N-acylethanolamine Acid Amidase Research Articles

Pharmacological inhibition of N-Acylethanolamine acid amidase (NAAA) mitigates intestinal fibrosis through modulation of macrophage activity.

Intestinal fibrosis, a frequent complication of inflammatory bowel disease, is characterized by stricture formation with no pharmacological treatment to date. N-acylethanolamine acid amidase (NAAA) is responsible of acylethanolamides (AEs, e.g., palmitoylethanolamide and oleoylethanolamide) hydrolysis. Here, we investigated NAAA and AEs signalling in gut fibrosis. NAAA and AEs signalling were evaluated in human intestinal specimens from stenotic Crohn's diseases (CD) patients. Gut fibrosis was induced by TNBS, monitored by colonoscopy and unascertained by qRT-PCR, histological analyses, and confocal microscopy. Immune cells were analysed in mesenteric lymph nodes by FACS. Colonic fibroblasts were cultured in conditioned media derived from polarized or not bone marrow-derived macrophages (BMDM). IL-23 signalling was evaluated by qRT-PCR, ELISA, FACS, and western blot in BMDM and in lamina propria CX3CR1+ cells. In ileocolonic human CD strictures, increased transcript expression of NAAA was observed with a decrease of its substrates OEA and PEA. NAAA inhibition reduced intestinal fibrosis in vivo, as revealed by decrease in inflammatory parameters, collagen deposition and fibrosis genes, including epithelial to mesenchymal transition. More in-depth studies revealed modulation of the immune response related to IL-23 following NAAA inhibition. The antifibrotic actions of NAAA inhibition are mediated by Mφ and M2 macrophages that indirectly affect fibroblast collagenogenesis. NAAA inhibitor AM9053 normalized IL-23 signalling in BMDM and in lamina propria CX3CR1+ cells. Our findings provide new insights into the pathophysiological mechanism of intestinal fibrosis and identify NAAA as a promising target for the development of therapeutic treatments to alleviate CD fibrosis.

CSF N-acylethanolamine acid amidase level and Parkinson's disease risk: A mendelian randomization study

BackgroundNeuroinflammation is involved in the progression of Parkinson's disease (PD), and N-acylethanolamine acid amidase (NAAA) is involved in regulating inflammation by hydrolyzing bioactive lipid mediators called N-acylethanolamines (NAEs). However, the causal relationship between cerebrospinal fluid (CSF) NAAA protein levels and the risk of PD remains unclear. This study aimed to explore the causal effect of CSF NAAA levels on PD risk through Mendelian randomization (MR) analysis. MethodGenome-wide association study (GWAS) summary statistics for CSF NAAA protein quantitative trait loci (pQTL) and GWAS summary statistics for PD were obtained from publicly available databases. Inverse-variance weighted (IVW) was the main causal estimation method for MR analysis. In addition, the maximum likelihood, MR Egger regression, and weighted median were used to supplement the IVW results. Finally, various sensitivity tests were performed to verify the reliability of the MR findings. ResultsIn the initial MR analysis, the IVW showed that CSF NAAA protein levels significantly increased PD risk (odds ratio [OR] = 1.17, 95% confidence interval [CI]: 1.01–1.35, P = 0.031). This finding was further validated in a replicate MR analysis (OR = 1.20, 95% CI: 1.02–1.41, P = 0.027). Sensitivity analysis showed that MR results were stable and not affected by heterogeneity and horizontal pleiotropy. ConclusionThe present MR study supports a causal relationship between elevated CSF NAAA protein levels and increased PD risk.

NAAA-regulated lipid signaling in monocytes controls the induction of hyperalgesic priming in mice

Circulating monocytes participate in pain chronification but the molecular events that cause their deployment are unclear. Using a mouse model of hyperalgesic priming (HP), we show that monocytes enable progression to pain chronicity through a mechanism that requires transient activation of the hydrolase, N-acylethanolamine acid amidase (NAAA), and the consequent suppression of NAAA-regulated lipid signaling at peroxisome proliferator-activated receptor-α (PPAR-α). Inhibiting NAAA in the 72 hours following administration of a priming stimulus prevented HP. This effect was phenocopied by NAAA deletion and depended on PPAR-α recruitment. Mice lacking NAAA in CD11b+ cells – monocytes, macrophages, and neutrophils – were resistant to HP induction. Conversely, mice overexpressing NAAA or lacking PPAR-α in the same cells were constitutively primed. Depletion of monocytes, but not resident macrophages, generated mice that were refractory to HP. The results identify NAAA-regulated signaling in monocytes as a control node in the induction of HP and, potentially, the transition to pain chronicity.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing β-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses.To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing β-oxidation and preventing inflammation.Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition

Ethnopharmacological relevanceRamulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. Aim of the studyTo investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. Materials and methodsRCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). ResultsWe established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 μg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 μg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. ConclusionsRCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

An Investigation on the Relationships between Mass Spectrometric Collisional Data and Biological Activity/Stability of Some N-Acylethanolamine Acid Amidase (NAAA) β-Lactone Inhibitors

The rationale of the present study is that relationships between in vitro biological activity and mass spectrometric (MS) collisional data of molecular species have been already reported in the literature. Herein, the same approach has been employed to investigate possible correlations between MS stability and biological activity/stability data of a series of β-lactone amides and carbamates N-acylethanolamine acid amidase (NAAA) inhibitors. Electrospray ionization MS experiments were performed using an LCQ Deca ion trap and samples were introduced by direct infusion. Mass spectra of positive and negative ions have been obtained, and collisional experiments were performed on selected ionic species. Collisional-induced fragmentation pathways of molecular species related to β-lactone amide inhibitors are different in comparison to those of carbamates, being the former species more stable than the latter, due to β-lactone reactivity. Correlations were found between the characteristic collision energy (CE50) obtained by the breakdown curves and in vitro NAAA inhibitory potency of the β-lactone amides and carbamates analyzed. In the case of carbamates, a relationship between CE50 values and bovine serum albumin (BSA) stability data was also found, while any correlation was not found for amides due to their instability to BSA. β-Lactone NAAA inhibitors’ activity can be qualitatively associated with their lability, as measured by CE50 values. The results obtained could suggest that MS may be used as a preliminary experimental tool to identify carbamate NAAA inhibitors endowed with good biological stability.

Inhibition of triple negative breast cancer-associated inflammation and progression by N- acylethanolamine acid amide hydrolase (NAAA)

Triple-negative breast cancer (TNBC) is associated with high mortality due to the high expression of pro-inflammatory cytokines and lack of targeted therapies. N-acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that promotes inflammatory responses through the deactivation of Palmitoylethanolamide (PEA), an endogenous bioactive lipid mediator. Here, we examined NAAA expression in TNBC cells (MDA-MB-231 and MDA-MB-BrM2 cells) and the effects of NAAA inhibition on TNBC tumor growth, using a selective NAAA inhibitor AM11095 (IC50 = 20 nM). TNBC cells expressed elevated levels of full-length and splice mRNAs naaa variants. TNBC cells also express the N-acyl ethanol amides and elevated levels of the two fatty acid cores arachidonic (AA) and docosahexaenoic (DHA). PEA or AM11095 inhibited the secretion of IL-6 and IL-8, reduced the activation of the NF-kB pathway, decreased the expression of VEGF and Placental growth factor (PLGF) in TNBCs, and inhibited tumor cell migration in vitro. Using cellular magnetic resonance imaging (MRI), body images of mice administered with human MDA-MB-BrM2 cells treated with AM11095 showed a significant decrease in tumor numbers with a lower volume of tumors and increased mice survival. Mice untreated or treated with vehicle control showed a high number of tumors with high volumes in multiple organs. Thus, NAAA inhibition may constitute a potential therapeutic approach in the management of TNBC-associated inflammation and tumor growth.

N-Acylethanolamine acid amidase (NAAA) exacerbates psoriasis inflammation by enhancing dendritic cell (DCs) maturation

Psoriasis is an incurable autoimmune disease that affects 2–3% of the world’s population. Limited understanding of its pathogenesis hinders the development of therapies for the disease. Herein, we reported that N-acylethanolamine acid amidase (NAAA), a cysteine enzyme that catalyzes the hydrolysis of fatty acid ethanolamides (FAEs), was upregulated in psoriasis patients and imiquimod (IMQ)-induced mouse model of psoriasis. The upregulated NAAA contributes to the progression of psoriasis via enhancing dendritic cell (DCs) maturation. Transgenic expression of NAAA in mice accelerated the development of psoriasis, whereas genetic ablation of NAAA or local administration of NAAA inhibitor F96 ameliorated psoriasis. NAAA expressed in dendritic cells (DCs), but not in macrophages, T cells, or keratinocytes plays a critical role in psoriasis development. In addition, the results showed that NAAA degrades palmitoylethanolamide (PEA) and reduces PEA-PPARα-mediated dissociation of NF-κB p65 from Sirtuin 1 (SIRT1), subsequently, repressing the acetylation of p65 and down-regulating IL10 production. The decreased IL10 then leads to the maturation of DCs, thus promoting the development of psoriasis. These results provide new insights into the pathophysiological mechanism of psoriasis and identify NAAA as a novel target for the treatment of psoriasis.

N-acylethanolamide metabolizing enzymes are upregulated in human neural progenitor-derived neurons exposed to sub-lethal oxidative stress.

N-acyl amides (NAAs) are a class of lipids that consist of an acyl group N-linked to an amino acid, neurotransmitter, taurine or ethanolamide group (N-acylethanolamines or NAEs) and include some endocannabinoids (eCB) such as anandamide. These lipids are synthesized in a wide variety of organisms and in multiple cell types, including neurons. NAEs are involved in numerous cellular and physiological processes and their concentrations are elevated in response to ischemia and physical trauma to play a role in neuroprotection. The neuroprotective properties of eCB NAEs make the protein targets of these compounds attractive targets for clinical intervention for a variety of conditions. The most promising of these targets include cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA), and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). Further characterization of these targets in a more contemporary model system of neurodegeneration and neuroprotection will allow us to fully describe their role and mechanism of action in neuroprotection against oxidative stress leading to better utilization in the clinical setting. Human stem cell-derived or human neural progenitor cell-derived cells, such as ReN cells, have become more utilized for the study of human neuronal development and neurodegenerative diseases. ReN cells can be easily differentiated thereby circumventing the need for using transformed cell lines and primary neurons as cell model systems. In this study, we determined whether ReN cells, a superior cell model system for studying neurodevelopment, differentiation, and neuroprotection, express proteins involved in canonical eCB NAE signaling and whether oxidative stress can induce their expression. We determined that sublethal oxidative stress upregulates the expression of all eCB proteins tested. In addition, we determined that oxidative stress increases the nuclear localization of FAAH, and to a lesser extent, NAAA and NAPE-PLD. This study is a first step toward determining how oxidative stress affects CB1, CB2, FAAH, NAAA, and NAPE-PLD expression and their potential defense against oxidative stress. As such, our data is important for further determining the role of eCB metabolizing proteins and eCB receptors against oxidative stress.

Synergistic antinociceptive effects of concomitant NAAA and peripheral FAAH inhibition

The intracellular lipid amidases, fatty acid amide hydrolase (FAAH) and N-acylethanolamine acid amidase (NAAA), terminate the actions of anandamide and palmitoylethanolamide (PEA), two antinociceptive and anti-inflammatory lipid-derived mediators. Here we show, confirming prior research, that small-molecule inhibitors of peripheral FAAH (compound URB937) and systemic NAAA (compound ARN19702) individually attenuate, in male CD-1 mice, pain-related behaviors and paw inflammation in the formalin and carrageenan tests. More importantly, isobolographic analyses revealed that the combination of URB937 and ARN19702 produced substantial synergistic (greater than additive) antinociceptive effects in both models as well as additive anti-inflammatory effects in the carrageenan test. Together, the findings uncover a functional interplay between FAAH and NAAA substrates in the control of nociception, which might be exploited clinically to develop safe and effective pain management strategies

Targeting NAAA counters dopamine neuron loss and symptom progression in mouse models of parkinsonism

The lysosomal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) deactivates palmitoylethanolamide (PEA), a lipid-derived PPAR-α agonist that is critically involved in the control of pain and inflammation. In this study, we asked whether NAAA-regulated PEA signaling might contribute to dopamine neuron degeneration and parkinsonism induced by the mitochondrial neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro experiments showed that 6-OHDA and MPTP enhanced NAAA expression and lowered PEA content in human SH-SY5Y cells. A similar effect was observed in mouse midbrain dopamine neurons following intra-striatal 6-OHDA injection. Importantly, deletion of the Naaa gene or pharmacological inhibition of NAAA activity substantially attenuated both dopamine neuron death and parkinsonian symptoms in mice treated with 6-OHDA or MPTP. Moreover, NAAA expression was elevated in postmortem brain cortex and premortem blood-derived exosomes from persons with Parkinson’s disease compared to age-matched controls. The results identify NAAA-regulated PEA signaling as a molecular control point for dopaminergic neuron survival and a potential target for neuroprotective intervention.

α-Acylamino-β-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-β-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-β-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time.

Genetic Blockade of NAAA Cell-specifically Regulates Fatty Acid Ethanolamides (FAEs) Metabolism and Inflammatory Responses.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme responsible for the hydrolysis of fatty acid ethanolamides (FAEs). However, the role of NAAA in FAEs metabolism and regulation of pain and inflammation remains mostly unknown. Here, we generated NAAA-deficient (NAAA-/-) mice using CRISPR-Cas9 technique, and found that deletion of NAAA increased PEA and AEA levels in bone marrow (BM) and macrophages, and elevated AEA levels in lungs. Unexpectedly, genetic blockade of NAAA caused moderately effective anti-inflammatory effects in lipopolysaccharides (LPS)-induced acute lung injury (ALI), and poor analgesic effects in carrageenan-induced hyperalgesia and sciatic nerve injury (SNI)-induced mechanical allodynia. These data contrasted with acute (single dose) or chronic NAAA inhibition by F96, which produced marked anti-inflammation and analgesia in these models. BM chimera experiments indicated that these phenotypes were associated with the absence of NAAA in non-BM cells, whereas deletion of NAAA in BM or BM-derived cells in rodent models resulted in potent analgesic and anti-inflammatory phenotypes. When combined, current study suggested that genetic blockade of NAAA regulated FAEs metabolism and inflammatory responses in a cell-specifical manner.

Insights Into the Prognostic Value and Immunological Role of NAAA in Pan-Cancer.

N-Acylethanolamine Acid Amidase (NAAA) is an N-terminal cysteine hydrolase and plays a vital physiological role in inflammatory response. However, the roles of NAAA in tumor immunity are still unclear. By using a series of bioinformatics approaches, we study combined data from different databases, including the Cancer Genome Atlas, the Cancer Cell Line Encyclopedia, Genotype Tissue-Expression, cBioPortal, Human Protein Atlas, TIMER, and ImmuCellAI to investigate the role of NAAA expression in prognosis and tumor immunity response. We would like to reveal the potential correlations between NAAA expression and gene alterations, tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, tumor microenvironment (TME), immune infiltration levels, and various immune-related genes across different cancers. The results show that NAAA displayed abnormal expression within most malignant tumors, and overexpression of NAAA was associated with the poor prognosis of tumor patients. Through gene set enrichment analysis (GSEA), we found that NAAA was significantly associated with cell cycle and immune regulation-related signaling pathways, such as in innate immune system, adaptive immune system, neutrophil degranulation, and Toll-like receptor signaling pathways (TLRs). Further, the expression of NAAA was also confirmed to be correlated with tumor microenvironment and diverse infiltration of immune cells, especially tumor-associated macrophage (TAM). In addition to this, we found that NAAA is co-expressed with genes encoding major histocompatibility complex (MHC), immune activation, immune suppression, chemokine, and chemokine receptors. Meanwhile, we demonstrate that NAAA expression was correlated with TMB in 4 cancers and with MSI in 10 cancers. Our study reveals that NAAA plays an important role in tumorigenesis and cancer immunity, which may be used to function as a prognostic biomarker and potential target for cancer immunotherapy.

N-Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth.

Background and Purpose N‐Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N‐acylethanolamines (NAEs) and its pharmacological inhibition has beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism, whereas its contribution to colorectal cancer (CRC) is unknown to date.Experimental ApproachCRC xenograft and azoxymethane models were used to assess the in vivo effect of NAAA inhibition. Further, the tumour secretome was evaluated by an oncogenic array, CRC cell lines were used for in vitro studies, cell cycle was analysed by cytofluorimetry, NAAA was knocked down with siRNA, human biopsies were obtained from surgically resected CRC patients, gene expression was measured by RT‐PCR and NAEs were measured by LC–MS.Key ResultsThe NAAA inhibitor AM9053 reduced CRC xenograft tumour growth and counteracted tumour development in the azoxymethane model. NAAA inhibition affected the composition of the tumour secretome inhibiting the expression of EGF family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR‐α and TRPV1. AM9053 induced cell cycle arrest in the S phase associated with cyclin A2/CDK2 down‐regulation. NAAA knock‐down mirrored the effects of NAAA inhibition with AM9053. NAAA expression was down‐regulated in human CRC tissues, with a consequential augmentation of NAE levels and dysregulation of some of their targets.Conclusion and ImplicationsOur results show novel data on the functional importance of NAAA in CRC progression and the mechanism involved. We propose that this enzyme is a valid drug target for the treatment of CRC growth and development.

Nogo-a Pathway Contributes to Neural Injury and Pain in Sickle Cell Disease

Sickle cell disease (SCD) is characterized by chronic pain and bouts of extreme acute pain from vasoocclusive crises (VOC). Sickle pain has both neuropathic and inflammatory features (Tran et al., Blood 2017). Mechanisms underlying neural injury remain unknown in SCD. Neurite outgrowth inhibitor (NOGO-A/reticulon-4) and its receptor NGR1 contribute to pain, neuronal damage, and inhibition of neurite outgrowth (Hu et al., FASEB J 2019). We examined if NOGO-A pathway is activated in a sickle microenvironment and if its inhibition will ameliorate hyperalgesia in BERK sickle mice. We used Rho kinase activity (ROCK) downstream of NGR1 as a readout of activation of NOGO-A/NGR1 pathway. We observed increased expression of NOGO-A (~260%, p<0.05) and NGR1 (~180%, p<0.05) in the dorsal root ganglia, and increased NOGO-A and ROCK activity in spinal cords of sickle mice compared to control mice expressing normal human hemoglobin A. Earlier, we found that an endogenous cannabinoid, palmitoylethanolamide (PEA) inhibits spinal NOGO-A expression and ROCK activity in sickle mice (Argueta et al., ASH 2020 #225). We hypothesized that sickle microenvironment with cell-free heme and inflammation activates NOGO-A/NGR1-ROCK pathway leading to nerve injury and pain, and inhibition of this pathway will ameliorate hyperalgesia in sickle mice.Using terminally differentiated rat pheochromocytoma (PC12) cells, we simulated a sickle microenvironment with hemin (40 µM) and TNFα (1 ng/ml) (H+T). H+T elevated ROCK activity compared to vehicle (~40%, p<0.05). PEA (30 uM) and 2 µM NEP (1-40), a competitive antagonist of NGR1, attenuated H+T-induced ROCK activity (both p<0.01); co-treatment had no additive effect, indicating a common pathway. As well, siRNA (10 nM) knockdown of NGR1 reversed H+T-induced ROCK activity (p<0.001), which was equally effective with 30 µM PEA co-treatment. Functionally, treatment with 30 µM PEA or 2 µM NEP (1-40) enhanced neurite outgrowth in H+T-treated PC-12 cells (~120%, p<0.001). NEP (1-40) at 5 mg/kg reduced mechanical and cold (both ~50%, p<0.001) hyperalgesia in sickle mice compared to baseline (BL) and/or vehicle treatment. Together, these data demonstrate that NOGO-A/NGR1 pathway activation may underlie nerve injury, and inhibition of this pathway with a NGR1 antagonist or PEA promotes neurite outgrowth and reduces hyperalgesia in a sickle microenvironment.We next examined the contribution of exogenously administered and endogenously produced PEA in ameliorating hyperalgesia. Mass spectrometry revealed that spinal PEA is reduced in female (p<0.05) and male (p<0.001) sickle mice compared to age/sex-matched control mice. Exogenous PEA (i.p. 20 mg/kg/d) reduced cold avoidance over a 3-day treatment period, showing significantly more time in the cold chamber compared to BL or vehicle at 1 h, 24 h, and 72 h (p<0.05). The analgesic effect of PEA was maintained for 9 days of treatment without developing tolerance. We next increased endogenous PEA by inhibiting its degradative enzyme, N-acylethanolamine acid amidase, with ARN19702 (i.p. 3, 10, & 30 mg/kg/d), which reduced mechanical (~50%, p<0.001) and cold hyperalgesia (~40%, p<0.001) over 72 hours in a dose-dependent manner. Since hypoxia and ischemia reperfusion injury contribute to acute VOC pain, we incited hypoxia-reoxygenation (HR; 3 h @ 8% O 2, 92% N 2, followed by 1 h @ normoxia) to simulate acute VOC pain. We observed that 5-day pretreatment with PEA (i.p. 20 mg/kg/d) before HR prevented mechanical and cold hyperalgesia following HR in sickle mice. Moreover, treatment with PEA after HR incitement significantly reduced hyperalgesia for 24 h after HR compared to BL (~30%, p<0.001) and vehicle treated (~66%, p<0.001) sickle mice.NGR1 antagonism reduces spinal microglial injury/activation. Heme and TNFα have been shown to cause microglial injury in vitro, while spinal microglial activation has been demonstrated in sickle mice (Lei et al., Antioxid Redox Signal 2021). Thus, NGR1/ROCK cascade may contribute to both neuronal injury and inflammation in the central nervous system leading to neuropathic pain. Our data suggest that PEA and targeting NOGO-A pathway may prevent/reduce chronic and acute hyperalgesia in sickle mice. We speculate that interventions targeting NOGO-A pathway may prevent/reduce neuropathic pain and that PEA has the translational potential for the treatment of chronic and acute pain in SCD. DisclosuresGupta: Tautona Group: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 1910 Genetics: Other: Grantee; Grifols: Other: Grantee; SCDAA: Membership on an entity's Board of Directors or advisory committees; CSL Behring LLC: Honoraria; NIH: Other: Grantee; University of Minnestoa Foundation: Other: Philanthropic Funding; Southern California Institute for Research and Education Foundation: Other: Philanthropic Funding; Cyclerion: Research Funding; UCI Foundation: Other: Philanthropic Funding.

NAAA inhibitor F96 attenuates BBB disruption and secondary injury after traumatic brain injury (TBI)

Traumatic brain injury (TBI) is a leading cause of death worldwide, for which there is currently no comprehensive treatment available. Preventing blood–brain barrier (BBB) disruption is crucial for TBI treatment. N-acylethanolamine acid amidase (NAAA)-regulated palmitoylethanolamide (PEA) signaling play an important role in the control of inflammation. However, the role of NAAA in BBB dysfunction following TBI remains unclear. In the present study, we found that TBI induces the increase of PEA levels in the injured cortex, which prevent the disruption of BBB after TBI. TBI also induces the infiltration of NAAA-contained neutrophils, increasing the contribution of NAAA to the PEA degradation. Neutrophil-derived NAAA weakens PEA/PPARα-mediated BBB protective effects after TBI, facilitates the accumulation of immune cells, leading to secondary expansion of tissue injury. Inactivation of NAAA increased PEA levels in injured site, prevents early BBB damage and improves secondary injury, thereby eliciting long-term functional improvements after TBI. This study identified a new role of NAAA in TBI, suggesting that NAAA is a new important target for BBB dysfunction related CNS diseases.

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

Endocannabinoid System: Overview and Therapeutic Relevance

The endocannabinoid system (ECS) modulates a variety of physiological processes, attracting considerable attention as a potential target for therapeutic intervention. This complex system is activated by the lipid-derived mediators anandamide and 2-arachidonoyl-sn-glycerol (2-AG), which mainly engage the cannabinoid receptor subtypes 1 (CB1) and 2 (CB2). The biological actions of anandamide and 2-AG are terminated by internalization and intracellular enzymatic hydrolysis catalyzed primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), respectively. Here, we provide an overview of ECS and discuss the implications for advancing pharmacological tools that interfere with such a system as next-generation therapeutics. This review contains 4 figures, 3 tables and 41 references Keywords: Endocannabinoid; anandamide; 2-Arachidonoyl-sn-glycerol; fatty acid amide hydrolase; monoacylglycerol lipase; cannabinoid receptors; N-acylethanolamine acid amidase; Δ9-tetrahydrocannabinol.

Antinociceptive Profile of ARN19702, (2-Ethylsulfonylphenyl)-[(2S)-4-(6-fluoro-1,3-benzothiazol-2-yl)-2-methylpiperazin-1-yl]methanone, a Novel Orally Active N-Acylethanolamine Acid Amidase Inhibitor, in Animal Models.

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that stops the physiologic actions of palmitoylethanolamide, an endogenous lipid messenger that activates the transcription factor, peroxisome proliferator-activated receptor-α We have previously reported that the compound ARN19702 [(2-ethylsulfonylphenyl)-[(2S)-4-(6-fluoro-1,3-benzothiazol-2-yl)-2-methylpiperazin-1-yl]methanone] is an orally active, reversible NAAA inhibitor (IC50 on human NAAA = 230 nM) that produces remarkable protective effects against multiple sclerosis in mice. In the present study, we assessed the profile of ARN19702 in mouse and rat models of acute and neuropathic pain. Oral administration in male mice attenuated in a dose-dependent manner the spontaneous nocifensive response elicited by intraplantar formalin injection and the hypersensitivity caused by intraplantar carrageenan injection, paw incision, or sciatic nerve ligation. In male rats, ARN19702 reduced nociception associated with paclitaxel-induced neuropathy without development of subacute antinociceptive tolerance. Finally, ARN19702 (30 mg/kg, oral) did not produce place preference or alter exploratory motor behavior in male mice. The findings support the conclusion that NAAA is a suitable molecular target for the discovery of efficacious analgesic drugs devoid of rewarding potential. SIGNIFICANCE STATEMENT: This study evaluated the pharmacological profile of the orally bioavailable N-acylethanolamine acid amidase (NAAA) inhibitor (2-ethylsulfonylphenyl)-[(2S)-4-(6-fluoro-1,3-benzothiazol-2-yl)-2-methylpiperazin-1-yl]methanone (ARN19702) in mouse and rat models of neurogenic and inflammatory pain. The compound's potential rewarding and sedative effects were also examined. It is concluded that ARN19702 exhibits a broad analgesic profile that can be generalized across rodent species. The findings point to NAAA as a control node in the processing of neuropathic and inflammatory pain and to ARN19702 as a lead to uncover novel pain therapeutics devoid of addictive potential .