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Hypofunctioning of NMDA receptors, and the resulting shift in the balance between excitation and inhibition, is considered a key process in the pathophysiology of schizophrenia. One important manifestation of this phenomenon is changes in neural oscillations, those above 30 Hz (i.e., gamma-band oscillations), in particular. Although both preclinical and clinical studies observed increased gamma activity following acute administration of NMDA receptor antagonists, the relevance of this phenomenon has been recently questioned given the reduced gamma oscillations typically observed during sensory and cognitive tasks in schizophrenia. However, there is emerging, yet contradictory, evidence for increased spontaneous gamma-band activity (i.e., at rest or under baseline conditions). Here, we use the sub-chronic phencyclidine (PCP) rat model for schizophrenia, which has been argued to model the pathophysiology of schizophrenia more closely than acute NMDA antagonism, to investigate gamma oscillations (30–100 Hz) in the medial prefrontal cortex of anesthetized animals. While baseline gamma oscillations were not affected, oscillations induced by train stimulation of the posterior dorsal CA1 (pdCA1) field of the hippocampus were enhanced in PCP-treated animals (5 mg/kg, twice daily for 7 days, followed by a 7-day washout period). This effect was reversed by pharmacological enhancement of endocannabinoid levels via systemic administration of URB597 (0.3 mg/kg), an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Intriguingly, the pharmacological blockade of CB1 receptors by AM251 unmasked a reduced gamma oscillatory activity in PCP-treated animals. The findings are consistent with the observed effects of URB597 and AM251 on behavioral deficits reminiscent of the symptoms of schizophrenia and further validate the potential for cannabinoid-based drugs as a treatment for schizophrenia. [ABSTRACT FROM AUTHOR]

Abstract Background Frontotemporal dementia (FTD) is a heterogeneous group of early onset and progressive neurodegenerative disorders, characterized by degeneration in the frontal and temporal lobes, which causes deterioration in cognition, personality, social behavior and language. Around 45% of the cases are characterized by the presence of aggregates of the RNA-binding protein TDP-43. Methods In this study, we have used a murine model of FTD that overexpresses this protein exclusively in the forebrain (under the control of the CaMKIIα promoter) for several biochemical, histological and pharmacological studies focused on the endocannabinoid system. Results These mice exhibited at postnatal day 90 (PND90) important cognitive deficits, signs of emotional impairment and disinhibited social behaviour, which were, in most of cases, maintained during the first year of life of these animals. Motor activity was apparently normal, but FTD mice exhibited higher mortality. Their MRI imaging analysis and their ex-vivo histopathological evaluation proved changes compatible with atrophy (loss of specific groups of pyramidal neurons: Ctip2- and NeuN-positive cells) and inflammatory events (astroglial and microglial reactivities) in both cortical (medial prefrontal cortex) and subcortical (hippocampus) structures at PND90 and also at PND365. The analysis of the endocannabinoid system in these mice proved a decrease in the hydrolysing enzyme FAAH in the prefrontal cortex and the hippocampus, with an increase in the synthesizing enzyme NAPE-PLD only in the hippocampus, responses that were accompanied by modest elevations in anandamide and related N-acylethanolamines. The potentiation of these elevated levels of anandamide after the pharmacological inactivation of FAAH with URB597 resulted in a general improvement in behaviour, in particular in cognitive deterioration, associated with the preservation of pyramidal neurons of the medial prefrontal cortex and the CA1 layer of the hippocampus, and with the reduction of gliosis in both structures. Conclusions Our data confirmed the potential of elevating the endocannabinoid tone as a therapy against TDP-43-induced neuropathology in FTD, limiting glial reactivity, preserving neuronal integrity and improving cognitive, emotional and social deficits.

The endocannabinoid system is prominently implicated in the control of cocaine reinforcement due to its relevant role in synaptic plasticity and neurotransmitter modulation in the mesocorticolimbic system. The inhibition of fatty acid amide hydrolase (FAAH), and the resulting increase in anandamide and other N-acylethanolamines, represents a promising strategy for reducing drug seeking. In the present study, we aimed to assess the effects of the FAAH inhibitor URB597 (1 mg/kg) on crucial features of cocaine addictive-like behaviour in mice. Therefore, we tested the effects of URB597 on acquisition of cocaine (0.6 mg/kg/inf) self-administration, compulsive-like cocaine intake and cue-induced drug-seeking behaviour during withdrawal. URB597 reduced cocaine intake under conditioned punishment while having no impact on acquisition. This result was associated to increased cannabinoid receptor 1 gene expression in the ventral striatum and medium spiny neurons activation in the nucleus accumbens shell. Moreover, URB597 mitigated cue-induced drug-seeking behaviour during prolonged abstinence and prevented the withdrawal-induced increase in FAAH gene expression in the ventral striatum. In this case, URB597 decreased activation of medium spiny neurons in the nucleus accumbens core. Our findings evidence the prominent role of endocannabinoids in the development of cocaine addictive-like behaviours and support the potential of FAAH inhibition as a therapeutical target for the treatment of cocaine addiction.

Background and Purpose: Devising novel strategies to therapeutically favour inflammation resolution and provide neuroprotection is an unmet clinical need. Enhancing endocannabinoid tone by inhibiting the catabolic enzyme fatty acid amide hydrolase (FAAH), or stimulating melatonin receptors has therapeutic potential to treat neuropathological states in which neuroinflammation plays a central role. Experimental Approach: A rodent hippocampal explant model of inflammatory injury was used to assess the effects of UCM1341, a dual‐acting compound with FAAH inhibitory action and agonist activity at melatonin receptors, against neuroinflammatory damage. FAAH activity was measured by a radiometric assay, and N‐acylethanolamine levels were assessed by HPLC–MS/MS methods. FAAH distribution, evolution of inflammation and the contribution of UCM1341 to the expression of proteins controlling macrophage behaviour were investigated by biochemical and confocal analyses. Key Results: UCM1341 exhibited greater neuroprotection against neuroinflammatory degeneration, compared with the reference compounds URB597 (FAAH inhibitor) and melatonin. During neuroinflammation, UCM1341 augmented the levels of anandamide and N‐oleoylethanolamine, but not N‐palmitoylethanolamine, up‐regulated PPAR‐α levels, attenuated demyelination and prevented the release of TNF‐α. UCM1341 modulated inflammatory responses by contributing to microglia/macrophage polarization, stimulating formation of lipid‐laden macrophages and regulating expression of proteins controlling cholesterol metabolism and efflux. The neuroprotective effects of UCM1341 were prevented by PPARα, TRPV1 and melatonin receptor antagonists. Conclusion and Implications: UCM1341, by enhancing endocannabinoid and melatoninergic signalling, benefits neuroprotection and stimulates inflammation resolution pathways. Our findings provide an encouraging prospect of therapeutically targeting endocannabinoid and melatoninergic systems in inflammatory demyelinating states in the CNS. [ABSTRACT FROM AUTHOR]

Background: Frontotemporal dementia (FTD) is a heterogeneous group of early onset and progressive neurodegenerative disorders, characterized by degeneration in the frontal and temporal lobes, which causes deterioration in cognition, personality, social behavior and language. Around 45% of the cases are characterized by the presence of aggregates of the RNA-binding protein TDP-43. Methods: In this study, we have used a murine model of FTD that overexpresses this protein exclusively in the forebrain (under the control of the CaMKIIα promoter) for several biochemical, histological and pharmacological studies focused on the endocannabinoid system. Results: These mice exhibited at postnatal day 90 (PND90) important cognitive deficits, signs of emotional impairment and disinhibited social behaviour, which were, in most of cases, maintained during the first year of life of these animals. Motor activity was apparently normal, but FTD mice exhibited higher mortality. Their MRI imaging analysis and their ex-vivo histopathological evaluation proved changes compatible with atrophy (loss of specific groups of pyramidal neurons: Ctip2- and NeuN-positive cells) and inflammatory events (astroglial and microglial reactivities) in both cortical (medial prefrontal cortex) and subcortical (hippocampus) structures at PND90 and also at PND365. The analysis of the endocannabinoid system in these mice proved a decrease in the hydrolysing enzyme FAAH in the prefrontal cortex and the hippocampus, with an increase in the synthesizing enzyme NAPE-PLD only in the hippocampus, responses that were accompanied by modest elevations in anandamide and related N-acylethanolamines. The potentiation of these elevated levels of anandamide after the pharmacological inactivation of FAAH with URB597 resulted in a general improvement in behaviour, in particular in cognitive deterioration, associated with the preservation of pyramidal neurons of the medial prefrontal cortex and the CA1 layer of the hippocampus, and with the reduction of gliosis in both structures. Conclusions: Our data confirmed the potential of elevating the endocannabinoid tone as a therapy against TDP-43-induced neuropathology in FTD, limiting glial reactivity, preserving neuronal integrity and improving cognitive, emotional and social deficits. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) plays an important role in pain processing and modulation. Since the specific effects of endocannabinoids within the orofacial area are largely unknown, we aimed to determine whether an increase in the endocannabinoid concentration in the cerebrospinal fluid (CSF) caused by the peripheral administration of the FAAH inhibitor URB597 and tooth pulp stimulation would affect the transmission of impulses between the sensory and motor centers localized in the vicinity of the third and fourth cerebral ventricles. The study objectives were evaluated on rats using a method that allowed the recording of the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation and URB597 treatment. The amplitude of ETJ was a measure of the effect of endocannabinoids on the neural structures. The concentrations of the endocannabinoids tested (AEA and 2-AG) were determined in the CSF, along with the expression of the cannabinoid receptors (CB1 and CB2) in the tissues of the mesencephalon, thalamus, and hypothalamus. We demonstrated that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was significantly increased in the CSF after treatment with a FAAH inhibitor, while tooth pulp stimulation had no effect on the AEA and 2-AG concentrations in the CSF. We also found positive correlations between the CSF AEA concentration and cannabinoid receptor type 1 (CB1R) expression in the brain, and between 2-AG and cannabinoid receptor type 2 (CB2R), and negative correlations between the CSF concentration of AEA and brain CB2R expression, and between 2-AG and CB1R. Our study shows that endogenous AEA, which diffuses through the cerebroventricular ependyma into CSF and exerts a modulatory effect mediated by CB1Rs, alters the properties of neurons in the trigeminal sensory nuclei, interneurons, and motoneurons of the hypoglossal nerve. In addition, our findings may be consistent with the emerging concept that AEA and 2-AG have different regulatory mechanisms because they are involved differently in orofacial pain. We also suggest that FAAH inhibition may offer a therapeutic approach to the treatment of orofacial pain.

Early life stress (ELS) increases predisposition to depression. We compared the effects of treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597, and the selective serotonin reuptake inhibitor paroxetine, on ELS-induced depressive-like behavior and the expression of microRNAs (miRs) associated with depression in the medial prefrontal cortex (mPFC), hippocampal CA1 area, lateral habenula and dorsal raphe in rats. We also examined the mRNA expression of serotonergic (htr1a and slc6a4) and endocannabinoid (cnr1, cnr2 and faah) targets in the mPFC following ELS and pharmacological treatment. Adult males and females exposed to the 'Limited Bedding and Nesting' ELS paradigm demonstrated a depressive-like phenotype and late-adolescence URB597 treatment, but not paroxetine, reversed this phenotype. In the mPFC, ELS downregulated miR-16 in males and miR-135a in females and URB597 treatment restored this effect. In ELS females, the increase in cnr2 and decrease in faah mRNAs in the mPFC were reversed by URB597 treatment. We show for the first time that URB597 reversed ELS-induced mPFC downregulation in specific miRs and stress-related behaviors, suggesting a novel mechanism for the beneficial effects of FAAH inhibition. The differential effects of ELS and URB597 on males and females highlight the importance of developing sex-specific treatment approaches. [ABSTRACT FROM AUTHOR]

The complexity of neuropathic pain and its associated comorbidities, including dysautonomia, make it difficult to treat. Overlap of anatomical regions and pharmacology of sympathosensory systems in the central nervous system (CNS) provide targets for novel treatment strategies. The dorsal periaqueductal gray (dPAG) is an integral component of both the descending pain modulation system and the acute stress response and is critically involved in both analgesia and the regulation of sympathetic activity. Local manipulation of the endocannabinoid signaling system holds great promise to provide analgesia without excessive adverse effects and also influence autonomic output. Inhibition of fatty acid amide hydrolase (FAAH) increases brain concentrations of the endocannabinoid N-arachidonoylethanolamine (AEA) and reduces pain-related behaviors in neuropathic pain models. Neuropathic hyperalgesia and reduced sympathetic tone are associated with increased FAAH activity in the dPAG, which suggests the hypothesis that inhibition of FAAH in the dPAG will normalize pain sensation and autonomic function in neuropathic pain. To test this hypothesis, the effects of systemic or intra-dPAG FAAH inhibition on hyperalgesia and dysautonomia developed after spared nerve injury (SNI) were assessed in male and female rats. Administration of the FAAH inhibitor PF-3845 into the dPAG reduces hyperalgesia behavior and the decrease in sympathetic tone induced by SNI. Prior administration of the CB1 receptor antagonist AM281, attenuated the antihyperalgesic and sympathetic effects of FAAH inhibition. No sex differences were identified. These data support an integrative role for AEA/CB1 receptor signaling in the dPAG contributing to the regulation of both hyperalgesia behavior and altered sympathetic tone in neuropathic pain. [ABSTRACT FROM AUTHOR]

Rationale: The endocannabinoid modulation of fear and anxiety due to the on-demand synthesis and degradation is supported by a large body of research. Although it has been proposed that anandamide (AEA) in the substantia nigra pars reticulata (SNpr) seems to be important for the organisation of innate fear-related behaviours, a role for endogenous AEA has yet to be clarified. Methods: Mice were treated with the fatty acid amide hydrolase (FAAH) selective inhibitor URB597 at different concentrations (0.01, 0.1, 1 nmol/0.1 µL) in the SNpr and confronted by rattlesnakes (Crotalus durissus terrificus). The most effective dose of URB597 (1 nmol) was also preceded by microinjections of the CB1 receptor antagonist AM251 (0.1 nmol) into the SNpr, and mice were then confronted by the venomous snake. Results: URB597 (0.1 and 1 nmol) in the SNpr decreased the expression of defensive behaviours such as defensive attention, escape, and time spent inside the burrow of mice confronted by rattlesnakes. Moreover, pretreatment of SNpr with AM251 suppressed these antiaversive effects of URB597 in this midbrain structure. Conclusion: Overall, these data clearly indicate that the panicolytic consequences of endogenous AEA enhancement in the SNpr are mediated by CB1 receptor signalling. [ABSTRACT FROM AUTHOR]

Inhibition of fatty acid amide hydrolase (FAAH), which increases anandamide levels, has been suggested as a potential treatment for stress-related conditions. We examined whether the stress-preventing effects of the FAAH inhibitor URB597 on behavior are mediated via β-catenin in the nucleus accumbens (NAc). Male rats were exposed to the shock and reminders model of PTSD and then treated with URB597 (0.4 mg/kg; i.p.). They were tested for anxiety- (freezing, startle response), depression-like behaviors (despair, social preference, anhedonia), and memory function (T-maze, social recognition). We also tested the involvement of the CB1 receptor (CB1r), β-catenin, and metabotropic glutamate receptor subtype 5 (mGluR5) proteins. URB597 prevented the shock- and reminders-induced increase in anxiety- and depressive-like behaviors, as well as the impaired memory via the CB1r-dependent mechanism. In the NAc, viral-mediated β-catenin overexpression restored the behavior of rats exposed to stress and normalized the alterations in protein levels in the NAc and the prefrontal cortex. Importantly, when NAc β-catenin levels were downregulated by viral-mediated gene transfer, the therapeutic-like effects of URB597 were blocked. We suggest a potentially novel mechanism for the therapeutic-like effects of FAAH inhibition that is dependent on β-catenin activation in the NAc in a PTSD rat model. [ABSTRACT FROM AUTHOR]

NMDA receptor blockade in rodents is commonly used to induce schizophrenia-like behavioral abnormalities, including cognitive deficits and social dysfunction. Aberrant glutamate and GABA transmission, particularly in adolescence, is implicated in these behavioral abnormalities. The endocannabinoid system modulates glutamate and GABA transmission, but the impact of endocannabinoid modulation on cognitive and social dysfunction is unclear. Here, we asked whether late-adolescence administration of the anandamide hydrolysis inhibitor URB597 can reverse behavioral deficits induced by early-adolescence administration of the NMDA receptor blocker MK-801. In parallel, we assessed the impact of MK-801 and URB597 on mRNA expression of glutamate and GABA markers. We found that URB597 prevented MK-801-induced novel object recognition deficits and social interaction abnormalities in adult rats, and reversed glutamate and GABA aberrations in the prelimbic PFC. URB597-mediated reversal of MK-801-induced social interaction deficits was mediated by the CB1 receptor, whereas the reversal of cognitive deficits was mediated by the CB2 receptor. This was paralleled by the reversal of CB1 and CB2 receptor expression abnormalities in the basolateral amygdala and prelimbic PFC, respectively. Together, our findings show that interfering with NMDA receptor function in early adolescence has a lasting impact on phenotypes resembling the negative symptoms and cognitive deficits of schizophrenia and on glutamate and GABA marker expression in the PFC. Prevention of behavioral and molecular abnormalities by late-adolescence URB597 via CB1 and CB2 receptors suggests that endocannabinoid stimulation may have therapeutic potential in addressing treatment-resistant symptoms. [ABSTRACT FROM AUTHOR]

Endocannabinoids act as analgesic agents in a number of headache models. However, their effectiveness varies with the route of administration and the type of pain. In this study, we assessed the role of the fatty acid amide hydrolase inhibitor URB597 in an animal model of orofacial pain based on tooth pulp stimulation. More specifically, we assessed the effects of intracerbroventricular (i.c.v.) and intraperitoneal (i.p.) administration of URB597 on the amplitude of evoked tongue jerks (ETJ) in rats. The levels of the investigated mediators anandamide (AEA), 2-arachidonyl glycerol (2-AG), Substance P (SP), calcitonin-gene-related peptide (CGRP), endomorphin-2 (EM-2) and fatty acid amide hydrolase (FAAH) inhibitor by URB597 and receptors cannabinoid type-1 receptors (CB1R), cannabinoid type-2 receptors (CB2R) and µ-opioid receptors (MOR) were determined in the mesencephalon, thalamus and hypothalamus tissues. We have shown that increasing endocannabinoid AEA levels by both central and peripheral inhibition of FAAH inhibitor by URB597 has an antinociceptive effect on the trigemino-hypoglossal reflex mediated by CB1R and influences the activation of the brain areas studied. On the other hand, URB597 had no effect on the concentration of 2-AG in the examined brain structures and caused a significant decrease in CB2R mRNA expression in the hypothalamus only. Tooth pulp stimulation caused in a significant increase in SP, CGRP and EM-2 gene expression in the midbrain, thalamus and hypothalamus. In contrast, URB597 administered peripherally one hour before stimulation decreased the mRNA level of these endogenous neuropeptides in comparison with the control and stimulation in all examined brain structures. Our results show that centrally and peripherally administered URB597 is effective at preventing orofacial pain by inhibiting AEA catabolism and reducing the level of CGRP, SP and EM-2 gene expression and that AEA and 2-AG have different species and model-specific regulatory mechanisms. The data presented in this study may represent a new promising therapeutic target in the treatment of orofacial pain. [ABSTRACT FROM AUTHOR]

Parkinson disease (PD) is a progressive neurodegenerative disorder that is often accompanied by motor and psychiatric symptoms. Various approaches have been proposed for the treatment of PD. Here, we investigated the effect of a low dose of fatty acid amide hydrolase inhibitor URB597 (as an enhancer of endocannabinoid anandamide levels), exercise or their combination on some behavior alterations in PD mice lesioned by 6-hydroxydopamine (6-OHDA). The impact of swimming exercise (5×/week for 4 weeks) and URB597 (0.1 mg/kg, 2×/week for 4 weeks) on the anxiety-related behavior (elevated plus maze; EPM), depression-related behavior (tail suspension test; TST), and passive avoidance memory (step-down task) was examined in the sham and male NMRI mouse of PD model. The results show that URB597 prevented memory deficits and elicited antidepressant- and anxiolytic-like effects but did not affect hypolocomotion in the PD mice. However, URB597 did not have a significant effect on the performance of the sham mice in the performed tests. Moreover, swimming training abolished depressive- and anxiogenic-like behaviors and increased locomotion without affecting memory deficits in the PD mice. Meanwhile, swimming decreased immobility time and increased locomotion in the sham mice. Furthermore, URB597 in association with swimming training prevented all deficits induced in the PD mice, while this combination impaired memory and produced the positive effects on depression- and anxiety-related behaviors and locomotion of the sham mice. It is concluded that although URB597 or exercise alone had positive effects on most behavioral tests, their combination improved all parameters in the PD mice. [ABSTRACT FROM AUTHOR]

Early life stress (ELS) increases predisposition to depression. We compared the effects of treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597, and the selective serotonin reuptake inhibitor paroxetine, on ELS-induced depressive-like behavior and the expression of microRNAs (miRs) associated with depression in the medial prefrontal cortex (mPFC), hippocampal CA1 area, lateral habenula and dorsal raphe in rats. We also examined the mRNA expression of serotonergic (htr1a and slc6a4) and endocannabinoid (cnr1, cnr2 and faah) targets in the mPFC following ELS and pharmacological treatment. Adult males and females exposed to the ‘Limited Bedding and Nesting’ ELS paradigm demonstrated a depressive-like phenotype and late-adolescence URB597 treatment, but not paroxetine, reversed this phenotype. In the mPFC, ELS downregulated miR-16 in males and miR-135a in females and URB597 treatment restored this effect. In ELS females, the increase in cnr2 and decrease in faah mRNAs in the mPFC were reversed by URB597 treatment. We show for the first time that URB597 reversed ELS-induced mPFC downregulation in specific miRs and stress-related behaviors, suggesting a novel mechanism for the beneficial effects of FAAH inhibition. The differential effects of ELS and URB597 on males and females highlight the importance of developing sex-specific treatment approaches.

Inhibition of fatty acid amide hydrolase (FAAH), which increases anandamide levels, has been suggested as a potential treatment for stress-related conditions. We examined whether the stress-preventing effects of the FAAH inhibitor URB597 on behavior are mediated via β-catenin in the nucleus accumbens (NAc). Male rats were exposed to the shock and reminders model of PTSD and then treated with URB597 (0.4 mg/kg; i.p.). They were tested for anxiety- (freezing, startle response), depression-like behaviors (despair, social preference, anhedonia), and memory function (T-maze, social recognition). We also tested the involvement of the CB1 receptor (CB1r), β-catenin, and metabotropic glutamate receptor subtype 5 (mGluR5) proteins. URB597 prevented the shock- and reminders-induced increase in anxiety- and depressive-like behaviors, as well as the impaired memory via the CB1r-dependent mechanism. In the NAc, viral-mediated β-catenin overexpression restored the behavior of rats exposed to stress and normalized the alterations in protein levels in the NAc and the prefrontal cortex. Importantly, when NAc β-catenin levels were downregulated by viral-mediated gene transfer, the therapeutic-like effects of URB597 were blocked. We suggest a potentially novel mechanism for the therapeutic-like effects of FAAH inhibition that is dependent on β-catenin activation in the NAc in a PTSD rat model.

Endocannabinoid-based therapies constitute an emerging tool for the potential treatment of neurodegenerative disorders, requiring characterization at the experimental level. The effects of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH), were tested against the quinolinic acid (QUIN)-induced early toxic effects in rat cortical slices, and compared with those effects exerted by the endocannabinoid anandamide (AEA). URB597 prevented the QUIN-induced loss of mitochondrial function/cell viability and lipid peroxidation, while reduced necrosis, and to a lesser extent, apoptosis. The protective effects of URB597 were mediated by activation of cannabinoid receptor 1 (CB1r), as evidenced by their inhibition by the selective CB1r antagonist AM281. Similar effects were observed when testing AEA against QUIN toxicity. Our findings demonstrate the neuroprotective properties of URB597 during the early stages of excitotoxic damage to cortical tissue, suggesting that these properties are mediated by FAAH inhibition, and might be linked to the protective effects of AEA, or the combination of endocannabinoids. [ABSTRACT FROM AUTHOR]

Endocannabinoids act as analgesic agents in a number of headache models. However, their effectiveness varies with the route of administration and the type of pain. In this study, we assessed the role of the fatty acid amide hydrolase inhibitor URB597 in an animal model of orofacial pain based on tooth pulp stimulation. More specifically, we assessed the effects of intracerbroventricular (i.c.v.) and intraperitoneal (i.p.) administration of URB597 on the amplitude of evoked tongue jerks (ETJ) in rats. The levels of the investigated mediators anandamide (AEA), 2-arachidonyl glycerol (2-AG), Substance P (SP), calcitonin-gene-related peptide (CGRP), endomorphin-2 (EM-2) and fatty acid amide hydrolase (FAAH) inhibitor by URB597 and receptors cannabinoid type-1 receptors (CB1R), cannabinoid type-2 receptors (CB2R) and µ-opioid receptors (MOR) were determined in the mesencephalon, thalamus and hypothalamus tissues. We have shown that increasing endocannabinoid AEA levels by both central and peripheral inhibition of FAAH inhibitor by URB597 has an antinociceptive effect on the trigemino-hypoglossal reflex mediated by CB1R and influences the activation of the brain areas studied. On the other hand, URB597 had no effect on the concentration of 2-AG in the examined brain structures and caused a significant decrease in CB2R mRNA expression in the hypothalamus only. Tooth pulp stimulation caused in a significant increase in SP, CGRP and EM-2 gene expression in the midbrain, thalamus and hypothalamus. In contrast, URB597 administered peripherally one hour before stimulation decreased the mRNA level of these endogenous neuropeptides in comparison with the control and stimulation in all examined brain structures. Our results show that centrally and peripherally administered URB597 is effective at preventing orofacial pain by inhibiting AEA catabolism and reducing the level of CGRP, SP and EM-2 gene expression and that AEA and 2-AG have different species and model-specific regulatory mechanisms. The data presented in this study may represent a new promising therapeutic target in the treatment of orofacial pain.

• Early life stress (ELS) induced depressive-like behaviors in male and female rats. • FAAH inhibition (i.e. URB597) at mid-adolescence induced depressive-like behaviors. • ELS and URB597 decreased PFC and BLA CB1r as well as PFC and BLA GRs in males. • ELS and URB597 decreased BLA CB1r as well as CA1 and BLA GRs in females. • ELS-induced decrease in CB1r and GRs was sex-dependent and associated with behavior. Early-life stress (ELS) is associated with later onset of depression. Early cannabis use may be a risk factor that interacts with environmental factors to increase the risk of psychopathologies. We aimed to examine the long-term effects of ELS on depression- and anxiety-like behavior, and examine whether chronic fatty acid amide hydrolase (FAAH) inhibition during mid-adolescence could ameliorate or exacerbate ELS effects on behavior. Male and female rats were exposed to ELS during post-natal days (P) 7–14, injected with the FAAH inhibitor URB597 (0.4 mg/kg, i.p.) or vehicle for 2 weeks during mid-adolescence (P30–45) or late-adolescence (P45–60). Rats were tested in adulthood for behavior and alterations in CB1 receptors (CB1r) and glucocorticoid receptors (GRs) in the brains' stress circuit. ELS produced decreased social preference, impaired social recognition, increased learned helplessness and anxiety-like behavior. Administering URB597 during mid-adolescence did not prevent the deleterious long-term effects of ELS on behavior in males and females. When URB597 was administered during late-adolescence , it ameliorated ELS-induced depression- and anxiety-like behavior. Moreover, in males, ELS and URB597 decreased CB1r levels in the prefrontal cortex (PFC) and CA1 and GRs in the PFC and basolateral amygdala (BLA). In females, ELS and URB decreased CB1r in the BLA and GRs in the CA1 and BLA. The findings suggest that mid-adolescence , as opposed to late-adolescence , may not be a potential developmental period for chronic treatment with FAAH inhibitors and that sex-dependent alterations in CB1r and GRs expression in the BLA–PFC–CA1 circuit may contribute to the depressive behavioral phenotype. [ABSTRACT FROM AUTHOR]

Background: Fatty-acid amide hydrolase (FAAH) is an intracellular serine hydrolase that catalyzes the cleavage of endogenous fatty-acid amides, including the endocannabinoid anandamide (AEA). We previously reported that the peripherally restricted FAAH inhibitor URB937, which selectively increases AEA levels outside the central nervous system, reduces hyperalgesia and c-Fos expression in the trigeminal nucleus caudalis (TNC) and the locus coeruleus in an animal model of migraine based on nitroglycerin (NTG) administration. Aim: To further investigate the relevance of FAAH inhibition in the NTG animal model of migraine by testing the effects of the globally active FAAH inhibitor URB597. Methods: Our experimental approach involved mapping neuronal c-Fos protein expression, measurement of AEA levels in brain areas and in trigeminal ganglia, evaluation of pain-related behavior and quantification of molecular mediators in rats that received URB597 (2 mg/kg i.p.) either before or after NTG administration (10 mg/kg, i.p.). Results: Pre-treatment with URB597 significantly reduced c-Fos immunoreactivity in the TNC and inhibited NTG-induced hyperalgesia in the orofacial formalin test. This behavioral response was associated with a decrease in neuronal nitric oxide synthase, calcitonin gene-related peptide and cytokine gene expression levels in central and peripheral structures. Administration of URB597 after NTG had no such effect. Conclusions: The findings suggest that global FAAH inhibition may offer a therapeutic approach to the prevention, but not the abortive treatment, of migraine attacks. Further studies are needed to elucidate the exact cellular and molecular mechanisms underlying the protective effects of FAAH inhibition.

Primary and secondary hypertension is associated with kidney redox imbalance resulting in enhanced reactive oxygen species (ROS) and enzymes dependent phospholipid metabolism. The fatty acid amide hydrolase inhibitor, URB597, modulates the levels of endocannabinoids, particularly of anandamide, which is responsible for controlling blood pressure and regulating redox balance. Therefore, this study aimed to compare the effects of chronic URB597 administration to spontaneously hypertensive rats (SHR) and rats with secondary hypertension (DOCA-salt rats) on the kidney metabolism associated with the redox and endocannabinoid systems. It was shown fatty acid amide hydrolase (FAAH) inhibitor decreased the activity of ROS-generated enzymes what resulted in a reduction of ROS level. Moreover varied changes in antioxidant parameters were observed with tendency to improve antioxidant defense in SHR kidney. Moreover, URB597 administration to hypertensive rats decreased pro-inflammatory response, particularly in the kidneys of DOCA-salt hypertensive rats. URB597 had tendency to enhance ROS-dependent phospholipid oxidation, estimated by changes in neuroprostanes in the kidney of SHR and reactive aldehydes (4-hydroxynonenal and malondialdehyde) in DOCA-salt rats, in particular. The administration of FAAH inhibitor resulted in increased level of endocannabinoids in kidney of both groups of hypertensive rats led to enhanced expression of the cannabinoid receptors type 1 and 2 in SHR as well as vanilloid receptor 1 receptors in DOCA-salt rats. URB597 given to normotensive rats also affected kidney oxidative metabolism, resulting in enhanced level of neuroprostanes in Wistar Kyoto rats and reactive aldehydes in Wistar rats. Moreover, the level of endocannabinoids and cannabinoid receptors were significantly higher in both control groups of rats after URB597 administration. In conclusion, because URB597 disturbed the kidney redox system and phospholipid ROS-dependent and enzymatic-dependent metabolism, the administration of this inhibitor may enhance kidney disorders depending on model of hypertension, but may also cause kidney disturbances in control rats. Therefore, further studies are warranted.

Background/Aims: Recent interest in the use of cannabinoids as therapeutic agents has revealed the involvement of the endogenous cannabinoid system (ECS) in the regulation of the cardiovascular system in hypertension. Abnormalities in glucose metabolism and insulin action are commonly detected in hypertensive animals. Thus, potential antihypertensive drugs should be investigated with respect to modulation of glucose homeostasis. Therefore, the aim of the present study was to evaluate the effects of the ECS activation after chronic fatty acid amide hydrolase inhibitor (URB597) administration on plasma glucose and insulin concentrations as well as parameters of myocardial glucose metabolism in the deoxycorticosterone acetate (DOCA)-salt hypertensive rats, an animal model of secondary hypertension. Methods: Hypertension was induced by DOCA (25mg/kg) injections and addition of 1% NaCl in the drinking water for six weeks. Chronic activation of the ECS was performed by URB597 (1mg/kg) injections for two weeks. We examined fasting plasma levels of insulin (ELISA), glucose and intramyocardial glycogen (colorimetric method). Expressions of glucose transporters (GLUT1, 4) and selected proteins engaged in GLUT translocation as well as glucose metabolism were determined using Western blotting. Results: Hypertension induced hypoinsulinemia with concomitant lack of significant changes in glycemia, reduced intramyocardial glycogen content and increased pyruvate dehydrogenase (PDH) expression in the cardiac muscle. Importantly, chronic URB597 administration in the hypertensive rats increased insulin concentration, elevated plasmalemmal GLUT1 and GLUT4 expression and concomitantly improved myocardial glycogen storage. Conclusion: Chronic administration of fatty acid amide hydrolase (FAAH) inhibitor has potential protective properties on myocardial glucose metabolism in hypertension.

Recent studies using full-field electroretinography (ffERG) that triggers a non-specific mass response generated by several retinal sources have attributed an important role for cannabinoid receptors in mediating vision in primates. Specific cone-mediated responses evoked through the photopic flicker ERG appear to be a better way to validate the assumption that endogenous cannabinoids modulate the cone pathway, since FAAH is mainly expressed in the vervet monkey cone photoreceptors. The aim of this study is two-fold: (1) to use the photopic flicker ERG to target the cone pathway specifically, and (2) use URB597 as a selective inhibitor of the endocannabinoid degrading enzyme Fatty Acid Amide Hydrolase (FAAH) to enhance the levels of fatty acid amides, particularly anandamide. We recorded ERGs under four different flicker frequencies (15, 20, 25, and 30 Hz) in light-adapted conditions after intravitreal injections of URB597. Our results show that intravitreal injections of URB597, compared to the vehicle DMSO, increased significantly ffERG amplitudes at 30 Hz, a frequency that solely recruits cone activity. However, at 15 Hz, a frequency that activates both rods and cones, no significant difference was found in the ERG response amplitude. Additionally, we found no differences in implicit times after URB597 injections compared to DMSO vehicle. These results support the role of molecules degraded by FAAH in cone-mediated vision in non-human primates.

Clinical studies have demonstrated that the NMDA receptor antagonist ketamine produces rapid antidepressant responses. There are safety concerns and adverse effects that limit the utilization of ketamine in psychiatry. Some studies have suggested combination therapy for optimal ketamine use. In this study, we evaluated the potential for combination therapy of ineffective doses of ketamine and fatty acid amide hydrolase inhibitor URB597 for the treatment of depression and pain in male NMRI mice. Intraperitoneal administration of ketamine (10 mg/kg) at the time intervals of 115, 145, and 160 min and ketamine (5 mg/kg) at the time interval of 160 min after administration increased the tail-flick latency, indicating an antinociceptive effect. The same doses of ketamine decreased immobility time in the forced swim test (FST), showing an antidepressant-like effect. Moreover, URB597 at the doses of 0.5 and 1 mg/kg induced an antinociceptive effect, while it at the dose of 1 mg/kg produced an antidepressant-like response. Furthermore, co-administration of the ineffective doses of ketamine (2.5 mg/kg) and URB597 (0.1 mg/kg) caused antinociceptive and antidepressant-like effects, while each one of them alone did not alter the performance of mice in the FST and tail-flick tests. It should be noted that none of the treatments alter animal locomotor activity compared to the control group. Therefore, the combined administration of ineffective doses of ketamine and URB597 might be an effective strategy in the therapy of depression and pain. [ABSTRACT FROM AUTHOR]

The endocannabinoid system modulates neurotransmission at inhibitory and excitatory synapses in brain regions relevant to the regulation of pain, emotion, motivation, and cognition. This signaling system is engaged by the active component of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC), which exerts its pharmacological effects by activation of G protein-coupled type-1 (CB1) and type-2 (CB2) cannabinoid receptors. During frequent cannabis use a series of poorly understood neuroplastic changes occur, which lead to the development of dependence. Abstinence in cannabinoid-dependent individuals elicits withdrawal symptoms that promote relapse into drug use, suggesting that pharmacological strategies aimed at alleviating cannabis withdrawal might prevent relapse and reduce dependence. Cannabinoid replacement therapy and CB1 receptor antagonism are two potential treatments for cannabis dependence that are currently under investigation. However, abuse liability and adverse side-effects may limit the scope of each of these approaches. A potential alternative stems from the recognition that (i) frequent cannabis use may cause an adaptive down-regulation of brain endocannabinoid signaling, and (ii) that genetic traits that favor hyperactivity of the endocannabinoid system in humans may decrease susceptibility to cannabis dependence. These findings suggest in turn that pharmacological agents that elevate brain levels of the endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol (2-AG), might alleviate cannabis withdrawal and dependence. One such agent, the fatty-acid amide hydrolase (FAAH) inhibitor URB597, selectively increases anandamide levels in the brain of rodents and primates. Preclinical studies show that URB597 produces analgesic, anxiolytic-like and antidepressant-like effects in rodents, which are not accompanied by overt signs of abuse liability. In this article, we review evidence suggesting that (i) cannabis influences brain endocannabinoid signaling and (ii) FAAH inhibitors such as URB597 might offer a possible therapeutic avenue for the treatment of cannabis withdrawal.

BackgroundCB(1) cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors. However, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys.MethodsWe investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Delta(9)-tetrahydrocannabinol (THC), anandamide, or cocaine and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay.ResultsURB597 (.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597, and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine, even though, as expected, it significantly potentiated anandamide self-administration.ConclusionsIn the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse.

RationaleA major clinical concern with the use of cannabinoid receptor 1 (CB1) direct agonists is that these compounds increase alcohol drinking and drug abuse-related behaviours. As an alternative approach, CB1-receptor-mediated activity can be facilitated by increasing anandamide levels with the use of hydrolase fatty acid amide hydrolase (FAAH) inhibitors.ObjectiveUsing the selective FAAH inhibitor URB597, we investigated whether activation of the endogenous cannabinoid tone increases alcohol abuse liability, as what happens with the CB1 receptor direct agonists.Materials and methodsURB597 was tested on alcohol self-administration in Wistar rats and on homecage alcohol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. In Wistar rats, URB597 effects on alcohol-induced anxiety and on stress-, yohimbine- and cue-induced reinstatement of alcohol seeking were also evaluated. For comparison, the effect of the CB1 receptor antagonist rimonabant on ethanol self-administration was also tested.ResultsUnder our experimental condition, intraperitoneal (IP) administration of URB597 (0.0, 0.3 and 1.0 mg/kg) neither increased voluntary homecage alcohol drinking in msP rats nor facilitated fixed ratio 1 and progressive ratio alcohol self-administration in nonselected Wistars. In the reinstatement tests, the compound did not have effects on cue-, footshock stress- and yohimbine-induced relapse. Conversely, URB597 completely abolished the anxiogenic response measured during withdrawal after an acute IP administration of alcohol (3.0 g/kg). Rimonabant (0.0, 0.3, 1.0 and 3.0 mg/kg) significantly reduced ethanol self-administration.ConclusionsResults demonstrate that activation of the endocannabinoid anandamide system by selective inhibition of FAAH does not increase alcohol abuse risks but does reduce anxiety associated to alcohol withdrawal. We thus can speculate that medication based on the use of endocannabinoid system modulators such as URB597 may offer important advantages compared to treatment with direct CB1 receptor activators.

RationaleAnticipatory nausea (AN) experienced by chemotherapy patients is resistant to current anti-nausea treatments. In this study, the effect of manipulation of the endocannabinoid (EC) system on a rat model of nausea (conditioned gaping) was determined.ObjectiveThe potential of cannabidiol (CBD) and the fatty acid amide hydrolase (FAAH) inhibitor, URB597 (URB) to reduce conditioned gaping in rats were evaluated.Materials and methodsIn each experiment, rats received four conditioning trials in which they were injected with lithium chloride immediately before placement in a distinctive odor-laced context. During testing, in experiment 1, rats were injected with vehicle (VEH), 1, 5 or 10 mg/kg CBD 30 min before placement in the context previously paired with nausea and in experiment 2, rats were injected with VEH, 0.1 or 0.3 mg/kg URB 2 h before placement in the context. Additional groups evaluated the ability of the CB(1) antagonist/inverse agonist, SR141716A, to reverse the suppressive effects of URB. Experiment 3 measured the potential of URB to interfere with the establishment of conditioned gaping.ResultsWhen administered before testing, CBD (1 and 5, but not 10 mg/kg) and URB (0.3, but not 0.1 mg/kg) suppressed conditioned gaping. The effect of URB was reversed by pre-treatment with the CB(1) antagonist/inverse agonist, SR141716A. When administered before conditioning, URB also interfered with the establishment of conditioned gaping.ConclusionsManipulations of the EC system may have therapeutic potential in the treatment of AN.

Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, we evaluated motivational effects of intravenously administered anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague-Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03-3 mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester; 0.3 mg/kg intraperitoneal), which blocks anandamide's metabolic degradation, anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50-300 microg/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of anandamide and URB597 were evaluated in a light/dark box, both a low anandamide dose (0.3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of anandamide (3 mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of anandamide plus URB597 and development of place aversions with URB597 plus anandamide were prevented by the CB1-receptor antagonist AM251 (3 mg/kg intraperitoneal). Thus, additive interactions between the effects of anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597.

Ischemic stroke is a leading cause of death and disability, and medical treatments for ischemic stroke are very limited. URB597 is a potent and selective inhibitor of fatty acid amide hydrolase (FAAH). However, the effect of URB597 on ischemic stroke and the underlying molecular mechanisms remain little known. In this study, focal cerebral ischemia was induced by transient middle cerebral artery occlusion in mice. Our results showed that URB597 dose-dependently improved neurological function and reduced brain infarct volume and brain edema 24 h after brain ischemia. The most effective dose was 1 mg/kg and the therapeutic time window was within 3 h after ischemic stroke. To further investigate the underlying mechanism, necroptosis and autophagy flux were detected by Western blot and/or immunofluorescence staining with or without chloroquine, an autophagic flux inhibitor. Our results showed that URB597 promoted autophagic flux and reduced neuronal necroptosis after brain ischemia and these effects could be abolished by chloroquine. In addition, we found that peroxisome proliferator-activated receptor α (PPARα) antagonist GW6471 partly abolished the effect of URB597 against brain ischemia and URB597 upregulated the expressions of PPARα. In conclusion, URB597 exerts a neuroprotective effect in a dose- and time-dependent manner, and this effect may be related to its restoration of autophagic flux and inhibition of neuronal necroptosis. PPARα is involved in the neuroprotective effect of URB597. This study provides novel evidence that URB597 may be a promising agent for the clinical treatment of ischemic stroke. • URB597 exerts a neuroprotective effect against brain ischemia in a dose- and time-dependent manner. • URB597 restores autophagic flux and inhibited neuronal necroptosis after brain ischemia. • URB597 exerts its neuroprotective effect partly by activating PPARα. [ABSTRACT FROM AUTHOR]

BackgroundThe 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.MethodsWe 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.ResultsDaily 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.ConclusionsURB597 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.

RationaleThe endogenous cannabinoid system plays a vital role in the control of nausea and emesis. Because of the rapid breakdown and hydrolysis of endocannabinoids, such as anandamide, the therapeutic effects may be enhanced by prolonging their duration of action.ObjectiveThe present experiment evaluated the potential of various doses of URB597, a fatty acid amide hydrolase (FAAH) inhibitor, alone and in combination with systemic administration of anandamide to modulate the establishment of lithium-induced conditioned taste reactivity responses in rats.Materials and methodsIn experiment 1, on the conditioning day, rats first received an injection of 0.3 mg/kg URB597, 0.15 mg/kg URB597, or vehicle and then received a second injection of anandamide (5 mg/kg) or vehicle, before a 3-min exposure of 0.1% saccharin by intraoral infusion. Immediately after the saccharin exposure, the rats were injected with lithium chloride. On each of three test days, rats received a 3-min intraoral infusion of saccharin solution, and the taste reactivity responses were videotaped and monitored. In experiment 2, the effects of pretreatment with the CB(1) antagonist, AM-251, on URB597 and anandamide-induced suppressed aversion was evaluated.ResultsAdministration of URB597 alone and in combination with anandamide reduced active rejection reactions elicited by a LiCl-paired saccharin solution; both effects were reversed by pretreatment with AM-251, suggesting that they were CB(1) receptor mediated.ConclusionsThe results suggest that prolonging the action of anandamide by pretreatment with the FAAH inhibitor, URB597, suppresses lithium-induced nausea in the rat.

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer’s disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-β peptide (Aβ). The morphological evaluation showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aβ. Moreover, URB597 reduced both the increase of Rho protein activation in Aβ-treated BV-2 cells and the Aβ-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.

The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, delta9-tetrahydrocannabinol, acts by binding to brain CB1 cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, we show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB1 antagonist rimonabant, are accompanied by increased brain anandamide levels, and are maintained upon repeated URB597 administration. Unlike direct CB1 agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of delta9-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs.

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB1 receptor (CB1R) expression were elevated. The CB1R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB1R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.

Complex regional pain syndrome (CRPS) describes an array of painful conditions that are characterized by continuing regional pain. CRPS comprises severe and inappropriate pain in cases of complete recovery after trauma. Research on the pharmacological treatment of CRPS, however, has not been well investigated. In this study, we compared the pain relief effects of different drugs (URB597, pyrrolidine dithiocarbamate, and hydralazine) in a rat model of chronic post-ischemic pain-induced CRPS. After drug injection, CRPS-induced mechanical allodynia was significantly recovered. After three repetitive drug injections, mechanical sensitivity generally improved as hyper-nociception subsided. Reduced Nav1.7 expression at dorsal root ganglions (DRGs) was observed in the drug treatment groups. Neural imaging analysis revealed decreased neural activity for each drug treatment, compared to vehicle. In addition, treatments significantly reduced IL-1β, IL-6, and TNFα expression in DRGs. These results indicated that drugs could reduce the expression of inflammatory factors and alleviate the symptoms of chronic post-ischemic pain-induced CRPS.

The maturation of attentional control during adolescence might influence later functional outcome or predisposition to psychiatric disorders. During adolescence, the cannabinoid system is particularly sensitive to pharmacological challenges, with potential impact on cognitive functions. Here, we used a recently validated five-choice serial reaction time task protocol to test adolescent C57BL/6J mice. We showed that the pharmacological inhibition (by URB597) of the fatty acid amide hydrolase (FAAH), the major enzyme implicated in anandamide degradation, prevented cognitive disruptions induced by distracting cues in adolescent mice. In particular, these protective effects were indicated by increased accuracy and correct responses and decreased premature responses selectively in the distractor trials. Notably, at the relatively low dose used, we detected no effects in other cognitive, motor, or incentive measures nor long-lasting or rebound effects of FAAH inhibition in cognitive functions. Overall, these data provide initial evidence of selective procognitive effects of FAAH inhibition in measures of attentional control in adolescent mice.

The maturation of attentional control during adolescence might influence later functional outcome or predisposition to psychiatric disorders. During adolescence, the cannabinoid system is particularly sensitive to pharmacological challenges, with potential impact on cognitive functions. Here, we used a recently validated five-choice serial reaction time task protocol to test adolescent C57BL/6J mice. We showed that the pharmacological inhibition (by URB597) of the fatty acid amide hydrolase (FAAH), the major enzyme implicated in anandamide degradation, prevented cognitive disruptions induced by distracting cues in adolescent mice. In particular, these protective effects were indicated by increased accuracy and correct responses and decreased premature responses selectively in the distractor trials. Notably, at the relatively low dose used, we detected no effects in other cognitive, motor, or incentive measures nor long-lasting or rebound effects of FAAH inhibition in cognitive functions. Overall, these data provide initial evidence of selective procognitive effects of FAAH inhibition in measures of attentional control in adolescent mice. [ABSTRACT FROM AUTHOR]

Abstract Hypertension is a civilization disease leading to remodeling and damage of blood vessels, impaired renal function and premature death. The aim of this study was to compare the effect of chronic administration of URB597, the FAAH (fatty acid amide hydrolase) inhibitor, to rats with primary (SHRs) and secondary (DOCA-salt hypertensive rats) hypertension on electrical and physicochemical properties of kidney cells membranes. Changes in the electrical charge of the membrane may affect the cell functions. The electrical properties of the kidney cells (surface charge density, zeta potential) were measured by electrophoresis. Qualitative and quantitative composition of the membrane (phospholipids and proteins) was determined by HPLC and lipid peroxidation product (4-hydroxy-2E-hexenal; 4-HHE) level was examined by GCMSMS, while the sialic acid content was measured by resorcinol method. In rats with primary hypertension (SHR) and secondary hypertension (DOCA-salt), changes in electrical properties (increase of electric charge and zeta potential) and membrane composition (increase in sialic acid and protein concentration and decrease in phospholipid level) of kidney cells are observed in comparison to control animals. Greater changes were observed in DOCA-salt hypertensive rats. Changes in membrane properties caused by URB597 depend on the type of hypertension. The administration of URB597 to rats with primary hypertension partially prevents changes in the electrical properties (electrical charge, zeta potential) of the membrane caused by hypertension as well as in the sialic acid and proteins content. However, there is no reduction in oxidative stress, assessed by the level of 4-HHE, which may affect the metabolic function of the kidneys. URB597 administered to rats with DOCA salt does not prevent, but rather intensifies, changes caused by hypertension in the kidney. In conclusion, URB597 given to individuals with hypertension, particularly with secondary hypertension, enhancing some disturbances in electric and physicochemical properties of kidney cells observed in hypertension what may lead to additional kidney disorders. Therefore, further researches are necessary. Highlights • In hypertension, the physicochemical properties of cell membrane change. • Greater changes were observed in DOCA-salt hypertensive rats. • Changes in membrane properties caused by URB597 depend on the type of hypertension. • URB597 administered to SHR partially prevents changes in the electrical properties. • URB597 administered to DOCA-salt rats does not prevent changes caused by hypertension. [ABSTRACT FROM AUTHOR]

Highlights • Fatty acid amide hydrolase inhibition by URB597 treatment decreases blood pressure in spontaneously hypertensive rats. • URB597 upregulates intramyocardial fatty acids metabolism in normotensive as well as hypertensive conditions. • URB597 enhances myocardial fatty acids uptake but not oxidation in spontaneously hypertensive rats. Abstract Pressure overload, which is typical of hypertension, is known to evoke alterations not only in the morphology of the heart but also in the preference of myocardial energetic substrates usage. Nowadays, the endocannabinoid system (ECS) serves as a potential therapeutic target for cardiovascular disorders and, simultaneously, affects whole body metabolism homeostasis. Therefore, an open question is whether ECS, apart from decreasing blood pressure, also affects cardiac muscle metabolism in hypertensive conditions. All experiments were conducted on a genetic model of primary hypertension i.e. spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) served as a normotensive control. ECS was chronically activated by 2-weeks intraperitoneal injections of fatty acid amide hydrolase (FAAH) inhibitor – URB597. Lipid analyses in the left ventricle and serum were based on ex vivo heart perfusion in Langendorff perfusion system, thin layer chromatography, and gas liquid chromatography. The total expression of selected proteins was determined using Western blot as well as immunohistochemical techniques. As expected, URB597 markedly reduced systolic as well as mean blood pressures in SHRs. Moreover, prolonged FAAH inhibition resulted in stimulation of 3H-palmitate uptake and incorporation into different lipid fractions in cardiomyocytes in the hypertensive as well as normotensive conditions. An increase in fatty acid oxidation caused by URB597 treatment was observed only in WKY rats, but not SHRs, and was accompanied by an elevation in peroxisome proliferator-activated receptor alpha (PPARα) and β-hydroxyacyl-CoA dehydrogenase (β-HAD) expressions. Chronic activation of ECS significantly upregulates palmitate uptake and its esterification but not oxidation in the SHR's myocardium. [ABSTRACT FROM AUTHOR]

The insular cortex is an important region of brain involved in the processing of pain and emotion. Recent studies indicate that lesions in the insular cortex induce pain asymbolia and reverse neuropathic pain. Endogenous cannabinoids (endocannabinoids), which have been shown to attenuate pain, are simultaneously degraded by fatty acid amide hydrolase (FAAH) that halts the mechanisms of action. Selective inhibitor URB597 suppresses FAAH activity by conserving endocannabinoids, which reduces pain. The present study examined the analgesic effects of URB597 treatment in the insular cortex of an animal model of neuropathic pain. Under pentobarbital anesthesia, male Sprague-Dawley rats were subjected to nerve injury and cannula implantation. On postoperative day 14, rodents received microinjection of URB597 into the insular cortex. In order to verify the analgesic mechanisms of URB597, cannabinoid 1 receptor (CB1R) antagonist AM251, peroxisome proliferatoractivated receptor alpha (PPAR alpha) antagonist GW6471, and transient receptor potential vanilloid 1 (TRPV1) antagonist Iodoresiniferatoxin (I-RTX) were microinjected 15 min prior to URB597 injection. Changes in mechanical allodynia were measured using the von-Frey test. Expressions of CB1R, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), and TRPV1 significantly increased in the neuropathic pain group compared to the sham-operated control group. Mechanical threshold and expression of NAPE-PLD significantly increased in groups treated with 2 nM and 4 nM URB597 compared with the vehicle-injected group. Blockages of CB1R and PPAR alpha diminished the analgesic effects of URB597. Inhibition of TRPV1 did not effectively reduce the effects of URB597 but attenuated expression of NAPE-PLD compared with the URB597-injected group. In addition, optical imaging demonstrated that neuronal activity of the insular cortex was reduced following URB597 treatment. Our results suggest that microinjection of FAAH inhibitor into the insular cortex causes analgesic effects by decreasing neural excitability and increasing signals related to the endogenous cannabinoid pathway in the insular cortex. [ABSTRACT FROM AUTHOR]

Background In rodent models, chronic exposure to cannabis' psychoactive ingredient, Δ9-tetrahydrocannabinol, during adolescence leads to abnormal behavior in adulthood. In female rats, this maladaptive behavior is characterized by endophenotypes for depressive-like and psychotic-like disorders as well as cognitive deficits. We recently reported that most depressive-like behaviors triggered by adolescent Δ9-tetrahydrocannabinol exposure can be rescued by manipulating endocannabinoid signaling in adulthood with the anandamide-inactivating enzyme FAAH inhibitor, URB597. However, the molecular mechanisms underlying URB597's antidepressant-like properties remain to be established. Methods Here we examined the impact of adult URB597 treatment on the cellular and functional neuroadaptations that occurred in the prefrontal cortex and dentate gyrus of the hippocampus upon Δ9-tetrahydrocannabinol during adolescence through biochemical, morphofunctional, and electrophysiological studies. Results We found that the positive action of URB597 is associated with the rescue of Δ9-tetrahydrocannabinol-induced deficits in endocannabinoid-mediated signaling and synaptic plasticity in the prefrontal cortex and the recovery of functional neurogenesis in the dentate gyrus of the hippocampus. Moreover, the rescue property of URB597 on depressive-like behavior requires the activity of the CB1 cannabinoid receptor. Conclusions By providing novel insights into the cellular and molecular mechanisms of URB597 at defined cortical and hippocampal circuits, our results highlight that positive modulation of endocannabinoid-signaling could be a strategy for treating mood alterations secondary to adolescent cannabis use. [ABSTRACT FROM AUTHOR]

The endocannabinoid system is prominently implicated in the control of cocaine reinforcement due to its relevant role in synaptic plasticity and neurotransmitter modulation in the mesocorticolimbic system. The inhibition of fatty acid amide hydrolase (FAAH), and the resulting increase in anandamide and other N-acylethanolamines, represents a promising strategy for reducing drug seeking. In the present study, we aimed to assess the effects of the FAAH inhibitor URB597 (1 mg/kg) on crucial features of cocaine addictive-like behaviour in mice. Therefore, we tested the effects of URB597 on acquisition of cocaine (0.6 mg/kg/inf) self-administration, compulsive-like cocaine intake and cue-induced drug-seeking behaviour during withdrawal. URB597 reduced cocaine intake under conditioned punishment while having no impact on acquisition. This result was associated to increased cannabinoid receptor 1 gene expression in the ventral striatum and medium spiny neurons activation in the nucleus accumbens shell. Moreover, URB597 mitigated cue-induced drug-seeking behaviour during prolonged abstinence and prevented the withdrawal-induced increase in FAAH gene expression in the ventral striatum. In this case, URB597 decreased activation of medium spiny neurons in the nucleus accumbens core. Our findings evidence the prominent role of endocannabinoids in the development of cocaine addictive-like behaviours and support the potential of FAAH inhibition as a therapeutical target for the treatment of cocaine addiction. • FAAH inhibition does not interfere with cocaine self-administration. • URB597 decreases cocaine intake under the risk of punishment. • URB597 increases activation of MSN in the NAc shell and increases CB1R gene expression during conditioned punishment. • URB597 mitigates cue-induced drug-seeking behaviour during abstinence. • URB597 during abstinence decreases activation of MSN in the NAc core and prevents FAAH gene expression increase. [ABSTRACT FROM AUTHOR]

Hypertension is accompanied by oxidative stress, which can be modified by the functioning of the endocannabinoid system playing a prominent modulatory role in the brain. The present study tested whether chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl) phenyl]N-cyclohexylcarbamate (URB597) to rats with primary hypertension (SHR) can modify redox balance and consequently brain phospholipid metabolism. Experiments were conducted using SHRs and normotensive control Wistar–Kyoto rats treated by intraperitoneal injection with URB597 for 14 days. The biochemical parameters were assayed in the rats’ brains. Inhibition of FAAH activity by URB597 resulted in an increase in anandamide and GPR55 receptor levels, as well as a decrease in CB2 receptor expression. However, there was a simultaneous increase in Nrf2 expression, as well as Cu, Zn-SOD, GSH-Px, glutathione reductase activity, and vitamin E levels in brain tissue of SHR rats. Consequently, URB597 caused a decrease in levels of phospholipid fatty acids and MDA, and an increase in free fatty acids. Given the importance of maintaining redox balance for brain function, the results of this study point to endocannabinoids as a potential therapeutic target for preventing brain metabolic disorders in hypertension.

Hypertension coincides with myocardial alternations in lipid (including sphingolipids) and glucose metabolism. The latest data indicate that accumulation of metabolically active lipids, especially ceramide (CER) and diacylglycerol (DAG) significantly influences intracellular signaling pathways along with inducing insulin resistance. Since, it was demonstrated that the endocannabinoid system (ECS) affects myocardial metabolism it seems to be a relevant tool in alleviating metabolic disturbances within the cardiac muscle due to hypertension. All designed experiments were conducted on the animal model of primary hypertension, i.e., spontaneously hypertensive rat (SHR) with chronic ECS activation by injections of fatty acid amide hydrolase (FAAH) inhibitor—URB597. Lipid analyses were performed using chromatography techniques (gas liquid, thin layer, and high performance liquid chromatography). Colorimetric and immunoenzymatic testes were applied in order to determine plasma concentrations of insulin and glucose. Total myocardial expression of selected proteins was measured by Western blotting and/or immunohistochemistry methods. SHRs exhibited significantly intensified myocardial de novo pathway of CER synthesis as well as DAG accumulation compared to the control Wistar Kyoto rats. Besides, intramyocardial level of potentially cardioprotective sphingolipid, i.e., sphingosine-1-phosphate was considerably decreased in SHRs, whereas URB597 treatment restored the level of this derivative. Unexpectedly, ECS upregulation protected overloaded cardiac muscle against CER and DAG accumulation. Moreover, chronic URB597 treatment improved intramyocardial insulin signaling pathways in both normotensive and hypertensive conditions. It seems that the enhanced ECS triggers protective mechanisms in the heart due to decreasing the level of lipid mediators of insulin resistance.

Fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) may influence redox balance and blood pressure through the modulation of endocannabinoids levels. Therefore, this study aimed to compare changes in oxidative metabolism and apoptosis in the hearts of rats with spontaneous hypertension (SHR) and secondary hypertension (11-deoxycorticosterone acetate; DOCA-salt rats) treated by URB597 via intraperitoneal injection for 14 days. The results showed that URB597 decreased the activity of NADPH and xanthine oxidases in both groups of rats. Moreover, in the heart of SHR rats, URB597 led to an increase of enzymatic and nonenzymatic antioxidant activity and levels (catalase, vitamin C, glutathione/glutathione disulfide [GSH/GSSG]) and upregulation of the thioredoxin system; however, NRf2 expression was downregulated. The opposite effect in relation to Nrf2 activity and the thioredoxin system was observed in DOCA-salt rats after URB597 administration. Despite improvement in antioxidant parameters, URB597 enhanced oxidative modifications of phospholipids (4-hydroxynonenal and isoprostanes) and proteins (carbonyl groups) in SHR heart, whereas 4-hydroxynonenal and carbonyl groups levels decreased in the heart of DOCA-salt rats. Obtained results suggest that examined lipid mediators are involved in peroxisome proliferator-activated receptors (PPAR)-independent and PPAR-dependent modulation of cardiac inflammatory reactions. Furthermore, decreased expression of pro-apoptotic proteins (Bax and caspase 3 and 9) was observed after URB597 administration in the heart of both groups of hypertensive rats, whereas expression of the antiapoptotic protein (Bcl-2) increased in SHR rats. Long-term administration of URB597 altered cardiac redox status depending on the type of hypertension. URB597 enhanced oxidative metabolism and reduced pro-apoptotic factors in the heart of SHR rats, increasing the probability of heart metabolic disorders occurrence or progression. [ABSTRACT FROM AUTHOR]