Since the discovery of the first cannabinoid receptor 12 years ago1,2, important advances have been made in several areas of cannabinoid pharmacology. Endocannabinoid compounds and their pathways of biosynthesis and inactivation have been identified, and the molecular structures and anatomical distribution of cannabinoid receptors have been investigated in detail. Pharmacological agents that interfere with various aspects of the endocannabinoid system have been developed, and pathophysiological circumstances in which this system might be active have begun to emerge. The manner in which these discoveries might impact our understanding of endocannabinoid signaling and help unlock its potential for developing novel therapeutic agents will be discussed.The two endocannabinoids isolated so far – anandamide and 2-arachidonylglycerol 3–5 – are lipid in nature but differ from amino acid, amine and peptide transmitters in ways other than just their chemical structures. Classical and peptide transmitters are synthesized in the cytosol of neurons and stored in synaptic vesicles, from where they are secreted by exocytosis following excitation of nerve terminals by action potentials. By contrast, anandamide and 2-AG can be produced upon demand by receptor-stimulated cleavage of membrane lipid precursors and released from cells immediately after their production. Anandamide can be produced from the hydrolysis of an N-acylated species of phosphatidylethanolamine N-arachidonyl PE,growers equipment a process catalysed by phospholipase D. The stimulation of neurotransmitter receptors appears to play a determinant role in initiating this reaction, as indicated by the finding that anandamide release in the striatum is strongly enhanced by activation of dopamine D2 receptors.
Once released, anandamide can act on cannabinoid receptors or accumulate back into cells via an energy- and Na1 -independent transport system. The selectivity of this system for anandamide has been documented but its molecular structure remains uncharacterized. Inside cells, anandamide can be catalytically hydrolysed by an amidohydrolase, whose gene has been cloned. The most likely route of 2-AG biosynthesis involves the same enzymatic cascade that catalyses the formation of the second messengers inositol -trisphosphate and 1,2- diacylglycerol . Phospholipase C , acting on phosphatidylinositol -bisphosphate, generates DAG, which is converted to 2-AG by DAG lipase.Regardless of the mechanism involved, 2-AG formation can be triggered by neural activity or by occupation of membrane receptors. Following its release, 2-AG can be taken up by cells via the anandamide transport system and hydrolysed by an unknown monoacylglycerol lipase activity. Thus, anandamide and 2-AG can be released from neuronal and non-neuronal cells when the need arises, utilizing analogous but distinct receptor-dependent pathways. The nonsynaptic release mechanisms and short life spans of anandamide and 2-AG suggest that these compounds might act near their site of synthesis to regulate the effects of primary messengers, such as neurotransmitters and hormones.Drugs that block the formation or inactivation of anandamide and 2-AG should help identify the physiological functions of these compounds and might be beneficial in disease states in which regulation of endocannabinoid levels might produce more selective responses than those elicited by cannabinoid receptor ligands. Although this area of pharmacology is still largely unexplored, inhibitors of the two main steps of anandamide disposition have recently become available.
Anandamide transport is inhibited by the compound AM404 . This drug potentiates various responses elicited by exogenous anandamide and interacts very poorly with cannabinoid CB1 receptors For example, AM404 enhances anandamide-induced hypotension without producing direct vasodilatory effects. Furthermore, when applied alone, AM404 decreases motor activity and elevates the levels of circulating anandamide . However, AM404 can accumulate in cells where it might reach concentrations that are sufficient to inhibit anandamide amidohydrolase. Anandamide amidohydrolase is blocked reversibly by transition state analogs such as arachidonyltrifluoromethylketone , which might act by forming a stable intermediate with a serine residue at the enzyme active site18 . Moreover, irreversible inhibition can be achieved with a variety of compounds including the fatty acid sulfonyl fluoride AM374 . AM374, one of the most potent anandamide amidohydrolase inhibitors identified thus far, potentiates anandamide responses in vitro and in vivo, but its specificity is limited by a relatively high affinity for CB1 receptors.The two cannabinoid receptor sub-types characterized so far, CB1 and CB2, belong to the super family of G-protein-coupled membrane receptors. Their molecular and pharmacological properties have recently been reviewed21. Three issues that might be relevant to the use of cannabinoid agents in medicine will be discussed: the apparently exclusive role of CB1 receptors in mediating central cannabinoid effects; the rapid tolerance that results from repeated cannabinoid administration; and the possible existence of multiple cannabinoid receptors in peripheral tissues. Although CB1 receptors are expressed throughout the body, they are particularly abundant in the CNS where, despite a great deal of effort, no other cannabinoid receptor sub-type has yet been found. This unusual situation – most neurotransmitters act on multiple CNS receptors – accords with data that indicate that a single pharmacological site accounts for all central effects of cannabimimetic drugs, whether therapeutically favorable or harmful . Consequently, although potent CB1 receptor agonists have been available for some time , the therapeutic development of these compounds has been very limited. Given this situation, how might centrally active cannabinoid agents that are more selective than those currently available be developed? One possibility is to target the mechanisms of endocannabinoid inactivation.
Blocking such mechanisms might cause an activity-dependent accumulation of anandamide and 2-AG at their sites of release, which might in turn result in a more localized activation of cannabinoid receptors than that elicited by direct receptor agonists. Another important issue that should be considered in the development of cannabinoid agonists for therapeutic use is receptor desensitization. This process, which might be mediated by the GPCR-kinase–b -arrestin pathway, causes a pharmacological tolerance that limits the prolonged use of cannabinoid receptor agonists. Partial agonists might offer a clue as to how to circumvent this obstacle. Evidence indicates that the CNS contains a large reserve of CB1 receptors; thus partial CB1 receptor agonists, which are expected to cause less receptor desensitization than full agonists, might produce adequate therapeutic responses with diminished tolerance liability. Although CB1 receptors are thought to mediate the effects of cannabinoid receptor agonists in the CNS, several peripheral effects of cannabimimetic drugs might only depend partially on CB1 receptor activation. The high expression of CB2 receptors in B cells and natural killer cells suggests that this sub-type contributes to the potential immuno suppressant and anti-inflammatory effects of cannabinoids. Additional tests of this hypothesis will be facilitated by the recent availability of selective CB2 receptor agonists and antagonists .Furthermore, cannabinoid-like receptors that are distinct from the CB1 and CB2 sub-types might participate in the vasodilatatory and analgesic effects of cannabinoids. Although the hypotensive actions of anandamide are mostly mediated by CB1 receptors, the endothelium-dependent vasorelaxation produced by this compound in mesenteric arteries appears to require a receptor that is pharmacologically distinct from CB1 and CB2 . Furthermore, the peripheral analgesic effects exerted by the endogenous anandamide analog palmitylethanolamide might also involve a novel cannabinoidlike receptor . In addition to acting on cannabinoid receptors, anandamide has been suggested to act on a variety of other targets, including capsaicin receptors. The concentrations required to attain these effects are, however, too high to be considered physiologically relevant and claims that vascular effects of anandamide might be mediated by vanilloid receptors appear unwarranted.The endocannabinoid system might serve important regulatory functions in physiological processes; thus, cannabinoid agents might prove useful in the treatment of pathological conditions that are associated with such processes. Exhaustive evaluations of the medicinal potential of cannabis and its derivatives in other therapeutic areas can be found elsewhere.Cannabinoid drugs strongly reduce pain responses by interacting with CB1 receptors in brain, spinal cord and peripheral sensory neurons . Brain sites that participate in cannabinoid-induced analgesia include the amygdala, thalamus, superior colliculus, periaqueductal gray and rostral ventromedial medulla. In the spinal cord, CB1 receptors are found in the dorsal horn and lamina X ,plant benches where they are located on intrinsic spinal neurons, nerve terminals of afferent sensory neurons and terminals of efferent supraspinal neurons. CB1 receptors are also expressed in the dorsal root ganglia by a subset of small- and large-diameter sensory neurons that contain the pain-stimulating peptides, substance P and a-calcitonin gene-related peptide. Although quantitatively small, the presence of CB1 receptors on CGRP containing neurons appears to be functionally significant because CB1 receptor agonists effectively reduce CGRP release from dorsal horn tissue. Immunohisto chemical experiments suggest that CB1 receptors are present not only on central terminals of primary sensory afferents, but also on their peripheral counterparts. In agreement with these findings, local applications of CB1 receptor agonists to skin reduce the responses to formalin and other irritants. The clinical impact of these advances is still modest but worth noting. Since a previous literature review, new studies have documented the analgesic effects of CB1 receptor agonists in humans , providing additional impetus for a re-evaluation of the endocannabinoid system as a target for analgesic drugs.Cannabinoids are potent in alleviating two hallmarks of neuropathic pain: allodynia and hyperalgesia. Indeed, in a rat model of neuropathic pain , the CB1 receptor agonist WIN552122 attenuates such responses at doses that do not cause overt side-effects. In this model, the CB1 receptor antagonist SR141716A reverses the analgesic response to WIN552122 and exacerbates pain behaviors when administered alone. One possible explanation for the pain-inducing effects of SR141716A is that nerve injury might be associated with an increase in endocannabinoid levels and/or a sensitization of CB1 receptors. Plastic modifications in endocannabinoid signaling during persistent pain can also be inferred from experiments conducted in a rat model of inflammation. In this model, SR141716A enhances the sensitivity to mechanical stimuli applied to the paw contralateral to the inflammatory focus, which suggests that inflammation can be accompanied by an increased cannabinergic activity that can be unmasked by the CB1 receptor antagonist. Furthermore, the peripheral administration of formalin stimulates anandamide release in the periaqueductal gray, a brain region involved in pain control. Whether CB1 receptor function and/or endocannabinoid levels are changed in neuropathic pain is unknown. If this syndrome is accompanied by a hypersensitivity of CB1 receptors in injured tissues, partial CB1 receptor agonists could alleviate pain at doses that might exert few undesirable effects and produce little tolerance. By contrast, if neuropathic pain is associated with elevated endocannabinoid release, drugs that interfere with the inactivation of these substances might offer an alternative to direct CB1 receptor agonists.
Elucidating the alterations in endocannabinoid function associated with neuropathic pain should be instrumental to define the value of these strategies.The finding that cannabinoid receptor agonists can alleviate pain by acting at peripheral CB1 receptors has both theoretical and practical ramifications. Theoretically, this observation emphasizes the notion that nociceptive signals can be modulated at the first stage of neural processing by a peripheral ‘gate’ mechanism in which endogenous cannabinoid lipids can act in concert with opioid peptides. Practically, it points to the possibility of achieving an effective control of peripheral pain without causing the psychotropic effects that follow the recruitment of brain CB1 receptors. The antinociceptive effects of palmitylethanolamide add a new dimension to this hypothesis. Palmitylethanolamide is produced in tissues through an enzymatic route similar to that of anandamide synthesis6. When administered as a drug, palmitylethanolamide potently reduces peripheral pain through a mechanism that is synergistic with anandamide and is blocked by the CB2 receptor antagonist SR144528 . However, palmitylethanolamide does not interact with the CB2 receptor , which suggests that the compound might produce its analgesic effects by activating an as-yet uncharacterized CB2-like receptor.CB1 receptors are densely expressed in the basal ganglia and cortex, CNS regions that are critical for movement control21. This distribution provides an anatomical substrate for functional interactions between the endocannabinoid system and ascending dopaminergic pathways. Several observations suggest that these interactions might indeed occur. First, in the striatum of freely moving rats anandamide release is stimulated by activation of dopamine D2 receptors. Second, the CB1 receptor antagonist SR141716A, which has little effect on motor activity when administered alone, potentiates the motor hyperactivity produced by the D2 receptor agonist quinpirole. Third, D2 and CB1 receptor agonists produce opposing behavioral responses after injection into the basal ganglia. These and other findings suggest that anandamide might modulate dopamine-induced facilitation of psychomotor activity. In further support of this hypothesis, disruption of the gene encoding the CB1 receptor profoundly affects motor control, decreasing locomotor activity.