Monoacylglycerol Lipase Inhibitor Research Articles

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.

High-frequency repetitive transcranial magnetic stimulation regulates neural oscillations of the hippocampus and prefrontal cortex in mice by modulating endocannabinoid signalling

BackgroundNeural oscillations play a role in the antidepressant effects of repetitive transcranial magnetic stimulation (rTMS). However, the effects of high-frequency rTMS on the neural oscillations of the medial prefrontal cortex (mPFC) and hippocampus (HPC) and its molecular mechanism have not been fully clarified. MethodsThe depressive-like behaviours, local field potentials (LFPs) of the ventral HPC (vHPC)-mPFC, and alternations of endocannabinoid system (ECS) in the HPC and mPFC were observed after rTMS treatment. Meanwhile, depressive-like behaviours and LFPs were also observed after cannabinoid type-1 receptor (CB1R) antagonist AM281 or monoacylglycerol lipase inhibitor JZL184 injection. Moreover, the antidepressant effect of rTMS was further assessed in glutamatergic-CB1R and gamma-amino butyric acid (GABA)-ergic -CB1R knockout mice. ResultsAlternations of endocannabinoids and energy value and synchronisation of mPFC-vHPC, especially the decrease of theta oscillation induced by CUMS, were alleviated by rTMS. JZL184 has similar effects to rTMS and AM281 blocked the effects of rTMS. GABAergic-CB1R deletion inhibited CUMS-induced depressive-like behaviours whereas Glutaminergic-CB1R deletion dampened the antidepressant effects of rTMS. LimitationsThe immediate effect of rTMS on field-potential regulation was not observed. Moreover, the role of region-specific regulation of the ECS in the antidepressant effect of rTMS was unclear and the effects of cell-specific CB1R knockout on neuronal oscillations of the mPFC and vHPC should be further investigated. ConclusionEndocannabinoid system mediated the antidepressant effects and was involved in the regulation of LFP in the vHPC-mPFC of high-frequency rTMS.

The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

Anti-nociceptive potential of an isatin-derived dual fatty acid amide hydrolase-monoacylglycerol lipase inhibitor.

Recently, we have reported an isatin-derived carbohydrazone, 5-chloro-N'-(6-chloro-2-oxoindolin-3-ylidene)-2-hydroxybenzohydrazide (SIH 3) as dual nanomolar FAAH (fatty acid amide hydrolase)-MAGL (monoacylglycerol lipase) inhibitor with good CNS penetration and neuroprotective activity profile. In this study, we further investigated the pharmacological profile of compound SIH 3 in the neuropathic pain model along with acute toxicity and ex vivo studies. Chronic constrictive injury (CCI) was used to induce neuropathic pain in male Sprague-Dawley rats and the anti-nociceptive activity of the compound SIH 3 was investigated at 25, 50, and 100mg/kg ip. Subsequently, locomotor activity was measured by rotarod and actophotometer experiments. The acute oral toxicity of the compound was assessed as per the OECD guidelines 423. Compound SIH 3 showed significant anti-nociceptive activity in the CCI-induced neuropathic pain model without altering the locomotor activity. Furthermore, compound SIH 3 showed an excellent safety profile (up to 2000mg/kg, po) in the acute oral toxicity study and was also non-hepatotoxic. Further, ex vivo studies revealed that the compound SIH 3 produces a significant antioxidant effect in oxidative stress induced by CCI. Our findings suggest that the investigated compound SIH 3 has the potential to be developed as an anti-nociceptive agent.

2-Arachidonoylglycerol Activity in Over Ischemia Reperfusion Damage: Can Endocannabinoids Protect Ovarian Reserve?

Objective: The present study aimed to demonstrate the possible effects of increased 2-arachidonoylglycerol (2-AG) by applying the monoacylglycerol lipase inhibitor KML-29 on rats with ovarian ischemia-reperfusion (IR) model. Methods: Forty-eight female Wistar albino rats were divided into six groups. Group 1: Sham, Group 2: Ischemia, Group 3: IR, Group 4: IR + KML-29 (2 mg/kg), Group 5: IR + KML-29 (20 mg/kg), and Group 6: IR + vehicle (dimethyl sulfoxide). Three hours of ischemia followed by 3 h of reperfusion. Two different doses of KML-29 (2 and 10 mg/kg) were administered intraperitoneally in Groups 4 and 5, 30 min before reperfusion. Ovarian IR injury and ovarian reserve were evaluated histopathological and by using nuclear factor (NF)-κB, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, superoxide dismutase, glutathione peroxidase pre-/postoperative blood antimullerian hormone, and inhibin B. Results: In the KML-1 and KML-2 groups, this damage was significantly reduced compared to the ischemia group. NF-κB, IL-1β, TNF-α, and TGF-β1 immunoreactivities increased statistically significantly in the ischemia group compared to the control group (p<0.001). Immunoreactivities of these proteins were significantly decreased in the KML-1 and KML-2 groups (p<0.001). It was observed that the number of these apoptotic cells decreased significantly in the KML-1 and KML-2 groups compared to the ischemia group (p<0.001). The postoperative inhibin level showed a significant decrease in the ischemia group compared to the sham group, while a significant increase was observed in the KML-1 and KML-2 groups compared to the ischemia group. Conclusion: It was seen that anti-inflammatory, antioxidant, and antiapoptotic activity was formed, and the ovarian reserve was preserved with 2-AG in ovarian IR damage. The protective effect of endocannabinoids on the ovaries may create a promising new treatment strategy for many pathologies that will affect the ovarian reserve.

Endocannabinoid 2-Arachidonoylglycerol Synthesis and Metabolism at Neuronal Nuclear Matrix Fractions Derived from Adult Rat Brain Cortex.

In this report, we describe the kinetics characteristics of the diacylglycerol lipase-α (DGLα) located at the nuclear matrix of nuclei derived from adult cortical neurons. Thus, using high-resolution fluorescence microscopy, classical biochemical subcellular fractionation, and Western blot techniques, we demonstrate that the DGLα enzyme is located in the matrix of neuronal nuclei. Furthermore, by quantifying the 2-arachidonoylglycerol (2-AG) level by liquid chromatography and mass spectrometry when 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) was exogenously added as substrate, we describe the presence of a mechanism for 2-AG production through DGLα dependent biosynthesis with an apparent Km (Kmapp) of 180 µM and a Vmax of 1.3 pmol min-1 µg-1 protein. We also examined the presence of enzymes with hydrolytic and oxygenase activities that are able to use 2-AG as substrate, and described the localization and compartmentalization of the major 2-AG degradation enzymes, namely monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), α/β-hydrolase domain 12 protein (ABHD12) and cyclooxygenase-2 (COX2). Of these, only ABHD12 exhibited the same distribution with respect to chromatin, lamin B1, SC-35 and NeuN as that described for DGLα. When 2-AG was exogenously added, we observed the production of arachidonic acid (AA), which was prevented by inhibitors (but not specific MGL or ABHD6 inhibitors) of the ABHD family. Overall, our results expand knowledge about the subcellular distribution of neuronal DGLα, and provide biochemical and morphological evidence to ensure that 2-AG is produced in the neuronal nuclear matrix. Thus, this work paves the way for proposing a working hypothesis about the role of 2-AG produced in neuronal nuclei.

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.

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.

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.

The monoacylglycerol lipase inhibitor, JZL184, has comparable effects to therapeutic hypothermia, attenuating global cerebral injury in a rat model of cardiac arrest

Post-resuscitation cerebral ischemia-reperfusion injury (IRI) is a vital contributor to poor neurological prognosis. Exploring novel therapeutics that attenuate cerebral IRI is of great significance. Inflammation plays a role in the development of cerebral IRI after successful cardiopulmonary resuscitation (CPR). Monoacylglycerol lipase (MAGL) is an enzyme that is predominantly responsible for the metabolism of endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) metabolites, which are associated with inflammation. Therefore, we investigated the efficacy of the MAGL inhibitor, JZL184, on cerebral IRI and further compared the effects to therapeutic hypothermia (TH). Thirty-six rats were randomized into three groups: 1) JZL184; 2) Control; 3) TH (N = 12 for each group). Animals underwent 6 min of ventricular fibrillation (VF) followed with 8 min of CPR. After return of spontaneous circulation (ROSC), rats received an intraperitoneal injection of JZL184 (16 mg/kg) or DMSO (20 mg/ml) or body cooling was initiated. Cerebral microcirculation, brain edema, blood brain barrier (BBB) permeability, serum neuron-specific enolase (NSE), S-100β, interleukin-6 (IL-6) and interleukin-10 (IL-10) were quantified at 6 h post ROSC. Compared to control, treatment with JZL184 or TH was associated with significantly ameliorated cerebral microcirculation, mitigated brain edema, attenuated BBB permeability, decreased serum levels of NSE, S-100β and IL-6, and increased serum IL-10 levels (p < 0.05). There was no significant difference in the above measurements between JZL184 and TH. JZL184 has comparable neuroprotective effects to therapeutic hypothermia on global cerebral IRI in a rat model of cardiac arrest (CA).

2-Arachidonoylglycerol-mediated endocannabinoid signaling modulates mechanical hypersensitivity associated with alcohol withdrawal in mice.

Alcohol use disorder(AUD) commonly occurs in patients with chronic pain, and a major barrier to achieving abstinence and preventing relapse is the emergence of hyperalgesia during alcohol withdrawal. Elucidating novel therapeutic approaches to target hyperalgesia associated with alcohol withdrawal could have important implications for treating AUD. Here, we examined the role of 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid (eCB) signaling in the regulation of hyperalgesia associated with alcohol withdrawal in mice. We tested the hypothesis that pharmacological augmentation of 2-AG signaling could reduce hyperalgesia during withdrawal. Male and female C57BL/6J mice were tested during withdrawal from a continuous access two-bottle choice (2BC) paradigm to investigate how eCB signaling modulates mechanical and thermal sensitivity during withdrawal. Mice were pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184 to elevate levels of 2-AG. Rimonabant or AM630 were given to block CB1 and CB2 receptor activity, respectively. DO34 was given to reduce 2-AG by inhibiting the 2-AG synthetic enzyme diacylglycerol lipase (DAGL). After 72 h of withdrawal, male and female mice exhibited increased mechanical, but not thermal, hypersensitivity, which normalized by 7 days. This effect was reversed by pretreatment with JZL184. The effects of JZL184 were prevented by coadministration of either the CB1 or the CB2 antagonist. DO34, Rimonabant, and AM630 exacerbated mechanical hypersensitivity during alcohol withdrawal, causing an earlier onset and persistent hypersensitivity even 1 week into withdrawal. Our findings demonstrate the critical role of 2-AG signaling in the bidirectional regulation of mechanical sensitivity during alcohol withdrawal, with enhancement of 2-AG levels reducing sensitivity, and inhibition of 2-AG signaling exacerbating sensitivity. These data suggest that 2-AG augmentation represents a novel approach to the treatment of alcohol withdrawal-associated hyperalgesia and AUD in patients with comorbid pain disorders.

Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare genetic multisystemic disorder characterized by mild-to-moderate intellectual disability and hypersocial phenotype, while the most life-threatening features are cardiovascular abnormalities. Nowadays, there are no pharmacological treatments to directly ameliorate the main traits of WBS. The endocannabinoid system (ECS), given its relevance for both cognitive and cardiovascular function, could be a potential druggable target in this syndrome. We analyzed the components of the ECS in the complete deletion (CD) mouse model of WBS and assessed the impact of its pharmacological modulation in key phenotypes relevant for WBS. CD mice showed the characteristic hypersociable phenotype with no preference for social novelty and poor short-term object-recognition performance. Brain cannabinoid type-1 receptor (CB1R) in CD male mice showed alterations in density and coupling with no detectable change in main endocannabinoids. Endocannabinoid signaling modulation with subchronic (10 days) JZL184, a selective inhibitor of monoacylglycerol lipase, specifically normalized the social and cognitive phenotype of CD mice. Notably, JZL184 treatment improved cardiovascular function and restored gene expression patterns in cardiac tissue. These results reveal the modulation of the ECS as a promising novel therapeutic approach to improve key phenotypic alterations in WBS.

Multi-parameter optimization: Development of a morpholin-3-one derivative with an improved kinetic profile for imaging monoacylglycerol lipase in the brain

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation.

A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature.

Chronic inflammation and blood–brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer’s disease and Parkinson’s disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood–brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood–brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood–brain barrier dysfunction caused by inflammatory insults.

Discovering monoacylglycerol lipase inhibitors by a combination of fluorogenic substrate assay and activity-based protein profiling.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly metabolized by monoacylglycerol lipase (MAGL) in the brain. Selective inhibitors of MAGL provide valuable insights into the role of 2-AG in a variety of (patho)physiological processes and are potential therapeutics for the treatment of diseases such as neurodegenerative disease and inflammation, pain, as well as cancer. Despite a number of MAGL inhibitors been reported, inhibitors with new chemotypes are still required. Here, we developed a substrate-based fluorescence assay by using a new fluorogenic probe AA-HNA and successfully screened a focused library containing 320 natural organic compounds. Furthermore, we applied activity-based protein profiling (ABPP) as an orthogonal method to confirm the inhibitory activity against MAGL in the primary substrate-based screening. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene (23, cryptotanshinone) and β-carbolines (82 and 93, cis- and trans-isomers), with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Moreover, these compounds also showed antiproliferative activities against multiple cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Remarkably, 23 achieved complete inhibition towards endogenous MAGL in most cancer cells determined by ABPP. Our results demonstrate the potential utility of the substrate-based fluorescence assay in combination with ABPP for rapidly discovering MAGL inhibitors, as well as providing an effective approach to identify potential targets for compounds with significant biological activities.

O-13: INHIBITION OF THE ENDOCANNABINOID REGULATING ENZYME MONOACYLGLYCEROL LIPASE AMELIORATES THE HYPER-NOCICEPTIVE PHENOTYPE IN THE BERKELEY MOUSE MODEL OF SICKLE CELL DISEASE

Purpose: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Recurrent episodes of acute, vaso-occlusive pain crisis, a hallmark of the disease, lead to chronic pain for many patients, though the mechanisms of this transition are poorly understood. Although opioids remain the standard of care to treat SCD chronic pain, their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion (DRG) neurons are peripheral sensory neurons essential for the transmission of nociceptive afferents to the CNS. In rodent models of neuropathic pain, harvested DRG neurons show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. As MAGL inhibitors have yet to be tested in BERK mice, here we test whether this approach will ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice. Materials and methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Nociceptive behaviors were assessed using the von Frey and Hot Plate tests. Motor-functional behavior was assessed using the Grip Strength and Nesting assays. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 DRG neurons. Data were analyzed as two- and three-way ANOVAs followed by Tukey post-hoc analysis when appropriate (p < 0.05 considered significant). Results: HbSS-BERK mice displayed profound mechanical, thermal and deep tissue/musculoskeletal hypersensitivity. HbSS-BERK mice also exhibited deficits in nest-building behavior. Patch clamp studies revealed that DRG neurons harvested from HbSS-BERK mice displayed extremely hyperexcitability. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia, and ameliorated deficits in grip strength in HbSS-BERK mice. Importantly, seven days of daily injections of MJN-110 (5 mg/kg) continued to ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice, which did not undergo tolerance. Moreover, DRG harvested from the MJN-treated HbSS-BERK mice showed similar DRG neuronal activity as seen in control mice. Conclusion: These findings validate that HbSS-BERK mice show hypersensitive responses to mechanical and heat stimuli, and exhibit motor-functional deficits in grip strength and nest building behavior. Additionally, this work demonstrates hyperexcitability of sensory neurons from the lower lumbar region of the spine of HbSS-BERK mice. Finally, the observations that MJN110 reduces these hyper-nociceptive behaviors and ameliorates neuronal hyperexcitability in HbSS-BERK mice suggest that MAGL inhibition represents a viable strategy to reduce chronic pain related to sickle cell disease. The authors do not declare any conflict of interest

Pristimerin, a triterpene that inhibits monoacylglycerol lipase activity, prevents the development of paclitaxel-induced allodynia in mice.

Background: Triterpenes such as euphol and pristimerin, which are plant secondary metabolites, were the first to be characterized as monoacylglycerol lipase (MAGL) inhibitors. MAGL inhibitors alleviate chemotherapy-induced neuropathic pain (CINP) in rodent models. Pristimerin has been shown to have additive anticancer activity with paclitaxel, a chemotherapeutic drug. However, the activity of pristimerin on CINP has not been evaluated. Objectives: The aims of this study were to evaluate whether various triterpenes had activity against recombinant human MAGL and MAGL activity in mouse tissues, and whether pristimerin could prevent development of paclitaxel-induced mechanical allodynia. Methods: The effects of four triterpenes betulinic acid, cucurbitacin B, euphol, and pristimerin on the activity human recombinant MAGL and MAGL activity of mice brain and paw skin tissues were evaluated using MAGL inhibitor screening and MAGL activity assay kits. The effects of treatment of female BALB/c mice with pristimerin intraperitoneally on the development of paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer and on nuclear factor-2 erythroid related factor-2 (Nrf2) gene expression in the paw skin were evaluated by real time polymerase chain reaction. Results: Pristimerin inhibited the human recombinant MAGL activity in a concentration-dependent manner like JZL-195, a MAGL inhibitor. Betulinic acid, cucurbitacin B and euphol inhibited human recombinant MAGL activity but their effects were not concentration dependent and were less to that of pristimerin. Pristimerin inhibited both mouse brain and paw skin MAGL activity in a concentration-dependent manner. Paclitaxel induced mechanical allodynia and increase in MAGL activity in the paw skin. Treatment with pristimerin prevented the development of paclitaxel-induced mechanical allodynia and the paclitaxel-induced increase in MAGL activity. Pristimerin significantly upregulated mRNA expression of Nrf2, a regulator of endogenous antioxidant defense. Conclusion: These results indicate that triterpenes inhibit human recombinant MAGL activity with varying degrees. Pristimerin inhibits both mouse brain and paw skin MAGL activity in a concentration-dependent manner, prevents both the development of paclitaxel-induced mechanical allodynia and the associated increase in MAGL activity in the paw skin, and might protect against paclitaxel-induced oxidative stress. Co-treatment with pristimerin and paclitaxel could be useful in the treatment of cancer and prevention of CINP.

MAGL inhibitor NanoMicellar formulation (MAGL-NanoMicellar) for the development of an antiglaucoma eye drop

The ocular endocannabinoid system (ECS) including enzymes and CB1/CB2 receptors determines various substantial effects, such as anti-inflammatory activity and reduction of the intraocular pressure (IOP). The modulation of 2-arachidonoylglycerol (2-AG) levels obtained via MAGL inhibition is considered as a promising pharmacological strategy to activate the ECS. Within the scope of this study, the effect of a selective monoacylglycerol lipase (MAGL) inhibitor (MAGL17b) was investigated by measuring the IOP reduction in normotensive rabbits after performing a solubilisation process of the molecule with non-ionic surfactants, to produce suitable eye drops containing the highest possible concentration of the drug. Furthermore, the study involved the evaluation of cytotoxicity and of in vitro/ex vivo corneal permeation of MAG17b of selected formulations based on polyoxyl(35)castor oil (C-EL) and polyethylene glycol (80) sorbitan monolaurate (TW80).The solubilisation of 0.5 mM MAGL17b with 3 % w/w TW80 (TW80/3-17b), through the formation of NanoMicellar structures (diameter of 12.3 nm), determined a significant permeation of MAGL17b, both through excised rabbits corneas and reconstituted corneal epithelium, with a limited corneal epithelial cells death. The blockade of MAGL activity induced a IOP reduction up to 4 mmHg in albino and pigmented rabbits after topical instillation, thus confirming the potential efficacy of the MAGL inhibition approach in the treatment of ocular pathologies.

Synthesis and evaluation of carbamate derivatives as fatty acid amide hydrolase and monoacylglycerol lipase inhibitors.

Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are the primary catabolic enzymes for endocannabinoids, anandamide (AEA), and 2-arachidonoyl glycerol. Numerous studies have shown that FAAH and MAGL play an important role in modulating various central nervous system activities; hence, the development of small molecule FAAH/MAGL inhibitors is an active area of research. Several small molecules possessing the carbamate scaffold are documented as potential FAAH/MAGL inhibitors. Here, we designed and synthesized a series of open chain and cyclic carbamates and evaluated their dual FAAH-MAGL inhibition properties. Phenyl [4-(piperidin-1-ylmethyl)phenyl]carbamate (2e) emerged as the most potent MAGL inhibitor (IC50 = 19 nM), benzyl (1H-benzo[d]imidazol-2-yl)carbamate (3h) was the most potent FAAH inhibitor (IC50 = 55 nM), and phenyl (6-fluorobenzo[d]thiazol-2-yl)carbamate (2i) egressed as a nonselective dual FAAH-MAGL inhibitor (FAAH: 82 nM, MAGL: 72 nM). The enzyme kinetics experiments revealed that the compounds inhibit FAAH/MAGL in a covalent-reversible manner, with a mixed binding mode of action. Moreover, the lead compounds were found suitable for blood-brain permeation in the parallel artificial membrane permeation assay. Furthermore, docking simulation experiments suggested that the potency of the lead compounds was governed by hydrogen bonds and hydrophobic interactions with the enzyme active sites. In silico drug-likeness and ADMETox prediction studies provided useful information on the compounds' oral absorption, metabolism, and toxicity profiles. In summary, this study afforded potent multifunctional carbamates with appreciable pharmacokinetic profiles meriting further investigation.

Attenuation of allodynia and microglial reactivity by inhibiting the degradation of 2-arachidonoylglycerol following injury to the trigeminal nerve in mice

Endocannabinoids have an important role for the regulation of neuropathic pain. In our previous study, we observed that preventing the degradation of a endocannabinoid, 2-arachidonoylglycerol (2-AG), using an inhibitor of monoacylglycerol lipase (JZL184), attenuated neuropathic orofacial pain (NOP). The present study aimed to investigate mechanisms underlying JZL184-induced attenuation of NOP. We hypothesized that JZL184 may suppress microglial reactivity in the trigeminal spinal subnucleus caudalis (Vc) under NOP. The infraorbital nerve (ION) was hemisected to model NOP in mice, resulting in a significant reduction of mechanical head-withdrawal threshold (MHWT) on day 4 following the ION hemisection. Chronic systemic application of JZL184 at a concentration of 8 or 16 mg/kg/day for 4 days significantly attenuated the reduction of MHWT in mice exposed to NOP. Administering JZL184 at 4 mg/kg/day or its vehicle, however, did not attenuate the MHWT of mice with NOP. The reactivity of microglial cells in the Vc increased in mice with NOP compared to sham-operated controls. The application of JZL184 at 8 or 16 mg/kg/day for 4 days significantly reduced the increased microglial reactivity in the Vc. The changes of microglia under NOP were, by contrast, not reduced by application of the drug at 4 mg/kg/day or its vehicle. The results indicate that preventing 2-AG degradation may increase its accumulation in the Vc and normalize microglial reactivity under NOP, which may contribute to suppressing NOP.