Treatment of Chronic Neuropathic Pain

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1 ,and 13 Adenosine * ,P2Y b, -triphosphates, 12 9 78,185 Therefore, often, ). ,P2Y 26,124 Fig. 1 11 Nucleotide-induced pain is ,P2Y 32 -triphosphate-gated P2X ion 6 9 onged anti-inflammatory effect protein-coupled P2Y receptors sequent activation of adenosine -diphosphates, 5 9 AR agonists offer a novel approach ), in general tends to put the brakes ,P2Y 3 3 4 Thus,inmanycases,actionofP2Rs , Daniela Salvemini neuroplasticity, and their antagonists b 32 ,P2Y ,andA 2 2B ing this pathway to clinically treat pain has not ,A ,P2Y 2A 1 ng system (bicyclo [3.1.0] hexane) in place of ribose ,A 1 , Fatma Mufti us in models of chronic neuropathic pain. Furthermore, b ) and 7 P2X receptor subunits (P2X1-7) that comprise an 14 hich reflected both pharmacokinetic and pharmacodynamic Purine receptors belong to a ubiquitous signaling system in the -triphosphate and other adenine nucleotides are released during 9 ate native agonist levels. Purinergic signaling modulators, such as body that has been termed the “purinome” ( active trimeric channel. The P2Xthe and immune P2Y response in receptors response tend toacting to nucleotides, as boost with immediate their ligands danger signals. increase, and ARs decrease, pain signaling. included in this schemea critical as survival a mechanism. beneficial Sub receptors function (ARs), also because known pain assubtypes is P1 (A receptors, of which there are 4 on the immune response,nously and their to activationa functions suppress temporal endoge- sequence pain. ofby nucleotides, first followed This activation by the ofof prol signaling adenosine, proinflammatory mainly P2Rs system formedreleased gradually ATP, by represents at enzymatic the hydrolysis ARs. of P2Y but not always, P2Rcontrolling antagonists pain. and RNA-seq AR (RNA-sequencing) agoniststhat analysis are the has sought expression shown for of many of these genes in the purinome is 5 physiological stress, stemming fromtion, oxygen inflammation, and cancer, nutrient tissue depriva- catabolized injury, to etc., adenosine. The which immediate areactivation response ultimately to by the stress ATP is ofchannels. Each P2X functional receptors P2X ion toor channel heterotrimer open consists of of P2X ligand-gated a subunits. cation homomer Auracil more nucleotides diverse activate set the of 8 adenine(P2YRs), and G- including nucleoside 5 and uridine diphosphate (UDP)-sugars.subtypes There are (P2Y 8 P2Y receptor pain. Adenosine and its analogues potently suppress nociception in ship of nucleoside derivatives at this subtype using a computational or modulation is a promising approach for future pain therapeutics. s in responding to physiological stress. The “purinome” encompasses , Filomena Lauro Rs play critical roles in maladaptive pain b AR) was achieved with the aid of receptor homology models based on related 12 are less 3 -aminobutyric ine, 8 GPCRs activated by nucleotides, 7 adenosine 5 g 26,151 s, MO, United States adenosine receptors (ARs), but safely harness 3 AR agonists for pain control containing a bicyclic ri diseases and pharmacological 3 and/or A Nevertheless, the existing treat- 1 of Medicine, St. Loui 31 10,000-fold selective for A . AR agonists are well tolerated in vivo and highly efficacio , Luigino Antonio Giancotti 3 a Number 00 www.painjournalonline.com AR agonists and antagonists are efficacious in pain models. Highly selective A · 2A Adenosine receptor, P2Y receptor, P2X receptor, Pain, Agonist, Antagonist -triphosphate [ATP]-gated channels) Volume 00 9 ·

2020 International Association for the Study of Pain Keywords: 4 G-protein-coupled receptors (GPCRs) for adenos Abstract Extracellular nucleosides and nucleotides have widespread function been achieved. Both A were designed andparameters. screened High specificity using ( an in vivo phenotypic model, w signaling molecules acting at P2X3, P2X4, P2X7, and P2Y channels, as well as the associated enzymesreceptor and agonists transporters and that antagonists, regul have potential for treating chronic preclinical models by activating A GPCR structures. These A reduce chronic or inflammatory pain, and, therefore, purine recept Structurally novel antagonists for these nucleotide receptors were discovered recently. structure-based approach. Novel A to treat chronic pain. We have explored the structure activity relation Narrative Review Department of Pharmacology and Physiology, Henry and Amelia Nasrallah Center for Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Neuroscience, Saint Louis University School *Corresponding author. Address: Department ofSaint Pharmacology Louis and University Physiology, School of63104, Medicine, United 1402 States. South Tel.: Grand 314-977-6430; Blvd,[email protected] fax: St. (D. 314-977-6411. Louis, Salvemini). E-mail MO address: PAIN 00 (2020) 1–17 © http://dx.doi.org/10.1097/j.pain.0000000000001857 Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, United States, Month 2020 a b Sponsorships or competing interests that mayat be the relevant end to of content this are article. disclosed interventions. Ion channels, G-protein-coupled receptorsand (GPCRs), kinases areHowever, common modulators targetsadenosine for of analgesic 5 purine drug discovery. receptors (either GPCRs or 1. Introduction Chronic pain treatment remains oneneeds of and the also major accompanies unsolved many medical

receptor modulation Kenneth A. Jacobson Treatment of chronic neuropathic pain: purine often considered intreatments: sodium and pain calcium channelacid research, blockers, compared (GABA) toreceptors, modulators, and widely kinase ligands inhibitors. used of opioid and cannabinoid ments are not generallytreatments effective in develop most patients, toleranceliabilities and or the upon current have prolongedeffective in other acute use. pain than serious in For chronicdesensitization, pain side-effect and example, can and lead to opiates addiction, approaches even for are chronic hyperalgesia. neuropathic more pain Thus, are needed. novel treatment Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

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Figure 1. Purinergic signaling pathways for purine nucleosides and nucleotides, and pyrimidine nucleotides. Extracellular ATP and other nucleotides originate from intracellular sources through cell damage, cotransmission, pannexin hemichannels, and other mechanisms. These nucleotides act on P2Y (GPCRs, activated by triphosphates, diphosphates, and UDP-sugars) and P2X (ion channels, mainly by ATP) receptors. Ectonucleotidases (CD39 and CD73) are largely responsible for the formation, from ATP, of adenosine that activates its 4 receptors. In general, adenosine receptor agonists and P2X/P2Y receptor antagonists induce pain relief in various models.

enhanced in the dorsal root ganglia (DRG) and the spinal cord (SC) microenvironment can promote activation of the P2XRs by known (Table 1).146 phenomenon of pH modulation of the receptor protein.39 Many compounds are now available as selective modulators of the purinergic system, either as directly acting agonists and 2.1. P2X3R antagonist antinociceptive effects antagonists of P2Rs (Fig. 2)orARs(Figs. 3 and 4),78 or as inhibitors Both pharma and academic laboratories have maintained a long-term of associated enzymes and transporters that regulate native levels of effort to produce a P2X3R antagonist that might be effective in adenosine (Fig. 4) or nucleotides. Inhibitors of enzymes involved in controlling pain.23,75,165,172 However, there are currently no P2X3 processing of adenosine include the widely used adenosine kinase antagonists in clinical trials for pain, but one is in trials for chronic inhibitors ABT-702 38, a pyridopyrimidine, and the nucleoside A- cough.133 Various P2X3 antagonists have been reported,64 including 134974 39 (Fig. 3).87 Inhibitors of these enzymes and transporters, A-317491 9, which is potent and selective but not orally bioavailable. in some cases, are approved drugs. For example, adenosine This first-reported selective P2X3R antagonist reduces neuropathic, deaminase (ADA) inhibitor, pentostatin (deoxycoformycin) 44,isan inflammatory, and chemogenic pain after intrathecal or intraplantar anticancer drug. Dipyridamole 43, a clinical vasodilator, inhibits administration.128 P2X3R antagonists have been shown to be equilibrative nucleoside transporters 1 and 2 (ENT1/2) and also acts efficacious in reducing chronic pain in animal models of cancer54,93 as a phosphodiesterase (PDE) inhibitor. and in several animal models of neuropathic pain such as chronic constriction injury (CCI) of sciatic nerve,152 spinal nerve injury,66 partial ligation of the unilateral infraorbital nerve,154 chronic pancreatitis,188 2. P2XR ligands and their use in relieving chronic pain maternal separation,198 and inflammatory pain.86,90 Recently, nucle- 15 otide derivatives with the ribose 29,39-hydroxyl groups in a chemically Various P2XR antagonists have shown efficacy in pain models. 43 For example, involvement of P2XRs has been explored in protected state were reported to be P2X3R antagonists, eg, 10. 106,169 Upregulation of various purine receptors in pain pathways is acupuncture analgesia and central pain syndrome. In 122 neuropathic pain, adrenomedullary chromaffin cells overexpress a consequence of the chronic pain state. Peripheral inflammation P2X3 and P2X7.5 Early studies of the mechanism of pain is associated with plasticity of purinergic signaling within sensory induction by ATP focused on the observation that the P2X3R ganglia. The P2X3R in the DRG and trigeminal ganglia (TG, responsible and P2X2/3R are localized and function in neurotransmission at for sensation in the face and cranium and motor functions in the mouth) are located on the neurons, at the presynaptic or postsynaptic the DRG (Table 1), the main conduit for pain signaling by the 25,134,138,157,186 peripheral nerve nociceptors.36,111 Adenosine 59-triphosphate is terminals. Neuronal plasticity leading to upregulation of produced by inflammatory cells, tumor cells, endothelial cells, the P2X3R on these ganglia has been observed in conditions of inflammation.48 Novel P2X3R antagonists such as 11 were studied in sympathetic neurons, and Merkel cells in the skin and other cells. 92 A local elevation of the extracellular levels of ATP can act to models of chronic pain. A methyl ester prodrug derivative seems to stimulate peripheral sensory nerves. It was demonstrated that be cleaved in vivo to form the active carboxylic acid. clorodonate, a first-generation bisphosphonate acting as a ve- 2.2. P2X4R antagonist antinociceptive effects sicular ATP release inhibitor, was able to attenuate hyperalgesia in rodent models of carrageenan- and complete Freund’s adjuvant P2X4R, P2X7R, and P2Y12R are upregulated in chronic pain states (CFA)-evoked inflammatory pain through inhibition of vesicular leading to hypersensitivity of the microglia and associated neurons.63 ATP release, as well as in a partial sciatic nerve injury model of This is a function of peripheral nerve injury, but not peripheral tissue chronic neuropathic pain.97 Furthermore, the acidification inflammation. P2X4R activation has an additional consequence of the that accompanies both inflammation and the hypoxic tumor release of brain-derived neurotrophic factor (BDNF) from microglial

Table 1 Table 1 (continued) Message for purinergic receptors and selected associated DRG SC enzymes and a transporter in DRG and SC, expressed as ENTPD1 7.077 23.276 transcripts per million (TPM) using RNA-seq data.146 NT5E 6.377 5.71 DRG SC ENPP1 1.307 0.683 A. Mouse gene ADK 61.783 207.371 ADORA1 96.424 143.604 SLC29A1 29.553 23.028 ADORA2A 6.218 1.342 Other names for enzymes listed below are: ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1, ADORA2B 0.969 6.501 CD39), NT5E (ecto-59-nucleotidase, CD73), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), SLC29A1 (equilibrative nucleoside transporter 1, ENT1). ADORA3 0.01 1.411 SC, spinal cord; DRG, dorsal root ganglion. P2RY1 25.792 10.238 P2RY2 29.923 0.521 cells, which act upon neighboring neurons to interfere with the P2RY4 0.01 0.01 GABAergic antinociceptive system. Fibronectin acts upon pain- activated microglia to upregulate the expression of P2X4.12,127 BDNF P2RY6 2.968 4.65 binds to neuronal tropomyosin receptor kinase B (TrkB) in spinal P2RY12 3.069 11.464 lamina I to interfere with K1-Cl2 cotransporter (KCC2), which P2RY13 0.343 4.303 normally maintains a low level of intracellular chloride in the SC neurons.42,110 Consistent with a lack of microglial role in the P2RY14 1.383 1.862 development and maintenance of neuropathic pain in females, this P2RX1 0.091 0.01 mechanism was found to be essential only in male mice156 because P2RX2 2.332 0.37 the P2rx4 gene was upregulated only in dorsal horn of male, but not female mice, after peripheral nerve injury.46,94,147,156 Despite P2RX3 120.479 1.932 qualitative sex differences in neuroimmune signaling after nerve P2RX4 48.646 27.775 injury, neurons in males and females ultimately experience a similar P2RX5 26.276 13.083 reduction in KCC2 function, leading to comparable levels of pain 126 P2RX6 40.086 15.987 hypersensitivity. The rise in intracellular chloride ions as a result of macrophage P2X4R activation diminishes the ability of drugs that P2RX7 23.197 5.529 directly or indirectly activate GABAA ion channels, to reduce chronic ENTPD1 9.469 96.8 pain. Therefore, if a clinical P2X4R antagonist were to be made NT5E 7.621 2.776 available, it might make the existing pain medicines more effective as well as have its own antinociceptive activity. Furthermore, a peripheral ENPP1 6.814 8.653 action of P2X4R antagonists on macrophages reduces the release of ADK 422.075 531.732 prostaglandin E2 (PGE-2) by a p38-mitogen-activated protein kinase SLC29A1 93.294 57.227 (MAPK)-dependent mechanism, which acts as a pronociceptive signal.179 Therefore, both spinal and peripheral sites of action of such B. Human gene antagonists could contribute to an analgesic effect.26 ADORA1 16.475 48.6 Thus, P2X4R and P2X7R antagonists have been considered for ADORA2A 2.237 8.659 pain control through their action on spinal microglial and other cells. Pharmaceutical companies and academic laboratories have de- ADORA2B 0.847 1.49 veloped numerous compounds to antagonize these receptors that ADORA3 65.881 33.548 are drug-like molecules and well tolerated in the body (Fig. P2RY1 1.275 0.621 2).34,44,137,168 PSB-12062 12 is a P2X4R antagonist of mM affinity 161 P2RY2 1.484 0.838 (human 1.38 mM, rat 0.928 mM, and mouse 1.76 mM), but it has low aqueous solubility. P2X4R knockout (KO) mice displayed normal P2RY4 0.01 0.124 acute pain responses and tissue damage-induced pain, but P2RY6 3.889 1.924 reduced chronic pain, especially tactile allodynia.181 P2X4R P2RY11 15.252 15.424 antagonists have been shown to be efficacious in reducing chronic pain in diabetes-induced neuropathic mechanical hyperalgesia P2RY12 23.365 10.179 (PSB-15417, structure not shown)170 and in a model of trigeminal P2RY13 4.851 3.693 allodynia.117 IgG#191 is a potent and selective antibody binding the P2RY14 6.429 2.638 head domain of P2X4 and able to reverse mechanical hyperalgesia in a mouse model of neuropathic pain induced by the Seltzer partial P2RX1 0.575 2.017 sciatic nerve ligation (PSNL). IgG#191 does not cross the blood– P2RX2 0.115 0.093 brain barrier (BBB) but a chimeric construct containing a domain that P2RX3 53.911 0.01 can be transported across the BBB into the central nervous system (CNS) showed dose-dependent reversal of mechanical hyper- P2RX4 21.389 24.89 algesia, indicating that with appropriate molecular modification, P2RX5 36.934 1.334 IgG#191 could access the SC from the periphery.190 The selective P2RX6 22.026 11.886 P2X4R antagonist NP-1815-PX 13 displayed antiallodynic effects in P2RX7 1.464 18.807 traumatic nerve damage and herpetic mouse models without affecting acute pain; however, it does not readily cross the BBB.127

Figure 2. Structures of representative P2YR and P2XR antagonists that have been used in pain studies.

NP-1815-PX was discovered as a result of a screen of chemical at Kyushu Univ. and academic and pharma collaborators (Nippon libraries and displays high aqueous solubility. NC-2600 (structure not Chemipharma) and entered a phase 1 clinical trial for chronic disclosed) was developed in a collaboration between Prof. K. Inoue neuropathic pain in Japan, in June 2016.

Figure 3. Structures of representative AR agonists that have been used in pain studies.

2.3. P2X7R antagonist antinociceptive effects cancer pain by releasing proinflammatory cytokine interleukin-18 (IL-18).197 However, there have been clinical trials for P2X7R Various P2X7R antagonists have been reported to be active antagonists in inflammatory diseases, cancer, and depression. 30 against pain and inflammation. P2X7R antagonists A-438079 JNJ 47965567 16 is a brain penetrant P2X7R (human, mouse, 14 and A-740003 15 have been shown to be efficacious in and rat) antagonist of nearly nM affinity. Several positron- 70,136 reducing chronic neuropathic and inflammatory pain. emission tomography ligands (17, 18) for brain imaging of the Furthermore, the more potent antagonist A-740003 is highly P2X7R have been reported. P2X7R antagonist GSK1482160 19 P2X7R-selective in both human and rodent species. Microglial developed to target peripheral pain and has been in a clinical trial P2X7R activation contributes to maintaining advanced bone for inflammatory pain (Table 2). It was also radiolabeled with 11C

Figure 4. Structures of representative AR antagonists that have been used in pain studies.

57,191 for positron-emission tomography imaging. AZ 11645373 P2Y1R, P2Y12RandP2Y13R. UDP is the principal endogenous 20 is a highly selective human P2X7R antagonist but does not agonist of P2Y6R. UDP-glucose is the principal endogenous bind to rodent P2X7Rs. agonist of P2Y14R. Pannexin-1 (Panx1) hemichannels are permeable to small molecules (,1 KD) and are the source of much extracellular ATP in the SC subsequent to morphine treatment, which can activate 3.1. P2Y R antagonist antinociceptive effects P2XRs.24 Adenosine 59-triphosphate originating in spinal micro- 1 glia in the dorsal horn contributes to long-term synaptic facilitation P2Y1R antagonists have been shown to be efficacious in that is characteristic of opioid withdrawal. Pharmacological reducing chronic and acute pain in several animal models such inhibition of ATP breakdown exacerbates opioid withdrawal, as neuropathic pain model of spinal nerve ligation (SNL), CCI, suggesting its detrimental action occurs through P2Rs, although spared nerve injury (SNI),9 formalin and carrageenan-induced a potential beneficial effect of adenosine produced from ATP inflammatory pain,8,109 visceral hypersensitivity in rats with hydrolysis was not addressed. experimental irritable bowel syndrome,193 cancer-induced bone pain,37 and migraine.123 The different cell types involved include: dorsal root, trigeminal, nodose sensory neurons, SGCs,145 and in 3. P2Y receptor ligands and their use in relieving the SC, astrocyte,58 and some of the dorsal horn neurons.101 chronic pain Potent and selective P2Y1R antagonist MRS2500 2 reduces Certain P2Rs present on the DRG and on the associated peripheral interleukin-1b (IL-1b)-mediated thermal hypersensitiv- 122 109 satellite glial cells (SGCs) are upregulated in pain states. The ity. P2Y1R activation stimulates visceral nociceptors, an effect receptors that are upregulated or activated in sensory neurons, blocked by MRS2500.67 The upregulation of neuronal purine such as the DRG and sciatic nerve, include: neuronal and glial receptors in pain states can be interrupted by receptor P2Y1R, P2Y2R, P2Y6R, P2Y11R (all Gq-coupled) as well as antagonists. For example, MRS2500 reduced upregulation of 8,132,207 32 P2Y12RandP2Y14R(Gi-coupled). Adenosine-59-di- P2Y1R in DRG induced by nerve injury. Also, the P2Y1R phosphate (ADP) is the principal endogenous agonist of intensifies the thermal hyperalgesia mediated by transient

Table 2

Representative recent clinical trials of AR agonists, an A1AR PAM, and P2R antagonists in pain conditions (data from clinicaltrials.gov, accessed May 8, 2019). Receptor Condition Compound (route) Years Phase, NCT# References

A1 Neuropathic pain Adenosine 2014-2018 2, 00349921 208 (i.t.) Perioperative pain Adenosine 2006 2, 00298636 91 (i.v.) Neuropathic pain GW493838 2002-2003 2, 00376454 47 24 Postherpetic neuralgia T-62 2008-2012 2, 00809679 129 40

A2A Diabetic nerve pain BVT.115929 2007-2014 2, 00452777 100 27 Diabetic foot ulcers Sonedenoson 2006-2012 2, 00318214 131 28

A2B None — — 164

A3 None — — 85 P2X4 Neuropathic pain NC-2600 2016-2017 1 www.chemiphar.co.jp P2X7 Inflammatory pain GSK1482160 2009-2017 1, 00849134 57 19 The only ongoing clinical development is P2X4R antagonist NC-2600 (unpublished). PAM, positive allosteric modulators.

receptor potential cation channel subfamily V member 1 (TRPV1) hypotheses in humans that are not yet feasible for most other in peripheral nerves, through its protein kinase C-dependent purine receptors. However, a liability of P2Y12R antagonists phosphorylation, leading to a pronociceptive TRPV1 as shown in would be the potential for increased bleeding. Currently, there is 108 an ischemic model. This process was blocked by P2Y1R no basis to separate the two-mechanism based actions of antagonist MRS2179 1. P2Y12R antagonists, ie, analgesic and antithrombotic effects. Numerous P2Y12R antagonists and prodrug inhibitors have been developed as antithrombotics. Among the clinically accepted 3.2. P2Y12R antagonist antinociceptive effects antagonists of the P2Y12R, cangrelor (ARC-69931MX 6)is

The Gi-coupled P2Y subfamily consists of 3 subtypes: P2Y12R, a competitive P2Y12R antagonist of sub-nM affinity. However, P2Y13R, and P2Y14R. P2Y12R already has antagonists that are used as a nucleotide analogue, it is not orally bioavailable, but it is active clinically for the prevention of thrombosis. This receptor is considered upon injection in vivo. Ticagrelor 7 is also a competitive P2Y12R a primary sensor of ATP and ADP in vivo, is present on neurons of antagonist, but it additionally inhibits ENT1, thus raising the levels peripheral sensory ganglia, and its presence in SGCs is induced in of endogenous adenosine. Other P2Y12R antagonists, eg, 96 pain states. AneuronalP2Y12R is also involved in excitability. 3 thienopyridines clopidogrel and prasugrel (structures not shown), days of successive administration of uncharged P2Y12R antagonist used widely in the clinic act noncompetitively by binding to the MRS2395 8 reversed glial cell activation in rat SGCs of the TG and receptor after preactivation in the body; thus, they are prodrugs. 124 alleviated mechanical and heat sensitivity of the tongue. Also, MRS2395 8 is a weak, uncharged P2Y12R antagonist that has 96,116,167 PSB-0739 5, a competitive, polyanionic antagonist of P2Y12Rhas been used in pain studies. proven useful in studies of pain.71 The microglial cells are the resident macrophage-like immune cells in the brain and SC, accounting for 3.3. P2Y R antagonist antinociceptive effects roughly 10% to 15% of the total cell numbers. Naturally, purine 6 receptors have prominent roles in the function of microglial cells. In Other microglial P2YRs that have a role in pain are P2Y4R, P2Y6R, 122 the SC, the microglial cells tend to amplify the pain signals by and P2Y13R. P2Y6R serves to induce phagocytosis in micro- 103 producing proinflammatory cytokines and other nociceptive mod- glial cells. Satellite glial cells express P2Y14R, which is ulators. Potential mechanisms have been described for purine associated with inflammatory cytokine release.113 There are few receptors on activated microglial cells to induce hyperexcitability in known antagonists of the P2Y6R, but agonist structure–activity the spinal dorsal horn, to increase neuropathic pain.146,180 relationships (SAR) has been extensively explored. MRS2578 3 is P2Y12R antagonists have been shown to be efficacious in a potent, but hydrophobic and likely noncompetitive P2Y6R reducing chronic and acute pain in the CFA-induced inflamma- antagonist, and the more recently reported TIM-38 4 is a weak 77 tory pain model, in some models of neuropathic pain induced by antagonist. P2Y6R activation suppresses the P2X4R-induced PSNL,71 SNI,102 and spinal nerve transection,63 in cancer- current amplitude, activation, and channel permeability in micro- 116 induced bone pain and in some orofacial pain models (tongue glia, suggesting that a P2Y6R agonist might provide therapeutic cancer pain167 and neuropathic tongue pain96,163). This likely benefit.14 However, a recent article reports the opposite effect involves action at multiple cell types: neurons, microglia, and based on a protective effect elicited by P2Y6 antagonist 163,178,177,189 satellite cells of the dorsal and TG. P2Y12R serves as MRS2578 but not P2Y6 agonist UDP in a model of neuropathic a find-me signal for microglial cells, ie, it induces motility.69 pain. In particular, intraperitoneal administration of MRS2578 Because P2Y12R antagonists are already in widespread use in alleviated CCI-induced hyperalgesia. Conversely, treatment of the clinic, there are opportunities for testing purine-based UDP on CCI rats increased pain intensity.16

3.4. P2Y14R antagonist antinociceptive effects unclear, because the same compound tested clinically, ie, A1AR PAM T-62 40, produced significant benefit in extensive preclinical P2Y R is widely expressed throughout the body,33 and it is 14 tests of efficacy in pain and related conditions. The side effects, in found in multiple parts of the nervous system.132 Its mRNA is general, may or may not be AR mechanism-related. Therefore, it expressed in various immune cells as well as glia and neuronal may not be appropriate to rule out testing any new potential cells,33,132 therefore P2Y R is speculated to play a role in 14 analgesic agents acting at the A AR or the A AR, simply based neuroimmune responses. Evidence supporting this was ob- 1 2A on the unsuccessful trials so far. However, it must be noted that served after acute challenge of rats with lipopolysaccharide A AR or A AR agonists would likely display cardiovascular side where an increase in P2Y R mRNA expression across various 1 2A 14 effects that would limit the dose range and utility of these agents in regions of the brain occurred.35,132 In addition, P2Y R seems to 14 the general population. Only 2 AR agonists have been approved be involved in chronic pain. One study observed increase in for clinical use, but not for pain: adenosine itself and regadenoson P2Y R mRNA levels in the dorsal horn of SC from days 3 to 14 14 31, but both lack oral bioavailability. Several nonselective AR after an SNI.102 Moreover, the in situ hybridization of dorsal horn antagonists are used clinically, but the only selective AR sections showed that expression of P2Y R mRNA was elevated 14 antagonist currently approved for human use is istradefylline selectively on the ipsilateral side and that it colocalized with Iba1, 51.20 Nonselective AR antagonist caffeine 47 is sometimes which indicates that the upregulation happened in microglia.102 present in over-the-counter pain medications. The same study found that the administration of antisense-locked A AR KO mice (A AR2/2) were used to evaluate the role of the nucleic acid (AS-LNA) to inhibit P2Y R for 7 days reversed SNI- 1 1 14 A AR in nociception. Under normal conditions, as well as during induced mechanical allodynia on days 5 and 7.102 The P2Y Ris 1 14 inflammatory or neuropathic pain, A AR2/2 animals showed expressed on both neurons and SGCs in the rat TG.113 Its 1 hypersensitivity to heat in comparison to the wild-type (WT) mice. upregulation in TG was observed in an inflammatory pain model No significant differences were found in terms of mechanical (CFA-induced pain) and that was associated with increased withdrawal threshold and cold response. A AR2/2 mice also release of anti-inflammatory cytokines and the activation of MAP 1 showed reduced antinociceptive effect of morphine given kinases.114 In addition, using PPTN, which is a selective P2Y R 14 intrathecally, but not systemically.192 The basis for the analgesic antagonist, attenuated mechanical hyperalgesia in this inflam- actions of A AR activation is multifold, as reviewed by Sawy- matory pain model.114 1 nok.151 A AR is highly expressed in the nervous system, including Functional modal shifts of purine receptors occur upon 1 on the central terminals of primary afferent neurons of the SC and microglial activation: P2X4R and A AR are upregulated, and 2A cell bodies in the dorsal horn. A AR activation produces P2Y R, A AR, and A AR are downregulated.104 Thus, when 1 12 1 3 presynaptic inhibition of primary afferent neurotransmission onto considering the application of purinergic modulation to chronic dorsal horn neurons by decreasing release of glutamate, neuropathic pain, it is important to consider the relationships in substance P, and other transmitters from primary afferents. the pathological state, as distinct from the normal state of A AR also hyperpolarizes dorsal horn neurons by increasing K1 a tissue. 1 conductance and potentiates the inhibitory postsynaptic trans- 7 mission mediated by glycine receptors. A1AR activation on 4. Adenosine receptor ligands and their use in nociceptive neurons by adenosine generated from locally re- relieving chronic pain leased ATP has also been suggested as a mechanism of action of acupuncture.61 Consistently, it is reported that caffeine, a non- The ARs are activated by endogenous adenosine, the ubiquitous selective AR antagonist, and 8-cyclopentyl-1,3-dipropylxanthine agonist produced by all cells. Adenosine receptors activity can (DPCPX 49), a selective A1AR antagonist, reduce the antinoci- also be modulated by exogenous agonists and antagonists or by ceptive effect of acupuncture.55,205 Central or peripheral inhibition of proteins that control adenosine transport, formation, administration of A AR agonist CPA 21 in the mouse improved 3,32 1 and degradation. Adenosine receptors agonists and antag- mechanical allodynia resulting from diabetic neuropathy.98 Data onists can be competitive with adenosine by binding to the same on A1AR antagonists or A1AR KO mice in diabetic neuropathy common site conserved among the 4 ARs (the orthosteric binding models have not been reported. N6-cyclopentyladenosine (CPA) site), or they can interact noncompetitively by binding to showed the ability to attenuate the mechanical hyperalgesia a separate, allosteric site (Fig. 3). Each of the 4 ARs has induced by PGE2, but when injected repeatedly, it causes numerous selective orthosteric agonists and antagonists, and tolerance, dependence, and changes in nociceptor function 88 their ligand binding SAR have been reviewed elsewhere. producing mechanical hyperalgesia similar to the effect of Extensive molecular modeling of the ARs has also been 4 m-opioid receptor agonists. Downregulation of A1AR contributes performed based on X-ray structures of the A2AAR and more to neuropathic pain in a mouse model of neuropathy induced by 60 recently of the A1AR. This modeling is informative in interpreting resiniferatoxin, a capsaicin analogue that acts on TRPV1.95 the SAR at any given subtype and guiding the discovery of new Luongo et al.120 demonstrated the analgesic properties of an AR ligands.88 Allosteric enhancers, ie, positive allosteric modu- A1AR agonist in a mouse model of neuropathic pain (SNI) that lators (PAMs), for the A1AR and the A3AR have been well were antagonized by the A AR-selective antagonist DPCPX. The 81 1 characterized pharmacologically. agonist used was Cl-(6)-ENBA 3, which is particularly potent and 29 selective (.3000-fold selective in binding to the mouse A1AR). However, Cl-(6)-ENBA is a mixture of 2 diastereoisomers, each 4.1. A AR antinociceptive effects 1 of which can have distinct pharmacokinetic and pharmacody- A number of clinical trials of potential analgesic agents acting as namic properties. An A1AR agonist of similar affinity, selectivity, 176 agonists at the Gi/o-coupled A1AR (eg, GW493838 24) and the and in vivo activity is MRS7469 26, which is a pure isomer. A Gs-coupled A2AAR (eg, BVT.115959 27 and sonedenoson 28) caveat in the use of A1AR agonists in vivo is that even compounds have been performed in recent years (Table 2 and Fig. 3).208 that have only 100 seconds of fold selectivity in binding to the However, all the trials were discontinued due to lack of efficacy or A1AR compared to A2AAR and A3AR can display nonselective the presence of side effects. The reason for the lack of efficacy is activity at the latter subtypes. For example, Reitman et al. found

that A1AR agonist CPA 1 activates mast cell A3AR to produce mice. This effect was prevented by caffeine and ZM241385 53, 27 hypothermia in mice at the same modest doses (eg, 0.3 mg/kg, aspecificA2AAR antagonist. In another study, LASSBio-1359 intraperitoneal [i.p.]) at which a central A1AR activation was 32,anA2A AR agonist of atypical structure, inhibits the hyperalgesic seen.29 Thus, it is essential to use antagonists to probe the response caused by acute and chronic inflammation induced by receptor subtype selectivity of an assumed selective agonist in formalin, carrageenan, or CFA injection.130 a given model. The use of AR subtype KO mice also can strengthen the conclusion that a particular AR subtype is 4.3. A AR pronociceptive effects involved. 2A Paeoniflorin 45, a natural phytochemical and a component of A2AAR activation might enhance the release and stimulatory Chinese traditional medicine, protected against mechanical and effects of excitatory neurotransmitters such as glutamate.153 It thermal pain in the PSNL mouse model at high doses. This has been demonstrated that KO mice lacking the A2AAR are less protection was blocked by moderately selective A1AR antagonist sensitive to nociceptive stimuli; this is due to a large reduction in 199 73 CPT 48 and in A1AR KO mice. However, the mechanism of the density of NMDA glutamate receptors as well as a reduced 74 A1AR activation by paeoniflorin was not established. Neuropep- neuronal activity in the SC. In A2AAR KO mice, carrageenan- tide S injected intracerebroventricularly significantly reduced induced hyperalgesia was significantly reduced compared to WT formalin-induced nociception during both phases of the formalin controls; furthermore, a selective A2AAR antagonist ZM241385, test by activating both A1AR and A2AAR during phase 1, but only when injected into the hind paw, reduced the mechanical 68 112 the A2AAR during phase 2. UNC32A 25 is an A1AR agonist hyperalgesia in female mice, but not in males. A2AAR KO mice structurally related to adenosine 59-monophosphate (59-AMP), also showed a significant decrease of mechanical allodynia in a nucleotide able to activate A1AR. UNC32A did not show a model of neuropathic pain induced by sciatic nerve injury when cardiovascular side effects and caused dose-dependent anti- compared to the WT group.22 nociceptive effects in WT mice when administered orally.105 In acetic acid-induced writhing and tail immersion tests in the Allosteric modulation of the ARs is one means of potentially mouse, a series of novel A2AAR-selective antagonists adminis- 76,184 183 avoiding their cardiovascular side effects. A1AR PAM tered i.p. decreased acute pain. These antagonists were TRR469 42, an aminothiophene, was antiallodynic in a chronic described as promising for the treatment of chronic pain as well. neuropathic pain model without locomotor or cataleptic side In another study, the A2AAR agonist CGS21680 induced effects. It attenuated nociceptive responses in the formalin and mechanical sensitization of esophageal C fibers, which was writhing tests, similarly to morphine’s effects. Coadministration of abolished by pretreatment with selective A2A antagonist 21 TRR469 and an A1AR agonist CCPA 2 produced additive SCH58261 54. Moreover, thermal and chemical stimuli, protection in a pain model. The effects of coadministered CCPA induced by hot plate and acetic acid, respectively, showed that and TRR469, or each agent alone, were antagonized by PSB36 an i.p. injection of the agonist CGS21680 produces pronocicep- 11 50, a highly A1AR-selective antagonist. The antinociceptive effect tive effects. Therefore, although most studies showed analge- of TRR469 was additive with those of morphine. VCP171 41,an sic effects, it seems that A2AAR activation might in some cases A1AR PAM of the same chemotype as 40 and 42, reduced the intensify response to nociceptive stimuli. The role of A2AAR in pain evoked excitatory postsynaptic current amplitude in the SC transmission needs to be explored further. dorsal horn to a larger degree in the nerve-injured state than in A few studies have examined the involvement of the A2BAR in control rats, suggesting that an elevated adenosinergic tone in pain; however, it is known that activation of the A2BAR has both 76 164 that tissue would enable the use of A1AR PAMs in pain therapy. proinflammatory and anti-inflammatory effects. The lack of studies in pain is partly a result of the fact that selective agonists for this subtype are not as plentiful or well-characterized as 4.2. A AR antinociceptive effects 2A agonists of the other ARs. Nevertheless, a study of ADA KO mice The role of the A2AAR in pain is somewhat controversial in that showed that prolonged increase in plasma adenosine activates pronociceptive and antinociceptive effects have been docu- A2BAR on myeloid cells that results in increased sensitivity and 72 mented. A2AAR activation as well as A1AR activation can reduce chronic pain. Furthermore, the A2BAR-selective adenosine the release of excitatory neurotransmitters such as glutamate.62 receptor antagonists PSB-1115 56, PSB-50, PSB-53, PSB-55, Watkins et al. showed that selective A2AAR agonist ATL-313 30 and enprofylline (structures not shown) displayed a dose- infused in the spinal column decreased chronic pain in a manner dependent analgesic effect in the hot-plate test in mice, an acute 2,72 that was long-lasting, and repeated administrations in four-week animal pain model. The selective A2BAR antagonist PSB-603 intervals remained efficacious.119 They also demonstrated that 57 has also proven useful as a pharmacological probe of this AR a single intrathecal injection of A2AR agonists CGS21680 29 or subtype. Although limited, such data suggest a pronociceptive ATL313 administered between 1 and 7 weeks after SC injury role of this receptor. reversed spinal neuropathic avulsion pain for up to 6 weeks.107 A single i.p. injection of CGS21680 was also able to significantly 4.4. A AR antinociceptive effects decrease the formalin-induced licking behavior in both the early (0- 3 15 minutes) and late (15-60 minutes) phase in a mouse model of Until recently, the dogma was that the pain-relieving effects of 135 formalin-induced inflammatory pain. Furthermore, a single adenosine were mediated predominantly by actions at the A1AR 208 peripheral injection of the potent but nonselective A2A-agonists, and perhaps at the A2AAR. These conclusions were made 85 5’-(N-ethyl)-carboxamido-adenosine and 2- without examining the contributions of the Gi-coupled A3AR. A phenylaminoadenosine, induced a decrease in mechanical noci- focus on the A1AR and A2AAR failed to harness the potent ceptive threshold in the hind paw of the rat that was antagonized by analgesic effects of adenosine.18,208 Thus, despite demonstrated 166 an A2AAR antagonist, PD 081360-0002 (HTQZ, 52). Intra- preclinical efficacy in several pain models, agonists of A1AR and cerebroventricular injection of adonis, an agonist-like monoclonal A2AAR have not been the focus of clinical trials due to significant 208 antibody with high specificity for the A2AAR, led to a significant cardiovascular side effects. A3AR mRNA is predominantly dose-dependent increase in hot-plate and tail-flick latencies in expressed in human and rodent testis, lung, kidney, placenta,

heart, brain, spleen, liver, uterus, bladder, jejunum, proximal Between 2004 and 2006, the early literature examining the role 19,146 colon, eye, DRG, and SC (Table 1). Early in the study of the of A3AR in pain was confounded by results from 3 contradictory 150,206 A3AR, soon after its cloning in 1992 to 1993, it was thought articles gathered from A3AR-targeted compounds with poor 148 6 that the A3AR was absent in the brain. However, subsequent specificity, e.g., N -benzyl–NECA (structure not shown) or from 2/2 85 studies established its neuronal expression in the thalamus, a single study performed in A3AR mice. In 2007, we hypothalamus, cortex, cerebellum, and other brain regions in revisited the A3AR hypothesis and demonstrated in a series of 195 rodents. Based on exonal RNA sequencing, in human studies using pharmacological tools and genetic A3AR KO mice 2/2 cadaver-derived tissue, A3AR mRNA is highly expressed in (A3AR ) that activation of A3AR exerts potent antinociceptive testes, SC, substantia nigra, adrenal gland, spleen, small effects in models of traumatic nerve-injury induced neuropathic intestine, amygdala, hypothalamus, tibial nerve, tibial nerve, and chemotherapy-induced neuropathic pain and bone cancer hippocampus, bladder, lung, adipose tissue, whole blood, pain38,53,115,160,175,187 validating the observations in models of transverse colon, and coronary arteries.80 When compared to non-neuropathic pain states.200 Pharmacological probes used A1R and A2AAR, A3AR is expressed at much lower levels in the included moderately selective agonists IB-MECA 33, Cl-IB- 20 CNS. However, A3AR has higher expression on many immune MECA 34, and highly selective A3AR agonists such as cell types, including glial cells.1,139,144 In rodents, it is expressed MRS5698 and MRS5980, as well as an antagonist for the mouse in astrocytes, neurons, oligodendrocyte precursor cells, newly A3AR, MRS1523 60. It is to be emphasized that human A3AR formed oligodendrocyte, myelinating oligodendrocyte, microglia/ antagonist MRS1220 59 is not selective when applied in rodent 203 macrophage, and endothelial cells. In humans, it was found in species. Noteworthy, A3AR agonists do not interfere with fetal and mature astrocytes, oligodendrocyte, microglia/ antitumor effects of widely used chemotherapeutics but instead macrophage, and endothelial cells.204 are themselves antitumor agents.6,40,52,49,51,182 Cl-IB-MECA is 149 A3AR can be found on both peripheral and central currently in phase II clinical trials for hepatocellular carcinoma as 59,79,118,202 19,65 28,142,159 neurons of the brain and SC. In pain-processing an anticancer agent. Therefore, the use of A3AR centers in rodents, A3AR transcript and protein have been agonists may provide dual benefits in the treatment of a variety identified in the lumbar SC and rostral ventromedial medulla of cancer-related pain states. 115 (RVM). The A3AR is significantly upregulated in settings of In addition to traumatic-nerve injury and chemotherapy- 38,53,115,160,171,175,187 inflammation and cancer in numerous cell types and both in induced neuropathic pain, A3AR agonists rodents and humans19,51,80,139; noteworthy activation of phos- are effective in formalin-induced inflammatory pain,141 breast phoinositide 3-kinases (PI3K), cAMP response element-binding cancer bone metastasis,182 and diabetic neuropathy.196 protein (CREB), and nuclear factor-kB (NFkB) plays critical roles The beneficial effects of A3AR agonists are exerted in the 40 in A3AR transcription. The intracellular signaling pathways peripheryandintheCNS(SCandRVM);wehaveproposedthat associated with the A3AR include the inhibition of adenylate their high degree of potency may be the outcome of their 85 cyclase through the Gi protein and also activation or deactivation potential synergy at multiple pain transmission sites, just like of a wide range of kinases, such as MAPK, extracellular signal- opioids.17 We are currently examining this possibility. More- regulated kinase (ERK), p38 mitogen-activated protein kinase over, A3AR agonists exert their effects through multiple (p38), jun N-terminal kinases (JNK), PI3K, protein kinase B (AKT), molecular mechanisms; we postulate that such polypharma- and glycogen synthase kinase 3b (GSK3b).80 These kinases and cology may also account for their potency and efficacy. In the transcription factors that they modulate are relevant to neuropathic pain models where nerve damage is induced by cancer, cell survival and proliferation, as well as CCI or chemotherapy treatment (oxaliplatin), A3AR agonists 89,125,143,155,194,201 pain. In general, A3AR agonists tend to restore exert their beneficial effects by suppressing spinal microglial a balance in signaling that is subject to pathological deviation in and astrocytic activation, respectively.84,171,187 In animal disease states, such as cancer and autoimmune inflammatory models of chemotherapy-induced neuropathic pain, A3AR diseases.80 This generality applies to chronic pain states as seem to be disease-modifying, i.e., achieving not only well.84 symptomatic relief, but also reversing some of the pathological The development of highly selective A3AR agonists such as processes in pain signaling pathways that contribute to vicious 173,174 38,52,84,115,187 MRS5698 35 and MRS5980 37 provide exquisite tools to cycles. A3AR signaling inhibits neuropathic pain now probe the mechanistic roles of the A3AR axis in pain; their by attenuating the production of proinflammatory cytokines selectivity for A3AR is .1000-fold when compared to A1AR and such as tumor necrosis factor and IL-1b, and increasing 173,174 A2AAR. The high affinity and selectivity of MRS5698 was formation of the anti-inflammatory interleukin-10 (IL-10) and observed for several species, with Ki values of 3 nM at both interleukin-4 (IL-4) in the SC.84,187 The functional role of IL-10 in 175 human and mouse A3ARs. MRS5841 16, another member of the beneficial effects exerted by A3AR has been estab- 140 10,187 the 2-arylethynyl series, was selective at both human and lished. A3AR agonists can also inhibit activation of MAPKs mouse A3ARs and was excluded from diffusing across biological (p38 kinase) and of NFkBinSCinresponsetonerve 83,99,171 membranes. Thus, MRS5841 was a useful tool in separating injury. Noteworthy, A3AR agonists have been reported effects of A3AR activation in the peripheral and the CNS because to inhibit MAPKs, NFkB activation, and release of inflammatory of its permanently charged sulfonate group. Another means of cytokines in models of inflammation (ie, rheumatoid arthritis) distinguishing classes of GPCR agonists is according to their and in cells and tissues harvested from patients with preferred signaling pathway, which is potentially a means of rheumatoid arthritis, psoriasis, and Crohn disease.40 reducing side effects. Several studies have addressed the A3AR agonists modulate redox-dependent events in the SC, question of biased A3AR agonists. Although a high degree of reducing the activation of nicotinamide adenine dinucleotide bias has not yet been found, some agonists tend to favor the Gi- phosphate (NADPH) oxidase and avoid the posttranslational dependent pathway over the b-arrestin pathway.56,162 These modification and inactivation of glutamate transporter glutamate ligand tools, some of which are now commercially available, are transporter-1 (GLT-1) and glutamine synthetase, which regulate 83 anticipated to further our understanding of the role of the A3AR synaptic glutamate homeostasis. Also, inhibitory neurotrans- axis in pain states. mission can be triggered by A3AR agonists. In a model of CCI,

115,204 both IB-MECA and MRS5698 were able to reverse neuropathic pathways. Moreover, A3AR agonists produce conditioned pain through a spinal mechanism of action that modulates GABA place preference only in nerve injured rodents, with no effects in 53 activity directly or through KCC2 function. An in vivo study sham animals, suggesting that A3AR agonists provide relief of 115 conducted on sensory DRG neurons isolated from rats revealed spontaneous pain. Therefore, A3AR agonists have the A3AR expression in DRG neurons and showed that A3AR potential to selectively modify pathological but not protective agonists Cl-IB-MECA and the highly selective MRS5980 are able pain, while avoiding the tolerance and abuse potential associated 21 41 85 to inhibit Ca currents evoked by a voltage-ramp protocol. The with opioid therapy. To this end, antinociceptive effects of A3AR pain-relieving effects observed after A3AR activation in the rat agonists persists even with prolonged treatment (at least up to 2 could be mediated by DRG neurons, which underscores the weeks of treatment),115,160,187 suggesting that tolerance does potential of A3AR agonists as effective therapies to relieve pain in not develop to A3AR agonism. Similar findings have been different pathologies. observed in animal models of autoimmune disorders and cancer, A3AR agonists are also mitoprotective because they preserve where chronic administration of A3AR agonists maintains anti- the activity of the major mitochondrial enzyme, manganese inflammatory/anticancer effects even during A3AR downregula- 121 superoxide dismutase, preventing the sustained formation of tion. Fishman postulated that the functionality of A3AR oxygen and nitrogen-derived free radicals (superoxide and agonists in inflammation/tumor growth may be dependent on peroxynitrite); here, mitoprotection in peripheral sensory afferents the downregulation of A3AR to inhibit downstream regulatory may contribute to their ability to attenuate increased abnormal proteins.50 spontaneous discharge observed in models of chemotherapy- In addition to their potential use as standalone analgesics, 13,82,83 induced neuropathic pain. Some of the mechanisms of A3AR agonists may be able to be given in conjunction with action depicted so far for A3AR are captured in Figure 5. The currently used drugs because these can increase the analgesic 38 development of highly selective A3AR agonists such as MRS5698 potency of morphine, gabapentin, and amitriptyline. Recently, and MRS5980173,174 provide exquisite tools to now probe the Kim et al.99 demonstrated that the antiallodynic effects of mechanistic roles of the A3AR axis in pain; their selectivity for amitriptyline and its inhibitory effects on the activation of ERK 173,174 A3AR is .1000-fold when compared to A1AR and A2AAR. and CREB and inflammatory cytokines in the SC in a rat model of These tools that are now commercially available are anticipated to SNL-induced neuropathic pain was abrogated by an A3AR further our understanding of the role of the A3AR axis in pain antagonist MRS1191 58. This suggests that part of the action states. mediated by amitriptyline is mediated through activation of the 99 The antinociceptive effects of A3AR agonists are not de- A3AR. The link between amitriptyline and the adenosine/A3AR pendent upon endogenous opioid or endocannabinoid axis remains to be elucidated.

21 Figure 5. Schematic representation of the molecular signaling pathways of A3AR agonists. Ca , calcium ions; ERK, extracellular signal-regulated kinase; GABA, gamma-aminobutyric Acid; GAD65, glutamic acid decarboxylase 65; GAT1, GABA transporter type 1; GLT-1, glutamate transporter 1; GS, glutamine synthetase; IL-1b, interleukin-1b; IL-4, interleukin-4; IL-10, interleukin-10; KCC2, potassium-chloride cotransporter protein; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; NF-kB, nuclear factor-kB; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; NOS, nitric oxide synthase; p38, p38 mitogen-activated protein kinase; PN, peroxynitrite; RVM, rostral ventromedial medulla; TNFa, tumor necrosis factor alpha; SO, superoxide.

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