cbd cb1 cb2

December 15, 2021 By admin Off

Moving on to experiments performed with hCB 2 -CHO cell membranes, Thomas et al. (2007) found the mean apparent K B value of CBD for antagonism of CP55940 in the [ 35 S]GTPγS-binding assay (65 n M ) to be markedly less than its K i value for displacing [ 3 H]CP55940 from these membranes ( Table 1 ). As in mouse brain membranes, so too in hCB 2 -CHO cell membranes CBD administered by itself inhibits [ 35 S]GTPγS binding (MacLennan et al. , 1998b; Thomas et al. , 2007). Since it is inhibitory in this bioassay at 1 μ M , the concentration at which it also antagonizes CP559540, it is possible that CBD produces this antagonism of CP55940 in a non-competitive manner by ‘physiologically’ opposing the ability of this agonist to stimulate CB 2 receptors. This hypothesis is supported by the findings first, that 1 μ M CBD produces a marked downward displacement of the CP55940 log concentration–response curve in the [ 35 S]GTPγS-binding assay and second, that this downward displacement appears to account entirely for this antagonism of CP55940 by CBD (Thomas et al. , 2007). Further experiments are now required to establish whether CBD also behaves as an inverse agonist in a tissue in which CB 2 receptors are expressed naturally and whether, as in brain experiments, there is any indication of an additional pharmacological target in such a tissue through which CBD can also act to produce signs of CB 2 inverse agonism. If CBD does indeed interact with more than one target to produce its inverse effect in brain tissue and/or in a tissue that expresses CB 2 receptors naturally, it will also be important to establish whether these interactions take place in an additive or synergistic manner.

Although Δ 9 -THCV may not be a CB 2 receptor inverse agonist, evidence has emerged recently that it is a CB 2 receptor partial agonist. This came from experiments with eΔ 9 -THCV in which the measured response used to indicate CB 2 receptor activation was inhibition of forskolin-induced stimulation of cyclic AMP production by hCB 2 -CHO cells (Gauson et al. , 2007). This is a bioassay that detects cannabinoid receptor activation with greater sensitivity than the [ 35 S]GTPγS-binding assay, probably because adenylate cyclase is located further along the cannabinoid receptor signalling cascade than G protein (reviewed in Pertwee, 1999; Howlett et al. , 2002). Additional experiments are now required to establish whether Δ 9 -THCV also activates CB 2 receptors in vivo . If it does, then it will be important to determine whether Δ 9 -THCV is effective against chronic liver diseases, there being evidence that one effective strategy for managing these disorders in the clinic may be to administer a medicine that simultaneously blocks CB 1 receptors and activates CB 2 receptors (Mallat et al. , 2007).

It was research in the 1960s and early 1970s that led to the discovery that the psychotropic effects of cannabis are produced mainly by (−)- trans -Δ 9 -tetrahydrocannabinol (Δ 9 -THC; Figure 1 ), to the pharmacological characterization of this plant cannabinoid (phytocannabinoid) and to the development of synthetic cannabinoids (reviewed in Pertwee, 2006). These advances led on to the introduction into the clinic in the 1980s of Δ 9 -THC (dronabinol, Marinol, Solvay Pharmaceuticals, Brussels, Belgium) and of one of its synthetic analogues, nabilone (Cesamet, Valeant Pharmaceuticals, Aliso Viejo, CA, USA), for the suppression of nausea and vomiting produced by chemotherapy and, in 1992, of Marinol for the stimulation of appetite in AIDS patients (reviewed in Robson, 2005; Pertwee and Thomas, 2007). Importantly, they also led on to the discovery that many of the effects produced by Δ 9 -THC and its synthetic cousins depend on the ability of these ligands to target a new family of receptors (reviewed in Howlett et al. , 2002; Pertwee, 2005a, 2006). Two types of these cannabinoid receptors have so far been identified and both are members of the superfamily of G-protein-coupled receptors. These are the CB 1 receptor, first cloned in 1990 (Matsuda et al. , 1990), and the CB 2 receptor, cloned in 1993 (Munro et al. , 1993).

There is evidence too that both CB 1 and CB 2 receptors are more highly expressed in human hepatocellular carcinoma tumour samples than in matched non-tumorous tissues, that this increased expression may prolong survival (Xu et al. , 2006) and that ‘protective’ increases in the densities of both these receptor types occur in human prostate cancer cells (Sarfaraz et al. , 2005). Increases that are apparently protective have also been detected in CB 1 receptor expression within the brain in rodent models of stroke (Jin et al. , 2000) and temporal-lobe epilepsy (Wallace et al. , 2003) and in the density or expression of intestinal CB 1 receptors in mouse models of intestinal inflammation, colitis and diarrhoea (Izzo et al. , 2001, 2003; Massa et al. , 2004; Kimball et al. , 2006) and of CB 2 receptors in colonic-infiltrated immune cells in mouse models of colitis (Kimball et al. , 2006) and in macrophages and T lymphocytes located in human and murine atherosclerotic plaques (Steffens et al. , 2005). It is noteworthy, however, that although CB 1 -receptor-coupling efficiency has been reported to increase in certain brain areas of rats with experimental autoimmune encephalomyelitis (EAE), this increase was accompanied by a decrease in CB 1 receptor density in the same brain areas (Berrendero et al. , 2001). Moreover in EAE mice, decreases have been detected in both central CB 1 receptor density (cerebellum, globus pallidus and lateral caudate–putamen) and coupling efficiency (cerebellum) (Cabranes et al. , 2006). In contrast, CB 2 receptor expression levels have been reported to increase in regions of human post-mortem spinal cord affected by multiple sclerosis or amyotrophic lateral sclerosis (Yiangou et al. , 2006) and in the central nervous systems of EAE mice (Maresz et al. , 2005). These increases have been shown to result from an accumulation of microglial cells and peripheral macrophages and there is evidence from the mouse experiments that activation of the CB 2 receptors expressed by these cells leads to an amelioration of EAE inflammation and possibly also to a slowing of EAE progression (Maresz et al. , 2007).

The finding that Δ 9 -THCV exhibits less potency against CP55940 or R -(+)-WIN55212 in mouse whole-brain membranes than in the vas deferens (Thomas et al. , 2005; Pertwee et al. , 2007b) indicates that it displays not only agonist dependence as an antagonist, but also tissue dependence. Further evidence for such tissue dependence was recently obtained by Dennis et al. (2007), who found that eΔ 9 -THCV antagonizes R -(+)-WIN55212-induced stimulation of [ 35 S]GTPγS binding more potently in mouse cerebellar membranes (apparent K B =7 n M ) than in mouse piriform cortical membranes (apparent K B =54 n M ). Clearly, further experiments are now required to establish why eΔ 9 -THCV does not display the same potency against CP55940 or R -(+)-WIN55212 in all CB 1 -expressing tissues and brain areas. It will also be important to investigate why, according to Schild analysis, Δ 9 -THCV appears to antagonize R -(+)-WIN55212 competitively in the mouse isolated vas deferens (Thomas et al. , 2005) but non-competitively in both mouse cerebellar and piriform cortical membranes (Dennis et al. , 2007).

Tolerance to Δ 9 -THC.

There is evidence that like established CB 1 receptor antagonists such as SR141716A and AM251 (reviewed in Pertwee, 2005b), Δ 9 -THCV can block CB 1 -mediated effects of endogenously released endocannabinoids when administered in vivo . This evidence has come from recent experiments showing that eΔ 9 -THCV shares the ability of AM251 to reduce the food intake and body weight of non-fasted and fasted ‘non-obese’ mice when administered once (Robinson et al. , 2007) and of dietary-induced obese mice when given repeatedly over 28 days (Cawthorne et al. , 2007). It has also been found that like AM251, eΔ 9 -THCV can reduce the body fat content and plasma leptin concentration and increase the 24-h energy expenditure and thermic response to food of dietary-induced obese mice (Cawthorne et al. , 2007), the data obtained suggesting that eΔ 9 -THCV produces its antiobesity effects more by increasing energy expenditure than by reducing food intake. In addition, both eΔ 9 -THCV and AM251 have been shown to reduce the time that ‘non-obese’ mice spend close to a food hopper (Robinson et al. , 2007). These experiments were prompted by conclusive evidence that established CB 1 receptor antagonists suppress feeding and body weight in animals and humans (reviewed in Matias and Di Marzo, 2007) and by the introduction into the clinic of SR141716A (rimonabant; Acomplia, Sanofi-Aventis, Paris, France) in 2006 as an antiobesity agent. Further research is now required to determine whether Δ 9 -THCV would also be effective as a medicine for the management of obesity, and indeed for drug-dependence therapy, experiments with drug-dependent animals and human subjects having shown that CB 1 receptor blockade can reduce signs of drug dependence and the incidence of relapse after drug withdrawal (reviewed in Le Foll and Goldberg, 2005).

Additional in vitro evidence that Δ 9 -THCV can block the activation of neuronal CB 1 receptors has come recently from experiments with murine cerebellar slices (Ma et al. , 2006). The results obtained suggest first, that eΔ 9 -THCV can block CB 1 -mediated inhibition of GABA release from basket-cell interneurons caused by R -(+)-WIN55212 and second, that by itself eΔ 9 -THCV shares the ability of the CB 1 receptor antagonist/inverse agonist, AM251, to increase GABA release from these neurons. These effects were observed at a concentration (5.8 μ M ) below any at which eΔ 9 -THCV has been found to induce signs of inverse agonism in the [ 35 S]GTPγS-binding assay when this is performed with murine cerebellar membranes (Dennis et al. , 2007). It will now be important to establish whether eΔ 9 -THCV is increasing GABA release by opposing activation of basket-cell CB 1 receptors by endogenously released endocannabinoid molecules, not least because such an effect could explain why eΔ 9 -THCV has also been found to disrupt the spread of epileptiform activity induced in rat piriform cortical slices by Mg 2+ -free Krebs medium (Weston et al. , 2006), an observation that does of course raise the possibility that Δ 9 -THCV may display anticonvulsant activity in vivo .

Some K i values of (−)-Δ 9 -THC and certain other phytocannabinoids for the in vitro displacement of [ 3 H]CP55940 or [ 3 H]HU-243 from CB 1 – and CB 2 -specific binding sites.

Although Δ 9 -THCV seems to be capable of eliciting CB 1 -receptor-mediated responses in vivo , there is also evidence that it can behave as a CB 1 receptor antagonist both in vivo and in vitro . Thus, when administered to mice in vivo at doses below those at which it produces signs of CB 1 receptor agonism, O-4394 has been found to block effects of Δ 9 -THC that are thought to be CB 1 receptor mediated. Moreover, when administered in vitro , both O-4394 and eΔ 9 -THCV antagonize established CB 1 /CB 2 receptor agonists in a surmountable manner (Thomas et al. , 2005; Pertwee et al. , 2007b). More specifically, O-4394 has been found to attenuate Δ 9 -THC-induced hypothermia at 0.3 and 3 mg kg −1 i.v. and Δ 9 -THC-induced antinociception in the tail-flick test at 3 mg kg −1 i.v., and both O-4394 and eΔ 9 -THCV antagonize CP55940-induced stimulation of [ 35 S]GTPγS binding to mouse whole-brain membranes with mean apparent K B values (82 and 93 n M, respectively) that do not deviate significantly from their CB 1 K i values for displacement of [ 3 H]CP55940 from these membranes ( Table 1 ; Thomas et al. , 2005; Pertwee et al. , 2007b). In contrast to SR141716A and CBD (Thomas et al. , 2007), Δ 9 -THCV (O-4394) lacks detectable inverse agonist activity in the [ 35 S]GTPγS-binding assay performed with mouse whole-brain membranes and also fails to produce any detectable stimulation of [ 35 S]GTPγS binding to such membranes (Pertwee et al. , 2007b). Even so, it would be premature to conclude that Δ 9 -THCV lacks significant efficacy as a CB 1 receptor inverse or partial agonist until its actions have been investigated in other in vitro bioassays that display greater sensitivity than the [ 35 S]GTPγS-binding assay to ligands of this kind.

Some non-CB 1 , non-CB 2 actions of Δ 9 -THC can also be produced by certain other cannabinoid receptor agonists at concentrations of 1 μ M or less. For example, like Δ 9 -THC, both anandamide and 2-arachidonoylglycerol can activate GPR55 (Ryberg et al. , 2007) and modulate conductance in ligand-gated ion channels of glycine receptors (reviewed in Oz, 2006), and the phytocannabinoid, cannabinol, can activate putative non-CB 1 , non-CB 2 , non-transient receptor potential vanilloid receptor 1 (non-TRPV1) peripheral neuronal receptors, though 11-hydroxy-Δ 9 -THC, Δ 9 -THC-11-oic acid, HU-210 and CP55940 cannot (Zygmunt et al. , 2002). Some cannabinoids have been found to share the ability of Δ 9 -THC to reduce conductance in ligand-gated ion channels of human 5-HT 3A receptors at submicromolar concentrations (Barann et al. , 2002). Importantly, Δ 9 -THC is the most potent of these cannabinoids as an inhibitor of these ion channels, the rank order of potency being Δ 9 -THC> R -(+)-WIN55212>anandamide>(2-methyl-1-propyl-1 H -indol-3-yl)-1-naphthalenylmethanone>CP55940, and consequently quite unlike that for CB 1 or CB 2 receptor agonism. It is also known that Δ 9 -THC-like antioxidant activity is exhibited by several other phenolic cannabinoids, for example CBD ( Table 3 ) and HU-210 (reviewed in Pertwee, 2005a).

Cannabis sativa is the source of a unique set of compounds known collectively as plant cannabinoids or phytocannabinoids. This review focuses on the manner with which three of these compounds, (−)- trans -Δ 9 -tetrahydrocannabinol (Δ 9 -THC), (−)-cannabidiol (CBD) and (−)- trans -Δ 9 -tetrahydrocannabivarin (Δ 9 -THCV), interact with cannabinoid CB 1 and CB 2 receptors. Δ 9 -THC, the main psychotropic constituent of cannabis, is a CB 1 and CB 2 receptor partial agonist and in line with classical pharmacology, the responses it elicits appear to be strongly influenced both by the expression level and signalling efficiency of cannabinoid receptors and by ongoing endogenous cannabinoid release. CBD displays unexpectedly high potency as an antagonist of CB 1 /CB 2 receptor agonists in CB 1 – and CB 2 -expressing cells or tissues, the manner with which it interacts with CB 2 receptors providing a possible explanation for its ability to inhibit evoked immune cell migration. Δ 9 -THCV behaves as a potent CB 2 receptor partial agonist in vitro . In contrast, it antagonizes cannabinoid receptor agonists in CB 1 -expressing tissues. This it does with relatively high potency and in a manner that is both tissue and ligand dependent. Δ 9 -THCV also interacts with CB 1 receptors when administered in vivo , behaving either as a CB 1 antagonist or, at higher doses, as a CB 1 receptor agonist. Brief mention is also made in this review, first of the production by Δ 9 -THC of pharmacodynamic tolerance, second of current knowledge about the extent to which Δ 9 -THC, CBD and Δ 9 -THCV interact with pharmacological targets other than CB 1 or CB 2 receptors, and third of actual and potential therapeutic applications for each of these cannabinoids.

The discovery that Δ 9 -THCV can antagonize cannabinoid receptor agonists was made in experiments with the mouse isolated vas deferens (Thomas et al. , 2005), a tissue in which such agonists are thought to inhibit electrically evoked contractions by acting on prejunctional neuronal CB 1 receptors to inhibit contractile transmitter release (Howlett et al. , 2002). These experiments showed eΔ 9 -THCV to behave as a competitive surmountable antagonist of CP55940 and other established cannabinoid receptor agonists at a concentration (100 n M ) at which it did not affect clonidine- or capsaicin-induced inhibition of evoked contractions of the vas deferens or produce any sign of CB 1 receptor activation or inverse agonism. Unexpectedly, the antagonism displayed by eΔ 9 -THCV in the vas deferens was found to be ligand dependent. Thus, the mean apparent K B values of eΔ 9 -THCV for its antagonism of anandamide, R -(+)-WIN55212, methanandamide, CP55940 and Δ 9 -THC were 1.2, 1.5, 4.6, 10.3 and 96.7 n M, respectively. The mean apparent K B values of eΔ 9 -THCV for its antagonism of anandamide, R -(+)-WIN55212, methanandamide and CP55940 in this tissue preparation are significantly less than the K i values of eΔ 9 -THCV for its displacement of [ 3 H]CP55940 from mouse brain membranes (Thomas et al. , 2005). So too is the apparent K B value of O-4394 against R -(+)-WIN55212 in the vas deferens (4.8 n M ) (Pertwee et al. , 2007b). The questions of why Δ 9 -THCV exhibits such potency as an antagonist of these cannabinoid receptor agonists in the vas deferens and of why it produces antagonism in this tissue that is ligand-dependent have yet to be answered.

R -(+)-WIN55212, anandamide and/or 2-arachidonoylglycerol to modulate ion currents in various voltage-gated or ligand-gated ion channels (reviewed in Oz, 2006).

That CBD can behave as a CB 2 receptor inverse agonist may account, at least in part, for its well-documented anti-inflammatory properties (Pertwee, 2004b) as there is evidence that CB 2 inverse agonism can inhibit immune cell migration and reduce clinical signs of inflammation (Lunn et al. , 2006) and that CBD is a potent inhibitor of evoked migration in the Boyden chamber both of murine microglial cells and macrophages (Walter et al. , 2003; Sacerdote et al. , 2005) and of human neutrophils (McHugh and Ross, 2005). However, as indicated in Table 3 and elsewhere (Pertwee, 2004b), CBD has a number of other actions, some of which are also expected to reduce inflammation. Moreover, it has already been proposed that CBD modulates murine microglial cell migration by targeting the putative abnormal CBD receptor (Walter et al. , 2003). Another possibility that CBD inhibits immune cell migration, at least in part, by activating CB 2 receptors should also not be excluded at present, as CBD-induced inhibition of chemotaxis of murine macrophages can be prevented by SR144528 (Sacerdote et al. , 2005) and CBD has been found to display high potency though low efficacy as an inhibitor of forskolin-stimulated cyclic AMP production by hCB 2 -expressing CHO cells (Gauson et al. , 2007). Clearly, additional research is needed to establish which of the many actions of CBD contribute most to its anti-inflammatory effects. Also urgently required is further research directed at identifying the mechanisms that underlie some of the other potentially beneficial effects of CBD, for example its anticonvulsant, antipsychotic, anxiolytic, antiemetic, neuroprotective, anticancer and sleep-promoting effects (Pertwee, 2004b, 2005c; Parker et al. , 2005).

The CB 2 receptor pharmacology of Δ 9 -THCV.

Such upregulation of cannabinoid CB 1 or CB 2 receptors is expected to improve the selectivity and effectiveness of a cannabinoid receptor agonist as a therapeutic agent, especially when it is a partial agonist such as Δ 9 -THC. Thus, although an increase in receptor density will augment the potencies of both full and partial agonists, it will sometimes also increase the size of the maximal response to a partial agonist without affecting the maximal response to a full agonist. This difference between the pharmacology of full and partial agonists is well illustrated by results obtained with cannabinol, which is also a partial CB 1 receptor agonist (reviewed in Pertwee, 1999), and with CP55940 in experiments in which an increase in the intestinal expression of CB 1 receptors (and in intestinal inflammation) had been induced in mice by oral croton oil, the measured response being cannabinoid-induced CB 1 -receptor-mediated inhibition of upper gastrointestinal transit of a charcoal suspension (Izzo et al. , 2001). It was found that this increase in CB 1 expression level was accompanied not only by a leftward shift in the log dose–response curve of cannabinol but also by an increase in the size of its maximal effect. In contrast, CP55940, which has higher CB 1 efficacy than cannabinol (reviewed in Pertwee, 1999), exhibited an increase in its potency but no change in its maximal effect. There has also been a recent report that in rats displaying signs of inflammatory thermal hyperalgesia in response to an intraplantar injection of complete Freund’s adjuvant, CB 1 expression in dorsal root ganglion neurons undergoes a transient elevation that is accompanied by a marked increase in the antinociceptive potency of the CB 1 -selective agonist, 2-arachidonyl-2-chloroethylamide, when this is injected directly into the inflamed paws (Amaya et al. , 2006).

Some CB 1 – and CB 2 -receptor-independent actions of Δ 9 -THC.

Turning first to the experiments performed in this investigation with brain membranes, these showed that the mean apparent K B values of CBD for antagonism of CP55940- and R -(+)-WIN55212-induced stimulation of [ 35 S]GTPγS binding to these membranes are 79 and 138 n M, respectively, both well below the K i value of CBD for its displacement of [ 3 H]CP55940 from specific binding sites on these membranes ( Table 1 ). In these experiments, CBD produced parallel dextral shifts in the log concentration–response curves of both agonists. Even so, the unexpectedly high potency with which these shifts were induced by CBD raises the possibility that this antagonism is non-competitive in nature. This hypothesis is supported by the finding that CBD can behave as a CB 1 receptor ‘inverse agonist’ at concentrations below those at which it undergoes significant binding to the CB 1 orthosteric site. Thus, when administered by itself at a concentration (1 μ M ) at which it has been shown to antagonize CP559540 and R -(+)-WIN55212, CBD inhibits [ 35 S]GTPγS binding to mouse brain membranes. CBD-induced inhibition of [ 35 S]GTPγS binding has also been detected in hCB 1 -CHO cell membranes (MacLennan et al. , 1998b; Thomas et al. , 2007). No such inhibition was detected by Thomas et al. (2007) in untransfected CHO cell membranes, suggesting that the inverse effect of CBD in mouse brain tissue may be at least partly CB 1 receptor mediated. It remains possible, however, that this inverse effect also has a CB 1 -receptor-independent component since CBD was found in the same investigation to be no less effective in inhibiting [ 35 S]GTPγS binding to CB 1 −/− than to wild-type mouse brain membranes. Although the nature of this putative non-CB 1 pharmacological target remains to be elucidated, there is already evidence that it is not present in all G-protein-coupled receptors as CBD does not reduce [ 35 S]GTPγS binding to mouse brain membranes when this is being stimulated by the opioid receptor agonist, morphine (Thomas et al. , 2007). The finding that CBD antagonizes CP55940 and R -(+)-WIN55212 in mouse brain and hCB 1 -CHO cell membrane experiments is consistent with previous reports first, that CBD at 10 μ M antagonizes CP55940-induced stimulation of [ 35 S]GTPγS binding to rat cerebellar membranes (Petitet et al. , 1998) second, that it antagonizes CP55940 and R -(+)-WIN55212 in the mouse isolated vas deferens with apparent K B values in the low nanomolar range (Pertwee et al. , 2002) and third, that it can block various in vivo responses to Δ 9 -THC in rabbits, rats, mice and human subjects (reviewed in Pertwee, 2004b).

(−)- trans -Δ 9 -Tetrahydrocannabinol shares the ability of anandamide and 2-arachidonoylglycerol to activate both CB 1 and CB 2 receptors. More particularly, as discussed in greater detail elsewhere (Pertwee, 1997, 1999, 2005a; Howlett et al. , 2002; Childers, 2006), it binds to cannabinoid CB 1 and CB 2 receptors with K i values in the low nanomolar range ( Table 1 ) that indicate it to have higher affinity for these receptors than its corresponding (+)- cis (6a S , 10a S ) enantiomer ((+)-Δ 9 -THC), but lower CB 1 and CB 2 affinity than certain synthetic CB 1 /CB 2 receptor agonists, for example HU-210, CP55940 and R -(+)-WIN55212. Δ 9 -THC also exhibits lower CB 1 and CB 2 efficacy than these synthetic agonists, indicating it to be a partial agonist for both these receptor types. In contrast, the affinity of Δ 9 -THC for CB 1 and CB 2 receptors does match or exceed that of the phytocannabinoids (−)-Δ 8 -THC, Δ 9 -THCV, CBD, cannabigerol and cannabinol ( Table 1 ). It has also been found that Δ 9 -THC resembles anandamide in its CB 1 affinity, in behaving as a partial agonist at CB 1 receptors, albeit with less efficacy than anandamide, and in displaying even lower efficacy at CB 2 than at CB 1 receptors in vitro . Although 2-arachidonoylglycerol also possesses Δ 9 -THC-like CB 1 affinity, it has been found in several investigations to display higher efficacy than anandamide and hence Δ 9 -THC at both CB 1 and CB 2 receptors.

It is now well established that Δ 9 -THC is a cannabinoid CB 1 and CB 2 receptor partial agonist and that depending on the expression level and coupling efficiency of these receptors it will either activate them or block their activation by other cannabinoids. Further research is now required to establish in greater detail the extent to which the in vivo pharmacology of Δ 9 -THC is shaped by these opposing actions both in healthy organisms, for example following a decrease in cannabinoid receptor density or signalling caused by prior cannabinoid administration, and in animal disease models or human disorders in which upward or downward changes in CB 1 /CB 2 receptor expression, CB 1 /CB 2 -receptor-coupling efficiency and/or in endocannabinoid release onto CB 1 or CB 2 receptors have occurred in cells or tissues that mediate unwanted effects or determine syndrome/disease progression. The extent to which the balance between cannabinoid receptor agonism and antagonism following in vivo administration of Δ 9 -THC is influenced by the conversion of this cannabinoid into the more potent cannabinoid receptor agonist, 11-OH-Δ 9 -THC, also merits investigation.

There is evidence that in addition to eliciting responses in healthy animals, cannabinoid receptor activation by Δ 9 -THC can also ameliorate clinical signs or delay syndrome progression in animal models of certain disorders (reviewed in Pertwee, 2005b, 2007a; Pertwee and Thomas, 2007). This it does in a manner that not only supports the established clinical applications of Δ 9 -THC and nabilone for appetite stimulation and antiemesis and of the Δ 9 -THC- and CBD-containing medicine, Sativex (GW Pharmaceuticals, Salisbury, Wiltshire, UK), for the symptomatic relief of neuropathic pain in patients with multiple sclerosis and of cancer pain, but has also identified potential additional therapeutic uses for Δ 9 -THC, nabilone or other cannabinoid receptor agonists ( Table 2 ). Clinical evidence supporting the introduction of Δ 9 -THC or other cannabinoid receptor agonists into the clinic, for example for the management of disorders such as glaucoma and cancer, and for the relief of postoperative pain, spasms and spasticity caused by multiple sclerosis and painful spasticity triggered by spinal cord injury has also been obtained (Tomida et al. , 2004, 2006; Robson, 2005; Guzmán et al. , 2006; Pertwee, 2007a; Pertwee and Thomas, 2007).

Introduction.

Important recent findings with Δ 9 -THCV have been that it can induce both CB 1 receptor antagonism in vivo and in vitro and signs of CB 2 receptor activation in vitro at concentrations in the low nanomolar range. Further research is now required to establish whether this phytocannabinoid also behaves as a potent CB 2 receptor agonist in vivo . Thus, a medicine that blocks CB 1 receptors but activates CB 2 receptors has potential for the management of certain disorders that include chronic liver disease and also obesity when this is associated with inflammation. The bases for the ligand and tissue dependency that Δ 9 -THCV displays as an antagonist of CB 1 /CB 2 receptor agonists in vitro also warrant further research. In addition, in view of the structural similarity of Δ 9 -THCV to Δ 9 -THC, it will be important to determine the extent to which Δ 9 -THCV shares the ability of Δ 9 -THC, and indeed of CBD, to interact with pharmacological targets other than CB 1 or CB 2 receptors at concentrations in the nanomolar or low micromolar range. It will also be important to establish the extent to which CB 1 – and CB 2 -receptor-independent actions contribute to the overall in vivo pharmacology of each of these phytocannabinoids and give rise to differences between the in vivo pharmacology of Δ 9 -THC or Δ 9 -THCV and other cannabinoid receptor ligands such as CP55940, R -(+)-WIN55212 and SR141716A.

(−)- trans -Δ 9 -Tetrahydrocannabinol can also produce antagonism at the CB 2 receptor. Thus, Bayewitch et al. (1996) have found Δ 9 -THC (0.01–1 μ M ) to exhibit only marginal agonist activity in COS-7 cells transfected with human CB 2 (hCB 2 ) receptors when the measured response was inhibition of cyclic AMP production stimulated by 1 μ M forskolin. Instead, Δ 9 -THC behaved as a CB 2 receptor antagonist in this bioassay at both 0.1 and 1 μ M with an apparent K B value against HU-210 of 25.6 n M . More recently, Kishimoto et al. (2005) found that Δ 9 -THC (1 μ M ) shares the ability of the CB 2 -selective antagonist, N -[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), to abolish 2-arachidonoylglycerol-induced migration of human leukaemic natural killer cells.

In addition, there is the possibility that Δ 9 -THC may share actions that have so far only been shown to be exhibited by other CB 1 /CB 2 receptor agonists (reviewed in Pertwee, 2004c, 2005a). These include the ability of.

anandamide to activate putative non-CB 1 , non-CB 2 , non-TRPV1 neuronal receptors in guinea-pig small intestine (Mang et al. , 2001);

Among the effects that Δ 9 -THC seems to produce in vivo in healthy animals by activating neuronal CB 1 receptors are several that are frequently used as measured responses in bioassays for CB 1 receptor agonists (reviewed in Howlett et al. , 2002; Pertwee, 2006). For mice, these include a ‘tetrad’ of effects, suppression of locomotor activity, hypothermia, immobility in the ring test and antinociception in the tail-flick or hot-plate test. That the production of these effects by Δ 9 -THC depends on CB 1 receptor activation is supported by findings that this is readily antagonized by the selective CB 1 receptor antagonist, N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride (SR141716A), that most of the tetrad effects are not produced by Δ 9 -THC in mice from which the CB 1 receptor has been genetically deleted, that Δ 9 -THC produces these effects with a potency (half-maximal effective dose=1–1.5 mg kg −1 intravenous (i.v.)) that is consistent with its CB 1 receptor affinity and that they are also produced by a wide range of other established CB 1 receptor agonists (Martin et al. , 1991; Zimmer et al. , 1999; Di Marzo et al. , 2000; Wiley et al. , 2001; Varvel et al. , 2005). It is noteworthy, however, that deletion of the CB 1 receptor from mice bred on a C57BL/6J background does not affect the ability of Δ 9 -THC to induce antinociception in the tail-flick test, though it does abolish HU-210-induced antinociception in this bioassay and also Δ 9 -THC-induced antinociception in the hot-plate test (Zimmer et al. , 1999).

HU-210 to increase 5-HT binding to the 5-HT 2 receptor (Cheer et al. , 1999);

Finally, cannabis is a source not only of Δ 9 -THC, CBD and Δ 9 -THCV but also of at least 67 other phytocannabinoids and as such can be regarded as a natural library of unique compounds. The therapeutic potential of many of these ligands still remains largely unexplored prompting a need for further preclinical and clinical research directed at establishing whether phytocannabinoids are indeed ‘a neglected pharmacological treasure trove’ (Mechoulam, 2005). As well as leading to a more complete exploitation of Δ 9 -THC and CBD as therapeutic agents and establishing the clinical potential of Δ 9 -THCV more clearly, such research should help to identify any other phytocannabinoids that have therapeutic applications per se or that constitute either prodrugs from which semisynthetic medicines might be manufactured or lead compounds from which wholly synthetic medicines might be developed.

The cannabinoid receptors are part of the endocannabinoid signaling system which also includes the enzymes that synthesize and degrade endocannabinoids, as well as possible transporters. The molecular mechanisms for regulating lipid-based signaling events such as cannabinoid receptor signaling are not yet completely understood, although significant progress has been made [104]. Because lipids and their derivatives can readily partition into and diffuse throughout cellular membranes, lipid messengers such as the endocannabinoids are not easily contained by such physical boundaries as those of neurotransmitter membrane vesicles. 2-AG [37, 64], is synthesized on demand from lipid in a two step process in which phospholipase C-β hydrolyses phosphatidylinositol-4,5-bisphosphate to generate diacylglycerol, which is then hydrolyzed by diacylglycerol lipase (DAGL-α) to yield 2-AG [105, 106]. 2-AG has been shown to mediate the retrograde signaling of the endocannabinoid system in the brain [104]. The biosynthetic enzymes for 2-AG are localized on post-synaptic neurons in dendritic spines and somatodendritic compartments. Released 2-AG controls the activity of the complementary pre-synaptic neuron, by binding to the CB1 receptor which is often expressed there [107]. It is still unclear for retrograde signaling how newly synthesized 2-AG is induced to leave the post-synaptic cell plasma membrane to interact with CB1 pre-synaptically. 2-AG may be secreted by simple diffusion; alternatively, passive (energy-independent) carrier proteins may be required to extrude 2-AG. Once 2-AG has reached CB1, it will bind within the binding site crevice formed by the seven transmembrane helices of CB1. The subsequent activation of CB1 by 2-AG results in the inhibition of neurotransmitter release in the presynaptic cell via inhibition of voltage-activated Ca 2+ channels and the enhancement of inwardly rectifying K + channels in the cell [108–114]. Degradation of 2-AG is then accomplished presynaptically, principally by a membrane associated enzyme, monoacylglycerol lipase [115].

CoMFA QSAR pharmacophore models for AEA and its analogs have focused on folded conformations, such as a J-shape [159] or a helical shape [160]. Di Marzo and coworkers designed, synthesized and evaluated the CB1 binding affinity of a number of new conformationally restricted lipopeptides. All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Some of the analogues showed CB1 affinity, albeit SO-SO fold less than AEA [161]. Barnett-Norris and co-workers [147] performed Monte Carlo/ simulated annealing studies of anandamide, 2-AG, a dimethylheptyl analog of AEA with higher CB1 affinity (16,16-dimethyldocosa- cis -5,8,11,14-tetraenoylethanolamine), and N -(2-hydroxyethyl)prostagl-andin-B 2 -ethanolamide (PGB 2 -EA, 14 ), a prostanoid ligand which does not bind to the CB1 receptor. They found that the highest conformer populations for AEA and 2-AG were angle-iron (extended) and U shaped conformations, with the predominant population influenced by the environment (aqueous vs. CHC1 3 ). For the dimethylheptyl analog of AEA, a U shape and an angle-iron (extended) conformation was favored, however, the inactive PGB 2 -EA was found to be incapable of forming angle-iron (extended conformations). It instead favored an L shaped conformation. The investigator’s concluded that the low probability of PGB 2 -EA adopting an extended conformation may be why PGB 2 -EA shows such low affinity for the CB1 receptor.

The Sugiura group has reported that 2-glycerols with fatty acid chains of 20–22 carbons showed the strongest CB2 agonist activity. For C 2 o fatty acids, activity was best for the 20:3 Δ 5,8,11 and 20:4 Δ 5,8,11,14 (2-AG, 8 ), suggesting that like the 2-AG SAR generated for CB1, a double bond at Δ 5 was important [153]. More recently, the Sugiura group has reported a series of 2-AG analogs with additional variations in the fatty acid moiety [154]. These include an analog containing an isomer of arachidonic acid with migrated olefins, an analog containing a one-carbon shortened fatty acyl moiety and an analog containing a one-carbon elongated fatty acyl moiety. These analogs exhibited only weak agonistic activities toward either the CB1 receptor or the CB2 receptor, which is in good contrast to 2-AG which acted as a full agonist at these cannabinoid receptors [154].

III. ENDOCANNABINOID SAR.

Helix net representations of the amino acid sequences of the CB1 and CB2 receptors are presented here.

In Tuccinardi and co-workers [188] CB2 receptor model, AEA binds in the TM3–4–5–6 region. It does not interact with K.3.28(109), but it forms a H-bond with S3.31(112) through the amide oxygen atom, and this is in agreement with mutagenesis studies [183]. Moreover, the hydroxy group interacts with the oxygen backbone of L3.27(108). The AEA aliphatic chain interacts principally with W5.43(194) and W6.48(25 8). The AEA docking results seem to support the validity of these CB1 and CB2 models since they are in good agreement with the main mutagenesis data available for this ligand. Brizzi and co-workers recently used these models to analyze the binding of hybrid resorcinol/anandamide hybids [207].

Mutation and chimera studies are excellent ways to gather information on ligand binding sites. Although the CB1 binding sites for SR141716A [169–177], WIN55212-2 [169, 172, 176, 178–180] and CP55940 [176, 177, 179, 180]/HU-210 [172, 174, 175, 178, 180] and the CB2 binding site for WIN55212-2 [63, 181–183], CP55940 [182, 183], HU-210 [182] and SR144528 [184, 185] have been explored (many quite extensively) via mutation studies, very few mutations have shed light on the endocannabinoid binding sites at CB1 or CB2. Song and Bonner reported that a K3.28A mutation in CB1 leads to severe loss of binding for anan-damide, HU-210 and CP-55940 [178]. Mutations of aromatic residues on TMH3,4,5,6 of CB1 revealed that the binding of anandamide was affected by the mutation of one aromatic on TMH3, F3.25 [169, 176]. Recently, the Kendall lab has reported that mutations of F268W, P269A and 1271A in the EC-2 loop of CB1 have a profound effect on the binding of R-methanandamide ( 13 , Chart 3 ) at CB1. These results are suggestive of a steric effect on R-methanandamide binding and imply that anandamide binds high enough in the CB1 binding pocket to be impacted by changes in the EC-2 loop [175]. Surprisingly, there have been no mutation studies that explored the 2-AG binding pocket at CB1 or CB2.

The Sugiura group has also reported that a hydroxy group-containing 2-AG analog, a ketone group-containing analog, and a methylene-linked analog exhibited only weak agonistic activities toward either the CB1 receptor or the CB2 receptor [154]. The Makriyannis group has reported a series of head group constrained and conformationally restricted analogues of 2-AG which are 2-AG esters of 1,2,3-cyclohexanetriol. Resolution of one of these (AM5503) into stereoisomers yielded (+)AM4434 and (−)AM4435 both of which exhibited CB1 K i ,= 360 nM and CB2 K i =770nM binding [155].

In comparison to the state of knowledge concerning 2-AG and its role in retrograde signaling, the molecular and neuroanatomical organization of synaptic AEA signaling has remained largely unknown. Nylias and co-authors have shown that N -acylphosphatidylethanolaminehydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of AEA [116], is concentrated presynaptically in several types of hippocampal excitatory axon terminals and is associated with intracellular calcium stores. This indicates that, in contrast to 2-AG, endocannabinoids like AEA may have a presynaptic origin and their production may reflect the status of axon terminal [Ca 2+ ] (in part following release from intracellular stores) [117]. After acting at pre-synaptic CB1 receptors, AEA is taken up by post-synaptic cells via possible transport proteins on both neurons and glia that mediate endocannabinoid uptake [118–120]. After being transported into the cell, AEA is subsequently broken down into arachidonic acid and ethanolamine by a membrane-bound enzyme called fatty-acid amide hydrolase (FAAH) [119, 121, 122] that has been shown by immunohistochemistry to be localized to the endoplasmic reticulum [123, 124].

GPR55 was originally isolated in 1999 as an orphan GPCR with high levels of expression in human striatum [74](Genbank accession # > NM_005683). This receptor exhibits low amino acid identity to CB1 (13.5%) or CB2 (14.4%) receptors. The closest related proteins to GPR55 are GPR35 (27%), P2Y (29%), GPR23 (30%), and CCR4 (23%) [74]. GPR55 was first identified as a putative cannabinoid receptor in two patent applications [75, 76]. Drmota and coworkers [76] isolated a variant of GPR55, GPR55a, which contains three amino acid substitutions (F3.33(102)L, G5.52(195)S, C7.47(281)R). The ability of GPR55 to recognize cannabinoids was first described in a yeast expression system, where the CB1 antagonists AM251 and SR141716A acted as agonists at micromolar concentrations [75, 77]. However, Sjogren and co-workers have expressed GPR55 in HEK293 cells; there, nanomolar concentrations of many cannabinoid agonists stimulated GTPγS binding [78]. Most of the endocannabinoids, including anandamide, 2-AG, vi-rodhamine, noladin ether and palmitoylethanolamide, as well as the agonists CP55940 and Δ 9 -THC, stimulated GTPγS binding, which was not antagonized by AM281, but was blocked with 450 nM Cannabidiol (CBD) [78]. AM251 produced an agonist response in HEK293 cells, similar to that found in the yeast expression system [76, 78]. However, WIN55212-2 did not produce an agonist response at GPR55 [76, 78]. On the other hand, palmitoylethanolamide (PEA), a potent anti-inflammatory, anti-excitotoxic and anti-hyperalgesic compound [79, 80], was a potent agonist at this receptor [78], raising the possibility that GPR55 may be a receptor for this endocannabinoid. More recently, GPR55 has been tested against a number of cannabinoid ligands with mixed results. Observations using a GTPγS functional assay indicate that GPR55 is activated by nanomolar concentrations of the endocannabinoids 2-AG, virodhamine, noladin ether, and palmitoylethanolamine [81]; and the atypical cannabinoids abn-CBD and O-1602 [82], as well as by the classical cannabinoids CP55940, HU210, and (−)-Δ 9 -THC [83]. Oka et al . [84] reported that GPR55 is not a typical cannabinoid receptor as numerous endogenous and synthetic cannabinoids, including many mentioned above, had no effect on GPR55 activity. They presented compelling data suggesting that the endogenous lipid, lysophosphatidylinositol (LPI) and its 2-arachidonyl analogs are agonists at GPR55 as a result of their abilities to phosphorylate extracellular regulated kinase and induce calcium signaling [85, 86]. Kapur and co-workers recently examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a beta-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Their data demonstrate that AM251 and SR141716A, both cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. These ligands possess comparable efficacy in inducing (β-arrestin trafficking, and moreover, activate the G-protein dependent signaling of PKCβII. Conversely, the potent synthetic cannabinoid agonist CP55940 acts as a GPR55 antagonist/partial agonist. CP55940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2 [87]. Kapur and co-workers have concluded that at best, GPR55 is an atypical cannabinoid responder. Because of the clearly controversial identification of GPR55 as a cannabinoid receptor at this time, this review will focus on the CB1 and CB2 receptors and their endogenous mediators only.

Relationship Between Endocannabinoid Conformation and Productive Receptor Interaction: Dynamic Plasticity Appears Key.

Arachidonic acid is the most preferred fatty acid moiety, although the activity of eicosatrienoic acid (n-9)-containing species was almost comparable to that of the arachidonic acid containing species. Because the activities of 2-eico-satrienoy 1(20:3 Δ 8,11,14, n-6) glycerol, 2-eicosatrienoy 1(20:3 Δ 11,14,17 , n-3) glycerol and 2-docosatetraenoyl(22:4 Δ 10,13,16, n-6) glycerol are lower than those of 2-eicosatrienoyl(20:3 Δ 5,8,11, n-9) and 2-eicosapentaenoyl(20:5 Δ 5,8,11,14,17, n-3) glycerol, it appears that the presence of a double bond at the Δ 5 position, rather than further towards the end of the acyl chain, is crucially important, probably for folding or curvature nearer the head group.

Sugiura and co-workers [150] have found that glycerol is the most suitable head group, and the 2-isomer is preferable over the l(3)-isomer. Parkkari and co-workers also explored the effect of alpha-methylation of 2-AG as a way to improve its enzymatic stability [151]. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.

One of the striking facts that emerges from endocannabinoid SAR for CB1 and CB2 is that for AEA as well as 2-AG, the moiety with the smallest number of viable substitutions is the fatty acid moiety . The arachidonic acid (AA, 15 ) moiety in AEA, 2-AG and congeners confers on the molecule “dynamic plasticity”. The arachidonic acid acyl chain contains four homoallylic double bonds (i.e. cis double bonds separated by methylene carbons). Rabinovich and Ripatti [156] reported that polyunsaturated acyl chains in which double bonds are separated by one methylene group are characterized by the highest equilibrium flexibility compared with other unsaturated acyl chains. Rich [157] reported that a broad domain of low-energy conformational freedom exists for these C-C bonds. Results of the Biased Sampling phase from Conformational Memories calculations of AA are consistent with Rich’s and with Rabinovich and Ripatti’s results [147], as they revealed a relatively broad distribution of populated torsional space about the classic skew angles of 119°(s) and −119°(s’) for the C8-C9-C10-C11 torsion angle in AEA, for example (see Chart 2 , 5 for numbering system).

Sugiura and co-workers have reported that 2-AG ( 8 ) and other cannabinoid ligands such as AEA ( 5 ) and Δ 9 -THC ( 1 ) induce rapid transient increases in [Ca 2+ ] in NG108-15 cells through a cannabiniod CB1 receptor-dependent mechanism [148–150]. 2-AG was the most potent compound for inducing these transient increases, as its activity was detectable from as low as 0.3 nM. The maximal response induced by 2-AG exceeded responses induced by other CB1 agonists. Activities of the CB1 agonists, HU-210 and CP 55,940 ( 2 ) were also detectable from as low as 0.3 nM, whereas, the maximal responses induced by these compounds were low compared with 2-AG. AEA was also found to act as a partial agonist in this system. Arachidonic acid ( 15 , Chart 3 ) failed to elicit a response, while noladin ether ( 9 ) possessed appreciable activity, although its activity was apparently lower than that of 2-AG [150].

IV. ENDOCANNABINOID CONFORMATION.

The cannabinoid CB2 receptor (see Fig. 1 ) also belongs to the Class A (rhodopsin (Rho) family) of G protein-coupled receptors (GPCRs). The second cannabinoid receptor sub-type, CB2 was first cloned from a human promyelocytic leukemia cell HL60 cDNA library [44]. The human CB2 receptor exhibits 68% identity to the human CB1 receptor within the transmembrane regions, 44% identity throughout the whole protein [44]. Unlike the CB1 receptor, which is highly conserved across human, rat and mouse, the CB2 receptor is much more divergent. Sequence analysis of the coding region of the rat CB2 genomic clone indicates 93% amino acid identity between rat and mouse and 81% amino acid identity between rat and human.

The CB1 receptor transduces signals in response to CNS-active constituents of Cannabis sativa, such as the classical cannabinoid ((−)-Δ 9 -THC, 1 , Chart 1 ) and to three other structural classes of ligands, the non-classical cannabinoids typified by CP55940 ( 2 , Chart 1 ) [17, 18], the aminoalkylin-doles (AAIs) typified by WIN55212-2 ( 3 , Chart 1 ) [19–21] and the endogenous cannabinoids. The non-classical cannabinoids clearly share many structural features with the classical cannabinoids, e.g. a phenolic hydroxyl at C-1 (C2’), and alkyl side chain at C-3 (C-4’), as well as, the ability to adopt the same orientation of the carbocyclic ring as that in classical cannabinoids [22]. The AAIs, on the other hand, bear no obvious structural similarities with the classical/non-classical cannabinoids.

An analogy has been drawn in the literature between the C16-C20 portion of AEA (see 5 , Chart 2 ) and the C-3 pentyl side chain of the classical cannabinoid, Δ 9 -THC (see 1 , Chart 1 ). Consistent with this hypothesis, replacement of the pentyl tail of AEA with a dimethylheptyl chain results in enhanced affinity (although not to the same degree as seen in the classical cannabinoids) [143, 144].

Enlargement of the ethanolamine head group by insertion of methylene groups revealed that the N-propanol analog had slightly higher CB1 affinity than AEA, while higher ho-mologs had reduced CB1 affinity [126, 135]. Alkyl branching of the alcoholic head group lead to lower affinity analogs [135]. N-(propyl) arachidonylamide possessed higher CBI affinity (K i ; = 7.3nM) than anandamide itself (K i ,=22nM) [126, 135]. Substitution of an N-cyclopropyl group for the ethanolamine head group of AEA lead to a very high CB 1 affinity compound [136]. These results suggest that there may exist a hydrophobic sub-site for the AEA head group such that the hydroxyl of AEA may not be necessary for receptor interaction [127]. Replacement of the hydroxyl group of AEA with a halogen such as F or CI increased CBI affinity as well [127, 136, 137]. Substitution of the 2-hydroxyethyl group of AEA with a phenolic group, however, greatly decreased affinity for CBI [138–141].

While the fatty acid literature indicates that unsaturated fatty acids that possess multiple homoallylic double bonds, such as AA, exhibit a high degree of flexibility, this literature also indicates that saturated fatty acids tend to be significantly less flexible and adopt primarily extended conformations. Fatty acids with decreasing amounts of unsaturation tend to show a decreasing tendency to form folded structures, but still tend to curve in acyl chain regions in which unsaturation is present [158]. A correlation has been drawn between this acyl chain conformation trend and the SAR of the anandamide (AEA) acyl chain [142].