The results of this study, which have been confirmed in several subsequent reports , demarcate the pharmacological profile of AM404 from those of direct-acting cannabinoid drugs. This distinction may result from the ability of AM404 to enhance anandamide signaling in an activity-dependent manner, causing anandamide to accumulate in discrete regions of the brain and only when appropriate stimuli initiate its release. Pharmacological activation of D2 receptors may represent one such stimulus, suggesting that blockade of anandamide transport might offer an innovative strategy to correct abnormalities associated with dysfunction in dopaminergic transmission. Initial tests of this hypothesis have shown that systemic administration of AM404 normalizes movement in spontaneously hypertensive rats , an inbred line in which hyperactivity and attention deficits have been linked to a defective regulation of mesocorticolimbic dopamine pathways .FAAH was first identified as an amide hydrolase activity present in rat liver tissue, which catalyzes the hydrolysis of the fatty-acid ethanolamides palmitoylethanolamide and oleoylethanolamide . That anandamide serves as a substrate for this activity was first suggested on the basis of biochemical evidence and later demonstrated by molecular cloning, heterologous expression and generation of FAAHnull mice by homologous recombination . FAAH belongs to a group of enzymes known as ‘amidase signature family’ and catalyzes the hydrolysis not only of anandamide and other fatty-acid ethanolamides,vertical cannabis but also of primary amides such as oleamide and even of fatty-acid esters such as 2-AG . Elegant site-directed mutagenesis and X-ray diffraction studies have demonstrated that this unusually broad substrate preference is 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 .
Three serine residues that are conserved in all amidase signature enzymes also may be essential for enzymatic activity: serine 241 may serve as the enzyme’s catalytic nucleophile, while serine 217 and 218 may modulate catalysis through an as-yet-unidentified mechanism . Electron microscopy experiments in the rat and mouse brain have shown that FAAH is predominantly, if not exclusively localized to intracellular membrane compartments, particularly to the endoplasmic reticulum and the mitochondria . Although FAAH appears to be the predominant route of anandamide hydrolysis in the brain, other enzymes are likely to participate in the breakdown of this endocannabinoid in peripheral tissues. An acid amide hydrolase activity catalytically distinct from FAAH has been characterized in human megakaryoblastic cells and shown to be highly expressed in the rat thymus, lungs and intestine .The search for small-molecule inhibitors of intracellular FAAH activity has led to the emergence of several potent and selective agents, which include substituted sulfonyl fluorides , alpha-keto-oxazolopyridines an d carbamic acid esters . The latter were identified during structure– activity relationship studies aimed at determining whether esters of carbamic acid such as the insecticide carbaryl inhibit FAAH activity. It was found that, although carbaryl is ineffective in this regard, variations in its template result in significant inhibitory potencies. Further structural optimizations yielded a group of highly potent inhibitors, a representative example of which is provided by the compound URB597 . Kinetic and dialysis experiments indicate that URB597 interacts non-competitively with FAAH, which is suggestive of anirreversible or slowly reversible association with the enzyme. Importantly, URB597 has no notable effect on CB1 or CB2 binding, anandamide transport, or rat brain monoglyceride lipase , a cytosolic serine hydrolase that catalyzes the hydrolysis of the second endocannabinoid, 2-arachidonoylglycerol . Following administration to rats in vivo, URB597 produces profound, dose-dependent inhibition of brain FAAH activity.
After injection of a maximal dose of compound , FAAH inhibition is rapid , persistent and associated with a 3-fold increase in brain anandamide levels. Furthermore, the inhibitor intensifies and prolongs the effects produced by exogenous anandamide, yet it elicits no overt cannabinoid-like actions when administered alone; for example, it does not cause hypothermia, hot-plate analgesia, or hyperphagia .Although URB597 does not display a typical cannabinoid profile in live animals, it exerts several pharmacological effects that might be therapeutically relevant. One such effect, the ability to reduce anxiety-like behaviors in rats, was demonstrated in two distinct experimental models: the elevated ‘zero maze’ test, and the isolation-induced ultrasonic emission test . The ‘zero maze’ consists of an elevated annular platform with two open and two closed quadrants and is based on the conflict between an animal’s instinct to explore its environment and its fear of open spaces where it may be attacked by predators . Benzodiazepines and other clinically used anxiolytic drugs increase the proportion of time spent in, and the number of entries made into, the open compartments. In a similar fashion, URB597 elicits anxiolytic-like responses at a dose that corresponds to those required to inhibit brain FAAH activity. Moreover, these effects are prevented by the CB1-selective antagonist rimonabant. Analogous results were obtained in the ultrasonic vocalizationemission test, which measures the number of stress-induced vocalizations emitted by rat pups removed from their nest . If con- firmed in further behavioral models, these findings would suggest that inhibition of intracellular FAAH activity might offer an innovative target for the treatment of anxiety , which is also a feature of marijuana withdrawal .2-AG was identified as a second endocannabinoid substance in 1995 . The multiple roles of this lipid compound incell metabolism and its high levels inbraintissue— about 200-fold higher than those of anandamide—suggest that much of cellular 2-AG may be involved in housekeeping functions. The diversity of roles played by this compound also complicates our efforts to establish biochemical route involved in its physiological formation. Nevertheless, one pathway has emerged as the most likely candidate .
This pathway starts with the phospholipase-mediated generation of 1, 2- diacylglycerol . This serves as a substrate for two enzymes: DAG kinase, which catalyzes DAG phosphorylationto phosphatidic acid; and DAG lipase , which hydrolyzes DAG to monoacylglycerol . Pharmacological inhibition of phospholipase C and DGL prevent the Ca2+-dependent accumulation of 2-AG in rat cortical neurons, which suggests a key role of this pathway in2-AG generation . However, additional routes of 2-AG synthesis also may exist, including phospholipase A1 ,cannabis drying racks hormone-sensitive lipase or a lipid phosphatase . In neurons and glia, 2-AG synthesis may be initiated by a rise incytosolic Ca2+ levels. For example, incultures of rat cortical neurons, the Ca2+ ionophore ionomycin and the glutamate receptor agonist N-methyld-aspartate stimulate 2-AG productionina Ca2+-dependent manner . Similarly, infreshly dissected hippocampal slices, electrical stimulation of the Schaffer collaterals, a glutamatergic fiber tract that connects neurons in the CA3 and CA1 fields, causes a Ca2+-dependent increase in 2-AG content . This stimulation has no effect on the levels of non-cannabinoid monoacylglycerols, such as 1-palmitoylglycerol, which indicates that 2-AG formation may not be attributed to a broad, non-specific increase in lipid turnover. Furthermore, electrical stimulationof the Scheffer collaterals does not modify hippocampal anandamide levels, suggesting that the biochemical pathways leading to the production of 2-AG and anandamide may be independently controlled . In further support of this idea, activation of D2 receptors, a potent stimulus for anandamide formation in the rat striatum, has no effect on striatal 2-AG levels .Neuronal and glial cells internalize 2-AG through a mechanism apparently similar to that implicated in anandamide transport. Thus, human astrocytoma and other tumor cells accumulate [3 H]anandamide and [ 3 H]2-AG with similar kinetic properties and this process is blocked by the anandamide transport inhibitor AM404 . In addition, anandamide and 2-AG prevent each other’s transport . Nevertheless, there also appear to be differences between anandamide and 2-AG accumulation. For example, [3 H]2-AG internalization in astrocytoma cells is reduced by exogenous arachidonic acid, whereas [3 H]anandamide internalization is not. This discrepancy may be explained in two ways: arachidonic acid may directly interfere with a 2- AG carrier distinct from anandamide’s; or the fatty acid may indirectly prevent the facilitated diffusion of 2-AG by inhibiting its enzymatic conversion to arachidonic acid. If the latter explanation is correct, agents that interfere with the arachidonic acid esterification into phospholipids, such as triacsinC , should decrease [ 3 H]2-AG uptake. This was found indeed to be the case, at least inastrocytoma cells . Thus, while anandamide and 2-AG may be internalized through similar transport mechanisms, they appear to differ in how their intracellular breakdown can affect the rate of transport into cells.After removal from the external medium, 2-AG is hydrolyzed to arachidonic acid and glycerol.
In cellfree preparations, FAAH cleaves anandamide and 2- AG at similar rates, which has led to suggest that this enzyme may contribute to the elimination of both compounds. This appears to be unlikely, however, for three reasons. First, pig brain tissue contains two distinct 2- AG-hydrolase activities, both of which are chromatographically different from FAAH . Second, inhibition of FAAH activity in intact neurons and astrocytoma cells prevents the hydrolysis of anandamide, but has no effect on 2-AG degradation . Finally, 2-AG hydrolysis is entirely preserved in FAAH-null mice . These findings suggest that, although 2-AG can be hydrolyzed by FAAH in vitro, different enzyme may be responsible for its degradation in vivo. A possible candidate for this role is MGL, a cytosolic serine hydrolase that cleaves 2- and 1-monoglycerides into fatty acid and glycerol .MGL is abundantly expressed in discrete areas of the rat brain—including the hippocampus, cortex, and cerebellum—where CB1 receptors are also found. Moreover, adenovirus-induced over expression of MGL enhances the hydrolysis of endogenously produced 2-AG in primary cultures of rat brain neurons . Finally, recent experiments indicate that silencing the MGL gene through RNA interference markedly impairs 2-AG degradation in intact HeLa cells . Although these results strongly support a role of MGL in2-AG hydrolysis, the development of additional experimental tools will be needed to demonstrate such a role unambiguously.Currently, in the United States, there are more annual deaths and disabilities from substance abuse than from any other preventable health condition . A robust body of literature suggests that prevention interventions that delay and minimize marijuana and other substance use during childhood and adolescence could have a considerable impact on morbidity and mortality in addition to reducing associated social and economic costs . Adolescent substance use is also a problem that is ideal for intervention within a public health framework, as it is both widespread and amenable to population-level approaches designed to reduce youth exposure to harmful influences. While various community-level prevention approaches have been implemented to protect underage youth from exposure to factors that encourage them to use alcohol and drugs at an age when substance use can seriously disrupt their social, physiological, and emotional development , an issue addressed in this dissertation is whether policies that restrict access to marijuana by adolescents enacted at the local level have any measurable impact. One important question is whether restrictions on marijuana have any impact on youth use in the context of high vehicle ownership, marijuana delivery services, and contrasting marijuana regulations between neighboring cities. The research literature shows that local regulations on legal substances can have a significant, if not always dramatic, effect . Given the large numbers of people impacted, if city policies cause even an incremental reduction in substance use it can have a qualitatively larger impact on a community than more intensive interventions carried out with less people . Throughout the United States , marijuana policy has been changing rapidly and following a consistent national trend of less restrictive state laws controlling access to marijuana . In an enduring legal paradox, the marijuana plant and its products remain illegal under U.S. Federal law, but the Federal government has also allowed regulations on marijuana to be determined by each state independently and these laws now contrast with Federal marijuana law in the majority of U.S. states. California’s marijuana laws are among the most permissive in the U.S., allowing for home delivery of psychoactive marijuana products, storefront medical and recreational marijuana outlets, and no limits on the THC potency of products sold . An important feature of California’s state marijuana laws, however, is that they do not preempt local regulations, meaning that local jurisdictions like cities and counties have the prerogative to enact local ordinances that further restrict access to marijuana within their borders.