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Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN.Significance:This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer.See related commentary by Conejo-Garcia and Curiel, p. 1841.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Figure from Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer

Recognition of pathogen-associated molecular patterns can trigger the IRE1α arm of the endoplasmic reticulum (ER) stress response in immune cells. IRE1α activation has been shown to maintain ER homeostasis while simultaneously coordinating diverse immunomodulatory programs in the setting of bacterial and viral infections. However, the role of IRE1α signaling in innate immune responses to fungal pathogens is unknown. Here we report that systemic infection with the fungusCandida albicanscauses severe renal immunopathology by triggering inflammatory IRE1α hyperactivation in host myeloid cells. Mechanistically, sensing of fungal β-glucans by the C-type lectin receptor Dectin-1 induced Src–Syk–NOX-dependent accumulation of intracellular reactive oxygen species and the ensuing generation of lipid peroxidation byproducts that sustained IRE1α activation. Selective deletion of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, reduced detrimental inflammatory responses in the kidney and extended survival in mice systemically infected withC. albicans. Hence, controlling IRE1α overactivation may be useful to impede the fatal immunopathogenic progression of disseminated candidiasis.One sentence summaryInnate IRE1α signaling in disseminated candidiasis

IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1β and increased transforming growth factor-β1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-β1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1β transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Lindsay Amann,1 Elizabeth Kruse,1 Allison J Lazard,2,3 Beth A Reboussin,4 Kimberly G Wagoner,5 E Alfonso Romero-Sandoval1 1Department of Anesthesiology, Pain Mechanisms Laboratory; Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA; 2Hussman School of Journalism and Media, University of North Carolina, Chapel Hill, NC, 27514, USA; 3Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel HIll, NC, 27599, USA; 4Department of Biostatistics and Data Science, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA; 5Department of Social Sciences and Health Policy, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USACorrespondence: E Alfonso Romero-Sandoval, Pain Mechanisms Laboratory, Anesthesiology, 1 Medical Center Boulevard, Winston-Salem, NC, 27157, USA, Tel +1 336-716-2725, Fax +1 336-713-2616, Email earomero.sandoval@gmail.comIntroduction: Cannabidiol (CBD) products are available nearly nationwide in the US and can coexist with medical or recreational programs. North Carolina (NC) is an example of a state with a program dedicated to integrating hemp cultivation and medicinal CBD exclusively, containing a multitude of retailers selling it as a primary product. The Food and Drug Administration (FDA) mandates that non-FDA approved CBD products cannot be marketed using medical or health-related claims and has sent warning letters to retailers violating these terms. We aim to characterize the online content of the NC CBD market by analyzing retailers’ websites to determine whether hemp/CBD shops comply with FDA regulations in terms of medical claims and analyze the claimed CBD content and price of products offered online.Methods: We randomly selected three CBD retailers from the ten most populated cities of NC. We analyzed their website content: product type, medical claims, other disclaimers, price, and CBD content.Results: We found that edible, oral, inhalable, and topical products are offered in similar proportions. Word analysis of product description revealed that “pain” and “pain relief” were the most common medical claim, followed by inflammation and anxiety. Health claims were mostly related to wellbeing. Other attributes indicate that products are associated with pleasant flavors or sensations (ie, cool, lavender, delicious, honey, menthol), which resembles the strategies used for tobacco advertisement. Most products (61%) claimed to contain less than 1000 mg of CBD. The median price of products ranged from $15– 30 per 300 mg. We found a positive correlation between CBD content and price.Discussion: Our data demonstrate that the NC online CBD market does not comply with FDA regulations, primarily targets patients with pain, inflammation, or anxiety, and offers products with low CBD concentration and high prices. New policies should limit the access and online promotion of non-pharmaceutical grade CBD products.Keywords: cannabidiol, hemp, medical marijuana, dispensary, cannabis market, misleading claims

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN. Significance: This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer. See related commentary by Conejo-Garcia and Curiel, p. 1841. This article is highlighted in the In This Issue feature, p. 1825

Enriqueta Munoz-Islas,1 Cecilia Esther Elizondo-Martinez,1 Mariela Gutierrez-Lopez,1 Rosa Issel Acosta-Gonzalez,1 Veronica Zaga-Clavellina,2 Addy Cecilia Helguera-Repetto,3 Martha Beatriz Ramirez-Rosas,1 E Alfonso Romero-Sandoval,4 Juan Miguel Jimenez-Andrade1 1Unidad Académica Multidisciplinaria Reynosa-Aztlán, Universidad Autónoma de Tamaulipas, Reynosa, Tamaulipas, Mexico; 2Departamento de Fisiología y Desarrollo Celular, Instituto Nacional de Perinatología, Ciudad de México, Mexico; 3Departamento de Inmunobioquímica, Instituto Nacional de Perinatología, Ciudad de México, Mexico; 4Department of Anesthesiology, Wake Forest University School of Medicine, Winston Salem, NC, USACorrespondence: Enriqueta Munoz-Islas Calle 16 y Lago de Chapala, Col. Aztlán C.P.88740, Reynosa, Tamaulipas, MexicoTel +52 5532076849Fax +52 8999230622Email e.munoz.islas@gmail.comPurpose: Gestational diabetes mellitus (GDM) induces cardiovascular and metabolic disturbances in offspring. However, the effects of GDM in pain processing in offspring and whether male and female offspring are equally affected is not well known. Thus, we determined: i) whether GDM in mice affects offspring hindpaw mechanical sensitivity, capsaicin-induced spontaneous pain-like behaviors, and epidermal nerve fiber density (ENFD); and ii) whether there is sexual dimorphism in these parameters in offspring from GDM dams.Methods: GDM was induced in pregnant ICR mice via i.p. streptozotocin (STZ). Then, glucose levels from dams and offspring were determined. Male and female offspring 2– 3 months of age were evaluated for: a) baseline mechanical sensitivity of the hind paw by using von Frey filaments; b) number of flinches and time spent guarding induced by intraplantar capsaicin (0.1%); and c) density of PGP-9.5 and CGRP axons in the epidermis from the hind paw glabrous skin.Results: Prepartum levels of glucose in STZ-treated dams were significantly increased compared to vehicle-treated dams; however, GDM or vehicle offspring displayed normal and similar blood glucose levels. Male and female GDM offspring showed significantly greater mechanical sensitivity and capsaicin-induced pain behaviors compared to vehicle offspring. Male GDM offspring displayed a slightly more intense nociceptive phenotype in the capsaicin test. PGP-9.5 and CGRP ENFD in hind paw glabrous skin were greater in male and female GDM offspring versus their controls. Sexual dimorphism was generally not observed in GDM offspring in most of the studied parameters.Conclusion: These results suggest GDM induced greater pain-like behaviors in adult offspring regardless of sex along with an increased ENFD of PGP-9.5 and CGRP in the hind paw glabrous skin. We show that GDM peripheral neuropathy differs from diabetic peripheral neuropathy acquired in adulthood and set the foundation to further study this in human babies exposed to GDM.Keywords: offspring, long-term effects, nociception, CGRP

While cancer patients may have chemotherapeutics to thank for being cured of their malignancy, they are often left to suffer a disabling neuropathy induced by that same cancer treatment. This neuropathy, known as chemotherapy-induced peripheral neuropathy, or CIPN, is one of the most debilitating survivorship concerns for patients, with many citing hallmark symptoms of hyperalgesia, allodynia, and numbness, and subsequently reducing their dose or even ceasing treatment altogether. Investigations into this interplay between the antineoplastic activity of chemotherapeutic agents and the preservation of peripheral nerve health are therefore crucial for the development of CIPN treatment and prevention methods. Responding to need, current literature is inundated with varying preclinical models of CIPN. This chapter thus seeks to provide a detailed and reliable methodology for the induction and assessment of CIPN in mice, using a preclinical model that is both reproducible and translatable to several aspects of the clinical phenotype. Specifically, this chapter lays out a model for intermittent low-dose paclitaxel induction of CIPN in C57BL/6J mice, and a testing of this induction via von Frey filament mechanical hypersensitivity assays, a mechanical hyposensitivity (numbness) assay, and a cold-thermal allodynia assay (acetone test). These protocols can easily be adjusted to fit the needs of individual CIPN experiments, as stated throughout the chapter.

Due to the large volume of surgeries and the subsequent incidence of postsurgical pain, it is vital that the underlying mechanisms of postsurgical pain are thoroughly understood. The intensity of acute postsurgical pain is typically dependent on the severity of tissue damage the surgery produces, and the development of chronic pain is more frequent in major surgeries than in minor ones. It is therefore important that postsurgical pain studies are conducted with the differences between major and minor surgeries in mind. To this end, the paw incision and skin muscle incision and retraction models are the focus of this chapter as they feature aspects observed in minor and major surgeries in humans, respectively. Several elements of these models translate to humans with some limitations, as they allow for the measurement of reflexive, spontaneous, and functional pain-like behavior. For these attributes, the SMIR and paw incision surgeries are widely used in postsurgical pain research. Here we layout detailed protocols to instruct experienced as well as inexperienced researchers studying postsurgical pain in rats and mice.

Background: Cannabidiol (CBD) products are increasingly available to consumers in the United States and are subject to regulation by the U.S. Food and Drug Administration (FDA). CBD products cannot be marketed as unapproved new drugs with claims of therapeutic benefit. In addition, because CBD is the active ingredient in a FDA-approved CBD product, Epidiolex, CBD cannot be marketed as, or in, food products or dietary supplements. The FDA has issued Warning Letters to promote voluntary regulatory compliance. These letters provide insights as to the types of violations for CBD products detected in the U.S. market. Objective: The goal of this retrospective study was to content analyze Warning Letters issued by the FDA to identify illicit marketing of CBD products. Design: Warning Letters issued by the FDA between 2015 and 2019 were content analyzed using a deductive approach. We extracted year of issuance, issuing office, and claim types that are currently prohibited by the FDA, including (i) unapproved new drug, (ii) misbranded drug, (iii) false and/or misleading, (iv) FDA-approved/endorsed, (v) dietary supplement, and (vi) adulterated food product. In addition, we documented the disease or conditions the product claimed to affect, pharmacological effects, and location of violation. Results: Of the 39 Warning Letters issued, 97% were for violations made on company websites and 56% were for social media accounts. Almost all letters (97%) cited violations of marketing CBD as an unapproved new drug. These illicit therapeutic claims were made for >125 unique health problems, including cancer (87.2%), diabetes (71.8%), inflammation (66.7%), pain (66.7%), and arthritis (66.7%). The majority of letters (79.5%) also cited illicit marketing of CBD as a dietary supplement or food product. CBD was promoted as having 16 unique pharmacological effects, including anti-inflammatory (53.8%), anticancer (43.6%), and antipsychotic (30.8%). Conclusions: CBD products have been unlawfully advertised online as unauthorized drugs with health claims that promote therapeutic benefits and as dietary supplements. Efforts are needed to regulate and monitor illicit advertising so consumers are not misled about the risks and benefits of CBD use.

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.

Increased public access to cannabis calls for a deeper understanding of cannabis's constituents and how they interact to induce clinical effects. Whereas trans-Δ9 -tetrahydrocannabinol (THC) is considered the main psychoactive component in cannabis, producing the associated "high" or "euphoria," various findings demonstrate medical potential for cannabidiol (CBD), from anxiolytic to antiepileptic implications. This has translated into a public optimism and given way to the popular opinion that CBD can provide countless other therapeutic benefits, including the potential to mitigate some of the adverse side effects of THC, such as intoxication, psychomotor impairment, anxiety, and psychotic symptoms. This is particularly relevant for patients seeking to garner therapeutic benefits from cannabis without experiencing the burden of a significant subjective high. Thus, this article analyzes the scientific evidence available to support or disprove the idea that presence of CBD is beneficial and can exude a protective effect against THC. A thorough review of relevant literature, a basis from which to interpret such evidence through a critical mechanistic discussion, and the implications for patients are presented in this article.

Large tissue damage or wounds cause serious comorbidities and represent a major burden for patients, families, and health systems. Due to the pivotal role of immune cells in the proper resolution of inflammation and tissue repair, we focus our current study on the interaction of macrophages with skin cells, and specifically on the effects of CD163 gene induction in macrophages in wound healing. We hypothesize that the over-expression of the scavenger receptor gene CD163 in human macrophages would result in a more efficient wound healing process. Using 3D human wounded skin organotypic tissues, we observed that CD163 overexpression in THP-1 and human primary macrophages induced a more efficient re-epithelization when compared to control cells. Using human primary skin cells and an in vitro scratch assay we observed that CD163 overexpression in THP-1 macrophages promoted a more rapid and efficient wound healing process through a unique interaction with fibroblasts. The addition of CD163-blocking antibody, but not isotype control, blocked the efficient wound healing process induced by CD163 overexpression in macrophages. We found that the co-culture of skin cells and CD163 overexpressing macrophages reduced monocyte chemoattractant protein (MCP)-1 and enhanced tumor growth factor (TGF)-α, without altering interleukin (IL)-6 or TGF-β. Our findings show that CD163 induces a more efficient wound healing and seems to promote a wound milieu with a pro-resolution molecular profile. Our studies set the foundation to study this approach in in vivo clinically relevant settings to test its effects in wound healing processes such as acute major injuries, large surgeries, or chronic ulcers.

The National Academies of Sciences, Engineering, and Medicine has found substantial evidence that cannabis (plant) is effective for the treatment of chronic pain in adults, and moderate evidence that oromucosal cannabinoids (extracts, especially nabiximols) improve short-term sleep disturbances in chronic pain. The paradoxical superiority of the cannabis plant over cannabinoid molecules represents a challenge for the medical community and the established processes that define modern pharmacy. The expanding and variable legalization of cannabis in multiple states nationwide represents an additional challenge for patients and the medical community because recreational and medicinal cannabis are irresponsibly overlapped. Cannabis designed for recreational use (containing high levels of active ingredients) is increasingly available to patients with chronic pain who do not find relief with current pharmacologic entities, which exposes patients to potential harm. This article analyzes the available scientific evidence to address controversial questions that the current state of cannabis poses for health care professionals and chronic pain patients and sets the basis for a more open discussion about the role of cannabis in modern medicine for pain management. A critical discussion on these points, the legal status of cannabis, and considerations for health care providers is presented.

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5 μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes.

Inositol-requiring enzyme 1[α] (IRE1[α])–X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1a–XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox-2) and prostaglandin E synthase (Ptges/mPGES-1) was compromised in IRE1a-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE2), was decreased in myeloid cells that lack IRE1a or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human PTGS2 and PTGES genes to enable optimal PGE2 production. Mice that lack IRE1α–XBP1 in leukocytes, or that were treated with IRE1a inhibitors, demonstrated reduced pain behaviors in PGE2-dependent models of pain. Thus, IRE1a–XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.

Abnormal outgrowth of sensory nerves is one of the important contributors to pain associated with cancer and its treatments. Primary neuronal cultures derived from dorsal root ganglia (DRG) have been widely used to study pain-associated signal transduction and electrical activity of sensory nerves. However, there are only a few studies using primary DRG neuronal culture to investigate neurite outgrowth alterations due to underlying cancer-related factors and chemotherapeutic agents. In this study, primary DRG sensory neurons derived from mouse, non-human primate, and human were established in serum and growth factor-free conditions. A bovine serum albumin gradient centrifugation method improved the separation of sensory neurons from satellite cells. The purified DRG neurons were able to maintain their heterogeneous subpopulations, and displayed an increase in neurite growth when exposed to cancer-derived conditioned medium, while they showed a reduction in neurite length when treated with a neurotoxic chemotherapeutic agent. Additionally, a semi-automated quantification method was developed to measure neurite length in an accurate and time-efficient manner. Finally, these exogenous factors altered the gene expression patterns of murine primary sensory neurons, which are related to nerve growth, and neuro-inflammatory pain and nociceptor development. Together, the primary DRG neuronal culture in combination with a semi-automated quantification method can be a useful tool for further understanding the impact of exogenous factors on the growth of sensory nerve fibers and gene expression changes in sensory neurons.

Beta 2 adrenergic receptor (β2 AR) activation in the central and peripheral nervous system has been implicated in nociceptive processing in acute and chronic pain settings with anti-inflammatory and anti-allodynic effects of β2-AR mimetics reported in several pain states. In the current study, we examined the therapeutic efficacy of the β2-AR agonist clenbuterol in a rat model of persistent postsurgical hypersensitivity induced by disruption of descending noradrenergic signaling in rats with plantar incision. We used growth curve modeling of ipsilateral mechanical paw withdrawal thresholds following incision to examine effects of treatment on postoperative trajectories. Depletion of spinal noradrenergic neurons delayed recovery of hypersensitivity following incision evident as a flattened slope compared to non-depleted rats (-1.8 g/day with 95% CI -2.4 to -1.085, p

Cannabis related online searches are associated with positive attitudes toward medical cannabis, particularly when information is obtained from dispensaries. Since pain is the main reason for medicinal cannabis use, information from dispensary websites has the potential to shape the attitude of pain patients towards cannabis. This is relevant because cannabis has demonstrated efficacy in neuropathic pain with low tetrahydrocannabinol (THC) concentrations (< 5–10%), in contrast to potent cannabis (>15% THC), which is highly rewarded in the recreational realm. The role of CBD in pain is not clear, however it has gained popularity. Thus, we hypothesize that the potency of medical cannabis that is advertised online is similar to the cannabis advertised for recreational purposes, which would potentially create a misconception towards medical cannabis. The current lack of knowledge surrounding advertised potencies in the legal cannabis market limits the ability to generate clear policies regarding online advertising to protect patients that are willing to use cannabis for their condition. Thus, we evaluated the advertised THC and CBD content of cannabis products offered online in dispensaries in the United States to determine products’ suitability to medicinal use and compare the strength of products offered in legal medical and recreational programs. We recorded THC and CBD concentrations for all herb cannabis products provided by dispensary websites and compared them between or within states. Four Western states (CA, CO, NM, WA) and five Northeastern states (ME, MA, NH, RI, VT) were included. A total of 8,505 cannabis products across 653 dispensaries were sampled. Despite the clear differences between medicinal and recreational uses of cannabis, the average THC concentration advertised online in medicinal programs was similar (19.2% ±6.2) to recreational programs (21.5% ±6.0) when compared between states with different programs, or between medicinal and recreational programs within the same states (CO or WA). Lower CBD concentrations accompanied higher THC products. The majority of products, regardless of medicinal or recreational programs, were advertised to have >15% THC (70.3% - 91.4% of products). These stated concentrations seem unsuitable for medicinal purposes, particularly for patients with chronic neuropathic pain. Therefore, this information could induce the misconception that high potency cannabis is safe to treat pain. This data is consistent with reports in which THC and CBD in products from legal dispensaries or in nationwide products from the illegal market were actually measured, which indicates that patients consuming these products may be at risk of acute intoxication or long-term side effects. Our study offers grounds to develop policies that help prevent misconceptions toward cannabis and reduce risks in pain patients.

Rachel Grosick,1 Perla Abigail Alvarado-Vazquez,2 Amy R Messersmith,1 E Alfonso Romero-Sandoval2 1Department of Pharmaceutical and Administrative Science, Presbyterian College School of Pharmacy, Clinton, SC, USA; 2Department of Anesthesiology, Wake Forest University School Medicine, Winston-Salem, NC, USA Introduction: Painful diabetic neuropathy is associated with chronic inflammation, in which macrophages are the key effectors. We utilized an in vitro approach to determine the effects of high glucose on macrophage phenotype. Materials and methods: We exposed human THP-1 macrophages to normal glucose (5 mM) and a clinically relevant high glucose environment (15 mM) and measured the expression and concentration of molecules associated with a diabetic cellular phenotype. Results: We found that THP-1 macrophages in high glucose conditions did not influence the basal expression of cyclooxygenase-2, Toll-like receptor-4, or class A scavenger receptor mRNA, or the concentrations of the cytokines interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and IL-10, but induced a priming effect on tumor necrosis factor (TNF)-α. Then, we stimulated THP-1 macrophages with a strong pro-inflammatory stimulus lipopolysaccharide (LPS; 5 µg/mL). After stimulation with LPS, we observed an exacerbated increase in TNF-α, IL-6, and MCP-1 concentration in the high glucose condition compared to the normal glucose environment. THP-1 macrophages in high glucose conditions developed tolerance to IL-10 anti-inflammatory effects (TNF-α production) when challenged with LPS. Conclusion: Our in vitro approach allows the study of macrophages as potential targets for therapeutic purposes since it compares them to primary human macrophages exposed to high glucose and macrophages from patients with diabetes or complications of painful diabetic neuropathy (i.e. ulcers, adipocytes, and pancreas). Keywords: diabetes, hyperglycemia, THP-1, monocyte, TNF, inflammation

The use of cannabinoids for the treatment of chronic diseases has increased in the United States, with 23 states having legalized the use of marijuana. Although currently available cannabinoid compounds have shown effectiveness in relieving symptoms associated with numerous diseases, the use of cannabis or cannabinoids is still controversial mostly due to their psychotropic effects (e.g., euphoria, laughter) or central nervous system (CNS)-related undesired effects (e.g., tolerance, dependence). A potential strategy to use cannabinoids for medical conditions without inducing psychotropic or CNS-related undesired effects is to avoid their actions in the CNS. This approach could be beneficial for conditions with prominent peripheral pathophysiologic mechanisms (e.g., painful diabetic neuropathy, chemotherapy-induced neuropathy). In this article, we discuss the scientific evidence to target the peripheral cannabinoid system as an alternative to cannabis use for medical purposes, and we review the available literature to determine the pros and cons of potential strategies that can be used to this end.

The purpose of this study was to provide the most up-to-date scientific evidence of the potential analgesic effects, or lack thereof, of the marijuana plant (cannabis) or cannabinoids, and of safety or tolerability of their long-term use. We found that inhaled (smoked or vaporized) cannabis is consistently effective in reducing chronic non-cancer pain. Oral cannabinoids seem to improve some aspects of chronic pain (sleep and general quality of life), or cancer chronic pain, but they do not seem effective in acute postoperative pain, abdominal chronic pain, or rheumatoid pain. The available literature shows that inhaled cannabis seems to be more tolerable and predictable than oral cannabinoids. Cannabis or cannabinoids are not universally effective for pain. Continued research on cannabis constituents and improving bioavailability for oral cannabinoids is needed. Other aspects of pain management in patients using cannabis require further open discussion: concomitant opioid use, medical vs. recreational cannabis, abuse potential, etc.

Sladjana Skopelja-Gardner,1,* Madhurima Saha,1,* Perla Abigail Alvarado-Vazquez,2 Brenna S Liponis,1 Elena Martinez,1 E Alfonso Romero-Sandoval2 1Department of Anesthesiology, Geisel School of Medicine at Dartmouth, Lebanon, NH, 2Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of Pharmacy, Clinton, SC, USA *These authors contributed equally to this work. Abstract: Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) and its substrates (extracellular signal-regulated kinase [ERK] and p38) play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT) and MKP-3 knockout (KO) mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2) inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08g day 1), which resolved normally by postoperative day 12 (0.46±0.08g, N=6). In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6). KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm2) than WT mice (8.7±1.2 cells/0.1 mm2) on postoperative day 1 (vs 5–6cells/0.1mm2 at baseline) that returned to baseline 12 days after surgery (10–12cells/0.1mm2). In WT mice, peripheral p-p38 and p-ERK-1/2 expression increased (5- and 3-fold, respectively) on postoperative days 1 and 5, and returned to basal levels 7–12 days after surgery (N=3 per group). Peripheral p-p38 levels in MKP-3 KO mice followed a similar expression pattern as WT mice. Peripheral p-ERK-1/2 levels in MKP-3 KO mice remained elevated 12 days after surgery (2.5-fold, N=3 per group). Administration of PD98059 (MEK inhibitor, N=8, vehicle N=9) reduced p-ERK-1/2 expression in the incised tissue and blocked hypersensitivity in MKP-3 KO mice (N=6). The findings of this study suggest that MKP-3 is pivotal for normal resolution of acute postoperative allodynia, through the regulation of peripheral p-ERK-1/2. Keywords: DUSP-6, p38, ERK

M1 macrophages release pro-inflammatory factors during inflammation. They transition to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra, and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive and practical approach for inflammatory conditions that could lead to persistent pain, i.e. major surgeries, burns, rheumatoid arthritis, etc.

Mitogen-activated protein kinases (MAPKs) play critical roles in the central nervous system immune responses through glial function, which are regulated with relative selectivity (or preference) by MAPK phosphatases (MKP). Phosphorylated extracellular signal-regulated protein kinase (p-ERK) is preferentially dephosphorylated by MKP-3, which display little activity over p-p38 and p–c-Jun NH2-terminal kinases (p-JNK). It has been proposed that these substrate preferences may vary depending on tissue or functional cellular processes. Since astrocytes display a prominent activity of JNK > ERK under stressed or reactive phenotype, we hypothesize that MKP-3 possess a similar or differential substrate preference in astrocytes for JNK and ERK (ERK = JNK or JNK > ERK). We generated transient expression of MKP-3 by transfecting a specific cDNA in primary rat neonatal brain cortex astrocytes. Cells were stimulated with lipopolysaccharide (LPS), and MAPKs and downstream pro-inflammatory products were measured by Western blot and ELISA analyses. MKP-3 expression in primary astrocytes reduced LPS-induced p-ERK and p-p38 by ~50%, and p-JNK by ~75%, and moderately reduced nitrite oxide (NO), while completely blocked Interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). We confirmed MKP-3 specific activity by developing a BV-2 microglia cell line stably overexpressing MKP-3 and using a specific siRNA against MKP-3. Our data demonstrate MKP-3 has differential substrate preference in astrocytes compared to other cells types, since it preferentially dephosphorylated p-JNK over p-ERK. Our results indicate also that astrocytic immune functions can be modulated by MKP-3 induction, a strategy that could be beneficial in neurological conditions in which astrocytes play a pathophysiological role, i.e. persistent pain.

The role of central glial cells in the mechanisms underlying pain has been intensively studied in the last two decades. Most studies on glia and pain focused on the potential detrimental role of glial cells following noxious stimulus/insults manifested as an "activation" or a "reactive" state (increase in glial marker expression and production of proinflammatory/nociceptive molecules). Therefore, "activated" or "reactive" glial cells became a target for the future generation of drugs to treat chronic pain. Several glial modulators that reduce the activation of glial cells have shown great efficacy in multiple animal (rodents mostly) models of pain (acute, subacute, chronic, inflammatory, neuropathic, surgical, etc.). These encouraging findings inspired clinical trials that have been completed in the last 5 years. Unfortunately, all clinical trials with these glial modulators have failed to demonstrate efficacy for the treatment of pain. New lines of investigation and elegant experimental designs are shedding light on alternative glial functions, which demonstrate that "glial reactivity" is not necessarily deleterious in some pathological conditions. New strategies to validate findings through our current animal models are necessary to enhance the translational value of our preclinical studies. Also, more studies using human subjects would enhance our understanding of glial cells in the context of pain. This chapter explores the available literature to objectively ponder the potential role of glial cells in human pain conditions.

The role of central glial cells in the mechanisms underlying pain has been intensively studied in the last two decades. Most studies on glia and pain focused on the potential detrimental role of glial cells following noxious stimulus/insults manifested as an "activation" or a "reactive" state (increase in glial marker expression and production of proinflammatory/nociceptive molecules). Therefore, "activated" or "reactive" glial cells became a target for the future generation of drugs to treat chronic pain. Several glial modulators that reduce the activation of glial cells have shown great efficacy in multiple animal (rodents mostly) models of pain (acute, subacute, chronic, inflammatory, neuropathic, surgical, etc.). These encouraging findings inspired clinical trials that have been completed in the last 5 years. Unfortunately, all clinical trials with these glial modulators have failed to demonstrate efficacy for the treatment of pain. New lines of investigation and elegant experimental designs are shedding light on alternative glial functions, which demonstrate that "glial reactivity" is not necessarily deleterious in some pathological conditions. New strategies to validate findings through our current animal models are necessary to enhance the translational value of our preclinical studies. Also, more studies using human subjects would enhance our understanding of glial cells in the context of pain. This chapter explores the available literature to objectively ponder the potential role of glial cells in human pain conditions.

New generations of cyclooxygenase (COX) inhibitors are more potent and efficacious than their traditional parent compounds. They are also safer than the classic non-steroidal anti-inflammatory drugs (NSAIDs) and are starting to be used not only for low to moderate intensity pain, but also for high intensity pain. Three different strategies have been followed to improve the pharmacological profile of COX inhibitors: 1. Development of COX-2 selective inhibitors. This is based on the initial hypothesis that considered COX-2 as the enzyme responsible for the generation of prostaglandins only in inflammation, and, therefore, uniquely responsible for inflammation, pain and fever. Initial expectations gave rise to controversial results, still under discussion. The second generation of these compounds is being developed and should contribute to clarifying both their efficacy and the specific functions of the COX enzymes. 2. Modified non-selective COX inhibitors. Molecules like nitro-NSAIDs or tromethamine salt derivatives have been synthesized considering that both COX-1 and COX-2 are responsible for the synthesis of prostaglandins involved either in homeostatic functions or inflammation. Nitroaspirin, nitroparacetamol or dexketoprofen trometamol are some examples of molecules that are already showing an important clinical efficacy. The modifications performed in their structures seem to lower the unwanted side effects as well as to enhance their analgesic efficacy. 3. Combined therapy of classic NSAIDs with other drugs. This strategy looks for improvements in the incidence of adverse effects or to take advantage of the synergistic enhancement of their therapeutic effects. Some of the molecules resulting from these strategies are very valuable as therapeutic agents and open a wide range of possibilities in the treatment of high intensity pain, including neuropathic pain, and opiate sparing therapy.

Non-steroidal anti-inflammatory drugs (NSAIDs) are effective anti-inflammatory and analgesic drugs although they also induce unwanted side effects due to the inhibition of the physiological effects regulated by prostaglandins. This has led to the search for new compounds with fewer side effects, such as the nitro-NSAIDs (NO-NSAIDs). Paracetamol is an analgesic drug devoid of some of the side effect of the NSAIDs but without anti-inflammatory activity. NCX-701 is a nitric oxide releasing version of paracetamol with anti-inflammatory and analgesic properties. We have tested, in the single motor unit technique, the antinociceptive actions of intravenous cumulative doses of NCX-701 vs paracetamol, studying their antinociceptive effects in responses to noxious mechanical and electrical stimulation (wind-up). Paracetamol did not induce any significant effect at the doses tested (maximum of 480 μmol kg−1, 72.5 mg kg−1). NCX-701 however was very effective in reducing responses to noxious mechanical stimulation (32±10% of control response) and wind-up (ED50 of 147±1 μmol kg−1, 41.5±0.3 mg kg−1). The inhibition was not reversed by 1 mg kg−1 of the opioid antagonist naloxone. In control experiments performed with either the vehicle or the NO donor NOC-18, no significant changes were observed in the nociceptive responses studied. We conclude that NCX-701 is a very effective non-opioid antinociceptive agent in normal animals and its action is located mainly at central areas. The antinociceptive effect was not due solely to the release of NO. British Journal of Pharmacology (2002) 135, 1556–1562; doi:10.1038/sj.bjp.0704589

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

Peripheral nerve injury generally results in spinal neuronal and glial plastic changes associated with chronic behavioral hypersensitivity. Spinal mitogen-activated protein kinases (MAPKs), eg, p38 or extracellular signal-regulated kinases (ERKs), are instrumental in the development of chronic allodynia in rodents, and new p38 inhibitors have shown potential in acute and neuropathic pain patients. We have previously shown that the cannabinoid type 2 receptor agonist JWH015 inhibits ERK activity by inducing MAPK phosphatase (MKP)-1 and MKP-3 (the major regulators of MAPKs) in vitro in microglial cells. Therefore, we decided to investigate the role of these phosphatases in the mechanisms of action of JWH015 in vivo using the rat L5 nerve transection model of neuropathic pain. We observed that peripheral nerve injury reduced spinal MKP-1/3 expression and activity and that intrathecal JWH015 reduced established L5 nerve injury-induced allodynia, enhanced spinal MKP-1/3 expression and activity, and reduced the phosphorylated form of p38 and ERK-1/2. Triptolide, a pharmacological blocker of MKP-1 and MKP-3 expression, inhibited JWH015’s effects, suggesting that JWH015 exerts its antinociceptive effects by modulating MKP-1 and MKP-3. JWH015-induced antinociception and MKP-1 and MKP-3 expression were inhibited by the cannabinoid type 2 receptor antagonist AM630. Our data suggest that MKP-1 and MKP-3 are potential targets for novel analgesic drugs. Perspective MAPKs are pivotal in the development of chronic allodynia in rodent models of neuropathic pain. A cannabinoid type 2 receptor agonist, JWH015, reduced neuropathic allodynia in rats by reducing MAPK phosphorylation and inducing spinal MAPK phosphatases 1 and 3, the major regulators of MAPKs.

Matthew S. Alkaitis1,2, Christian Ndong1,3, Russell P. Landry III1,3, Joyce A. DeLeo1,3,4 and E. Alfonso Romero-Sandoval1,3,4 1Neuroscience Center at Dartmouth, Dartmouth Medical School, 2Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, 3Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, 4Department of Pharmacology and Toxicology, Dartmouth-Hitchcock Medical Center, 1,3,4USA 2UK

Background An alarming portion of patients develop persistent or chronic pain following surgical procedures, but the mechanisms underlying the transition from acute to chronic pain states are not fully understood. In general, endocannabinoids (ECBs) inhibit nociceptive processing by stimulating cannabinoid receptors type 1 (CB1) and type 2 (CB2). We have previously shown that intrathecal administration of a CB2 receptor agonist reverses both surgical incision-induced behavioral hypersensitivity and associated over-expression of spinal glial markers. We therefore hypothesized that endocannabinoid signaling promotes the resolution of acute postoperative pain by modulating pro-inflammatory signaling in spinal cord glial cells. Methodology/Principal Findings To test this hypothesis, rats receiving paw incision surgery were used as a model of acute postoperative pain that spontaneously resolves. We first characterized the concentration of ECBs and localization of CB1 and CB2 receptors in the spinal cord following paw incision. We then administered concomitant CB1 and CB2 receptor antagonists/inverse agonists (AM281 and AM630, 1 mg.kg−1 each, i.p.) during the acute phase of paw incision-induced mechanical allodynia and evaluated the expression of glial cell markers and phosphorylated p38 (a MAPK associated with inflammation) in the lumbar dorsal horn. Dual blockade of CB1 and CB2 receptor signaling prevented the resolution of postoperative allodynia and resulted in persistent over-expression of spinal Glial Fibrillary Acidic Protein (GFAP, an astrocytic marker) and phospho-p38 in astrocytes. We provide evidence for the functional significance of these astrocytic changes by demonstrating that intrathecal administration of propentofylline (50 µg, i.t.) attenuated both persistent behavioral hypersensitivity and over-expression of GFAP and phospho-p38 in antagonist-treated animals. Conclusions/Significance Our results demonstrate that endocannabinoid signaling via CB1 and CB2 receptors is necessary for the resolution of paw incision-induced behavioral hypersensitivity and for the limitation of pro-inflammatory signaling in astrocytes following surgical insult. Our findings suggest that therapeutic strategies designed to enhance endocannabinoid signaling may prevent patients from developing persistent or chronic pain states following surgery.

Anti-nociceptive tolerance to opioids is a well-described phenomenon, which severely limits the clinical efficacy of opioids for the treatment of chronic pain syndromes. The mechanisms that drive anti-nociceptive tolerance, however, are less well understood. We have previously shown that glia have a central role in the development of morphine tolerance and that administration of a glial modulating agent attenuated tolerance formation. Recently, we have demonstrated that morphine enhances microglial Iba1 expression and P2X4 receptor-mediated microglial migration via direct mu opioid receptor signaling in in vitro microglial cultures. We hypothesize that P2X4 receptors drive morphine tolerance and modulate morphine-induced spinal glial reactivity. Additionally, we hypothesize that perivascular microglia play a role in morphine tolerance and that P2X4 receptor expression regulates perivascular microglia ED2 expression. To test these hypotheses, rats were implanted with osmotic minipumps releasing morphine or saline subcutaneously for seven days. Beginning three days prior to morphine treatment, P2X4 receptor antisense oligonucleotide (asODN) was injected intrathecally daily, to selectively inhibit P2X4 receptor expression. P2X4 receptor asODN treatment inhibited morphine-induced P2X4 receptor expression and blocked anti-nociceptive tolerance to systemically administered morphine. P2X4 receptor asODN treatment also attenuated the morphine-dependent increase of spinal ionized calcium binding protein (Iba1), glial fibrillary acidic protein (GFAP) and mu opioid receptor protein expression. Chronic morphine also decreased perivascular microglial ED2 expression, which was reversed by P2X4 receptor asODN. Together, these data suggest that the modulation of P2X4 receptor expression on microglia and perivascular microglia may prove an attractive target for adjuvant therapy to attenuate opioid-induced anti-nociceptive tolerance.

The combination of numerous classic drugs with nitric oxide donors has led to the development of new compounds with promising therapeutic activities in a great variety of situations, including cardiovascular and respiratory systems, ocular pressure, inflammation, and pain. One of the first compounds developed was NCX-701 or nitroparacetamol, resulting from the combination of paracetamol, a classic and popular analgesic used in a great number of over-the-counter medications because of its antipyretic and analgesic properties, and a nitrooxybutyroyl moiety, which releases nitric oxide at a low but steady level. Although paracetamol is devoid of most of the gastrointestinal toxicity associated with aspirin-like drugs, this type of compounds was first designed to take advantage of the cytoprotective properties of nitric oxide when released at low concentrations. However, the combination of these molecules also resulted in an unexpected enhancement of the analgesic activity of paracetamol. In fact, NCX-701 has been shown to be effective in acute nociception as well as in neuropathic pain, situations in which paracetamol and other COX inhibitors are devoid of any effect. In addition, NCX-701 is more potent and, in some circumstances, more effective than its parent compound in different models of inflammatory pain. Furthermore, whereas paracetamol lacks any effective antiinflammatory action, NCX-701 might reduce inflammation. All these results taken together imply that the mechanism of action of NCX-701 is different from that of paracetamol, although it is not yet established for either molecule. NCX-701 appears to be a promising compound in the treatment of different types of pain, with a likely better profile of side effects than its parent molecule, paracetamol. Although recent clinical trials provided data consistent with the preclinical profile of NCX-701, further studies are needed to support its clinical use.

Neuropathic pain may be primarily driven by immune responses in peripheral nerves. Peripherally released catecholamines may exacerbate neuropathic pain and also modulate immune responses in a complex and sometimes opposing manner by actions on multiple adrenoceptor subtypes. We showed previously that injection of the α2-adrenoceptor agonist clonidine at the site of peripheral nerve injury reduces pain behavior and local tissue pro-inflammatory cytokine content in rats. The current study used a model of acute inflammatory neuritis to test the efficacy and mechanisms of action ofα2-adrenoceptor stimulation to reduce pain. Zymosan, injected on the sciatic nerve, caused hypersensitivity to mechanical stimuli ipsilateral to injection and contralaterally, so-called mirror image pain. Ipsilateral hypersensitivity was inhibited dose-dependently by perineural injection of clonidine. Zymosan increased leukocyte number at the site of injection 3 d later as well as their content of interleukin 1α (IL-1α), IL-1β, and IL-6. Perineural clonidine prevented both the increase in leukocyte number and cytokine expression induced by zymosan. Additionally, clonidine reduced the capacity of leukocytes to express pro-inflammatory cytokines as assessed by treatment of cellsex vivowith lipopolysaccharide, whereas no repression of IL-10 production occurred. Clonidine reduced the number of macrophages and lymphocytes as well as their expression of tumor necrosis factor α. All of the effects of clonidine were prevented by coadministration of anα2A-adrenoceptor-preferring antagonist. These results suggest that α2-adrenoceptor stimulation transforms cytokine gene expression, especially in macrophages and lymphocytes from a pro- to an anti-inflammatory profile in the setting of neuritis, likely relieving neuritis-induced pain by this mechanism.

All-trans retinoic acid (ATRA), the active metabolite of vitamin A, is involved in the inflammatory reaction and modulates the expression of cyclooxygenase (COX) enzymes and nitric oxide (NO) activity. Since COX enzymes are the substrate of action of COX inhibitors, we studied the analgesic activity of paracetamol (PAR) and its NO-derivative nitroparacetamol (NOP) in the presence and absence of oral ATRA. Nociceptive responses were studied using the recording of single motor units technique in alpha-chloralose anesthetized normal and monoarthritic male Wistar rats. Intravenous PAR was not effective in normal rats. However, after pre-treatment with ATRA, PAR reduced dose-dependently the responses to noxious mechanical stimulation (ID50: 60+/-7 micromol/kg; 9.1 mg/kg), but not wind-up. The analgesic activity of NOP was enhanced after pre-treatment with ATRA either on responses to noxious mechanical stimulation (ID50s: 147+/-2 vs. 46+/-2 micromol/kg) or wind-up (maximal effect of 46+/-1% with 480 micromol/kg vs. 33+/-3% of control with 240 micromol/kg). The administration of ATRA did not modify the effect of PAR and NOP in monoarthritic rats. We conclude that pre-treatment with oral ATRA enhances the analgesic activity of PAR and NOP in acute pain, probably due to a positive modulation of their activity on spinal cord COX enzymes.

Nitric oxide (NO)-releasing NSAIDs have been shown to be safer and more potent as antinociceptive and anti-inflammatory agents than their parent compounds. NCX-701 (nitroparacetamol), in contrast to paracetamol, is an effective antinociceptive drug in normal animals but their effectiveness in monoarthritis has not been compared. We have now investigated this question by comparing the antinociceptive effects of i.v. NCX-701 and paracetamol in monoarthritic rats under alpha-chloralose anesthesia. The influence of spinalization on the effects of NCX-701 was also studied. NCX-701 and paracetamol were equipotent in reducing single motor unit responses to noxious mechanical stimulation, ID50s of 320+/-1.2 and 305+/-1.2 micromol/kg, respectively. The mechanism of action seems to be different since NCX-701, but not paracetamol, reduced wind-up. This effect suggests a central action, probably within the spinal cord. Sham spinalization reduced the effect of NCX-701 on nociceptive responses drastically. In spinalized animals, however, the effect was similar to that observed in intact animals, indicating a strong effect of NCX-701 at spinal sites, which counterbalances the decrease in the activity induced by the surgery. We conclude that NCX-701 is an effective antinociceptive drug in arthritic animals, with a mechanism of action located in the spinal cord, and different to that of paracetamol.