1521-01033582315ndash323$2500 httpdxdoiorg101124jpet115231241THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358315ndash323 August 2016Copyright ordf 2016 by The American Society for Pharmacology and Experimental Therapeutics

Treatment with Adenosine Receptor Agonist Ameliorates PainInduced by Acute and Chronic Inflammation

Guilherme Carneiro Montes Nathalia Hammes Miguel Divino da RochaTadeu Lima Montagnoli Carlos Alberto Manssour Fraga Eliezer J BarreiroRoberto Takashi Sudo and Gisele Zapata-SudoPrograma de Pesquisa em Desenvolvimento de Faacutermacos Instituto de Ciecircncias Biomeacutedicas Universidade Federal do Rio deJaneiro Rio de Janeiro Brazil

Received December 3 2015 accepted May 17 2016

ABSTRACTRheumatoid arthritis is an inflammatory autoimmune conditionand tumor necrosis factor-a (TNF-a) plays an important role in itspathophysiology In vitro (E)-N9-(34-dimethoxybenzylidene)-N-methylbenzohydrazide (LASSBio-1359) has exhibited anti-TNF-a properties and in vivo these effects are mediated via activationof adenosine receptor This work investigates the antinocicep-tive action of LASSBio-1359 in murine models of acute andchronic inflammatory pain Male mice received an intraperitonealinjection of LASSBio-1359 and then were evaluated in formalin-and carrageenan-induced paw edema assays CompleteFreundrsquos adjuvant (CFA) was used to induce a mouse model ofmonoarthritis These mice were treated with LASSBio-1359 byoral gavage to evaluate thermal and mechanical hyperalgesiaTNF-a and inducible nitric oxide synthase (iNOS) expression aswell as histologic features were analyzed The time of reactivityto formalin in the neurogenic phase was reduced from 5636 60

seconds to 327 6 22 seconds and 238 6 26 seconds aftertreatment with LASSBio-1359 at doses of 10 mgkg and20 mgkg respectively A reversal of the antinociceptive actionof LASSBio-1359 was observed in the inflammatory phase aftertreatment with ZM 241385 [4-(2-[7-amino-2-(2-furly)[124]-triazolo[23-a][135]triazin-5-ylamino]ethyl)phenol] an aden-osine A2A antagonist Carrageenan-induced thermal andmechanical hyperalgesia were reduced after treatment withLASSBio-1359 Similarly CFA-induced thermal and mechanicalhyperalgesia were reduced after treatment with LASSBio-1359(25 and 50 mgkg) Levels of TNF-a and iNOS expressionincreased in the monoarthritis model and were normalized inanimals treated with LASSBio-1359 which was also associatedwith beneficial effects in the histologic analysis These resultssuggest that LASSBio-1359 represents an alternative treatmentof monoarthritis

IntroductionPain an unpleasant feeling that is often caused by intense

or damaging stimuli is an essential sensation that usuallysignals that tissue injury has occurred as a result of externaland internal damaging events Tissue injury can lead to theactivation of nociceptors (sensitizing peripheral sensory neu-rons) by mediators whose identification has contributed to ourunderstanding of the pathophysiology of pain (Woolf and Ma2007 Pavin et al 2011) In the 1970s clinical observations thatpatientswith chronic pain exhibited other symptoms in additionto hyperalgesia indicated that pain and the immune systemmaybe associated beyond an acute response Moreover the othersymptoms paralleled the classic systemic sickness response thatis commonly observed which includes lethargy depression andanxiety Therefore the concomitant presence of sickness behav-iors and chronic pain is considered suggestive of underlyingimmune activity (Grace et al 2014 McMahon et al 2015)

Cytokines are small regulatory proteins that are producedby white blood cells and a variety of other cells including cellsof the central nervous system The activation or dysregulationof cytokine production is implied in a variety of disease statesincluding acute (bacterial viral fungal and parasitic infec-tions tissue necrosis tissue injury by foreign bodies and hyper-sensitivity reactions) and chronic inflammation (Sommer andKress 2004 Staud 2015) In particular the latter includesrheumatoid arthritis (RA)Tumor necrosis factor-a (TNF-a) a principal proinflamma-

tory cytokine that is produced by the immune system is alsoproduced in the peripheral and the central nervous systemunder pathologic conditions (Zhang et al 2011) Several studieshave correlated high tissue levels of TNF-a with the pain andhyperalgesia associated with a number of diseases (Barneset al 1992 Shafer et al 1994 Tak et al 1997 Lindenlaub andSommer 2003 Uceyler et al 2015 Yunus 2015)RA is a chronic disease that leads to inflammation in the

thin synovial membrane that surrounds joints This mem-brane normally produces lubricating and nutritive synovialdxdoiorg101124jpet115231241

ABBREVIATIONS A2A-R A2A adenosine receptor ANOVA analysis of variance ASA acetylsalicylic acid CFA Complete Freundrsquos adjuvantDMSO dimethylsulfoxide IFA incomplete Freundrsquos adjuvant IL interleukin iNOS inducible nitric oxide synthase LASSBio-1359 (E)-N9-(34-dimethoxybenzylidene)-N-methylbenzohydrazide LASSBio-294 (29-thienylidene)34-methylenedioxybenzoylhydrazine RA rheumatoid arthritis SEMstandard error of the mean TNF-a tumor necrosis factor-a ZM 241385 4-(2-[7-amino-2-(2-furly)[124]triazolo[23-a][135]triazin-5-ylamino]ethyl)phenol

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fluid However during RA the synovial layer is invaded byneutrophilsmast cells dendritic cellsmacrophage and T andB cells After extensive angiogenesis and the proliferation ofsynoviocytes during disease progression hyperplastic syno-vial tissue becomes invasive and destroys cartilage and boneIn the chronic phase of RA TNF-a and interleukin 1 (IL-1) areresponsible for the maturation of osteoclasts and bone re-sorption that impair the rheumatic joint (Strand et al 2007Schett and Teitelbaum 2009 Komatsu and Takayanagi2015)Chronic pain can occur during RA and the inflammation

involved is usually followed by increased behavioral responsesto innocuous (allodynia) and noxious (hyperalgesia) stimula-tion (Pinto et al 2007 Edwards et al 2011) Hyperalgesia isinduced by the action of mediators released in response toinflammation in the inflamed tissue Patients with RA showincreased TNF-a levels consequently it has been hypothe-sized that blocking the release of TNF-a represents a strategyfor reducing acute and chronic inflammation (Cunha et al2005 Verri et al 2006 Strand et al 2007 Silveira et al2013 Walsh and McWilliams 2014 Buttgereit et al 2015Wei et al 2015)Currently our research group is studying a series of

new N-acylhydrazone derivatives that exhibit potent anti-inflammatory and analgesic activities These compounds weresynthesized via molecular simplification of the lead com-pound LASSBio-294 [(29-thienylidene)34-methylenedioxy-benzoylhydrazine] by replacing the rings linked to the acyl orimine subunits andor modifying the stereoelectronic behav-ior of the acylhydrazone group (previously described as apharmacophore group for analgesic and anti-inflammatorypurposes) by N-alkylation (Kummerle et al 2012)One of these analogs LASSBio-1359 [(E)-N9-(34-dimethoxy-

benzylidene)-N-methylbenzohydrazide] (Fig 1) has exhibitedanti-TNF-a activity in vitro and in vivo (Kummerle et al 2012)and been described as an adenosine receptor (A2A-R) agonist(Alencar et al 2013 Moura et al 2015) Therefore we inves-tigated the antinociceptive and anti-inflammatory activities ofLASSBio-1359 with the goal of reversing the mechanical andthermal hyperalgesia that can be induced in an animal model ofRA (monoarthritis)

Material and MethodsAnimals Experimental protocols were approved by the Animal

Care and Use Committee of the Universidade Federal do Rio deJaneiro Brazil CEUA (DFBCICB069) Male Swissmice (25ndash35g) werehoused under controlled temperature (21 6 1degC) and humidity (60)conditions with a 12-hour lightdark cycle Food and water wereavailable ad libitum Animals were acclimated for at least 30 minutesbefore the beginning of the experiment

Drugs LASSBio-1359 (LASSBioUniversidade Federal do Rio deJaneiro Brazil) was synthesized and characterized as previouslydescribed elsewhere (Kummerle et al 2012) LASSBio-1359 mor-phine sulfate (Cristaacutelia Itapira Brazil) ZM 241385 [4-(2-[7-amino-2-(2-furly)[124]triazolo[23-a][135]triazin-5-ylamino]ethyl)phenol](Tocris BioscienceRampD Systems Minneapolis MN) and acetylsali-cylic acid (ASA Sigma-Aldrich St Louis MO) were dissolved indimethylsulfoxide (DMSO Cristaacutelia) Ethylenediaminetetraace-tic acid (EDTA Sigma-Aldrich) and formaldehyde (Isofar Duquede Caxias Brazil) were dissolved in distilled water Carrageenan(Sigma-Aldrich) was dissolved in saline Indomethacin (Sigma-Aldrich) was dissolved in a mixture of ethanolTween 80saline(2890)

Formalin-Induced Hind Paw Licking Thirty minutes afterreceiving an ip injection of either vehicle morphine (10 mgkg)acetylsalicylic acid (150 mgkg) or LASSBio-1359 (5 10 or 20 mgkg)mice were administered 20 ml of formalin (25) via the intraplantarroute The nociceptive behavior in response to a formalin injection ischaracterized by licking or biting the paw which causes a classicbiphasic nociceptive response (Abbott et al 1995) The initial acuteneurogenic phase (0ndash5 minutes) is followed by a quiescent period(5ndash15 minutes) before a prolonged tonic inflammatory response(15ndash30 minutes) The effect of the adenosine pathway was investi-gated by pretreatment with a specific antagonist of the A2A-R ZM241385 (3 mgkg ip) which was injected 20 minutes before theLASSBio-1359 administration

Carrageenan-Induced Pain Subacute paw inflammation inmice was induced by injection of 20 ml of carrageenan (1 intra-plantar) The antinociceptive effects of LASSBio-1359 (10 or 20mgkgip) and indomethacin (4mgkg ip) were evaluated 150minutes afterthe injection of carrageenan

Complete Freundrsquos AdjuvantndashInduced MonoarthritisChronic inflammation was induced by two subcutaneous injectionsof 15ml of Complete Freundrsquos adjuvant (CFA) which contained 5mgmlheat-killed mycobacterium butyricum (Becton Dickinson FranklinLakes NJ) Mice under anesthesia (sevoflurane 2 Cristaacutelia) wereinjected with CFA in the vicinity of the tibiotarsal joint (Chillingworthand Donaldson 2003) The control group received an injection ofincomplete Freundrsquos adjuvant (IFA without bacteria) After 7 daysthemice were treatedwith vehicle (DMSO) LASSBio-1359 (10 25 50or 100 mgkg) or ASA (300 mgkg) by oral gavage

Paw Immersion Test Thermal hyperalgesia observed in miceafter carrageenan and CFA injection was evaluated through the pawimmersion test as previously described (Lolignier et al 2011) Brieflythe injected hind paw was immersed in a 46degC water bath untilwithdrawal was observed Control latency was determined as theaverage of three observations In the subacute inflammation modelthe latency of each animal was obtained every 15 minutes during thefirst 2 hours and measured 24 hours after carrageenan injectionHowever in the chronic inflammation model the latency was observedevery 3 to 4 days until the end of treatment

Paw Pressure Test Mechanical hyperalgesia was investi-gated through the paw pressure test using the RandallndashSelitto device(analgesimeter Ugo-Basile Varese Italy) in both subacute andchronic models Increasing pressure (expressed in grams) was appliedto the hind paw until vocalization or paw withdrawal The maximalpressure was set at 250 g (Almela et al 2009)

Membrane Preparation and Western Blot Analysis Thetreated animals were euthanized on day 21 and the soft paw tissueswere removed stored in lysis buffer containing protease inhibitorsand frozen in liquid nitrogen The tissues were homogenized in an ice-cold lysis buffer that consisted of 20 mM HEPES (pH 74) 150 mMNaCl 2 mM EDTA 1 mM MgCl 1 mM phenylmethanesulfonyl fluo-ride 1 mM benzamidine 1 mM dithiothreitol 1 mgmL polypeptideprotease inhibitor solution (pepstatin chymostatin aprotininleupeptin and antipain Sigma-Aldrich) 1 sodium dodecyl sul-fate and 1 Triton X-100 (Bio-Rad Laboratories Hercules CA)The homogenate was centrifuged for 5 minutes at 1000g and the

Fig 1 (E)-N9-(34-dimethoxybenzylidene)-N-methylbenzohydrazide (LASS-Bio-1359)

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supernatant (sim05 ml) was collected and stored at 280degC (Okorokovand Lehle 1998) The total protein concentration for each sample wasdetermined spectrophotometrically by using the Lowry method(Lowry et al 1951)

Proteins (14 mg) were separated by electrophoresis in 10 poly-acrylamide gel and were transferred onto nitrocellulose membranes(Bio-Rad Laboratories) Membranes were blocked with 5 nonfat drymilk in phosphate-buffered saline containing 01 Tween 20 andthen were incubated with primary antibodies against TNF-a andinducible nitric oxide synthase (iNOS Abcam Cambridge MA) andglyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technol-ogy Beverly MA) The secondary antibodies used were anti-rabbitand anti-mouse both of which were horseradish peroxidase labeled(Abcam) Bound antibodies were visualized with a Super Signal WestPico Chemiluminescence Kit (Pierce Rockford IL) and an AmershamImager 600 (GE Healthcare Little Chalfont United Kingdom) Thedensity of each bandwasdetermined andnormalized to glyceraldehyde-3-phosphate dehydrogenase usingNational Institutes ofHealth ImageJsoftware (httpsimagejnihgovij)

Histologic Analysis of Inflammation Animals were sacrificedat the end of the treatment and their hind paws were immediatelyremoved fixed in 10 neutral buffered formalin and decalcified in10 EDTA (pH 72) over 14 days Tissues were dehydrated overnightin 70 ethanol then 3 times (40 minutes each) in absolute alcoholfollowed by 3 times (40 minutes each) in xylol After that the tissueswere embedded in paraffin (60degC) and sections (3 mm thick) werestained with hematoxylin and eosin or safranin Ofast green FCFfollowed by the examination by blinded experts in a light microscopy

Statistical Analysis All data were expressed as the mean 6standard error of the mean (SEM) Differences among groups wereconsidered statistically significant when P 005 using either one-way analysis of variance (ANOVA) followed by a post-hoc Dunnettrsquostest or two-way ANOVA followed by Tukeyrsquos test

ResultsEffect of LASSBio-1359 in Formalin-Induced Hind-

Paw Licking Test Swiss mice received a single intraplan-tar injection of 20 ml of formalin When LASSBio-1359 was

administered before the formalin the animals spent less timelickingbiting their paws during the neurogenic (0ndash5 minutes)and inflammatory (15ndash30 minutes) phases In the neuro-genic nociception phase the administration of LASSBio-1359(10 mgkg and 20 mgkg) resulted in an attenuation of thelickingbite time response of the control group (DMSO) from56 6 6 seconds to 37 6 2 seconds and 24 6 2 secondsrespectively In the inflammatory phase the animals treatedwith LASSBio-1359 (10 mgkg and 20 mgkg) exhibited adecrease in lickingbite time from 307 6 44 seconds (for thecontrol group) to 129 6 21 seconds and 140 6 16 secondsrespectively Pretreatment with ASA a nonsteroidal anti-inflammatory agent significantly reduced reactivity in thesecond phase but not in the neurogenic phase (Fig 2) The admin-istration of the A2A-R antagonist ZM 241385 (3 mgkg ip)inhibited the antinociceptive effect of LASSBio-1359 duringthe inflammatory phase (Fig 3)Effect of LASSBio-1359 in Thermal Hyperalgesia of

Carrageenan-Induced Pain Test Initially carrageenan-induced thermal hyperalgesia was evaluated for Swiss micebased on their paw withdrawal latency in response to a 46degCwater bath The thermal hyperalgesia threshold was reducedfrom 1356 03 seconds to 566 06 seconds 150 minutes afterthe injection of carrageenanWhen themice were treated withLASSBio-1359 (10 mgkg and 20 mgkip) the carrageenan-induced thermal hyperalgesia thresholds were 115 6 15seconds and 138 6 06 seconds respectively These effectswere observed at different time points of the evaluation Simi-larly treatment with indomethacin attenuated carrageenan-induced thermal hyperalgesia (Fig 4)Effect of LASSBio-1359 in Mechanical Hyperalgesia

of Carrageenan-Induced Pain Test Treatment withLASSBio-1359 (10 mgkg and 20 mgkg ip) reduced themechanical hyperalgesia induced by carrageenan with thethreshold increasing from 797 6 66 g to 2067 6 177 g and19786 216 g respectively 150 minutes after the injection of

Fig 2 Effects of ip administration of LASSBio-1359 (510 and 20 mgkg) morphine (10 mgkg) and acetyl salicylicacid (150 mgkg) in formalin-induced hind-paw licking testData represent the time spent by the animal licking orbiting the formalin-injected paws Values are expressed asmean 6 SEM (n = 10) P 001 or P 0001 whencompared with the vehicle-treated group using one-wayANOVA followed by Dunnetrsquos test

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carrageenan Indomethacin produced a total reversal of thecarrageenan-induced mechanical hyperalgesia as well asLASSBio-1359 (Fig 5)Effect of LASSBio-1359 in Monoarthritis CFA in-

duced chronic inflammation around the tibiotarsal joint ofthe Swiss mice after 7 day and this was accompanied byreduced thresholds for both thermal and mechanical hyper-algesia from 1366 03 seconds to 696 06 seconds and from2457 6 19 g to 1099 6 87 g respectively Significantreductions in both thermal and mechanical hyperalgesia wereobserved during 13 days of treatment In contrast injections ofIFA did not reduce either threshold Animals that receivedCFA andwere subsequently treated withDMSO exhibited lowthresholds for mechanical and thermal hyperalgesia (Figs 6and 7) In contrast the mice that were treated with LASSBio-1359 at doses of 25 50 and 100 mgkg were found to exhibitimproved thermal and mechanical hyperalgesia over the

treatment period Similar results were observed after treat-ment with ASA which increased the thermal and mechanicalthreshold in mice with monoarthritisEffect of LASSBio-1359 on TNF-a and iNOS Expres-

sion in Paw Tissues of Monoarthritis Mouse Model Ina Western blot analysis of paw tissues obtained from themonoarthritis animal model higher levels of TNF-a expres-sion were detected (Fig 8A) In contrast the monoarthritisanimals that received LASSBio-1359 expressed lower levels ofTNF-a detected in their paw tissues A similar expressionprofile was observed for iNOS in the paw tissues without andwith LASSBio-1359 treatment respectively (Fig 8B) How-ever TNF-a and iNOS were overexpressed in paws of animalswith monoarthritis treated with ASAHistopathologic Evaluations of Paw Tissues Obtained

from Monoarthritis Mice Sections of the tibiotarsal jointfrom the animals treated with IFA were stained with HampE andwere evaluated histologically (Fig 9A) IFA-treated mice (egthe normal group) exhibited a minimal inflammatory responsearound the cartilage and bone in the synovium tissues Incontrast the CFA group that was treated with DMSO (Fig 9B)exhibited classic features of arthritis Specifically a severeinflammatory response was observed that included synovial-cell hyperplasia moderate to severe fibrosis pannus formationcartilage destruction and extensive bone lysis Figure 9 D andE shows that oral treatment with LASSBio-1359 at doses of25 mgkg and 50 mgkg resulted in a significant suppression ofhistopathologic changes in the cartilage preservation of boneand an absence of pannus or fibrosis In addition minorinflammatory cells were observed Despite these results anoral treatment regimen of 10 mgkg LASSBio-1359 was foundto be associated with an infiltrate of inflammatory cells (Fig9C) suggesting that a transient inflammatory responseoccurred in the treated groupCartilage tissues were also analyzed (Fig 10) The thickness

of the cartilage tissue from the monoarthritis group treatedwith DMSO (Fig 10B) was less than that of the control groupthat was treated with IFA (Fig 10A) For the animals treatedwith LASSBio-1359 (50 mgkg) no decrease in cartilagethickness was observed thereby suggesting that cartilagedegradation was reversed (Fig 10D) The ImageJ programrevealed that the area of cartilage stained with safranin was

Fig 3 Effects of LASSBio-1359 (10 mgkg ip) on formalin-induced hind-paw licking test in the absence or presence ofZM 241385 Data are expressed as mean 6 SEM (n =6ndash10) P 005 and P 001 versus control xP 005versus LASSBio-1359 (10 mgkg ip) using one-wayANOVA followed by Dunnetrsquos test

Fig 4 Effects of LASSBio-1359 (10 and 20 mgkg ip) or indomethacin(4 mgkg ip) in the paw immersion test Carrageenan induced thermalhyperalgesia in the ipsilateral paw after 150 minutes Data are expressedas the mean 6 SEM (n = 10) P 001 when compared with DMSOusing two-way ANOVA followed by Tukeyrsquos test

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reduced by approximately 40 in the CFA-induced monoar-thritis group indicating a severe proteoglicans (in red) de-ficiency which was prevented after treatment with LASSBio-1359 (50 mgkg) prevented this reduction

DiscussionThis study demonstrated that LASSBio-1359 a novel agonist

of adenosine A2A receptor (A2A-R) reversed the hyperalgesicresponse induced by stimulation of peripheral receptor modu-lated by activation of inflammatory process in mice A decreasein the expression levels of both TNF-a and iNOS or damage tothe tibiotarsal joint in the paw of animals with CFA-inducedmonoarthritis was noted after treatment with LASSBio-1359The behavioral response of mice to formalin was evaluated

after intraplantar injection of formalin which produced abiphasic behavioral response that involved both central andperipheral components The first phase includes a directstimulation of nociceptors by formalin while the second phasemay be associated with the release of inflammatory mediators

into tissues (Yano et al 2006) In the present study treatmentwith LASSBio-1359 inhibited both the first and second phasesof formalin-induced nociception Similarly morphine has beenreported to inhibit nociception in both phases of the formalintest (Shibata et al 1989)When the mechanisms associated with the antinociceptive

action observed during the second phase of the formalin testwere evaluated pretreatment with an antagonist for A2A-RZM 241385 was found to reverse the antinociceptive effect ofLASSBio-1359 LASSBio-1359 has also been reported to be anA2A-R agonist with a capacity to stimulate adenylate cyclaseactivity (Alencar et al 2013) As a result ATP is converted tocAMP thereby leading to the intracellular accumulation ofthis second messenger and the activation of protein kinase AThis in turn leads to inhibition of the formation and releaseof proinflammatory cytokines such as TNF-a and IL-1b(Jacobson and Gao 2006 Varani et al 2010 Chen et al2013) and it also leads to inhibition nuclear factor-kB nucleartranslocation (Mediero et al 2013) Those anti-inflammatoryeffects were observed after treatment with LASSBio-1359The inflammation in the hind paw of the animals caused

by carrageenan usually is used to investigate antinociceptiveand anti-inflammatory activities (Randall and Selitto 1957Sugishita et al 1981 Henriques et al 1987 Hargreaveset al 1988 Shibata et al 1989 Petersson et al 2001Mendes et al 2009 Shah and Shah 2015 Sudo et al 2015)Carrageenan may induce inflammation in two phases In theearly phase (1ndash2 hours after the injection of carrageenan)inflammation is mostly mediated by histamine serotonin andthe increased synthesis of prostaglandins in the surroundingdamaged tissues The later phase of inflammation (2 hours afterthe injection of carrageenan) prostaglandins are released bypolymorphonuclear cells and macrophages are engaged in pro-cess of vascular permeability (Gupta et al 2006 Prajapatiet al 2014) Usually the local inflammation is elevated withproinflammatory cytokines TNF-a IL-1 and IL-6 (Cuzzocreaet al 1999 Ogata et al 1999) Besides inflammation carra-geenan can induce hyperalgesia (Zhang et al 2004)TNF-a has been shown to initiate inflammatory responses

and have an important role in pain and central sensitization(Zhang et al 2011) The decreased sensitization induced byhigh levels of TNF-a (secreted by macrophages) needs to bereduced Reduced TNF-a expression has been demonstratedas responsible for the anti-inflammatory responses observed

Fig 5 Effects of LASSBio-1359 (10 and 20 mgkg ip) or indomethacin(4 mgkg ip) in the paw pressure test Carrageenan induced mechanicalhyperalgesia in the ipsilateral paw after 150 minutes Data are expressedas mean 6 SEM (n = 10) P 001 when compared with DMSO usingtwo-way ANOVA followed by Tukeyrsquos test

Fig 6 Effects of LASSBio-1359 (10 25 50 or100 mgkg oral administration) or acetylsalicylicacid (300 mgkg oral administration) in the mono-athritis model CFA induced mechanical hyper-algesia in the ipsilateral paw after 3 days Data areexpressed as mean 6 SEM (n = 7ndash10) P 001P 0001 P 001 P 0001 comparedwith CFA + DMSO using two-way ANOVA followedby Tukeyrsquos test

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in animal models (Xu et al 2012 Wang et al 2015a) In ourpresent study the thermal and mechanical hyperalgesiainduced by carrageenanwere suppressed after treatment withLASSBio-1359 This approach has been widely employed toassess the effects of new analgesic and anti-inflammatorydrugs (Posadas et al 2004 Radhakrishnan et al 2004Quintao et al 2005)To mimic a chronic inflammatory pain state that is accom-

panied by increased behavioral responses to noxious (hyper-algesia) stimulation monoarthritis was induced by CFA Thismodel differs from neuropathic pain because the hyperalgesiais deeply associated with inflammation furthermore there isno time for healing of the pathologic tissues CFA-inducedbehavioral changes are usually observed in animals after3 days (early phase) then they typically continue for at least3 weeks (Ohsawa et al 2000 Hong et al 2009 Aoki et al

2014b) In the present study strong hypersensitivity to heatand mechanical responses were characterized by a reductionin paw withdrawal latencies between day 3 and day 7Moreover both mechanical and thermal hyperalgesia wereattenuated after treatment with LASSBio-1359Nociceptors may be activated through noxious thermal

mechanical or chemical responses Some studies report thatthe heat threshold for cutaneous nociceptors is sensitized firstin monoarthritis (Danziger et al 1999 Morell et al 2014)After that joint and surrounding deep nociceptors are in-volved in the sensitization in the chronic process (Danzigeret al 1999 Morell et al 2014) The joint nerves have thickmyelinated Ab thinly myelinated Ad and a high proportionunmyelinated C fiber When a joint undergoes an inflamma-tory process the fibers Ab and Ad are more sensitive toapplied pressure and to movements of the joint C fibers

Fig 7 Effects of LASSBio-1359 (10 25 50 or100 mgkg oral administration) or acetylsalicylicacid (300 mgkg oral administration) in monoathri-tis model CFA induced thermal hyperalgesia in theipsilateral paw after 3 days Data are expressed asmean 6 SEM (n = 7ndash10) P 001 P 005P 001 P 0001 compared with CFA+DMSO using two-way ANOVA followed by Tukeyrsquostest

Fig 8 Expression of TNF-a (A) and iNOS (B) in paws frommice treated with IFA or CFA after oral administration ofLASSBio-1359 (10 25 50 or 100 mgkg) Graphs showprotein quantification and each column represents themean 6 SEM (n = 3ndash4) P 005 compared with controlP 005 compared with DMSO using one-way ANOVAfollowed by Dunnetrsquos test

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which modulate the hyperalgesia become sensitized to re-spond to light pressure and movements in the working rangeof the joint These changes provide a mechanical afferentsensory response that indicates joint pain (Cline et al 1989Schaible and Grubb 1993 Aoki et al 2014a) Exacerbatedmechanical and thermal hyperalgesic responses were ob-served in the arthritis murine model and antinociceptiveaction of LASSBio-1359 was demonstratedPrevious studies characterized LASSBio-1359 as an agonist

of A2A-R which is coupled with a stimulatory G-protein (Scottand Kingsley 2006) culminating in an increase of adenylylcyclase activity mediating an increase of cAMP accumulationA2A-R are widely expressed in immune system cells and havea crucial role in inflammatory conditions such as arthritisHuntingtonrsquos disease and Parkinsonrsquos disease (Ohta andSitkovsky 2001 Bilzer and Gerbes 2002 Sebastiatildeo andRibeiro 2009 Neumann et al 2014) Studies have describedhigh expression of A2A-R in patients with RA TNF-a producedby macrophage monocytes and T cells has an important role

in the appearance spreading and systemicmanifestation of thedisease its increased levels might increase the expression andactivity of iNOS (Choy and Panayi 2001 Scott and Kingsley2006 Haskoacute et al 2008 Moore et al 2008 Gonzalez-Gay et al2009 Nowak et al 2010 Varani et al 2011 Vincenzi et al2013) Additionally the activation of A2A-R can inhibit nuclearfactor-kB nuclear translocation and osteoclast differentiationpreventing bone destruction This event has a beneficial effectfor inflammatory diseases such as arthritis (Haskoacute and Cron-stein 2013 Mediero et al 2013) LASSBio-1359 binding andstimulating A2A-R can lead to the inhibition of the release ofproinflammatory cytokines such as TNF-a which provides adecrease in the transcription of inflammatory proteins andiNOS preventing bone destructionPain the principal symptom in patients with RA is a

common cause of disability more than one-third of patientseventually experience work disability Currently nonsteroi-dal anti-inflammatory drugs with acetaminophen are pre-scribed as the first-line therapy for the treatment of pain in

Fig 9 Representative HampE sections oftibiotarsal joints (n = 3) Arthritic jointsshowed synovial hyperplasia (arrow) de-struction of cartilage and bone () andinflammatory cell infiltrate () A = IFAB = CFA + DMSO C = CFA + LASSBio-1359 (10 mgkg) D = CFA + LASSBio-1359(25 mgkg) E = CFA + LASSBio-1359(50 mgkg) Cart = Cartilage Horizontalbar = 100 mm

Fig 10 Safranin OFast green FCF staining ofarticular cartilages (n = 3) Arthritic-joints treatedwith DMSO showed lower amount of proteoglycanscompared with animals injected with IFA Micetreated with LASSBio-1359 (10 or 25 mgkg) showeda red-staining pattern indicating partially reversalof proteoglycan loss A = IFA B = CFA + DMSO C =CFA + LASSBio-1359 (10 mgkg) D = CFA +LASSBio-1359 (25 mgkg) Dashed line indicatesthe cartilage (Morelli et al 2009) border Horizontalbar = 100 mm

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inflammatory arthritis Meanwhile the use of antidepres-sants as an analgesic therapy for inflammatory arthritisremains controversial (Heiberg et al 2005 Allaire et al2008 Saag et al 2008 Lee 2013) It is also important to notethat some patients experience adverse effects from the drugsused to treat arthritis including gastrointestinal toxicitymainly during treatment with nonsteroidal anti-inflammatoryagents (Farrell et al 1991 Carrasco-Pozo et al 2011 Guthrie2011 Carrasco-Pozo et al 2016)The adenosine receptors which are distributed in the spinal

cord and some brain areas are involved in themodulation of pain(Ribeiro et al 2002 Sawynok and Liu 2003 Morelli et al 2009Burnstock et al 2011 Zylka 2011) because their activation byadenosine or others agonists can inhibit pain Activation of theA2A-R in the spinal cord precisely in lamina II neurons promotesinhibition N-methyl-D-aspartate currents and could modulateantinociceptive activity (Guntz et al 2008) its blockade byantagonists reverses this effect (DeLander et al 1992 De Landerand Keil 1994 Borghi et al 2002 Yoon et al 2005 2006 Leeet al 2010 Ng et al 2015 Wang et al 2015b) Intrathecaladministration of the A2AR agonist was effective to attenuatemechanical allodynia and thermal hyperalgesia in a pain model(Loram et al 2009) Additionally the activation of A2A-R onmicroglia and astrocytes cells can control chronic pain Thus theadenosine pathway could represent an important target for thesuppression of pain as well as for anti-inflammatory action andneuroprotection (Eisenach et al 2003 Haskoacute et al 2005 Loramet al 2009 2013 Melani et al 2014 Sawynok 2015)In conclusion this study has provided evidence that the

novel adenosine A2A agonist LASSBio-1359 can effectivelyreverse the pain response associated with an inflammatoryreaction modulated by the production and release of cytokinein CFA-induced arthritis in mice

Authorship Contributions

Participated in research designMontes Hammes Sudo Zapata-SudoConducted experiments Montes Hammes MontagnoliContributed new reagents or analytic tools da Rocha Fraga BarreiroPerformed data analysis Fraga Barreiro Sudo Zapata-SudoWrote or contributed to writing of the manuscript Montes Sudo

Zapata-Sudo

References

Abbott FV Franklin KB and Westbrook RF (1995) The formalin test scoring prop-erties of the first and second phases of the pain response in rats Pain 6091ndash102

Alencar AK Pereira SL Montagnoli TL Maia RC Kuumlmmerle AE Landgraf SSCaruso-Neves C Ferraz EB Tesch R Nascimento JH et al (2013) Beneficialeffects of a novel agonist of the adenosine A2A receptor on monocrotaline-inducedpulmonary hypertension in rats Br J Pharmacol 169953ndash962

Allaire S Wolfe F Niu J and Lavalley MP (2008) Contemporary prevalence andincidence of work disability associated with rheumatoid arthritis in the US Ar-thritis Rheum 59474ndash480

Almela P Garciacutea-Nogales P Romero A Milaneacutes MV Laorden ML and Puig MM(2009) Effects of chronic inflammation and morphine tolerance on the expression ofphospho-ERK 12 and phospho-P38 in the injured tissue Naunyn SchmiedebergsArch Pharmacol 379315ndash323

Aoki Y Mizoguchi H Watanabe C Sakurada T and Sakurada S (2014a) Differentialalternation of the antinociceptive effect of narcotic analgesics on the inflammatorypain state Neurosci Lett 560122ndash125

Aoki Y Mizoguchi H Watanabe C Takeda K Sakurada T and Sakurada S (2014b)Potential involvement of m-opioid receptor dysregulation on the reduced anti-nociception of morphine in the inflammatory pain state in mice J Pharmacol Sci124258ndash266

Barnes PF Chatterjee D Brennan PJ Rea TH and Modlin RL (1992) Tumor ne-crosis factor production in patients with leprosy Infect Immun 601441ndash1446

Bilzer M and Gerbes AL (2002) [Role of G-protein-coupled adenosine receptors indownregulation of inflammation and protection from tissue damage] Z Gastro-enterol 40543ndash544

Borghi V Przewlocka B Labuz D Maj M Ilona O and Pavone F (2002) Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modu-lated by A1 and A2a adenosine agonists in mice Brain Res 956339ndash348

Burnstock G Fredholm BB and Verkhratsky A (2011) Adenosine and ATP receptorsin the brain Curr Top Med Chem 11973ndash1011

Buttgereit F Smolen JS Coogan AN and Cajochen C (2015) Clocking in chronobi-ology in rheumatoid arthritis Nat Rev Rheumatol 11349ndash356

Carrasco-Pozo C Castillo RL Beltraacuten C Miranda A Fuentes J and Gotteland M (2016)Molecular mechanisms of gastrointestinal protection by quercetin against indomethacin-induced damage role of NF-kB and Nrf2 J Nutr Biochem 27289ndash298

Carrasco-Pozo C Speisky H Brunser O Pastene E and Gotteland M (2011) Applepeel polyphenols protect against gastrointestinal mucosa alterations induced byindomethacin in rats J Agric Food Chem 596459ndash6466

Chen JF Eltzschig HK and Fredholm BB (2013) Adenosine receptors as drug targetsmdashwhat are the challenges Nat Rev Drug Discov 12265ndash286

Chillingworth NL and Donaldson LF (2003) Characterisation of a Freundrsquos completeadjuvant-induced model of chronic arthritis in mice J Neurosci Methods 12845ndash52

Choy EH and Panayi GS (2001) Cytokine pathways and joint inflammation inrheumatoid arthritis N Engl J Med 344907ndash916

Cline MA Ochoa J and Torebjoumlrk HE (1989) Chronic hyperalgesia and skin warmingcaused by sensitized C nociceptors Brain 112621ndash647

Cunha TM Verri WA Jr Silva JS Poole S Cunha FQ and Ferreira SH (2005) Acascade of cytokines mediates mechanical inflammatory hypernociception in miceProc Natl Acad Sci USA 1021755ndash1760

Cuzzocrea S Sautebin L De Sarro G Costantino G Rombolagrave L Mazzon E Ialenti ADe Sarro A Ciliberto G and Di Rosa M et al (1999) Role of IL-6 in the pleurisyand lung injury caused by carrageenan J Immunol 1635094ndash5104

Danziger N Weil-Fugazza J Le Bars D and Bouhassira D (1999) Alteration ofdescending modulation of nociception during the course of monoarthritis in the ratJ Neurosci 192394ndash2400

De Lander GE and Keil GJ 2nd (1994) Antinociception induced by intrathecal co-administration of selective adenosine receptor and selective opioid receptor ago-nists in mice J Pharmacol Exp Ther 268943ndash951

DeLander GE Mosberg HI and Porreca F (1992) Involvement of adenosine in anti-nociception produced by spinal or supraspinal receptor-selective opioid agonists dis-sociation from gastrointestinal effects in mice J Pharmacol Exp Ther 2631097ndash1104

Edwards RR Cahalan C Mensing G Smith M and Haythornthwaite JA (2011) Paincatastrophizing and depression in the rheumatic diseases Nat Rev Rheumatol 7216ndash224

Eisenach JC Rauck RL and Curry R (2003) Intrathecal but not intravenous aden-osine reduces allodynia in patients with neuropathic pain Pain 10565ndash70

Farrell B Godwin J Richards S and Warlow C (1991) The United Kingdom transientischaemic attack (UK-TIA) aspirin trial final results J Neurol Neurosurg Psy-chiatry 541044ndash1054

Gonzalez-Gay MA Garcia-Unzueta MT Berja A Vazquez-Rodriguez TR Miranda-Filloy JA Gonzalez-Juanatey C de Matias JM Martin J Dessein PH and Llorca J(2009) Short-term effect of anti-TNF-alpha therapy on nitric oxide production inpatients with severe rheumatoid arthritis Clin Exp Rheumatol 27452ndash458

Grace PM Hutchinson MR Maier SF and Watkins LR (2014) Pathological pain andthe neuroimmune interface Nat Rev Immunol 14217ndash231

Guntz E Dumont H Pastijn E drsquoExaerde AdeK Azdad K Sosnowski M SchiffmannSN and Gall D (2008) Expression of adenosine A 2A receptors in the rat lumbarspinal cord and implications in the modulation of N-methyl-d-aspartate receptorcurrents Anesth Analg 1061882ndash1889

Gupta M Mazumder UK Gomathi P and Selvan VT (2006) Antiinflammatoryevaluation of leaves of Plumeria acuminata BMC Complement Altern Med 636

Guthrie R (2011) Review and management of side effects associated with antiplatelettherapy for prevention of recurrent cerebrovascular events Adv Ther 28473ndash482

Hargreaves K Dubner R Brown F Flores C and Joris J (1988) A new and sensitivemethod for measuring thermal nociception in cutaneous hyperalgesia Pain 3277ndash88

Haskoacute G and Cronstein B (2013) Regulation of inflammation by adenosine FrontImmunol 485

Haskoacute G Linden J Cronstein B and Pacher P (2008) Adenosine receptors therapeuticaspects for inflammatory and immune diseases Nat Rev Drug Discov 7759ndash770

Haskoacute G Pacher P Vizi ES and Illes P (2005) Adenosine receptor signaling in thebrain immune system Trends Pharmacol Sci 26511ndash516

Heiberg T Finset A Uhlig T and Kvien TK (2005) Seven year changes in healthstatus and priorities for improvement of health in patients with rheumatoid ar-thritis Ann Rheum Dis 64191ndash195

Henriques MG Silva PM Martins MA Flores CA Cunha FQ Assreuy-Filho Jand Cordeiro RS (1987) Mouse paw edema A new model for inflammation Braz JMed Biol Res 20243ndash249

Hong Y Liu Y Chabot JG Fournier A and Quirion R (2009) Upregulation of adre-nomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats Pain 146105ndash113

Jacobson KA and Gao ZG (2006) Adenosine receptors as therapeutic targets Nat RevDrug Discov 5247ndash264

Komatsu N and Takayanagi H (2015) Regulatory T cells in arthritis Prog Mol BiolTransl Sci 136207ndash215

Kuumlmmerle AE Schmitt M Cardozo SV Lugnier C Villa P Lopes AB Romeiro NCJustiniano H Martins MA and Fraga CA et al (2012) Design synthesis andpharmacological evaluation of N-acylhydrazones and novel conformationally con-strained compounds as selective and potent orally active phosphodiesterase-4 in-hibitors J Med Chem 557525ndash7545

Lee HG KimWM Choi JI and Yoon MH (2010) Roles of adenosine receptor subtypes onthe antinociceptive effect of sildenafil in rat spinal cord Neurosci Lett 480182ndash185

Lee YC (2013) Effect and treatment of chronic pain in inflammatory arthritis CurrRheumatol Rep 15300

Lindenlaub T and Sommer C (2003) Cytokines in sural nerve biopsies from in-flammatory and non-inflammatory neuropathies Acta Neuropathol 105593ndash602

Lolignier S Amsalem M Maingret F Padilla F Gabriac M Chapuy E Eschalier ADelmas P and Busserolles J (2011) Nav19 channel contributes to mechanical andheat pain hypersensitivity induced by subacute and chronic inflammation PLoSOne 6e23083

322 Montes et al

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T Journals on D

ecember 23 2020

jpetaspetjournalsorgD

ownloaded from

Loram LC Harrison JA Sloane EM Hutchinson MR Sholar P Taylor FR BerkelhammerD Coats BD Poole S and Milligan ED et al (2009) Enduring reversal of neuro-pathic pain by a single intrathecal injection of adenosine 2A receptor agonists anovel therapy for neuropathic pain J Neurosci 2914015ndash14025

Loram LC Taylor FR Strand KA Harrison JA Rzasalynn R Sholar P Rieger JMaier SF and Watkins LR (2013) Intrathecal injection of adenosine 2A receptoragonists reversed neuropathic allodynia through protein kinase (PK)APKC sig-naling Brain Behav Immun 33112ndash122

Lowry OH Rosebrough NJ Farr AL and Randall RJ (1951) Protein measurementwith the Folin phenol reagent J Biol Chem 193265ndash275

McMahon SB La Russa F and Bennett DL (2015) Crosstalk between the nociceptiveand immune systems in host defence and disease Nat Rev Neurosci 16389ndash402

Mediero A Perez-Aso M and Cronstein BN (2013) Activation of adenosine A(2A)receptor reduces osteoclast formation via PKA- and ERK12-mediated suppressionof NFkB nuclear translocation Br J Pharmacol 1691372ndash1388

Melani A Corti F Cellai L Vannucchi MG and Pedata F (2014) Low doses of theselective adenosine A2A receptor agonist CGS21680 are protective in a rat model oftransient cerebral ischemia Brain Res 155159ndash72

Mendes TC Raimundo JM Nascimento-Junior NM Fraga CA Barreiro EJ SudoRT and Zapata-Sudo G (2009) Sedation and antinociception induced by a newpyrazolo[34-b]pyrrolo[34-d]pyridine derivative (LASSBio-873) is modulated byactivation of muscarinic receptors Pharmacol Biochem Behav 9470ndash74

Moore CC Martin EN Lee GH Obrig T Linden J and Scheld WM (2008) An A2Aadenosine receptor agonist ATL313 reduces inflammation and improves survivalin murine sepsis models BMC Infect Dis 8141

Morell M Camprubiacute-Robles M Culler MD de Lecea L and Delgado M (2014) Cor-tistatin attenuates inflammatory pain via spinal and peripheral actions NeurobiolDis 63141ndash154

Morelli M Carta AR and Jenner P (2009) Adenosine A2A receptors and Parkinsonrsquosdisease Handb Exp Pharmacol 193589ndash615

Moura VJ Alencar AK Calasans-Maia Jde A da Silva JS Fraga CA Zapata-Sudo GBarreiro EJ and Sudo RT (2015) Novel agonist of adenosine receptor inducesrelaxation of corpus cavernosum in guinea pigs an in vitro and in vivo studyUrology 851217ndash1221

Neumann E Khawaja K and Muumlller-Ladner U (2014) G protein-coupled receptors inrheumatology Nat Rev Rheumatol 10429ndash436

Ng SK Higashimori H Tolman M and Yang Y (2015) Suppression of adenosine 2areceptor (A2aR)-mediated adenosine signaling improves disease phenotypes in amouse model of amyotrophic lateral sclerosis Exp Neurol 267115ndash122

Nowak M Lynch L Yue S Ohta A Sitkovsky M Balk SP and Exley MA (2010) TheA2aR adenosine receptor controls cytokine production in iNKT cells Eur JImmunol 40682ndash687

Ogata M Matsui T Kita T and Shigematsu A (1999) Carrageenan primes leukocytesto enhance lipopolysaccharide-induced tumor necrosis factor alpha productionInfect Immun 673284ndash3289

Ohsawa M Narita M Mizoguchi H Suzuki T and Tseng LF (2000) Involvement ofspinal protein kinase C in thermal hyperalgesia evoked by partial sciatic nerveligation but not by inflammation in the mouse Eur J Pharmacol 40381ndash85

Ohta A and Sitkovsky M (2001) Role of G-protein-coupled adenosine receptors indownregulation of inflammation and protection from tissue damage Nature 414916ndash920

Okorokov LA and Lehle L (1998) Ca(21)-ATPases of Saccharomyces cerevisiae di-versity and possible role in protein sorting FEMS Microbiol Lett 16283ndash91

Pavin NF Donato F Cibin FW Jesse CR Schneider PH de Salles HD Soares LdoAAlves D and Savegnago L (2011) Antinociceptive and anti-hypernociceptive effectsof Se-phenyl thiazolidine-4-carboselenoate in mice Eur J Pharmacol 668169ndash176

Petersson M Wiberg U Lundeberg T and Uvnaumls-Moberg K (2001) Oxytocin de-creases carrageenan induced inflammation in rats Peptides 221479ndash1484

Pinto M Lima D and Tavares I (2007) Neuronal activation at the spinal cord andmedullary pain control centers after joint stimulation a c-fos study in acute andchronic articular inflammation Neuroscience 1471076ndash1089

Posadas I Bucci M Roviezzo F Rossi A Parente L Sautebin L and Cirino G (2004)Carrageenan-induced mouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expression Br J Pharmacol 142331ndash338

Prajapati VD Maheriya PM Jani GK and Solanki HK (2014) Carrageenan a nat-ural seaweed polysaccharide and its applications Carbohydr Polym 10597ndash112

Quintatildeo NL Medeiros R Santos AR Campos MM and Calixto JB (2005) The effectsof diacerhein on mechanical allodynia in inflammatory and neuropathic models ofnociception in mice Anesth Analg 1011763ndash1769

Radhakrishnan R Bement MK Skyba D Sluka KA and Kehl LJ (2004) Models ofmuscle pain carrageenan model and acidic saline model Curr Protoc PharmacolChapter 5Unit 535

Randall LO and Selitto JJ (1957) A method for measurement of analgesic activity oninflamed tissue Arch Int Pharmacodyn Ther 111409ndash419

Ribeiro JA Sebastiatildeo AM and de Mendonccedila A (2002) Adenosine receptors in thenervous system pathophysiological implications Prog Neurobiol 68377ndash392

Saag KG Teng GG Patkar NM Anuntiyo J Finney C Curtis JR Paulus HEMudano A Pisu M and Elkins-Melton M et al American College of Rheumatol-ogy (2008) American College of Rheumatology 2008 recommendations for the use ofnonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoidarthritis Arthritis Rheum 59762ndash784

Sawynok J (2015) Adenosine receptor targets for pain Neuroscience DOI [publishedahead of print]

Sawynok J and Liu XJ (2003) Adenosine in the spinal cord and periphery release andregulation of pain Prog Neurobiol 69313ndash340

Schaible HG and Grubb BD (1993) Afferent and spinal mechanisms of joint painPain 555ndash54

Schett G and Teitelbaum SL (2009) Osteoclasts and arthritis J Bone Miner Res 241142ndash1146 DOI

Scott DL and Kingsley GH (2006) Tumor necrosis factor inhibitors for rheumatoidarthritis N Engl J Med 355704ndash712

Sebastiatildeo AM and Ribeiro JA (2009) Adenosine receptors and the central nervoussystem Handb Exp Pharmacol 193471ndash534

Shafer DM Assael L White LB and Rossomando EF (1994) Tumor necrosis factor-alpha as a biochemical marker of pain and outcome in temporomandibular jointswith internal derangements J Oral Maxillofac Surg 52786ndash791

Shah SM and Shah SM (2015) Phytochemicals antioxidant antinociceptive and anti-inflammatory potential of the aqueous extract of Teucrium stocksianum biossBMC Complement Altern Med 15351

Shibata M Ohkubo T Takahashi H and Inoki R (1989) Modified formalin testcharacteristic biphasic pain response Pain 38347ndash352

Silveira KD Coelho FM Vieira AT Barroso LC Queiroz-Junior CM Costa VVSousa LF Oliveira ML Bader M and Silva TA et al (2013) Mechanisms of theanti-inflammatory actions of the angiotensin type 1 receptor antagonist losartan inexperimental models of arthritis Peptides 4653ndash63

Sommer C and Kress M (2004) Recent findings on how proinflammatory cytokinescause pain peripheral mechanisms in inflammatory and neuropathic hyperalgesiaNeurosci Lett 361184ndash187

Staud R (2015) Cytokine and immune system abnormalities in fibromyalgia andother central sensitivity syndromes Curr Rheumatol Rev 11109ndash115

Strand V Kimberly R and Isaacs JD (2007) Biologic therapies in rheumatologylessons learned future directions Nat Rev Drug Discov 675ndash92

Sudo RT Neto ML Monteiro CE Amaral RV Resende AC Souza PJ Zapata-Sudo Gand Moura RS (2015) Antinociceptive effects of hydroalcoholic extract from Euterpeoleracea Mart (Accedilaiacute) in a rodent model of acute and neuropathic pain BMCComplement Altern Med 15208

Sugishita E Amagaya S and Ogihara Y (1981) Anti-inflammatory testing methodscomparative evaluation of mice and rats J Pharmacobiodyn 4565ndash575

Tak PP Smeets TJ Daha MR Kluin PM Meijers KA Brand R Meinders AEand Breedveld FC (1997) Analysis of the synovial cell infiltrate in early rheumatoidsynovial tissue in relation to local disease activity Arthritis Rheum 40217ndash225

Uumlccedileyler N Riediger N Kafke W and Sommer C (2015) Differential gene expressionof cytokines and neurotrophic factors in nerve and skin of patients with peripheralneuropathies J Neurol 262203ndash212

Varani K Padovan M Govoni M Vincenzi F Trotta F and Borea PA (2010) The roleof adenosine receptors in rheumatoid arthritis Autoimmun Rev 1061ndash64

Varani K Padovan M Vincenzi F Targa M Trotta F Govoni M and Borea PA (2011)A2A and A3 adenosine receptor expression in rheumatoid arthritis upregulationinverse correlation with disease activity score and suppression of inflammatorycytokine and metalloproteinase release Arthritis Res Ther 13R197

Verri WA Jr Cunha TM Parada CA Poole S Cunha FQ and Ferreira SH (2006)Hypernociceptive role of cytokines and chemokines targets for analgesic drugdevelopment Pharmacol Ther 112116ndash138

Vincenzi F Padovan M Targa M Corciulo C Giacuzzo S Merighi S Gessi S Govoni MBorea PA and Varani K (2013) A(2A) adenosine receptors are differentially modulatedby pharmacological treatments in rheumatoid arthritis patients and their stimulationameliorates adjuvant-induced arthritis in rats PLoS One 8e54195

Walsh DA and McWilliams DF (2014) Mechanisms impact and management of painin rheumatoid arthritis Nat Rev Rheumatol 10581ndash592

Wang HL Li YX Niu YT Zheng J Wu J Shi GJ Ma L Niu Y Sun T and Yu JQ(2015a) Observing anti-inflammatory and anti-nociceptive activities of glycyrrhizinthrough regulating COX-2 and pro-inflammatory cytokines expressions in miceInflammation 382269ndash2278

Wang ML Yu G Yi SP Zhang FY Wang ZT Huang B Su RB Jia YX and Gong ZH(2015b) Antinociceptive effects of incarvillateine amonoterpene alkaloid from Incarvilleasinensis and possible involvement of the adenosine system Sci Rep 516107

Wei ST Sun YH Zong SH and Xiang YB (2015) Serum levels of IL-6 and TNF-a maycorrelate with activity and severity of rheumatoid arthritisMed SciMonit 214030ndash4038

Woolf CJ and Ma Q (2007) Nociceptorsndashnoxious stimulus detectors Neuron 55353ndash364

Xu Z Zhou J Cai J Zhu Z Sun X and Jiang C (2012) Anti-inflammation effects ofhydrogen saline in LPS activated macrophages and carrageenan induced paw oe-dema J Inflamm (Lond) 92

Yano S Suzuki Y Yuzurihara M Kase Y Takeda S Watanabe S Aburada Mand Miyamoto K (2006) Antinociceptive effect of methyleugenol on formalin-induced hyperalgesia in mice Eur J Pharmacol 55399ndash103

Yoon MH Bae HB and Choi JI (2005) Antinociception of intrathecal adenosine re-ceptor subtype agonists in rat formalin test Anesth Analg 1011417ndash1421

Yoon MH Bae HB Choi JI Kim SJ Chung ST and Kim CM (2006) Roles of aden-osine receptor subtypes in the antinociceptive effect of intrathecal adenosine in arat formalin test Pharmacology 7821ndash26

Yunus MB (2015) Editorial review an update on central sensitivity syndromes andthe issues of nosology and psychobiology Curr Rheumatol Rev 1170ndash85

Zhang H Zhang YQ Qiu ZB and Zhao ZQ (2004) Inhibitory effect of intrathecalmeptazinol on carrageenan-induced thermal hyperalgesia in rats Neurosci Lett3569ndash12

Zhang L Berta T Xu ZZ Liu T Park JY and Ji RR (2011) TNF-a contributes tospinal cord synaptic plasticity and inflammatory pain distinct role of TNF receptorsubtypes 1 and 2 Pain 152419ndash427

Zylka MJ (2011) Pain-relieving prospects for adenosine receptors and ectonucleoti-dases Trends Mol Med 17188ndash196

Address correspondence to Dr Gisele Zapata-Sudo Av Carlos ChagasFilho 373 Cidade Universitaacuteria bloco J sala J1 -14 Programa de Pesquisaem Desenvolvimento de Faacutermacos Instituto de Ciecircncias BiomeacutedicasUniversidade Federal do Rio de Janeiro Rio de Janeiro Brazil E-mailgzsudooicombr

Anti-inflammatory and Antinociceptive Action of LASSBio-1359 323

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