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New developments in the pharmacology of cannabinoids
H. van der Goot (Editor) Trends in Drug Research III © 2002 Elsevier Science B.V. All rights reserved
249
New Developments in the Pharmacology of Cannabinoids Roger G. Pertwee Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.
1. Introduction Cannabis sativa is the source of a set of more than sixty oxygen-containing aromatic hydrocarbon compounds called cannabinoids and one of these, A^tetrahydrocannabinol (A^-THC), is the mam psychotropic constituent of this plant [1]. A^-THC is also of interest because it is one of just two cannabinoids to be licensed fcr medical use. Thus, as the oral preparation dronabinol (Marinol), it is available in the USA for the suppression of nausea and vomiting provoked by anticancer drugs and for the reversal, through appetite stimulation, of body weight loss experienced by AIDS patients. The other cannabinoid that it is permissible to use clinically is nabilone (Cesamet), a synthetic analogue of A^-THC that is also given by mouth and that is licensed for use in the UK, again to suppress nausea and vomiting produced by cancer chemotherapy. Because cannabinoids have high lipid solubility and low water solubility they were long thought to owe their pharmacological properties to an ability to perturb the phospholipid constituents of biological membranes. However, all this changed in the late 1980's with the discovery of specific cannabinoid receptors. The existence of endogenous ligands for these receptors ("endocannabinoids") has also been demonstrated, suggesting that cannabinoid receptors have physiological as well as pharmacological significance. Cannabinoid receptors and their endogenous ligands constitute the "endocannabinoid system". 2. The endocannabinoid system Mammalian tissues contain at least two types of cannabinoid receptors, CBi and CB2 (see references 2 and 3 for reviews). Both receptor types are coupled through Gi/o proteins, negatively to adenylate cyclase and positively to MAP kinase. CBi receptors are also coupled through Gi/o proteins to ion channels, positively to A-type and inwardly rectifying potassium channels and negatively to N-type and P/Q type calcium channels and to D-type potassium channels. There is also evidence that CBi receptors are negatively coupled to M-type in rat hippocampal CAl pyramidal neurones and to voltage gated L-type calcium channels in cat cerebral arterial smooth muscle cells. Experiments with CBi and CB2-transfected cells have revealed other signalling mechanisms for cannabinoid receptors [3]. However, the physiological significance of these remains to be established. CBi receptors are present in the central nervous system and also in some peripheral tissues including pituitary gland, immune cells, reproductive tissues, gastromtestinal tissues, sympathetic ganglia, heart, lung, urinary bladder and adrenal gland [2]. Many
250 CBi receptors are to be found at central and peripheral nerve terminals and an important function of these receptors is to suppress the release of a range of excitatory and inhibitory neurotransmitters [3]. For example, CBi receptors are known to mediate inhibition of evoked release of acetylcholine from neurones in rat hippocampal slices and guinea-pig intestinal tissue and of dopamine in rat striatal slices and guinea-pig retinal discs. CBi receptors have also been found to mediate inhibition of evoked release of noradrenaline, 5-hydroxytryptamine, y-ammobutyric acid, glutamate and aspartate in various brain areas or in the peripheral nervous system. Much less is known about the role of CB2 receptors although it is very likely that this includes immunomodulation as CB2 receptors are expressed mainly by immune cells, particularly B-cells and natural killer cells [2, 3]. One important role of CB2 receptors may be to regulate cytokine release in health or disease [3, 4]. If this is true, then a common property of CBi and CB2 receptors would be the ability to modulate ongoing release of various chemical messengers, CBi receptors from neurones and CB2 receptors from immune cells. Within the bram, the distribution of CBi receptors is heterogeneous, brain areas that express this receptor type including the cerebral cortex, hippocampus, caudate-putamen, substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus, cerebellum, periaqueductal grey, rostral ventromedial medulla, superior coUiculus and certain nuclei of the thalamus and amygdala [2, 3, 5]. This distribution pattern accounts for several prominent pharmacological properties of CBi receptor agonists, for example their ability to impair cognition and memory and to alter the control of motor function. It also accounts for the ability of these agonists to produce analgesia in humans and antinociception in anunal models both of acute pam and of tonic pain induced by nerve damage or by the injection of an inflammatory agent. More specifically, as detailed elsewhere [3], CBi receptors that mediate the analgesic/antinociceptive effects of cannabmoids seem to be located not only in the brain but also on the terminals of neurones that project from the brain stem to the spinal cord and/or on intrmsic spinal neurones. There are also CBi receptors at the central and peripheral terminals of primary afferent neurones, both on C-fibres and on larger diameter Ap/A5-fibres. The presence of significant numbers of CBi receptors on these larger diameter primary afferent fibres helps to explain the eflBcacy shown by CBi receptor agonists against signs of neuropathic pain in animals since this kind of pain is thought to be elicited in part by abnormal spontaneous discharges of myelinated AjJ- and A8-fibres. CB2 receptors, and possibly other types of cannabmoid receptors yet to be characterized, may also contribute to the analgesic/antinociceptive effects of cannabinoids [3]. The most important endocannabinoids so fer identified are arachidonoylethanolamide (anandamide), 2-arachidonoyl glycerol (2-AG) [6-9] and probably also arachidonoyl glyceryl ether (noladin ether), the discovery of which has only just been announced [10]. Of these, anandamide behaves as a partial cannabinoid receptor agonist with margmally greater CBi than CB2 affinity but much less CB2 than CBi eflBcacy [11]. The pharmacological properties of 2-arachidonoyl glycerol and arachidonoyl glyceryl ether have been less well characterized. The available data suggests that both are cannabinoid receptor agonists, that the affinity of 2-AG for CBi and CB2 receptors is similar to that of anandamide and that noladin ether has significantly higher affinity for CBi receptors (Ki= 21.2 nM) than for CB2 receptors (Ki > 3 ]xM) [10, 11]. Anandamide
251 and 2-AG both serve as neurotransmitters or neuromodulators as there is evidence that they are synthesized by neurones ("on demand"), that they can undergo depolarizationinduced release from neurones and that once released they are rapidly removed from the extracellular space by a membrane transport process yet to be ftilly characterized [7, 1215]. Once within the cell, anandamide is thought to be hydrolysed to arachidonic acid and ethanolamine by the microsomal enzyme, fatty acid amide hydrolase (FAAH) [7, 14, 16]. 2-AG can also be hydrolysed enzymically, both by FAAH and by intracellular lipases [7, 17]. Mechanisms underlying the release and fate of noladin ether remain to be identified. There is firm evidence that anandamide activates not only cannabinoid receptors but also vanilloid VRl receptors, a property not shared by non-eicosanoid CBi or CB2 receptor agonists. The existence of an SR144528-sensitive non-CB2 cannabinoid receptor ('CB2-like' receptor) has also been proposed [18]. The evidence for this receptor type is based on the observation that even though pahnitylethanolamide lacks significant affinity for CBi or CB2 receptors [11, 19], its ability to produce signs of antihyperalgesia m the mouse formalin paw test is readily attenuated by the CB2-selective antagonist/inverse agonist, SR144528 but not by the CBi-selective antagonist/inverse agonist, SR141716A [18]. The existence of CB2-like receptors in the mouse vas deferens has also been proposed [20]. There is also evidence for the presence in vascular endothelium of an SR141716A-sensitive non-CBi, non-CB2, non-vanilloid receptor that is unresponsive to established non-eicosanoid CB1/CB2 receptor agonists but that can be activated both by the eicosanoid cannabinoids, anandamide and methanandamide, and by certain classical cannabinoids that do not act through CBi or vanilloid receptors ("abnormal cannabidiol" and its more potent analogue, 0-1602) [21, 22]. Interestingly, two effects of abnormal cannabidiol, hypotension and mesenteric vasodilation, were found to be antagonized by the non-psychotropic classical cannabinoid, cannabidiol. So too was anandamide-induced mesenteric vasodilation. Finally, the existence in the brain of non-CBi, non-CB2, SR141716A-insensitive G protein-coupled receptors for anandamide has recently been proposed to explain results obtained from experiments with CBi knockout [23]. 3. Inhibitors of endocannabinoid membrane transport or enzymic hydrolysis The discovery that the actions of anandamide and 2-AG are terminated by tissue uptake and intracellular enzymic hydrolysis has led to the development of inhibitors of these processes. The first of these to have been developed is A^-(4-hydroxyphenyl) arachidonylamide (AM404). When administered to rats by itself, AM404 increases plasma levels of anandamide and shares the ability of this endocannabinoid to decrease locomotor activity, depress plasma levels of prolactin and alter tyrosine hydroxylase activity in the hypothalamus (increase) and substantia nigra (decrease) [24-26]. The inhibitory effect of AM404 on locomotor activity is susceptible to antagonism by SR141716A [24, 25]. AM404 does not, however, elicit two other typical responses to CBi receptor agonists in rats: catalepsy and signs of analgesia in the hot plate test [24]. At concentrations at which it inhibits the membrane transport of endocannabmoids, AM404 bmds both to CBi receptors and to vanilloid VRl receptors. However, whilst it is known to activate vanilloid receptors, there are no reports that AM404 behaves as a
252 CBi receptor agonist or antagonist. A recently developed analogue of AM404, VDM11, retains the ability to inhibit endocannabinoid membrane transport but shows markedly less eflBcacy than AM404 as a vanilloid receptor agonist [27]. However, like AM404, VDM-11 does bind to CBi receptors at inhibitory concentrations [27]. The compound that has been most widely used to inhibit the enzymic hydrolysis of endocannabinoids in non-clmical experiments is the non-specific serine protease inhibitor, phenyhnethylsulphonyl fluoride. However, inhibitors with much greater potency are now available. Among these are two irreversible inhibitors of FAAH, palmitylsulphonyl fluoride (AM374) and stearylsulphonyl fluoride (AM381). Both of these inhibitors show good separation between potency for FAAH inhibition and ability to bind to CBi receptors [28]. AM374 potentiates both anandamide-induced inhibition of evoked [^H]acetylcholine release in rat hippocampal slices [29] and anandamideinduced suppression of rat operant lever pressing and open field locomotor activity [30]. Even more potent inhibitors of FAAH are to be found in a series of a-keto bicyclic heterocycles with alkyl or phenylaUcyl side chains. These inhibit the enzyme competitively, some with Ki values m the picomolar or low nanomolar range [31]. Other pharmacological properties of these inhibitors, for example their ability to interact with cannabmoid or vanilloid receptors or to potentiate endocannabinoids have yet to be reported. 4. Ligands for CBi and CBi receptors There are several established cannabinoid receptor agonists that bind more or less equally well to CBi and CB2 receptors, the best known examples being A^-THC, the Pfizer compound, CP55940 and the Sterlmg Winthrop compound, WIN55212-2, which has only marginally greater CB2 than CBi affinity [11, 32]. However, since the discovery of cannabinoid receptors, agonists with significant selectivity for CBi or CB2 receptors have emerged, hnportant CBi-selective agonists include the anandamide analogues, methanandamide, 0-689, arachidonyl-2'-chloroethylamide (ACEA) and arachidonylcyclopropylamide (ACPA) [11, 33]. Of these both ACEA and ACPA share the susceptibility of anandamide to enzymic hydrolysis whilst methanandamide and O689 are metabolically more stable than anandamide. This is presumably because methanandamide and 0-689 are protectedfromenzymic hydrolysis by the presence of a methyl substituent on the T or 2 carbon whilst anandamide, ACPA and ACEA are not. In line with this hypothesis, it was recently shown that the addition of a methyl group to the 1' carbon of ACEA markedly decreases the susceptibility of this molecule to FAAH-mediated hydrolysis [34]. This structural change also reduces the affinity of ACEA for CBi receptors by about 14-fold. The best CBi-selective agonists to have been developed so fer are all structural analogues of THC. They include L-759633, L759656, JWH-133 and HU-308 [32, 35, 36]. The discovery of cannabinoid receptors also prompted a search for selective CBi and CB2 receptor antagonists. The most promising compounds so fer developed are both Sanofi compounds. These are the CBi-selective SR141716A and the CB2-selective SR144528 [11, 37, 38]. There is convincing evidence, however, that both these compounds are not "silent" antagonists. Thus, as well as attenuating effects of CBi or CB2 receptor agonists, both agents can by themselves elicit responses in some
253 cannabinoid receptor-containing tissues that are opposite in direction from those elicited by CBi or CB2 receptor agonists. Whilst some of these "inverse cannabimimetic effects" may be attributable to a direct antagonism of responses elicited at cannabinoid receptors by released endocannabinoids, there is evidence that this is not the only possible mechanism and that SR141716A and SR144528 are in fact both inverse agonists [36, 38-41]. Thus both agents may produce inverse cannabimimetic effects in at least some tissues by somehow reducing the constitutive activity of cannabinoid receptors (the coupling of these receptors to their effector mechanisms that it is thought can occur in the absence of exogenously added or endogenously produced agonists). Two cannabinoid receptor ligands that are closer to being silent cannabinoid receptor antagonists at CBi and/or CB2 receptors are 6'-azidohex-2'-yne-A*-THC (0-1184) and 6-iodopravadoline (AM630) [42, 43]. 0-1184 behaves as a high-affinity low-eflBcacy agonist at CBi receptors and as a high-affinity low-efficacy inverse agonist at CB2 receptors. AM630 is a potent CBa-selective antagonist/inverse agonist which resembles 0-1184 appears in having less inverse efficacy at CB2 receptors than SR144528. AM630 also interacts with CBi receptors, albeit with significantly less potency. Results from several investigations when taken together suggest that AM630 has mixed agonistantagonist properties and that it is a low-affinity partial CBi agonist [11, 36, 44-46]. There is also one report that it can behave as a low-potency inverse agonist at CBi receptors [47]. 5. The therapeutic potential of cannabinoids Several potential therapeutic applications have been suggested for CBi receptor antagonists/inverse agonists [48]. These include appetite suppression [49-51], the reduction of L-Dopa-induced dyskinesia in patients with Parkinson's disease [52], the management acute schizophrenia [53] and the amelioration of cognitive/memory dysfunctions associated with disorders such as Alzheimer's disease [54]. The prospect of exploiting CBi receptor antagonists/inverse agonists for clinical purposes remains particularly attractive to the pharmaceutical industry as such agents do not produce the unwanted central effects for which CBi receptor agonists are so renowned. Even so, there is growing evidence that in addition to their recognized uses in the clmic as appetite stimulants and anti-emetics, CBi receptor agonists may have therapeutic potential as neuroprotective agents through CBi-mediated inhibition of glutamate release [55], as anticancer agents [56, 57] and for the management of glaucoma [58], pain [3, 59] and various kinds of motor dysfunction that include the muscle spasticity/spasm/tremor associated with multiple sclerosis and spinal cord injury, the tics and psychiatric signs and symptoms of Tourette's syndrome and the dyskinesia that is produced by L-Dopa in patients with Parkinson's disease [60-64]. Of these potential therapeutic targets for CBi receptor agonists, pain and motor disorders associated with multiple sclerosis and spinal cord injury, are currently attractmg particular attention. Some cannabinoids that do not act through CBi or CB2 receptors also have pharmacological properties that may come to be exploited in the clinic. One of these is (+)-ll-hydroxy-A^-THC-dimethylheptyl(HU-211, dexanabinol) which, as discussed in greater detail elsewhere [65], shows potential as a neuroprotective agent and also for the management of neuropathic pain and septic shock. The psychotropically inactive plant
254 cannabinoid, cannabidiol, also has neuroprotective activity [66]. Another cannabinoid that merits special mention is r,r-dimethylheptyl-A^-THC11-oic acid (DMH-A^-THC-11-oic acid). This is a cannabinoid receptor ligand with therapeutic potential as an anti-inflammatory/analgesic [3, 67]. In spite of its ability to interact with CBi and CB2 receptors [68], it remains possible that DMH-A^-THC-11oic acid produces its anti-inflammatory effects by suppressing eicosanoid synthesis in inflamed tissue through inhibition of inducible cyclooxygenase (COX-2), an enzyme that is thought to be activated during inflammation and to be responsible for the production of eicosanoids that act as inflammatory mediators [69]. DMH-A*-THC-11oic acid is more potent as an inhibitor of COX-2 than of COX-1 (constitutive cyclooxygenase), raising the possibility that it may be able to relieve inflammation without producing gastrointestinal or kidney toxicity [67, 69, 70]. It is unwanted effects of this kind that limit the clinical usefiibiess of less selective non-steroidal antiinflammatory drugs that inhibit COX-1 more or less as effectively as they inhibit COX2. 6. Future Clinical Research One challenge for future clinical research is to develop cannabinoid formulations and modes of administration that produce more reliable cannabinoid absorption than has so far been realisable, at least by the oral route. Possible solutions are to develop improved oral formulations or to use other routes for cannabinoid delivery, for example administration by rectal suppository [71], by aerosol/vapour inhalation, by injection, by skin patch or by the sublingual or intrathecal route, all modes of administration that avoid first-pass metabolism of the absorbed drug. Some success has ateady been achieved by GW Pharmaceuticals in phase I studies with a sub-lingual cannabinoid spray (personal communication from Dr. Geoffrey Guy). The emergence of better modes of cannabinoid administration should be facilitated by the development of a centrallyactive water-soluble cannabmoid [72]. The availability of better delivery systems fir cannabinoids should facilitate the gathering of more conclusive clinical data both about the efficacy of cannabinoids and about then- unwanted effects than has hitherto been achievable. Another important area for future clmical research must be the development of strategies that maxunize separation between the sought-after therapeutic effects of CBi receptor agonists and the unwanted effects of these drugs, particularly their psychotropic effects. One strategy may be to use agents that activate the endogenous cannabinoid system indirectly by increasing extracellular levels of endocannabinoids through inhibition of their membrane transport or enzymic hydrolysis. This approach is based on the expectation that drugs with this kind of action would be more selective than direct CBi receptor agonists. This is because they are unlikely to affect all parts of the endocannabinoid system at one time, producing instead effects only at sites where ongoing production of endocannabinoids is taking place. The success of this strategy depends on whether endogenous cannabinoids are released to a greater extent at sites at which they produce sought-after effects than at sites at which they provoke unwanted effects; In line with this possibility is evidence obtained from experiments using an autoimmune model of multiple sclerosis that is set up by mjecting Biozzi ABH mice
255 subcutaneously with an emulsion of mouse spinal cord homogenate in Freund's complete adjuvant. These showed that inhibitors of endocannabinoid membrane transport (AM404) or enzymic hydrolysis (AM374) share the ability of direct cannabinoid receptor agonists to ameliorate spasticity in CREAE mice and that spastic CREAE mice have elevated concentrations of the endocannabinoids anandamide and 2arachidonoyl glycerol in their brains and spinal cords [73]. There is also evidence fix)m animal experiments that peripheral inflammatory pain triggers increased release of anandamide in at least one area of the brain at which cannabinoids can induce signs of analgesia in animals, the periaqueductal grey area of the midbrain [74]. Because the unwanted central effects of cannabmoids are probably mediated largely or entirely by CBi receptors within the brain, a second strategy for reducing the unwanted effects of a CBi receptor agonist, at least for pain relief, might be to inject a CBi receptor agonist directly into the spinal cord. This approach exploits the presence of CBi receptors on nociceptive neurones within the spinal cord and also takes account of the ability of CBi receptor agonists to induce antinociception in anunals when these agents are administered intrathecally. The same strategy is sometimes adopted to reduce the incidence of adverse responses of multiple sclerosis patients to the anti-spasticity agent, baclofen. Yet another strategy would be to administer a cannabinoid in combination with a second agent that augments only the sought-after effects of the cannabinoid. Indeed, there is evidence fi-om animal experiments that synergistic interactions can occur between cannabinoids and opioids for analgesia [3, 75] and between cannabinoids and benzodiazepines for depressant effects on motor function [76, 77]. As there are claims by multiple sclerosis patients that cannabis can relieve their symptoms at dose levels that do not induce a 'high', another strategy may be to admmister an agonist (partial agonist) with a reduced ability (efficacy) to activate CBi receptors. This approach assumes that it should be possible to develop a partial agonist that has sufficient efficacy to relieve muscle spasticity/spasm and pam but insufficient efficacy to produce a full range of cannabhnimetic psychotropic effects even when it occupies all available CBi receptors. A possible lead compound is 0-1184 (see section 4). 7. References 1. Pertwee, R.G., 1988, Pharmacol. Ther., 36, 189. 2. Pertwee, R.G., 1997, Pharmacol. Then, 74, 129. 3. Pertwee, R.G., 2001, Prog. NeurobioL, 63, 569. 4. Molina-Holgado, E., Guaza, C , Borrell, J. and Molina-Holgado, F., 1999, Biodrugs, 12, 317. 5. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R. and Rice, K.C., 1991, J. Neurosci., 11, 563. 6. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R., 1992, Science, 258, 1946. 7. Di Marzo, V., Melck, D., Bisogno, T. and De Petrocellis, L., 1998, Trends Neurosci., 21, 521. 8. Mechoulam, R., Fride, E. and Di Marzo, V., 1998, Eur. J. Pharmacol., 359, 1.
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249
New Developments in the Pharmacology of Cannabinoids Roger G. Pertwee Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.
1. Introduction Cannabis sativa is the source of a set of more than sixty oxygen-containing aromatic hydrocarbon compounds called cannabinoids and one of these, A^tetrahydrocannabinol (A^-THC), is the mam psychotropic constituent of this plant [1]. A^-THC is also of interest because it is one of just two cannabinoids to be licensed fcr medical use. Thus, as the oral preparation dronabinol (Marinol), it is available in the USA for the suppression of nausea and vomiting provoked by anticancer drugs and for the reversal, through appetite stimulation, of body weight loss experienced by AIDS patients. The other cannabinoid that it is permissible to use clinically is nabilone (Cesamet), a synthetic analogue of A^-THC that is also given by mouth and that is licensed for use in the UK, again to suppress nausea and vomiting produced by cancer chemotherapy. Because cannabinoids have high lipid solubility and low water solubility they were long thought to owe their pharmacological properties to an ability to perturb the phospholipid constituents of biological membranes. However, all this changed in the late 1980's with the discovery of specific cannabinoid receptors. The existence of endogenous ligands for these receptors ("endocannabinoids") has also been demonstrated, suggesting that cannabinoid receptors have physiological as well as pharmacological significance. Cannabinoid receptors and their endogenous ligands constitute the "endocannabinoid system". 2. The endocannabinoid system Mammalian tissues contain at least two types of cannabinoid receptors, CBi and CB2 (see references 2 and 3 for reviews). Both receptor types are coupled through Gi/o proteins, negatively to adenylate cyclase and positively to MAP kinase. CBi receptors are also coupled through Gi/o proteins to ion channels, positively to A-type and inwardly rectifying potassium channels and negatively to N-type and P/Q type calcium channels and to D-type potassium channels. There is also evidence that CBi receptors are negatively coupled to M-type in rat hippocampal CAl pyramidal neurones and to voltage gated L-type calcium channels in cat cerebral arterial smooth muscle cells. Experiments with CBi and CB2-transfected cells have revealed other signalling mechanisms for cannabinoid receptors [3]. However, the physiological significance of these remains to be established. CBi receptors are present in the central nervous system and also in some peripheral tissues including pituitary gland, immune cells, reproductive tissues, gastromtestinal tissues, sympathetic ganglia, heart, lung, urinary bladder and adrenal gland [2]. Many
250 CBi receptors are to be found at central and peripheral nerve terminals and an important function of these receptors is to suppress the release of a range of excitatory and inhibitory neurotransmitters [3]. For example, CBi receptors are known to mediate inhibition of evoked release of acetylcholine from neurones in rat hippocampal slices and guinea-pig intestinal tissue and of dopamine in rat striatal slices and guinea-pig retinal discs. CBi receptors have also been found to mediate inhibition of evoked release of noradrenaline, 5-hydroxytryptamine, y-ammobutyric acid, glutamate and aspartate in various brain areas or in the peripheral nervous system. Much less is known about the role of CB2 receptors although it is very likely that this includes immunomodulation as CB2 receptors are expressed mainly by immune cells, particularly B-cells and natural killer cells [2, 3]. One important role of CB2 receptors may be to regulate cytokine release in health or disease [3, 4]. If this is true, then a common property of CBi and CB2 receptors would be the ability to modulate ongoing release of various chemical messengers, CBi receptors from neurones and CB2 receptors from immune cells. Within the bram, the distribution of CBi receptors is heterogeneous, brain areas that express this receptor type including the cerebral cortex, hippocampus, caudate-putamen, substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus, cerebellum, periaqueductal grey, rostral ventromedial medulla, superior coUiculus and certain nuclei of the thalamus and amygdala [2, 3, 5]. This distribution pattern accounts for several prominent pharmacological properties of CBi receptor agonists, for example their ability to impair cognition and memory and to alter the control of motor function. It also accounts for the ability of these agonists to produce analgesia in humans and antinociception in anunal models both of acute pam and of tonic pain induced by nerve damage or by the injection of an inflammatory agent. More specifically, as detailed elsewhere [3], CBi receptors that mediate the analgesic/antinociceptive effects of cannabmoids seem to be located not only in the brain but also on the terminals of neurones that project from the brain stem to the spinal cord and/or on intrmsic spinal neurones. There are also CBi receptors at the central and peripheral terminals of primary afferent neurones, both on C-fibres and on larger diameter Ap/A5-fibres. The presence of significant numbers of CBi receptors on these larger diameter primary afferent fibres helps to explain the eflBcacy shown by CBi receptor agonists against signs of neuropathic pain in animals since this kind of pain is thought to be elicited in part by abnormal spontaneous discharges of myelinated AjJ- and A8-fibres. CB2 receptors, and possibly other types of cannabmoid receptors yet to be characterized, may also contribute to the analgesic/antinociceptive effects of cannabinoids [3]. The most important endocannabinoids so fer identified are arachidonoylethanolamide (anandamide), 2-arachidonoyl glycerol (2-AG) [6-9] and probably also arachidonoyl glyceryl ether (noladin ether), the discovery of which has only just been announced [10]. Of these, anandamide behaves as a partial cannabinoid receptor agonist with margmally greater CBi than CB2 affinity but much less CB2 than CBi eflBcacy [11]. The pharmacological properties of 2-arachidonoyl glycerol and arachidonoyl glyceryl ether have been less well characterized. The available data suggests that both are cannabinoid receptor agonists, that the affinity of 2-AG for CBi and CB2 receptors is similar to that of anandamide and that noladin ether has significantly higher affinity for CBi receptors (Ki= 21.2 nM) than for CB2 receptors (Ki > 3 ]xM) [10, 11]. Anandamide
251 and 2-AG both serve as neurotransmitters or neuromodulators as there is evidence that they are synthesized by neurones ("on demand"), that they can undergo depolarizationinduced release from neurones and that once released they are rapidly removed from the extracellular space by a membrane transport process yet to be ftilly characterized [7, 1215]. Once within the cell, anandamide is thought to be hydrolysed to arachidonic acid and ethanolamine by the microsomal enzyme, fatty acid amide hydrolase (FAAH) [7, 14, 16]. 2-AG can also be hydrolysed enzymically, both by FAAH and by intracellular lipases [7, 17]. Mechanisms underlying the release and fate of noladin ether remain to be identified. There is firm evidence that anandamide activates not only cannabinoid receptors but also vanilloid VRl receptors, a property not shared by non-eicosanoid CBi or CB2 receptor agonists. The existence of an SR144528-sensitive non-CB2 cannabinoid receptor ('CB2-like' receptor) has also been proposed [18]. The evidence for this receptor type is based on the observation that even though pahnitylethanolamide lacks significant affinity for CBi or CB2 receptors [11, 19], its ability to produce signs of antihyperalgesia m the mouse formalin paw test is readily attenuated by the CB2-selective antagonist/inverse agonist, SR144528 but not by the CBi-selective antagonist/inverse agonist, SR141716A [18]. The existence of CB2-like receptors in the mouse vas deferens has also been proposed [20]. There is also evidence for the presence in vascular endothelium of an SR141716A-sensitive non-CBi, non-CB2, non-vanilloid receptor that is unresponsive to established non-eicosanoid CB1/CB2 receptor agonists but that can be activated both by the eicosanoid cannabinoids, anandamide and methanandamide, and by certain classical cannabinoids that do not act through CBi or vanilloid receptors ("abnormal cannabidiol" and its more potent analogue, 0-1602) [21, 22]. Interestingly, two effects of abnormal cannabidiol, hypotension and mesenteric vasodilation, were found to be antagonized by the non-psychotropic classical cannabinoid, cannabidiol. So too was anandamide-induced mesenteric vasodilation. Finally, the existence in the brain of non-CBi, non-CB2, SR141716A-insensitive G protein-coupled receptors for anandamide has recently been proposed to explain results obtained from experiments with CBi knockout [23]. 3. Inhibitors of endocannabinoid membrane transport or enzymic hydrolysis The discovery that the actions of anandamide and 2-AG are terminated by tissue uptake and intracellular enzymic hydrolysis has led to the development of inhibitors of these processes. The first of these to have been developed is A^-(4-hydroxyphenyl) arachidonylamide (AM404). When administered to rats by itself, AM404 increases plasma levels of anandamide and shares the ability of this endocannabinoid to decrease locomotor activity, depress plasma levels of prolactin and alter tyrosine hydroxylase activity in the hypothalamus (increase) and substantia nigra (decrease) [24-26]. The inhibitory effect of AM404 on locomotor activity is susceptible to antagonism by SR141716A [24, 25]. AM404 does not, however, elicit two other typical responses to CBi receptor agonists in rats: catalepsy and signs of analgesia in the hot plate test [24]. At concentrations at which it inhibits the membrane transport of endocannabmoids, AM404 bmds both to CBi receptors and to vanilloid VRl receptors. However, whilst it is known to activate vanilloid receptors, there are no reports that AM404 behaves as a
252 CBi receptor agonist or antagonist. A recently developed analogue of AM404, VDM11, retains the ability to inhibit endocannabinoid membrane transport but shows markedly less eflBcacy than AM404 as a vanilloid receptor agonist [27]. However, like AM404, VDM-11 does bind to CBi receptors at inhibitory concentrations [27]. The compound that has been most widely used to inhibit the enzymic hydrolysis of endocannabinoids in non-clmical experiments is the non-specific serine protease inhibitor, phenyhnethylsulphonyl fluoride. However, inhibitors with much greater potency are now available. Among these are two irreversible inhibitors of FAAH, palmitylsulphonyl fluoride (AM374) and stearylsulphonyl fluoride (AM381). Both of these inhibitors show good separation between potency for FAAH inhibition and ability to bind to CBi receptors [28]. AM374 potentiates both anandamide-induced inhibition of evoked [^H]acetylcholine release in rat hippocampal slices [29] and anandamideinduced suppression of rat operant lever pressing and open field locomotor activity [30]. Even more potent inhibitors of FAAH are to be found in a series of a-keto bicyclic heterocycles with alkyl or phenylaUcyl side chains. These inhibit the enzyme competitively, some with Ki values m the picomolar or low nanomolar range [31]. Other pharmacological properties of these inhibitors, for example their ability to interact with cannabmoid or vanilloid receptors or to potentiate endocannabinoids have yet to be reported. 4. Ligands for CBi and CBi receptors There are several established cannabinoid receptor agonists that bind more or less equally well to CBi and CB2 receptors, the best known examples being A^-THC, the Pfizer compound, CP55940 and the Sterlmg Winthrop compound, WIN55212-2, which has only marginally greater CB2 than CBi affinity [11, 32]. However, since the discovery of cannabinoid receptors, agonists with significant selectivity for CBi or CB2 receptors have emerged, hnportant CBi-selective agonists include the anandamide analogues, methanandamide, 0-689, arachidonyl-2'-chloroethylamide (ACEA) and arachidonylcyclopropylamide (ACPA) [11, 33]. Of these both ACEA and ACPA share the susceptibility of anandamide to enzymic hydrolysis whilst methanandamide and O689 are metabolically more stable than anandamide. This is presumably because methanandamide and 0-689 are protectedfromenzymic hydrolysis by the presence of a methyl substituent on the T or 2 carbon whilst anandamide, ACPA and ACEA are not. In line with this hypothesis, it was recently shown that the addition of a methyl group to the 1' carbon of ACEA markedly decreases the susceptibility of this molecule to FAAH-mediated hydrolysis [34]. This structural change also reduces the affinity of ACEA for CBi receptors by about 14-fold. The best CBi-selective agonists to have been developed so fer are all structural analogues of THC. They include L-759633, L759656, JWH-133 and HU-308 [32, 35, 36]. The discovery of cannabinoid receptors also prompted a search for selective CBi and CB2 receptor antagonists. The most promising compounds so fer developed are both Sanofi compounds. These are the CBi-selective SR141716A and the CB2-selective SR144528 [11, 37, 38]. There is convincing evidence, however, that both these compounds are not "silent" antagonists. Thus, as well as attenuating effects of CBi or CB2 receptor agonists, both agents can by themselves elicit responses in some
253 cannabinoid receptor-containing tissues that are opposite in direction from those elicited by CBi or CB2 receptor agonists. Whilst some of these "inverse cannabimimetic effects" may be attributable to a direct antagonism of responses elicited at cannabinoid receptors by released endocannabinoids, there is evidence that this is not the only possible mechanism and that SR141716A and SR144528 are in fact both inverse agonists [36, 38-41]. Thus both agents may produce inverse cannabimimetic effects in at least some tissues by somehow reducing the constitutive activity of cannabinoid receptors (the coupling of these receptors to their effector mechanisms that it is thought can occur in the absence of exogenously added or endogenously produced agonists). Two cannabinoid receptor ligands that are closer to being silent cannabinoid receptor antagonists at CBi and/or CB2 receptors are 6'-azidohex-2'-yne-A*-THC (0-1184) and 6-iodopravadoline (AM630) [42, 43]. 0-1184 behaves as a high-affinity low-eflBcacy agonist at CBi receptors and as a high-affinity low-efficacy inverse agonist at CB2 receptors. AM630 is a potent CBa-selective antagonist/inverse agonist which resembles 0-1184 appears in having less inverse efficacy at CB2 receptors than SR144528. AM630 also interacts with CBi receptors, albeit with significantly less potency. Results from several investigations when taken together suggest that AM630 has mixed agonistantagonist properties and that it is a low-affinity partial CBi agonist [11, 36, 44-46]. There is also one report that it can behave as a low-potency inverse agonist at CBi receptors [47]. 5. The therapeutic potential of cannabinoids Several potential therapeutic applications have been suggested for CBi receptor antagonists/inverse agonists [48]. These include appetite suppression [49-51], the reduction of L-Dopa-induced dyskinesia in patients with Parkinson's disease [52], the management acute schizophrenia [53] and the amelioration of cognitive/memory dysfunctions associated with disorders such as Alzheimer's disease [54]. The prospect of exploiting CBi receptor antagonists/inverse agonists for clinical purposes remains particularly attractive to the pharmaceutical industry as such agents do not produce the unwanted central effects for which CBi receptor agonists are so renowned. Even so, there is growing evidence that in addition to their recognized uses in the clmic as appetite stimulants and anti-emetics, CBi receptor agonists may have therapeutic potential as neuroprotective agents through CBi-mediated inhibition of glutamate release [55], as anticancer agents [56, 57] and for the management of glaucoma [58], pain [3, 59] and various kinds of motor dysfunction that include the muscle spasticity/spasm/tremor associated with multiple sclerosis and spinal cord injury, the tics and psychiatric signs and symptoms of Tourette's syndrome and the dyskinesia that is produced by L-Dopa in patients with Parkinson's disease [60-64]. Of these potential therapeutic targets for CBi receptor agonists, pain and motor disorders associated with multiple sclerosis and spinal cord injury, are currently attractmg particular attention. Some cannabinoids that do not act through CBi or CB2 receptors also have pharmacological properties that may come to be exploited in the clinic. One of these is (+)-ll-hydroxy-A^-THC-dimethylheptyl(HU-211, dexanabinol) which, as discussed in greater detail elsewhere [65], shows potential as a neuroprotective agent and also for the management of neuropathic pain and septic shock. The psychotropically inactive plant
254 cannabinoid, cannabidiol, also has neuroprotective activity [66]. Another cannabinoid that merits special mention is r,r-dimethylheptyl-A^-THC11-oic acid (DMH-A^-THC-11-oic acid). This is a cannabinoid receptor ligand with therapeutic potential as an anti-inflammatory/analgesic [3, 67]. In spite of its ability to interact with CBi and CB2 receptors [68], it remains possible that DMH-A^-THC-11oic acid produces its anti-inflammatory effects by suppressing eicosanoid synthesis in inflamed tissue through inhibition of inducible cyclooxygenase (COX-2), an enzyme that is thought to be activated during inflammation and to be responsible for the production of eicosanoids that act as inflammatory mediators [69]. DMH-A*-THC-11oic acid is more potent as an inhibitor of COX-2 than of COX-1 (constitutive cyclooxygenase), raising the possibility that it may be able to relieve inflammation without producing gastrointestinal or kidney toxicity [67, 69, 70]. It is unwanted effects of this kind that limit the clinical usefiibiess of less selective non-steroidal antiinflammatory drugs that inhibit COX-1 more or less as effectively as they inhibit COX2. 6. Future Clinical Research One challenge for future clinical research is to develop cannabinoid formulations and modes of administration that produce more reliable cannabinoid absorption than has so far been realisable, at least by the oral route. Possible solutions are to develop improved oral formulations or to use other routes for cannabinoid delivery, for example administration by rectal suppository [71], by aerosol/vapour inhalation, by injection, by skin patch or by the sublingual or intrathecal route, all modes of administration that avoid first-pass metabolism of the absorbed drug. Some success has ateady been achieved by GW Pharmaceuticals in phase I studies with a sub-lingual cannabinoid spray (personal communication from Dr. Geoffrey Guy). The emergence of better modes of cannabinoid administration should be facilitated by the development of a centrallyactive water-soluble cannabmoid [72]. The availability of better delivery systems fir cannabinoids should facilitate the gathering of more conclusive clinical data both about the efficacy of cannabinoids and about then- unwanted effects than has hitherto been achievable. Another important area for future clmical research must be the development of strategies that maxunize separation between the sought-after therapeutic effects of CBi receptor agonists and the unwanted effects of these drugs, particularly their psychotropic effects. One strategy may be to use agents that activate the endogenous cannabinoid system indirectly by increasing extracellular levels of endocannabinoids through inhibition of their membrane transport or enzymic hydrolysis. This approach is based on the expectation that drugs with this kind of action would be more selective than direct CBi receptor agonists. This is because they are unlikely to affect all parts of the endocannabinoid system at one time, producing instead effects only at sites where ongoing production of endocannabinoids is taking place. The success of this strategy depends on whether endogenous cannabinoids are released to a greater extent at sites at which they produce sought-after effects than at sites at which they provoke unwanted effects; In line with this possibility is evidence obtained from experiments using an autoimmune model of multiple sclerosis that is set up by mjecting Biozzi ABH mice
255 subcutaneously with an emulsion of mouse spinal cord homogenate in Freund's complete adjuvant. These showed that inhibitors of endocannabinoid membrane transport (AM404) or enzymic hydrolysis (AM374) share the ability of direct cannabinoid receptor agonists to ameliorate spasticity in CREAE mice and that spastic CREAE mice have elevated concentrations of the endocannabinoids anandamide and 2arachidonoyl glycerol in their brains and spinal cords [73]. There is also evidence fix)m animal experiments that peripheral inflammatory pain triggers increased release of anandamide in at least one area of the brain at which cannabinoids can induce signs of analgesia in animals, the periaqueductal grey area of the midbrain [74]. Because the unwanted central effects of cannabmoids are probably mediated largely or entirely by CBi receptors within the brain, a second strategy for reducing the unwanted effects of a CBi receptor agonist, at least for pain relief, might be to inject a CBi receptor agonist directly into the spinal cord. This approach exploits the presence of CBi receptors on nociceptive neurones within the spinal cord and also takes account of the ability of CBi receptor agonists to induce antinociception in anunals when these agents are administered intrathecally. The same strategy is sometimes adopted to reduce the incidence of adverse responses of multiple sclerosis patients to the anti-spasticity agent, baclofen. Yet another strategy would be to administer a cannabinoid in combination with a second agent that augments only the sought-after effects of the cannabinoid. Indeed, there is evidence fi-om animal experiments that synergistic interactions can occur between cannabinoids and opioids for analgesia [3, 75] and between cannabinoids and benzodiazepines for depressant effects on motor function [76, 77]. As there are claims by multiple sclerosis patients that cannabis can relieve their symptoms at dose levels that do not induce a 'high', another strategy may be to admmister an agonist (partial agonist) with a reduced ability (efficacy) to activate CBi receptors. This approach assumes that it should be possible to develop a partial agonist that has sufficient efficacy to relieve muscle spasticity/spasm and pam but insufficient efficacy to produce a full range of cannabhnimetic psychotropic effects even when it occupies all available CBi receptors. A possible lead compound is 0-1184 (see section 4). 7. References 1. Pertwee, R.G., 1988, Pharmacol. Ther., 36, 189. 2. Pertwee, R.G., 1997, Pharmacol. Then, 74, 129. 3. Pertwee, R.G., 2001, Prog. NeurobioL, 63, 569. 4. Molina-Holgado, E., Guaza, C , Borrell, J. and Molina-Holgado, F., 1999, Biodrugs, 12, 317. 5. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R. and Rice, K.C., 1991, J. Neurosci., 11, 563. 6. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R., 1992, Science, 258, 1946. 7. Di Marzo, V., Melck, D., Bisogno, T. and De Petrocellis, L., 1998, Trends Neurosci., 21, 521. 8. Mechoulam, R., Fride, E. and Di Marzo, V., 1998, Eur. J. Pharmacol., 359, 1.
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