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The cannabinoid system is one of the most investigated neuromodulatory systems because of its involvement in multiple pathologies such as cancer, inflammation, and psychiatric diseases. Recently, the CB2 receptor has gained increased attention considering its crucial role in modulating neuroinflammation in several pathological conditions like neurodegenerative diseases. Here we describe the rational design of pyrrole-based analogues, which led to a potent and pharmacokinetically suitable CB2 full agonist particularly effective in improving cognitive functions in a scopolamine-induced amnesia murine model. Therefore, we extended our study by investigating the interconnection between CB2 activation and neurotransmission in this experimental paradigm. To this purpose, we performed a MALDI imaging analysis on mice brains, observing that the administration of our lead compound was able to revert the effect of scopolamine on different neurotransmitter tones, such as acetylcholine, serotonin, and GABA, shedding light on important networks not fully explored, so far., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)- 5a - v , (±)- 6a - j , and (±)- 7a - d ) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)- 5d , (±)- 5l , and (±)- 5r . X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)- 5c , (±)- 5d , and (±)- 5v ) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl- sn -glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3 R ,4 S )- 5v is one of the most potent irreversible inhibitors of h / m MAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

The endocannabinoid system (ECS) constitutes a broad-spectrum modulator of homeostasis in mammals, providing therapeutic opportunities for several pathologies. Its two main receptors, cannabinoid type 1 (CB1) and type 2 (CB2) receptors, mediate anti-inflammatory responses; however, their differing patterns of expression make the development of CB2-selective ligands therapeutically more attractive. The benzo[ d ]imidazole ring is considered to be a privileged scaffold in drug discovery and has demonstrated its versatility in the development of molecules with varied pharmacologic properties. On the other hand, the main psychoactive component of Cannabis sativa , delta-9-tetrahydrocannabinol (THC), can be structurally described as an aliphatic terpenoid motif fused to an aromatic polyphenolic (resorcinol) structure. Inspired by the structure of this phytocannabinoid, we combined different natural product motifs with a benzo[ d ]imidazole scaffold to obtain a new library of compounds targeting the CB2 receptor. Here, we synthesized 26 new compounds, out of which 15 presented CB2 binding and 3 showed potent agonist activity. SAR analysis indicated that the presence of bulky aliphatic or aromatic natural product motifs at position 2 of the benzo[ d ]imidazoles ring linked by an electronegative atom is essential for receptor recognition, while substituents with moderate bulkiness at position 1 of the heterocyclic core also participate in receptor recognition. Compounds 5 , 6 , and 16 were further characterized through in vitro cAMP functional assay, showing potent EC50 values between 20 and 3 nM, and compound 6 presented a significant difference between the EC50 of pharmacologic activity (3.36 nM) and IC50 of toxicity (30-38 µM).

Selective ligands of the CB2 receptor are receiving considerable attention due to their potential as therapeutic agents for a variety of diseases. Recently, 7-hydroxy-5-oxopyrazolo[4,3- b ]pyridine-6-carboxamide derivatives were shown to act at the CB2 receptor either as agonists or as inverse agonists/antagonists in vitro and to have anti-osteoarthritic activity in vivo. In this article, we report the synthesis, pharmacological profile, and molecular modeling of a series of twenty-three new 7-hydroxy-5-oxopyrazolo[4,3- b ]pyridine-6-carboxamides with the aim of further developing this new class of selective CB2 ligands. In addition to these compounds, seven other analogs that had been previously synthesized were included in this study to better define the structure-activity relationship (SAR). Ten of the new compounds studied were found to be potent and selective ligands of the CB2 receptor, with K i values ranging from 48.46 to 0.45 nM and CB1/CB2 selectivity indices (SI) ranging from >206 to >4739. In particular, compounds 54 and 55 were found to be high-affinity CB2 inverse agonists that were not active at all at the CB1 receptor, whereas 57 acted as an agonist. The functional activity profile of the compounds within this structural class depends mainly on the substitution pattern of the pyrazole ring.

Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disorder and a complication of SARS-CoV-2 infection. In patients with severe SARS-CoV-2 infection, the transition to ARDS is principally due to the occurrence of a cytokine storm and an exacerbated inflammatory response. The effectiveness of ultra-micronized palmitoylethanolamide (PEA-um) during the earliest stage of COVID-19 has already been suggested. In this study, we evaluated its protective effects as well as the effectiveness of its congener, 2-pentadecyl-2-oxazoline (PEA-OXA), using in vitro models of acute lung injury. In detail, human lung epithelial cells (A549) activated by polyinosinic-polycytidylic acid (poly-(I:C)) or Transforming Growth Factor-beta (TGF-β) were treated with PEA-OXA or PEA. The release of IL-6 and the appearance of Epithelial-Mesenchymal Transition (EMT) were measured by ELISA and immunofluorescence assays, respectively. A possible mechanism of action for PEA-OXA and PEA was also investigated. Our results showed that both PEA-OXA and PEA were able to counteract poly-(I:C)-induced IL-6 release, as well as to revert TGF-β-induced EMT. In addition, PEA was able to produce an "entourage" effect on the levels of the two endocannabinoids AEA and 2-AG, while PEA-OXA only increased PEA endogenous levels, in poly-(I:C)-stimulated A549 cells. These results evidence for the first time the superiority of PEA-OXA over PEA in exerting protective effects and point to PEA-OXA as a new promising candidate in the management of acute lung injury.

Cannabinoid type 1 (hCB1) and type 2 (hCB2) receptors are pleiotropic and crucial targets whose signaling contributes to physiological homeostasis and its restoration after injury. Being predominantly expressed in peripheral tissues, hCB2R represents a safer therapeutic target than hCB1R, which is highly expressed in the brain, where it regulates processes related to cognition, memory, and motor control. The development of hCB2R ligands represents a therapeutic opportunity for treating diseases such as pain, inflammation and cancer. Identifying new selective scaffolds for cannabinoids and determining the structural determinants responsible for agonism and antagonism are priorities in drug design. In this work, a series of N -[1,3-dialkyl(aryl)-2-oxoimidazolidin-4-ylidene]-aryl(alkyl)sulfonamides is designed and synthesized and their affinity for human hCB1R and hCB2R is determined. Starting with a scaffold selected from the NIH Psychoactive Drug Screening Program Repository, through a combination of molecular modeling and structure-activity relationship studies, we were able to identify the chemical features leading to finely tuned hCB2R selectivity. In addition, an in silico model capable of predicting the functional activity of hCB2R ligands was proposed and validated. The proposed receptor activation/deactivation model enabled the identification of four pure hCB2R-selective agonists that can be used as a starting point for the development of more potent ligands.

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aβ 42 ) was assessed. Compounds 2 and 3 , bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Aβ 42 self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.

Palmitoylethanolamide (PEA) is an endogenous anti-inflammatory lipid mediator and a widely used nutraceutical. In this study, we designed, realized, and tested a drug-carrier conjugate between PEA (the active drug) and glucuronic acid (the carrier). The conjugate, named GLUPEA, was characterized for its capability of increasing PEA levels and exerting anti-inflammatory activity both in vitro and in vivo. GLUPEA treatment, compared to the same concentration of PEA, resulted in higher cellular amounts of PEA and the endocannabinoid 2-arachidonoyl glycerol (2-AG), and increased 2-AG-induced transient receptor potential vanilloid type 1 (TRPV1) channel desensitization to capsaicin. GLUPEA inhibited pro-inflammatory monocyte chemoattractant protein 2 (MCP-2) release from stimulated keratinocytes, and it was almost as efficacious as ultra-micronized PEA at reducing colitis in dinitrobenzene sulfonic acid (DNBS)-injected mice when using the same dose. GLUPEA is a novel pro-drug able to efficiently mimic the anti-inflammatory and endocannabinoid enhancing actions of PEA.

Background: Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a "dietary food for special medical purposes" against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the "entourage" effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model., Methods: RBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and β-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -β enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively., Results: SP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -β activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188., Conclusions: Activation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

Background: The nuclear Peroxisome Proliferator Activated Receptors (PPARs) are ligand-activated transcription factors playing a fundamental role in energy homeostasis and metabolism. Consequently, functional impairment or dysregulation of these receptors lead to a variety of metabolic diseases. While some phytocannabinoids (pCBs) are known to activate PPARγ, no data have been reported so far on their possible activity at PPARα., Methods: The putative binding modes of pCBs into PPARα/γ Ligand Binding Domains were found and assessed by molecular docking and molecular dynamics. Luciferase assays validated in silico predictions whereas the biological effects of such PPARα/γ ligands were assessed in HepG2 and 3T3L1 cell cultures., Results: The in silico study identified cannabigerolic acid (CBGA), cannabidiolic acid (CBDA) and cannabigerol (CBG) from C. sativa as PPARα/γ dual agonists, suggesting their binding modes toward PPARα/γ isoforms and predicting their activity as full or partial agonists. These predictions were confirmed by luciferase functional assays. The resulting effects on downstream gene transcription in adipocytes and hepatocytes were also observed, establishing their actions as functional dual agonists., Conclusions: Our work broadens the activity spectrum of CBDA, CBGA and CBG by providing evidence that these pCBs act as dual PPARα/γ agonists with the ability to modulate the lipid metabolism., General Significance: Dual PPARα/γ agonists have emerged as an attractive alternative to selective PPAR agonists to treat metabolic disorders. We identified some pCBs as dual PPARα/γ agonists, potentially useful for the treatment of dyslipidemia and type 2 diabetes mellitus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation, and anxiety, among others. We report a new multitarget drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a noncompetitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation, and mood disorders.

The unique role of fatty acid amide hydrolase (FAAH) in terminating endocannabinoid (EC) signaling supports its relevance as a therapeutic target. Inhibition of EC metabolizing enzymes elicits indirect agonism of cannabinoid receptors (CBRs) and therapeutic efficacy devoid of psychotropic effects. Based on our previous ligands, and aiming at the discovery of new selective FAAH inhibitors, we developed a series of 12 new compounds characterized by functionalized tricyclic scaffolds. All the developed compounds display negligible activity on monoacylglycerol lipase (MAGL) and CBRs. The most potent FAAH inhibitors of the newly developed series, 6-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-6-phenylhexylcarbamate (5 h) and 4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(6-phenylhexyl)carbamate (5 i) (nanomolar FAAH inhibitors, the latter of which also shows micromolar affinity at the CB 1 R), were selected for further studies. Results of cell-based studies on a neuroblastoma cell line (IMR32) demonstrated 5 h, 5 i, and our reference compound 3 ([3-(3-carbamoylpyrrol-1-yl)phenyl] N-(5-phenylpentyl)carbamate) to lack any cytotoxic effect, while all three showed the ability to decrease oxidative stress by reducing the expression of the redox-sensitive transcription factor NF-κB. Encouraged by these data, these compounds were studied in vivo and were dosed orally in a mouse model of neuropathic pain. At 10 mg kg -1 all the compounds were able to relieve the hypersensitivity induced by oxaliplatin., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The increasing manufacture of plastics and their mismanagement has turned plastic into a ubiquitous waste in the marine environment. Among all the substances conforming the plastic items, the effects of a dietary Bisphenol A (BPA) and Di-isononyl phthalate (DiNP) have been evaluated in adult male gilthead sea bream, focusing on their effects in the modulation of the Endocannabinoid System (ECS). In zebrafish, the ECS has been recently chosen as a new target for the activity of some Endocrine Disrupting Chemicals (EDC), since it represents a complex lipid signaling network essential for the well-being of the organisms. The results obtained in gilthead seabream showed that BPA and DiNP altered the structure and the biochemical composition of liver, increasing the presence of lipids and triglycerides and decreasing the glycogen and phospholipids. Moreover, the addition of BPA or DiNP in the gilthead sea bream diet altered the levels of endocannabinoids (EC) and EC-like mediators in the liver. These alterations were also associated to changes at the transcriptomic level of genes involved in lipid biosynthesis and ECS metabolism. At the central level, both BPA and DiNP reduced the expression of the endocannabinoid receptor type I (cnr1) and the neuropeptide Y (npy) as well as the levels of the endocannabinoid Anandamide (AEA), suggesting a downregulation of appetite. The results herein reported highlighted the negative effects of chronic dietary exposure to DiNP or BPA on ECS functions and lipid metabolism of male gilthead sea bream liver, showing a similar disruptive activity of these contaminants at metabolic level. Moreover, the novelty of the biomarkers used evidenced possible innovative endpoints for the development of novel OEDCS test guidelines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We have previously shown that endocannabinoids (eCBs) (e.g., anandamide) are involved in the maintenance of homeostatic sebaceous lipid production in human sebaceous glands and that eCB treatment dramatically increases sebaceous lipid production. Here, we aimed to investigate the expression of the major eCB synthesizing and degrading enzymes and to study the effects of eCB uptake inhibitors on human SZ95 sebocytes, thus exploring the role of the putative eCB membrane transporter, which has been hypothesized to facilitate the cellular uptake and subsequent degradation of eCBs. We found that the major eCB synthesizing (N-acyl phosphatidylethanolamine-specific phospholipase D, and diacylglycerol lipase-α and -β) and degrading (fatty acid amide hydrolase, monoacylglycerol lipase) enzymes are expressed in SZ95 sebocytes and also in sebaceous glands (except for diacylglycerol lipase-α, the staining of which was dubious in histological preparations). eCB uptake-inhibition with VDM11 induced a moderate increase in sebaceous lipid production and also elevated the levels of various eCBs and related acylethanolamides. Finally, we found that VDM11 was able to interfere with the proinflammatory action of the TLR4 activator lipopolysaccharide. Collectively, our data suggest that inhibition of eCB uptake exerts anti-inflammatory actions and elevates both sebaceous lipid production and eCB levels; thus, these inhibitors might be beneficial in cutaneous inflammatory conditions accompanied by dry skin., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB 2 ) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB 1 , CB 2 , or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor- α in a manner reversed by CB 2 and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Phthalates, used as plasticizers, have become a ubiquitous contaminant and have been reported for their potential to induce toxicity in living organisms. Among them, di-isononyl phthalate (DiNP) has been recently used to replace di(2-ethylhexyl) phthalate (DEHP). Nowadays, there is evidence that DiNP is an endocrine-disrupting chemical; however, little is known about its effects on the endocannabinoid system (ECS) and lipid metabolism. Hence, the aim of our study was to investigate the effects of DiNP on the ECS in zebrafish liver and brain and on hepatic lipid storage. To do so, adult female zebrafish were exposed to three concentrations (0.42 µg/L, 4.2 µg/L, and 42 µg/L) of DiNP via water for 3 weeks. Afterwards, we investigated transcript levels for genes involved in the ECS of the brain and liver as well as liver histology and image analysis, Fourier-transform infrared spectroscopy imaging, and measurement of endocannabinoid levels. Our results demonstrate that DiNP upregulates orexigenic signals and causes hepatosteatosis together with deregulation of the peripheral ECS and lipid metabolism. A decrease in the levels of ECS components at the central level was observed after exposure to the highest DiNP concentration tested. These findings suggest that replacement of DEHP with DiNP should be considered with caution because of observed adverse DiNP effects on aquatic organisms., (Copyright © 2017 Endocrine Society.)

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system., (Copyright © 2017, American Association for the Advancement of Science.)

N -acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, with N- palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic and neuroprotective activities. Because PEA is produced on demand and exerts pleiotropic effects, the modulation of specific amidases for NAEs (and in particular NAE-hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here we investigate the effect of 2-Pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA, on human recombinant NAAA in vitro and in an established model of Carrageenan (CAR)-induced rat paw inflammation. PEA-OXA dose-dependently significantly inhibited recombinant NAAA and, orally administered to rats (10 mg/kg), limiting histological damage, thermal hyperalgesia and the increase of infiltrating inflammatory cells after CAR injection in the rat right hindpaw, compared to ultramicronized PEA given orally at the same dose (10 mg/kg). These effects were accompanied by elevation of paw PEA levels. Moreover, PEA-OXA markedly reduced neutrophil infiltration and pro-inflammatory cytokine release and prevented CAR-induced IκB-α degradation, nuclear translocation of NF-κB p65, the increase of inducible nitric oxide synthase, cyclooxygenase-2, intercellular adhesion molecule-1, and mast cell activation. Experiments in PPAR-α knockout mice showed that the anti-inflammatory effects of PEA-OXA were not dependent on the presence of PPAR-α receptors. In conclusion, NAAA modulators as PEA-OXA could help to maximize the tissue availability of PEA by increasing its levels and anti-inflammatory effects.

Herein we report the design, synthesis, bioinformatic and biological studies of benzimidazole and benzothiophene derivatives as new cannabinoid receptor ligands. To test the hypothesis that the lack of a hydrogen bond interaction between benzimidazole and benzothiophene derivatives with Lys192 reduces their affinity for CB1 receptors (as we previously reported) and leads to CB2 selectivity, most of the tested compounds do not exhibit hydrogen bond acceptors. All compounds displayed mostly CB2 selectivity, although this was more pronounced in the benzimidazoles derivatives. Furthermore, docking assays revealed a ∏-cation interaction with Lys109 which could play a key role for the CB2 selectivity index. The series displayed low toxicity on five different cell lines. Derivative 8f presented the best binding profile (Ki = 0.08 μM), high selectivity index (KiCB1/KiCB2) and a low citoxicity. Interestingly, in cell viability experiments, using HL-60 cells (expressing exclusively CB2 receptors), all synthesised compounds were shown to be cytotoxic, suggesting that a CB2 agonist response may be involved., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki = 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (∼70 and ∼200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

4-(Thiophen-2-yl)butanoic acid was identified as a cyclic substitute of the unsaturated alkyl chain of the natural ligand, capsaicin. Accordingly, a new class of amides was synthesized in good yield and high purity and their molecular recognition against the target was investigated by means of docking experiments followed by molecular dynamics simulations, in order to rationalize their geometrical and thermodynamic profiles. The pharmacological properties of these new compounds were expressed as activation (EC50) and desensitization (IC50) potencies. Several compounds were found to activate TRPV1 channels, and in particular, derivatives 1 and 10 behaved as TRPV1 agonists endowed with good efficacy as compared to capsaicin. The most promising compound 1 was also evaluated for its protective role against oxidative stress on keratinocytes and differentiated human neuroblastoma cell lines expressing the TRPV1 receptor as well as for its cytotoxicity and analgesic activity in vivo.

The xenoestrogen bisphenol A (BPA) is a widespread plasticizer detectable within several ecosystems. BPA is considered a metabolic disruptor, affecting different organs; however, little is known about its mechanism of action in the liver, in which it triggers triglyceride accumulation. Adult zebrafish (Danio rerio) exposed to BPA developed hepatosteatosis, which was associated with an increase in the liver levels of the obesogenic endocannabinoids 2-arachidonoylglycerol and anandamide and a concomitant decrease in palmitoylethanolamide. These changes were associated with variations in the expression of key endocannabinoid catabolic and metabolic enzymes and an increase in the expression of the endocannabinoid receptor cnr1. Acute and chronic in vitro treatments with nano- and micromolar BPA doses showed increased anandamide levels in line with decreased activity of fatty acid amide hydrolase, the main anandamide hydrolytic enzyme, and induced triglyceride accumulation in HHL-5 cells in a CB1-dependent manner. We conclude that BPA is able to produce hepatosteatosis in zebrafish and human hepatocytes by up-regulating the endocannabinoid system.

A series of 31 arylboronic acids designed on the basis of the pharmacophore model for a variety of TRPV1 antagonists was prepared and tested on FAAH and TRPV1 channel. Four of them, that is, compounds 3c, 4a, 5a,b acted as dual FAAH/TRPV1 blockers with IC50 values between 0.56 and 8.11μM whereas ten others (compounds 1c,f-i, 2c-f, 4b) inhibited FAAH and activated/desensitized TRPV1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

4-Quinolone-3-carboxamide derivatives have long been recognized as potent and selective cannabinoid type-2 receptor (CB2R) ligands. With the aim to improve their physicochemical properties, basically aqueous solubility, two different approaches were followed, entailing the substitution of the alkyl chain with a basic replacement or scaffold modification to 4-hydroxy-2-quinolone structure. According to the first approach, compound 6d was obtained, showing slightly reduced receptor affinity (K(i) = 60 nM) compared to the lead compound 4 (0.8 nM) but greatly enhanced solubility (400-3400 times depending on the pH of the medium). On the other hand, shifting from 4-quinolone to 4-hydroxy-2-quinolone structure enabled the discovery of a novel class of CB2R ligands, such as 7b and 7c, characterized by K(i) < 1 nM and selectivity index [SI = K(i)(CB1R)/K(i)(CB2R)] > 1300. At pH 7.4, compound 7c resulted by 100-fold more soluble than 4.

Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.

The CB2 receptor is a therapeutic target of increasing importance for several diseases, including pain, inflammation, neurodegeneration, cancer and osteoporosis. While several compounds showing CB2-selective agonist or inverse agonist properties have been developed, only few CB2 receptor selective neutral antagonists are actually known. Such type of compounds could be useful to study more in depth the role of the CB2 receptor, because they lack the ability to counteract its "constitutive" activity. Here we describe the synthesis and biological activity of a series of biphenylic carboxamides as a new class of CB2 receptor selective ligands. In binding assays, one of these compounds showed good CB2 receptor affinity and selectivity (Ki = 11.48 nM; Selectivity Index = 130). Furthermore, in functional assays, the same compound showed a very interesting pharmacological profile as CB2 receptor selective neutral antagonist. These results pave the way to further developments, including structural optimization, with the aim to obtain more potent CB2 receptor ligands with this peculiar feature., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)