Fatty Acid Amide Hydrolase Inhibitor Research Articles

Pharmacological inhibition of the endocannabinoid anandamide metabolism alleviates cisplatin-induced acute kidney injury

Fatty acid amide hydrolase (FAAH) is the primary enzyme that degrades the endocannabinoid anandamide (AEA). Inhibition of FAAH by pharmacology or genetic approach has been shown to reduce inflammation in the brain, colon, heart and kidneys. Kidney medullary infusion of a FAAH inhibitor exerts diuretic and natriuretic effects. Faah knockout mice are protected from both post ischemia reperfusion injury and cisplatin-induced acute kidney injury (AKI) but through distinct mechanisms. The presented study tested the hypothesis that pharmacological inhibition of FAAH activity mitigates cisplatin-induced AKI and explored the potential renoprotective mechanism. Male wild type C57BL/6 (WT) were treated with oral gavage of a FAAH inhibitor (PF-04457845, PF, 5mg/kg) or vehicle (10% PEG200+5% Tween80+normal saline) at 72, 48, 24, 2-hr before and 24, 48-hr after a single dose of intraperitoneal injection of cisplatin (Cis, 25 mg/kg). Mice were euthanatized 72 hours after cisplatin treatment. PF-treated mice showed a decrease of cisplatin-induced plasma creatinine levels compared with vehicle (Veh)-treated mice (mean: 0.23±0.17, 1.98± 1.09, 0.31±0.16 and 1.11±0.56 mg/dL in Veh, Veh+Cis, PF and PF+Cis, respectively, p=0.02). Western blot analysis revealed that increased kidney injury molecule-1 (KIM-1) levels in kidney tissue of Veh-mice by cisplatin were reduced in PF-treated mice (2-fold increase vs. 1.1-fold in Veh+Cis vs. PF+Cis compared to the Veh-mice, respectively, p=0.04). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that PF+Cis mice displayed enhanced AEA tone compared with Veh+Cis mice (1.3-fold increase vs. 2.9-fold in Veh+Cis vs. PF+Cis compared to the Veh-mice, respectively, p=0.03). These results suggest that the renal protection from oral gavage of the FAAH inhibitor against cisplatin nephrotoxicity may be associated with enhanced tone of AEA and that pharmacological inactivation of FAAH could be a novel strategy to prevent cisplatin-induced AKI. NIH grant R01HL145163 and P30DA033934 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combination treatment with FAAH inhibitors/URB597 and ferroptosis inducers significantly decreases the growth and metastasis of renal cell carcinoma cells via the PI3K-AKT signaling pathway

Ferroptosis, a nonapoptotic form of programmed cell death characterized by significant iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various cancer-related signaling pathways. Recently, considerable progress has been made in demonstrating the critical role of lipid metabolism in regulating ferroptosis, indicating the potential of combinational strategies for treating cancer in the future. In this study, we explored the combinational effects of lipid metabolism compounds and ferroptosis inducers on renal cell carcinoma (RCC) cells. We found potent synergy of the fatty acid amide hydrolase (FAAH) inhibitor URB597 with ferroptosis inducer (1S, 3R)-RSL3 (RSL3) in inhibiting the growth and metastasis of RCC cells both in vitro and in vivo via induction of G1 cell cycle arrest and promotion of the production of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and cytosolic reactive oxygen species (ROS). In addition, inhibition of FAAH increased the sensitivity of RCC cells to ferroptosis. Genome-wide RNA sequencing indicated that the combination of URB597 and RSL3 has more significant effects on regulation of the expression of genes related to cell proliferation, the cell cycle, cell migration and invasion, and ferroptosis than either single agent alone. Moreover, we found that combinational treatment modulated the sensitivity of RCC cells to ferroptosis via the phosphatidylinositol 3 kinase (PI3K)-AKT signaling pathway. These data demonstrate that dual targeting of FAAH and ferroptosis could be a promising strategy for treating RCC.

Fatty acid amide hydrolase inhibition alleviates anxiety-like symptoms in a rat model used to study post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD), a chronic debilitating condition that affects nearly 5-10% of American adults, is treated with a handful of FDA-approved drugs that provide at best symptomatic relief and exert multiple side effects. Preclinical and clinical evidence shows that inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which deactivates the endocannabinoid anandamide, exhibit anxiolytic-like properties in animal models. In the present study, we investigated the effects of two novel brain-permeable FAAH inhibitors - the compounds ARN14633 and ARN14280 - in a rat model of predator stress-induced long-term anxiety used to study PTSD. We exposed male Sprague-Dawley rats to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a volatile constituent of fox feces, and assessed anxiety-like behaviors in the elevated plus maze (EPM) test seven days later. We measured FAAH activity using a radiometric assay and brain levels of FAAH substrates by liquid chromatography/tandem mass spectrometry. Rats challenged with TMT developed persistent (≥ 7days) anxiety-like symptoms in the EPM test. Intraperitoneal administration of ARN14633 or ARN14280 1h before testing suppressed TMT-induced anxiety-like behaviors with median effective doses (ED50) of 0.23 and 0.33mg/kg, respectively. The effects were negatively correlated (ARN14663: R2 = 0.455; ARN14280: R2 = 0.655) with the inhibition of brain FAAH activity and were accompanied by increases in brain FAAH substrate levels. The results support the hypothesis that FAAH-regulated lipid signaling serves important regulatory functions in the response to stress and confirm that FAAH inhibitors may be useful for the management of PTSD.

The Estrous Cycle Influences the Effects of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibition in the Anxiety-Like Behavior in Rats.

Background: Sex differences in the response to the anxiety-related effects of cannabinoid drugs have been reported, with females being more sensitive than males. Evidence suggests that, according to sex and estrous cycle phase (ECP), the content of the endocannabinoids (eCBs) N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) varies in brain areas involved in the anxiety-like behavior. Methods: Considering the lack of studies evaluating sex and ECP differences in the eCB system in anxiety, using URB597, a fatty acid amide hydrolase inhibitor, or MJN110, a monoacylglycerol lipase inhibitor, we explored the effects of increasing AEA or 2-AG levels, respectively, in cycling and ovariectomized (OVX) female adult Wistar rats, as well as males, subjected to the elevated plus maze. Results: The administration of URB597 (0.1 or 0.3mg/kg; intraperitoneally) either increased or reduced the percentage of open arms time (%OAT) and open arms entries (%OAE), being anxiolytic in diestrus and anxiogenic in estrus. No effects were observed in proestrus or when all ECPs were analyzed together. Both doses produced anxiolytic-like effects in males. In OVX females, the anxiolytic-like effect of URB597 0.1 was associated with low levels of estradiol, whereas the anxiogenic-like effect of URB597 0.3 was spared by estradiol pretreatment. The systemic administration of MJN110 3.0 mg/kg reduced the risk assessment behavior (RAB), suggesting an anxiolytic-like effect independent of the ECP. When considering the ECP, MJN110 3.0 increased the %OAT and reduced the RAB, being anxiolytic in estrus and diestrus. No effects were observed in proestrus. Both doses of MJN110 were anxiogenic in males. In OVX females, the anxiolytic-like effect of MJN110 was dependent on low estradiol levels. Conclusion: Together, our findings support the evidence that females react differently to the effects of cannabinoids in the anxiety-like behavior; in addition, AEA and 2-AG modulation elicits anxiety-like responses that are closely influenced by hormone levels, mainly estradiol.

Neuropharmacological Modulation of N-methyl-D-aspartate, Noradrenaline and Endocannabinoid Receptors in Fear Extinction Learning: Synaptic Transmission and Plasticity

Learning to recognize and respond to potential threats is crucial for survival. Pavlovian threat conditioning represents a key paradigm for investigating the neurobiological mechanisms of fear learning. In this review, we address the role of specific neuropharmacological adjuvants that act on neurochemical synaptic transmission, as well as on brain plasticity processes implicated in fear memory. We focus on novel neuropharmacological manipulations targeting glutamatergic, noradrenergic, and endocannabinoid systems, and address how the modulation of these neurobiological systems affects fear extinction learning in humans. We show that the administration of N-methyl-D-aspartate (NMDA) agonists and modulation of the endocannabinoid system by fatty acid amide hydrolase (FAAH) inhibition can boost extinction learning through the stabilization and regulation of the receptor concentration. On the other hand, elevated noradrenaline levels dynamically modulate fear learning, hindering long-term extinction processes. These pharmacological interventions could provide novel targeted treatments and prevention strategies for fear-based and anxiety-related disorders.

Anxiety associated with palatable food withdrawal is reversed by the selective FAAH inhibitor PF-3845: A regional analysis of the contribution of endocannabinoid signaling machinery.

Consumption of energy-dense palatable "comfort" food can alleviate stress and negative emotions, while abrupt withdrawal from a palatable diet can worsen these symptoms, causing difficulties with adherence to weight-loss diets. Currently, no pharmacological treatment is effective for obesity-related anxiety, so we investigated the endocannabinoid system (ECS), and specifically the fatty acid amidehydrolase (FAAH), as an interesting emerging target in this context because of its key role in the regulation of both energy homeostasis and emotional behavior. Rats were subjected to exposure and subsequent abstinence from a palatable cafeteria diet. During abstinence period, rats were treated with the selective FAAH inhibitor PF-3845 (10mg/kg; intraperitoneal administration every other day). Abstinent rats displayed an anxiogenic-like behavior and changes in the proteins of ECS signaling machinery in brain areas involved both in anxiety and food intake regulation. In particular, withdrawal caused a reduction of the expression of cannabinoid receptors in the nucleus accumbens and of enzymes diacylglycerol lipase alpha and monoacylglycerol lipase (MAGL) in the amygdala. Pharmacological inhibition of FAAH exerted an anxiolytic-like effect in abstinent animals and increased both MAGL expression in amygdala and CB2 expression in prefrontal cortex. Overall, our results suggest that emotional disturbances associated with dieting are coupled with region-specific alterations in the cerebral expression of the ECS and that the enhancement of the endocannabinoid signaling by FAAH inhibition might represent a novel pharmacological strategy for the treatment of anxiety related to abstinence from palatable food. The present study focused on evaluating the role of the endocannabinoid system in modulating withdrawal from naturally rewarding activities that have an impact on mood, such as feeding. The variations observed in the emotional behavior of abstinent rats was linked to neuroadaptations of the ECS in specific brain areas.

Phytochemical, GC-MS, FTIR and Amino acid profile of methanol extract of Tetrapleura tetraptera fruit

The methanolic fruit extract of Tetrapleura tetraptera was analyzed for the presence of phytocompounds, their bioactivity, the functional groups involved in this activity, and its amino acid profile using standard procedures. Phytochemicals such as tannins, phenols, flavonoids, and alkaloids were identified as being highly present. Gas chromatographic-mass spectrometric (GC-MS) analysis identified 16 bioactive compounds, with 2-thiopheneethanol (58.77%) being the most abundant. Curcumin, with the most diverse pharmacological role, and other bioactive compounds such as cedren-13-ol, 8 (1.56%), N-benzyl stearamide (4.46%), a prominent fatty acid amide hydrolase (FAAH) inhibitor; pthalic acid, butyl undecyl ester (1.49%); and phenol, 2, 6-bis (1,1 dimethyl ethyl) (1.46%), were also present. Fourier transform infrared (FTIR) analysis confirmed the presence of alkanes, esters, benzene rings, aliphatic, sulfonic acid, and methylene chains. Also, the amino acid analysis of the T. tetraptera revealed that the fruit contains 18 amino acids. Leucine (4.20%), phenylalanine (3.37%), and valine (3.25%) were the most abundant essential amino acids identified, with glutamic (7.20%) and aspartic acid (5.61%) having the highest concentrations as non-essential amino acids. This therefore indicates that T. tetraptera fruit could be used as a pharmacological or therapeutic agent as well as a dietary condiment, particularly at this time when there is a demand for novel protein sources.
 Keywords: Tetrapleura tetraptera, phytochemicals, GC-MS, FTIR, amino acid, curcumin

Fatty acid amide hydrolase inhibitor URB597 suppressed conditioned and sensitized fear responses in a rat model of post-traumatic stress disorder

BackgroundPost-traumatic stress disorder (PTSD) is a debilitating disorder that results from exposure to various traumatic events. Endocannabinoids levels change during PTSD. Inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide degradation, has shown potential in treating PTSD. MethodsWe used male Wistar rats and single prolonged stress plus shock as the experimental model of PTSD-like phenotype. Freezing times during conditioned fear response (CFR) and sensitized fear response (SFR) were recorded. The effect of URB597 (0.1, 0.3, and 0.6 mg/kg), a FAAH inhibitor, and fluoxetine (10 mg/kg) as control drug on CFR and SFR were evaluated for two weeks. After the test, the anxiety-like and depression-like behaviors of the animals were assessed by elevated plus-maze and forced swim test, respectively. The serum concentration of corticosterone was also measured at the end of the experiments. ResultsThe results showed that fluoxetine and URB597 suppressed freezing time in both CFR and SFR procedures. The drugs showed apparent anxiolytic-like and antidepressant-like effects. URB597 at the dose of 0.6 mg/kg decreased corticosterone level. ConclusionsThe results of the present study showed that URB597 induced a protective effect against experimental PTSD-like phenotype. Regarding its anxiolytic-like and antidepressant-like effects, the drug may be suggested as a potential treatment for PTSD.

Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates Cisplatin-Induced Nephropathy in Mice.

Cisplatin is a potent first-line therapy for many solid malignancies, such as breast, ovarian, lung, testicular, and head and neck cancer. However, acute kidney injury (AKI) is a major dose-limiting toxicity in cisplatin therapy, which often hampers the continuation of cisplatin treatment. The endocannabinoid system, consisting of anandamide (AEA) and 2-arachidonoylglycerol and cannabinoid receptors, participates in different kidney diseases. Inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme for the degradation of AEA and AEA-related N-acylethanolamines, elicits anti-inflammatory effects; however, little is known about its role in cisplatin nephrotoxicity. The current study tested the hypothesis that genetic deletion of Faah mitigates cisplatin-induced AKI. Male wild-type C57BL6 (WT) and Faah-/- mice were administered a single dose of intraperitoneal injection of cisplatin (30 mg/kg) and euthanatized 72 hours later. Faah-/- mice showed a reduction of cisplatin-induced blood urea nitrogen, plasma creatinine levels, kidney injury markers, and tubular damage in comparison with WT mice. The renal protection from Faah deletion was associated with enhanced tone of AEA-related N-acylethanolamines (palmitoylethanolamide and oleoylethanolamide), attenuated nuclear factor-κB/p65 activity, DNA damage markers p53 and p21, and decreased expression of the inflammatory cytokine interleukin-1β, as well as infiltration of macrophages and leukocytes in the kidneys. Notably, a selective FAAH inhibitor (PF-04457845) did not interfere with or perturb the antitumor effects of cisplatin in two head and neck squamous cell carcinoma cell lines, HN30 and HN12. Our work highlights that FAAH inactivation prevents cisplatin-induced nephrotoxicity in mice and that targeting FAAH could provide a novel strategy to mitigate cisplatin-induced nephrotoxicity. SIGNIFICANCE STATEMENT: Mice lacking the Faah gene are protected from cisplatin-induced inflammation, DNA damage response, tubular damage, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to mitigate cisplatin-induced nephrotoxicity.

Combined targeting of fatty acid amide hydrolase and melatonin receptors promotes neuroprotection and stimulates inflammation resolution in rats.

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. 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. 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. 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.

Multi-targeted anti-inflammatory drugs for the treatment of neurological disorders.

Multi-targeted anti-inflammatory drugs for the treatment of neurological disorders.

Unraveling the effects of cannabidiol and the fatty acid amide hydrolase inhibitor URB597 on cocaine-seeking under conditioned punishment

Unraveling the effects of cannabidiol and the fatty acid amide hydrolase inhibitor URB597 on cocaine-seeking under conditioned punishment

Protective effect of fatty acid amide hydrolase inhibitor URB597 and monoacylglycerol lipase inhibitor KML29 on renal ischemia-reperfusion injury via toll-like receptor 4/nuclear factor-kappa B pathway.

Arachidonoyl ethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the most studies endocannabinoids. AEA and 2-AG are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes, respectively. FAAH and MAGL enzymes are widely expressed in many tissues, including kidney. Recent works have depicted that AEA and 2-AG levels are associated with ischemia-reperfusion (IR) injury. In this study, we investigated the effects of MAGL inhibitor KML29 and FAAH inhibitor URB597 against kidney IR injury. The kidneys of the rats underwent ischemia for 45min and then reperfusion for 24h. KML29 and URB597 were administered intraperitoneally with kidney IR to two different treatment groups. IR application increased serum blood urea nitrogen (BUN), creatinine (Cre), interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels, while these parameters were decreased following KML29 and URB597 administration. KML29 and URB597 administration also reduced the increased toll-like receptor-4 (TRL-4), phosphorylated-NF-κB, phosphorylated-IκB-α, tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), caspase-3 levels and histopathological damage in kidney tissue. Our results reveal that MAGL inhibitor KML29 and FAAH inhibitor URB597 have a protective effect on kidney IR injury by preventing apoptosis and inflammation. Inhibition of MAGL and FAAH may be a new therapeutic strategy to prevent kidney IR injury.

The endocannabinoid systemʼs involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling

The endocannabinoid system (eCS) plays critical roles in locomotor function and motor development; however, the roles of non-canonical cannabinoid receptor systems such as transient receptor potential (TRP) channels and the Sonic Hedgehog (SHH) signaling pathway in conjunction with the eCS in sensorimotor development remains enigmatic. To investigate the involvement of canonical and non-canonical cannabinoid receptors, TRP channels, and the SHH pathway in the development of sensorimotor function in zebrafish, we treated developing animals with pharmacological inhibitors of the CB1R, CB2R, TRPA1/TRPV1/TRPM8, and a smoothened (SMO) agonist, along with inhibitors of the eCS catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) during the first ~24 h of zebrafish embryogenesis. Locomotor function was examined by assessing touch-evoked escape swimming at 2 days post-fertilization. We report that FAAH inhibition had no effect on swimming while MAGL inhibition using JZL 184 reduced swimming distance and the dual FAAH/MAGL inhibitor JZL 195 impaired swimming distance and mean swimming velocity. The CB1R antagonist AM 251 prevented locomotor deficits caused by eCS perturbation but the CB2R antagonist AM 630 did not. Inhibition of TRPA1/TRPV1/TRPM8 using AMG 9090 rescued the locomotor reductions caused by FAAH/MAGL inhibition, but not by MAGL inhibition alone. The SMO agonist purmorphamine attenuated the effects of JZL 184 and JZL 195 on swimming distance, but not mean velocity. Together, these findings provide one of the first investigations examining the interactions between the eCS and its non-canonical receptor systems in vertebrate motor development.

Development of N-(1-Adamantyl)benzamides as Novel Anti-Inflammatory Multitarget Agents Acting as Dual Modulators of the Cannabinoid CB2 Receptor and Fatty Acid Amide Hydrolase.

Cannabinoid type 2 receptor (CB2R), belonging to the endocannabinoid system, is overexpressed in pathologies characterized by inflammation, and its activation counteracts inflammatory states. Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the degradation of the main endocannabinoid anandamide; thus, the simultaneous CB2R activation and FAAH inhibition may be a synergistic anti-inflammatory strategy. Encouraged by principal component analysis (PCA) data identifying a wide chemical space shared by CB2R and FAAH ligands, we designed a small library of adamantyl-benzamides, as potential dual agents, CB2R agonists, and FAAH inhibitors. The new compounds were tested for their CB2R affinity/selectivity and CB2R and FAAH activity. Derivatives 13, 26, and 27, displaying the best pharmacodynamic profile as CB2R full agonists and FAAH inhibitors, decreased pro-inflammatory and increased anti-inflammatory cytokines production. Molecular docking simulations complemented the experimental findings by providing a molecular rationale behind the observed activities. These multitarget ligands constitute promising anti-inflammatory agents.

Potential effects of cannabinoids on audiovestibular function: A narrative review.

The growing interest in the development of drugs that target the endocannabinoid system has extended to conditions that affect the audiovestibular pathway. The expression of cannabinoid (CB) receptors in that pathway has been widely demonstrated, indicating a therapeutic potential for drug development at this level. These medications may be beneficial for conditions such as noise-induced hearing loss, ototoxicity, or various forms of vertigo of central or peripheral origin. The therapeutic targets of interest include natural or synthetic compounds that act as CB1/CB2 receptor agonists/antagonists, and inhibitors of the endocannabinoid-degrading enzymes FAAH and MAGL. Furthermore, genetic variations implicated in the response to treatment and the development of related disorders such as epilepsy or migraine have been identified. Direct methods of administering these medications should be examined beyond the systemic strategy.

FAAH Inhibition Restores Early Life Stress-Induced Alterations in PFC microRNAs Associated with Depressive-Like Behavior in Male and Female Rats.

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.

Structure-activity relationship studies of benzothiazole-phenyl analogs as multi-target ligands to alleviate pain without affecting normal behavior

Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are potential targets for several diseases. Previous studies have reported that concomitant selective inhibition of sEH and FAAH produced antinociception effects in an animal model of pain. However, the co-administration of a selective sEH inhibitor and a selective FAAH inhibitor might produce serious side effects due to drug-drug interactions that could complicate drug development in the long term. Thus, discovering dual sEH/FAAH inhibitors, single small molecules that can simultaneously inhibit both sEH and FAAH, would be a significant accomplishment in the medicinal chemistry field. Herein, we report the synthesis and biological evaluation of benzothiazole-phenyl-based analogs as potential dual sEH/FAAH inhibitors. This work represents a follow-up structure-activity relationship (SAR) and metabolic-stability studies of our best dual sEH/FAAH inhibitor identified previously, as well as in vivo evaluation of its effects on voluntary locomotor behavior in rats. Our SAR study indicates that trifluoromethyl groups on the aromatic rings are well tolerated by the targeted enzymes when placed at the ortho and para positions; however, they, surprisingly, did not improve metabolic stability in liver microsomes. Our behavioral studies indicate that doses of dual sEH/FAAH inhibitors that alleviate pain do not depress voluntary behavior in naïve rats, which is a common side effect of currently available analgesic drugs (e.g., opioids). Thus, dual sEH/FAAH inhibitors may be a safe and effective approach to treat pain.

Endocannabinoid metabolism inhibition has no effect on spontaneous fear recovery or extinction resistance in Lister hooded rats.

Endocannabinoid transmission is emerging as a target for treating anxiety-related disorders, given its regulation of fear extinction. Boosting anandamide levels via inhibition of its metabolism by fatty acid amide hydrolase (FAAH) can enhance extinction, whereas inhibiting monoacylglycerol lipase (MAGL) to elevate 2-arachidonoylglycerol levels can impair extinction. However, whether endocannabinoids regulate fear relapse over time or extinction resistance remains unclear. In two experiments using auditory fear conditioned rats, we examined the effects of the FAAH inhibitor URB597 and the MAGL inhibitor JZL184 administered systemically on 1) spontaneous fear recovery after delayed extinction, and 2) extinction resistance resulting from immediate extinction [the immediate extinction deficit (IED)]. In Experiment 1, URB597 or JZL184 was given immediately after delayed extinction occurring 24h after conditioning. Extinction recall and spontaneous fear recovery were tested drug-free 1 and 21 days later, respectively. We found no effects of either drug on extinction recall or spontaneous fear recovery. In Experiment 2, URB597 or JZL184 was given before immediate extinction occurring 30min after conditioning and extinction recall was tested drug-free the next day. We also examined the effects of propranolol, a beta-adrenoceptor antagonist that can rescue the IED, as a positive control. JZL184 enhanced fear expression and impaired extinction learning but we found no lasting effects of URB597 or JZL184 on cued extinction recall. Propranolol reduced fear expression but, unexpectedly, had no enduring effect on extinction recall. The results are discussed in relation to various methodological differences between previous studies examining endocannabinoid and adrenergic regulation of fear extinction.

Molecular Basis for Non-Covalent, Non-Competitive FAAH Inhibition

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.