Cannabinoid Receptor Pharmacology: The Endocannabinoid System

Cannabinoid Receptor Pharmacology
Sponsored Content by Tocris Bioscience Oct 28 2019
The Endocannabinoid System
Cannabinoid receptors that have been identified so far are of two types1,2:
the CB1 receptor cloned in 19903 and the CB2 receptor cloned in 19934. Both
receptors belong to the superfamily of Gproteincoupled receptors (GPCRs).
The cloning of these receptors was the motivation for developing mice in
which cannabinoid CB1 and/or CB2 receptors have been deleted genetically.
Currently, these transgenic animals, specifically CB1 knockout mice, are
extensively used to investigate the pathological and physiological functions of
cannabinoid receptors1,5,6.
CB1 receptors are predominantly located at the terminals of peripheral and
central neurons where they often mediate inhibition of the release of
neurotransmitters. They also exist in certain nonneuronal cells, such as
immune cells. CB2 receptors are mainly found in immune cells, both inside
and outside the central nervous system. These receptors perform functions
within immune cell migration and modulation cytokine release. In the brain,
these receptors are expressed by blood vessels and microglia,7 and by certain
neurons8,9. Yet, researchers are still unaware of the role played by neuronal
CB2 receptors.
CB1 receptors’ central distribution pattern is heterogeneous and accounts for
various notable pharmacological properties of CB1 agonists, for instance, their
potential to affect memory and cognition as well as to modify the control of
motor function. Hence, specifically high concentrations of CB1 receptors are
found in the hippocampus, cerebral cortex, substantia nigra pars reticulata,
lateral caudateputamen, entopeduncular nucleus, globus pallidus, and the
molecular layer of the cerebellum1,10. In accordance with the analgesic
properties of cannabinoid receptor agonists, CB1 receptors are also found on
pain pathways in the spinal cord and brain as well as at the peripheral
terminals of primary sensory neurons11,12.
Despite the fact that the population of CB1 receptors is significantly less in
peripheral tissues compared to their concentration in the central nervous
system, this does not imply that peripheral CB1 receptors are insignificant. In
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certain peripheral tissues, discrete regions. like nerve terminals that
constitute only a trivial portion of the total tissue mass, are known to possess
high concentrations of CB1 receptors. CB1 receptors are also expressed on
neurons in peripheral tissues such as the urinary bladder, vas deferens, the
heart, and small intestine10,13.
Gi/o proteins couple to both CB1 and CB2 receptors, positively modulating
mitogenactivated protein kinase (MAPK) and negatively modulating adenylyl
cyclase (AC)1,14. They also couple CB1 receptors to ion channels: negatively
to CaV2.2 (Ntype) and CaV2.1 (P/Qtype) calcium channels and positively to
Atype voltagegated (KV), and inwardly rectifying (Kir) potassium
channels1,13,14.
In addition, CB1 receptors can activate adenylyl cyclase by coupling to Gs
proteins15–17. The extent to which this takes place is probably governed by
where these receptors are located or by crosstalk between CB1 receptors and
colocalized Gproteincoupled nonCB1 receptors15,16,18,19. There is a
likelihood that CB1 receptors occur as two distinct subpopulations, one of
which is coupled to Gi/o proteins and the other coupled to Gs proteins15.
Additional signaling mechanisms for cannabinoid CB1 and CB2 receptors have
also been proposed elsewhere1,14.
After cannabinoid receptors were cloned, it was discovered that mammalian
tissues synthesize compounds with the potential to activate these receptors.
Narachidonoyl ethanolamine (anandamide) identified in 1992 and 2
arachidonylglycerol identified in 1995 were the firstever endogenous
cannabinoids (endocannabinoids) identified20–22. Both of these are produced
on demand as a reaction to an increase in intracellular calcium23. Nacyl
phosphatidylethanolamineselective phospholipase D (NAPEPLD) catalyzes
the process in which anandamide is produced from Narachidonoyl
phosphatidylethanolamine. However, it is thought that the production of 2
arachidonylglycerol is dependent on the transformation of 2arachidonate
containing phosphoinositides to diacylglycerols, and on their subsequent
conversion to 2arachidonylglycerol by DAGLα and DAGLβ, two diacylglycerol
lipase (DAGL) isozymes23,24.
Once these endocannabinoids are produced and released, cellular uptake
removes them from their sites of action and they are degraded by enzymes;
2arachidonylglycerol mainly by monoacylglycerol lipase (MAGL) but also by
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fatty acid amide hydrolase (FAAH), and anandamide by FAAH and/or by
palmitoylethanolamidepreferring acid amidase (PAA), cyclooxygenase2,
lipoxygenases, and cytochrome P4505,23–25.
Oarachidonoyl ethanolamine (virodhamine), 2arachidonylglyceryl ether
(noladin ether), Ndocosatetraenoyl ethanolamine, Ndihomoγlinolenoyl
ethanolamine, Narachidonoyl dopamine (NADA), oleamide, and Noleoyl
dopamine (OLDA) are other ligands that could be endocannabinoids5. Along
with their receptors, endocannabinoids form what is now termed the
“endocannabinoid system”.
Although it is usually agreed that endocannabinoids pass through cell
membranes, one problem that is now a matter of dispute is whether a
transporter mediates the cellular uptake of endocannabinoids, like
anandamide25–27. On the other hand, currently, FAAH is well characterized. As
a matter of fact, it has been cloned28 and FAAH knockout mice have been
developed29,30. In addition, NAPE/PLD31, MAGL32–34, and DAGLα and DAGLβ35
have been cloned and mice with a genetic deletion of NAPE/PLD have been
developed36.
It has been found that at least some of the effects brought about by
endogenously released anandamide and 2arachidonylglycerol seem to be
elevated by the socalled “entourage effect.” This is dependent on the co
release of other endogenous fatty acid derivatives, including oleamide and
palmitoylethanolamide, which can potentiate anandamide, and 2
palmitoylglycerol and 2linoleoylglycerol, which can potentiate 2
arachidonylglycerol37. The mechanism(s) that cause the entourage effect
have not yet been determined.
It is most likely that endocannabinoids have immunomodulatory as well as
neuromodulatory roles, including the suppression of ongoing transmitter
release via retrograde signaling38 and the regulation of cytokine release and
immune cell migration39,40. Moreover, it is also widely acknowledged that in
some disorders there is an increase in endocannabinoid release in specific
tissues, and furthermore, in certain cases this upregulation of the
endocannabinoid system results in the inhibition of unwanted signs and
symptoms and so is “autoprotective”, while in others it leads to undesirable
effects5. Hence, there is evidence that the release of endocannabinoids in one
way ameliorates spasticity in inflammatory pain and multiple sclerosis, and in
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another way it leads to obesity in certain individuals or affects fertility in some
women. Consequently, at present, there is huge interest not just in
cannabinoid receptor agonists and antagonists that act directly but also in
compounds that can have an indirect effect on the activity of the
endocannabinoid system through allosteric modulation of endocannabinoid
induced activation of cannabinoid receptors, or by modifying the
concentration of endocannabinoids at their receptors by means of effects on
endocannabinoid production or degradation.
The rest of this article deals with the main pharmacological actions of several
such direct and indirect cannabinoid receptor agonists and antagonists. The
focus is specifically on those compounds that are most extensively used as
experimental tools in cannabinoid research. Where possible, citations to
previous review articles have been included that offer more detailed
information.
Mixed CB1/CB2 Receptor Agonists
In earlier studies1,2,41, it has been reported that compounds known to
activate CB1 and CB2 receptors with almost equal potency are most often
used in the lab as CB1/CB2 receptor agonists, and typically fall into one of
four chemical categories: aminoalkylindole, classical cannabinoid, nonclassical
cannabinoid, and eicosanoid. Several CB1/CB2 receptor agonists that are
extensively used consist of chiral centers and normally present signs of
stereoselectivity in pharmacological assays where the measured response is
CB1 or CB2 receptormediated1,2,41. In general, (–)trans (6aR, 10aR)
classical and nonclassical cannabinoids present considerably greater potency
as cannabinoid receptor agonists when compared to their (+)cis (6aS, 10aS)
enantiomers. Three prominent examples of such compounds are (–)Δ9
tetrahydrocannabinol (Δ9THC), (–)11hydroxyΔ 8THCdimethylheptyl (HU
210), and CP 55,940.
With regards to WIN 55,212, an aminoalkylindole, its (R)(+)isomer (WIN
55,2122) presents considerable agonist activity at both CB1 and CB2
receptors, but its (S)(–)isomer (WIN 55,2123) does not. In fact, upon
administering WIN 55,2123 in vitro at concentrations in the low micromolar
range, it has been observed to act as a partial inverse agonist at CB1
receptors and as a neutral CB2 receptor antagonist44. Anandamide, an
eicosanoid cannabinoid, does not containg any chiral centers. Yet, some of its
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synthetic equivalents—for example, the CB1selective agonist
methanandamide (see next section)—do contain chiral centers. The (R)(+)
isomer of methanandamide exhibits nine times increased affinity for CB1
receptors when compared to the (S)(–)isomer45.
The low water solubility and high lipophilicity of the majority of CB1 and CB2
receptor ligands are major practical challenges within cannabinoid research,
both in vivo and in vitro, as this demands the use of a nonaqueous vehicle
like polyvinylpyrrolidone, ethanol, Emulphor, Cremophor, dimethyl sulfoxide,
Tween 80, bovine serum albumin, or the watersoluble emulsion Tocrisolve
100, which is a blend of Pluronic F68, soya oil, and water1,46,47. As a result,
the Organix compound 3(5′cyano 1′,1′dimethylpentyl)1(4Nmorpho
linobutyryloxy)Δ8THC hydrochloride (O1057) 48 is one other cannabinoid
CB1/CB2 receptor agonist that deserves special reference as it is readily
watersoluble. O1057’s in vitro potency when compared to that of CP 55,940
is just 2.9 times less at CB1 receptors and 6.5 times less at CB2 receptors.
CB1Selective Agonists
The anandamide molecule was the starting point for the development of the
first CB1selective agonists. The marginal CB1 selectivity of this molecule can
be considerably improved by introducing a fluorine atom on the terminal 2′
carbon to produce O585 and/or by substituting a methyl group in the place
of a hydrogen atom on the 1′ or 2 carbon to produce (R)(+)
methanandamide, its cyano equivalent O1812, or O6891,2,41. Another
significant outcome of introducing a methyl group on the 1′ or 2 carbon is
enhanced resilience to the hydrolytic action of FAAH. In fact, (R)(+)
methanandamide was first produced in Dr Alexandros Makriyannis’ lab to
satisfy the need for an anandamide equivalent that is metabolically more
stable. Apart from O181249, arachidonyl2′chloroethylamide (ACEA) and
arachidonylcyclopropylamide (ACPA) have been the most potent CB1selective
agonists developed so far. Both of these present fairly high CB1 efficacy.50
In contrast to O1812, or in fact O689 or methanandamide, neither ACEA nor
ACPA exhibit any resilience to enzymic hydrolysis.1,2,49 This is possibly due to
the fact that they lack a methyl substituent on the 1′ or 2 carbon and,
actually, it has been demonstrated that the introduction of a methyl group to
the 1′ carbon of ACEA evidently reduces the vulnerability of this molecule to
FAAHmediated hydrolysis51. In addition, this structural change decreases
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ACEA’s affinity for CB1 receptors by nearly 14 times. 2arachidonylglyceryl
ether (noladin ether) is one more arachidonic acid derivative that is worth
mentioning as a CB1selective agonist, more so as it is a putative
endocannabinoid52. When compared to CP 55,940, this ligand presents CP
55,940like CB1 efficacy yet less CB1 potency53,54.
CB2Selective Agonists
JWH 133, the classical cannabinoid, and JWH 015, the less selective
aminoalkylindole have been the CB2selective agonists most extensively used
as experimental tools. Both of these were synthesized by Dr John
Huffman1,2,72. Aside from more readily binding to CB2 than to CB1 receptors,
each of these agents also acts as a potent CB2selective agonist in functional
assays. GW 405833, the GlaxoSmithKline compound that acts as a potent
partial agonist at the CB2 receptor67, as well as AM 1241, HU 308, and the
Merck Frosst compounds L759,633 and L759,656 are other prominent CB2
selective agonists1,2. Fascinatingly, AM 1241 could be a “protean agonist”
since it has been found to act as an agonist in tissues naturally expressing
CB2 receptors, but not in tissues where CB2 receptors are genetically inserted
and are hence possibly overexpressed73.
Selective CB1 Receptor Antagonists/Inverse
Agonists
SR141716A, the diarylpyrazole, was the first one to be developed among
these56. This selective and highly potent CB1 receptor ligand readily blocks or
reverses in vitro and in vivo CB1mediated effects1,2,41. AM 251, AM 281, and
LY 320135 are other notable CB1selective antagonists. Both AM 251 and AM
281 were developed by Dr Alexandros Makriyannis. When compared to AM
251, AM 281, or SR141716A, LY 320135 has less affinity for CB1 receptors
and at in the low micromolar range concentrations, it also binds to muscarinic
and 5hydroxytryptamine (5HT2) receptors1,2,41.
As described in other studies,2,74 there is evidence that AM 251, AM 281, LY
320135, and SR141716A are not “neutral” antagonists. Hence, in addition to
mitigating the effects of CB1 receptor agonists, they can by elicit responses
on their own in certain CB1 receptorcontaining tissues that are in a direction
opposite from those elicited by CB1 receptor agonists. Although in some cases
“inverse cannabimimetic effects” such as these might be due to a direct
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antagonism of responses evoked at CB1 receptors by released
endocannabinoids, there is research indicating that this is not always the
underlying mechanism, and that AM 251, AM 281, LY 320135, and
SR141716A are actually inverse agonists74. In particular, they seem to
generate inverse cannabimimetic effects in certain tissues by reducing the
constitutive activity of CB1 receptors in some way (the coupling of CB1
receptors to their effector mechanisms that, it is considered, can take place
without endogenously released or exogenously added CB1 agonists).
Selective CB2 Receptor Antagonists/ Inverse
Agonists
SR14452869, the SanofiAventis diarylpyrazole, and 6iodopravadoline (AM
630)71 are the most prominent CB2selective antagonists/inverse agonists.
Both the compounds bind to CB2 with considerably higher affinity compared
to CB1 receptors, present pronounced potency similar to CB2 receptor
antagonists, and act as inverse agonists that have the ability to bring about
inverse cannabimimetic effects at CB2 receptors on their own1,2,41. It has
been reported that AM 630 reverses CP 55,940induced suppression of the
production of forskolinstimulated cyclic AMP by human CB2transfected CHO
cell preparations at nanomolar concentrations (EC50 = 129 nM). It has also
been observed to increase the production of forskolinstimulated cyclic AMP
by the same cell line upon being administered by itself (EC50 = 230 nM)71,
though with efficacy that seems to be slightly less than the inverse efficacy
exhibited by SR144528 in this bioassay75. In certain tests, AM 630 has been
observed to act as a lowpotency partial agonist41,71,76–78 at the CB1
receptor; however, in others it acts as a lowpotency inverse agonist79,80.
Neutral Cannabinoid Receptor Antagonists
At present, there is much attention toward the possibility of creating potent
neutral CB1 and CB2 receptor antagonists—that is, highaffinity ligands for
CB1 or CB2 receptors that are deficient in essential agonist or inverse agonist
efficacy. One reason for this is that, in contrast to the CB1 and CB2selective
antagonists/inverse agonists currently available (see earlier sections), it is
possible to use a neutral antagonist to differentiate between tonic
cannabimimetic activity that occurs due to ongoing release of
endocannabinoid onto CB1 or CB2 receptors, which it will oppose, as well as
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tonic activity that occurs due to the existence of intrinsically active CB1 or CB2
receptors, which it will not oppose.
Despite the fact that it has not been possible to develop neutral antagonists
that selectively target the CB2 receptor, some advancement has been made
with respect to the CB1 receptor. There is some evidence that 6″azidohex2″
ynecannabidiol (O2654), O2050, a sulfonamide equivalent of Δ8THC that
has an acetylenic side chain, and VCHR, an equivalent of SR141716A, are
neutral CB1 receptor antagonists2,74. Since this proof is somewhat
preliminary, there should be discretion while using any of these ligands as a
pharmacological tool. There exists more comprehensive proof that NESS
0327, another SR141716A equivalent, is a neutral CB1 receptor antagonist55.
Yet, at present, this compound is not available commercially and hence has
not been used to a large extent in research on cannabinoids.
Radiolabeled Cannabinoid Receptor Ligands
CB1selective [3H]SR141716A (CB1 Kd = 0.19–1.24 nM) as well as [3H]CP
55,940, [3H]WIN 55,2122, and [3H]HU 243, are the tritiated cannabinoid
receptor ligands most extensively used in binding assays or for
autoradiography. The latter three compounds bind almost equally well to CB1
and CB2 receptors. Standard Kd values for [3H]CP 55,940, [3H]WIN 55,2122,
and [3H]HU 243 are 0.07–4 nM, 1.9–16.2 nM, and 0.045 nM, respectively, at
CB1 receptors and 0.2–7.4 nM, 2.1–3.8 nM, and 0.061 nM, respectively, at
CB2 receptors2,41. [3H]HU 243—that is structurally quite similar to HU 210—
has a specifically high affinity for these receptors.
In addition, radiolabeled ligands have been created as prospective probes for
human positron emission tomography (PET) or single photon emission
computed tomography (SPECT) experiments. These are 123I labeled
equivalents of AM 251 (CB1 Kd = 0.23–0.62 nM) and AM 28181–83 and an 18F
labeled equivalent of SR141716A, i.e., SR144385.84 Notably favorable
outcomes have been observed in animal experiments when [123I]AM 281 is
used.82,85
Additional Pharmacological Targets for CB1
and CB2 Receptor Ligands
At present, it is widely accepted that certain cannabinoid receptor agonists
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At present, it is widely accepted that certain cannabinoid receptor agonists
are fairly potent at stimulating the TRPV1 (vanilloid VR1) receptor. These
agonists include eicosanoids like ACEA, anandamide, NADA,
methanandamide, and certain anandamide metabolites, as well as OLDA, the
putative endocannabinoid. The list excludes 2arachidonylglycerol as well as
classical, aminoalkylindole, and nonclassical cannabinoid receptor agonists
like HU 210, WIN 55,2122, and CP 55,9402,5,88,92,93.
Several other nonCB1 and nonCB2 pharmacological targets for certain
CB1/CB2 receptor agonists have been put forward, such as a number of
targets that seem to respond to agonist concentrations of 1 μM or less and
thus to have sensitivity to these ligands of the same order as that presented
by CB1 or CB2 receptors. For instance, anandamide interacts with different
types of voltagegated and ligandgated ion channels at submicromolar
concentrations, where some (but not all) of these channels are also sensitive
to Δ9THC, 2arachidonylglycerol, WIN 55,2122, CP 55,940, and/or JWH 015.
There is also evidence that Δ9THC also interacts strongly with neuronal
transporters of 5hydroxytryptamine, noradrenaline, and dopamine, and that
there are several less sensitive pharmacological targets for Δ9THC and/or for
some other cannabinoid receptor agonists. These targets only appear to react
to >1 μM cannabinoid concentrations and include TRPA1 and PPARγ
receptors; shaker Kv1.2 K+ and Ltype Ca2+ channels; putative nonTRPV1,
nonCB1, and nonCB2 neuronal receptors in the small intestine; sites at gap
junctions between cells; and sites on glutamate GLUA1 and GLUA3 receptors
and on muscarinic M1 and M4 receptors2,86,88,94,95.
While anandamide and methanandamide also act as agonists for the putative
abnormalcannabidiol (abnormalCBD) receptor2,88,96, WIN 55,2122, Δ9
THC, or 2arachidonylglycerol do not (see also section on other notable
ligands). SR141716A and AM 251—the CB1 receptor antagonists/inverse
agonists—also have the ability to interact with nonCB1 and nonCB2 targets,
though only at concentrations in the micromolar range and thus above
concentrations at which these ligands have the potential to produce CB1
receptor antagonism. Hence, for instance, as discussed in other studies74,88,
it has been observed that at micromolar concentrations:
Neuronal voltagesensitive Na+ channels can be blocked by AM 251
Adenosine A1 receptor activation can be blocked by AM 251 and
SR141716A
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Activation of putative abnormalCBD receptors on mesenteric arteries
and of putative nonI1 and nonI2 imidazoline receptors can be blocked
by SR141716A
WIN 55,2122induced activation of central presynaptic putative TRPV1
like receptors can be antagonized by SR141716A and not AM 251
Ca2+activated (BK) K+ channels, Ltype Ca2+ channels, sites at gap
junctions between cells, and ATPsensitive K+ channels can be blocked
by SR141716A
It is most likely that future studies will uncover additional targets for CB1 and
CB2 receptor ligands. In fact, in AstraZeneca and GlaxoSmithKline patents, it
has already been claimed that certain standard cannabinoid receptor
agonists, as well as antagonists, stimulate GPR55, the Gproteincoupled
orphan receptor97,98.
Since cannabinoid receptor agonists vary by how much they interact with the
standard or proposed nonCB1 and nonCB2 targets, it follows that certain
ligands that activate CB1 and/or CB2 receptors with comparable potencies will
most likely have pharmacological profiles that differ from each other. It is also
worth mentioning that despite the fact that there exist several established
cannabinoid receptor ligands presenting pronounced selectivity as agonists or
antagonists/inverse agonists for CB1 or CB2 receptors (see earlier sections),
none of these ligands are completely CB1 or CB2specific.
Hence, it is anticipated that each of these ligands will block or activate both
types of receptors equally well upon being delivered at a high enough
concentration and hence will display selectivity only upon being delivered at
lower concentrations or doses lying within its CB1 or CB2 “selectivity window”.
Inhibitors of 2Arachidonylglycerol
Biosynthesis
As 2arachidonylglycerol and anandamide are produced on demand and not
stored, and because there is proof for elevated production and release of
either or both of these endocannabinoids to be responsible for undesirable
signs and symptoms of specific disorders (see section on the endocannabinoid
system), selective suppressors of their enzymic biosynthesis would not just
constitute vital experimental tools but also exhibit potential as therapeutic
agents.
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Selective inhibitors of NAPEPLD have not been discovered so far, but
inhibitors are available for DAGLα and DAGLβ, the enzymes that catalyze the
conversion of diacylglycerols to 2arachidonylglycerol. Tetrahydrolipstatin is
one such inhibitor that suppresses DAGLα (IC50 = 60 nM) and DAGLβ (IC50 =
100 nM) much more potently than it suppresses NAPEPLD (IC50 = 10 μM). It
does not suppress MAGL even at micromolar concentrations of 25 μM35,99.
O3841 is another prominent DAGL inhibitor that suppresses DAGLα at
nanomolar concentrations (IC50 = 160 nM). However, at concentrations of up
to 25 μM, it lacks any noticeable inhibitory effect on FAAH, NAPEPLD,
triacylglycerol lipase, or MAGL activity or on the particular binding of [3H]CP
55,940 to human CB1 or CB2 receptors99. Although both O3841 and
tetrahydrolipstatin suppress DAGL in membrane preparations, thus far, only
tetrahydrolipstatin has been observed to synthesize noticeable signs of DAGL
inhibition in intact cells99.
Inhibitors of the Enzymic Hydrolysis of
Endocannabinoids
The occurrence of MAGL and FAAH in various tissues has led to the need for
selective inhibitors of these enzymes that can be employed to promote
studies investigating the pharmacological actions of endocannabinoids upon
being administered exogenously as well as their pathological and physiological
roles during their endogenous release. Somewhat as a consequence of
experiments with MAGL and FAAH inhibitors, there already exists evidence
that endogenous release of cannabinoid increases in certain some disorders in
a way that results in an amelioration of undesirable signs and symptoms (see
the section on the endocannabinoid system). As a result, such inhibitors have
therapeutic potential.
After anandamide was discovered, the compound most extensively employed
to irreversibly suppress its enzymic hydrolysis was phenylmethylsulfonyl
fluoride (IC50 for FAAH inhibition = 290 nM to 15 μM), a nonselective serine
protease inhibitor that also suppresses MAGL, though less potently (IC50 ≥
155 μM)100.
Currently, additional inhibitors of FAAH have been discovered5,23,100, where
O1887, URB532, URB597, and AM 374 (the palmitylsulfonyl fluoride
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equivalent) are most probably the best among these for use as research tools
for irreversible FAAH inhibition, as well as OL135 for reversible FAAH
inhibition. All these compounds can bring about their inhibitory effects in vitro
at concentrations in the low nanomolar range, which lack the potential to
displace radiolabeled ligands from CB1 receptors or (where this has been
analyzed) from CB2 receptors. However, it is important to note that the
pharmacological characterization of a majority of these inhibitors is
incomplete.
The compound O1887 is a structural equivalent of methyl arachidonyl
fluorophosphonate (MAFP), which is an irreversible FAAH inhibitor (IC50 = 1–3
nM) by itself that additionally suppresses both DAGL (IC50 = 800 nM) and
MAGL (IC50 = 2–800 nM) but not NAPEPLD, and potently displaces [3H]CP
55,940 irreversibly from particular binding sites on rat brain membranes (IC50
= 20 nM)5,99,100,104. In one instance, it has been reported that MAFP acts as
an irreversible CB1 receptor antagonist118, and in another instance, that it
does not54.
Looking now at compounds blocking the metabolism of 2arachidonylglycerol
by suppressing MAGL, several of these have currently been
identified99,100,119,120. The most characterized inhibitor of this enzyme is
URB602, which induces noncompetitive inhibition of MAGL at micromolar
concentrations (IC50 = 28 μM) and lacks any noticeable potential to suppress
FAAH at 100 μM or to displace [3H]WIN 55,2122 from CB1 or CB2 receptors
at 5 μM119. It has been reported that another compound—URB754—inhibits
MAGL with considerably greater potency when compared to URB602120.
However, it is now evident that t.his inhibition was produced by an impurity
that exists in a commercially available sample of URB754, and the pure
compound is deficient of substantial activity as an MAGL inhibitor at
concentrations of up to 100 μM (personal communication from Dr Daniele
Piomelli).
Inhibitors of the Cellular Uptake of
Anandamide
N(4hydroxyphenyl) arachidonylamide (AM 404) was the first inhibitor of the
cellular uptake of anandamide to be created. Yet, this compound is not
specifically selective as it also binds to CB1 receptors, suppresses FAAH, and
activates TRPV1 receptors at concentrations at or lesser than those at which it
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has been found to suppress anandamide uptake. Crucially, it is presently not
evident whether any of the compounds identified to suppress the cellular
uptake of anandamide thus far do so by targeting an anandamide transport
protein or by attenuating FAAHmediated metabolism of anandamide to
induce an intracellular aggregation of this fatty acid amide that is adequate to
oppose its penetration into the cell through diffusion25–27.
Some Other Notable Ligands
Apart from the CB1 and CB2 receptor ligands discussed already, there are
several other compounds noteworthy because they have the ability to
modulate certain effects of standard CB1/CB2 receptor agonists through
apparently new mechanisms or because they share the evident potential of
such agonists to target specific putative nonCB1 and nonCB2 receptors.
These compounds are:
Δ9tetrahydrocannabivarin, the plant cannabinoid that displaces [3H]CP
55,940 from CB1 and CB2 receptors at concentrations in the low
nanomolar range (Ki = 75.4 and 62.8 nM, respectively) and acts as a
CB1 and CB2 receptor competitive antagonist, presenting higher potency
against CP 55,940 in the mouse isolated vas deferens and in membranes
acquired from human CB2transfected cells (apparent KB = 10 nM) than
in mouse brain membranes (apparent KB = 93 nM)121.
(–)Cannabidiol (CBD), the nonpsychoactive plant cannabinoid that has
therapeutic potential (for example, as an antiinflammatory agent), lacks
substantial affinity for CB1 or CB2 receptors, has anti
oxidant/neuroprotective properties, and blocks, activates, or inhibits (at
submicromolar concentrations) several standard or putative
pharmacological targets including an adenosine transporter122 as well as
delayed rectifier K+ and Ltype Ca2+ channels, acyltransferase, CYP
enzymes, a neuronal nonCB1 site of action in the mouse vas deferens,
and the putative nonCB1, nonCB2, and nonTRPV1 “abnormalCBD
receptor” that has been hypothesized to exist in tissues like mesenteric
arteries and in microglial cells2,88,123.
a set of CBD equivalents lacking substantial affinity for the CB1 receptor
and acting as antagonists (O1918) or agonists (abnormalCBD and O
1602) for the putative abnormalCBD receptor2,88,96.
NarachidonoylLserine, the endogenous compound that may be an
endogenous agonist for the abnormalCBD receptor as it seems to
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activate this putative receptor upon being added exogenously (EC50 =
550 or ca 1200 nM), and which binds just weakly to CB1 receptors (Ki >
10 μM) and does not bind to TRPV1 or CB2 receptors at concentrations of
up to 30 μM124.
Nvanillyl arachidonyl amide (arvanil), the anandamide/capsaicin
structural hybrid that binds to CB1 receptors and suppresses the cellular
uptake of anandamide at concentrations in the low micromolar range,
binds to TRPV1 receptors at concentrations in the low nanomolar range,
and may also have one or more as yet unidentified nonCB1 and non
TRPV1 sites of action125,126.
Org 27569, Org 29647, and Org 27759, the synthetic indole derivatives,
experiments on which have showed the existence of an allosteric site on
the cannabinoid CB1 receptor that includes a new target through which
CB1 receptor activation could be modulated by endocannabinoids that
are endogenously released, for instance, to combat nicotine dependence,
obesity, or inflammatory pain127.
It has been observed that although Org 27569, Org 29647, and Org 27759
act as CB1 allosteric enhancers in binding assays, they act as CB1 allosteric
inhibitors in functional in vitro bioassays127, thereby restricting their use as
experimental tools and establishing a need for additional CB1 allosteric
modulators.
There are three other ligands that are worth mentioning: HU 211, the (+)
enantiomer of the potent CB1/CB2 receptor agonist HU 210; Sch.336; and
palmitoylethanolamide, the endogenous ligand.
Sch.336, which is a CB2selective antagonist/inverse agonist, presents much
greater potency and efficacy as a CB2 receptor inverse agonist when
compared to SR144528128. Sch.336’s high inverse efficacy may be the reason
behind its potential to suppress leukocyte migration/trafficking, an effect that
could be useful in the clinic for managing inflammatory disorders128.
Although HU 211 lacks substantial affinity for CB1 or CB2 receptors, it has
neuroprotective properties that could be due to its potential to reduce the
synthesis of tumor necrosis factorα, to act as a noncompetitive antagonist
at the NmethylDaspartate (NMDA) receptor, to scavenge oxygenderived
free radicals, and/or to suppress depolarizationevoked calcium fluxes2,129.
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Palmitoylethanolamide has gained attention since it lacks substantial affinity
for CB1 or CB2 receptors and yet is vulnerable to antagonism by SR144528, a
discovery that has led to the postulate that this fatty acid amide could be the
endogenous agonist for a “CB2like” receptor2,88. There is also proof, firstly,
that palmitoylethanolamide is a PPARα receptor agonist130, secondly that it is
metabolized both by PAA and FAAH5 and thirdly, that it may potentiate
anandamide via what is called the “entourage effect” (see section on the
endocannabinoid system).
Future Directions
The focus of this article has specifically been on ligands that are most
extensively used as experimental tools to directly target cannabinoid CB1
and/or CB2 receptors or to modulate endocannabinoids’ tissue levels once
they are endogenously released. There is a possibility that future studies in
the area of cannabinoid pharmacology will focus on:
evaluating the therapeutic potential of CB1 and/or CB2 receptor allosteric
modulators and neutral antagonists
investigating the structureactivity relationships of ligands targeting the
CB1 allosteric site or acting as neutral CB1 and/or CB2 receptor
antagonists
determining the pharmacological profiles of fresh and current modulators
of endocannabinoid biosynthesis, metabolism, or cellular uptake
collecting more convincing evidence against or for the occurrence of an
endocannabinoid transporter in mammalian cells
characterizing and validating nonCB1 and nonCB2 targets for specific
cannabinoids and creating compounds with the ability to selectively
activate or block such targets with credible potency
learn why CB2 receptors appear to be expressed by central neurons
gaining a more comprehensive knowledge of the role played by the
endocannabinoid system in ameliorating the underlying pathology and/or
the symptoms of specific disorders
monitoring early indications for the occurrence of cannabinoid receptors
as homodimers or that they form oligomers or heterodimers with one or
more classes of noncannabinoid receptor2
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