suspensions were incubated at 37 1C for 20 min
with variable concentrations of test ligands or 10 mM CP
55,940 as a positive control in the presence of a fixed
concentration of forskolin (18 mM for the rat CB2 line; and
37 mM for human CB1 and CB2, and rat CB1 lines) in
D-phosphate-buffered saline (PBS) buffer (Invitrogen) sup-
plemented with BSA (0.01% final concentration). The
reactions were terminated by the addition of lysis buffer
and the luminescence was detected following the procedure
according to the manufacturer’s instructions. The positive
control, CP 55,940 10 mM, produced significant inhibition of
cAMP levels induced by forskolin in the cell lines expressing
the human CB1 (84% inhibition; n ¼ 10), human CB2 (71%
inhibition; n ¼ 10), rat CB1 (90% inhibition; n ¼ 10) and rat
CB2 receptors (63% inhibition; n ¼ 10). Receptor activation
by ligands is expressed as % response compared to that of
10 mM CP 55,940. EC50 values were calculated by sigmoidal
dose–response curve fitting using Prism (GraphPad) software.
Cl
N
H
Me
N
H
Me
Me
Me
O
OSO2Me
NaH
N
1.
2.
Me
•TsOH
O
SO3H
N
N
O
Cell culture
Reagents for cell culture were purchased from Invitrogen
(Carlsbad, CA, USA) unless indicated otherwise. Human
embryonic kidney (HEK; American Type Culture Collection,
Rockville, MD, USA) cells stably expressing the human CB2,
rat CB2 or rat CB1 receptor were grown in Dulbecco’s
modified Eagle’s medium containing high glucose supple-
mented with 10% fetal bovine serum and 25 mg mlÀ1 zeocin
in a 37 1C incubator in the presence of 5% CO2. HEK cells
stably co-expressing the chimeric Gaq/o5 protein with either
the human or rat CB2 receptor were grown under similar
conditions, except that in addition to the components
described above, the growth medium was further supple-
mented with 200 mg mlÀ1 hygromycin. The Chinese hamster
ovary cell lines stably expressing the human CB1 receptor
were purchased from Euroscreen (Brussels, Belgium), and the
cells were grown under the conditions recommended by the
vendor.
Fluorescence imaging plate reader functional assays
Fluorescence imaging plate reader (FLIPR) assays were
performed using HEK cells stably co-expressing the chimeric
Gaq/o5 protein with either human or rat CB2 receptors
(Mukherjee et al., 2004). Briefly, cells were seeded at 75 000
cells per well 1 day prior to the assay and the assays were
performed with no-wash dye (FLIPR Calcium Assay Kit;
Molecular Device, Sunnyvale, CA, USA) following the
manufacturer’s instructions. Variable concentrations of test
compounds (0.3 nM to 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 and 0.05% BSA), and fluorescence responses were
measured immediately with a FLIPR machine. Net peak
responses were compared with that of 10 mM CP 55,940 and
expressed as percentages of the CP 55,940-evoked response.
EC50 values were analysed with sigmoidal dose–response
curve fitting using Prism.
Radioligand-binding assays
Membrane samples prepared from HEK cells stably expres-
sing the human CB2, rat CB2 or rat CB1 receptor, and Chinese
hamster ovary cells stably expressing the human CB1
receptor were used to perform radioligand-binding assays
using [3H]-CP 55,940 ligand as previously described
(Mukherjee et al., 2004). Briefly, competition experiments
were conducted using 0.5 nM [3H]-CP 55,940 in the presence
of variable concentrations of test compounds in an assay
buffer containing 50 mM Tris-HCl, pH 7.4, 2.5 mM EDTA,
5 mM MgCl2 and 0.05% fatty acid free BSA. After 90 min
incubation at 30 1C, 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
Animals
Adult Sprague–Dawley rats (male, 250–300 g body weight;
obtained from Charles River, Portage, MI, USA) were used for
all experiments. All animals were housed in Association for
Assessment and Accredidation of Laboratory Animal Care
(AALAC) approved facilities at Abbott Laboratories in a
temperature-regulated environment under
a controlled
British Journal of Pharmacology (2008) 153 390–401

A-796260: a selective CB2 receptor agonist
BB Yao et al
393
12 h:12 h light–dark cycle, with lights on at 0600 h. Food and
water were available ad libitum at all times except during
testing. All testing was carried out following procedures
outlined in protocols approved by Abbott’s Institutional
Animal Care and Use Committee.
effect in this assay. Drug effects in this model were
conducted in a randomized, blind manner.
Postoperative pain model. A model of postoperative pain was
performed as described by Brennan et al. (1996). The plantar
aspect of the rat left hind paw was exposed through a hole in
a sterile plastic drape, and a 1 cm longitudinal incision was
made through the skin and fascia, starting 0.5 cm from the
proximal edge of the heel and extending towards the toes.
The plantaris muscle was elevated and incised longitudin-
ally, leaving the muscle origin and insertion points intact.
After homoeostasis by application of gentle pressure, the
skin was apposed with two mattress sutures using 5-0 nylon.
Animals were then allowed to recover for 2 h after surgery, at
which time tactile allodynia was assessed as described above.
Drug effects in this model were conducted in a randomized,
blind manner.
In vivo pain models
Chronic inflammatory pain model. Chronic inflammatory
thermal hyperalgesia was induced by injection of 150 ml of
CFA solution (50%) in PBS into the plantar surface of the
right hind paw of the rats. Thermal hyperalgesia was assessed
48 h post-CFA injection.
Thermal hyperalgesia was determined using a commer-
cially available thermal paw stimulator (UARDG; University
of California, San Diego, CA, USA) described by Hargreaves
et al. (1988). Rats were placed in individual plastic cubicles
mounted on a glass surface maintained at 30 1C, and allowed
a 20 min habituation period. A thermal stimulus, in the form
of radiant heat emitted from a focused projection bulb, was
then applied to the plantar surface of each hind paw. The
stimulus current was maintained at 4.50 0.05 A, and the
maximum time of exposure was set at 20.48 s to limit
possible tissue damage. The elapsed time until a brisk
withdrawal of the hind paw from the thermal stimulus was
recorded automatically using photodiode motion sensors.
The right and left hind paws of each rat were tested in three
sequential trials at approximately 5-min intervals. Paw
withdrawal latency (PWL) was calculated as the mean of
the two shortest latencies. PWL was measured 30 min post-A-
796260 administration in both the CFA-treated and unin-
jected paws.
Knee joint osteoarthritic pain model. Unilateral knee joint
osteoarthritis was induced in the rats by a single intra-
articular (i.a.). injection of sodium monoiodoacetate (MIA)
(Sigma-Aldrich, St Louis, MO, USA) (3 mg in 0.05 ml sterile
isotonic saline) into the joint cavity under light (1–3%)
isoflurane anaesthesia using a 26 G needle (Guzman et al.,
2003; Pomonis et al., 2005). Following the injection, the
animals were allowed to recover from anaesthesia (usually
5–10 min) before being returned to their home cages. To
maintain uniformity across the study, the right knee joint of
each animal was injected with MIA. Hindlimb grip force
assessment was carried out 20 days following MIA injection
as discussed below. The dose of the MIA (3 mg per i.a.
injection) was selected based on results obtained from
preliminary studies wherein optimal pain behaviour was
observed at this dose.
Neuropathic pain model. A model of chronic constriction
injury (CCI)-induced neuropathic pain was produced by
following the method of Bennett and Xie (1988). The right
common sciatic nerve was isolated at mid-thigh level, and
loosely ligated by four chromic gut (5-0) ties separated by an
interval of 1 mm. All animals were left to recover for at least 2
weeks and no more than 5 weeks prior to testing tactile
allodynia.
Pain behavioural assessment of hindlimb grip force
following unilateral injection of MIA was used as
a
behavioural measure of activity-induced pain in adult
osteoarthritic (OA) rats (body weight 20 days following
MIA injection: 325–350 g). Measurements of peak hindlimb
grip force were conducted by recording the maximum
compressive force exerted on the hindlimb strain gauge set
up, in a commercially available grip force measurement
system (Columbus Instruments, Columbus, OH, USA).
During testing, each rat was gently restrained by grasping
it around its rib cage and then allowed to grasp the wire
mesh frame attached to the strain gauge. The experimenter
then moved the animal in a rostral-to-caudal direction until
the grip was broken. Each rat was sequentially tested twice at
approximately 2–3 min interval to obtain a raw mean grip
force (CFmax). This raw mean grip force data were converted
to a maximum hindlimb cumulative compressive force
(CFmax) (g force) per kg body weight for each animal. For
assessing the effects of A-796260, the evaluation of hindlimb
grip force was conducted 20 days following the i.a. injection
of MIA. A group of age-matched naive animals were added to
each experiment and the data obtained from the different
dose groups for the compound being tested were compared
to the naive group. Experiments were conducted in a
A-796260 was injected i.p. 30 min before the test for tactile
allodynia. Tactile allodynia was measured using calibrated
von Frey filaments (Stoelting, Wood Dale, IL, USA) as
previously described (Chaplan et al., 1994). Rats were
placed into inverted individual plastic containers
(20 cm  12.5 cm  20 cm) on top of a suspended wire mesh
grid, and acclimatized to the test chambers for 20 min. von
Frey filaments were presented perpendicularly to the plantar
surface of the selected hind paw, and then held in this
position for approximately 8 s with enough force to cause a
slight bend in the filament. Positive responses included an
abrupt withdrawal of the hind paw from the stimulus, or
flinching behaviour immediately following removal of the
stimulus. A 50% withdrawal threshold was determined using
an up–down procedure (Dixon, 1980). Only rats with a
baseline threshold score of less than 5 g were used in this
study, and animals demonstrating motor deficit were
excluded. A 15 g threshold was used as the maximal possible
British Journal of Pharmacology (2008) 153 390–401

A-796260: a selective CB2 receptor agonist
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BB Yao et al
randomized, blind manner, and drug effects are expressed as
raw mean grip force (CFmax)/body weight (kg).
In vitro ligand characterization in cyclase and FLIPR functional
assays
To assess agonist properties of these ligands, receptor-
coupled adenylyl cyclase activity and calcium influx
response were measured, respectively, in cyclase and FLIPR
functional assays, employing cell lines that express the
recombinant cannabinoid receptors. In cyclase assays, A-
796260 behaved as a potent and efficacious agonist at
human and rat CB2 receptors with EC50 values of 0.710 nM
(Emax of 78%) and 1.63 nM (Emax of 69%), respectively
(Table 2). A-796260 is highly selective at CB2 receptors,
exhibiting 1380- and 175-fold selectivity at human and rat
CB2 receptors compared to CB1 receptors of the respective
species. In contrast, AM1241 did not exhibit either agonist
or inverse agonist activity at human and rat CB2 receptors
in these assays, but it was shown to produce a full agonist
efficacy at both human and rat CB1 receptors at high
concentrations (human CB1 EC50 ¼ 2650 nM, Emax ¼ 101%;
and rat CB1 EC50 ¼ 1970 nM, Emax ¼ 81%). L-768,242 was a
potent inverse agonist at both human (EC50 ¼ 44.4 nM) and
rat (EC50 ¼ 3.26 nM) CB2 receptors, producing maximum
(–30% (human) and –39% (rat)) negative efficacy relative to
that of CP 55,940. In contrast, it was a weak, but efficacious,
agonist at the rat CB1 receptor. GW-842166X was inactive at
human and weakly efficacious at rat CB1 receptors; however,
it exhibited full agonist activity at human and rat CB2
receptors. Reference standards, WIN 55,212-2, CP 55,940,
SR144528 and SR141716A, exhibited properties similar to
previously published results (Felder et al., 1995; Bonhaus
et al., 1998; Tao and Abood, 1998; Lunn et al., 2006).
In FLIPR assays, where a chimeric Gaq/o protein was
employed to facilitate redirection of the Gai/o signalling to
intracellular calcium release responses (Yao et al., 2006),
receptor activation was readily measured by the use of a
FLIPR machine. To characterize A-796260 and other canna-
binoid receptor ligands further, FLIPR functional assays
were performed. A-796260 displayed high potency and full
efficacy at the human CB2 receptor. In contrast, AM1241
and L-768,242 failed to exhibit agonist efficacy, and
GW-842166X exhibited weak agonist potency with an EC50
of 7780 nM and Emax of 84%. Non-selective agonists, WIN
55,212-2 and CP 55,940, exhibited high potencies and full
efficacy, whereas SR144528 and SR141716A were inactive.
WIN 55,212-2, a partial agonist at the rat CB2 receptor in
cyclase assays, was inactive in this assay. Similar to the result
with the human CB2 receptor, AM1241 was also inactive at
the rat CB2 receptor in FLIPR assays (Figure 1).
Locomotor activity. To determine potential CNS side-effects
of A-796260, spontaneous exploratory behaviour was exam-
ined in naive rats (30 min post-compound injection). Rats
were individually placed into test chambers, and horizontal
(locomotion) activity was recorded by a photo beam detector
system for 30 min (AccuScan Instruments, Columbus, OH,
USA).
Data analysis. Statistical analyses were carried out using
Graph Pad Prism (version 4.03; Graph Pad Software Inc., San
Diego, CA, USA). The results are presented as mean
s.e.mean. Statistical significance between group means was
measured by one-way ANOVA, followed by Bonferroni post
hoc analysis. In all cases, Po0.05 was assumed as the level for
statistical significance. ED50 values were also calculated
by linear regression analysis and presented with CI 95%
(Graph Pad).
Results
Ligand affinities and selectivity at CB2 receptors in
radioligand-binding assays
The receptor-binding potencies of A-796260, WIN 55,212-2,
CP 55,940, AM1241, SR144528, SR141716A, GW-842166X
and L-768,242 (GW405833) were determined in [3H]-CP
55,940 competition binding assays performed with mem-
brane preparations generated from recombinant cell lines
expressing the human CB1, human CB2, rat CB1 or rat CB2
receptor, respectively. The results are summarized in Table 1.
A-796260 exhibited high binding potencies at both human
and rat CB2 receptors with Ki values of 4.37 and 13.0 nM,
respectively. Selectivity for the CB2- over the CB1-binding
site was 193-fold in human and 30-fold in rat samples. This is
comparable to the potencies and selectivity exhibited by
AM1241. GW-842166X did not demonstrate detectable [3H]-
CP 55,940 displacement at either human or rat CB1 (up to
10 mM) and had weak affinities at the human and rat CB2
receptors (2000 and 2580 nM, respectively) in the current
assays, whereas L-768,242 showed good binding affinity at
the human and rat CB2 receptors. The binding potencies of
the non-selective agonists WIN 55,212-2 and CP 55,940,
CB1-selective antagonist SR141716A and CB2-selective an-
tagonist SR144528 displayed binding properties similar to
those published previously (Howlett et al., 2002).
In a recent publication (Ibrahim et al., 2005), AM1241 has
been shown to stimulate b-endorphin release via a m-opioid
receptor-dependent pathway. To investigate if CB2-selective
ligands directly interact with the m-opioid receptor, [3H]-
DAMGO-binding assays were performed according to the
method described by Spetea et al. (1998). Neither A-796260,
AM1241 nor SR144528 exhibited affinity at the m-opioid
receptor, up to ligand concentrations of 10 mM, in this assay,
whereas m-opioid receptor ligand naloxone had a Ki value of
17 nM in these experiments.
Effects of A-796260 on CFA-induced chronic inflammatory
thermal hyperalgesia
Two days following CFA injection into the hind paw, a
significant decrease in PWL to thermal stimulation was
observed (PWL uninflamed: 10.7 0.3 s vs PWL inflamed:
5.3 0.1 s, Po0.01), demonstrating the development of
thermal hyperalgesia (Figure 2a). A-796260 dose-depen-
dently attenuated CFA-induced thermal hyperalgesia
(Figure 2a; F7,88 ¼ 68.2, Po0.0001, ED50 ¼ 2.8 mg kgÀ1 i.p.,
CI 95% ¼ 2.1–3.4 mg kgÀ1), demonstrating full efficacy
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BB Yao et al
395
Table 1 Radioligand competition binding assays using [3H]CP 55,940
Ligands
Mean Ki, nM (n)
95% CI of Ki values, nM
hCB1
hCB2
hCB2/hCB1 selectivity
rCB1
rCB2
rCB2/rCB1 selectivity
A-796260
845 (13)
410–1740
13.3 (27)
7.5–23.6
2.11 (71)
1.62–2.77
1270 (10)
354–4545
264 (12)
4.37 (14)
1.87–10.2
1.30 (44)
0.971–1.74
1.08 (99)
0.925–1.27
11.5 (21)
6.99–18.8
5.66 (17)
3.86–8.31
429 (13)
270–682
2000 (7)
561–7140
7.62 (4)
2.23–26.1
193
395 (5)
173–899
4.47 (11)
3.02–6.65
1.54 (29)
1.15–2.07
115 (4)
7.75–1710
428 (6)
159–1150
2.80 (23)
1.94–4.03
410 000 (2)
13.0 (5)
6.35–26.6
1.64 (19)
0.871–3.11
0.987 (56)
0.764–1.28
3.37 (15)
1.24–9.15
1.80 (7)
0.477–6.81
127 (4)
35.4–452
2580 (8)
30
WIN 55,212-2
CP 55,940
10
2.7
2.0
1.6
AM1241
110
34
SR144528
47
238
175–398
SR141716A
2.36 (29)
1.41–3.95
410 000 (2)
182 Â CB1 selective
45 Â CB1 selective
GW-842166X
L-768,242 (GW405833)
NA
NA
993–6700
11.1 (4)
4.74–25.9
282 (2)
1370–5830
37
ND
NA
Abbreviation: NA, not available.
Table 2 Cyclase and FLIPR functional assays
Ligands
Mean agonist EC50, nM (n), efficacy % relative to CP 55,940 (%)
95% CI of EC50 values, nM
Cyclase assays
FLIPR assays
hCB1
hCB2
rCB1
rCB2
hCB2
rCB2
A-796260
983 (7), 118
618–1560
47.2 (5), 113
23.5–94.9
1.81 (12), 103
0.777–4.21
2650 (5), 101
1730–4050
9420 (4), 52
4340–20 400
21.3 (4), À67
5.73–79.1
0.710 (6), 78
0.315–1.60
0.650 (5), 98
0.156–2.72
0.864 (41), 106
0.626–1.19
41000 (9)
286 (8), 97
206–397
16.5 (5), 114
3.74–72.5
4.12 (17), 103
2.51–6.76
1970 (6), 81
842–4610
5960 (3), 39
1780–20 000
31.6 (7), À61
16.2–61.8
26 000 (4), 33
24 200–27 900
916 (2), 70
839–1000
1.63 (7), 69
0.505–5.28
1.16 (6), 46
0.367–3.65
0.682 (42), 99
0.444–1.05
41000 (8)
17.3 (8), 71
10.64–28.2
119 (10), 74
57.94–245
31.3 (212), 99
28.3–34.5
31.0 (4), 39
7.87–125
410 000 (13)
WIN 55,212-2
CP 55,940
13.0 (29), 106
9.44–17.8
410 000 (9)
AM1241
410 000 (18)
SR144528
73.4 (20), À106
48.4–111
43000 (4)
34.3 (17), À75
24.9–47.3
43000 (2)
410 000 (12)
410 000 (3)
410 000 (6)
410 000 (6)
ND
SR141716A
GW-842166X
L-768,242 (GW405833)
425 000 (2)
133 (4), 101
94–187
96.2 (6), 95
65.5–141
7780 (5), 84
2700–22 400
410 000 (4)
ND
44.4 (2), À30
3.26 (4), À39
ND
22.4–88.1
1.09–9.78
Abbreviation: ND, not determined.
(94 7%) at 11 mg kgÀ1. No effect was observed in the
uninflamed hind paw.
antagonist (Rinaldi-Carmona et al., 1994), had no effect on
the analgesia induced by A-796260 (F7,40 ¼ 13.2, Po0.0001).
The rats treated with CB2 receptor antagonists alone at the
doses used in the present studies did not exhibit any change
in PWL as compared with the vehicle-treated animals.
Release of endogenous opioids has been proposed as a
mechanism of action for the analgesic activities of CB2
agonists, and the effects of the CB2 ligand AM1241 have
been shown to be dependent on b-endorphin release and
require a functional m-opioid receptor (Ibrahim et al., 2005).
To investigate whether the analgesic effects of A-796260 are
mediated via m-opioid receptor-dependent activity, CFA-
treated rats were injected with the opioid receptor antagonist
naloxone (10 mg kgÀ1 i.p.) prior to the administration of
Using this in vivo inflammatory pain model, the receptor
specificity was investigated using receptor-selective antago-
nists (Figure 2b). Pretreatment with SR144528 (4.8 mg kgÀ1
i.p.), a CB2 receptor-selective antagonist (Rinaldi-Carmona
et al., 1998), significantly reversed the analgesic efficacy
induced by treatment with A-796260 at 11 mg kgÀ1 i.p.
(F7,40 ¼ 112.2, Po0.0001). AM630, another CB2 receptor-
selective antagonist (Hosohata et al., 1997; Ross et al., 1999),
when pre-administered at 3 mg kgÀ1 i.p., also significantly
blocked the antihyperalgesic activity induced by A-796260
(F7,38 ¼ 77.7, Po0.0001). In contrast, pretreatment with
SR141716A (14 mg kgÀ1 i.p.),
a selective CB1 receptor
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BB Yao et al
Figure 1 Characterization of cannabinoid ligands in recombinant rat CB1 and CB2 cyclase assay systems. A-796260 (a), WIN 55,212-2 (b) and
CP 55,940 (c) were tested in rat CB1 and CB2 cyclase assays. Levels of receptor activation are calculated as the reduction of cAMP levels and are
expressed as a percentage relative to the response of 10 mM CP 55,940. Typically, 10 mM CP 55,940 inhibits forskolin-induced cAMP levels in rat
CB1 and CB2 cyclase assays by 90 and 63%, respectively; these responses were normalized to 100% activation of receptor activity. (d) The
effects of A-796260, AM1241, GW-842166X and L-768,242 (GW405833) at the rat CB2 receptor.
A-796260 (11 mg kgÀ1 i.p.). Naloxone pretreatment had no
effect on the antihyperalgesic activity of A-796260
(Figure 2c; F7,52 ¼ 14.1, Po0.0001). However, under similar
conditions, pretreatment with naloxone significantly re-
versed the analgesic effects induced by AM1241 at
15 mg kgÀ1 i.p. (Figure 2c; F7,52 ¼ 51.5, Po0.0001).
the behavioural testing, and no difference was seen between
the two treatment groups (data not shown). In contrast to
the results with A-796260, the analgesic effects of morphine
were almost completely abolished after the repeated admin-
istration with the same dosing regime (Figure 3b;
F5,41 ¼ 29.4, Po0.0001).
To examine whether the analgesic effects of A-796260 in
the skin incision model of postoperative pain are mediated
via CB2 receptors, studies were conducted in conscious rats
treated with A-796260, 35 mg kgÀ1 i.p., before and after
pretreatment with the CB2 antagonist SR144528. A-796260
produced 75 11% analgesia (Po0.01 vs vehicle) when given
alone, whereas the same dose of A-796260 had no significant
effect (26 6%) in rats pretreated with SR144528 4.8 mg kgÀ1
i.p. (Figure 3c; F3,20 ¼ 29.9, Po0.0001).
Effects of A-796260 in the skin incision model of acute
postoperative pain
Two hours following skin incision surgery, tactile allodynia
was observed on the injured paw (PWTvonfrey ¼ 1.91 0.17 g).
Skin incision did not affect tactile PWT in the uninjured paw.
A-796260, administered i.p. 1.5 h post-surgery, dose-depen-
dently attenuated tactile allodynia (Figure 3a; F4,48 ¼ 39.8,
Po0.0001, ED50 18 mg kgÀ1, CI 95% ¼ 13–25 mg kgÀ1), with
a maximal efficacy of 68 7% (Po0.01 vs vehicle) at the
highest dose tested (Figure 3a). Intraperitoneal administra-
tion of morphine (6 mg kgÀ1) also produced a statistically
significant reversal of allodynia 30 min post-dosing.
A separate study was conducted to determine whether the
analgesic effect of A-796260 was maintained after repeat
administration (twice daily for 5 days). The results, shown in
Figure 3b, indicate that tolerance to the analgesic effect of A-
796260, 35 mg kgÀ1 i.p., did not develop following repeated
administration for 5 days (64 4%, Po0.01 vs vehicle for
acute dosing vs 60 10%, Po0.01 vs vehicle for repeated
dosing). The plasma levels of A-796260 were measured after
Effects of A-796260 on tactile allodynia in a neuropathic pain
model
In rats, CCI of the sciatic nerve (Bennett and Xie, 1988)
produced a decrease in PWT to mechanical stimulation 2
weeks following surgery (PWTvonfrey ¼ 3.7 0.4 g), demon-
strating the development of tactile allodynia. A-796260
treatment attenuated CCI-induced tactile allodynia in a
dose-related manner with an ED50 of 15 mg kgÀ1 i.p. (CI
95% ¼ 5.3–26 mg kgÀ1) and an efficacy of 66 9% (Po0.01 vs
vehicle) at the highest dose tested (Figure 4; F4,54 ¼ 18.0,
Po0.0001). Under the same conditions, morphine
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397
Figure 2 Effects of A-796260 (i.p.) on complete Freund’s adjuvant (CFA)-induced chronic inflammatory thermal hyperalgesia. (a) At 48 h
following CFA injection, A-796260 was injected 30 min before testing. Data represent mean s.e.mean. *Po0.05, **Po0.01 as compared to
vehicle-treated rats (n ¼ 12 per group). (b) Reversal of A-796260-evoked analgesia by pretreatment with CB2-selective antagonists SR144528
or AM630 (SR2, 4.8 mg kgÀ1 i.p.; AM630, 3.0 mg kgÀ1 i.p.), but the CB1-selective antagonist SR141716A (SR1, 14 mg kgÀ1 i.p) had no effect.
The antagonists were administered 15 min before A-796260 injection. (c) Effects of the opioid antagonist naloxone (Nal) on the analgesic
activity induced by A-796260 or AM1241. Naloxone (10 mg kgÀ1 i.p.) was administered 15 min before the injection of A-796260 (11 mg kgÀ1
i.p.) or AM1241 (15 mg kgÀ1 i.p.). In panels b and c data are expressed as mean s.e.mean (n ¼ 6). **Po0.01 as compared to vehicle-treated
rats, þ þ Po0.01 as compared to A-796260 control.
(6 mg kgÀ1 i.p.) also produced
a
statistically significant
haloperidol (1 mg kgÀ1 i.p.) significantly decreased locomo-
reversal of allodynia 30 min post-administration.
tion in rats (Figure 6; F5,42 ¼ 7.10, Po0.0001).
Effects of A-796260 in an OA pain model
Discussion and conclusions
The analgesic effects of A-796260 on activity-induced pain
behaviour were evaluated in a rat model of MIA-induced OA
joint pain (Guzman et al., 2003; Pomonis et al., 2005),
observed 20 days following the i.a. injection of MIA.
Previously we showed that i.a. administration of MIA
produces a long lasting decrease in hindlimb grip force
when compared to saline-injected animals (Chandran et al.,
2006). A-796260 (i.p.) reversed, in a dose-related manner,
MIA-induced decrease in the grip force with 56 7% effect at
35 mg kgÀ1 (Po0.01 vs the vehicle CFmax 370 87 g). The
effects were comparable to the 62% efficacy elicited by
celecoxib (38 mg kgÀ1 i.p.), a clinically effective analgesic for
OA pain (Figure 5; F5,30 ¼ 31.6, Po0.0001).
Selective activation of the cannabinoid CB2 receptor has
been proposed as a new mechanism for the treatment of
pain, with potential for the development of broad-spectrum
analgesics devoid of the CNS-mediated side-effects associated
with non-selective cannabinoid ligands. The evidence sup-
porting this hypothesis has been largely derived from the
study of a handful of CB2-selective ligands that share efficacy
across multiple rodent pain models, but have significantly
different in vitro pharmacological profiles at the CB2 receptor
and also differ with respect to the potential involvement of
the m-opioid receptor in their mechanism of action. In the
present study, we describe the in vitro and in vivo properties of
a new CB2-selective ligand, A-796260.
Similar to the structurally related indole derivatives
AM1241 and L-768,242 (GW405833), A-796260 is character-
ized by high affinity for human and rat CB2 receptors and
moderate to excellent selectivity vs the CB1 receptor
subtypes. Previous studies on AM1241 have shown it has
affinities for both the human and mouse CB2 receptors in the
range from 1.6 (Makriyannis and Deng, 2002) to 28.7 nM
(Bingham et al., 2007) for the human receptor, and from 2
(Nackley et al., 2003b) to 28.8 nM (Bingham et al., 2007) for
Effects of A-796260 on locomotion
Locomotor activity in rats was evaluated by automatically
recording distance travelled in an open field. Systemic
administration of A-796260 (1–35 mg kgÀ1 i.p.) did not
significantly affect locomotor activity (Figure 6). The highest
dose tested in this study has been shown to produce full or
nearly full efficacy across models of chronic inflammatory,
postoperative, neuropathic and OA pain. In this study,
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BB Yao et al
Figure 3 Effects of A-796260 on skin incision-induced acute postoperative pain. (a) A-796260, administered 1.5 h post-surgery, dose-
dependently attenuated tactile allodynia (ED50 ¼ 18 mg kgÀ1 i.p.). Morphine (6 mg kgÀ1 i.p.) was included in the same study. Data represent
mean s.e.mean. **Po0.01 as compared to vehicle-treated animals (n ¼ 12 per group). (b) Analgesic effect of A-796260 (35 mg kgÀ1 i.p.) and
morphine (3 mg kgÀ1 s.c.) in rats after repeated administration for 5 days. Data are expressed as mean s.e.mean (n ¼ 6). **Po0.01 as
compared to vehicle-treated animals; þ þ Po0.01 as compared to the acute single dosing group. (c) Reversal of A-796260 analgesic effects by
CB2-selective antagonist. SR144528 (SR2, 4.8 mg kgÀ1 i.p.) was administered 15 min before A-796260 injection (35 mg kgÀ1 i.p.). Data are
expressed as mean s.e.mean (n ¼ 6). **Po0.01 as compared to vehicle-treated animals; þ þ Po0.01 as compared to A-796260 control.
Figure 5 Effects of A-796260 on hindlimb grip force in rats 20 days
following injection of monoiodoacetate (MIA). A-796260 demon-
strated dose-related reversal of activity-induced pain behaviour in
osteoarthritic (OA) rats with effects comparable to celecoxib
(38 mg kgÀ1 i.p.), a clinically relevant analgesic for OA pain. Data
are expressed as mean s.e.mean. **Po0.01 vs vehicle-treated
group, n ¼ 6 per group.
Figure
4 Effects of A-796260 on tactile allodynia in a rat
neuropathic pain model, sciatic nerve chronic constriction injury
(CCI). A-796260 dose-dependently attenuated neuropathic pain in
this model (ED50 ¼ 12 mg kgÀ1 i.p.), which is seen as an increase in
the withdrawal threshold of the nerve-injured paw. Data are
expressed as mean s.e.mean. **Po0.01 vs vehicle-treated group,
n ¼ 12 per group.
the human CB1 site. The values obtained in this study are
intermediate between these two results (Table 1). Based on
radioligand-binding experiments, A-796260 exhibits a com-
parable level of affinity and selectivity for the human and rat
CB2 receptors relative to previously described CB2-selective
ligands.
the mouse receptor, and 26.7 nM for the rat CB2 receptor
(Bingham et al., 2007). In this study, we observed comparable
affinities and selectivity ratios to these values for AM1241
(see Table 1). However, in previous studies discrepancies in
binding affinity data for L-768,242 have been obtained;
Valenzano et al. (2005) reported a Ki of 3.92 nM at the human
CB2-binding site and 41200-fold selectivity vs the human
CB1 site, whereas Gallant et al. (1996) reported an affinity of
12 nM at the human CB2 receptor and 160-fold selectivity vs
In in vitro functional assays, A-796260 exhibits a profile
distinct from other available CB2 ligands, characterized by
high potencies and full or near full agonist efficacy at human
and rat CB2 receptors, and a high degree of selectivity vs CB1
receptors (Table 2). This is in contrast to structurally related
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399
In this study, A-796260 exhibited robust analgesic activity
in rat models of inflammatory, postoperative, neuropathic
and OA pain. In the CFA model of chronic inflammatory
pain (Figure 2a), A-796260 produced a dose-dependent
antihyperalgesic effect, but did not increase the response
time to thermal stimulus in the uninjured paw. The
antihyperalgesic effects of A-796260 were reversed by the
CB2-selective antagonists AM630 and SR144528 but not by
the CB1-selective antagonist SR141716A (Figure 2b), support-
ing a CB2-mediated mechanism of action. However, unlike
AM1241, whose analgesic activity requires
a functional
m-opioid receptor, A-796260-mediated analgesic effects were
not dependent on m-opioid receptors. Extending the previous
studies by Ibrahim et al. (2005), we demonstrated that the
effects of AM1241 in the CFA model are blocked by the
m-opioid antagonist naloxone (Figure 2c), whereas the anti-
hyperalgesic effects of A-796260 were unaffected by naloxone
under identical conditions, suggesting that the mechanisms
of action of these two CB2-selective indole derivatives are
clearly distinct. However, although the mechanism whereby
m-opioid receptors are involved in AM1241-evoked analgesic
activity is not clearly understood, it does not appear to
involve a direct receptor interaction as AM1241 has no
affinity for the m-opioid receptor.
Figure 6 Effects of A-796260 treatment on spontaneous horizontal
locomotor activity. Average ( s.e.mean; n ¼ 8 rats per group) levels
of horizontal locomotor activity exhibited by rats 30 min following
compound injection (i.p.) are shown. Haloperidol (1 mg kgÀ1 i.p.)
was included as a positive control. **Po0.01, vs vehicle control.
ligands that, in this study, either exhibit no agonist efficacy
(AM1241) at either the human or rat CB2 receptor or display
inverse agonist activity (L-768,242) at the rat CB2 receptor.
Bingham et al. (2007) showed that AM1241 exhibits partial
agonist efficacy when a low (1 mM) concentration of forskolin
was used to stimulate the cAMP release, a finding consistent
with that of Yao et al. (2006), where it was demonstrated that
at the human CB2 receptor, AM1241 exhibits no antagonistic
activity or very low efficacy as an inverse agonist at high
forskolin concentrations (37 mM), but is a partial agonist at
lower (8 mM) forskolin concentrations. Further, the effects of
AM1241 are species-dependent; it is a partial agonist at
human CB2 receptor and an inverse agonist at the rat CB2
receptor (Bingham et al., 2007). L-768,242 has been shown to
exhibit partial agonist efficacy at the recombinant human
CB2 receptor (Valenzano et al., 2005), but its effects on rat
CB1 and CB2 receptors had not been characterized pre-
viously. In this study, L-768,242 exhibited inverse agonist
activities at both human and rat CB2 receptors. The
pharmacological properties of AM1241 and L-768,242 are
consistent with those of protean agonists. In contrast, the
fully efficacious agonist CP 55,940 and the inverse agonist
SR144528 are unaffected by the assay conditions used and
the level of receptor constitutive receptor activity (Yao et al.,
2006). GW-842166X, despite its low potency for displace-
ment of [3H]-CP 55,940 from the human and rat CB2
receptors (2000 and 2580 nM, respectively), did exhibit full
agonist efficacy in cyclase assays in the present study
(Table 2), exhibiting potencies similar to those found by
Giblin et al. (2007) in a yeast reporter gene assay (human
CB2 EC50 ¼ 63 nM, Emax ¼ 95%; rat CB2 EC50 ¼ 91 nM,
Emax ¼ 100%). It is possible that GW-842166X may activate
signalling through another site in addition to the CP 55,940-
binding site on the CB2 receptor. Compared with the CB2
ligands studied previously, the consistent pharmacology,
robust and fully efficacious agonist activities, as well as
selectivity at CB2 receptors displayed by A-796260 demon-
strate that this compound may be a useful tool for studying
CB2 receptor pharmacology and for evaluating the thera-
peutic potential of CB2 agonists in rodent models.
Similarly, in a skin-incision model of postoperative pain
(Brennan et al., 1996), A-796260 was efficacious in reversing
allodynia (2 h post-incision), with
a maximal efficacy
comparable to that observed with morphine (Figure 3a).
This effect of A-796260 was completely inhibited by the
CB2-selective antagonist SR144528, indicating that CB2
receptors are involved in this model as well (Figure 3c).
The efficacy of cannabinoid agonists has previously been
demonstrated in
a postoperative pain model (Romero-
Sandoval and Eisenach, 2007); it was demonstrated that
both CB1 and CB2 activation can contribute to reversal of
incisional pain and the antiallodynic effects of the non-
selective ligand CP 55,940 were partially reversed by both
CB1 and CB2 antagonists, whereas the effects of the
CB2-selective agonist JWH-015 were antagonized by
the CB2 antagonist and not the CB1 antagonist. Consistent
with this work, we also observed that systemic administra-
tion of the CB1 full agonist and CB2 partial agonist WIN
55,212 (see Table 2) produced a reversal of allodynia in the
skin incision model, and that these effects were preferen-
tially blocked by the CB1 antagonist SR141716A (data not
shown). In addition, L-768,242 has also been shown to be
active in this model at a 24 h time point post-surgical
incision (Valenzano et al., 2005). These results provide
further support for the potential of CB2 agonists in the
management of post-surgical pain.
Tolerance develops rapidly to the analgesic effects of
morphine, particularly in animal models. In the present
study, tolerance to the effects of A-796260 did not develop at
a dosing protocol shown to induce tolerance to the analgesic
effects of morphine. Although chronic dosing studies of a
substantially longer duration are required before it can be
concluded that tolerance to the analgesic effects of A-796260
does not occur, these preliminary data suggest that this
compound has less propensity for the development of
tolerance than opioid agonists.
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A-796260: a selective CB2 receptor agonist
400
BB Yao et al
In the CCI of the sciatic nerve model of neuropathic pain
(Bennett and Xie, 1988), A-796260 produced dose-
In this study, we describe the properties of a new ligand,
A-796260, with attributes suggesting it could be useful
compound for the further exploration of CB2-selective
receptor agonists in the treatment of pain. A-796260 was
found to be a potent and selective CB2 agonist with broad-
spectrum antihyperalgesic and antiallodynic activity across
models of chronic inflammatory, postoperative, neuropathic
and OA pain, and these effects were shown to be mediated
through the CB2 receptor and independent of an interaction
with the m-opioid receptor. These properties differentiate A-
796260 from many of the existing CB2 agonists both with
respect to its agonist efficacy profile in vitro and lack of
opioid receptor dependence in vivo. The studies described
here also add additional support for the hypothesis that
selective CB2 receptor agonists may be an attractive
approach for the development of new drugs for the
treatment of chronic pain; they are less likely to cause side-
effects compared to the currently available treatments.
a
dependent reversal of tactile allodynia, suggesting that CB2
receptor agonists have potential as a treatment of neuro-
pathic pain (Figure 4). These findings are consistent with the
effects observed of other CB2-selective ligands, such as
AM1241 (Ibrahim et al., 2003a) and L-768,242 (Valenzano
et al., 2005) in models of neuropathic pain.
I.a. injection of sodium monoiodoacetate into the knee
joint of rats has been used to induce morphological and
behavioural changes that mimic OA in humans (Guingamp
et al., 1997; Guzman et al., 2003; Combe et al., 2004). A
behavioural end point that measures grip force in rats has
been characterized for several classes of drugs with demon-
strated clinical efficacy in the management of pain asso-
ciated with OA (Chandran et al., 2006). In this model,
A-796260 exhibited dose-dependent restoration of perfor-
mance comparable to the clinically effective COX-2 inhibi-
tor celecoxib. To our knowledge, this is the first time a CB2
agonist has been demonstrated to be an effective analgesic in
a model of chronic OA pain. Compounds, such as morphine
and non-selective agonists, which produce sedation or
decreased motor coordination, also have a negative effect
on the grip force task that the animals are required to
perform even in uninjured animals (data not shown). In
contrast, the present findings indicate that, within the dose
range used, A-796260 has minimal effects that could be
attributed to activation of central CB1 receptors.
Conflict of interest
All authors are employees of Abbott Laboratories, which
funded the research.
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