An updated patent review of TRPA1 antagonists (2020 - present).

INTRODUCTION: TRPA1 is a nonselective calcium channel, a member of the transient receptor potential (TRP) superfamily, also referred to as the 'irritant' receptor, being activated by pungent and noxious exogenous chemicals as well as by endogenous algogenic stimuli, to elicit pain, itching, and inflammatory conditions. For this reason, it is considered an attractive therapeutic target to treat a wide range of diseases including acute and chronic pain, itching, and inflammatory airway diseases. AREAS COVERED: The present review covers patents on TRPA1 antagonists disclosed from 2020 to present, falling in the following main classes: i) novel therapeutic applications for known or already disclosed antagonists, ii) identification and characterization of TRPA1 antagonists from natural sources, and iii) synthesis and evaluation of novel compounds. EXPERT OPINION: Despite the limited number of TRPA1 antagonists in clinical trials, there is an ever-growing interest on this receptor-channel as therapeutic target, mainly due to the relevant outcomes from basic research, which unveiled novel physio-pathological mechanisms where TRPA1 is believed to play a pivotal role, for example the Alzheimer's disease or ocular diseases, expanding the panel of potential therapeutic applications for TRPA1 modulators.

Eukaryotic Initiation Translation Factor 2A activation by cannabidiolic acid alters the protein homeostasis balance in glioblastoma cells.

Eukaryotic Initiation Translation Factor 2A (EIF2A) is considered to be primarily responsible for the initiation of translation when a cell is subjected to stressful conditions. However, information regarding this protein is still incomplete. Using a combination of proteomic approaches, we demonstrated that EIF2A is the molecular target of the naturally occurring bioactive compound cannabidiolic acid (CBDA) within human glioblastoma cells. This finding allowed us to undertake a study aimed at obtaining further information on the functions that EIF2A plays in tumor cells. Indeed, our data showed that CBDA is able to activate EIF2A when the cells are in no-stress conditions. It induces conformational changes in the protein structure, thus increasing EIF2A affinity towards the proteins participating in the Eukaryotic Translation Machinery. Consequently, following glioblastoma cells incubation with CBDA we observed an enhanced neosynthesis of proteins involved in the stress response, nucleic acid translation and organization, and protein catabolism. These changes in gene expression resulted in increased levels of ubiquitinated proteins and accumulation of the autophagosome. Our results, in addition to shedding light on the molecular mechanism underlying the biological effect of a phytocannabinoid in cancer cells, demonstrated that EIF2A plays a critical role in regulation of protein homeostasis.

Flipping the GPCR Switch: Structure-Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists.

We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB2R) selective inverse agonists (S)-1 and (R)-1, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem-dimethylheptyl side chain. Epimer (R)-1 exhibits high affinity for CB2R with Kd = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-mediated cAMP as well as G protein recruitment assays and do not trigger ß-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp2586.48 and thereby stabilize CB2R in its inactive state.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Phase I Trial of Viral Vector-Based Personalized Vaccination Elicits Robust Neoantigen-Specific Antitumor T-Cell Responses.

PURPOSE: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T-cell response to overcome tumor heterogeneity. NOUS-PEV is a vector-based personalized vaccine, expressing 60 nAgs and consists of priming with a nonhuman Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara. Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment-naïve patients with metastatic melanoma (NCT04990479). PATIENTS AND METHODS: The feasibility of this approach was demonstrated by producing, releasing, and administering to 6 patients 11 of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. RESULTS: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen-specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced T-cell receptor (TCR) clonotypes was observed in the posttreatment biopsies of patients with clinical response, providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cells. CONCLUSIONS: These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor-reactive T cells to empower a diverse, potent, and durable antitumor immune response. Finally, a gene signature indicative of the reduced presence of activated T cells together with very poor expression of the antigen-processing machinery genes has been identified in pretreatment biopsies as a potential biomarker of resistance to the treatment.

Phytochemical Characterization and TRPA1/TRPM8 Modulation Profile of the Cannabigerol-Rich Cannabis sativa L. Chemotype IV.

The first detailed phytochemical analysis of the cannabigerol (CBG)-rich chemotype IV of Cannabis sativa L. resulted in the isolation of the expected cannabigerolic acid/cannabigerol (CBGA/CBG) and cannabidiolic acid/cannabidiol (CBDA/CBD) and of nine new phytocannabinoids (5-13), which were fully characterized by HR-ESIMS and 1D and 2D NMR. These included mono- or dihydroxylated CBGA/CBG analogues, a congener with a truncated side chain (10), cyclocannabigerol B (11), and the CBD derivatives named cannabifuranols (12 and 13). Cyclocannabigerol B and cannabifuranols are characterized by a novel phytocannabinoid structural architecture. The isolated phytocannabinoids were assayed on the receptor channels TRPA1 and TRPM8, unveiling a potent dual TRPA1 agonist/TRPM8 antagonist profile for compounds 6, 7, and 14. Chiral separation of the two enantiomers of 5 resulted in the discovery of a synergistic effect of the two enantiomers on TRPA1.

PPARα/γ-Targeting Amorfrutin Phytocannabinoids from Aerial Parts of Glycyrrhiza foetida.

An LC-MS/MS-guided analysis of the aerial parts of Glycyrrhiza foetida afforded new phenethyl (amorfrutin)- and alkyl (cannabis)-type phytocannabinoids (six and four compounds, respectively). The structural diversity of the new amorfrutins was complemented by the isolation of six known members and the synthesis of analogues modified on the aralkyl moiety. All of the compounds so obtained were assayed for agonist activity on PPARα and PPARγ nuclear receptors. Amorfrutin A (1) showed the highest agonist activity on PPARγ, amorfrutin H (7) selectively targeted PPARα, and amorfrutin E (4) behaved as a dual agonist, with the pentyl analogue of amorfrutin A (11) being inactive. Decarboxyamorfrutin A (2) was cytotoxic, and modifying its phenethyl moiety to a styryl or a phenylethynyl group retained this trait, suggesting an alternative biological scenario for these compounds. The putative binding modes of amorfrutins toward PPARα and PPARγ were obtained by a combined approach of molecular docking and molecular dynamics simulations, which provided insights on the structure-activity relationships of this class of compounds.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.

Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Marine Natural and Nature-Inspired Compounds Targeting Peroxisome Proliferator Activated Receptors (PPARs).

Peroxisome proliferator-activated receptors α, γ and ß/δ (PPARα, PPARγ, and PPARß/δ) are a family of ligand-activated transcriptional factors belonging to the superfamily of nuclear receptors regulating the expression of genes involved in lipid and carbohydrate metabolism, energy homeostasis, inflammation, and the immune response. For this reason, they represent attractive targets for the treatment of a variety of metabolic diseases and, more recently, for neurodegenerative disorders due to their emerging neuroprotective effects. The degree of activation, from partial to full, along with the selectivity toward the different isoforms, greatly affect the therapeutic efficacy and the safety profile of PPAR agonists. Thus, there is a high interest toward novel scaffolds with proper combinations of activity and selectivity. This review intends to provide an overview of the discovery, optimization, and structure-activity relationship studies on PPAR modulators from marine sources, along with the structural and computational studies that led to their identification and/or elucidation, and rationalization of their mechanisms of action.

Fluorescence-Based Assay for TRPV1 Channels.

The transient receptor potential vanilloid 1 ion channel (TRPV1) is a ligand-gated nonselective calcium-permeant cation channel involved in the detection of a wide variety of chemical and physical noxious stimuli, ranging from exogenous and endogenous ligands to noxious heat (>42 °C) and low pH (pH < 5.2). Due to its central role in pain and hyperalgesia, TRPV1 is considered a relevant therapeutic target for the development of analgesic and anti-inflammatory drugs potentially useful to relieve chronic, neuropathic, and inflammatory pain and to treat disorders such as inflammatory bowel disease. In this view, the availability of in vitro assays for the screening of novel TRPV1 modulators is highly desirable. Since TRPV1 activation leads to an increase in the intracellular calcium (Ca2+) levels, the use of Ca2+ fluorescent indicators represent a valuable and sensitive tool for monitoring such intracellular changes. In this chapter, we describe methods for recording and monitoring Ca2+ signals through the fluorescent indicators Fluo-4 acetoxymethyl (AM) and Fura-2 AM in HEK-293 cells transfected with TRPV1 or other thermoTRP channels.

Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion.

BACKGROUND: A number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the 'Cancer Immunity Cycle'. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance. METHODS: The antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs. RESULTS: The addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs. CONCLUSION: These data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

Biosynthesis of the Novel Endogenous 15-Lipoxygenase Metabolites N-13-Hydroxy-octodecadienoyl-ethanolamine and 13-Hydroxy-octodecadienoyl-glycerol by Human Neutrophils and Eosinophils.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis inhibition with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15-30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Identification and Characterization of Cannabidiol as an OX1R Antagonist by Computational and In Vitro Functional Validation.

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 µM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.

The Combined Effect of Branching and Elongation on the Bioactivity Profile of Phytocannabinoids. Part I: Thermo-TRPs.

The affinity of cannabinoids for their CB1 and CB2 metabotropic receptors is dramatically affected by a combination of α-branching and elongation of their alkyl substituent, a maneuver exemplified by the n-pentyl -> α,α-dimethylheptyl (DMH) swap. The effect of this change on other cannabinoid end-points is still unknown, an observation surprising since thermo-TRPs are targeted by phytocannabinoids with often sub-micromolar affinity. To fill this gap, the α,α-dimethylheptyl analogues of the five major phytocannabinoids [CBD (1a), Δ8-THC (6a), CBG (7a), CBC (8a) and CBN (9a)] were prepared by total synthesis, and their activity on thermo-TRPs (TRPV1-4, TRPM8, and TRPA1) was compared with that of one of their natural analogues. Surprisingly, the DMH chain promoted a shift in the selectivity toward TRPA1, a target involved in pain and inflammatory diseases, in all investigated compounds. A comparative study of the putative binding modes at TRPA1 between DMH-CBC (8b), the most active compound within the series, and CBC (8a) was carried out by molecular docking, allowing the rationalization of their activity in terms of structure-activity relationships. Taken together, these observations qualify DMH-CBC (8b) as a non-covalent TRPA1-selective cannabinoid lead that is worthy of additional investigation as an analgesic and anti-inflammatory agent.

The (Poly)Pharmacology of Cannabidiol in Neurological and Neuropsychiatric Disorders: Molecular Mechanisms and Targets.

Cannabidiol (CBD), the major nonpsychoactive Cannabis constituent, has been proposed for the treatment of a wide panel of neurological and neuropsychiatric disorders, including anxiety, schizophrenia, epilepsy and drug addiction due to the ability of its versatile scaffold to interact with diverse molecular targets that are not restricted to the endocannabinoid system. Albeit the molecular mechanisms responsible for the therapeutic effects of CBD have yet to be fully elucidated, many efforts have been devoted in the last decades to shed light on its complex pharmacological profile. In particular, an ever-increasing number of molecular targets linked to those disorders have been identified for this phytocannabinoid, along with the modulatory effects of CBD on their cascade signaling. In this view, here we will try to provide a comprehensive and up-to-date overview of the molecular basis underlying the therapeutic effects of CBD involved in the treatment of neurological and neuropsychiatric disorders.

The Endocannabinoid System and PPARs: Focus on Their Signalling Crosstalk, Action and Transcriptional Regulation.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors including PPARα, PPARγ, and PPARß/δ, acting as transcription factors to regulate the expression of a plethora of target genes involved in metabolism, immune reaction, cell differentiation, and a variety of other cellular changes and adaptive responses. PPARs are activated by a large number of both endogenous and exogenous lipid molecules, including phyto- and endo-cannabinoids, as well as endocannabinoid-like compounds. In this view, they can be considered an extension of the endocannabinoid system. Besides being directly activated by cannabinoids, PPARs are also indirectly modulated by receptors and enzymes regulating the activity and metabolism of endocannabinoids, and, vice versa, the expression of these receptors and enzymes may be regulated by PPARs. In this review, we provide an overview of the crosstalk between cannabinoids and PPARs, and the importance of their reciprocal regulation and modulation by common ligands, including those belonging to the extended endocannabinoid system (or "endocannabinoidome") in the control of major physiological and pathophysiological functions.

N-palmitoyl-D-glucosamine, a Natural Monosaccharide-Based Glycolipid, Inhibits TLR4 and Prevents LPS-Induced Inflammation and Neuropathic Pain in Mice.

Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells; (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines; (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas; (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI); (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.

2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors.

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.