Inhibition of Fatty Acid Amide Hydrolase (FAAH) Regulates NF-kb Pathways Reducing Bleomycin-Induced Chronic Lung Inflammation and Pulmonary Fibrosis.

The deadly interstitial lung condition known as idiopathic pulmonary fibrosis (IPF) worsens over time and for no apparent reason. The traditional therapy approaches for IPF, which include corticosteroids and immunomodulatory drugs, are often ineffective and can have noticeable side effects. The endocannabinoids are hydrolyzed by a membrane protein called fatty acid amide hydrolase (FAAH). Increasing endogenous levels of endocannabinoid by pharmacologically inhibiting FAAH results in numerous analgesic advantages in a variety of experimental models for pre-clinical pain and inflammation. In our study, we mimicked IPF by administering intratracheal bleomycin, and we administered oral URB878 at a dose of 5 mg/kg. The histological changes, cell infiltration, pro-inflammatory cytokine production, inflammation, and nitrosative stress caused by bleomycin were all reduced by URB878. Our data clearly demonstrate for the first time that the inhibition of FAAH activity was able to counteract not only the histological alteration bleomycin-induced but also the cascade of related inflammatory events.

Fatty Acid Amide Hydrolase (FAAH) Inhibition Plays a Key Role in Counteracting Acute Lung Injury.

Acute lung injury (ALI) is a group of lung illnesses characterized by severe inflammation, with no treatment. The fatty acid amide hydrolase (FAAH) enzyme is an integral membrane protein responsible for the hydrolysis of the main endocannabinoids, such as anandamide (AEA). In pre-clinical pain and inflammation models, increasing the endogenous levels of AEA and other bioactive fatty acid amides (FAAs) via genetic deletion or the pharmacological inhibition of FAAH produces many analgesic benefits in several different experimental models. To date, nobody has investigated the role of FAAH inhibition on an ALI mouse model. Mice were subjected to a carrageenan injection and treated orally 1 h after with the FAAH inhibitor URB878 dissolved in a vehicle consisting of 10% PEG-400, 10% Tween-80 and 80% saline at different doses: The inhibition of FAAH activity was able to counteract not only the CAR-induced histological alteration, but also the cascade of related inflammatory events. URB878 clears the way for further studies based on FAAH inhibition in acute lung pathologies.

Inhibition of anandamide hydrolysis enhances noradrenergic and GABAergic transmission in the prefrontal cortex and basolateral amygdala of rats subjected to acute swim stress.

Limbic forebrain endocannabinoid (eCB) signaling is critically involved in stress integration by modulating neurotransmitters release. The purpose of this study was to examine, by brain microdialysis, the effects of fatty acid amide hydrolase (FAAH) inhibition on noradrenergic and γ-aminobutyric acid (GABA)-ergic neurotransmission in the prefrontal cortex (PFC) and basolateral amygdala (BLA) of rats subjected to a 20-min swim stress. Microdialysis started on stress- and drug-naïve rats that were treated with the FAAH inhibitor URB597 (0.1 or 0.3 mg/kg) 30 min before undergoing the stress procedure. Dialysate samples were collected every 20 min from the beginning of the experiment. Concentrations of noradrenaline (NA) and GABA were determined by HPLC coupled to electrochemical and fluorescence detection, respectively. We found that neither URB597 treatment nor 20 min of swim stress exposure per se altered NA and GABA extracellular levels in PFC or BLA. Interestingly, rats treated with 0.1 mg/kg of URB597 followed by 20 min of stress showed significantly higher NA and GABA levels in PFC and BLA. These effects were absent in rats treated with 0.3 mg/kg URB597, indicating a dose-specific effect. Moreover, we found that the pretreatment with the CB1 receptor antagonist rimonabant blocked the URB597 effects on NA and GABA release in PFC and BLA of animals subjected to forced swimming. The present study might provide an important first step toward understanding the mechanisms through which URB597 modulates stress-induced neuroendocrine secretion and behavioral coping strategies.

Chronic stimulation of the tone of endogenous anandamide reduces cue- and stress-induced relapse in rats.

BACKGROUND: The endogenous cannabinoid system plays an important role in motivation, stress, and drug abuse. Pharmacologically, the endocannabinoid system can be stimulated by either agonists of CB1 receptors or inhibition of metabolic degradation of endogenous cannabinoids and consequent increases in their brain levels. METHODS: Here, we investigated whether chronic administration during a period of withdrawal of the fatty acid amide hydrolase inhibitor URB597, which increases anandamide levels, would decrease the risks of relapse to cocaine seeking. Rats were allowed to self-administer cocaine and then they underwent forced withdrawal for 28 days, during which they were treated with URB597 or vehicle. One day after the last injection, we investigated cocaine seeking in one 6h extinction session and relapse triggered by re-exposure to drug-associated cues or a pharmacological stressor. RESULTS: We found that administration of URB597 significantly decreases cocaine-seeking behavior and cue- and stress-induced relapse. CONCLUSION: These results suggest that stimulation of the endocannabinoid system could be helpful to prevent relapse to cocaine addiction.

Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain.

The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

Characterization and Preliminary In Vitro Antioxidant Activity of a New Multidrug Formulation Based on the Co-Encapsulation of Rutin and the α-Acylamino-ß-Lactone NAAA Inhibitor URB894 within PLGA Nanoparticles.

A biodegradable and biocompatible polymeric matrix made up of poly(d,l-lactide-co-glycolide) (PLGA) was used for the simultaneous delivery of rutin and the (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide derivative (URB894). The goal was to exploit the well-known radical scavenging properties of rutin and the antioxidant features recently reported for the molecules belonging to the class of N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitors, such as URB894. The use of the compounds, both as single agents or in association promoted the development of negatively-charged nanosystems characterized by a narrow size distribution and an average diameter of ~200 nm when 0.2-0.6 mg/mL of rutin or URB894 were used. The obtained multidrug carriers evidenced an entrapment efficiency of ~50% and 40% when 0.4 and 0.6 mg/mL of rutin and URB894 were associated during the sample preparation, respectively. The multidrug formulation evidenced an improved in vitro dose-dependent protective effect against H2O2-related oxidative stress with respect to that of the nanosystems containing the active compounds as a single agent, confirming the rationale of using the co-encapsulation approach to obtain a novel antioxidant nanomedicine.

Characterization of URB Series Synthetic Cannabinoids by HRMS and UHPLC-MS/MS.

A large number of synthetic cannabinoids are included in new psychoactive substances (NPS) and constitute an open research area in analytical pharmaceutical and toxicology when methods are needed to unambiguously identify these substances and their metabolites in biological fluids. A full molecular characterization of five synthetic molecules of the URB series that is able to interact with the endocannabinoid system was achieved with a high-resolution mass spectrometry (HRMS) in positive ion electrospray ionization and collisional experiments on the protonated parent ions, obtaining characteristic fragmentation patterns. Ultra-high-performance liquid chromatography coupled with a triple quadrupole (UHPLC-MS/MS) has also been used, which can help develop methods for screening and confirming synthetic cannabinoids in biological fluids.

Synthesis and Biological Evaluation of 6-O-Sucrose Monoester Glycolipids as Possible New Antifungal Agents.

A small library of 6-O-sucrose monoester surfactants has been synthesized and tested against various microorganisms. The synthetic procedure involved a modified Mitsunobu reaction, which showed improved results compared to those present in the literature (higher yields and larger scope). The antifungal activities of most of these glycolipids were satisfactory. In particular, sucrose palmitoleate (URB1537) showed good activity against Candida albicans ATCC 10231, Fusarium spp., and Aspergillus fumigatus IDRAH01 (MIC value: 16, 32, 64 µg/mL, respectively), and was further characterized through radical scavenging, anti-inflammatory, and biocompatibility tests. URB1537 has been shown to control the inflammatory response and to have a safe profile.

Sugar-Based Monoester Surfactants: Synthetic Methodologies, Properties, and Biological Activities.

Glycolipids are biocompatible and biodegradable amphiphilic compounds characterized by a great scientific interest for their potential applications in various technological areas, including pharmaceuticals, cosmetics, agriculture, and food production. This report summarizes the available synthetic methodologies, physicochemical properties, and biological activity of sugar fatty acid ester surfactants, with a particular focus on 6-O-glucose, 6-O-mannose, 6-O-sucrose, and 6'-O-lactose ones. In detail, the synthetic approaches to this class of compounds, such as enzymatic lipase-catalyzed and traditional chemical (e.g., acyl chloride, Steglich, Mitsunobu) esterifications, are reported. Moreover, aspects related to the surface activity of these amphiphiles, such as their ability to decrease surface tension, critical micelle concentration, and emulsifying and foaming ability, are described. Biological applications with a focus on the permeability-enhancing effect across the skin or mucosa, antimicrobial and antifungal activities, as well as antibiofilm properties, are also presented. The information reported here on sugar-based ester surfactants is helpful to broaden the interest and the possible innovative applications of this class of amphiphiles in different technological fields in the future.

Endocannabinoid Degradation Enzyme Inhibitors as Potential Antipsychotics: A Medicinal Chemistry Perspective.

The endocannabinoid system (ECS) plays a very important role in numerous physiological and pharmacological processes, such as those related to the central nervous system (CNS), including learning, memory, emotional processing, as well pain control, inflammatory and immune response, and as a biomarker in certain psychiatric disorders. Unfortunately, the half-life of the natural ligands responsible for these effects is very short. This perspective describes the potential role of the inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), which are mainly responsible for the degradation of endogenous ligands in psychic disorders and related pathologies. The examination was carried out considering both the impact that the classical exogenous ligands such as Δ9-tetrahydrocannabinol (THC) and (-)-trans-cannabidiol (CBD) have on the ECS and through an analysis focused on the possibility of predicting the potential toxicity of the inhibitors before they are subjected to clinical studies. In particular, cardiotoxicity (hERG liability), probably the worst early adverse reaction studied during clinical studies focused on acute toxicity, was predicted, and some of the most used and robust metrics available were considered to select which of the analyzed compounds could be repositioned as possible oral antipsychotics.

Synthesis and Properties of Sucrose- and Lactose-Based Aromatic Ester Surfactants as Potential Drugs Permeability Enhancers.

The delivery of therapeutics across biological membranes (e.g., mucosal barriers) by avoiding invasive routes (e.g., injection) remains a challenge in the pharmaceutical field. As such, there is the need to discover new compounds that act as drug permeability enhancers with a favorable toxicological profile. A valid alternative is represented by the class of sugar-based ester surfactants. In this study, sucrose and lactose alkyl aromatic and aromatic ester derivatives have been synthesized with the aim to characterize them in terms of their physicochemical properties, structure-property relationship, and cytotoxicity, and to test their ability as permeability enhancer agents across Calu-3 cells. All of the tested surfactants showed no remarkable cytotoxic effect on Calu-3 cells when applied both below and above their critical micelle concentration. Among the explored molecules, lactose p-biphenyl benzoate (URB1420) and sucrose p-phenyl benzoate (URB1481) cause a reversible ~30% decrease in transepithelial electrical resistance (TEER) with the respect to the basal value. The obtained result matches with the increased in vitro permeability coefficients (Papp) calculated for FTIC-dextran across Calu-3 cells in the presence of 4 mM solutions of these surfactants. Overall, this study proposes sucrose- and lactose-based alkyl aromatic and aromatic ester surfactants as novel potential and safe permeation enhancers for pharmaceutical applications.

Synthetic Methodologies and Therapeutic Potential of Indole-3-Carbinol (I3C) and Its Derivatives.

Indole-3-carbinol (I3C) is a natural product contained in vegetables belonging to the Brassicaceae family and has been studied in recent decades for its biological and pharmacological properties. Herein, we will analyze: (1) the biosynthetic processes and synthetic procedures through which I3C and its main derivatives have been obtained; (2) the characteristics that lead to believe that both I3C and its derivatives are responsible for several important activities-in particular, antitumor and antiviral, through insights concerning in vitro assays and in vivo tests; (3) the mechanisms of action of the most important compounds considered; (4) the potential social impact that the enhancement of the discussed molecules can have in the prevention and treatment of the pathologies' examined field-first of all, those related to respiratory tract disorders and cancer.

The influence of mannose-based esters on the mesophase behaviour of lyotropic liquid crystalline nanosystems as drug delivery vectors.

Lyotropic Liquid Crystalline (LLC) nanoparticles represent an emerging class of smart, biocompatible, and biodegradable systems for the delivery of drugs. Among these, structures with complex 3D architectures such as cubosomes are of particular interest. These are non- lamellar assemblies having hydrophobic and hydrophilic portions able to carry drugs of different nature. They can further be modulated including suitable additives to control the release of the active payload, and to promote an active targeting. Starting from monoolein (GMO) cubic phase, different concentrations of mannose-based esters were added, and the eventual structural modifications were monitored to ascertain the effects of the presence of glycolipids. Moreover, the structural properties of these nanosystems loaded with Dexamethasone (DEX), a very well-known anti-inflammatory steroid, were also studied. Experiments were carried out by synchrotron Small Angle X-ray Scattering (SAXS), Raman Microspectroscopy (RMS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) measurements. The drug delivery potential (i.e. entrapment efficiency and release properties) of the obtained nanoparticles was evaluated. Finally, in vitro cytocompatibility and anti-inflammatory activity studies of the prepared formulations were carried out. Inclusion of mannose-based surfactants up to 10 mol% influenced the structural parameters of Im3m cubic phase and swollen cubic phases were obtained with the different glycolipids with lattice parameters significantly higher than GMO. A complete cytocompatibility and an increased DEX activity were observed, thus suggesting the possibility to use GMO/glycolipids nanoparticles to formulate innovative drug delivery systems.

Pretreatment with the monoacylglycerol lipase inhibitor URB602 protects from the long-term consequences of neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB602 can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury. METHODS: URB602 was administered into the right lateral ventricle 30 min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively. RESULTS: Pretreatment with URB602 reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB602-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage. CONCLUSION: These results demonstrate that a pretreatment with URB602 significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury.

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

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

The Synthetic Cannabinoid URB447 Exerts Antitumor and Antimetastatic Effect in Melanoma and Colon Cancer.

The endocannabinoid system is widespread through the body and carries out a wide variety of functions. However, its involvement in other pathologies, such as cancer, still needs further attention. We aim to investigate the role of CB2 receptor during melanoma and colorectal cancer (CRC) aggressiveness and metastatic growth in the liver. We used the synthetic cannabinoid URB447, a known CB2 agonist and CB1 antagonist drug, and studied prometastatic ability of mouse B16 melanoma and MCA38 CRC cells, by means of proliferation, apoptosis, cell cycle, migration and matrix degradation in vitro upon URB447 treatment. We reported a dose-dependent viability decrease in both tumor types. This result is partly mediated by apoptotic cell death and cell cycle arrest in G1/G0 phase, as observed through flow cytometry. Melanoma and CRC cell migration was affected in a dose-dependent fashion as observed through scratch assay, whereas the secretion of matrix degrading proteins metalloprotease 2 (MMP2) and 9 (MMP9) in tumor cells did not significantly change. Moreover, daily treatment of tumor bearing mice with URB447 decreased the development of liver metastasis in a melanoma model in vivo. This proof of concept study points out to the synthetic cannabinoid URB447 as a potential candidate for deeper studies to confirm its potential as antitumor therapy and liver metastasis treatment for CRC and melanoma.

α-Acylamino-ß-lactone N-Acylethanolamine-hydrolyzing Acid Amidase Inhibitors Encapsulated in PLGA Nanoparticles: Improvement of the Physical Stability and Protection of Human Cells from Hydrogen Peroxide-Induced Oxidative Stress.

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that preferentially catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide, which has been shown to exhibit neuroprotective and antinociceptive properties by engaging peroxisome proliferator-activated receptor-α. A few potent NAAA inhibitors have been developed, including α-acylamino-ß-lactone derivatives, which are very strong and effective, but they have limited chemical and plasmatic stability, compromising their use as systemic agents. In the present study, as an example of a molecule belonging to the chemical class of N-(2-oxo-3-oxetanyl)amide NAAA inhibitors, URB866 was entrapped in poly(lactic-co-glycolic acid) nanoparticles in order to increase its physical stability. The data show a monomodal pattern and a significant time- and temperature-dependent stability of the molecule-loaded nanoparticles, which also demonstrated a greater ability to effectively retain the compound. The nanoparticles improved the photostability of URB866 with respect to that of the free molecule and displayed a better antioxidant profile on various cell lines at the molecule concentration of 25 µM. Overall, these results prove that the use of polymeric nanoparticles could be a useful strategy for overcoming the instability of α-acylamino-ß-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time.

Synthesis and Biological Characterization of the New Glycolipid Lactose Undecylenate (URB1418).

As a follow-up to our previous studies on glycolipid surfactants, a new molecule, that is lactose 6'-O-undecylenate (URB1418), was investigated. To this end, a practical synthesis and studies aimed at exploring its specific properties were carried out. URB1418 showed antifungal activities against Trichophyton rubrum F2 and Candida albicans ATCC 10231 (MIC 512 µg/mL) and no significant antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. At the same time, it presented anti-inflammatory properties, as documented by the dose-dependent reduction in LPS-induced NO release in RAW 264.7 cells, while a low antioxidant capacity in the range of concentrations tested (EC50 > 200 µM) was also observed. Moreover, URB1418 offers the advantage of being more stable than the reference polyunsaturated lactose esters and of being synthesized using a "green" procedure, involving an enzymatic method, high yield and low manufacturing cost. For all these reasons and the absence of toxicity (HaCaT cells), the new glycolipid presented herein could be considered an interesting compound for applications in various fields.

The Endocannabinoid System as a Target for Neuroprotection/Neuroregeneration in Perinatal Hypoxic-Ischemic Brain Injury.

The endocannabinoid (EC) system is a complex cell-signaling system that participates in a vast number of biological processes since the prenatal period, including the development of the nervous system, brain plasticity, and circuit repair. This neuromodulatory system is also involved in the response to endogenous and environmental insults, being of special relevance in the prevention and/or treatment of vascular disorders, such as stroke and neuroprotection after neonatal brain injury. Perinatal hypoxia-ischemia leading to neonatal encephalopathy is a devastating condition with no therapeutic approach apart from moderate hypothermia, which is effective only in some cases. This overview, therefore, gives a current description of the main components of the EC system (including cannabinoid receptors, ligands, and related enzymes), to later analyze the EC system as a target for neonatal neuroprotection with a special focus on its neurogenic potential after hypoxic-ischemic brain injury.

A combination of sugar esters and chitosan to promote in vivo wound care.

In recent years, researchers are exploring innovative green materials fabricated from renewable natural substances to meet formulation needs. Among them, biopolymers like chitosans and biosurfactants such as sugar fatty acid esters are of potential interest due to their biocompatibility, biodegradability, functionality, and cost-effectiveness. Both classes of biocompounds possess the ability to be efficiently employed in wound dressing to help physiological wound healing, which is a bioprocess involving uncontrolled oxidative damage and inflammation, with an associated high risk of infection. In this work, we synthesized two different sugar esters (i.e., lactose linoleate and lactose linolenate) that, in combination with chitosan and sucrose laurate, were evaluated in vitro for their cytocompatibility, anti-inflammatory, antioxidant, and antibacterial activities and in vivo as wound care agents. Emphasis on Wnt/ß-catenin associated machineries was also set. The newly designed lactose esters, sucrose ester, and chitosan possessed sole biological attributes, entailing considerable blending for convenient formulation of wound care products. In particular, the mixture composed of sucrose laurate (200 µM), lactose linoleate (100 µM), and chitosan (1%) assured its superiority in terms of efficient wound healing prospects in vivo together with the restoring of the Wnt/ß-catenin signaling pathway, compared with the marketed wound healing product (Healosol®), and single components as well. This innovative combination of biomaterials applied as wound dressing could effectively break new ground in skin wound care.