The Endocannabinoid System and Pain

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CNS Neurol Disord Drug Targets. Author manuscript; available in PMC 2010 June 1.
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CNS Neurol Disord Drug Targets. 2009 December ; 8(6): 403–421.
The Endocannabinoid System and Pain
Josée Guindon and Andrea G. Hohmann*

Neuroscience and Behavior Program, Department of Psychology, University of Georgia, Athens,
GA 30602-3013
Abstract
The therapeutic potential of cannabinoids has been the topic of extensive investigation following the
discovery of cannabinoid receptors and their endogenous ligands. Cannabinoid receptors and their
endogenous ligands are present at supraspinal, spinal and peripheral levels. Cannabinoids suppress
behavioral responses to noxious stimulation and suppress nociceptive processing through activation
of cannabinoid CB1 and CB2 receptor subtypes. Endocannabinoids, the brain’s own cannabis-like
substances, share the same molecular target as Δ9-tetrahydrocannabinol, the main psychoactive
component in cannabis. Endocannabinoids serve as synaptic circuit breakers and regulate multiple
physiological and pathological conditions, e.g. regulation of food intake, immunomodulation,
inflammation, analgesia, cancer, addictive behavior, epilepsy and others. This review will focus on
uncovering the roles of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the two best
characterized endocannabinoids identified to date, in controlling nociceptive responding. The roles
of AEA and 2-AG, released under physiological conditions, in modulating nociceptive responding
at different levels of the neuraxis will be emphasized in this review. Effects of modulation of
endocannabinoid levels through inhibition of endocannabinoid hydrolysis and uptake is also
compared with effects of exogenous administration of synthetic endocannabinoids in acute,
inflammatory and neuropathic pain models. Finally, the therapeutic potential of the endocannabinoid
signaling system is discussed in the context of identifying novel pharmacotherapies for the treatment
of pain.
Keywords
anandamide; 2-arachidonoyl glycerol; FAAH; MGL; endocannabinoid transporter; analgesia;
inflammatory; neuropathic pain
INTRODUCTION
Cannabis has been used for more than twelve thousand years and for many different purposes
(i.e. fiber, medicinal, recreational). However, the endocannabinoid signaling system has only
recently been the focus of medical research and considered a potential therapeutic target [1–
3]. Endocannabinoids mimic the pharmacological actions of the psychoactive principle of
marijuana, Δ9-tetrahydrocannabinol (Δ9-THC) [4]. Endocannabinoids are endogenous lipid-
signaling molecules. They are generated in the cell membrane from phospholipid precursors
and possess cannabimimetic properties because they bind and activate one or more cannabinoid
receptor subtypes [5,6]. Endocannabinoids are implicated in different physiological and
*Author for Correspondence: Andrea G. Hohmann, Neuroscience and Behavior Program, Department of Psychology, University of
Georgia, Athens, GA 30602-3013, Tel: 706-542-2252, Fax: 706-542-3275, [email protected].
CONFLICT OF INTEREST
The authors state no conflict of interest.
Guindon and Hohmann Page 2
pathological functions (regulation of food intake, immunomodulation, inflammation,

analgesia, cancer, addictive behavior, epilepsy and others) [2,7]. The two best-studied
endocannabinoids isolated to date are arachidonoylethanolamine (anandamide or AEA) and
2-arachidonoylglycerol (2-AG). AEA is hydrolyzed by the enzyme fatty-acid amide hydrolase

(FAAH) whereas 2-AG is degraded by the enzyme monoacylglycerol lipase (MGL) [7,8]. The
main goal of this review will be to uncover the role of AEA and 2-AG in pain modulation. This
will be accomplished by reviewing studies examining mobilization of endocannabinoids under
physiological conditions or by using pharmacological tools that inhibit their uptake or
degradation. This review will also consider studies employing exogenous administration of
synthetic endocannabinoids in combination with other pharmacological approaches aimed at
regulating their uptake or degradation. The overall goal is to understand the physiological role
of the endogenous ligands at different levels of the pain pathway and in different models of
pathological pain.
CANNABINOID RECEPTOR PHARMACOLOGY

Cannabinoids produce their effects through the activation of distinct G protein-coupled
receptors identified as the cannabinoid CB1 [9,10] and CB2 receptors [11]. Cannabinoid CB1
and CB2 receptors are members of the superfamily of seven-transmembrane-spanning G
protein-coupled receptors and share 44 % identity at the protein level [11,12]. Similarity
increases to 68 % when only the transmembrane region is considered [11,12]. Activation of
both cannabinoid receptor subtypes inhibits adenylate cyclase activity by coupling to the α–
subunit of the G protein of the Gi/o family (Gi 1, 2 and 3, and Go 1 and 2) [13]. In contrast to
CB2 receptor activation, CB1 receptor activation modulates calcium or potassium conductance
[14,15], properties linked to the suppression of neuronal excitability and neurotransmitter
release. However, activation of MAPK and Krox-24 expression presumably through the
activation of G-protein βγ subunits is another signalling mechanism recruited by both CB1 and
CB2 receptors [16,17]. Furthermore, CB1 receptor activation can inhibit type 5-HT3 ion
channels [18], modulate the production of nitric oxide [for review see 19,20], alter sodium
channel conductance [21] and activate the Na+/H+ exchanger [22]. Signaling mechanisms
engaged by activation of CB1 and CB2 receptors have been recently reviewed [13,23].
Cannabinoid CB1 receptors are found mainly in the CNS and, to a lesser extent, in certain
peripheral tissues [24]. At the peripheral level, they are localized in adrenal gland, adipose
tissue, heart, liver, lung, prostate, uterus, ovary, testis, bone marrow, thymus, tonsils and
presynaptic nerve terminals [12,20,25–27]. Within the brain, they are found in the cerebral
cortex, hippocampus (with highest concentrations in the dentate gyrus), amygdala, basal
ganglia, substantia nigra pars reticulata, internal and external segment of the globus pallidus
and cerebellum (molecular layer) [15,20,24]. More significantly for the purposes of the present
review, they are found at central and peripheral levels of the pain pathways [28–32]. The
distribution of cannabinoid receptors provides an anatomical basis for the analgesic effects of
the cannabinoids. Activation of presynaptic CB1 receptors in different brain regions or on
primary afferents inhibits the release of neurotransmitters by decreasing calcium conductance
and by increasing the potassium conductance [26].
CB2 receptors are primarily localized to cells of the immune system. CB2 receptors are mainly
found in the spleen, tonsils and thymus, tissues responsible for immune cell production and
regulation [11,12,15]. These immune cells include mast cells, B cells, T4 and T8 cells,
microglial cells, macrophages, natural killer cells and, to a lesser extent, monocytes and
polymorphonuclear neutrophils [12,15,33]. Previous reports suggested that CB2 receptors were
absent in neurons of the central nervous system (CNS) [11,34]. However, recent studies suggest
that they are found in the brain, on dorsal root ganglia, in the lumbar spinal cord, on sensory
neurons, on microglia as well as in peripheral tissues [35,36].
A better understanding of the role of cannabinoid receptors in different physiological processes

has been obtained through research employing pharmacological and genetic tools such as
competitive antagonists and knockout mice with disrupted CB1 [37,38] and/or CB2 genes
[39,40]. Pharmacological evidence also supports the existence of one or more additional
receptors for cannabinoids distinct from CB1 and CB2 receptors (reviewed in [41,42]). Of
particular recent interest are the GPR55 receptor [43–45] and GPR3, GPR6 and GPR12 which
are sphingosine-1-phosphate lipid receptors [46–48]. More work is necessary to determine the
connection of novel receptor subtypes such as GPR55 to the endocannabinoid system using
more specific compounds and genetic tools.
ENDOCANNABINOIDS
The discovery of AEA [49], the first endocannabinoid isolated from brain, was followed a few
years later by the identification of 2-AG [50,51]. Since then, several putative endocannabinoids
have been isolated which include noladin ether [52], virodhamine [53] and N-
arachidonoyldopamine (NADA) [54,55]. Much less information is known about the
endocannabinoid-like properties of these latter putative endogenous ligands (see [56] for a
review). Indeed, elucidation of the endogenous function of these compounds in different
physiological processes and their precise mechanisms of action requires further investigation
[57]. Here, we will consider the roles of different cannabinoid receptors, different
endocannabinoids and the machinery responsible for their synthesis and degradation. In some
cases, functions of the endocannabinoid system are surmised following pharmacological

inhibition of endocannabinoid deactivation. Thus, FAAH and MGL inhibitors increase
endocannabinoid accumulation (AEA and 2-AG, respectively) by inhibiting hydrolysis of
fatty-acid amides and monoacylglycerols; these enzymes have multiple substrates. Both AEA
and 2-AG are derivatives of arachidonic acid and bind to cannabinoid CB1 and CB2 receptors,
although with different affinities and efficacies [58]. However, the variable affinity for
cannabinoid receptors may be due, in part, to the existence of distinct binding sites for the
different ligands on cannabinoid receptors, as documented by molecular modeling studies
[59].
SYNTHESIS AND RELEASE OF AEA AND 2-AG

Endocannabinoids are produced on demand either by activity-dependent or receptor-stimulated
cleavage of membrane phospholipid precursors. Endocannabinoids can be released
immediately from cells after their production since they are highly lipophilic and thus are poorly
suited for storage (for review see [8,60,61]). Endocannabinoid signaling is regulated by
synthesis, release, uptake and degradation. Membrane depolarization, increases in intracellular
calcium levels and receptor stimulation can all activate enzymatic processes leading to the
cleavage of membrane phospholipids precursors and subsequent synthesis of

endocannabinoids (see [8,60,61] for a review). Different enzymes are implicated in the
synthesis of AEA and 2-AG. AEA biosynthesis was originally believed to occur from
enzymatic cleavage of a phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine
(NAPE). NAPE is synthesized by the enzymatic transfer of arachidonic acid in the sn-1 position
of a phosphatidylcholine to the amide group of a phosphatidylethanolamine under the
supervision of the calcium-independent N-acyl-transferase (NAT) [62]. NAPE is then
hydrolyzed to AEA by a specific phospholipase D (NAPE-PLD) which has recently been
cloned and molecularly characterized [8,63–65]. However, NAPE-PLD knockout mice show
no deficit in AEA production, a finding which questions the role of this enzyme in anandamide
biosynthesis [66]. Thus, multiple enzymatic pathways may be involved in the biosynthesis of
anandamide and NAPE-PLD is unlikely to exclusively control its’ biosynthesis [66,67]. 2-AG
is synthesized in a two step process. First, the 2-AG precursor diacylglycerol (DAG) is formed
from enzymatic cleavage of membrane phospholipid precursors by the enzyme phospholipase
C (PLC) (for review see [68,69]). DAG is subsequently hydrolyzed by a diacylglycerol lipase
(DAGL) selective for the sn-1 position to generate 2-AG [8,68,70,71]. A detailed review of
these processes is available [7,60,61] (see Fig. 1). Subsequent to their on-demand synthesis,
endocannabinoids may activate cannabinoid receptors following their release into the
extracellular space or their movement directly into the cell membrane [72]. AEA preferentially
binds to CB1 receptors in vitro, and exhibits low affinity for the transient receptor potential
vanilloid 1 (TRPV1) [73–76]. 2-AG is known to activate both CB1 and CB2 receptors [50,
51]. This compound is found in the brain in concentrations 170-fold higher than those of
anandamide [77]. A role for endogenous 2-AG in pain modulation has only recently been
described [78,79].
In addition to activating metabotropic CB1 receptors, AEA can also activate ionotropic TRPV1
receptors as an endovanilloid. TRPV1 receptors are expressed in nociceptive sensory neurons
and can detect/respond to noxious mechanical, thermal (i.e. heat) and chemical (i.e. capsaicin)
stimuli [73,75,80–83]. Capsaicin and AEA share the same binding site [84], but AEA must be
found at high concentrations to activate TRPV1 receptors. Activation of TRPV1 receptors
increases intracellular levels of cations such as Ca2+ and depolarizes the cell; these effects can
also liberate calcitonin gene-related peptide (CGRP) and substance P to produce vasodilatation
[73]. At high concentrations, AEA can thus exert opposing effects through activation of
cannabinoid and TRPV1 receptors, respectively. A functional relationship exists between
TRPV1 and CB1 receptors in dorsal root ganglia [85], spinal cord and brain [86] and wherever
these two receptors may be co-expressed in the same cell. Antagonists of TRPV1 receptors are
implicated in anxiolytic effects in the brain [82]. Peripheral and central TRPV1 receptors
therefore remain a viable therapeutic target.
UPTAKE OF ENDOCANNABINOIDS
Reuptake of endcannabinoids, and most notably anandamide, in the synaptic space may be
facilitated by a transporter that has yet to be molecularly cloned. Pharmacological inhibitors
for endocannabinoid transport have nonetheless been developed (AM404, VDM11, and others)
[7,74,87,88]. AEA may accumulate in neurons and other cells by facilitated diffusion rather
than employing a specific transport mechanism [89,90]. This process is saturable, temperature-
dependent, does not require ATP and is driven by a transmembrane concentration gradient.
The existence of a specific endocannabinoid transporter remains controversial, and new
discoveries are necessary to establish beyond doubt the mechanism of endocannabinoid
transport [90–93]. However, it is noteworthy that AEA uptake is selectively inhibited by a
variety of pharmacological agents, consistent with the existence of a saturable component in
the transport of anandamide [87,94–96] (see Fig. 1).
Since endocannabinoids are produced on demand and can be released immediately from cells,
they can regulate synaptic transmission, both excitatory and inhibitory. In the CNS,
endocannabinoids act as neurotransmitters. Endocannabinoids are released from depolarized
postsynaptic neurons and travel to presynaptic terminals where they activate CB1 receptors
through a retrograde signaling mechanism [97–100] (see Fig. 1). The general effect is a
decrease in the release of neurotransmitters such as GABA (γ-amino butyric acid) or glutamate.
This retrograde signaling mechanism suggests an important modulatory role for
endocannabinoids in controlling neuronal excitability and maintaining homeostasis [101].
DEGRADATION OF AEA AND 2-AG

Endocannabinoid signaling is limited by efficient degradation processes involving enzymatic
hydrolysis mediated by specific intracellular enzymes. The enzymes which degrade
endocannabinoids are quite well characterized and include fatty-acid amide hydrolase (FAAH)
and monoacylglycerol lipase (MGL) ([60,61], for a review). Inhibitors for FAAH (AM374,
URB597, URB532 and others) or MGL (URB602, OMDM169, JZL184 and Compound 11)
enzymes have been described ([102]; see [7,103] for a review), although selectivity of some
agents may vary considerably. FAAH hydrolyzes AEA and related compounds [103–105]
whereas MGL metabolizes 2-AG [106,107]. FAAH, a membrane bound enzyme, hydrolyzes
AEA in neurons and astrocytes into breakdown products arachidonic acid and ethanolamine
[104,108]. The distribution of FAAH in organs of the rat has been described in detail; its activity
is highest in the liver followed by the small intestine, brain, and testis (see [109] for a review).
Immunohistochemical studies have mapped the distribution of FAAH in the brain. FAAH is
found in the termination zone of the spinothalamic tract in the ventral posterior lateral nucleus
of the thalamus [110–112]. This pathway is implicated in the transmission of nociceptive
information to the brain (for review see [113]). FAAH has also been found in Lissauer’s tract,
in neurons of the superficial dorsal horn of the spinal cord and in dorsal root ganglion cells.
Although FAAH can hydrolyze 2-AG in vitro [114], MGL is the predominant enzyme which
controls 2-AG hydrolysis. MGL, a serine hydrolase, hydrolyzes 2-AG into breakdown products
(arachidonic acid and glycerol). MGL is located on presynaptic [60,78,106] whereas FAAH
is found on post-synaptic [60,103] neurons. Northern blot, immunohistochemical and in situ
hybridization techniques have demonstrated that MGL, a presynaptic enzyme, is
heterogeneously distributed in the rat brain with the highest levels observed in regions
expressing CB1 receptors, such as the cortex, thalamus, hippocampus and cerebellum [106].
MGL is localized exclusively to axon terminals, where it colocalizes with CB1 [115]. By
contrast, FAAH is a postsynaptic enzyme and may regulate AEA levels near sites of synthesis
[60,103]. Although the biosynthesis and metabolism of AEA and 2-AG have been simplified
here to maintain the focus of this review, it is important to mention that, in addition to
hydrolysis, alternative metabolic pathways exist [67,116–118]. For example, in addition to
undergoing hydrolysis, endocannabinoids undergo oxidative metabolism, through which they
are transformed into other biologically active mediators [119]. Indeed, there is evidence for
the metabolism of AEA and 2-AG by cyclooxygenase (COX), lipoxygenase (LOX) and
cytochrome P450 enzymes, further adding to the complexity of endocannabinoid signalling
mechanisms [116,117,120,121].
ENDOCANNABINOIDS IN PAIN PATHWAYS

Cannabinoid receptors, endocannabinoids, and enzymes controling their synthesis and
degradation are localized to multiple levels of the neuraxis, from the periphery to the CNS
([122]; for review see [123]). The discovery of the endocannabinoid system, the availability
of antagonists for cannabinoid receptors (CB1 and CB2) and the generation of knockout mice
lacking CB1 and/or CB2 and FAAH have spurred research in this growing field. Sites of action
for endocannabinoids in suppressing pain were initially suggested by studies employing
synthetic cannabinoids targeted at CB1 and/or CB2 receptors. These studies have been recently
reviewed [123–126].
SUPRASPINAL LEVEL
The antinociceptive [127] and electrophysiological [128] effects produced by the systemic
administration of cannabinoids are attenuated following spinal transaction. These studies
implicate an important role for supraspinal sites in contributing to cannabinoid analgesic action.
Direct support for supraspinal sites of cannabinoid analgesic action was derived from studies
injecting synthetic cannabinoid agonists intraventricularly and locally into various brain
regions (for review see [126]). Structures targeted include the periaqueductal gray (PAG)
[129,130], thalamus [131], rostral ventromedial medulla (RVM) [132,133] and amygdala
[134,135], among others. These studies have permitted the identification of brain regions
responsible for the antinociceptive properties of cannabinoids. Activation of these sites by
endocannabinoids may, therefore, produce antinociception under physiological conditions.
Neurophysiological studies by Walker’s laboratory first documented that cannabinoids

suppress nociceptive processing ([131,132,136]; see [126] for a review). Cannabinoids,
administered systemically, suppress activity of nociceptive neurons in the spinal dorsal horn
[136] and ventralposterior lateral nucleus of the thalamus, without altering the activity of purely
nonnociceptive neurons [131]. Importantly, these neurophysiological effects correlate highly
with the antinociceptive effects of cannabinoids, and cannot be attributed to the motor effects
of the same compounds [131].
Walker’s group first identified a role for endogenous AEA, released under physiological
conditions, in pain modulation [137]. Electrical stimulation of the dorsolateral PAG produced
antinociception in the tail-flick test and mobilized endogenous AEA, as measured by
microdialysis. Importantly, this stimulation-produced analgesia was blocked by the CB1
antagonist SR141716A, demonstrating mediation by CB1. Intraplantar administration of
formalin was also shown to increase levels of endogenous AEA in the dorsolateral PAG. Thus,
noxious stimulation may produce endocannabinoid mobilization [137]. Exposure to an
environmental stressor (brief continous footshock) also produces endocannabinoid-mediated
stress-induced analgesia that is associated with mobilization of endogenous 2-AG and
anandamide [78]. Endocannabinoid mobilization was most pronounced in dorsal midbrain
fragments containing the intact PAG [78]. Endocannabinoid-mediated stress-induced
analgesia is blocked by CB1 but not by CB2 antagonists and is insensitive to blockade by opioid
(i.e. with naltexone) and TRPV1 (i.e. with capsazepine) antagonists [78,138]. Moreover, 2-
AG mobilization in the PAG correlates highly with endocannabinoid-mediated stress

antinociception [139]. These observations are also consistent with the ability of systemic and
locally administered FAAH inhibitors (e.g. URB597, arachidonoylserotonin),
endocannabinoid uptake inhibitors (e.g. VDM11) and locally administered MGL inhibitors
(URB602) to enhance endocannabinoid-mediated stress antinociception through a CB1-
dependent mechanism [78,79,138]. These effects were all observed at doses that do not alter
basal nociceptive thresholds. In the case of URB602, which is not appropriate for systemic use
as a selective MGL inhibitor, biochemical studies confirmed that URB602, injected into the
PAG, increased levels of 2-AG selectively without altering levels of AEA [78]. These studies
collectively suggest a functional role for endogenous AEA and 2-AG in suppressing pain under
physiological conditions.
Exogenous cannabinoids also modulate activity of ON and OFF cells in the rostral
ventromedial medulla; here, inactivation of the RVM suppresses exogenous cannabinoid
antinociception [133]. Pharmacological inactivation of the RVM also suppresses
endocannabinoid-mediated analgesia in a rodent model of stress-induced analgesia [138].
Endocannabinoid-mediated stress-induced analgesia is also enhanced in a CB1-dependent
manner by intra-RVM administration of a FAAH inhibitor, administered at doses that do not
alter the basal nociceptive threshold [138]. These studies support a role for endogenous AEA
in the RVM in endocannabinoid-mediated analgesia, although a role for 2-AG has not been
assessed.
Endocannabinoid levels are altered following nerve injury in specific brain regions implicated
in cannabinoid antinociceptive mechanisms. For example, injury of the sciatic nerve increases
AEA and 2-AG levels in the PAG and RVM [140], structures implicated in both the descending
modulation and the descending facilitation of pain (see [113] for review). AEA levels were
also increased in the dorsal raphe following chronic constriction injury (CCI) [140,141].
Systemic administration of inhibitors of endocannabinoid uptake (VDM-11, OMDM-2,

UCM-707 and LY2318912) increases AEA and/or 2-AG levels in brain [93,142]. Interestingly,
FAAH inhibition by N-arachidonoyl-serotonin (AA-5-HT) was shown to increase brain levels
of AEA and 2-AG [142]. These studies suggest that inhibitors of endocannabinoid uptake and
deactivation show therapeutic potential for increasing endocannabinoid levels.
SPINAL LEVEL
Intrathecal administration of cannabinoids produces antinociception [143–145], and
suppresses nociceptive neuronal activity [146]. These studies initially documented the
existence of spinal sites of cannabinoid antinociceptive actions. Indeed, behavioral [143,145],
electrophysiological [146–148] and neurochemical [128,149] studies have demonstrated that
cannabinoids act at the spinal level to modulate pain. Mixed cannabinoid agonists such as
levonantradol [145], WIN55,212-2 [150] and CP,55,940 [151], at the spinal level, produce
CB1-mediated antinociceptive effects. Moreover, cannabinoids suppress C-fiber-evoked
responses of dorsal horn neurons recorded in normal, inflamed and nerve injured rats [152–
155]. Furthermore, these data are consistent with the ability of cannabinoids to suppress Fos
protein expression, a neurochemical marker of sustained neuronal activation, in different
animal models of persistent pain through CB1 and CB2-specific mechanisms [128,156–159].
Cannabinoid receptors have been demonstrated on primary afferents neurons at pre- and post-
synaptic sites in the spinal cord using receptor binding and quantitative autoradiography
[160,161]. In the dorsal horn of the spinal cord, CB1 receptors have been found on interneurons
[29] and on astrocytes [162].
Upregulation of cannabinoid receptors is also observed in the spinal cord following nerve injury
[150,163], suggesting that both endocannabinoids and their receptors are regulated under
conditions of injury. Exposure to an acute environmental stressor increases 2-AG, but not
anandamide, accumulation in the lumbar spinal cord; 2-AG accumulation in the spinal cord
correlates highly with the appearance of stress antinociception [79]. Intrathecal administration
of inhibitors of both FAAH (URB597/AA5-HT) and MGL-preferring (URB602) also enhance
endocannabinoid-mediated stress-induced analgesia through a CB1-dependent mechanism.
AEA and 2-AG are also increased in the spinal cord following induction of a neuropathic pain
state produced by CCI of the sciatic nerve [140]. The endocannabinoid system is similarly
modulated in response to a spinal cord contusion in rats [164]. The early stages are marked by
increases in AEA levels, upregulation of the synthetic enzyme NAPE-PLD, and
downregulation of the degradative enzyme FAAH. The delayed stages are marked by increases
in 2-AG, a marked upregulation of the 2-AG synthesizing enzyme DAGL-α (i.e. in neurons,
astrocytes and immune infiltrates), and a moderate increase in levels of the degradative enzyme
MGL [164]. In this study, CB1 receptors were expressed in neurons, oligodendrocytes, and
astrocytes, whereas CB2 receptors were strongly upregulated after the lesion and expressed
mainly in immune infiltrates and astrocytes [164]. These studies highlight the importance of
the endocannabinoid system as a potential therapeutic target for treatment of both spinal cord
injury and neuropathic pain.
PERIPHERAL LEVEL
Peripheral antinociceptive actions of cannabinoids have been demonstrated in numerous
animal pain models (for review see [123–125]). Harnessing these mechanisms shows
considerable promise for separating the therapeutic effects of cannabinoids from unwanted
CNS side-effects. Cannabinoid receptors are synthesized in dorsal root ganglion (DRG) cells,
which are the source of primary afferent input to the spinal cord [30,31,85,165–167]. These
afferent nerve fibers transmit information about sensory stimulation to the spinal cord, thereby
enabling communication between the periphery and specific areas of the CNS that contribute
to pain perception [168,169]. Following the induction of neuropathy (by spinal nerve ligation),
cannabinoid receptors and their endogenous ligands (AEA and 2-AG) are increased in the DRG
on the ipsilateral side of the injury [170]. Cannabinoid CB1 [30,31,85,162] and CB2 receptors
[165,167] are also found in the DRG. DRG cells synthesize cannabinoid receptors, and
transport them to peripheral terminals of primary afferents [30,31]. Multiple approaches
support the presence of cannabinoid receptors on primary afferent neurons [85,166,171].
CB1 and CB2 receptors are found in large myelinated and small unmyelinated human cutaneous
nerve fibers [166]. Both cannabinoid receptor subtypes have also been found in different layers
of the skin, and in some adnexal structures (sweat glands, sebaceous cells and others) which
may contribute to peripheral antinociceptive actions [166,172–175]. Endocannabinoid levels
and FAAH activity have also been measured in rodent paw skin [176–179]. AEA is observed
in paw tissue [177–178] whereas a decrease in FAAH activity is observed in the inflamed paw
following carrageenan-induced inflammation [179]. In the formalin model, 2-AG hydrolysis
inhibitor, OMDM169, increased levels of 2-AG, but not AEA, in the ipsilateral paw [180].
However, Beaulieu and collaborators did not find an increase in AEA and 2-AG levels in the
formalin test, measured 2 h after formalin injection when pain behavior has subsided [176]. In
a model of bone cancer pain, intraplantar administration of exogenous AEA or the FAAH
inhibitor URB597 increased the local level of AEA [181]. These studies suggest that
manipulation of peripheral endocannabinoids may be promising strategy for the management
of pain.
MODULATION OF THE ENDOCANNABINOID SYSTEM IN ANIMAL MODELS

Studies evaluating the presence of hypersensitivity to pain (hyperalgesia) following
pharmacological blockade of CB1 receptors provided early physiological support for the
hypothesis that endocannabinoids suppress pain. Hyperalgesia has been observed in the
hotplate test following intrathecal administration of the CB1 antagonist/inverse agonist
SR141716A, and these effects are mimicked by CB1 antisense knockdown at the spinal level
[182,183]. Pharmacological blockade of CB1 receptors with SR141716A has also been
reported to produce hyperalgesia in the formalin test [177,184]. However, these findings have
not been observed by all investigators [176], suggesting that differences in the level of
endogenous analgesic tone may contribute to differences between the studies (see [126] for
review). Moreover, the inverse agonist properties of SR141716A complicate interpretation of
studies attempting to unmask tonic endocannabinoid activity using competitive antagonists.
Therefore, documentation of intrinsic effects of endocannabinoids released under

physiological conditions is critical for understanding the functional roles of endocannabinoids
in nociceptive processing. As described above, studies employing stimulation-produced
analgesia and stress-induced analgesia provide direct support for the hypothesis that
endogenous AEA and 2-AG suppress pain through a CB1-dependent mechanism. In these
studies, the tail-flick test was used to quantify the impact of electrical brain stimulation or
exposure to footshock stress on antinociception. Thus, it is important to emphasize that
treatment with CB1 antagonists [79,128,137,138,160] or modulators of endocannabinoid

transport or deactivation [79,128,138,160] lacked intrinsic effects in the tail-flick test in the
absence of a stimulus to trigger endocannabinoid mobilization (i.e. brain stimulation or
footshock exposure). Thus, it is important to emphasize that tail-flick stimulation is not the
trigger for endocannabinoid mobilization in these studies, and antagonists do not alter basal
nociceptive thresholds under testing conditions. A role for CB2 was not evaluated in studies
of endocannabinoid-mediated stimulation-produced analgesia, presumably due to the lack of
availability of a CB2 antagonist at the time the work was conducted [137]. Stress-induced
analgesia is also CB1-mediated; it is blocked by multiple CB1 antagonists, involves the
mobilization of endocannabinoids at supraspinal (2-AG and AEA; [78]) and spinal (2-AG
alone; [79]) levels and is enhanced by inhibitors of endocannabinoid deactivation (URB597,
AA-5-HT, URB602) or transport (VDM11). The failure of a CB2 antagonist to block
endocannabinoid-mediated stress-induced analgesia in these studies [78] may reflect the
absence of a CB2-mediated component in endocannabinoid-mediated stress analgesia or,
alternately, the failure of the spinally-mediated tail-flick test to detect a CB2-mediated

component of endocannabinoid analgesia. The existence of a cross-tolerance and cross-
sensitization between exogenous cannabinoid antinociception and endocannabinoid-mediated
stress-induced analgesia suggests that these phenomena are linked by a common mechanism
[185]. The development of drugs that inhibit the enzymatic degradation of endocannabinoids
(i.e. through inhibition of FAAH or MGL) or their transport has improved our understanding
of the functional roles of the endocannabinoid system in modulating pain under physiological
conditions.
Effects of exogenous administration of endocannabinoids (focusing on AEA and 2-AG) and

their modulation in models of acute, inflammatory and neuropathic pain models are reviewed
below. However, one limitation of studies employing exogenous endocannabinoids is that they
do not demonstrate that the endogenous ligands play similar roles under physiological
conditions.
ACUTE NOCICEPTION
Exogenous administration of endocannabinoids or their modulation via inhibition of
endocannabinoid deactivation or uptake can produce antinociception in acute pain models (see
Table 1 and Table 2). The magnitude of the observed antinociceptive effect may differ
depending upon the assay, the endocannabinoid used and/or the mechanism employed to alter
endocannabinoid levels. The tail flick test examines the latency for a rodent to “flick” its tail
away from a radiant heat source [186], or to remove the tail following immersion in hot water
(see Table 1). In this test, the endocannabinoid uptake inhibitors (VDM-11 and UCM707)
produce CB1-mediated antinociception [187] under conditions in which the endocannabinoid
system is activated [78]. Exogenous administration of AEA produces antinociception [188–
191], although few studies have evaluated whether this effect is mediated by cannabinoid
receptors. Several groups have evaluated a CB1 component in exogenous AEA antinociception
[192–194], but other studies have suggested that anandamide produces antinociception through
a CB1-independent mechanism [188,191]. All these studies assessed pharmacological
specificity using the CB1 antagonist/inverse agonist SR141716A antagonist. Thus, it is
important to emphasize that SR141716A acts as an inverse agonist at CB1 receptors and can
activate both CB2 and vanilloid TRPV1 receptors, albeit with low affinity (for review see
[195]). Moroever, a role for CB2 receptors cannot be discounted from contributing to the
antinociceptive effects of exogenous administration of AEA, because mediation by CB2
receptors was not assessed in these studies. MGL (URB602) and FAAH (AA-5-HT, PMSF,
PTK, URB597) inhibitors with varying degrees of selectivity also produce antinociceptive
effects in the tail flick test [189,196,197], and specifically under conditions in which the
endocannabinoid system is activated and basal nociceptive thresholds are not altered by the
same treatments ([79,138] for FAAH inhibitors only; [78] for MGL and FAAH inhibitors). In
these studies, cannabinoid receptor antagonists directed at CB1 (AA-5-HT, PTK, URB597 and
URB602 [78 79,138]) or at CB1/CB2 (URB597 [197]) were used to identify the receptor
mechanism underlying these effects. Indeed, studies employing FAAH knockout mice also
corroborate the previous results; a CB1-mediated component is observed in both the tail
immersion and hot plate tests under conditions in which both CB1 and CB2 antagonists were
evaluated [198]. The combination of exogenous AEA with FAAH (ibuprofen, indomethacin,
PMSF, URB597) inhibitors also produces antinociception [189,191,196] that is mediated by
CB1 receptors [189,191].
The hot plate test involves individually placing rodents on a metal surface typically maintained
at 52°C (Range: 52–58°C for these studies) and measures the latency for the rats to exhibit the
first sign of pain (i.e. licking the hind paws or jumping) [199] (see Table 2). In this procedure,
inhibitors of endocannabinoid uptake (UCM707, OMDM-2, VDM-11) produce
antinociception, although mediation by cannabinoid receptors has not been assessed using
competitive antagonists [200,201]. Moreover, exogenous administration of AEA produces an
antinociceptive effect in the hotplate test [192,202,203] that seems to be CB1-mediated [192]
(see Table 2). Consistent with this observation, FAAH inhibitors (URB597 and URB532)
produce CB1-mediated antinociception [204]. Endocannabinoid uptake inhibitors (UCM707
and OMDM-1) also potentiate the antinociceptive effect of exogenous anandamide at a dose
that did not produce an effect when given alone [200,201]. These observations are consistent
with the CB1-mediated enhancement of endocannabinoid-mediated stress analgesia produced
by the uptake inhibitor VDM11 in the tail-flick test [78].
The plantar test measures the latency for animals to remove their paws from a radiant heat
source that is focused onto the plantar surface of the paw through the floor of a glass platform
[205]. In this test, the FAAH inhibitor Compound 17 dose-dependently potentiates the effects
of exogenous AEA in the plantar test [206]. Finally, exogenous administration of AEA also
produces CB1-mediated antinociception in the paw pressure test [207], assessed using the
method of Randall and Selitto [208] (see Table 2). A role for cannabinoid CB2 receptors in
antinociception in otherwise naive animals has been studied in an attempt to optimize the
therapeutic potential of cannabinoid analgesic systems. CB2 agonists show therapeutic
potential because they are devoid of the unwanted central side-effects attributed to activation
of CB1 receptors ([124] for a review). However, previous studies assessing responsiveness to
acute nociceptive stimulation have either not typically examined the role of CB2 in mediating
effects linked to endocannabinoids (AEA and 2-AG), or have not supported the involvement
of CB2 mechanisms in endogenous analgesia [78]. It is therefore acknowledged that only
certain assays (e.g. the plantar test) are likely to be sensitive to detection of CB2-mediated
antinociceptive effects in the absence of inflammation or injury (for review see [124]). Thus,
animal models of persistent pain are likely to be differentially sensitive to CB2-mediated
components of cannabinoid antinociception. Thus, manipulation of endocannabinoid
accumulation through inhibition of metabolism or reuptake mechanisms may be employed to
elucidate a role for cannabinoid CB2 receptors under conditions of inflammation or injury.
PERSISTENT INFLAMMATORY NOCICEPTION

Cannabinoids produce antinociception in tissue injury models of persistent pain. Indeed,
behavioural, electrophysiological and neurochemical studies all support a role for cannabinoid
CB1 and CB2 receptors in modulating inflammatory nociception (for review see [126]). Effects
of exogenous administration of endocannabinoids and/or their modulators (i.e. inhibitors of
endocannabinoid uptake or hydrolysis) in different inflammatory pain models (formalin,
carrageenan, capsaicin, complete Freund’s adjuvant,) is discussed separately because the
mechanisms underlying the development and maintenance of distinct inflammatory pain states
differs (see Table 3 and Table 4).
The formalin test is a well-established model of persistent pain characterized by a transient,

biphasic pattern of pain behaviour [209]. The early phase is characterized by acute activation
of C and Aδ fibers. The late phase involves an inflammatory reaction in peripheral tissue
[210], the development of central nervous system sensitization [211,212] and additionally
involves activation of primary afferent nociceptors [213]. In the formalin test, endocannabinoid
uptake inhibitors (AM404, UCM707, LY2318912, LY2183240, OMDM132) produce
antinociception [93,214,215] (see Table 3). These antinociceptive effects are mediated either
exclusively by CB1 receptors [214,215] or by both CB1 and CB2 receptors [215]. Exogenous
AEA produces CB1-dependent antinociception in the formalin test [177,216] whereas
exogenous 2-AG predominantly produces CB2-dependent antinociception [217]. The formalin
test has also been used to assess antinociceptive effects produced by FAAH inhibitors (MAFP,
Flurbiprofen, Ibuprofen, Compound 17, propofol, AA-5-HT, OMDM106, LY2183240 and
others) [178,206,215,216,218–220]. Thus, it is noteworthy that the mechanism of action varies

with the compound employed. For example, AA-5-HT [219,220] and LY2183240 produce
CB1-mediated antinociception [215] whereas propofol, a widely used general anesthetic,
mediates its antinociceptive effects through activation of CB1 and CB2 receptors [221] (see
Table 3). FAAH knockout mice also exhibit CB1-mediated hypoalgesia in both phases of the
formalin test [198]. However, the nonsteroidal anti-inflammatory drug ibuprofen produces
antinociception in the formalin test that is not related to cannabinoid or TRPV1 receptors
[216]. Both CB1 and CB2 receptors are implicated in the antinociceptive effects of MGL
inhibitors (OMDM169 and URB602) in this test [180,217]. Furthermore, the combination of
AEA with nonselective FAAH inhibitors (ibuprofen or rofeocoxib) produces an
antinociceptive effect [178,216] that is CB1-mediated [216], whereas the combination of 2-
AG with URB602 produces antinociception whose mechanism of action remains to be
determined [217].
The carrageenan model involves intraplantar injection of the inflammatory agent, carrageenan,
which produces paw swelling (edema) and hypersensitivity to mechanical or thermal
stimulation [205]. Carrageenan also induces the expression of Fos, a nonspecific marker of
neuronal activation, in the lumbar spinal cord [222]. In this model, exogenous administration
of anandamide produces antinociception [183,223,224] which is likely to be CB1-mediated
[183] (see Table 4). FAAH inhibitors (URB597 and JNJ-1661010) also produce
antinociception in this model ([179,225,226] using URB597; [227] using JNJ-1661010).
However, this antinociceptive effect is likely to be independent of CB1 receptor activation

because a CB1 antagonist failed to reverse the observed antinociceptive effects [179,226]. A
role for both CB2 receptors [179] and peroxisome proliferator-activated receptor-α (PPAR-α)
receptors has been implicated in the antinociceptive effects of URB597 in this model [226]. A
role for CB2 but not CB1 receptors in thermal anti-hyperalgesic effects exhibited by FAAH
knockout mice has also been demonstrated; however, neither CB1 nor CB2 receptors are
implicated in the anti-edemic effects of FAAH−/− mice [198]. Although highly specific MGL
inhibitors have only recently been described, MGL-selective inhibitors (URB602 and
compound 21) nonetheless exhibit antinociception in this model [228,229; respectively), an
effect which involves CB2 receptors [228]. However, caution must be exerted in interpreting
effects of URB602, which also inhibits FAAH, and thus, is unlikely to act as a selective MGL
inhibitor following systemic administration.
Capsaicin, the pungent ingredient in hot chili peppers, produces hypersensitivity to mechanical
and thermal stimulation as well as spontaneous pain following intradermal administration
[230]. Hyperalgesia evoked in capsaicin model refers to an increase in pain behavior evoked
by suprathreshold stimuli and/or a lowered threshold for pain [230,231]. Only one study has
assessed antinociceptive effects following exogenous administration of AEA [202] without
investigating the cannabinoid (CB1 and/or CB2) receptor mechanism of action (see Table 4).
Complete Freund’s adjuvant (CFA), administered in the plantar hindpaw surface, produces
peripheral edema as well as hypersensitivity to mechanical and thermal stimulation in rodents
[232–234]. The inflammation appears approximately two hours following injection of
complete Freund’s adjuvant, produces its maximal effect after six to eight hours and can persist
for weeks following injection [233,235]. Exogenous administration of AEA produces
antinociception in the CFA model, but this effect does not involve CB1 receptors [207]. A
CB2 mechanism of action was not investigated in this study, likely due to the lack of available
CB2-selective antagonists at the time of testing. In this model, the antinociceptive effect of the
FAAH inhibitor URB597 is mediated by both CB1 and CB2 receptors [236]. Furthermore,
AM404, an inhibitor of endocannabinoid uptake, produces CB1-mediated antinociception in
the CFA model [214,237] (see Table 4). These observations are consistent with the ability of
exogenous anandamide to produce antinociception in other inflammatory pain models (acid
acetic writhing test, kaolin writhing test, and other models) through a CB1-dependent
mechanism [202,238] (see Table 4).
NEUROPATHIC NOCICEPTION
Animal models of neuropathic pain have been developed to mimic symptoms associated with
nerve injury observed clinically. Neuropathic pain can be induced by traumatic nerve injury
[239–241], toxic insults and metabolic challenges. Pharmacotherapies used to treat neuropathic
pain produce inadequate pain relief and/or unwanted side-effects (for review see [124,242]),
which reinforce the importance of identifying and validating novel therapeutic approaches
which suppress neuropathic pain, including those targeting the endocannabinoid system (see
Table 5). The chronic constriction injury model is a widely used animal model of neuropathic
pain that is produced by loosely placing three constrictive ligatures around the common sciatic
nerve [239]. In this model, inhibition of endocannabinoid uptake with AM404 produces
antinociceptive effects which are mediated by CB1 [141,214,243,244] and partially by CB2
receptors [243]. However, discrepancies between studies are also apparent [214,244] (see
Table 5). The endocannabinoid uptake inhibitor VDM11 also produces antinociceptive effects,
but involvement of cannabinoid receptors in these effects has not been evaluated [243]. FAAH
inhibitors (URB597, AA-5-HT, OL-135) also produce antinociception in the CCI model
[219,245]. The FAAH inhibitor URB597 produces antihyperalgesic effects in this model that
are CB1-mediated and partially CB2-mediated. By contrast, another FAAH inhibitor (AA-5-
HT) has been shown to produce antihyperalgesia that is mediated exclusively by CB1 receptors.
No genotype differences in pain behavior were observed between FAAH−/− and wildtype mice
subjected to a chronic constriction injury [198]. However, nerve injury may promote adaptive
changes in these animals because CCI was found to obliterate the phenotypic hypoalgesia
displayed by FAAH−/− mice in the tail immersion and hot plate tests [198].
Pharmacological modulation of endocannabinoid levels also suppresses neuropathic pain

behavior in other models of surgically-induced traumatic nerve injury. For example, AM404
produces CB1-dependent antinociception [237] in a model of unilateral hind limb neuropathy
induced by partial sciatic nerve ligation (PSNL) [240]. Exogenous administration of
anandamide similarly produces CB1-dependent antinociceptive effects [246,247] whereas the
antinociceptive effects of 2-AG, administered via the same route, are CB1/CB2 mediated
[248] (see Table 5). FAAH inhibitors (URB597, Ibuprofen, Rofecoxib) are also antinociceptive
in this model [246,248]. URB597 produces antinociception through a local peripheral
mechanism that is mediated by CB1/CB2 cannabinoid receptors [248]. However, systemic
administration of the same compound does not reliably produce antinociception [236].
Moreover, antinociception produced by local injection of ibuprofen and rofecoxib in the paw
does not involve CB1 or CB2 cannabinoid receptors [246]. Local administration of URB602
also produces a CB1/CB2 antinociception in this model [248]. The combination of FAAH or
MGL inhibitors with the exogenous administration of endocannabinoids (AEA or 2-AG) also
enhances the antinociceptive effects of the putative endocannabinoid [246,248], but the
mechanism of action remains to be determined. The combination of AEA with either ibuprofen
or rofecoxib produces antinociception that is mediated exclusively by CB1 receptors, although
the mechanism of action for these other combinations remains to be investigated [246].
Effects of modulation of the endocannabinoid system on neuropathic pain behavior have

recently been evaluated using a spinal nerve ligation model (SNL). Neuropathic pain was
induced by ligating the L5 and L6 spinal nerves according to the procedures described by Kim
and Chung [241]. In this model, FAAH inhibitors (URB597, Compound 17, JNJ-1661010 and
Compound 34) have been studied exclusively [206,227,249,250] (see Table 5). The
antinociceptive effects produced by these agents may involve non-cannabinoid receptor
mechanisms (e.g. PPAR-α). However, antinociception produced by URB597 has been shown
to involve CB1 receptors [249]. Thus, antinociception produced by FAAH/MGL/

endocannabinoid uptake inhibitors are influenced by the compound employed, the animal
model used and potentially the level of endocannabinoid tone produced by the injury. Thus,
systemic administration of URB597 produces CB1-mediated enhancements of stress

antinociception at doses that do not alter basal nociceptive thresholds in the tail flick test (Table
1). However, systematically administered URB597 produces CB2-mediated antinociception
in the carrageenan model and CB1/CB2-mediated antinociception in complete Freund’s
adjuvant, partial sciatic nerve ligation (local injection) and chronic constriction injury models.
Interpretation of effects of URB602 is more complicated as this compound is not MGL-

selective, and can inhibit FAAH; URB602 produces CB2-mediated antinociception in the
carrageenan model (systemic injection) and CB1/CB2-mediated antinociception in the formalin
test (i.e. following local injection) and partial sciatic nerve ligation (i.e. following local
injection) models (see Table 3–Table 5). Thus, effects of URB602 are only likely to be mediated
by MGL under conditions in which it is documented that local administration of URB602
increases 2-AG accumulation without altering levels of AEA [78]. Systemic administration of
AM404 produces CB1-mediated antihyperalgesic effects in inflammatory pain models such as
complete Freund’s adjuvant and formalin models but involves CB1/CB2 receptors in the CCI
model. Moreover, local exogenous administrations of 2-AG produce CB2-mediated
antinociception in the formalin test and CB1/CB2-mediated antinociception in the partial sciatic
nerve ligation model. However, local administration of AEA produces CB1-mediated
antinociception in both of these models (see Table 3–Table 5). A local route of agonist
administration may unmask CB2-mediated components in the antinociceptive effects produced
by pharmacological inhibitors of endocannabinoid uptake and degradation. However, URB597
produces antinociceptive effects with largely consistent pharamacological specificity
following either systemic or local routes of administration. It is also important to emphasize
that inhibitors of FAAH elevate levels of fatty-acid amides that do not bind to cannabinoid
receptors (e.g. palmitoylethanolamine) and have targets (e.g. PPAR-α) that are distinct from
CB1 and CB2 receptors. Thus, the contribution of non-cannabinoid receptor mechanisms of
action in the in vivo pharmacological effects of FAAH and MGL inhibitors must also be
considered.
LIMITATIONS
This review focuses on understanding the functional consequences of increasing
endocannabinoid accumulation through blockade of endocannabinoid deactivation or
transport, with the caveat that many of these agents employed (e.g. FAAH or MGL inhibitors)
are not selective for the endocannabinoid system. Moreover, increasing specific
endocannabinoids (e.g. anandamide) or fatty-acid amides (e.g. palmiotylethanolamine) can
activate other non-cannabinoid receptors (e.g. TRPV1 or PPAR-α, respectively). Entourage

effects may also be produced by manipulations that elevate levels of endogenous lipid
mediators that do not bind to cannabinoid receptors but, nonetheless, compete for the same
enzymes for hydrolysis [251]. Thus, not all effects of these modulators can be attributed to
actions at cannabinoid receptors, and assessment of pharmacological specificity is critical for
interpretation of in vivo actions of any compound. Palmitoylethanolamide (PEA), an
endogenous fatty-acid ethanolamide, is an agonist at PPAR-α receptors, but does not bind to
cannabinoid receptors [252,253]. However, effects of this compound can nonetheless be
blocked by the CB2 antagonist SR144528 [177]. Inhibition of FAAH by URB597 can also
produce antinociceptive effects in inflammatory pain models that are mediated by the activation
of PPAR-α receptors [225,226,254]. Synergistic interactions between anandamide and
GW7647 (PPAR-α agonist) have been demonstrated in the formalin test [255]. Thus,
modulation of the endocannabinoid system by FAAH/MGL/uptake inhibitors and their
possible interaction with non-cannabinoid receptors requires further investigation. Even
though increases in endocannabinoid accumulation are produced by inhibition of the

degradative enzymes described in this review, differences in selectivity or potency and
heretofore uncharacterized off-target effects may complicate interpretation of results.
Therefore, the reader should be aware of these limitations when interpreting the results of any
specific study.
CONCLUSION
Endocannabinoids modulate pain under physiological conditions. Pharmacological approaches
that enhance levels of endocannabinoids by inhibiting enzymes controling endocannabinoid
deactivation or by blocking their reuptake consequently exhibit therapeutic potential. It is clear
that the endocannabinoid system is regulated following conditions of injury. Therefore, more
work is necessary to better understand the broad consequences of pharmcological approaches
that modulate endocannabinoid levels. Inhibition of endocannabinoids deactivation is likely
to show a more beneficial and circumscribed spectrum of biological effects compared to direct
activation of CB1 receptors; effects of these inhibitors are limited to sites where
endocannabinoids are mobilized under physiological conditions in a stimulation-contingent
fashion. Limitations to therapeutic approaches which modulate the endocannabinoid system
(e.g. in immunosuppressive diseases) should also be considered when assessing the therapeutic
potential of any approach. The impact of long term treatment should be assessed. Multimodal
approaches combining modulation of endocannabinoid with other conventional analgesics
(e.g. NSAIDs) should also be evaluated for their therapeutic potential. Adjunctive approaches
show strong promise for improving the efficacy of existing pharmacotherapies for pain and
limiting unwanted side-effect profiles.
Acknowledgments
JG is supported by a Fonds de la Recherche en Santé du Québec (FRSQ) postdoctoral fellowship. AGH is supported
by DA021644, DA022478, and DA022702.
ABBREVIATIONS
2-AG 2-arachidonoylglycerol
AA arachidonic acid
AA-5-HT N-arachidonoyl serotonine
AEA anandamide
Ca2+ calcium
CB1 cannabinoid receptor 1

CB2 cannabinoid receptor 2
CCI chronic constriction injury
CGRP calcitonin gene-related peptide
CNS central nervous system
COX cyclooxygenase
Δ9-THC delta 9-tetrahydrocannabinol
DAG diacylglycerol
DAGL diacylglycerol lipase
DR dorsal raphe
DRG dorsal root ganglion

ET endocannabinoid membrane transporter

FAAH fatty acid amide hydrolase
GABA γ-amino butyric acid
LOX lipoxygenase
MAFP methyl arachidonyl fluorophosphonate
MAPK mitogen-activated protein kinase
MGL monoacylglycerol lipase
NADA N-arachidonoyldopamine
NAPE N-arachidonoyl-phosphatidylethanolamine
NAT N-acyl transferase
NT not tested
PAG periaqueductal gray
PG prostaglandins
PLC phospholipase C
PLD phospholipase D
PMSF phenylmethylsulfonyl fluoride
PSNL partial sciatic nerve ligation
PTK palmitoyltrifluoromethylketone
RVM rostral ventromedial medulla
SIA stress-induced analgesia
SNL spinal nerve ligation
TRPV1 transient receptor potential vanilloid 1
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Figure 1.
Formation and inactivation of anandamide and 2-arachidonoylglycerol
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 1
Antinociceptive effects of modulators of the endocannabinoid system in the tail flick model
Mediated by: Studies

Compounds
Pain Anti-
Model nociception CB1 CB2 References
Endocannabinoid VDM-11 Yes during SIA Yes NT 78

Guindon and Hohmann
uptake inhibitors
UCM707 Yes Yes NT 187
Yes NT NT 190
Yes NT NT 256
Yes NT NT 203
Yes No NT 188
Yes Yes NT 194

Exogenous Yes trend Yes NT 192
AEA
endocannabinoids
Yes Yes NT 193
Yes NT NT 257
Yes No NT 191
Yes NT NT 189
Tail flick Yes NT NT 224
FAAH inhibitors PMSF Yes NT NT 196
PTK Yes during SIA Yes NT 138
AA-5-HT Yes during SIA Yes NT 79,138
URB597 Yes during SIA Yes NT 78
Yes during SIA Yes NT 79
Yes NT NT 189
Yes Yes No 197

Page 31

Compounds
Pain Anti-
MGL inhibitors URB602 Yes during SIA Yes NT 78
Yes during SIA Yes NT 79

Guindon and Hohmann
AEA + PMSF Yes NT NT 196
Yes Yes NT 191
Exogenous AEA + Yes NT NT 191

Endocannabinoids+ Ibuprofen
FAAH inhibitors
AEA + Yes NT NT 191
Indomethacin
AEA + Yes Yes No 189
URB597
Page 32
Table 2
Antinociceptive effects of modulators of the endocannabinoid system in acute pain models

Compounds
Pain Anti-
Hot plate UCM707 Yes NT NT 200

Guindon and Hohmann
OMDM-2 Yes NT NT 201

Endocannabinoid
uptake inhibitors VDM-11 Yes NT NT 201
O-2093 No NT NT 258
AEA Yes NT NT 203
Yes NT NT 87
Exogenous
endocannabinoids Yes trend Yes NT 192
Yes NT NT 202
FAAH inhibitors URB597 Yes Yes NT 204
URB532 Yes Yes NT 204
Endocannabinoid AM404 Yes NT NT 87

uptake inhibitors + AEA
+ Exogenous
endocannabinoids UCM707 Yes NT NT 200
+ AEA
OMDM-1 Yes NT NT 201
+ AEA
Plantar Exogenous AEA + Yes NT NT 206
Endocannabinoids+ Compound 17
FAAH inhibitors
Paw Exogenous AEA Yes Yes NT 207

Pressure Endocannabinoids
Test
Page 33
Table 3
Antinociceptive effects of modulators of the endocannabinoid system in the formalin model of inflammation

Compounds
Pain Anti-
Formalin Endocannabinoid uptake LY2318912 Yes NT NT 93

Guindon and Hohmann
Test inhibitors
UCM707 Yes NT NT 214
AM404 Yes Yes No 214
LY2183240 Yes Yes No 215
OMDM132 Yes Yes Yes 215
Exogenous Yes NT NT 259

Endocannabinoids
Yes Yes No 177
AEA Yes Yes No 216
Yes NT NT 255
2-AG Yes No Yes 217
FAAH inhibitors MAFP Yes Yes NT 218
Flurbiprofen Yes Yes NT 218
Ibuprofen Yes No No 216
Rofecoxib Yes NT NT 178
Propofol Yes Yes Yes 221
AA-5-HT Yes Yes No 219
OMDM106 Yes Yes NT 220
Compound 17 Yes Yes NT 206
OMDM119 Yes No NT 215
OMDM122 Yes No NT 215

Page 34

Compounds
Pain Anti-
LY2183240 Yes Yes No 215
MGL inhibitors URB602 Yes Yes Yes 217

Guindon and Hohmann
OMDM169 Yes Yes Yes 180
Exogenous AEA + Yes Yes No 216

Endocannabinoids Ibuprofen
+ FAAH inhibitors
AEA + Yes NT NT 178
Rofecoxib
Exogenous 2-AG + Yes NT NT 217

Endocannabinoids+ URB602
MGL inhibitors
Page 35
Table 4
Antinociceptive effects of modulators of the endocannabinoid system in inflammatory pain models

Compounds
Pain Anti-
Carrageenan Exogenous AEA Yes NT NT 223

Guindon and Hohmann
Endocannabinoids
Yes Yes NT 183
Yes NT NT 224
FAAH inhibitors URB597 Yes No Yes 179
Yes NT NT 225
Yes No NT 226
JNJ-1661010 Yes NT NT 227
MGL inhibitors URB602 Yes No Yes 228
Compound 21 Yes NT NT 229
Capsaicin Exogenous AEA Yes NT NT 202

Endocannabinoids
Complete Endocannabinoid AM404 Yes Yes No 214

Freund’s uptake inhibitors
Adjuvant Yes trend NT NT 237
Exogenous AEA Yes No NT 207
Endocannabinoids
FAAH inhibitors URB597 Yes Yes Yes 236
Acetic Acid Exogenous AEA Yes Yes No 202

Writhing Endocannabinoids
Test
Page 36

Compounds
Pain Anti-
MGL inhibitors Compound 21 Yes NT NT 229
Kaolin Exogenous AEA Yes Yes No 202

Writhing Endocannabinoids
Guindon and Hohmann
Test
NGF Exogenous AEA Yes Yes No 238

inflammatory Endocannabinoids
hyperalgesia
p-phenyl- Exogenous AEA Yes NT NT 203

quinone Endocannabinoids
stretch test
Turpentine Exogenous AEA Yes NT NT 259

bladder Endocannabinoids
inflammation
Page 37
Table 5
Antinociceptive effects of modulators of the endocannabinoid system in neuropathic pain models

Compounds
Pain Anti-
CCI Endocannabinoids AM404 Yes Yes Yes 243

Guindon and Hohmann
uptake inhibitors
Yes Yes No 214,244
Yes Yes NT 141
VDM11 Yes NT NT 243
FAAH inhibitors URB597 Yes NT NT 219
Yes Yes Yes 245
OL-135 Yes NT NT 219
PSNL Endocannabinoids AM404 Yes Yes NT 237

uptake inhibitors
Exogenous AEA Yes Yes No 247

Endocannabinoids
Yes Yes No 246
2-AG Yes Yes Yes 248
URB 597 No NT NT 236
Yes Yes Yes 248
FAAH inhibitors
Ibuprofen Yes No No 246
Rofecoxib Yes No No 246
MGL inhibitors URB602 Yes Yes Yes 248

Page 38

Compounds
Pain Anti-

Ibuprofen
Exogenous
Endocannabinoids+
Rofecoxib
FAAH inhibitors
Guindon and Hohmann
2-AG + Yes NT NT 248

URB597
Exogenous 2-AG + Yes NT NT 248

Endocannabinoids+ URB602
MGL inhibitors
Exogenous 2-AG Yes NT NT 248

Endocannabinoids+ +URB597+
FAAH + MGL URB602
inhibitors
SNL FAAH inhibitors URB597 Yes Yes NT 249
JNJ-1661010 Yes NT NT 227
Page 39

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CNS Neurol Disord Drug Targets. 2009 December ; 8(6): 403–421.

The Endocannabinoid System and Pain

Josée Guindon and Andrea G. Hohmann*

pathological functions (regulation of food intake, immunomodulation, inflammation,

2-arachidonoylglycerol (2-AG). AEA is hydrolyzed by the enzyme fatty-acid amide hydrolase

CANNABINOID RECEPTOR PHARMACOLOGY

A better understanding of the role of cannabinoid receptors in different physiological processes

cases, functions of the endocannabinoid system are surmised following pharmacological

SYNTHESIS AND RELEASE OF AEA AND 2-AG

cleavage of membrane phospholipids precursors and subsequent synthesis of

DEGRADATION OF AEA AND 2-AG

ENDOCANNABINOIDS IN PAIN PATHWAYS

Neurophysiological studies by Walker’s laboratory first documented that cannabinoids

AG mobilization in the PAG correlates highly with endocannabinoid-mediated stress

Systemic administration of inhibitors of endocannabinoid uptake (VDM-11, OMDM-2,

MODULATION OF THE ENDOCANNABINOID SYSTEM IN ANIMAL MODELS

Therefore, documentation of intrinsic effects of endocannabinoids released under

treatment with CB1 antagonists [79,128,137,138,160] or modulators of endocannabinoid

alternately, the failure of the spinally-mediated tail-flick test to detect a CB2-mediated

Effects of exogenous administration of endocannabinoids (focusing on AEA and 2-AG) and

PERSISTENT INFLAMMATORY NOCICEPTION

differs (see Table 3 and Table 4).

The formalin test is a well-established model of persistent pain characterized by a transient,

others) [178,206,215,216,218–220]. Thus, it is noteworthy that the mechanism of action varies

However, this antinociceptive effect is likely to be independent of CB1 receptor activation

Pharmacological modulation of endocannabinoid levels also suppresses neuropathic pain

Effects of modulation of the endocannabinoid system on neuropathic pain behavior have

to involve CB1 receptors [249]. Thus, antinociception produced by FAAH/MGL/

systemic administration of URB597 produces CB1-mediated enhancements of stress

Interpretation of effects of URB602 is more complicated as this compound is not MGL-

activate other non-cannabinoid receptors (e.g. TRPV1 or PPAR-α, respectively). Entourage

though increases in endocannabinoid accumulation are produced by inhibition of the

CB1 cannabinoid receptor 1

DRG dorsal root ganglion

ET endocannabinoid membrane transporter

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Mediated by: Studies

Endocannabinoid VDM-11 Yes during SIA Yes NT 78

Yes Yes NT 194

Tail flick Yes NT NT 224

FAAH inhibitors PMSF Yes NT NT 196

PTK Yes during SIA Yes NT 138

URB597 Yes during SIA Yes NT 78

Yes during SIA Yes NT 79