Adjuvant Model Of Inflammatory Pain Research Articles

Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a β-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious β-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

Objective Quantification of Limb Position in Mouse Spared Nerve Injury Reveals New Motor Phenotypes

Difficulties translating basic animal research into novel pharmacotherapies for neuropathic pain can, in part, be attributed to a lack of predictive efficacy in preclinical pain models. Most animal models of neuropathic pain rely heavily on targeted nerve injuries quantified using reflexive measures in response to an applied noxious stimulus. However, data from these approaches fail to capture spontaneous and ongoing pain – a critical component of neuropathic pain in human patients. Furthermore, the common spared nerve injury (SNI) model damages the tibial and common peroneal nerves that should result in motor phenotypes not typically captured in preclinical literature. To overcome these limitations, we employed deep learning-based markerless pose estimation software (DeepLabCut) to quantify spontaneous limb position in C57BL/6J mice during a one-minute tail suspension following either SNI or sham surgery. Using this granular detail, we were able to characterize previously unidentified injury-induced motor behaviors that persist up to six weeks following SNI. In particular, we found a decrease in the median distance between body midline and injured limb position in the SNI group compared to sham controls. We also found some evidence for a decrease in toe movement relative to the heel movement. We repeated these analyses in subsequent cohorts of SNI mice to determine whether these outputs could be reversed by multiple analgesics with diverse mechanistic actions and thus far do not see evidence that these behaviors are reversed by analgesics. Furthermore, we do not see these phenotypes in the Complete Freund's Adjuvant model of inflammatory pain. Together these results suggest SNI causes previously unobserved motor phenotypes unrelated to altered sensation that are likely underappreciated while interpreting preclinical pain research. Grant support from R01 NS117899-01 F31 NS124301-01 Rita Allen Foundation.

Kappa opioid receptor blockade in the nucleus accumbens shell prevents sex-dependent alcohol deprivation effect induced by inflammatory pain.

Pain-induced negative affect reduces life quality of patients by increasing psychiatric comorbidities, including alcohol use disorders (AUDs). Indeed, clinical data suggest pain as a risk factor to suffer AUDs, predicting relapse drinking in abstinent patients. Here, we analyse the impact of pain on alcohol relapse and the role of kappa opioid receptor (KOR) activation in mediating these pain-induced effects because KORs play an important role in pain-driven negative affect and AUD. Female and male Sprague-Dawley rats underwent 2 alcohol intermittent access periods separated by a forced abstinence period. The complete Freund adjuvant model of inflammatory pain was introduced during abstinence, and alcohol intake before and after alcohol reintroduction was assessed. In addition, we used behavioural approaches to measure stress and memory impairment and biochemical assays to measure KOR expression in abstinence and reintroduction periods. Only female CFA-treated rats increased alcohol intake during the reintroduction period. Concomitantly, this group showed enhanced anxiety-like behaviour and increased KOR expression in the nucleus accumbens shell that was developed during abstinence and remained during the reintroduction period. Finally, KOR antagonist norbinaltorphimine was administered in the nucleus accumbens shell during abstinence to prevent a pain-induced alcohol deprivation effect, a phenomenon observed in CFA-female rats. The administration of norbinaltorphimine effectively blocked a pain-induced alcohol deprivation effect in female rats. Our data evidenced that inflammatory pain constitutes a risk factor to increase alcohol consumption during a reintroduction phase only in female rats by the rise and maintenance of stress probably mediated by KOR signalling in the nucleus accumbens.

Peripheral signaling mechanisms underlying mu‐delta opioid synergy

In response to the ongoing opioid epidemic, and in an effort to develop new analgesics with improved safety profiles, researchers have invested significant energy into further understanding the various downstream signaling cascades initiated by opioid receptor activation. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Further research has shown that bivalent or bifunctional opioid compounds can also result in differential signal transduction. We hypothesized that a combination of mu and delta opioid agonists would engage different signaling pathways than drugs that target only one opioid receptor. Therefore, we probed the signaling mechanisms underlying the behavioral analgesic synergy previously demonstrated by a 1:1 combination of loperamide, a peripherally-restricted mu-opioid agonist, and oxymorphindole, a delta-opioid agonist. Using the complete Freund's adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of loperamide-oxymorphindole in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M. Then, mice were given an injection of loperamide, oxymorphindole, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of G protein-coupled inwardly-rectifying potassium (GIRK) channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the loperamide-oxymorphindole combination (p<0.01) than either drug alone, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05). Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination's anti-nociceptive effect (p<0.0001). From these data we conclude that the loperamide-oxymorphindole combination binds to heterodimeric opioid receptors, activates a G protein signaling pathway, and opens GIRK channels.

494 Mechanisms underlying MOR-DOR analgesic synergy in epidermal nerve fibers

We recently published electrophysiological data showing that the combination of the mu-opioid (MOR) agonist loperamide and the delta-opioid (DOR) agonist oxymorphindole (Lo-OMI), applied topically to mouse toe pads, prevents activation of nociceptive epidermal nerve fibers with high potency and efficacy (Uhelski et al., 2020). The present study tested the hypotheeis that i) heterodimeric opioid receptors mediate this analgesic action, and ii) inhibitory G proteins mediate this action via activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Using the complete Freund’s adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of Lo-OMI in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M (Olson, Streicher et al., 2018). Then, mice were given an injection of Lo, OMI, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of GIRK channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the Lo-OMI combination (p<0.01) than either drug alone, supporting the heterodimeric opioid receptor hypothesis, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05), supporting the Gai/o signaling hypothesis. Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination’s anti-nociceptive effect (p<0.0001), supporting the requirement for GIRK channels.

C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund’s Adjuvant model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic constrictive nerve injury, a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-chronic constrictive nerve injury. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J × B6N-F2 cross (N = 164), we mapped a major quantitative trait locus underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (log of the odds [LOD] = 3.81, p < 0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression quantitative trait loci associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (false discovery rate < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain.

The central nucleus of the amygdala (CeA) encompasses the main output pathways of the amygdala, a temporal lobe structure essential in affective and cognitive dimensions of pain. A major population of neurons in the CeA send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. CeA-PAG neurons are topographically organized based on their targeted subregion within the PAG. PAG-projecting neurons in the central medial (CeM) and central lateral (CeL) regions of CeA are intrinsically distinct. CeL-PAG neurons are a homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered in the complete Freund's adjuvant model of inflammatory pain. A major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA-PAG pathway has proved to be a component of descending anti-nociceptive circuitry, the functional organization of CeA-PAG neurons remains unclear. We identified CeA-PAG neurons in C57BL/6 mice of both sexes using intracranial injection of a fluorescent retrograde tracer into the PAG. In acute brain slices, we investigated the topographical and intrinsic characteristics of retrogradely labelled CeA-PAG neurons using epifluorescence and whole-cell electrophysiology. We also measured changes to CeA-PAG neurons in the complete Freund's adjuvant (CFA) model of inflammatory pain. Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL-PAG neurons consist of a relatively homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered 1day after induction of the CFA inflammatory pain model. Collectively, our results provide insight into pain-induced changes to a specific population of CeA neurons that probably play a key role in the integration of noxious input with endogenous analgesia and behavioural coping response.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

A novel intrinsic analgesic mechanism: the enhancement of the conduction failure along polymodal nociceptive C-fibers

Although conduction failure has been observed in nociceptive C-fibers, little is known regarding its significance or therapeutic potential. In a previous study, we demonstrated that C-fiber conduction failure, which is regarded as an intrinsic self-inhibition mechanism, was reduced in circumstances of painful diabetic neuropathy. In this study, we extend this finding in the complete Freund's adjuvant model of inflammatory pain and validate that the degree of conduction failure decreased and led to a greater amount of pain signals conveyed to the central nervous system. In complete Freund's adjuvant-injected animals, conduction failure occurred in a C-fiber-selective, activity-dependent manner and was associated with an increase in the rising slope of the C-fiber after-hyperpolarization potential. To target conduction failure in a therapeutic modality, we used ZD7288, an antagonist of hyperpolarization-activated, cyclic nucleotide-modulated channels which are activated by hyperpolarization and play a pivotal role in both inflammatory and neuropathic pain. ZD7288 promoted conduction failure by suppressing Ih as a mechanism to reduce the rising slope of the after-hyperpolarization potential. Moreover, perineuronal injection of ZD7288 inhibited abnormal mechanical allodynia and thermal hyperalgesia without affecting motor function or heart rate. Our data highlight the analgesic potential of local ZD7288 application and identify conduction failure as a novel target for analgesic therapeutic development.

Chronic Inflammatory Injury Results in Increased Coupling of Delta Opioid Receptors to Voltage-Gated Ca2+ Channels

BackgroundOpioid receptors regulate a diverse array of physiological functions. Mu opioid receptor agonists are well-known analgesics for treating acute pain. In contrast, animal models suggest that chronic pain is more effectively relieved by delta opioid receptor agonists. A number of studies have shown that chronic pain results in increased function of delta opioid receptors. This is proposed to result from enhanced trafficking of the delta opioid receptor to the cell membrane induced by persistent tissue injury. However, recent studies have questioned this mechanism, which has resulted in some uncertainty as to whether delta opioid receptors are indeed upregulated in chronic pain states. To clarify this question, we have examined the effect of chronic inflammatory pain over time using both an ex vivo measure of delta function: receptor-Ca2+ channel coupling, and an in vivo measure; the relief of chronic pain by a delta opioid receptor agonist. In addition, as beta-arrestin 2 can regulate delta opioid receptor trafficking and signaling, we have further examined whether deleting this scaffolding and signal transduction molecule alters delta opioid receptor function.ResultsWe used the Complete Freund’s Adjuvant model of inflammatory pain, and examined the effectiveness of the delta agonist, SNC80, to both inhibit Ca2+ channels in primary afferent neurons and to attenuate mechanical allodynia. In naïve beta-arrestin 2 wildtype and knockout mice, SNC80 neither significantly inhibited voltage-dependent Ca2+ currents nor produced antinociception. However, following inflammatory pain, both measures showed a significant and long-lasting enhancement of delta opioid receptor function that persisted for up to 14 days post-injury regardless of genotype. Furthermore, although this pain model did not alter Ca2+ current density, the contribution of N-type Ca2+ channels to the total current appeared to be regulated by the presence of beta-arrestin 2.ConclusionsOur results indicate that there is an upregulation of delta opioid receptor function following chronic pain. This gain of function is reflected in the increased efficacy of a delta agonist in both behavioral and electrophysiological measures. Overall, this work confirms that delta opioid receptors can be enhanced following tissue injury associated with chronic pain.

Spinal activation of delta opioid receptors alleviates cancer-related bone pain

Over the past few years, significant progress has been made in cancer therapy. Indeed, the lifespan of cancer patients has significantly increased. Although patients live longer, cancer-related pain remains a daily problem affecting their quality of life, especially when metastases reach the bone. In patients coping with cancer-induced bone pain, morphine and NSAIDs, often used in combination with other medications, are the most commonly used drugs to alleviate pain. However, these drugs have dose-limiting side effects. Morphine and other routinely used opioids are mu opioid receptor (MOPR) agonists. The MOPR is responsible for most opioid-related adverse effects. In the present study, we revealed potent analgesic effects of an intrathecally-administered selective delta opioid receptor (DOPR) agonist, deltorphin II, in a recently developed rat bone cancer model. Indeed, we found that deltorphin II dose-dependently reversed mechanical allodynia 14 days post-surgery in this cancer pain model, which is based on the implantation of mammary MRMT-1 cells in the femur. This effect was DOPR-mediated as it was completely blocked by naltrindole, a selective DOPR antagonist. Using the complete Freund's adjuvant model of inflammatory pain, we further demonstrated that deltorphin II was equipotent at alleviating inflammatory and cancer pain (i.e. similar ED50 values). Altogether, the present results show, for the first time, that activation of spinal DOPRs causes significant analgesia at doses sufficient to reduce inflammatory pain in a rat bone cancer pain model. Our results further suggest that DOPR represents a potential target for the development of novel analgesic therapies to be used in the treatment of cancer-related pain.

Ecto-5′-Nucleotidase (CD73) Inhibits Nociception by Hydrolyzing AMP to Adenosine in Nociceptive Circuits

Ecto-5'-nucleotidase (NT5E, CD73) is a membrane-anchored protein that hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in diverse tissues but has not been directly studied in nociceptive neurons. We found that NT5E was located on peptidergic and nonpeptidergic nociceptive neurons in dorsal root ganglia (DRG) and on axon terminals in lamina II (the substantia gelatinosa) of spinal cord. NT5E was also located on epidermal keratinocytes, cells of the dermis, and on nociceptive axon terminals in the epidermis. Following nerve injury, NT5E protein and AMP histochemical staining were coordinately reduced in lamina II. In addition, AMP hydrolytic activity was reduced in DRG neurons and spinal cord of Nt5e(-/-) mice. The antinociceptive effects of AMP, when combined with the adenosine kinase inhibitor 5-iodotubericidin, were reduced by approximately 50% in Nt5e(-/-) mice and were eliminated in Adenosine A(1) receptor (A(1)R, Adora1) knock-out mice. Additionally, Nt5e(-/-) mice displayed enhanced sensitivity in the tail immersion assay, in the complete Freund's adjuvant model of inflammatory pain and in the spared nerve injury model of neuropathic pain. Collectively, our data indicate that the ectonucleotidase NT5E regulates nociception by hydrolyzing AMP to adenosine in nociceptive circuits and represents a new molecular target for the treatment of chronic pain. Moreover, our data suggest NT5E is well localized to regulate nucleotide signaling between skin cells and sensory axons.

A Peripherally Acting Nav1.7 Sodium Channel Blocker Reverses Hyperalgesia and Allodynia on Rat Models of Inflammatory and Neuropathic Pain

Voltage-gated sodium channels (Na(v)1) are expressed in primary sensory neurons where they influence excitability via their role in the generation and propagation of action potentials. Recently, human genetic data have shown that one sodium channel subtype, Na(v)1.7, plays a major role in pain. We performed these studies to characterize the antinociceptive effects of N-[(R)-1-((R)-7-chloro-1-isopropyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylcarbamoyl)-2-(2-fluorophenyl)-ethyl]-4-fluoro-2-trifluoromethyl-benzamide (BZP), a non-central nervous system (CNS) penetrant small molecule with high affinity and preferential selectivity for Na(v)1.7 over Na(v)1.8 and Na(v)1.5. BZP was evaluated in rat preclinical models of inflammatory and neuropathic pain and compared with standard analgesics. Two models were used: the complete Freund's adjuvant model of inflammatory pain and the spinal nerve ligation model of neuropathic pain. BZP was also evaluated in a motor coordination assay to assess its propensity for CNS side effects. In preclinical models of chronic pain, BZP displayed efficacy comparable with that of leading analgesics. In the complete Freund's adjuvant model, BZP produced reversal of hyperalgesia comparable with nonsteroidal antiinflammatory drugs, and in the spinal nerve ligation model, BZP produced reversal of allodynia comparable with gabapentin and mexiletine. Unlike the CNS penetrant compounds gabapentin and mexiletine, BZP did not induce any impairment of motor coordination. These data suggest that a peripherally acting sodium channel blocker, preferentially acting through Na(v)1.7, could provide clinical relief of chronic pain without the CNS side effects typical of many existing pain treatments.

Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain

Event Abstract Back to Event Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain Rongwen Li1, 2*, Andrew Barszczyk1 and Michael Tymianski1, 2 1 Toronto Western Hospital Research Institute, Canada 2 NoNO Inc, Canada NADH dehydrogenase subunit 2 (ND2) is one of several proteins that interacts with the unique domain of the Src tyrosine kinase. Acting as an adapter protein, ND2 anchors Src to the N-methyl-d-aspartate (NMDA) receptor complex in post synaptic densities (PSDs), allowing it to regulate NMDA receptor activity. While the peptide sequence comprising the ND2 interaction domain of Src has been characterized (Src40-49) and has been used as a cell-permeant peptide to treat pain (Src40-49Tat), the Src interaction domain of ND2 has not yet been determined, though it is known to reside in an ND2 segment known as ND2.1. To identify the Src interacting domain of ND2, four Glutathione-S-transferase (GST) fusion proteins containing 4 overlapping sequence fragments of ND2.1 were constructed (ND2.1.1 through ND2.1.4). ND2.1.4–GST was observed to bind directly to the biotinylated domains of Src 40-49. Dot blot and competitive ELISA assays demonstrated that the interaction of Src 40-49Tat with ND2 or ND2.1 is blocked by ND2.1.4–GST. Next, eight peptides containing different segments of ND2.1.4 were synthesized and used in binding assays for Src 40-49Tat. One segment of ND2 (ND2.1.4-7) demonstrated a strong dose-response relationship on binding to Src 40-49-Tat. The interaction of ND2 with Src 40-49 was significantly reduced by pre-incubation with the ND2.1.4-7 peptide in competitive ELISA assays and dot blot assays. Additionally, endogenous colocalization of ND2 with Src was significantly reduced in primary cultures of hippocampal neurons treated with ND2.1.4-7. It was also determined that ND2.1.4-7, when affixed to the cell-permeating HIV1 Tat domain, alleviated the reduction in paw withdrawal threshold induced by the Complete Freund’s Adjuvant model of inflammatory pain. This suggests that inhibiting the interaction between Src and ND2 with the Src-interacting domain of ND2 can alleviate the NMDAR-mediated central sensitization that contributes to inflammatory and neuropathic pain. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Li R, Barszczyk A and Tymianski M (2009). Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.029 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Dec 2009; Published Online: 17 Dec 2009. * Correspondence: Rongwen Li, Toronto Western Hospital Research Institute, Toronto, Canada, rongwen_li@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rongwen Li Andrew Barszczyk Michael Tymianski Google Rongwen Li Andrew Barszczyk Michael Tymianski Google Scholar Rongwen Li Andrew Barszczyk Michael Tymianski PubMed Rongwen Li Andrew Barszczyk Michael Tymianski Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Characterization of a Cannabinoid CB2Receptor-Selective Agonist, A-836339 [2,2,3,3-Tetramethyl-cyclopropanecarboxylic Acid [3-(2-Methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide], Using in Vitro Pharmacological Assays, in Vivo Pain Models, and Pharmacological Magnetic Resonance Imaging

Studies demonstrating the antihyperalgesic and antiallodynic effects of cannabinoid CB(2) receptor activation have been largely derived from the use of receptor-selective ligands. Here, we report the identification of A-836339 [2,2,3,3-tetramethyl-cyclopropanecarboxylic acid [3-(2-methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide], a potent and selective CB(2) agonist as characterized in in vitro pharmacological assays and in in vivo models of pain and central nervous system (CNS) behavior models. In radioligand binding assays, A-836339 displays high affinities at CB(2) receptors and selectivity over CB(1) receptors in both human and rat. Likewise, A-836339 exhibits high potencies at CB(2) and selectivity over CB(1) receptors in recombinant fluorescence imaging plate reader and cyclase functional assays. In addition A-836339 exhibits a profile devoid of significant affinity at other G-protein-coupled receptors and ion channels. A-836339 was characterized extensively in various animal pain models. In the complete Freund's adjuvant model of inflammatory pain, A-836339 exhibits a potent CB(2) receptor-mediated antihyperalgesic effect that is independent of CB(1) or mu-opioid receptors. A-836339 has also demonstrated efficacies in the chronic constrain injury (CCI) model of neuropathic pain, skin incision, and capsaicin-induced secondary mechanical hyperalgesia models. Furthermore, no tolerance was developed in the CCI model after subchronic treatment with A-836339 for 5 days. In assessing CNS effects, A-836339 exhibited a CB(1) receptor-mediated decrease of spontaneous locomotor activities at a higher dose, a finding consistent with the CNS activation pattern observed by pharmacological magnetic resonance imaging. These data demonstrate that A-836339 is a useful tool for use of studying CB(2) receptor pharmacology and for investigation of the role of CB(2) receptor modulation for treatment of pain in preclinical animal models.

A-317491, a selective P2X 3/P2X 2/3 receptor antagonist, reverses inflammatory mechanical hyperalgesia through action at peripheral receptors in rats

The effect of A-317491 (5-({(3-Phenoxybenzyl)[(1 S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino}carbonyl)-1,2,4-benzenetricarboxylic acid), a recently described selective P2X 3 and P2X 2/3 receptor antagonist, on inflammatory mechanical hyperalgesia was examined. In the rat Freund's complete adjuvant model of inflammatory pain, s.c. administration of A-317491 dose-dependently reversed mechanical hyperalgesia. Maximum percent reversal (72%) was seen 3 h after administration at 10 mg/kg. Substantial plasma concentrations were measured for A-317491 after s.c. dosing 3, 10 and 30 mg/kg. However, the brain-to-plasma concentration ratio, determined 1 h after a 10 mg/kg s.c. dose, indicated limited penetration of A-317491 into the central nervous system. As revealed by neural activity recorded from single C-fiber nociceptive afferent in a Freund's complete adjuvant-inflamed rat skin-nerve preparation, topical application of A-317491 completely blocked afferent activation and mechanical sensitization induced by α,β-methylene ATP, a P2X agonist. These results suggest that A-317491 is a peripherally acting P2X blocker. Its efficacy demonstrates the importance of peripheral P2X 3/P2X 2/3 receptors in mediating ATP-associated mechanical hyperalgesia following inflammation, confirming previous suggestions of a significant role for P2X 2/3.