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Professor R. G. Pertwee The central distribution pattern of CB1 receptors is Department of Biomedical Sciences, Institute heterogeneous and accounts for several prominent of Medical Sciences, University of Aberdeen, pharmacological properties of CB1 receptor , Foresterhill, Aberdeen AB25 2ZD, Scotland for example their ability to impair cognition and Roger Pertwee is Professor of and to alter the control of motor function. Neuropharmacology at the University of Aberdeen. Thus the cerebral cortex, , lateral His research interests include the pharmacology of caudate-putamen, substantia nigra pars reticulata, cannabinoid receptors, the physiological and globus pallidus, entopeduncular nucleus and the pathophysiological roles of endogenous molecular layer of the cerebellum are all populated ligands, and the therapeutic with particularly high concentrations of CB1 potential of . receptors.12,18 In line with the properties of cannabinoid receptor agonists, CB1 receptors are also found on pathways in brain and spinal cord and The at the peripheral terminals of primary sensory 14 neurones. Although the concentration of CB1 Two types of cannabinoid receptor have so far been receptors is considerably less in peripheral tissues 1 than in the , this does not imply identified, CB1 , cloned in 1990, and CB2 , cloned in 1993.2 These have now been detected in several that peripheral CB1 receptors are unimportant. This is species including man, rat and mouse3 and both CB because some peripheral tissues may contain high 1 concentrations of CB receptors, localized in discrete and CB2 receptor knockout mice have been 1 4-6 regions such as terminals that form only a small developed. The CB1 receptors of different mammalian species exhibit a high level of similarity. part of the total tissue mass. Peripheral tissues in which CB receptors are expressed on neurones For example, CB1 nucleotide sequences of human 1 and rat are 90% identical, those of human and mouse include the heart, vas deferens, urinary bladder and 91% identical and those of rat and mouse 96% small intestine.12 identical.7,8 CB receptors show greater interspecies 2 12,19 differences, the deduced sequence of the As detailed elsewhere, both CB12 and CB receptors are coupled through G proteins, negatively mouse CB2 receptor differing from that of the human i/o 9 to adenylate cyclase and positively to mitogen- CB2 receptor in 60 residues (82% similarity). These differences are apparent mainly in the N-terminal activated protein kinase. In addition, CB1 receptors are coupled to ion channels through G proteins, extramembrane region. A spliced variant of CB1 i/o cDNA, CB , has also been isolated.10,11 However, positively to A-type and inwardly rectifying potassium 1a channels and negatively to N-type and P/Q-type CB1a mRNA exists only as a minor transcript and there is no evidence for any notable difference between the calcium channels and to D-type potassium pharmacology of CB and CB receptors. channels.12,19,20 The coupling to A-type and D-type 11a potassium channels is thought to be through adenylate cyclase.20,21 Inwardly rectifying potassium CB1 mRNA has been detected both in the central nervous system and in certain peripheral tissues channels can also serve as a signalling mechanism including pituitary gland, immune cells, reproductive for the CB2 receptor, at least inXenopus oocytes that tissues, gastrointestinal tissues, superior cervical have been transfected with such channels together ganglion, heart, , urinary bladder and adrenal with this receptor type.22,23 In addition, there is 12 evidence from experiments with rat hippocampal CA1 gland. Some CB1 receptors are located at central and peripheral nerve terminals12,13,14 and, when pyramidal neurones that CB1 receptors are negatively 24 activated, these receptors seem to suppress the coupled to M-type potassium channels. CB1 neuronal release of one or other of a range of receptors may also mobilize arachidonic acid and 12 excitatory and inhibitory transmitters that include close 5-HT3 receptor ion channels and, under certain , noradrenaline, , 5 hydroxy- conditions, couple to Gs proteins to activate adenylate 25,26 ,g -aminobutyric acid, glutamate and cyclase and/or to reduce outward potassium K aspartate.14 CB mRNA is present mainly in immune current, possibly through arachidonic acid-mediated 2 27 cells with particularly high levels in B-cells and natural stimulation of protein kinase C. The questions of killer cells.15 Little is yet known in any detail about the whether CB1s receptor coupling to G proteins has physiological importance and of whether such physiological or pathophysiological roles of CB2 receptors. Most likely, these include coupling increases after Gi/o protein sequestration by immunomodulation which may depend at least in part co-localized non-cannabinoid Gi/o protein-coupled receptors have yet to be resolved. CB1 receptors have on CB2 receptor-mediated suppression of proinflammatory cytokine release and enhancement of also been reported to be positively coupled to antiinflammatory cytokine release from immune C through G proteins in COS7 cells co- 22 16,17 transfected with CB1 receptors and Ga subunits and cells. Thus one major role that CB1 and CB2 receptors may have in common is modulation of negatively coupled to voltage-gated L-type calcium ongoing release of chemical messengers. channels in cat cerebral arterial smooth muscle

Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Toll Free Tel: (800) 421-3701 Tel: (636) 207-7651 Fax: + 44 (0)117 982 6552 Toll Free Fax: (800) 483-1993 Fax: (636) 207-7683 e-mail: [email protected] e-mail: [email protected] e-mail: [email protected] 28 cells. One other recent finding is that CB1 receptors chemical groups: classical, nonclassical, on cultured cerebellar granule neurones can operate aminoalkylindole and ,45 (Figures 1 and 2 through a -sensitive mechanism to and Table 1). Classical cannabinoids consist of enhance NMDA-elicited calcium release from inositol dibenzopyran derivatives and are either plant-derived 1,4,5-triphosphate-gated intracellular stores.29 cannabinoids or their synthetic analogues. The most investigated of these include the psychotropic plant The discovery of cannabinoid receptors was followed cannabinoids,DD99 - ( -THC) and in 1992 by the demonstration that arachidonoyl D8-THC, and the synthetic cannabinoid, 11-hydroxy- ethanolamide () is an endogenous D8-THC-dimethylheptyl (HU-210). The nonclassical for these receptors.30 Other endogenous cannabinoids cannabinoids were developed by a Pfizer research (endocannabinoids) have since been identified, of team.48 They are quite similar in structure to classical which the most important is 2-arachidonoyl glycerol.31-33 cannabinoids, consisting as they do of bicyclic and Both anandamide and 2-arachidonoyl glycerol tricyclic analogues ofD9 -THC that lack a pyran ring. undergo depolarization-induced synthesis/release Important examples are CP 55940, CP 55244, from neurones and are removed from the extracellular CP 50556 (L-nantradol) and desacetyl-L-nantradol. space by a carrier-mediated, saturable uptake process The aminoalkylindole group was developed by a that is present in the membranes of neurones and Sterling Winthrop research team,49,50 the prototype of 31,34-37 astrocytes. There is also evidence that this group being WIN 55,212-2 (R -(+)-WIN55212). anandamide release in rat dorsal striatum can be This group contains compounds that are structurally triggered by the activation of dopamine D , D and/or 23 quite different from classical or nonclassical D receptors.38 Once within the cell, anandamide is 4 cannabinoids and, indeed, there is evidence that WIN hydrolysed to arachidonic acid and ethanolamine by 55,212-2 binds differently to the CB receptor than 31,36,39 1 hydrolase (FAAH). FAAH can classical and nonclassical cannabinoids, albeit it in a also catalyse the hydrolysis of 2-arachidonoyl manner that still permits mutual displacement between 31,40 glycerol, an indication that it has esterase as well WIN 55,212-2 and non-aminoalkylindole cannabinoids as amidase activity. The distribution in rat brain of 45 at CB12 and CB binding sites. The prototypic FAAH immunoreactivity is heterogeneous, as is the member of the eicosanoid group of cannabinoid brain distribution of FAAH mRNA and FAAH receptor agonists is the endocannabinoid, enzymatic activity. In line with the putative roles of anandamide (see above). Cannabinoid receptor anandamide and 2-arachidonoyl glycerol as agonists often contain chiral centres and these endogenous cannabinoids, these measures of FAAH generally confer marked stereoselectivity in brain distribution exhibit considerable though not pharmacological assays. WIN 55,212-2 is more active complete overlap with the brain distribution of CB 1 than WIN 55,212-3 (S -(-)-WIN55212) and classical 34,39,41-43 receptors. Unlike the endocannabinoid and nonclassical cannabinoids with the same absolute membrane transporter, which remains to be fully stereochemistry as (-)-D9 -THC at 6a and 10a (6aR , characterized, FAAH has been cloned44 and FAAH 10aR ) have the greater activity. Anandamide itself knockout mice developed (personal communication does not contain any chiral centres. However, some of from Dr Benjamin Cravatt). At least one other its synthetic analogues do, one example being anandamide-hydrolysing also exists, probably , theR -(+)-isomer of which has nine in lysozomes.43 Cannabinoid receptors and their times greater affinity for CB receptors than theS -(-)- endogenous ligands together constitute what is now 1 isomer.51 usually referred to as the ‘endocannabinoid system’. These receptors, together with FAAH and the The ability of cannabinoid receptor agonists that are endocannabinoid membrane transporter, constitute commonly used in the laboratory to interact with important new molecular targets and the remainder of cannabinoid receptors has been reviewed this review focuses on ligands that have been 45,52,53 9 developed to interact selectively with these targets. elsewhere. Essentially, (-)-D -THC binds equally well to CB12 and CB receptors and is a partial at both these receptor types. It has even less efficacy CB12 and CB receptor agonists at CB21 than at CB receptors and, indeed, has been Cannabinoid receptor agonists fall essentially into four reported in one CB2 bioassay system to behave as an

Table 1. Pharmacological properties of some important cannabinoid receptor agonists

Ligand CB1i K(nM) CB 2 Ki (nM) Likely Relative Efficacy

CB12 CB ACEA (Cat. Number 1319) 1.4††† > 2000 +++++ ? ACPA (Cat. Number 1318)2.2††† 715 ++++ ? Methanandamide (Cat. Number 1121)18† , 20 † 868 †† , 815 †† ++++ ? Anandamide (Cat. Number 1339)89* 371* ++++ + 2-AG 58.3, 472 145, 1400 ++++ ++ 2-AG 13.9** 58** ++++ ++ HU-210 (Cat. Number 0966)0.06, 0.73 0.52, 0.22 +++++ +++++ D9-THC 40.7 36.4 +++ + CP 55940 (Cat. Number 0949)0.58, 3.72, 5 0.69, 2.55, 1.8 +++++ +++++ WIN 55,212-2 (Cat. Number 1038)1.89, 62.3, 123 0.28, 3.3, 4.1 +++++ +++++ JWH-133 677† 3.4 ? +++++

2-AG, 2-arachidonoyl glycerol. See text and figures for full names or structures of other agonists. *PMSF present. **Inhibitors of the enzymic hydrolysis of 2-AG present.46 Binding assays were performed with [ 3 H]CP 55940 or [ 3 H]HU-243 (2-AG) using rat brain † and rat or mouse spleen †† membranes or membranes from cultured cells transfected with CB12 or CB receptors. For further information see text and references 45 and 47.

2 Figure 1. Some non-eicosanoid cannabinoid cannabinoid receptors can be largely attributed to the receptor agonists replacement of the pentyl side chain ofD8 -THC with a dimethylheptyl group. Me

OH One other classical cannabinoid receptor agonist that (-)-D9 -THC merits special mention is 3-(5´-cyano-1´,1´-dimethyl- pentyl)-1-(4-N-morpholinobutyryloxy)-D8 -THC Me O hydrochloride (O-1057; Figure 1).55 This stands out Me O-1057 from established cannabinoid receptor agonists in Me O O being readily soluble in water. The potency of O-1057 N+ O relative to that of CP 55940 is just 2.9 times less at H CB receptors and 6.4 times less at CB receptors. Cl- 12 The availability of a water-soluble cannabinoid will OH Me CN O facilitate cannabinoid delivery not only in the Me OH Me Me laboratory but also in the clinic, particularly where administration to patients is to be by injection or aerosol inhalation. Another important advance has been the development of cannabinoid receptor Me Me agonists that, unlike the agents already mentioned, OH CP 55,940 Me Me show marked differences in their abilities to interact (Cat. Number 0949) 45,47 n-Pr with CB12 and CB receptors. For the development H Me of CB1 -selective agonists, the starting point has been the anandamide molecule, the marginal CB1 selectivity O of which can be significantly enhanced by inserting a O H Me Me fluorine atom on the terminal 2´ carbon to form O-585 JWH-133 and/or by replacing a hydrogen atom on the 1´ or 2

Me N carbon with a methyl group to formR -(+)- 45 O methanandamide (Figure 2) or O-689. Another important consequence of inserting a methyl group on N WIN 55,212-2 the 1´ or 2 carbon is greater resistance to the (Cat. Number 1038) hydrolytic action of FAAH and, indeed,R -(+)- O methanandamide was first synthesized in order to meet the need for a metabolically more stable (Bold text denotes compounds available from Tocris) anandamide analogue. The most CB1 -selective agonists so far developed are arachidonyl-2´- 54 8 9 antagonist. (-)-DD -THC resembles (-)- -THC both in chloroethylamide (ACEA) and arachidonylcyclo- its affinities for CB12 and CB receptors and in its CB 1 propylamide (ACPA) (Figure 2), both of which behave receptor efficacy. CP 55940 and WIN 55,212-2 have as potent CB1 receptor agonists with reasonably high CB12 and CB affinities in the low nanomolar range and efficacy.56 However, unlike methanandamide and O- exhibit relatively high efficacy at both these receptor 689,45 neither ACEA nor ACPA show any sign of types. Results from binding experiments with CB1 - and reduced susceptibility to enzymic hydrolysis. This is CB2 -transfected cells indicate that CP 55940 has presumably because they lack a methyl substituent on essentially the same affinity for CB12 and CB the 1´ or 2 carbon and, indeed, it was recently shown receptors. WIN 55,212-2 has slightly greater affinity for that the addition of a methyl group to the 1´ carbon of CB21 than for CB receptors whilst anandamide binds ACEA does markedly decrease the susceptibility of marginally more readily to CB12 than to CB receptors this molecule to FAAH-mediated hydrolysis.57 This and, when protected from enzymic hydrolysis, exhibits a structural change also reduces the affinity of ACEA for CB affinity similar to that of (-)-D9 -THC. Anandamide 1 CB12 receptors by about 14-fold. As to CB -selective also resembles (-)-D9 -THC in behaving as a partial agonists, the best to have been developed so far are agonist at CB12 and CB receptors and in exhibiting all classical cannabinoids: L-759633, L-759656, JWH- lower CB21 than CB efficacy. One commercially 133 (Figure 1) and HU-308. Each of these agents not available classical cannabinoid, HU-210, has only binds more readily to CB21 than to CB receptors efficacies at CB and CB receptors that match those 12 but also behaves as a potent CB2 -selective agonist in of CP 55940 and WIN 55,212-2 and affinities for CB1 functional assays.47,58,59 Conformational requirements and CB2 receptors that exceed those of these other for the interaction of endocannabinoids not only with cannabinoids. As a result it is a particularly potent cannabinoid receptors but also with FAAH and with cannabinoid receptor agonist. Its pharmacological the anandamide membrane transporter (see below) effectsin vivo are also exceptionally long-lasting. The have been reviewed recently by Reggio and Traore.52 enhanced affinity and efficacy shown by HU-210 at

Figure 2. Some eicosanoid cannabinoid receptor agonists

OH O O

OH N O H

ACPA 2-Arachidonoyl glycerol (Cat. Number 1318)

O Me O O OH Cl N OH N N H H H

R-(+)-Methanandamide Anandamide ACEA (Cat. Number 1121) (Cat. Number 1339) (Cat. Number 1319)

(Bold text denotes compounds available from Tocris)

3 Figure 3. Some cannabinoid receptor antagonists Selective CB1 receptor antagonists/inverse agonists

AM 251 The first of these, the diarylpyrazole, SR141716A SR141716A O N (Cat. Number 1117) (Figure 3), was developed by Sanofi.45,60 This is a Me NH highly potent and selective CB1 receptor ligand (Table 2) that readily prevents or reverses CB -mediated N O N 1 N effects bothin vitro and in vivo . There is convincing Me NH Cl evidence that SR141716A is not a “silent” antagonist. Cl N Thus, as well as attenuating effects of CB1 receptor N agonists, SR141716A can by itself also elicit Cl Cl I responses in some CB1 receptor-containing tissues that are opposite in direction from those elicited by O CB1 receptor agonists. Whilst such “inverse Cl cannabimimetic effects” may in some instances be O N attributable to a direct antagonism of responses Me NH evoked at CB1 receptors by released AM 281 endocannabinoids, there is evidence that this is not N N (Cat. Number 1115) the only possible mechanism and that SR141716A is Me 63-65 Cl in fact an inverse agonist. Thus SR141716A may I Me produce inverse cannabimimetic effects in at least O SR144528 some tissues by somehow reducing the constitutive NH Me activity of CB receptors (the coupling of CB Cl 11 N receptors to their effector mechanisms that it is N thought can occur in the absence of exogenously O Cl added or endogenously produced CB1 agonists). Me Another notable CB1 -selective antagonist that exhibits Me OMe inverse CB1 receptor properties in some assay systems is LY320135.45 This agent, which was I NMe developed by Eli Lilly, shares the ability of SR141716A to bind much more readily to CB12 than CB receptors (Table 2). However it has less affinity for CB N 1 CN receptors than SR141716A and, at concentrations in LY320135 O the low micromolar range, also binds to muscarinic O AM 630 and 5HT2 receptors. Although neither SR141716A nor (Cat. Number 1120) LY320135 are commercially available, it is possible to OMe purchase structural analogues of SR141716A. These MeO O are AM 251 and AM 281 (Figure 3) which have been found to be respectively three and eight times less (Bold text denotes compounds available from Tocris) potent than SR141716A in displacing [3 H]SR141716A from binding sites on mouse cerebellar membranes.66 In somein vitro bioassay systems, for example rat and AM 281 has also been reported to bind more readily to guinea-pig isolated arteries and mouse isolated vas deferens, SR141716A has been reported to be less CB12 than CB receptors (Table 2) and to attenuate the ability of WIN 55,212-2 or CP 55940 to decrease rat potent against anandamide than against non- 71,72 locomotor activity, to inhibit single population spikes eicosanoid CB1 receptor agonists. There are also and evoked acetylcholine release in rat hippocampal reports that in certainin vivo bioassay systems, slices, to inhibit electrically-evoked contractions of the SR141716A fails to antagonize effects of anandamide myenteric plexus-longitudinal muscle preparation of altogether when it is given at doses that markedly guinea-pig ileum and to suppress guinea-pig intestinal antagonize the effects of other CB1 receptor agonists peristalsis.62,67-70 Like SR141716A, AM 281 can in the same bioassays.73-75 For the isolated tissue behave as an inverse agonist when administered preparations at least, this discrepancy often seems to alone.68-70 stem from the ability of anandamide to activate vanilloid as well as cannabinoid receptors.71,72,76,77

Table 2. Pharmacological properties of some important receptor partial agonists/antagonists/inverse agonists

Ligand CB1i K(nM) CB 2 Ki (nM) Pharmacological Classification

CB12 CB LY320135141 14900 A/I ? SR141716A 5.6, 11.8, 11.8, 12.3 > 1000, 973, 13200, 702A/I A/(I?) AM 281 (Cat. Number 1115) 12††† 4200 A/I ? O-1238 3.5 7.8 P A/P O-11845.2 7. 4 A/P A/I * AM 630 (Cat. Number 1120) 5152 31.2 A/P/IA/I * SR144528 437, > 10,0000.6, 5.6 A/(I?) A/(I?)

† MAFP 20(IC50 ) ?iA ?

A, surmountable antagonist; iA, irreversible antagonist; I, inverse agonist, P, partial agonist. Main pharmacological classification for each ligand is shown in bold. See text and figures for full names or structures of ligands. *Less inverse agonist efficacy at CB2 receptors than SR144528. MAFP is also a potent irreversible inhibitor of FAAH (see text). Binding assays were performed with [3† H]CP 55940 using rat forebrain and mouse †† spleen membranes or membranes from cultured cells transfected with CB12 or CB receptors. For further information see text and references 45, 47, 61 and 62.

4 Analogues of anandamide that share this ability to bind [33 H]WIN 55,212-2 and [ H]HU-243 are 0.07 to 4 nM, to both cannabinoid and vanilloid receptors include two 1.9 to 16.2 nM and 0.045 nM respectively at CB1 anandamide membrane transport inhibitors, AM 404 receptors and 0.2 to 7.4 nM, 2.1 to 3.8 nM and 71,78-80 45 and arvanil. Of these, arvanil is a hybrid of 0.061 nM respectively at CB2 receptors. Thus anandamide and the established vanilloid receptor [3 H]HU-243 is particularly potent. Three radiolabelled agonist, capsaicin. As is to be expected, the structural ligands have also been developed as potential probes requirements for activation of cannabinoid and vanilloid for human single photon emission computed receptors are not the same.71,72,76,77 Whether the ability tomography (SPECT) or positron emission of anandamide to activate vanilloid receptors has tomography (PET) experiments. These are 123 physiological as well as pharmacological significance I-labelled analogues of AM 251 (CB1d K = 0.23 to remains to be established. 0.62 nM) and AM 28166,88,89 and an 18 F-labelled analogue of SR141716A (SR144385).90 Particularly CB2 receptor antagonists/inverse agonists promising results have been obtained from animal experiments with [123 I]AM 281. 66 The most notable of these is the Sanofi compound, SR144528 (Figure 3), a diarylpyrazole that binds with Figure 5. HU-210 and [3 H]HU-243 markedly higher affinity to CB21 than CB receptors (Table 2).81 There is good evidence that, like SR141716A, SR144528 is not a “silent” antagonist but HO HU-210 [3 H]HU-243 (Cat. Number 0966) rather an inverse agonist that can, by itself, produce (Cat. Number R967) 59,81 OH inverse cannabimimetic effects at CB2 receptors. HO H Another CB2 -selective antagonist/inverse agonist is *H *H H 6-iodopravadoline (AM 630; Figure 3 and Table 2) OH Me H O which potently reverses CP 55940-induced inhibition of Me Me Me H forskolin-stimulated cyclic AMP production by human Me O CB250 -transfected CHO cell preparations (EC = Me 129 nM) and enhances forskolin-stimulated cyclic AMP Me Me production by the same cell line when administered by (Bold text denotes compounds available from Tocris) 59 itself (EC50 = 230 nM). The inverse efficacy of AM 630 at CB receptors appears to be less than that 2 Inhibitors of the enzymic hydrolysis of of SR144528.82 As to the ability of AM 630 to interact endocannabinoids with CB1 receptors, results from several investigations when taken together suggest that this ligand has mixed The presence of FAAH in many cannabinoid bioassay agonist-antagonist properties and that it is a low-affinity systems has created the need for FAAH inhibitors that partial CB agonist.45,59,83-85 There is also one report 1 can be used to protect endocannabinoids from that it can behave as a low-potency inverse agonist at enzymic hydrolysis.12 The need for FAAH inhibitors CB receptors.86 1 also stems from the possibility that FAAH inhibitors might have therapeutic potential as indirect One cannabinoid receptor ligand that is close to being cannabinoid receptor agonists, the expectation being a silent cannabinoid receptor antagonist, albeit at both that drugs that activate the endocannabinoid system CB and CB receptors, is the classical cannabinoid, 12 by increasing the concentration of endocannabinoids 8 6´-azidohex-2´-yne-D -THC (O-1184; Figure 4 and at cannabinoid receptors would be more selective Table 2). In addition to a terminal N group, the alkyl 3 than direct agonists. This is because they are unlikely side chain of this ligand contains a carbon-carbon triple to affect all parts of the endocannabinoid system at bond, a structural modification that reduces CB and 1 one time, producing instead effects only at sites where CB efficacy but not CB or CB affinity.82 O-1184 212 on-going production of endocannabinoids is taking behaves as a high-affinity low-efficacy agonist at CB 1 place. The drug that has been most widely used to receptors and as a high-affinity low-efficacy inverse inhibit the enzymic hydrolysis of anandamide is the agonist at CB receptors.82,87 O-1238 (Figure 4), in 2 non-specific protease inhibitor, phenylmethyl- which the carbon-carbon triple bond of O-1184 is sulphonyl fluoride.12 replaced by a carbon-carbon double bond, has higher efficacy than O-1184 at CB receptors and behaves as 1 FAAH inhibitors with much greater potency are now a high-affinity low-efficacy partial agonist at CB 2 available, two notable examples being 82 receptors (Table 2). palmitylsulphonyl fluoride (AM 374; Figure 6) and stearylsulphonyl fluoride (AM 381). Both ligands inhibit Radiolabelled cannabinoid receptor ligands FAAH irreversibly and show good separation between potency for FAAH inhibition and ability to bind to CB Tritiated cannabinoid receptor ligands that have been 1 receptors. Thus the EC50 value of AM 374 is 7 nM for most widely used in binding assays or for inhibition of FAAH and 520 nM for displacement of autoradiography are the CB -selective [3 H]SR141716A 1 [3 H]CP 55940 from specific binding sites on rat (CB K = 0.19 to 1.24 nM) and [3 H]CP 55940, 1d forebrain membranes whilst the corresponding EC [33 H]WIN 55,212-2 and [ H]HU-243 (Figure 5), all three 50 values of AM 381 are 4 nM and 18.5m M of which bind more or less equally well to CB and CB 12respectively.91 AM 374 potentiates both anandamide- receptors. Typical K values for [3 H]CP 55940, d induced inhibition of evoked [3 H]acetylcholine release in rat hippocampal slices92 and anandamide-induced Figure 4. O-1184 and O-1238 suppression of rat operant lever pressing and open field locomotor activity.93 When administered by Me O-1238 themselves, both AM 374 and the anandamide membrane transport inhibitor AM 404 (see below) OH share the ability of cannabinoid receptor agonists to O-1184 ameliorate in mice with chronic relapsing Me Me H N3 experimental allergic encephalomyelitis (CREAE), an Me O OH induced syndrome that serves as an animal model of .94,95 This they most likely do by Me H N3 augmenting levels of endogenously released Me O endocannabinoid(s) as brain and spinal cord

5 concentrations of both anandamide and 2-AG are produced FAAH inhibitors.32 This putative “entourage” greater in spastic CREAE mice than in control effect may well extend to 2-arachidonoyl glycerol animals.95 The ability of AM 381 to potentiate which can be protected from the esterase action of anandamide remains to be reported. FAAH (and so potentiated) by the endogenous fatty acid derivatives, 2-linoleyl glycerol and 2-palmitoyl Methyl arachidonyl fluorophosphonate (MAFP; Figure glycerol.32,46,107 Other inhibitors of FAAH have also 6) is another potent irreversible inhibitor of FAAH been identified and details of these appear 61 18,39,43,57,97,101,102,108-110 (EC50 = 2.5 nM). However, it also potently displaces elsewhere. [3 H]CP 55940 from specific binding sites on rat brain membranes in an irreversible manner (EC50 = Inhibitors of endocannabinoid membrane 20 nM)61 and produces insurmountable antagonism at transport 96 CB1 receptors (Table 2). One recently developed analogue of MAFP, O-1887 (Figure 6), shows much One notable membrane transport inhibitor to have greater separation of FAAH inhibitory potency (EC50 = been developed isN -(4-hydroxyphenyl) 15 nM) from potency for binding to CB11 receptors (CB arachidonylamide (AM 404; Figure 6). This has been 97 Ki > 10m M). Interestingly, this compound shares the reported to inhibit anandamide uptake by rat cultured ability of PMSF to elicit cannabimimetic responses cortical neurones (EC50 = 1m M) and astrocytes (EC 50 when administered to mice by itself.97,98 Whether this = 5m M) and to potentiate anandamide both in vitro is through inhibition of the enzymic hydrolysis of andin vivo .35,111 When administered to rats by itself, endogenously released cannabinoids has yet to be AM 404 increases plasma levels of anandamide and investigated. Other reasonably potent FAAH inhibitors shares the ability of this endocannabinoid to decrease 99 are diazomethyl arachidonyl ketone (EC50 = 520 nM) locomotor activity, depress plasma levels of prolactin and (E)-6-(bromomethylene) tetrahydro-3-(1- and alter tyrosine hydroxylase activity in the 100 naphthalenyl)-2H-pyran-2-one (EC50 = 800 nM), hypothalamus (increase) and substantia nigra which act irreversibly, and arachidonyl trifluoromethyl (decrease).112-114 The inhibitory effect of AM 404 on ketone (EC50 = 900 nM), which is a tight-binding but locomotor activity has been found to be susceptible to reversible FAAH inhibitor that also inhibits cytosolic antagonism by SR141716A.113,114 AM 404 does not, 91,101 phospholipase A2 . Arachidonyl trifluoromethyl however, elicit two other typical responses to CB1 ketone and diazomethyl arachidonyl ketone have receptor agonists in rats: catalepsy and signs of 113 been reported to bind to CB1i receptors with K values analgesia in the hot plate test. of 0.65 and 1.3m M respectively99,102 and there is also a report that the EC50 of arachidonyl trifluoromethyl Structure-activity experiments with AM 404 analogues 3 ketone for displacement of [ H]CP 55940 from CB1 have revealed major differences between the binding sites is 2.5m M.91 Significantly more potent as structural requirements of the transporter for ligand FAAH inhibitors are a series ofa -keto bicyclic recognition and those for ligand translocation.35 At heterocycles with alkyl or phenylalkyl side chains that concentrations in the low micromolar range, it have recently been developed by Boger et al.103 displaces [3 H]CP 55940 from specific binding sites on 115 These inhibit the enzyme competitively, some with Ki rat forebrain membranes (Ki = 1.76m M). Even so, values in the picomolar or low nanomolar range. The there are no reports that AM 404 behaves as a structure of the most potent of this new generation of cannabinoid receptor agonist or antagonist. There is FAAH inhibitors is shown in Figure 6 (Compound 59; also one report that AM 404 inhibits FAAH with an 57 Ki = 140 pM). Other pharmacological properties of EC50 of 0.5m M. However, this conflicts with other these inhibitors, for example their ability to interact reports that the EC50 of AM 404 for inhibition of FAAH with cannabinoid or vanilloid receptors or to potentiate is 22mm M116 or exceeds 30 M. 111 Submicromolar endocannabinoids, have yet to be reported. concentrations of AM 404 can activate vanilloid receptors,78,80 prompting the need for a more selective It has been proposed that since endogenous inhibitor of endocannabinoid membrane transport. substrates for FAAH such as linoleoylethanolamide, This need was recently addressed by the and serve as reversible development of the AM 404 analogue, VDM11 (Figure inhibitors of this enzyme,36,104-106 anandamide may be 6), which exhibits the same potency as AM 404 as an protected from enzymic hydrolysis by endogenously

Figure 6. Some inhibitors of FAAH (MAFP, O-1887, Compound 59 and AM374) or of endocannabinoid membrane transport

O O OMe N P N F Compound 59 O MAFP

O O OMe P OH S F O F O-1887 O N Palmitylsulphonyl fluoride H Me (AM 374)

OH VDM11 O Me O OOMe N H O O AM 404 (Cat. Number 1116) O O SKM 4-45-1 O OMe HN N O H O OH O N H Arvanil

(Bold text denotes compounds available from Tocris)

6 endocannabinoid membrane transport inhibitor in C6 rat Figure 7. , and O-1602 glioma cells (EC50 = 10m M) but markedly less efficacy than AM 404 at vanilloid receptors.116 Although Abnormal cannabidiol Me membrane transport-inhibiting concentrations of VDM11 Me OH undergo some degree of binding to CB1 receptors, these concentrations (5-10m M) displace Cannabidiol 3 [ H]SR141716A from CB1 binding sites by less than 116 OH 50%. VDM11 also exhibits relatively low potency as Me an inhibitor of FAAH (EC > 50m M). Another ligand OH OH 50 Me Me that inhibits the endocannabinoid membrane transporter (EC = 3.6m M) more readily than it inhibits FAAH Me 50 O-1602 (EC50 = 32m M), at least in RBL-2H3 cells, is arvanil (Figure 6).79 However this agent not only has OH OH reasonably high affinity for CB1i receptors (K = 0.5m M) Me but is also a potent vanilloid receptor agonist (human 71,79,116 and rat VR1 EC50 = 0.5 nM). Details of other membrane transport inhibitors are to be found Conclusions elsewhere.35,37,57,79,110,117 The field of cannabinoid research is now at a One other important recent advance has been the particularly exciting stage. Thus, whilst a number of development of a fluorescent substrate for anandamide major advances have recently been made, many uptake (SKM 4-45-1; Figure 6) that should serve as a important questions still remain unanswered or incompletely addressed so prompting the need for useful experimental tool for transporter studies.117 This agent is more potent as an inhibitor of endocannabinoid more research at both non-clinical and clinical levels. Of particular importance at the pharmacological level membrane transport (EC = 7.8m M) than as an FAAH 50 is the need: inhibitor (EC50 > 10m M). Whilst the ability of SKM 4-45- 1 to interact with vanilloid receptors remains to be ! to understand the modes of action of anandamide investigated, it is known that, at 3m M, it does not and 2-arachidonoyl glycerol more fully and to 3 displace [ H]CP 55940 from CB1 binding sites on rat characterize their effects and the processes that brain membranes.117 terminate these effects more completely, ! to develop silent cannabinoid receptor antagonists Other cannabinoid receptors? possibly through further elucidation of the structural features that attenuate agonist and Calignano et al118 have postulated the existence of an inverse agonist efficacy at cannabinoid receptors SR144528-sensitive non-CB2 cannabinoid receptor without reducing cannabinoid receptor affinity, (‘CB2 -like’ receptor). This hypothesis is based on ! to seek out any additional endocannabinoids or evidence that even though lacks novel types of cannabinoid receptor, 30,119-121 significant affinity for CB12 or CB receptors, its ! to achieve a fuller pharmacological ability to produce signs of anti-hyperalgesia in the characterization of some existing inhibitors of mouse formalin paw test is readily attenuated by the endocannabinoid membrane transport and 118 CB2 -selective antagonist/inverse agonist, SR144528. enzymic hydrolysis, The existence of CB2 -like receptors in the mouse vas ! to develop inhibitors of endocannabinoid deferens has also been proposed.122 More recently, membrane transport and enzymic hydrolysis that evidence was obtained for the presence in vascular show greater separation between their sought- of an SR141716A-sensitive non-CB1 , non- after inhibitory actions and other pharmacological CB2 , non-vanilloid receptor that is unresponsive to actions, particularly the ability to activate or block established non-eicosanoid CB12 /CB receptor agonists cannabinoid, vanilloid or other receptors or to but can be activated both by the eicosanoid inhibit other or transporters, cannabinoids, anandamide and methanandamide, and ! to obtain more detailed information about by certain classical cannabinoids that do not act endocannabinoid release in both health and through CB1 or vanilloid receptors (“abnormal disease and hence gain a better appreciation of cannabidiol” and its more potent analogue, O-1602; the therapeutic potential of drugs that inhibit Figure 7).123,124 Interestingly, two effects of abnormal endocannabinoid membrane transport and/or cannabidiol, hypotension and mesenteric , endocannabinoid enzymic hydrolysis, were found to be antagonized by the non-psychotropic ! to search for beneficial and unwanted classical cannabinoid, cannabidiol (Figure 7). So too consequences of selective inhibition of both was anandamide-induced mesenteric vasodilation. endocannabinoid membrane transport and the Finally, the existence in the brain of non-CB12 , non-CB , enzymic hydrolysis of endocannabinoids. SR141716A-insensitive -coupled receptors for anandamide has recently been proposed to explain Note added in proof: the presence in the brain of a results obtained from experiments with CB knockout 1 novel CB1 -selective endocannabinoid, 2-arachidonoyl mice.125 glyceryl ether (noladin ether), has just been announced.126

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CANNABINOIDS AVAILABLE FROM TOCRIS

1319 ACEA...... Potent, highly selective CB1 agonist 1318 ACPA...... Potent, selective CB1 agonist 1117 AM 251...... Potent, selective CB1 antagonist 1115 AM 281...... Potent, selectiveCB1 anta gonist / inverse agonist 1116 AM 404...... Anandamide transport inhibitor 1120 AM 630...... CB2 selective antagonist / inverse agonist 1017 Anandamide (in water soluble emulsion) ...... Endogenous CB receptor ligand 1339 Anandamide (pure oil)...... Endogenous CB receptor ligand 0949 CP 55,940 ...... CBB12 and C agonist 0966 HU 210 ...... Highly potent cannabinoid agonist R967 [3 H]-HU 243b ...... Potent for CB receptors 1341 JWH 015...... Selective CB2 agonist 1121 R-(+)-Methanandamide ...... Potent and selective CB1 ligand 0879 Palmitoylethanolamide ...... Putative endogenous CB2 agonist 1038 WIN 55,212-2 ...... Highly potent cannabinoid agonist Local regulations may restrict the sale of some of the above products in certain territories. Please consult your local Tocris Cookson office or distributor for further details.

Cannabinoid Receptor Ligands, Tocris Reviews No. 16, April 2001

©2001 Tocris Cookson Published and distributed by Tocris Cookson

Editor: Samantha Manley, Ph.D. Managing Editor: Duncan Crawford, Ph.D. Design and Production: Jane Champness; Lacia Ashman, MA

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