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Year:2014|Volume:5|Issue:3|Page:129133 SureshaRN
AmoghimathS
Evaluationofanalgesicactivityofperindoprilinalbinomice
VaibhaviPS
ShruthiSL
RNSuresha,SiddammaAmoghimath,PSVaibhavi,SLShruthi,MKJayanthi,HLKalabharathi
JayanthiMK
DepartmentofPharmacology,JSSMedicalCollege,Mysore,Karnataka,India
KalabharathiHL
DateofWebPublication 23Jul2014 SearchinGoogleScholar
for

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

Relatedarticles
SourceofSupport:None,ConflictofInterest:None
AngiotensinII
angiotensin(17)
endogenousopioidand
1
prostaglandinE2

DOI:10.4103/22314040.137423 AccessStatistics
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Abstract *Registrationrequired(free)

Theaimwastoevaluatetheanalgesicactivityofperindoprilinchemical,thermalandmechanicalpainonSwissalbino

mice.Atotalof54albinomice(Swissstrain)weighing2530gwereallocatedtoeachexperimentalmodelandineach
Inthisarticle
modeltherewerethreegroups.Thecontrolgroupreceivednormalsaline(25ml/kg)perorally,standardgroupreceived
pentazocine(10mg/kg)intraperitonealandtestgroupsreceivedperindopril(1mg/kg)perorally.Perindoprilandnormal Abstract
salinewasadministered2hbefore,whereasthepentazocinewasadministered15minpriortoEddy'shotplate,writhing Introduction
andtailclipmethods.Thedecreaseinnumberofwrithes,thedelayinreactiontimeintailclipandEddy'shotplate Materialsandme...
methoddenotedtheanalgesicactivity.Perindoprildecreasedthenumberofwrithes,delayedthereactiontimeintailclip Results
andEddy'shotplatemethodconsiderablywhencomparedwithcontrol(normalsaline),butlesswhencomparedwith Discussion
standard(pentazocine).Perindoprilexhibitsanalgesicactivityinthermal,chemical,andmechanicalpainmodelsin
Conclusion
albinomice.
References
Keywords:AngiotensinII,angiotensin(17),endogenousopioidandprostaglandinE2 ArticleFigures
ArticleTables

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SureshaRN,AmoghimathS,VaibhaviPS,ShruthiSL,JayanthiMK,KalabharathiHL.Evaluationofanalgesic
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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]
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.

References

1. HilalDandanR.Reninandangiotensin.In:BrutonLL,editor.GoodmanandGilmansthePharmacologicalBasis
ofTherapeutics.12thed.China:McGrawHill2011.p.72135.

2. KumarV,AbbasAK,FaustoN.Acuteandchronicinflammation.In:KumarV,editor.RobbinsandCotron
PathologicalBasisofDiseases.8thed.NewDelhi:Elsevier2008.p.4855.

3. DasUN.AngiotensinIIbehavesasanendogenousproinflammatorymolecule.JAssocPhysiciansIndia
200553:4726.

4. SiragyHM,CareyRM.Thesubtype2(AT2)angiotensinreceptorregulatesrenalcyclicguanosine3,5
monophosphateandAT1receptormediatedprostaglandinE2productioninconsciousrats.JClinInvest
199697:197882.

5. ChampePC.Drugsaffectingthecardiovascularsystem.In:HarveyRA,editor.LippincottsIllustratedReviews.4
thed.NewDelhi:LippincottWilliamsandWilkins2009.p.1849.

6. FurstDE,UlrichRD,VarkeyAltamiranongC.Nonsteroidalantiinflammatorydrugs.In:KatzungBG,editor.
BasicandClinicalPharmacology.11thed.Mumbai:McGrawHill2009.p.1815.

7. ScuacherMA,BasbaumAI,WayWL.Opoidanalgesicandantagonist.In:KatzungBG,editor.BasicandClinical
Pharmacology.11thed.Mumbai:McGrawHill2009.p.6229.

8. KumaeGS,RajeshK,SengottuveluS.Evaluationofanalgesicandantiinflammatoryactivityofmethanolicextract
 ofcocculushirsutosleaves.IntResJPharm20112:2304.

9. KosterR,AndersonM,DeBeerEJ.Aceticacidforanalgesicscreening.FedProc195918:4128.

10. BianchiC,FranceschiniJ.ExperimentalobservationsonHaffnersmethodfortestinganalgesicdrugs.BrJ
PharmacolChemother19549:2804.

11. TrivediD,AnandE,SinghGN,MohantyI,JaiswalJ,JaiPrakashetal.Analgesicagents.In:GuptaSK,editor.
DrugScreeningMethods.2nded.NewDelhi:Jaypee2009.p.4628.

12. HallJE.SomaticsensationsIIpain,headacheandthermalsensations.In:HallJE,editor.GuytonandHallText
BookofMedicalPhysiology.12thed.NewDelhi:Saunders2011.p.5838.

13. KellerS,FrishmanWH.Neuropsychiatriceffectsofcardiovasculardrugtherapy.CardiolRev200311:7393.

14. RabinowitzI,ReisS.Doctor,theresaflyinmysoup!Angiotensinconvertingenzymeinhibitors,endogenous
opioidsandvisualhallucinations.IsrMedAssocJ20013:9634.

15. OmarME,SalamA,ElShenaveyS,NofalSM.Effectoframiprilvalsartanandcandesartanonthermaland
visceralpaininmice.JPharmacolToxicol20072:53341.

16. XingJ,KongJ,LuJ,LiJ.Angiotensin(17)inhibitsneuronalactivityofdorsolateralperiaqueductalgrayviaa
nitricoxidepathway.NeurosciLett2012522:15661.

Figures

[Table3]

Tables

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

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