The fact that the cannabinoid antagonist SR141716A prevents these effects suggests that AM404 may act by preventing anandamide inactivation and enhancing its interactions with cannabinoid receptors. Importantly, however, AM404 also can be transported inside cells , where it may reach levels that are sufficient to inhibit anandamide degradation by FAAH . The target selectivity of AM404 has been investigated in some detail. Initial studies showed that AM404 has no affinity for a panel of 36 potential targets, including G protein-coupled receptors, ligand-gated channels, and voltage-dependent channels . Subsequent work suggested, however, that AM404 may activate the capsaicin sensitive VR1 vanilloid receptor in vitro . It is unlikely that this effect occurs in vivo, since AM404 does not display any of the pharmacological properties of a vanilloid agonist . Yet, these findings underscore the need to design novel inhibitors of anandamide transport endowed with greater target selectivity. Ongoing research efforts in this direction have led to the development of several arachidonic acid derivatives that are equivalent or slightly superior to AM404 in inhibiting anandamide transport in vitro and in vivo, with effects similar to those of AM404 . Consistent with its low affinity for CB1 receptors, AM404 does not act as a direct cannabinoid agonist when administered to live animals. The compound has no antinociceptive effects in the mouse hot-plate test and does not reduce arterial blood pressure in the urethaneanesthetized guinea pig . In the same models, however, AM404 magnifies the responses elicited by exogenous anandamide, actions that are prevented by the CB1 antagonist SR141716A . Furthermore, when administered alone, AM404 reduces motor activity , attenuates apomorphine-induced yawning , decreases the levels of circulating prolactin , and alleviates the motor hyperactivity induced in the rat by striatal 3-nitropropionic acid lesions . These actions resemble those of anandamide and are blocked by SR141716A ,growing cannabis outdoors suggesting that endogenous anandamide may be involved. In keeping with this notion, systemic administration of AM404 in the rat causes a time-dependent increase in circulating anandamide levels .
The participation of anandamide in the effects of AM404 in vivo has been questioned based on the ability of this compound to interact with vanilloid receptors in vitro . Yet, the fact that SR141716A blocks the motor inhibitory actions of AM404 at doses that are selective for CB1 receptors strongly argues for a predominant, if not unique, role of the endocannabinoid system in the behavioral response to AM404 administration. Furthermore, the pharmacological properties of AM404 are very different, often opposite to those of capsaicin and other vanilloid agonists. For example, capsaicin produces pain and bronchial smooth muscle constriction , whereas AM404 has no such effect when administered alone, and in fact enhances anandamide’s analgesic and bronchodilatory actions . The ability of intraperitoneal capsaicin to inhibit movement, described by Di Marzo et al. , superficially mimics one property of AM404, but should be viewed with caution, as it most likely results from the strong visceral pain and subsequent “freezing response” elicited by capsaicin. In conclusion, current evidence suggests that AM404 may magnify the actions of anandamide primarily by inhibiting the clearance of this compound from its sites of action.Almost a decade before anandamide was discovered, Schmid and collaborators identified a hydrolase activity in rat liver that catalyzes the hydrolysis of fatty acid ethanolamides to free fatty acid and ethanolamine . That anandamide may be a substrate for such an activity was first suggested by biochemical experiments and then demonstrated by molecular cloning, heterologous expression, and genetic disruption of the enzyme involved . FAAH is an intracellular membrane-bound protein whose primary structure displays significant homology with the “amidase signature family” of enzymes . It acts as a hydrolytic enzyme for fatty acid ethanolamides such as anandamide, but also for esters such as 2-AG and primary amides such as oleamide . Site-directed mutagenesis experiments indicate that this unusually wide substrate preference may be due to a novel catalytic mechanism involving the amino acid residue lysine-142. This residue may act as a general acid catalyst, favoring the protonation and consequent detachment of reaction products from the enzyme’s active site . This mechanism was recently confirmed by the solution of the crystal structure of FAAH complexed with the active site-directed inhibitor methoxy arachidonyl fluorophosphonate .
In addition to FAAH, other enzymes may participate in the breakdown of anandamide and its fatty acid ethanolamide analogs. A PEA-hydrolyzing activity distinct from FAAH was described in rat brain membranes and human megakaryoblastic cells . This activity was purified to homogeneity from rat lung and shown to possess a marked substrate preference for PEA over anandamide . PEA does not bind to any of the known cannabinoid receptors but produces profound analgesic and anti-inflammatory effects , which are prevented by the CB2-preferring antagonist SR144528 . Future studies will undoubtedly address the relative roles of FAAH and this newly discovered enzyme in the biological disposition of PEA and anandamide. The ability of FAAH to act in reverse has generated some confusion as to the mechanism of anandamide formation. Early reports of anandamide synthesis from free arachidonate and ethanolamine have now been unambiguously attributed to the reverse of the FAAH reaction . Because high concentrations of arachidonic acid and ethanolamine are needed to drive FAAH to work in reverse, it is unlikely that this reaction plays a physiological role in anandamide generation . One possible exception is represented by the rat uterus, where substrate concentrations in the micromolar range are required for the synthetic reaction to occur, implying that FAAH or a similar enzyme might contribute to anandamide biosynthesis in this tissue .Systematic structure-activity relationship investigations have identified several general requisites for substrate recognition by FAAH. First, FAAH accommodates a wide range of fatty acid amide substrates, but reducing the number of double bonds in the fatty acid chain generally results in a decrease in hydrolysis rate . Second, replacing the ethanolamine moiety with a primary amide yields excellent substrates. For example, the rate of hydrolysis of arachidonamide is two to three times greater than anandamide’s . Third, anandamide congeners containing a tertiary nitrogen in the ethanolamine moiety are poor substrates . Fourth,introduction of a methyl group at the C2, C1, or C2 positions of anandamide yields analogs that are resistant to hydrolysis, probably due to increased steric hindrance around the carbonyl group . Fifth, substrate recognition at the FAAH active site is stereoselective, at least with fatty acid ethanolamide congeners containing a methyl group in the C1 or C2 positions .
Finally, fatty acid esters such as 2-AG also are excellent substrates for FAAH activity in vitro .Early biochemical experiments showed that FAAH activity is abundant throughout the CNS, with particularly high levels in the neocortex,mobile vertical rack the hippocampus, and the basal ganglia . Subsequent investigations have confirmed this wide distribution. Thus, in situ hybridization studies in the rat have found that FAAH mRNA expression is higher in the neocortex and hippocampus; intermediate in the cerebellum, thalamus, olfactory bulb, and striatum; and lower in the hypothalamus, brain stem, and pituitary gland . Immuno histochemical experiments suggest that large principal neurons in the cerebral cortex, hippocampus, cerebellum, and olfactory bulb have the highest levels of FAAH immunoreactivity . For example, large pyramidal neurons in the neocortex are prominently stained together with their apical and basal dendrites in layer V . Moderate immunostaining is observed also in the amygdala, the basal ganglia, the ventral and posterior thalamus, the deep cerebellar nuclei, the superior colliculus, the red nucleus, and motor neurons of the spinal cord . A more recent study reported staining of principal cells and astrocytes in various regions of the human brain . However, the protein recognized by the antibody utilized in these experiments has an apparent molecular mass of 50 kDa , which does not correspond to that of native FAAH . Many FAAH-positive neurons throughout the brain are found in close proximity to axon terminals that contain CB1 cannabinoid receptors , providing important evidence for a role of FAAH in anandamide deactivation. Yet, there are multiple other regions of the brain where there is no such correlation, a discrepancy that likely reflects the participation of FAAH in the catabolism of other bio-active fatty acid ethanolamides, such as OEA and PEA .A number of inhibitors of anandamide hydrolysis have been described, including fatty acid trifluoromethylketones, fluorophosphonates, -keto esters and -keto amides , bromoenol lactones , and nonsteroidal anti-inflflammatory drugs . These compounds lack, in general, target selectivity and biological availability; thus attempts to use them in vivo should be interpreted with caution.The first are fatty acid sulfonyl flfluorides, such as palmitylsulfonylfluoride . AM374 irreversibly inhibits FAAH activity with an IC50 of 10 nM and displays a 50-fold preference for FAAH inhibition versus CB1 receptor binding . Systemic administration of AM374 enhances the operant lever-pressing response evoked by anandamide administration, but exerts no overt behavioral effect per se , raising the possibility that AM374 may protect anandamide from peripheral metabolism but may not have access to the brain. The second group of FAAH inhibitors is represented by a series of substituted -keto-oxazolopyridines, which are both reversible and extremely potent , but whose pharmacological selectivity and in vivo properties are not yet known. The third group is constituted by a class of aryl-substituted carbamate derivatives . The most potent member of this class, the compound URB597, inhibits FAAH activity with an IC50 value of 4 nM in brain membranes and an ID50 value of 0.1 mg/kg in live rats.
This compound has 25,000-fold greater selectivity for FAAH than cannabinoid receptors, which is matched by an apparent lack of cannabimimetic effects in vivo . The pharmacological profile of URB597, which is currently under investigation, includes profound antianxiety effects accompanied by modest analgesia .The generation of mutant mice in which the faah gene was disrupted by homologous recombination has shed much light on the role of FAAH in anandamide inactivation . FAAH /_x0005_ mice cannot metabolize anandamide and are therefore extremely sensitive to the pharmacological effects of this compound: doses of anandamide that are inactive in wild-type mice exert profound cannabimimetic effects in these mutants. FAAH /_x0005_ mice also have markedly elevated brain anandamide levels and reduced nociception . This finding is consistent with the roles of anandamide in the modulation of pain sensation and is supported by the analgesic activity of FAAH inhibitors . Recently, a single nucleotide polymorphism in the human gene encoding for FAAH, which produces a proteolysis-sensitive variant of the enzyme, was found to be strongly associated with street drug and alcohol abuse . This important observation reinforces the central role played by the endocannabinoid system in the control of motivation and reward .The fact that FAAH catalyzes the hydrolysis of 2-AG along with anandamide’s has prompted the suggestion that this enzyme may be responsible for eliminating both endocannabinoids. There is, however, strong evidence against this hypothesis. First, inhibitors of FAAH activity have no effect on [3 H]2-AG hydrolysis at concentrations that completely block anandamide degradation . Second, 2-AG hydrolysis is preserved in mutant FAAH /_x0005_ mice, which do not degrade either endogenous or exogenous anandamide . In agreement with these results, a 2-AG hydrolase activity distinct from FAAH has been identified and partially purified from porcine brain . This activity likely corresponds to monoglyceride lipase , a cytosolic serine hydrolase that converts 2- and 1-monoglycerides to fatty acid and glycerol . Several findings support this conclusion . First, heterologous expression of rat brain MGL confers strong 2-AG-hydrolyzing activity and MGL immunoreactivity to HeLa cells. Second, adenovirus mediated transfer of the MGL gene in intact neurons increases MGL expression and shortens the life span of endogenously produced 2-AG, without any effect on either 2-AG synthesis or anandamide degradation. Third, MGL mRNA and protein are discretely distributed in the rat brain, with highest levels in regions where CB1 receptors are also present . The distribution of MGL in the rat hippocampus is particularly noteworthy. The high density of MGL immunore activity in the termination zones of the glutamatergic Schaffer collaterals suggests a presynaptic localization of this enzyme at CA3-CA1 synapses.