2-Pentadecyl-2-oxazoline inhibits lipopolysaccharide-induced microglia activation interfering with TLR4 signaling.

AIM: 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of N-palmitoylethanolamine, exerts anti-inflammatory activity; however, very little is known about the molecular mechanisms underlying this effect. Here, we tested the anti-neuroinflammatory effect of PEA-OXA in primary microglia and we also investigated the possible interaction of the molecule with the Toll-like receptor 4 (TLR4)-myeloid differentiation protein-2 (MD-2) complex. MAIN METHODS: The anti-inflammatory effect of PEA-OXA was analyzed by measuring the expression and release of pro-inflammatory mediators in primary microglia by real-time PCR and ELISA, respectively. The effect of PEA-OXA on the activation of TLR4 signaling was assessed using two stably TLR4-transfected cell lines (i.e., HEK-293 and Ba/F3 cells). Finally, the putative binding mode of PEA-OXA to TLR4-MD-2 was investigated by molecular docking simulations. KEY FINDINGS: Treatment with PEA-OXA resulted in the following effects: (i) it down-regulated gene expression of several pro-inflammatory molecules and the secretion of pro-inflammatory cytokines in LPS stimulated microglia cells; (ii) it did not prevent microglia activation after stimulation with TLR2 ligands; (iii) it prevented TLR4/NF-κB activation triggered by LPS in HEK-Blue™ hTLR4 cells; and (iv) it interfered with the binding of LPS to TLR4-MD-2 complex. Furthermore, molecular docking studies suggested that PEA-OXA could bind MD-2 with a 1:3 (MD-2/PEA-OXA) stoichiometry. CONCLUSION: We show for the first time that the anti-neuroinflammatory effect of PEA-OXA involves its activity against TLR4 signaling, making this molecule a valuable tool for the development of new compounds directed to control neuroinflammation via inhibiting TLR4 signaling.

Co-Ultramicronized Palmitoylethanolamide/Luteolin-Induced Oligodendrocyte Precursor Cell Differentiation is Associated With Tyro3 Receptor Upregulation.

Remyelination in patients with multiple sclerosis frequently fails, especially in the chronic phase of the disease promoting axonal and neuronal degeneration and progressive disease disability. Drug-based therapies able to promote endogenous remyelination capability of oligodendrocytes are thus emerging as primary approaches to multiple sclerosis. We have recently reported that the co-ultramicronized composite of palmitoylethanolamide and the flavonoid luteolin (PEALut) promotes oligodendrocyte precursor cell (OPC) maturation without affecting proliferation. Since TAM receptor signaling has been reported to be important modulator of oligodendrocyte survival, we here evaluated the eventual involvement of TAM receptors in PEALut-induced OPC maturation. The mRNAs related to TAM receptors -Tyro3, Axl, and Mertk- were all present at day 2 in vitro. However, while Tyro3 gene expression significantly increased upon cell differentiation, Axl and Mertk did not change during the first week in vitro. Tyro3 gene expression developmental pattern resembled that of MBP myelin protein. In OPCs treated with PEALut the developmental increase of Tyro3 mRNA was significantly higher as compared to vehicle while was reduced gene expression related to Axl and Mertk. Rapamycin, an inhibitor of mTOR, prevented oligodendrocyte growth differentiation and myelination. PEALut, administered to the cultures 30 min after rapamycin, prevented the alteration of mRNA basal expression of the TAM receptors as well as the expression of myelin proteins MBP and CNPase. Altogether, data obtained confirm that PEALut promotes oligodendrocyte differentiation as shown by the increase of MBP and CNPase and Tyro3 mRNAs as well as CNPase and Tyro3 immunostainings. The finding that these effects are reduced when OPCs are exposed to rapamycin suggests an involvement of mTOR signaling in PEALut effects.

A Pharmacological Rationale to Reduce the Incidence of Opioid Induced Tolerance and Hyperalgesia: A Review.

Chronic pain is an important health and social problem. Misuse and abuse of opioids in chronic non-cancer pain management seem to be a huge problem, in some countries. This could probably affect the normal use of such analgesics in patients in need of them. Basic and clinical researches should find the solution to mitigate the potential damage. Dysregulation of mast cell and microglia activation plays an important role in the pathogenesis and management of chronic pain. Persistent mast cell activation sensitizes nociceptors and initiates central nervous system inflammatory processes, involving microglial cell activation and sensitization of spinal somatosensory neurons. Exposure of mast cells and microglia to opioids is well known to provoke activation of these non-neuronal immune cell populations, thereby contributing to an exacerbation of pro-inflammatory and pro-nociceptive processes and promoting, over the long-term, opioid-induced hyperalgesia and tolerance. This review is intended to provide the reader with an overview of the role for these non-neuronal cells in opioid-induced chronic pain and tolerance as a consequence of prolonged exposure to these drugs. In addition, we will examine a potential strategy with the aim to modulate opioid-induced over-activation of glia and mast cells, based on endogenous defense mechanisms and fatty acid amide signaling molecules.

Degenerative Joint Diseases and Neuroinflammation.

Rheumatic and joint diseases, as exemplified by osteoarthritis and rheumatoid arthritis, are among the most widespread painful and disabling pathologies across the globe. Given the continuing rise in life expectancy, their prevalence is destined to grow. Osteoarthritis, a degenerative joint disease, is, in particular, on its way to becoming the fourth leading cause of disability worldwide by 2020, with the rising incidence of obesity in addition to age being important factors. It is estimated that 25% of osteoarthritic individuals are unable to perform daily activities. Accompanying osteoarthritis is rheumatoid arthritis, which is a chronic systemic disease that often causes pain and deformity. At least 50% of those affected are unable to remain gainfully employed within 10 years of disease onset. A growing body of evidence now points to inflammation, locally and more systemically, as a promoter of damage to joints and bones, as well as joint-related functional deficits. The pathogenesis underlying joint diseases remains unclear; however, it is currently believed that cross-talk between cartilage and subchondral bone-and loss of balance between these two structures in joint diseases-is a critical element. This view is amplified by the presence of mast cells, whose dysregulation is associated with alterations of junction structures (cartilage, bone, synovia, matrix, nerve endings, and blood vessels). In addition, persistent activation of mast cells facilitates the development of spinal neuroinflammation mediated through their interaction with microglia. Unfortunately, current treatment strategies for rheumatic and articular disease are symptomatic and do little to limit disease progression. Research now should be directed at therapeutic modalities that target osteoarticular structural elements and thereby delaying disease progression and joint replacement.

PEA and luteolin synergistically reduce mast cell-mediated toxicity and elicit neuroprotection in cell-based models of brain ischemia.

The combination of palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N-acylethanolamines, and the flavonoid luteolin has been found to exert neuroprotective activities in a variety of mouse models of neurological disorders, including brain ischemia. Indirect findings suggest that the two molecules can reduce the activation of mastocytes in brain ischemia, thus modulating crucial cells that trigger the inflammatory cascade. Though, no evidence exists about a direct effect of PEA and luteolin on mast cells in experimental models of brain ischemia, either used separately or in combination. In order to fill this gap, we developed a novel cell-based model of severe brain ischemia consisting of primary mouse cortical neurons and cloned mast cells derived from mouse fetal liver (MC/9 cells) subjected to oxygen and glucose deprivation (OGD). OGD exposure promoted both mast cell degranulation and the release of lactate dehydrogenase (LDH) in a time-dependent fashion. MC/9 cells exacerbated neuronal damage in neuron-mast cells co-cultures exposed to OGD. Likewise, the conditioned medium derived from OGD-exposed MC/9 cells induced significant neurotoxicity in control primary neurons. PEA and luteolin pre-treatment synergistically prevented the OGD-induced degranulation of mast cells and reduced the neurotoxic potential of MC/9 cells conditioned medium. Finally, the association of the two drugs promoted a direct synergistic neuroprotection even in pure cortical neurons exposed to OGD. In summary, our results indicate that mast cells release neurotoxic factors upon OGD-induced activation. The association PEA-luteolin actively reduces mast cell-mediated neurotoxicity as well as pure neurons susceptibility to OGD.

Palmitoylethanolamide, a Special Food for Medical Purposes, in the Treatment of Chronic Pain: A Pooled Data Meta-analysis.

BACKGROUND: A growing body of evidence suggests that neuroinflammation, which is characterized by infiltration of immune cells, activation of mast cells and glial cells, and production of inflammatory mediators in the peripheral and central nervous systems, has an important role in the induction and maintenance of chronic pain. These findings support the notion that new therapeutic opportunities for chronic pain might be based on anti-inflammatory and pro-resolving mediators that act on immune cells, in particular mast cells and glia, to mitigate or abolish neuroinflammation. Among anti-inflammatory and pro-resolving lipid mediators, palmitoylethanolamide (PEA) has been reported to down-modulate mast cell activation and to control glial cell behaviors. OBJECTIVE: The aim of this study was to perform a pooled meta-analysis to evaluate the efficacy and safety of micronized and ultra-micronized palmitoylethanolamide (PEA) on pain intensity in patients suffering from chronic and/or neuropathic pain. STUDY DESIGN: Pooled data analysis consisting of double-blind, controlled, and open-label clinical trials. METHODS: Double-blind, controlled, and open-label clinical trials were selected consulting the PubMed, Google Scholar, and Cochrane databases, and proceedings of neuroscience meetings. The terms chronic pain, neuropathic pain, and micronized and ultra-micronized PEA were used for the search. Selection criteria included availability of raw data and comparability between tools used to diagnose and assess pain intensity. Raw data obtained by authors were pooled in one database and analyzed by the Generalized Linear Mixed Model. The changes in pain over time, measured by comparable tools, were also assessed by linear regression post-hoc analysis and the Kaplan-Meier estimate. Twelve studies were included in the pooled meta-analysis, 3 of which were double-blind trials comparing active comparators vs placebo, 2 were open-label trials vs standard therapies, and 7 were open-label trials without comparators. RESULTS: Results showed that PEA elicits a progressive reduction of pain intensity significantly higher than control. The magnitude of reduction equals 1.04 points every 2 weeks with a 35% response variance explained by the linear model. In contrast, in the control group pain, reduction intensity equals 0.20 points every 2 weeks with only 1% of the total variance explained by the regression. The Kaplan-Meier estimator showed a pain score = 3 in 81% of PEA treated patients compared to only 40.9% in control patients by day 60 of treatment. PEA effects were independent of patient age or gender, and not related to the type of chronic pain. LIMITATIONS: Noteworthy, serious adverse events related to PEA were not registered and/or reported in any of the studies. CONCLUSION: These results confirm that PEA might represent an exciting, new therapeutic strategy to manage chronic and neuropathic pain associated with neuroinflammation.

The anti-inflammatory mediator palmitoylethanolamide enhances the levels of 2-arachidonoyl-glycerol and potentiates its actions at TRPV1 cation channels.

BACKGROUND AND PURPOSE: Palmitoylethanolamide (PEA) is an endogenous congener of anandamide and potentiates its actions at cannabinoid CB1 and CB2 receptors, and at transient receptor potential vanilloid type-1 (TRPV1) channels. The other endocannabinoid, 2-arachidonoylglycerol (2-AG), was recently suggested to act as a TRPV1 channel agonist. We investigated if PEA enhanced levels of 2-AG in vitro or in vivo and 2-AG activity at TRPV1 channels. EXPERIMENTAL APPROACH: Endogenous lipid levels were measured by LC-MS in (i) human keratinocytes incubated with PEA (10-20 µM, 40 min, 6 and 24 h, 37°C); (ii) the blood of spontaneously Ascaris suum hypersensitive beagle dogs given a single oral dose of ultramicronized PEA (30 mg·kg(-1), 1, 2, 4 and 8 h from administration); (iii) the blood of healthy volunteers given a single oral dose of micronized PEA (300 mg, 2, 4 and 6 h from administration). Effects of 2-AG at TRPV1 channels were assessed by measuring intracellular Ca(2+) in HEK-293 cells over-expressing human TRPV1 channels. KEY RESULTS: PEA elevated 2-AG levels in keratinocytes (∼3-fold) and in human and canine plasma (∼2 and ∼20-fold respectively). 2-AG dose-dependently raised intracellular Ca(2+) in HEK-293-TRPV1 cells in a TRPV1-dependent manner and desensitized the cells to capsaicin. PEA only slightly enhanced 2-AG activation of TRPV1 channels, but significantly increased 2-AG-induced TRPV1 desensitization to capsaicin (IC50 from 0.75 ± 0.04 to 0.45 ± 0.02 µM, with PEA 2 µM). CONCLUSIONS AND IMPLICATIONS: These observations may explain why several effects of PEA are attenuated by cannabinoid receptor or TRPV1 channel antagonists. LINKED ARTICLES: This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.

Chronic Pain in the Elderly: The Case for New Therapeutic Strategies.

BACKGROUND: Elderly patients in general exhibit a higher incidence of chronic and neuropathic pain conditions. This group poses a particular clinical challenge due to age-related pharmacokinetic and pharmacodynamic issues, comorbid conditions, and polypharmacy, as well as frailty and cognitive decline. Poor control of pain has consistently been identified as an issue for older people. The identification of safe and efficacious treatments for chronic pain remains a critical public health concern, especially considering the progressive increase of the world's elderly population. OBJECTIVES: This narrative review deals with the principal alterations of the somatosensory system together with changes in non-neuronal cells in the course of aging. The possibility to control chronic pain based on an innovative strategy which addresses non-neuronal cell dysregulation control will also be discussed. STUDY DESIGN: Narrative review. RESULTS: Peripheral nerves display functional, structural, and biochemical changes with aging that mainly involve Aδ fibers. Alteration in the responses to heat pain in the middle insular cortex and primary somatosensory cortex are also observed in the elderly. In general, pain threshold increases with age while the threshold of pain tolerance remains unchanged or decreases. Additionally, other important modifications of the pain perception system in this age group consist in a clear reduction in the descending inhibitory capacity with an associated increase in central sensitization. Furthermore, different changes concern immune system cells, such as mast cells and microglia, that with age show an increase in their sensitivity to noxious stimuli and a decreased capability to be regulated by homeostatic endogenous systems. Since these cells are the primary interlocutors for pain neurons, their alterations lead to changes that promote persistent neuroinflammation, thereby impacting pain neuronal cell functionality. LIMITATION: This review is not an exhaustive review for the current evidence supporting the role of immune cells in influencing pain somatosensory neuron functions. It is also important to stress the small number of studies designed to determine the efficacy and safety of anti-pain therapies in elderly patients. CONCLUSION: Non-neuronal cells of immune system origin such as microglia and mast cells, along with astrocytes, are capable of influencing pain somatosensory neuron functions. These nervous system non-neuronal cells may thus be viewed as innovative targets for persistent pain control. Among therapies aiming at preserving the functionality of non-neuronal cells, palmitoylethanolamide, with its high efficacy/risk ratio, may be an excellent co-treatment for the ever-growing elderly population with chronic pain.

Reply to: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI:10.1007/s10787-014-0202-3.

This is a reply to a recently published Commentary: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI: 10.1007/s10787-014-0202-3 , written in relation to our review article: Skaper SD, Facci L, Fusco M, della Valle MF, Zusso M, Costa B, Giusti P (2014) "Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain" Inflammopharmacology 22:79-94 DOI: 10.1007/s10787-013-0191-7 . We believe that the Commentary by Kriek contains a number of erroneous statements and misinterpretations of the published scientific/medical literature which our reply shall elaborate on. Further, the writer of the Commentary has a direct connection to a company, JP Russell Science Ltd that sells palmitoylethanolamide. The take-home message of our review remains as originally stated: "Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain".

Mast cells in chronic inflammation, pelvic pain and depression in women.

Inflammatory and neuroinflammatory processes are increasingly recognized as critical pathophysiologic steps in the development of multiple chronic diseases and in the etiology of persistent pain and depression. Mast cells are immune cells now viewed as cellular sensors in inflammation and immunity. When stimulated, mast cells release an array of mediators to orchestrate an inflammatory response. These mediators can directly initiate tissue responses on resident cells, and may also regulate the activity of other immune cells, including central microglia. New evidence supports the involvement of peripheral and central mast cells in the development of pain processes as well as in the transition from acute, to chronic and neuropathic pain. That behavioral and endocrine states can increase the number and activation of peripheral and brain mast cells suggests that mast cells represent the immune cells that peripherally and centrally coordinate inflammatory processes in neuropsychiatric diseases such as depression and anxiety which are associated with chronic pelvic pain. Given that increasing evidence supports the activated mast cell as a director of common inflammatory pathways/mechanisms contributing to chronic and neuropathic pelvic pain and comorbid neuropsychiatric diseases, mast cells may be considered a viable target for the multifactorial management of both pain and depression.

Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain.

Persistent pain affects nearly half of all people seeking medical care in the US alone, and accounts for at least $80 billion worth of lost productivity each year. Among all types of chronic pain, neuropathic pain stands out: this is pain resulting from damage or disease of the somatosensory nervous system, and remains largely untreatable. With few available treatment options, neuropathic pain represents an area of significant and growing unmet medical need. Current treatment of peripheral neuropathic pain involves several drug classes, including opioids, gabapentinoids, antidepressants, antiepileptic drugs, local anesthetics and capsaicin. Even so, less than half of patients achieve partial relief. This review discusses a novel approach to neuropathic pain management, based on knowledge of: the role of glia and mast cells in pain and neuroinflammation; the body's innate mechanisms to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation. The discovery that palmitoylethanolamide, a member of the N-acylethanolamine family which is produced from the lipid bilayer on-demand, is capable of exerting anti-allodynic and anti-hyperalgesic effects by down-modulating both microglial and mast cell activity has led to the application of this fatty acid amide in several clinical studies of neuropathic pain, with beneficial outcome and no indication of adverse effects at pharmacological doses. Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain.

Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism.

It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show that modulation of CB1 function has anti-inflammatory effects and suggests that inverse agonism of CB1 block signal transduction mechanisms controlling encephalitogenic T cells adhesion in inflamed brain venules by a PKA-dependent mechanism.

Nimodipine selectively stimulates beta-amyloid 1-42 secretion by a mechanism independent of calcium influx blockage.

Several lines of evidence indicate that perturbed cellular Ca2+ homeostasis may play a prominent role in synaptic dysfunction and neuronal death in Alzheimer's disease (AD), suggesting a potential benefit of drugs capable to stabilize Ca2+ homeostasis. We here investigated the effects of a panel of L-type Ca2+ channel antagonists on the secretion of the amyloid beta-peptide (Abeta), which abnormally accumulates in the senile plaques of the brain of AD patients. We found that, in primary and immortalized neuronal cells in culture, nimodipine robustly stimulated secretion (up to about four-fold at 30 microM) of the highly amyloidogenic 42-residue isoform of Abeta (Abeta42), while leaving largely unaffected total Abeta secretion. An analogous effect was also observed in vivo, as the administration of a single dose of nimodipine (10 mg/kg i.p.) induced a significant rise of Abeta42 levels in plasma of Tg2576 mice. The effect of nimodipine was independent of blockage of L-type Ca2+ channels and capacitative calcium entry. Accordingly, nimodipine effect was largely Ca2+-independent, as neither depletion nor rise of extracellular Ca2+ abolished it. Hence, by showing that the effect of nimodipine on Abeta42 production is distinct from its ability to block Ca2+-influx pathways, we provide evidence for a previously uncharacterized effect of this long known molecule also used in clinical practice.

The presence of trace amines in postmortem cerebrospinal fluid in humans.

The postmortem levels of biogenic amines in cerebrospinal fluid may represent a useful tool in defining some pathological conditions; no information is available concerning the occurrence of trace amines in postmortem cerebrospinal fluid. Thus, the occurrence of octopamine, synephrine and tyramine were evaluated by using a HPLC system in 20 postmortem samples of cerebrospinal fluid (obtained from 11 males and 9 females) and their levels were compared with those of 20 living subjects (obtained from 11 males and 9 females). The results show that trace amines dramatically increase in the postmortem cerebrospinal fluid (100, 20, and 4 fold increase for tyramine, octopamine, and synephrine respectively). To our knowledge, our data represent the first time trace amines have been identified in postmortem cerebrospinal fluid and the dramatic increase observed for tyramine has the potential of becoming a new tool in forensic science for better defining the time of death.

Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts.

BACKGROUND: Hunter syndrome, mucopolysaccharidosis type II (MPS II), is a X-linked inherited disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS), involved in the lysosomal catabolism of the glycosaminoglycans (GAG) dermatan and heparan sulfate. Such a deficiency leads to the intracellular accumulation of undegraded GAG and eventually to a progressive severe clinical pattern. Many attempts have been made in the last two to three decades to identify possible therapeutic strategies for the disorder, including gene therapy and somatic cell therapy. METHODS: In this study we evaluated the intraperitoneal implantation of allogeneic myoblasts over-expressing IDS, enclosed in alginate microcapsules, in the MPS II mouse model. Animals were monitored for 8 weeks post-implantation, during which plasma and tissue IDS levels, as well as tissue and urinary GAG contents, were measured. RESULTS AND CONCLUSIONS: Induced enzyme activity occurred both in the plasma and in the different tissues analyzed. A significant decrease in urinary undegraded GAG between the fourth and the sixth week of treatment was observed. Moreover, a biochemical reduction of GAG deposits was measured 8 weeks after treatment in the liver and kidney, on average 30 and 38%, respectively, while in the spleen GAG levels were almost normalized. Finally, the therapeutic effect was confirmed by histolochemical examination of the same tissues. Such effects were obtained following implantation of about 1.5 x 10(6) recombinant cells/animal. Taken together, these results represent a clear evidence of the therapeutic efficacy of this strategy in the MPS II mouse model, and encourage further evaluation of this approach for potential treatment of human beings.