Joel E. Schlosburg

Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-AG, is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, intimating a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL, JZL184, that, upon administration to mice, raises brain 2-AG by 8-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia, and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system

Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.

Corticosteroid-Dependent Plasticity Mediates Compulsive Alcohol Drinking in Rats

Alcoholism is characterized by a compulsion to seek and ingest alcohol, loss of control over intake, and the emergence of a negative emotional state during abstinence. We hypothesized that sustained activation of neuroendocrine stress systems (e.g., corticosteroid release via the hypothalamic-pituitary-adrenal axis) by alcohol intoxication and withdrawal and consequent alterations in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation drive compulsive alcohol drinking. Our results showed that rats exposed to alcohol vapor to the point of dependence displayed increased alcohol intake, compulsive drinking measured by progressive-ratio responding, and persistent alcohol consumption despite punishment, assessed by adding quinine to the alcohol solution, compared with control rats that were not exposed to alcohol vapor. No group differences were observed in the self-administration of saccharin-sweetened water. Acute alcohol withdrawal was accompanied by downregulated GR mRNA in various stress/reward-related brain regions [i.e., prefrontal cortex, nucleus accumbens (NAc), and bed nucleus of the stria terminalis (BNST)], whereas protracted alcohol abstinence was accompanied by upregulated GR mRNA in the NAc core, ventral BNST, and central nucleus of the amygdala. No significant alterations in MR mRNA levels were found. Chronic GR antagonism with mifepristone (RU38486) prevented the escalation of alcohol intake and compulsive responding induced by chronic, intermittent alcohol vapor exposure. Chronic treatment with mifepristone also blocked escalated alcohol drinking and compulsive responding during protracted abstinence. Thus, the GR system appears to be involved in the development of alcohol dependence and may represent a potential pharmacological target for the treatment of alcoholism.

Targeting Fatty Acid Amide Hydrolase (FAAH) to Treat Pain and Inflammation

The endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA) produces most of its pharmacological effects by binding and activating CB1 and CB2 cannabinoid receptors within the CNS and periphery. However, the actions of AEA are short lived because of its rapid catabolism by fatty acid amide hydrolase (FAAH). Indeed, FAAH knockout mice as well as animals treated with FAAH inhibitors are severely impaired in their ability to hydrolyze AEA as well as a variety of noncannabinoid lipid signaling molecules and consequently possess greatly elevated levels of these endogenous ligands. In this mini review, we describe recent research that has investigated the functional consequences of inhibiting this enzyme in a wide range of animal models of inflammatory and neuropathic pain states. FAAH-compromised animals reliably display antinociceptive and anti-inflammatory phenotypes with a similar efficacy as direct-acting cannabinoid receptor agonists, such as Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa. Importantly, FAAH blockade does not elicit any apparent psychomimetic effects associated with THC or produce reinforcing effects that are predictive of human drug abuse. The beneficial effects caused by FAAH blockade in these models are predominantly mediated through the activation of CB1 and/or CB2 receptors, though noncannabinoid mechanisms of actions can also play contributory or even primary roles. Collectively, the current body of scientific literature suggests that activating the endogenous cannabinoid system by targeting FAAH is a promising strategy to treat pain and inflammation but lacks untoward side effects typically associated with Cannabis sativa.

Blockade of Endocannabinoid Hydrolytic Enzymes Attenuates Precipitated Opioid Withdrawal Symptoms in Mice

Δ9-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB1 and CB2 cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB1 receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB1 receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

A vaccine strategy that induces protective immunity against heroin.

Heroin addiction is a wide-reaching problem with a spectrum of damaging social consequences. A vaccine capable of blocking heroins effects could provide a long-lasting and sustainable adjunct to heroin addiction therapy. Heroin, however, presents a particularly challenging immunotherapeutic target, as it is metabolized to multiple psychoactive molecules. To reconcile this dilemma, we examined the idea of a singular vaccine with the potential to display multiple drug-like antigens; thus two haptens were synthesized, one heroin-like and another morphine-like in chemical structure. A key feature in this approach is that immunopresentation with the heroin-like hapten is thought to be immunochemically dynamic such that multiple haptens are simultaneously presented to the immune system. We demonstrate the significance of this approach through the extremely rapid generation of robust polyclonal antibody titers with remarkable specificity. Importantly, both the antinociceptive effects of heroin and acquisition of heroin self-administration were blocked in rats vaccinated using the heroin-like hapten.

Inhibitors of Endocannabinoid-Metabolizing Enzymes Reduce Precipitated Withdrawal Responses in THC-Dependent Mice

Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis-related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy using the psychoactive constituent of marijuana, Δ9-tetrahydrocannabinol (THC). However, new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoid ligands anandamide and 2-arachidonoylglycerol, respectively. In the present study, we investigated whether increasing endogenous cannabinoids levels, through the use of FAAH (−/−) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184, would reduce the intensity of withdrawal signs precipitated by the CB1 receptor antagonist rimonabant in THC-dependent mice. Strikingly, acute administration of either URB597 or JZL184 significantly attenuated rimonabant-precipitated withdrawal signs in THC-dependent mice. In contrast, FAAH (−/−) mice showed identical withdrawal responses as wild-type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB597 did not lead to cannabinoid dependence and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal.

Glucocorticoid receptor antagonism decreases alcohol seeking in alcohol-dependent individuals

Alcoholism, or alcohol use disorder, is a major public health concern that is a considerable risk factor for morbidity and disability; therefore, effective treatments are urgently needed. Here, we demonstrated that the glucocorticoid receptor (GR) antagonist mifepristone reduces alcohol intake in alcohol-dependent rats but not in nondependent animals. Both systemic delivery and direct administration into the central nucleus of the amygdala, a critical stress-related brain region, were sufficient to reduce alcohol consumption in dependent animals. We also tested the use of mifepristone in 56 alcohol-dependent human subjects as part of a double-blind clinical and laboratory-based study. Relative to placebo, individuals who received mifepristone (600 mg daily taken orally for 1 week) exhibited a substantial reduction in alcohol-cued craving in the laboratory, and naturalistic measures revealed reduced alcohol consumption during the 1-week treatment phase and 1-week post-treatment phase in mifepristone-treated individuals. Mifepristone was well tolerated and improved liver-function markers. Together, these results support further exploration of GR antagonism via mifepristone as a therapeutic strategy for alcoholism.

VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation

SUMMARY Dopaminergic neurons in the ventral tegmental area (VTA) are well known for their role in mediating the positive reinforcing effects of drugs of abuse. Here, we identify in rodents and humans a population of VTA dopamine neurons co-expressing corticotropin releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates CRF mRNA in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors, and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of CRF mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal, and limited the escalation of nicotine intake. These results link the brain reward and stress systems within the same brain region in signaling the negative motivational effects of nicotine withdrawal.Dopaminergic neurons in the ventral tegmental area (VTA) are well known for mediating the positive reinforcing effects of drugs of abuse. Here we identify in rodents and humans a population of VTA dopaminergic neurons expressing corticotropin-releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates Crh mRNA (encoding CRF) in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of Crh mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal and limited the escalation of nicotine intake. These results link the brain reward and stress systems in the same brain region to signaling of the negative motivational effects of nicotine withdrawal.

Long-term antagonism of κ opioid receptors prevents escalation of and increased motivation for heroin intake.

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.