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ARE P2X RECEPTORS SUITABLE DRUG TARGETS FOR THE TREATMENT OF CHRONICINFLAMMATORYANDNEUROPATHICPAIN?

DuuameneNyimanuMScMolecularMedicinestudent,UniversityofEastAnglia,NorwichNR47TJ

ABSTRACT

Several years ago, studiesdemonstrated that extracellularATP is important inpain signallingbothat theperipheryand in theCNS.This triggered significantadvancesinthisarearesultinginthediscoveryofthecell-surfacereceptor,P2Xreceptors,asATP-bindingreceptors.ItwasalsofoundthatATPbindingtothesereceptors results in their activation and signalling in different pain states,especially chronic (inflammatory and neuropathic) pain. Inflammatory pain iselicited following inflammatory responses to peripheral nerve injury or anunspecificimmuneresponse,whichaltersnervefunction.Generally,thistypeofpaincouldrespondtotreatmentbutneuropathicpain,whichdevelopsfollowingnervedamage resulting inhypersensitivity in the absenceof overt stimulus, isusually refractory to treatment. Several studies demonstrated that ATP-dependent activation of P2X receptors, particularly P2X3, P2X2/3, P2X4 andP2X7receptors,arerequiredforthedevelopmentofchronic inflammatoryandneuropathic pain, and that blocking these receptors with antagonists orantisense oligonucleotide silencing or knockout of these receptors in miceresultsinsignificantreductioninhypersensitivitytopain,suggestingthatthesereceptors could be a potential drug target in managing inflammatory andneuropathicpain.ThisreviewdescribesthelatestevidencesfortheroleofP2Xreceptors in chronic inflammatory and neuropathic pain, thereby establishingwhy theywould be a suitable drug target for painmanagement and concludewithareviewofdifferentdrug-likemoleculesthathavebeentestedinpreclinicalandclinicaltrialstudiesforthetreatmentofthesepainstates.

IntroductionPain isanunpleasantsensoryandemotionalexperienceassociatedwithactual

or potential tissue damage. It minimises contact with the injurious stimuli

therebypromotingaprotectiveresponsewhichincludesreflexwithdrawalanda

complexbehavioural strategy to avoid furtherpain [1]. Pain is transmittedvia

thesomatosensorysystem,apartof thenervoussystem,whichhasevolved to

integrate sensory inputs from the body including touch, heat and pain

sensations.Thesesensoryinputsareconductedbytheprimaryafferentneurons

on thedorsal sideof thespinal cord,dorsal rootganglion(DRG)neurons from

theperipheralsites(e.g.skin)tothedorsalhornofthespinalcord,fromwhere

theyaretransmittedtothebrainforperception[2],[3].StudiessuggestthatATP

released by activated microglia in sensory neurons promote nociceptor

signalling and produces fast excitatory potentials in the dorsal root ganglion

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(DRG) neurons [4], [5]. Consequently, Bleehen & Keele (1977), demonstrated

thatATPinducedpainwhenappliedtoablisterbaseinhumanskin.Thisreport

instigatedenormousinterestinthemolecularmechanismbywhichATPcauses

painresulting in thediscoveryofcell-surfacereceptors, thereby facilitatingthe

detectionofextracellularATPandothernucleotidesonsensoryneurons[7].For

instance,itwasobservedthatATPoritsanaloguesinprimaryafferentneurons

produceelectrophysiologicalandbiologicalresponsesthroughligand-gatedion-

channel receptors, called P2X receptors (P2XRs), and G protein-coupled

receptors, called P2Y receptors (P2YRs) [8]–[10]. However, pharmacological

inhibition or suppression of the expression of P2XRs or P2YRs on sensory

neurons or spinal cord had little effect on acute pain evoked by heat or

mechanical pressure in normal animals but inflammatory painwas attenuated

[11], [12], suggesting that the actions of ATP and its receptors may be more

prominentinchronicpainespeciallyinflammatoryandneuropathicpain,thanin

normalconditions.

Inflammatory pain develops from inflammatory responses to trauma in the

peripheraltissuesandmayhavephysiologicalimportanceinthatitcouldassist

woundrepairsincecontactwiththedamagedareaisminimised[13]butitcould

alsoresult fromnon-specific immuneresponsewhichaltersnervefunction[3].

Also,inflammatorypainmaygoawayafterdamageisrepairedandcangenerally

be managed by treatment with analgesics [13]. However, neuropathic pain

usually develops following nerve damage, which may be caused by surgery,

cancer,bonecompression,diabetesorinfection,butdoesnotresolveevenwhen

the damage has been healed [1]. It usually presents as hypersensitivity in the

absence of overt stimulus or can be evoked as in the case of allodynia (pain

resulting from innocuous stimulus) and hyperalgesia (exaggerated pain in

response to noxious stimulus), and is often refractory to treatments including

morphines[1],[14].Evidencessuggestthatthedamageresultsintheactivation

ofmicrogliacellsinthespinalcordleadingtocellhypertrophy,proliferationand

alteredgeneexpression[7],[15].Also,inresponsetoenvironmentalfactors,glia

cells evoke various cellular responses including production and release of

various cytokines and neurotrophic factors causing neuroanatomical and

neurochemicaltransformationintheCNSthatresultsinthehyperexcitabilityof

dorsal horn neurons [2], [13], [16]. Furthermore, several studies suggest that

P2X3,P2X4andP2X7receptorsareimportantinthepathophysiologyofchronic

inflammatory and neuropathic pain [15], [17]–[19]. For instance, it had been

reported that P2X3R knockout resulted in enhanced thermal hyperalgesia in

chronicinflammation[11].Additionally,ithasalsobeenshownthatstimulation

ofP2X4Rresultsinthereleaseofbrain-derivedneurotrophicfactor(BDNF)and

ashiftintheneuronalaniongradientinunderlyingneuropathicpain[20].

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Thisreviewwillattempttoanswerthequestion‘areP2Xreceptorssuitabledrug

targets for the treatment of chronic inflammatory and neuropathic pain?’ by

providingevidences for the roleofP2X2/3RandP2X3R,P2X4R,andP2X7R in

chronicinflammatoryandneuropathicpain.Butbeforethis,itwillprovidesome

information about the different P2X receptor subtypes and their signalling

mechanism.Thereviewwillthendiscussthesuccessandfailureofexperimental

antagonistsforthesereceptorsandconcludewiththefutureperspectiveonP2X

receptortargetedtherapies.

TheP2XReceptorsubtypesandSignallingTheP2X familyof receptorscomprisessevensubtypesofATP-gatedreceptors,

P2X1-7.TheywereinitiallydesignatedP2XbyBurnstockin1985basedontheir

agonistandantagonistselectivityindifferenttissues[21].ThiswasbecauseATP

analogs such as α,β-methylene-ATP selectively activated P2X receptors while

adenosine5’-diphosphatewithβ-sulfurwasmoreselectivefortheP2Yreceptors

[22]. ItthenbecameclearthatP2Xreceptors,wasactivatedselectivelybyATP,

much less activated by ADP, and insensitive to AMP or adenosine or other

purines and pyrimidines. Additionally, this family of receptors have about 40-

50% amino acid sequence identity and each subunit has two transmembrane

domains(TM1andTM2),whichareseparatedbyalargeextracellularcysteine-

rich domain with intracellular N-terminus and C-terminus of considerably

variable length [18], [22]. Also, the channel can form multimers of several

subunits but the most characterised following heterologous expression are

homomericP2X1,P2X2,P2X3,P2X4,P2X6andP2X7channels,andheteromeric

P2X2/3,andP2X1/5.Theyareabundantlyexpressedinneurons,glia,epithelia,

endothelial, bone, muscle and hematopoietic tissues and they are involved in

several physiological processes including cell proliferation, differentiation,

motilityanddeathindevelopment,woundhealing,restenosisandepithelialcell

turnoverasidepain[23],[24].

Furthermore, P2X receptors mediate ATP signalling mainly through three

mechanisms; by forming a ligand-gated Ca2+-permeable cationic channels,

inducing the formation of a large pore, and forming signalling complexeswith

interacting proteins andmembrane lipids [24]. For instance, as a ligand-gated

Ca2+-permeable cationic channel, ATP-mediated activation of P2XRs has been

found to induce more Ca2+ influx than glutamate ion channel and nicotic

acetycholine ionchannelswhileas largepore-formingchannel, somemembers

ofP2Xreceptor familyhavebeenshownto inducethemembranepermeability

orpore-formationuponprolongedstimulationand thesephenomenahasbeen

observedinP2X2,P2X4,P2X7,P2X2/3,andP2X2/5[25];andfinallyassignalling

complexes,ithasbeenshownthatP2Xreceptorscanassociatestructurallyand

functionallywithotherproteinsandlipidstoformATPsignallingcomplexes,an

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exampleofwhichiscalmodulininteractionwithP2X7receptorviaacalmodulin-

binding motif to form a signalling complex necessary for Ca2+-dependent

enhancementofreceptoractivityandmembraneblebbing[24],[26].

P2X3AndP2X2/3ReceptorsInThePathogenesisOfInflammatoryAndNeuropathicPain

TheP2X3receptorwasthefirstmemberoftheP2Xreceptorfamilytobecloned

and shown tobe localisedmainlyon small nociceptive sensoryneurons in the

dorsal root ganglia (DRG) [8]. It was first associated with pain through the

unifyinghypothesisfortheinitiationofpain[10],whichstatedthathighlevelsof

ATPreleasedfromtumourcellsduringabrasiveactivityreachesP2X3receptors

on nociceptive sensory neurons in the DRG [27]. Other studies later used

immunohistochemicalapproach to show thatP2X3receptorsareexpressedon

isolectinB4(IB4)bindingsubpopulationsofsmallnociceptiveneuronsandthat

it co-localiseswith the P2X2 receptors on large-diameter neurons in theDRG,

formingaheteromericP2X2/3receptor[3].ThebindingofATPtothereceptor,

depolarises the DRG by eliciting fast-inactivating currents mediated by the

homomericP2X3receptorswhiletheheteromericP2X2/3receptorswerefound

tomediate slow-desensitising currents [28]. Itwas also found inDRGneurons

isolated fromratswithperipheral inflammation inducedby completeFreund’s

adjuvant (CFA), that ATP application results in the induction of both fast- and

slow-inactivatingcurrents incontroland inflamedneurons,suggestingthat the

activation of this receptors in sensory neurons facilitates the transmission of

nociceptive signals from periphery to the spinal cord [28]. The loss of IB4-

bindingneuronsexpressingP2X3receptorsresultedindecreasedsensitivityto

noxiousstimulisuggestingacriticalrole for thesereceptors inacutepain[27].

However,P2X3andP2X2/3receptorshasnowbeenshowntoplayapivotalrole

in the signalling pathways involved in chronic inflammatory and neuropathic

pain[15],[29].

SeveralstudieshavereportedhighlevelsP2X3R-mediatednocifensivebehaviour

in rat and human models of inflammatory pain [6], [30]. The stimulation of

P2X3RwithATPor its analogue (α,β-methylene-ATP) in an in-vitro-skin-nervemodelresultedintheexcitationofC-mechanoheatpolymodalnociceptors,which

was enhanced in the carrageenan-inflamed skin [31], suggesting that not only

are the levels of ATP in inflamed tissues elevated but P2X3 receptors on the

peripheralnerveendingsininflamedtissuescouldmodulatepaintransmission.

Also,P2X2knockoutandP2X2/3knockoutmicestudiesrevealedthatthedouble

knockoutmicehadsignificantreductioninformalin-inducedinflammatorypain,

inability to code the intensity of non-noxious 'warming' stimuli, inability to

rapidlydesensitiseATP-inducedcurrentsinresponsetoATPapplicationaswell

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asdecreasednociceptivebehaviour compared towildtype [11], [32]. Similarly,

other studies showed that P2X3 antisense oligonucleotides prevented

hyperalgesia in CFA model of chronic inflammatory pain and spinal nerve

ligationmodelofneuropathicpain,whichwerecorrelatedwithdecreasedP2X3

expression in theDRG [15], [33]. This suggests that P2X3R and P2X2/3R are

importantreceptorsinnociceptivepainandthattherapeuticallytargetingthem

withaselectiveantagonistcouldmodulatepainstate.

Furthermore,increasingevidencesuggestthatpersistentinflammationbyCFAis

accompanied by upregulation of both P2X2 and P2X3 receptors in sensory

neurons. It was observed that ATP stimulation of these receptors in inflamed

DRG neurons resulted in elevated expression of P2X2 and P2X3 receptors

resulting in the development of large depolarisation above the threshold for

action potentials compared to control as well as receptor-induced increased

response in DRG neurons observed in vitro and at the peripheral terminals invivo [28]. Also, in another study itwas shown that intraperitoneal injection ofstreptozotocin, a potent P2X3 agonist, in a diabetic neuropathic pain model

results in increased membrane expression of P2X3 receptor and large

enhancement of mechanical allodynia, which was significantly attenuated

following peripheral administration of P2X3 receptor antagonist, pyridoxal-

phosphate-6-azophenyl-2’,4’-disulfonate (PPADs) and TNP-ATP [34]. Similarly,

usinghighlyselectiveP2X3andP2X2/3receptorantagonistA-317491,Jarviset

al. (2002) showed that intraplantar and intrathecal injection of A-317491 into

ratsresultedinantinociceptiveeffectsinCFA-inducedchronichyperalgesiaand

nerve injury-induced hyperalgesia. Hence, this collectively demonstrates the

criticalroleofP2X3receptorsinchronicinflammatoryandneuropathicpainand

that relief from these forms of pain could be achieved by pharmacologically

blockingP2X3orP2X2/3expressionand/oractivation.

However, thecellularmechanismbywhichP2X3Rexpressionand functionare

upregulatedinsensoryneuronsisnotfullyknownalthoughitisthoughtthatthis

could be mediated by interaction between P2X3 and P2X2/3 receptors, and

inflammatorymediators. This is because various inflammatorymediators such

assubstanceP,neurokininB,prostaglandinE2,protonsandbradykininstrongly

enhance P2X-mediated responses [7]. It has also been reported that P2X3

receptoractivationinperipheralnerveendingsofinflamedtissuesresultsinthe

activationofERK in theDRGneurons inratmodelsof inflammationbutnot in

normal rats and that administration of PPADs and TNP-ATP, significantly

decreasedthemechanicalstimulation-evokedactivationofERKinCFA-inflamed

ratsbutnotinnormalrats[35].Moreover,theupregulationofP2X3andP2X2/3

inpainstateshavealsobeenassociatedwithgrowthfactors.Forinstance,ithas

been shown that glial cell line-derived neurotrophic factor (GDNF) and nerve

growth factor (NGF) treatmentDRGneurons increases the expression of P2X3

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receptors, with evidence of NGF-mediated denoP2X3 expression in cells thatdoes not normally express the receptor, suggesting a mechanism of NGF-

mediated hypersensitivity that may contribute to chronic inflammatory pain

[36].

P2X4ReceptorsInThePathogenesisOfInflammatoryAndNeuropathicPain

The first clue to identifying the role of P2X4 receptors in the spinal cord in

neuropathic pain came from pharmacological investigation of pain behaviour

afternerveinjuryusingtheantagonistsTNP-ATPandPPADS[37].Theyreported

that marked tactile allodynia developed following nerve injury which was

reversed by acutely administering TNP-ATP intrathecally but unaffected by

administering PPADS, suggesting that the tactile allodynia depends on P2X4

receptors in the spinal cord. Also, immunohistochemical analysis showed that

manysmallcells,identifiedasmicroglia,inthedorsalhornofthenerve-injured

sidewerepositive forP2X4receptorprotein,andshowedhigh levelsofOX-42

labelling and morphological hypertrophy characteristic of activated microglia.

Additionally,P2X4receptorantisenseoligodeoxynucleotides(ASO)reducedthe

up-regulationofP2X4receptorprotein,therebypreventingthedevelopmentof

nerve-inducedtractileallodyniainmice[37].Moreover,otherearlystudiesina

ratmodelofneuropathicpaininducedbyspinalnerveligation(SNL)reportedan

upregulated expression of P2X4 receptor in activated spinal microglia that

mediate tactile allodynia but not in neurons [38]. They observed that P2X4KO

mice were insensitive to SNL-induced neuropathic pain experienced by wild-

type littermates. This collectively suggests that activation of microglia P2X4

receptor is necessary for pain hypersensitivity following nerve injury.

Consequently, efforts to determine how peripheral injury increases the

overexpression of P2X4 receptor inmicroglia suggest that fibronectinmay be

involved. Itwas observed thatmicroglia cultured on fibronectin-coated dishes

showed a marked increase in P2X4 receptor expression at both mRNA and

protein level while intrathetical delivery of ATP-stimulated microglia to a rat

lumbar spinal cord, showed that microglia treated with fibronectin more

effectively induced allodynia than control microglia [39]. Similarly, it was

observedinadorsalhornmodelofneuropathicpainthattheleveloffibronectin

proteinwaselevatedgreatlyafternerve injuryasP2X4protein level increased

andpharmacologicalinhibitionofthefibronectinreceptorresultedinattenuated

nerveinjury-inducedP2X4receptorupregulationandpainhypersensitivity[40].

Additionally, it was shown in Lyn tyrosine kinase knockoutmice studies, that

fibronectincouldnotinducetheupregulationofP2X4receptorinmicrogliacells

andneuropathicpain inLyn-deficientmice, suggesting that thiskinasemaybe

important in the molecular mechanism mediating the upregulation of P2X4

receptorsinmicroglia[41].

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Furthermore,Coulletal. (2005)showedusingspinalcordslices fromrats that

had displayed pain hypersensitivity following intrathetical administration of

P2X4R-stimulated microglia, that ATP-stimulated microglia positively shifted

the anion reversal potential (Eanion) in lamina I neurons and rendered GABA-receptor- and glycine-receptor-mediated effects depolarising rather than

hyperpolarising these neurons (fig. 1). Previously, it has been shown in a

peripheral nerve injury model of neuropathic pain that this shift in

transmembraneaniongradientwhichchanges inhibitorycurrentstoexcitatory

followingnerveinjury,wasduetotrans-synapticreductionintheexpressionof

the potassium-chloride exporter KCC2 [42]. Moreover, TNP-ATP which can

reversenerve-injury induced allodynia, acutely reverses thedepolarisingEanioninthelaminaIneuronsafterperipheralinjury[37].Therefore,thestimulationof

P2X4 receptor on spinalmicroglia causes neuropathic pain through increased

intracellularchloride(Cl-)inthespinallaminaIneurons(fig.1).

Fig.1.IllustrationofthemechanismbywhichP2X4Rcouldmodulateneuropathicpain [3]. Damaged sensory neurons release ATP, which binds to P2X4 receptorresulting in the release of Ca2+ and activation of p38 MAPK, which induces the

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releaseofbrainderivedneurotrophic (BDNF).TheBDNFactson itsreceptorTrkand the inhibitory interneurons to releaseGABA.Also, actionpotentials from theprimaryafferentterminalinducethereleaseofglutamateandconsequentopeningof AMPAandNMDA receptors. This collectively results in the depolarisation andhyperexcitabilityofthedorsalhornneuronsleadingtoneuropathicpain.

Furthermore,Coulletal.(2005)alsoobservedusingbrainderivedneurotrophic

factor (BDNF) administered intrathecally to normal rats that BDNF induced

tactileallodyniaanddepolarisingshiftinEanioninlaminaIneuronsbyperipheralnerve injury comparable to those produced by ATP-stimulated microglia.

Moreover, the interruption of signalling between BDNF and its receptor TrkB,

either by pharmacological inhibition or by BDNF-sequestering fusion protein

(TrkB-Fc) prevented tactile allodynia caused by peripheral injury or by

intrathecal administration of P2X4-stimulated microglia [7], [20]. Also, it was

observed that theapplicationofATP tomicroglia induced the releaseofBDNF

butthiswasabrogatedbyTNP-ATP,suggestingthatP2X4R-stimulatedmicroglia

releaseBDNFasasignallingfactorleadingtothecollapseofthetransmembrane

anion gradient and subsequent neuronal hyperexcitability observed in

neuropathicpain[3].Additionally, inastudyinvolvingP2X4receptorknockout

mice, primary cultures of dorsal horn microglia showed a reduction in BDNF

stainingafterATPstimulation inwild-typecultures,while in cultures fromthe

P2X4R-mutant mice, application of ATP failed to induce any change [38].

Similarly,otherstudiesinvolvingATP-stimulationofP2X4receptorsresultedin

SNARE-mediatedsynthesisandreleaseofBDNFthatwasdependentontheCa2+

influxthroughP2X4receptorsandsubsequentp38-MAPKactivation(fig.1)[1],

[43].Also,GABAreceptor-mediateddepolarisationcouldproduceanexcitation

through voltage sensitive Ca2+ channels and NMDA receptors [3], thereby

suggestingthatp38-MAPKaswellasGABAandNMDAreceptorsareimportant

in the molecular processes involved in P2X4R-mediated development of

neuropathicpain.

Finally,severalevidencessuggestthatP2X4receptorsareimportant inchronic

inflammatory pain development. For instance, P2X4R knockout mice studies

involving the injection of inflammatory stimuli such as formalin, carrageenan,

andCFAshowed the complete lossof tactileallodynia inP2X4R-deficientmice

compared to control [44], [45]. Also, it was observed that P2X4R deficiency

attenuatesinflammatorystimuli-inducedproductionofprostaglandinE2(PGE2),

whichusually inducespainhypersensitivitybysensitisingandoverexciting the

nociceptive neurons, from macrophages. Additionally, the injection of naïve

animalswithATP-primedmicrogliaormacrophageshasbeenshownto induce

neuropathic and chronic inflammatory pain respectively [45], suggesting that

P2X4 receptors mediate the cellular and molecular mechanisms involved in

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eliciting chronic neuropathic and inflammatory pain, and that selectively

targetingP2X4receptorscouldbeastrategyfortreatmentofchronicpain.

P2X7ReceptorsInThePathogenesisOfInflammatoryAndNeuropathicPain

P2X7receptorsareusuallyconsideredthemostunusualamongtheP2Xreceptor

superfamilyintermsoftheirmolecularandfunctionalcharacteristicsduetothe

presenceofadditional200aminoacidsintheirC-terminal,andthefactthataside

requiring high ATP concentration for activation, prolonged agonist exposure

results in the formation of a larger pore in themembrane [3], [16]. However,

they share a common transmembrane domainwith other P2X receptors. They

are predominantly expressed on immune cells including lymphocytes and

peripheral macrophages and have also been described on microglia and

astrocytes.LikeotherP2XRs,ATPbindingactivatesthereceptorresultinginthe

openingof the receptorpore forpermeationofCa2+,Na+ andK+,which causes

changes intheintracellularconcentrationofpotassiumandconsequentrelease

andactivationofinterleukin-1β(IL-1β),apotentproinflammatorycytokine(fig.

2) [46]. IL-1β induces a cytokine network resulting in the production of

superoxideproducts,nitricoxidesynthase(iNOS),cyclo-oxygenaseandtumour

necrosisfactor(TNF)-α,allofwhichhaveimportantrolesinthegenerationand

maintenanceofpain[17].Thus,manystudieshavebeenperformedtodetermine

itsroleinchronicinflammatoryandneuropathicpain.

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Fig.2ThemechanismofactionofP2X7receptor inIL-1β-mediatedinflammation[47].ATPbindingtotheP2X7receptoractivates itresulting intheopeningof itsnon-selective ionporeandpermeationof Ca2+ andK+.The consequent change inmembrane potential and intracellular Ca2+ andK+ results in the assembly of theinflammasomeandconversionofpro-caspase-1toactivecaspase-1.Pro-caspase-1convertstheinactiveIL-1βintoitsactiveforminthelysosome,beforeitissecretedoutofthecell.Itisalsobelievedthatlipopolysaccharide(LPS)actsonTollreceptor4 to activate NFκB and nuclear transcription of IL-1β, later translated into theinactiveformthatissecretedintothecytoplasm.

P2X7 receptor knockout mice studies have facilitated the investigation of the

roleofthisreceptorinchronicpain.Forinstance,usingP2X7R-/-micemodelsof

chronic inflammatory pain (intraplantar Freund’s complete adjuvant) and

neuropathic pain (partial ligation of the sciatic nerve), itwas shown that pain

hypersensitivitytobothmechanicalandthermalstimuliwascompletely lost in

receptor-deficient mice, while normal nociceptive processing was preserved

[17].Theyalsoreported that thereceptor isupregulated inhumandorsal root

ganglia and injurednerve obtained fromneuropathic painpatients, suggesting

that P2X7 receptor plays an important role in the development of chronic

inflammatory and neuropathic pain via regulation of IL-1β (fig. 2). In another

studyinvestigatingtheresponseofblood-derivedleukocytesfromwildtypeand

P2X7R-/-micetoATP,itwasshownthatP2X7R-deficiencyresultsinlossofATP-

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dependent leukocyte functions including IL-1β production [48]. They also

showed in a monoclonal antibody-induced arthritis model, that P2X7R-/- was

associated with significantly attenuated arthritis compared to the severe

arthriticphenotypeobserved inwildtypemice, suggesting thatATP-dependent

activationofP2X7receptorwasimportantinchronicinflammatorypain.

Furthermore, the pore-forming property of P2X7R has been associated with

development of chronic neuropathic pain. For instance, in a genome-wide

linkage study, it was shown that mice expressing P2X7 receptorsdeficient of

inducingporeformationwerelesssensitivetonerveinjury-inducedneuropathic

pain thanmice expressing the P2X7 receptors that can induce pore formation

[49]. The study also found that within two cohorts of patients; onewith pain

aftermastectomy and another cohort suffering fromosteoarthritis, individuals

expressingP2X7receptordeficientofporeformationreportedloweramountof

painthanthoseexpressingtheP2X7receptorwithpore-formingability.Also,the

administrationofapeptidewhichblocksporeformationbutnotchannelactivity

hasbeenshowntoselectivelyreducenervedinjuryandinflammatoryallodynia

in wildtypemice but not in P2X7R-deficientmice [16]. This suggests that the

pore-forming ability rather than the small ion channel opening alone is key to

thefunctionofP2X7Rinchronic inflammatoryandneuropathicpainsensitivity

andthatselectivelytargetingporeformationcouldbeastrategyfortreatmentof

chronicpain.

Additionally,asideitsinvolvementinP2X4R-mediatedallodynia,p38MAPKhas

also been shown tomediate P2X7R-induced production of IL-1β, cathespsin S

andTNF-α,whichfunctionsinthemaintenanceofmechanicalhypersensitivityin

thespinalcord.Recentstudiessuggestthatthephosphorylationofp38MAPKvia

P2X7receptorinducehyperalgesiainanorofacialpainmodelfollowingchronic

constrictioninjury(CCI)oftheinfraorbitalnerve(CCI-ION)mediatedbyTNF-α

releasefrommicroglia[50].TheyalsoobservedthattreatmentofratswithP2X7

receptor agonist, 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP),

inducedtactileallodyniathroughup-regulationofsolubleTNF-αandp38MAPK

in the trigeminal sensory nuclear complex (TNC) which was inhibited by

SB203580 (a phosphorylated p38 MAPK inhibitor) and Etanercept (a TNF-α

inhibitor). This suggests that the activation of p38 MAPK could be a possible

convergence point in the P2X4 and P2X7 receptor signalling pathways during

neuropathicpain. Indeed,althoughnot inpainmodels,evidenceofastructural

and functional interactionbetweenthe tworeceptorshadbeendescribed [51],

butitiscurrentlyunclearwhethertheheteromericinteractionofthesereceptors

is critical in chronic inflammatory or neuropathic pain. Nevertheless, the

expressionofthesereceptorsonvariouscelltypesinvolvedinpaintransmission,

suggestsapromisingtargetforpharmacologicalintervention.Anexampleofthis

was by Dell’Antonio et al. (2002) who demonstrated in a paw pressure

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experimentthatanirreversibleinhibitorofP2X7receptor,oxidisedATP,hadan

anti-hyperalgesiceffectonCFA-inducedmechanicalhyperalgesia.

In summary, this reviewhave thus fardiscussed thepresent evidences for the

importantroleplayedbyP2X3,P2X2/3,P2X4andP2X7receptorsinmediating

ATP signalling involved in the pathogenesis of chronic inflammatory and

neuropathicpain.This therefore,suggests that thesereceptorsareapromising

targetforpaintherapies.

P2XReceptorsasTherapeuticTargetsinChronicInflammatoryandNeuropathicPain

Several attempts have been made to develop molecules that can specifically

target ATP-mediated signalling through different members of P2X receptor

familyinvolvedinpainsensitivity.Initialattemptsresultedintheidentification

ofSuramin,alargepolysulfonatedmolecule,thatisactiveatmultipleP2receptor

subtypes and its derivatives such as NF023, which was reported to be ~10-

20fold more selective for P2X receptors; NF279 and NF449 which are potent

P2X1 receptor antagonists [53]. Later, PPADS, a potent coenzyme antagonist

againsthumanP2X1,P2X7andP2Y1werediscoveredalongwithitsderivatives

but thenon-selective interactionof these compounds limited theirprogress as

potential therapeutic agents. Other compounds developed as potential

antagonistsincludeoxidisedATP,BrilliantBlueG,KN-62,NF770andNF778but

these compoundswhere unsuccessful due to thewide diversity of recognition

sitesandactionsforwhichATPisacrucialligandresultingintheirnon-selective

interactionwith thereceptors[21].Otherantagonistsdevelopedto targeteach

ofthesereceptorsaredescribedbelow.

P2X3andP2X2/3ReceptorsNucleotide derivatives were developed to modulate the activity of these

receptors.Thus,TNP-ATP,anon-selectivebuthighlypotentantagonistofP2X1

and P2X3 receptors was developed and shown to block the pronociceptive

effects of P2X receptor agonists. However, the ability of TNP-ATP to enter

preclinicalpainstudies formanagementofP2X3-mediatedpainwas limitedby

itspoormetabolicstability in theplasma[21].A-317491 isanothercompound

which showed high capability to competitively block homomeric P2X3 and

heteromeric P2X3 receptors when administered in CFA-induced inflammatory

hyperalgesia although it had limited CNS penetration following systemic

administration thereby requiringhigherdoses or intrathecal administration to

effectively attenuate tactile allodynia following peripheral injury [12], [54].

Other potent P2X2/3 and P2X3 receptor antagonists have been identified

includingRO-4, reported tobe capableof crossing theblood-brainbarrier and

attenuate nerve injury-induced pain models, and has significantly high oral

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bioavailabilityandlowplasmabloodbindingaswellasgoodCNSprenetration;

MK-3901, reported to attenuate both neuropathic and chronic inflammatory

pain in experimental models [55]; AZ-2, reported to effectively reverse CFA-

induced mechanical allodynia following systemic and intraplantar dosing but

ineffective at intrathecal dosing; RO85, reported to have highMulti-parameter

optimization(MPO)scoreandoralbioavailability;andfinallyAF-219reportedto

have high antagonists potency and selectivity for P2X3 and P2X2/3 receptors,

with moderate protein binding and high oral bioavailability [53]. However,

majority of these drug-like P2X receptor antagonists were unsuccessful in

preclinicalstudiesdue tounsatisfactorypharmacologicalprofileswhileAF-219

entered advanced clinical trials for treatment of osteoarthritic knee pain and

bladderpain[24].

P2X4ReceptorsThe discovery of potent antagonist for P2X4 receptors is still in its infancy

althoughthecrystalstructureoftheproteinhasbeensolved.However,TNP-ATP

has been used as putative antagonists to block P2X4 receptor activation. Also,

Brilliant Blue G is also believed to have antagonistic effects on the receptor

activityaswellastheserotoninreuptakeinhibitor,paroxetine,aclinicallyused

antidepressant[53],[56].Interestingly,N-(benzyloxycarbonyl)phenoxazinewasrecently discovered as a potent and selective P2X4 receptor antagonist that

couldhavepotentialtherapeuticbenefit[57].

P2X7ReceptorsThe increased characterisation of the role of P2X7 receptor in different

inflammatory diseases not limited to chronic pain, resulted in different

companies initiating a search for selective receptor antagonists (table 1). This

search resulted in the identification of AACBA1, which has been shown to

attenuate collagen-induced arthritis following prophylactic dosing in rats and,

CE-224,535andAZD9056whichfailedphase IIaandphase IIb clinical trial for

the treatment of rheumatoid arthritic pain respectively for lack of efficacy

comparedtocontrolalthoughtheyhadacceptabletolerabilityandsafetyprofile

[58], [59].Also, systemic screening resulted in thediscoveryof other selective

antagonists includingAZ-11645373, ahighlypotentP2X7 receptorantagonists

whichhasbeenshowntoeffectivelyinhibitATP-andBz-ATP-elicitedcurrentsas

well asA-438079 andA-740003,which has been shown in inflammatory pain

models to reduce thermal hyperalgesia with some success [60], [61]. Similar

effectwasalsoobservedonexperimentalmodelsofneuropathicpain,aneffect

thatismediatedpartlybyatleastspinaland/orsupraspinalsitesofaction[62].

1N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, a hydrochloride salt.

14

Table 1. Recently tested compoundswith potential antagonistic activity onP2X7Receptor[63]Compound Study Trial

PhaseCompleted Observed

OutcomeSideeffects

A-438079 Neuropathicandinflammatorypain

Pre-clinical

Yes Inhibitedmechanicalallodynia andeffective inthe formalinpainmodel

Notevaluated.

A-740003 Inflammatoryandneuropathicpain

Pre-clinical

Yes Analgesiceffect ininflammatoryandneuropathicratmodels

Notevaluated

A-804598 Neuropathicandinflammatorypain

Pre-clinical

Yes Analgesiceffect ininflammatoryandneuropathicratmodels

Notevaluated

A847227 Inflammatoryandneuropathicpain

Pre-clinical

Yes Antiallodyniceffect anddecreased IL-1βreleasedinmousemodel

Notevaluated

AZ10606120 Ligandinteractionand bindingtoP2X7R

Pre-clinical

Yes Had allostericeffect ofreceptoractivity as itbinds non-ATP bindingsite

Notevaluated

AZD9056 RheumatoidArthritis

IIb Yes No significantefficacy

Gastrointestinal(vomiting,nausea anddiarhoea

GSK314118A

Inflammatorypain

Pre-clinical

Yes

Analgesiceffect in ratCFA model ofinflammatoryhyperalgesia

Notrelated

NaturalproductsasnovelsourceofanalgesicsfortargetingATP-mediatedP2Xreceptors

Attemptstoovercometheproblemofnon-selectivityofP2Xreceptorantagonists

resulted in the search for clues from natural products. Natural products have

been shown to be potentially good source of new specific molecules for the

15

treatmentofdifferentpainsyndromes.Thus,severalnaturalproductshavebeen

developed which has antagonistic properties on P2X receptors in chronic

neuropathic and inflammatory pain, including Emolin, Amentoflavone,

Ligunstrazine,puerarinandpurotoxin-1[64].Forinstance,itwasshowninrats

that following formalin-induced pain, the herbal product used in Chinese

medicine called, Ligunstrazine (tetramethypyrazine) derived from Ligusticumwallichii, antagonise P2X3 receptor resulting in the inhibition of membranedepolarisationinducedinDRGsneurons[65].Thesamegroupconfirmedthisin

anotherstudy,wheretheyalsofoundthatLigunstrazineinhibitedP2X3receptor

resulting in reduced ionic currents induced by ATP in theDRG neurons but it

wasnon-selectivesinceitalsoinducesPKCactivation.Similarly,inaneuropathic

painmodel itwas also found that theLigunstrazine inhibited the activationof

P2X3receptorsontheprimaryafferentneurons.

Furthermore, other natural products including puerarin were discovered and

showedto inhibitburn-associatedhyperalgesiabypreventing theupregulation

ofP2X3receptorexpressionintheDRGneurons,withsimilaranalgesiceffecton

neuropathic pain via the same mechanism [64]. Additionally, Emodin, an

anthraquinone obtained from rhubarb extract did not only show analgesic

activity on neuropathic pain through the inhibition of P2X3 receptors in the

primary sensory neurons, but also had antagonistic activity on the P2X7

receptors [64]. Also, a peptide isolated from the venom of the Asian spider

Geolyscosa species, Purotoxin, has been shown to be a potent and selectiveantagonist of P2X3 receptors, as it selectively blocks the P2X3 receptor ion

current in rat neurons [64]. Thus, these evidences collectively suggest that

natural products could offer more potent and selective compounds for

therapeutictargetingofP2Xreceptors.

Conclusion

Pain is the unpleasant sensory and emotional experience associated with the

actual or potential tissue damage. It can simply be classified into acute and

chronicpainwhereacutepaindescribestheactualpainsensationexperiencedin

response to injury or tissue damage. Chronic pain, which can further be

subdividedininflammatoryandneuropathicpain,occursduetoneurochemical

and phenotypic sensitisation of the peripheral central sensory nerves

characterisedby increased sensitivity topainful stimuli (hyperalgesia) and the

perception of pain in response to normally innocuous stimuli (allodynia). Pain

sensitisationusuallyoccursinresponsetotissuedamageorinflammationandis

mediatedbyseveralpronociceptiveneurotransmittersandneurotrophicfactors

suchasATPorBDNF.ExtracellularATPreleasehasnowbeenshowntoelicitand

maintain sensations following inflammation and nerve injury by activating

16

homomeric and heteromeric P2X receptors notably P2X2/3, P2X3, P2X4 and

P2X7 receptors on the peripheral nerves and glial cells (astrocytes and

microglia)inthespinalcord.

Furthermore,severalevidencessuggesttheinvaluableroleP2X2/3,P2X3,P2X4

andP2X7receptorsinthemolecularprocessesinvolvedinpainsensation,while

inhibition of receptor activity using ASO, gene knockout and chemical

compounds collectively showed that the absence of P2X receptors reverts the

ATP-inducednociceptivepain.Thus,suggestingthatinhibitionofthesereceptors

couldbe a potential therapeutic strategy for development of future analgesics.

Massive studies in this area have resulted in the discovery of P2XR-targetted

therapeutics or molecules. These P2XR antagonists include MK3901, AZ-2,

A740003, AZD9056, AZ10606120, A847227, etc which have been shown to

effectivelyblockreceptoractivityalthoughmajorityofthemfailedclinicaltrials.

This is mostly due to the widespread expression of all the different receptor

subtypes,whichrenders thediscriminationbetweenbeneficialandsideeffects

extremely complicated. For instance, studies showed that double-knockout

P2X2/P2X3receptorsinthegustatorynervesofmiceeliminatestasteresponses,

althoughthenervewasresponsivetotouch,temperatureandmenthol[66],[67]

whileP2X4R-/- inmicehadalsobeenshowntocause increasedbloodpressure

and decreased excretion of nitric oxide products in their urine compared to

wildtype [68]. Similarly, the lack of good subtype-selective agents, and when

selectiveligandsareavailable,thelackofanacceptablerouteofadministration

to humans are potential challenges to the development of novel P2XR targets.

Other challenges include the restrictive tissue distribution of some of these

receptors; the ability of channels to form heterotrimers, the ability of the

extracellular domain to undergo substantial conformational rearrangement on

channel opening as well as limited information on the role of P2XRs in

physiologicalandpathologicalprocesses,althoughthisrolesarenowbeginning

tobeidentifiedthroughknockoutmicestudies[21].However,thegoodnewsis

thatcrystalstructuresofsomeofthesereceptorsarenowavailableintheclosed

andATP-bound(open)states.Hence,inthenearestfuturewewillbegintofully

understand the mechanism of action of these receptors and how to

pharmacologicallyinhibitthem.Furtherworkcouldthus,explorethepotentialof

naturalproductsaspossiblesourceoffutureP2XR-targetedtherapeuticsaswell

asidentifyingpossiblemoleculesthatcouldpotentlyinhibitP2X4receptor.

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