Andrea Duranti

Modulation of anxiety through blockade of anandamide hydrolysis

The psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol, produces in humans subjective responses mediated by CB1 cannabinoid receptors, indicating that endogenous cannabinoids may contribute to the control of emotion. But the variable effects of Δ9-tetrahydrocannabinol obscure the interpretation of these results and limit the therapeutic potential of direct cannabinoid agonists. An alternative approach may be to develop drugs that amplify the effects of endogenous cannabinoids by preventing their inactivation. Here we describe a class of potent, selective and systemically active inhibitors of fatty acid amide hydrolase, the enzyme responsible for the degradation of the endogenous cannabinoid anandamide. Like clinically used anti-anxiety drugs, in rats the inhibitors exhibit benzodiazepine-like properties in the elevated zero-maze test and suppress isolation-induced vocalizations. These effects are accompanied by augmented brain levels of anandamide and are prevented by CB1 receptor blockade. Our results indicate that anandamide participates in the modulation of emotional states and point to fatty acid amide hydrolase inhibition as an innovative approach to anti-anxiety therapy.

An endocannabinoid mechanism for stress-induced analgesia

Acute stress suppresses pain by activating brain pathways that engage opioid or non-opioid mechanisms. Here we show that an opioid-independent form of this phenomenon, termed stress-induced analgesia, is mediated by the release of endogenous marijuana-like (cannabinoid) compounds in the brain. Blockade of cannabinoid CB1 receptors in the periaqueductal grey matter of the midbrain prevents non-opioid stress-induced analgesia. In this region, stress elicits the rapid formation of two endogenous cannabinoids, the lipids 2-arachidonoylglycerol (2-AG) and anandamide. A newly developed inhibitor of the 2-AG-deactivating enzyme, monoacylglycerol lipase, selectively increases 2-AG concentrations and, when injected into the periaqueductal grey matter, enhances stress-induced analgesia in a CB1-dependent manner. Inhibitors of the anandamide-deactivating enzyme fatty-acid amide hydrolase, which selectively elevate anandamide concentrations, exert similar effects. Our results indicate that the coordinated release of 2-AG and anandamide in the periaqueductal grey matter might mediate opioid-independent stress-induced analgesia. These studies also identify monoacylglycerol lipase as a previously unrecognized therapeutic target.

Antidepressant-like Activity of the Fatty Acid Amide Hydrolase Inhibitor URB597 in a Rat Model of Chronic Mild Stress

BACKGROUND The endocannabinoid anandamide may be involved in the regulation of emotional reactivity. In particular, it has been shown that pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of anandamide, elicits anxiolytic-like and antidepressant-like effects in rodents. METHODS We investigated the impact of chronic treatment with the selective FAAH inhibitor, URB597 (also termed KDS-4103), on the outcomes of the chronic mild stress (CMS) in rats, a behavioral model with high isomorphism to human depression. RESULTS Daily administration of URB597 (.3 mg kg(-1), intraperitoneal [IP]) for 5 weeks corrected the reduction in body weight gain and sucrose intake induced by CMS. The antidepressant imipramine (20 mg kg(-1), once daily, IP) produced a similar response, whereas lower doses of URB597 were either marginally effective (.1 mg kg(-1)) or ineffective (.03 mg kg(-1)). Treatment with URB597 (.3 mg kg(-1)) resulted in a profound inhibition of brain FAAH activity in both CMS-exposed and control rats. Furthermore, the drug regimen increased anandamide levels in midbrain, striatum, and thalamus. CONCLUSIONS URB597 exerts antidepressant-like effects in a highly specific and predictive animal model of depression. These effects may depend on the ability of URB597 to enhance anandamide signaling in select regions of the brain.

Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus–evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

Selective inhibition of 2-AG hydrolysis enhances endocannabinoid signaling in hippocampus

The functions of 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid found in the brain, remain largely unknown. Here we show that two previously unknown inhibitors of monoacylglycerol lipase, a presynaptic enzyme that hydrolyzes 2-AG, increase 2-AG levels and enhance retrograde signaling from pyramidal neurons to GABAergic terminals in the hippocampus. These results establish a role for 2-AG in synaptic plasticity and point to monoacylglycerol lipase as a possible drug target.

Selective N-acylethanolamine-hydrolyzing acid amidase inhibition reveals a key role for endogenous palmitoylethanolamide in inflammation

Identifying points of control in inflammation is essential to discovering safe and effective antiinflammatory medicines. Palmitoylethanolamide (PEA) is a naturally occurring lipid amide that, when administered as a drug, inhibits inflammatory responses by engaging peroxisome proliferator-activated receptor-α (PPAR-α). PEA is preferentially hydrolyzed by the cysteine amidase N-acylethanolamine-hydrolyzing acid amidase (NAAA), which is highly expressed in macrophages. Here we report the discovery of a potent and selective NAAA inhibitor, N-[(3S)-2-oxo-3-oxetanyl]-3-phenylpropanamide [(S)-OOPP], and show that this inhibitor increases PEA levels in activated leukocytes and blunts responses induced by inflammatory stimuli both in vitro and in vivo. These effects are stereoselective, mimicked by exogenous PEA, and abolished by PPAR-α deletion. (S)-OOPP also attenuates inflammation and tissue damage and improves recovery of motor function in mice subjected to spinal cord trauma. The results suggest that PEA activation of PPAR-α in leukocytes serves as an early stop signal that contrasts the progress of inflammation. The PEA-hydrolyzing amidase NAAA may provide a previously undescribed target for antiinflammatory medicines.

The Fatty Acid Amide Hydrolase Inhibitor URB597 (Cyclohexylcarbamic Acid 3′-Carbamoylbiphenyl-3-yl Ester) Reduces Neuropathic Pain after Oral Administration in Mice

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase that catalyzes the cleavage of bioactive fatty acid ethanolamides, such as the endogenous cannabinoid agonist anandamide. Genetic deletion of the faah gene in mice elevates brain anandamide levels and amplifies the antinociceptive effects of this compound. Likewise, pharmacological blockade of FAAH activity reduces nocifensive behavior in animal models of acute and inflammatory pain. In the present study, we investigated the effects of the selective FAAH inhibitor URB597 (KDS-4103, cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) in the mouse chronic constriction injury (CCI) model of neuropathic pain. Oral administration of URB597 (1–50 mg/kg, once daily) for 4 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single i.p. administration of the cannabinoid CB1 receptor antagonist rimonabant (1 mg/kg). The antihyperalgesic effects of URB597 were accompanied by a reduction in plasma extravasation induced by CCI, which was prevented by rimonabant (1 mg/kg i.p.) and attenuated by the CB2 antagonist SR144528 (1 mg/kg i.p.). Oral dosing with URB597 achieved significant, albeit transient, drug levels in plasma, inhibited brain FAAH activity, and elevated spinal cord anandamide content. The results provide new evidence for a role of the endocannabinoid system in pain modulation and reinforce the proposed role of FAAH as a target for analgesic drug development.

Synthesis and characterization of a peripherally restricted CB1 cannabinoid antagonist, URB447, that reduces feeding and body-weight gain in mice

Cannabinoid CB(1) receptor antagonists reduce body weight in rodents and humans, but their clinical utility as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed CB(1) antagonist/CB(2) agonist, URB447 ([4-amino-1-(4-chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl](phenyl)methanone), which lowers food intake and body-weight gain in mice without entering the brain or antagonizing central CB(1)-dependent responses. URB447 may provide a useful pharmacological tool for investigating the cannabinoid system, and might serve as a starting point for developing clinically viable CB(1) antagonists devoid of central side effects.

Endocannabinoids in the Treatment of Mood Disorders: Evidence from Animal Models

Among all mental disorders, major depression has the highest rate of prevalence and incidence of morbidity. Currently available antidepressant therapies have limited efficacies; consequently, research on new drugs for the treatment of mood disorders has become increasingly critical. Recent preclinical evidences that cannabinoid agonists and endocannabinoid enhancers, such as the fatty acid amide hydrolase (FAAH) inhibitors, can impact mood regulation have opened a new line of research in antidepressant drug discovery. However, the neurobiological mechanisms linking the endocannabinoid system with the pathophysiology of mood disorders and antidepressant action remain unclarified. In this review, we have presented an update on preclinical data indicating the antidepressant potential of cannabinoid agonists and endocannabinoid enhancers in comparison to standard antidepressants. Data obtained from CB(1) knockout (CB(1)-/-) and FAAH knockout (FAAH-/-) mice have also been examined within this context. We have illustrated how the various classes of antidepressants exert their therapeutic action. In particular, all antidepressants increase the neurotransmission of serotonin after long-term treatment, enhance the tonic activity of hippocampal 5-HT(1A) receptors, promote neurogenesis, and modulate (decrease or increase) the firing activity of noradrenergic neurons. Interestingly, cannabinoid agonists and endocannabinoid enhancers increase serotonin and noradrenergic neuronal firing activity, increase serotonin release in the hippocampus, as well as promote neurogenesis. Since cannabinoid-derived drugs potentiate monoaminergic neurotransmission and hippocampal neurogenesis through distinct pathways compared to classical antidepressants, they may represent an alternative drug class in the pharmacotherapy of mood and other neuropsychiatric disorders.

Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models.

TOC summary The N‐acylethanolamine acid amidase inhibitor ARN077 exerts profound antinociceptive effects in animal pain models by enhancing endogenous lipid signaling at peroxisome proliferator‐activated receptor‐α. N‐acylethanolamine acid amidase may be a new target for analgesic drugs. ABSTRACT Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator‐activated receptor‐α (PPAR‐α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N‐acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti‐inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA‐mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose‐dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR‐α antagonist GW6471 and did not occur in PPAR‐α–deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR‐α.