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ORIGINALARTICLE
Year:2014|Volume:5|Issue:3|Page:129133

Evaluationofanalgesicactivityofperindoprilinalbinomice
RNSuresha,SiddammaAmoghimath,PSVaibhavi,SLShruthi,MKJayanthi,HLKalabharathi
DepartmentofPharmacology,JSSMedicalCollege,Mysore,Karnataka,India
DateofWebPublication

23Jul2014

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SureshaRN
AmoghimathS
VaibhaviPS
ShruthiSL
JayanthiMK
KalabharathiHL
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AmoghimathS
VaibhaviPS
ShruthiSL
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CorrespondenceAddress:
SiddammaAmoghimath
DepartmentofPharmacology,JSSMedicalCollege,SSNagar,Mysore570015,Karnataka
India

Relatedarticles
SourceofSupport:None,ConflictofInterest:None

AngiotensinII
angiotensin(17)
endogenousopioidand
prostaglandinE2

DOI:10.4103/22314040.137423

Abstract

Theaimwastoevaluatetheanalgesicactivityofperindoprilinchemical,thermalandmechanicalpainonSwissalbino
mice.Atotalof54albinomice(Swissstrain)weighing2530gwereallocatedtoeachexperimentalmodelandineach
modeltherewerethreegroups.Thecontrolgroupreceivednormalsaline(25ml/kg)perorally,standardgroupreceived
pentazocine(10mg/kg)intraperitonealandtestgroupsreceivedperindopril(1mg/kg)perorally.Perindoprilandnormal
salinewasadministered2hbefore,whereasthepentazocinewasadministered15minpriortoEddy'shotplate,writhing
andtailclipmethods.Thedecreaseinnumberofwrithes,thedelayinreactiontimeintailclipandEddy'shotplate
methoddenotedtheanalgesicactivity.Perindoprildecreasedthenumberofwrithes,delayedthereactiontimeintailclip
andEddy'shotplatemethodconsiderablywhencomparedwithcontrol(normalsaline),butlesswhencomparedwith
standard(pentazocine).Perindoprilexhibitsanalgesicactivityinthermal,chemical,andmechanicalpainmodelsin
albinomice.
Keywords:AngiotensinII,angiotensin(17),endogenousopioidandprostaglandinE2
Howtocitethisarticle:
SureshaRN,AmoghimathS,VaibhaviPS,ShruthiSL,JayanthiMK,KalabharathiHL.Evaluationofanalgesic
activityofperindoprilinalbinomice.JAdvPharmTechnolRes20145:12933
HowtocitethisURL:
SureshaRN,AmoghimathS,VaibhaviPS,ShruthiSL,JayanthiMK,KalabharathiHL.Evaluationofanalgesic
activityofperindoprilinalbinomice.JAdvPharmTechnolRes[serialonline]2014[cited2016Dec8]5:12933.
Availablefrom:http://www.japtr.org/text.asp?2014/5/3/129/137423
Introduction

Angiotensinconvertingenzyme(ACE)catalyzetheformationofangiotensinIIfromangiotensinI.Thisenzymeoccurs
notonlyintheplasmabutalsointhekidneys,brain,adrenalglands,ovaries,andpossibleothertissues.AngiotensinIIis
apotentvasoconstrictorandblockadeofitssynthesisbyACEinhibitorsarecurrentlythemedicationofchoicein
managementofhypertensionandcardiacfailure.[1]

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Painisveryoftenassociatedwithinflammation.Inflammationisanormalresponsetoanynoxiousstimulusthat
threatensthehostandmayvaryfromlocalizedresponsetoageneralizedone.Itisacomplexprocessinvolvingreleaseof
chemicalsfromtissuesandmigratingcellsandvariousmediatorssuchasprostaglandins,leukotriene's,andplatelet
activatingfactors.[2]
AngiotensinIIregulatesvasculartone,stimulatesthereleaseofproinflammatorycytokines,activatesnuclearfactor
kappaB(NFkB),increasesoxidantstressandthus,itfunctionsasaninflammatorymolecule.AngiotensinIIincreases
thereleaseofreactiveoxygenspecies(ROS).ROSactivateNFkB(NFkB,knowntoinitiateinflammatoryprocess)that
increasesthetranscriptionofproinflammatorycytokines,adhesionmolecules,andnicotinamideadeninedinucleotide
phosphate(NADPH)oxidase.AngiotensinIIenhancesROSproductionbyactivatingNADPHoxidaseandstimulatesthe
DNAbindingactivityofNFkBinhumanneutrophils.AngiotensinIIincreasesthesynthesisandconcentrationoftumor
necrosisfactor,interleukin6,andchemokinemonocytechemoattractantprotein1.ItalsoelevatestissuelevelsofNF
kB,andresultsininflammatorycellinfiltration.[3]
AngiotensinIIincreasestheformationofprostaglandinE2(PGE2)byinhibitingtheenzymePGE9ketoreductaseand
increasingthecyclooxygenases2(COX2)activitybyincreasingcyclicadenosinemonophosphate.PGE2sensitizespain
receptorsatefferentnerveendingstomediatorsofpainandamplifyalgesia.[4]
Painisalsomediatedbyactivatingtheafferentfibersinsympatheticnerves.AngiotensinIIincreasesthereleaseof
epinephrineandnorepinephrinefromthesympatheticnerveterminalsandadrenalmedullabystimulatingautonomic
ganglia.[5]
Mostcommonlyemployedpharmacotherapiesforpainfulinflammatoryconditionsarenonsteroidalantiinflammatory
drugssuchasaspirin,ibuprofen,acetaminophen,naproxen,iterocoxibandsoon.Theseareknowntocausesideeffects
suchaserosivegastritis,pepticulceration,increaseinbleedingtime,worseningofrenalfunctionin
renal/cardiac/cirrhoticpatients,hyperkalemia,higherriskofstroke,myocardialinfarction,andosteoarthritis.[6]
Otherdrugsusedtoalleviatepainareopioids,whichareknownforsideeffectslikesedation,constipation,respiratory
depression,tolerance,anddependence.[7]Thus,thisstudyisaimedatevaluatingdrugsinthetreatmentofpainwithless
adverseeffectandmoreorequiefficaciouscomparedtotheexistingdrugs.
Perindopril(ACEinhibitor),whichdecreasestheangiotensinIIformationafterabsorptionisconvertedinto
perindoprilat,theactivemetabolite.Ithasthehalflifeof117h.Andthepeakplasmaconcentrationisattainedafter34
h.Perindopriliseliminatedviaurine(unchangeddrug,asmetabolite).[1]
Hypothesis
Thus,itmaybehypothesizedthatperindopril(ACEinhibitor)canexhibitanalgesicactivitybyinhibitingtheformation
ofangiotensinII,decreasingtheproductionofPGE2andCOX2,decreasingthecentralsympatheticactivity,increasing
angiotensin(17)andendogenousopioidmechanism.
Theaimofthisstudywas
1.Toevaluatetheanalgesicactivityofperindopril
2.Tocomparetheanalgesicactivitywiththatofstandarddrug(pentazocine).

ThethermalpainwasassessedthroughEddy'shotplateDeshBiologicalWorks,Ambala,chemicalpainthrough
Writhingmethodandmechanicalpainthroughtailclipmethod.
Materialsandmethods

ThestudywasconductedaftergettingapprovalfromInstitutionAnimalEthicalCommittee(IAEC).CPSEAapproval
numberfromIAECof:JSSMC/PR/IAEC/17/26(01)/201314.
Albinomiceofeithersexofaverageweight3050gaged34monthswereusedinexperiments.Thealbinomicewere
bredincentralanimalhouseofJSSMedicalCollege,Mysore.ThestudywasdoneinDepartmentofPharmacology
duringSeptember2013.Animalswereacclimatizedtothelaboratoryconditionsforatleast1hbeforetestingandwere
usedduringexperiments.Thedosesofdrugswerebasedonthehumandailydoseconvertedtothatofmiceaccordingto
PagetandBarnes(1962).
Drugsandchemicals
Perindopril1mg(SerdiaPharmaceuticals,India)wasdissolvedindistilledwaterimmediatelybeforeusedorally,glacial
aceticaciddilutedindistilledwatertoprovide0.06%solutionforintraperitonealinjection,pentazocine(Taj
Pharmaceuticals,India)andnormalsaline.
Themiceweredividedintothreegroupscontainingsixanimals(n=6)ineachgroup(control,standard,andtestgroup).
Thetestdrugperindopril1mg/kgandnormalsaline25ml/kgwasadministeredorally2hprior.Standarddrug
pentazocine10mg/kgwasadministeredintraperitoneal15minpriortotheexperiment.Significantanalgesiaof
pentazocineoccursbetween15and30min.
Group1:Normalsaline25ml/kg(oral)
Group2:Pentazocine10mg/kg(intraperitoneal)
Group3:Perindopril1mg/kg(oral).

Analgesicactivity
Eddy'shotplate
Miceweighing2030gwereused.Micewereplacedonthehotplate,whichconsistsofelectricallyheatedsurface.
Temperatureofthehotplatewasmaintainedat55C.Responsessuchasjumping,withdrawalofthepawsandlickingof
thepawswereobserved.Thetimeperiod(latencyperiod)whenanimalswereplacedanduntilresponsesoccurwas
recordedbythestopwatch.Perindoprilwasadministeredorallyandlatencyperiodwasrecordedafter0,30,60,90and
120min.Thesevalueswerecomparedwiththestandarddrugpentazocineandcontrolnormalsaline.Thismodel
evaluatesthecentralpain.[8]
Writhingmethod
Miceweighing2030gwereused.Aceticacid0.06%wasinjectedintraperitonallyineachanimal.Theanimalreacted
withacharacteristicstretchingbehaviorthatis,aseriesofconstrictionsoccurthattravelalongtheabdominalwall,
sometimesaccompaniedbyturningmovementsofthebodyandextensionofthehindlimbs.Thisresponseofwrithing
wasrecorded.Testgroupanimalswereadministeredperindoprilpriortoadministrationofaceticacidintraperitonally.
Later,micewereplacedindividuallyintoglasschambersandnumberofwritheswererecordedfor15min.Thismodel
evaluatesperipheralpain.[9]

Thetimeperiodwiththegreatestpercentageofinhibitionwasconsideredthepeaktime.
Tailclipmethod
Miceweighing2530gwereused.Haffner'sclipwasplacedattherootofthetailofthemicetoapplynoxiousstimulus.
Aquickresponseoftheanimalwasseenasbitingthecliportail,wheretheclipwasplaced.Thereactiontimebetween
applicationoftheclipandtheresponsewasnotedbyastopwatch.Testdrugperindoprilwasadministeredorally.After
15,30and60min,sameprocedurewasrepeatedandreactiontimewasmeasured.Thismodelevaluatesthecentralpain.
[10]

Statisticalanalysis
Theresultwasanalyzedbycalculatingthemeanvalues,standarddeviation,andanalysisofvariance,posthoctest
(Bonferroni).IBMSPSSstatisticsIBMCorporationandOther(s)1989,2012softwarewasusedforstatisticalanalysis
purpose.Totesttheresultsofstudyforthecorrespondingdegreesoffreedomthevalueswerecomparedat0.05levelof
significance.P<0.05wasconsideredassignificant.
Results

0Eddy'shotplate
Thus,thelatencyperiodofperindoprilwassignificantly(P<0.05)goodwhencomparedtocontrolattimeperiod30120
min,whereasthelatencyperiodofthestandardwasmoresignificant(P<0.05)whencomparedtoperindoprilatalltime
intervalsofexperimentation[Table1]a,band[Table4].
Table1:
Clickheretoview

Writhing
Perindoprilwhichwasgivenorally2hbeforeintraperitonealinjectionofaceticacidsignificantlyreducedthenumberof
writhes.Significantinhibitionofthewrithingresponsewasobservedaftertheadministrationofperindopril1mg/kgwhen
comparedtonormalsalinecontrolgroup.
Thenumberofwrithesofperindoprilwaslesswhencomparedtostandard,whereasthenumberofwrithesofstandard
drug(pentazocine)werelesswhencomparedtoperindoprilandnormalsaline.Whencomparedtocontrol,thepercentage
inhibitionofperindoprilwas56.39%andthatofthestandardwas84.35%[Table2].
Table2:
Clickheretoview

Tailclip
Thus,themeanreactiontimeofperindoprilwassignificantly(P<0.05)goodascomparedtocontrolattimeperiod30
120min,whereasthelatencyperiodofthestandardwasmoresignificant(P<0.05)whencomparedtoperindoprilatall
timeintervalsofexperimentation[Table3]a,band[Table5].

Table3:
Clickheretoview
Table4:ThepercentanalgesicactivityofperindoprilwhencomparedtocontrolEddy's
hotplatemethod
Clickheretoview
Table5:Thepercentanalgesicactivityofperindoprilwhencomparedtocontroltailclip
method
Clickheretoview

Discussion

Painisanunpleasantsensoryandemotionalexperienceassociatedwithactualandpotentialtissuedamage.Painis
producedbytheexcitationofnociceptorsortheirafferentfreenerveendings.Therearetwotypesofpain,fastpainand
slowpain,mediatedthroughAdfibersandCfibers.Nociceptionisthemechanism,wherebynoxiousperipheralstimuli
aretransmittedtothecentralnervoussystem.Nociceptivefibersterminateinthesuperficiallayersofthedorsalhorn,
formingsynapticconnectionswithtransmissionneuronsrunningtothethalamus.Nociceptorsreleaseglutamate,
substancePcontributingtoneurogenicinflammation.[11]
Transmissioninthedorsalhornissubjectedtovariousmodulationsconstitutingthegatecontroltheory.Descending
inhibitorypathwaysfromthemidbrain(periaqueductalgreyarea)andbrainstem(nucleusraphemagnus)exertastrong
inhibitoryeffectondorsalhorntransmission.Maintransmittersinthispathwayareenkephalinand5hydroxytryptamine.
ItcausesbothpresynapticandpostsynapticinhibitionofincomingTypeCandTypeAdpainfiberswheretheysynapsein
thedorsalhorn.[12]
Nervefibersderivedfromtheperiventricularnucleiandfromtheperiaqueductalgreyareasecreteenkephalinattheir
endings.Fibersoriginatinginthisareasendsignalstothedorsalhornsofthespinalcordneuronstosecreteenkephalin.
TheenkephalincausebothpresynapticandpostsynapticinhibitionofincomingTypeCandAdpainfiberswherethey
synapseinthedorsalhorns.Thus,theanalgesicsystemblockpainsignalsattheinitialentrypointtothespinalcord.[13]
AngiotensinIIactsasanalgesicandinflammatorymolecule(asdescribedearlier)andincreasesthesympathetictone
thusaidingtopain.Perindopril(ACEinhibitor)causesanalgesiceffectbydecreasingthecentralsympatheticactivity,
inhibitingthesynthesisofangiotensinIIandbyeliminatingtheeffectofangiotensinII.
Importantendogenousopioidsubstancesareendorphan,metenkaphalin,leuenkephalinanddynorphin.Thetwo
enkephalinsarefoundinthebrainstemandspinalcordareknowntoinvolveinanalgesia.Perindopril(ACEinhibitor)
inhibitenkephalinase,thisisthepeptidaseresponsibleforthehydrolysisofenkephalins,henceincreasingthe
endogenousopioids.[13],[14]StudieshaveshownthatACEinhibitorsexertanalgesiceffectduetotheactiononcentral
nervoussystem,whichincreasesenkephalinandendorphanlevels.ThevisceralantinoceptiveeffectofACEinhibitor
isduetoopioiddependentmechanism.[15]
Perindopril(ACEinhibitor)increasesthelevelsofangiotensin(17)byfollowingtwomechanisms,
1.BypassingtherequisiteproductionofangiotensinIIand,
2.Byinhibitingthehydrolysisofangiotensin(17).

Perindoprilsubstantiallyaugmentscirculatinglevelsofangiotensin(17)andincreasesthepeptidehalflife.Angiotensin
(17)increasesnnitricoxidesynthasederivednitricoxide(NO)levels.IncreasedNOsignificantlydecreasesthe
dischargerateofspontaneousactionpotentialindorsolateralperiaqueductalgray(PAG)neurons.ThemidbrainPAGisa
neuralsiteforseveralphysiologicalfunctionsrelatedtocardiovascularregulation,painmodulationandbehavioral
reactions.[16]Hence,angiotensin(17)isconsideredasanimportantbiologicallyactivecomponentoftherenin
angiotensinsystemthatplaysaninhibitoryroleinthedorsolateralPAGviaaNOdependentsignalingpathway.
Therefore,angiotensin(17)isinvolvedinpainmodulationbyactingonPAGthroughNOdependentsignaling.

Inthisstudy,threeanalgesicmodels,aceticacidinducedwrithingreflex,Eddy'shotplateandtailclipmethodwereused
toevaluatetheanalgesicactivityofperindopril.Thesemodelsinvolvedthelatencyperiod,percentofinhibitionand
meanreactiontimetoapainfulstimulus.Thestimulusinthesemodelsarethermal(Eddy'shotplate),chemical(acetic
acidinducedwrithing)andmechanical(tailclip).
InEddy'shotplatemodel,whencomparedtocontrolthelatencyperiodofperindoprilwasalmostequalat0minand
graduallyincreasingfrom30min,peakingat60minand90min.Andthepercentanalgesicactivityofperindoprilwas
graduallyincreasingfrom30min.Although,thelatencyperiodandpercentanalgesicactivityofstandard,when
comparedtoperindoprilwasmoreatalltimeperiods.
Inwrithingmodel,whencomparedtocontrolperindoprilsignificantlydecreasedthenumberofwrithesandthe
percentageinhibitionof54.39%wasobserved.
Intailclipmodel,whencomparedtocontrolthemeanreactiontimeandpercentanalgesicactivityofperindoprilstarted
increasinggraduallyat30min,peakingat60minand90mintherebydecreasingat120min.While,themeanreaction
timeandpercentanalgesicactivityofstandard,whencomparedtoperindoprilwasmoreatalltimeperiods.
Conclusion

Thetestdrugperindoprilshowssignificantanalgesicactivitywhencomparedtothatofcontrolinallthethreeestablished
experimentalmodelsofpain.Theanalgesicactivitywasmaximumat60minand90min.Thepossiblemechanismisdue
todecreasingthecentralsympathetictone,increaseinthereleaseofendorphanandenkephalinlevelsinthespinal
cord,increasingtheangiotensin17levelsanddecreasingPGE2andCOX2.
Thus,toconclude,perindoprilpossiblyexhibitsitsanalgesicactivitybothbycentralanalgesicactivity(Eddy'shotplate
andtailclip)throughreleaseofendorphanandenkephalinsandalsoperipheralanalgesicaction(writhingmethod)
throughinhibitionofCOX2andPGE2.

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RabinowitzI,ReisS.Doctor,theresaflyinmysoup!Angiotensinconvertingenzymeinhibitors,endogenous
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XingJ,KongJ,LuJ,LiJ.Angiotensin(17)inhibitsneuronalactivityofdorsolateralperiaqueductalgrayviaa
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Figures
[Table3]

Tables
[Table1],[Table2],[Table4],[Table5]

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