Review Article The mechanism of action of aspirin J - PDF document

ReviewArticleThemechanismofactionofaspirinJ.R.Vane,R.M.BottingTheWilliamHarveyResearchInstitute,St.Bartholomew’sandtheRoyalLondonSchoolofMedicine,CharterhouseSquare,LondonEC1M6BQ,UKThetherapyofrheumatismbeganthousandsofyearsagowiththeuseofdecoctionsorextractsofherbsorplantssuchaswillowbarkorleaves,mostofwhichturnedouttocontainsalicylates.Followingtheadventofsyntheticsalicylate,FelixHoffman,workingattheBayer *Correspondingauthor.Tel.:+44-207-882-6179;fax:+44-207-882-ThrombosisResearch110(2003)255–258 Ofcourse,itisquitepossiblethatDreserwasawareofbothpossiblederivationsandthattheambiguousnamewasadeliberateandfelicitouscontrivance.Ofinterest,‘‘Eusparin’’wassuggestedintheoriginaldocumentasanalternativename.1.EarlyexplanationsfortheactionofaspirinBefore1971,littlewasknownabouttherealmechanismofactionofaspirin-likedrugs.Theyproducedananti-inflammatoryeffectthatwasqualitativelyandquantitativelydifferentfromthatoftheanti-inflammatorysteroids,andtheiranalgesicactionwasofadifferentnaturethanthatproducedbyopiates.Aspirin-likedrugsareweakanalgesicscomparedwith‘‘strong’’narcoticanalgesicslikemorphine.Theyareeffectiveinclinicalpainoflowormoderatebutnothighintensitysuchaspostoperativepain,osteoarthritis,rheumatoidarthritis,ankylosingspondilytisandsomeformsofheadacheheadache.Aspirin-likedrugsareeffectiveinexperi-mentalmodelsinvolvingtheinductionofapreviousin-flammatorystateandblockthedelayedstretchingresponseinducedwithanintraperitonealinjectionofphenylbenzo-quinoneordiluteaceticacidinmice.Theyarenoteffectiveagainstnociceptionofshortdurationinducedbypinchingorstimulatingthetailortoesofmouse,ratorguineapig.Guzmanetal.al.andLimetal.al.provideddefinitiveevidenceoftheperipheralanalgesicactivityofaspirin-likedrugs.Whatthendoesaspirindointheperipherytodecreasenociceptionorpain?Manybiochemicaleffectsofaspirin-likedrugshavebeendocumentedandtheoriesbasedontheseeffectshavebeenabandoned.Itwasobserved,forexample,thatmostofthesedrugsuncoupledoxidativephosphoryla-tionandthatseveralsalicylatesinhibiteddehydrogenaseenzymes,particularlythosedependentonpyridinenucleo-tides.Someaminotransferasesanddecarboxylaseswerealsoinhibited,aswereseveralkeyenzymesinvolvedinproteinandRNAbiosynthesis.Alloftheseinhibitoryactionsonswereatsometimeinvokedtoexplainthetherapeuticactionsofaspirin.Aproblemwithmostofthesetheorieswasthattheconcentrationofthedrugrequiredforenzymeinhibitionwasinexcess(sometimesgreatlyinexcess)oftheconcentrationtypicallyfoundintheplasmaaftertherapy,andtherewasinvariablyalackofcorrelationbetweentheabilityofthesedrugstoinhibitparticularenzymesandtheiractivitiesasanti-inflammatoryagents.Perhapsthemostseriousimped-imentofalltoacceptanceofanyoftheaboveideaswasthattheirproponentscouldnotprovideaconvincingreasonwhyinhibitionofanyoftheseenzymesshouldproducetheanti-inflammatory,analgesicandantipyreticeffectsofaspirin.2.AspirinandtheprostaglandinsystemItwasagainstthisbackgroundofknowledgethattheinvestigationofaspirin’sactionwastakenoverbyprosta-glandin(PGs)researchers.PiperandVaneaneusedisolatedlungsperfusedwithKrebs’solutionfromsensitisedguineapigs.Thepurposewastodetectsubstancesreleasedduringtheanaphylacticreaction,includinghistamineandSRS-A,bothofwhichhadbeenknownformanyyearsaspossiblemediatorsofanaphylaxis.TheyusedthetechniqueofcontinuousbioassaywiththecascadebioassaysystemdevelopedbyVaneaneinthemiddle1960sforusewithbloodorartificialsaltsolution.Asexpected,PiperandVanefoundthereleaseduringanaphylaxisofhistamineandSRS-A,buttheyalsofoundsomepreviouslyunreportedsubstances:PGs,(mainlyPGEbutsomePGF)andanother,veryephemeralsubstancethattheycalled‘‘rabbitaortacontractingsubstance’’(RCS)basedontheassaytissuethatidentifiedit.InthelungperfusateRCShadahalf-lifeofabout2min;evenwhencooledtoafewdegreesabovefreezing,itremainedstablefornomorethan20min.Itwasidentifiedin1975asthromboxaneAbyHambergetal.al..ItwasRCSthatprovidedthefirstcluetotherelationbetweenaspirinandthePGs.InthecourseoffurtherexperimentsinvolvingRCS,PiperandVanediscoveredthatinsomepreparationsRCSwasreleasedbybradykinin.Thissuggestedthataspirin’sabilitytominimisesomeeffectsofbradykininmightbeduetoitsblockingofRCSrelease.ThisideawasconfirmedwhenPiperandVaneanepresentedexperimentalevidencethatthereleaseofRCSfromisolatedguineapiglungsduringanaphylaxiswasblockedbyaspirin.TheseguineapiglungexperimentsalsoindicatedthatwheneveraspirinblockedRCSrelease,therewasasmallercontractionofthetissuesthatassayedPGsandaself-evidentreductioninPGoutputafteraspirin.ThenaturalresultoftheseexperimentswastomovethefocusofVane’sattentionfromRCStowardPGs.‘‘WhileIwaswritingareviewpaperovertheweekend’’,herecalled,‘‘includingtheresultsofsomeoftheseexperiments,athoughtoccurredtomethatperhapsshouldhavebeenobviousearlieron.Inalltheseexperiments(andinthoseofmanyotherworkers),the‘release’ofPGsmustinfactamounttofreshsynthesisofPGs.Thatis,PGoutputintheseexperiments,thoughverylow,wasstillfarhigherthanthetissues’initialcontentofthehormones.Evidently,then,thevariousstimuli,mechanicalandchemical,whichre-leasedPGs,wereinfact‘turningon’thesynthesisofthesecompounds.AlogicalcorollarywasthataspirinmightwellbeblockingthesynthesisofPGs.’’VaneimmediatelytestedthisexcitingideaonthefollowingMondaymorning.Intheabsenceoframseminalvesicles,fromwhichthesynthetaseenzymewasusuallyobtained,heusedthesupernatantofabrokencellho-mogenatefromguineapiglung,thesamekindofprepa-rationinwhichAnggardandSamuelsson[11]haddetectedthegenerationofPGEandPGFin1965.Aliquotsofthesupernatantwereincubatedwitharachi-donicacidanddifferentconcentrationsofaspirin,indo-methacinorsodiumsalicylate.PGFgenerationwasJ.R.Vane,R.M.Botting/ThrombosisResearch110(2003)255–258 estimatedbybioassayafter30minofincubationat37Therewasadose-dependentinhibitionofPGformationbyallthreedrugs,indomethacinbeingthemostpotentandsodiumsalicylatetheleast.Threeotherdrugs,morphine(anopiateanalgesic),hydrocortisone(asteroidalanti-inflamma-tory)andmepyramine(anantihistamine),hadlittleornoVaneanepublishedtheresultsoftheseexperimentsinNaturein1971.Twootherreportsinthesameissuelentsupporttohisfindingsandextendedthemconsiderably.Bothstudiesoriginatedfromthesamedepartment,andbycoincidence,oneofthesestemmedfromanentirelyinde-pendentlineofinvestigation.SmithandWillisilliswereinvestigatingtheeffectsofaspirinonplateletbehaviour.Venousbloodsampleswereobtainedfromthreecolleaguesbeforeand1haftertaking600mgofaspirinorally.Plateletswereisolated,washedandincubatedwiththrombinandthesupernatantwastestedforthepresenceofvarioussubstancesincludingPGs.NoconsistentchangeswereseeninthereleaseofanyofthesubstancesexceptthePGs,whichweresubstantiallyinhibitedafteraspirin.IndomethacinalsoblockedPGre-leasewhentakenorallyorwhenaddeddirectlytotheplateletsinvitro.Theimportanceofthisstudylayinitsdemonstrationthatthesedrugswereactivenotonlyinguineapiglungsinvitrobutalsoinhumans,inplateletsandafteroraladministration.Inotherwords,theaspirineffectwasnotrestrictedbyspecies,tissueorrouteofadministration.TheseconclusionsderivedsupportfromthefinaltypeofexperimentreportedinthatissueofNature.Ferreiraetal.al.demonstratedthattheaspirin-likedrugsblockedPGreleasefromtheperfused,isolateddogspleen.Inthesameyear,CollierandFlower[15]reportedinLancetthatadministrationofaspirininhibitedhumanseminalPGproduction.3.Correlationofanti-enzymeactivityofaspirinwithitstherapeuticactivityThemajorimportanceofthesefindingswasthattheyprovidedasimpleexplanationofthemannerinwhichaspirin-likedrugsexertedtheirtherapeuticactions.Whenthereportswerepublishedin1971,therewasalreadyevidencesuggestingthatPGEwasanextremelypotentpyreticagentinseveralspeciesesandthatPGEorPGEmimickedtheinflammatoryresponsewheninjectedintra-dermally.PGshadalsobeendetectedininflammatoryexudateses,sothereweregroundsforspeculatingthatPGsmightberesponsible,atleastinpart,forthegenesisoffeverorinflammationandthattheaspirin-likedrugsmightowetheirtherapeuticactivitytotheirabilitytopreventPGbiosynthesis.Certainly,asFloweretal.al.pointedout,theconcentrationsofthesedrugsrequiredtoinhibitsynthesiswerewithintheplasmalevelsfoundduringtherapy,evenwhenproteinbindingwastakenintoaccount.4.InhibitionofcyclooxygenaseAhomogeneous,enzymaticallyactivecyclooxygenase(COX)orprostaglandinendoperoxidesynthase(PGHS)wasisolatedin19766.Thismembrane-boundhemoproteinandglycoproteinwithamolecularweightof72kDaisfoundingreatestamountsintheendoplasmicreticulumofprostanoid-formingcellss.ItexhibitsCOXactivitythatbothcyclizesarachidonicacidandaddsthe15-hydroperoxygrouptoformPGG.ThehydroperoxygroupofPGGreducedtothehydroxygroupofPGHbyaperoxidasethatusesawidevarietyofcompoundstoprovidetherequisitepairofelectrons.BothCOXandhydroperoxidaseactivitiesarecontainedinthesamedimericproteinmolecule.Aspirinselectivelyacetylatesthehydroxylgroupofoneserineresidue(Ser530)located70aminoacidsfromtheCterminusoftheenzymee.Acetylationleadstoirrevers-ibleCOXinhibition;thus,anewenzymemustbesynthe-sizedbeforemoreprostanoidsareproduced.Whenthepurifiedenzymeisacetylated,onlytheCOX,notthehydro-peroxidase,activityisinhibited.Thestoichiometryofthisreactionis1:1,withoneacetylgrouptransferredperenzymemonomerofthisdimericprotein.Atlowconcentrations,aspirinacetylatesPGHSrapidly(withinminutes)andselec-tively.Athighconcentrations,overlongertimeperiods,aspirinwillalsonon-specificallyacetylateavarietyofproteinsandnucleicacidsacids.AcetylationoftheenzymebyaspirinplacesabulkysubstituentontheSer530oxygenthatinhibitsbindingofarachidonicacidacid.5.DiscoveryofCOX-2andCOX-3Bythelate1980s,severalreportsappearedthatthesynthesisofPGHScouldbestimulatedbygrowthfactors,tumourpromoters,interleukin-1[24],lipopolysaccharideandtumournecrosisfactor.Interleukin-1exerteditseffectduringthetranscriptionalratherthanduringthetranslationalphaseofinducedsynthesisofPGHSPGHS.InductionofPGHSgeneexpressionbyserumfactorsoccurredafterapproximately2hinmouse3T3cells,inwhichPGsareessentialforcelldivision.ThesereportsculminatedwiththediscoverybyDanSimmonsSimmonsofasecond,distinctCOXgenewhichcouldbeinducedwithmitogens,growthfactors,tumourpromotersandlipopolysaccharide,andtheinductionofwhichcouldbeinhibitedwithglucocorticoids.ThisgeneexpressesCOX-2whichelaboratesPGs,mostlyPGEduringinflammatoryreactionsincontrasttoCOX-1,whichproducesPGsinvolvedinphysiologicalprocessessuchasprotectionofthestomachmucosa,plateletaggregationandkidneyfunction.Thereis60%homologybetweentheaminoacidstruc-turesofCOX-1andCOX-2andaspirinbindstoSer516intheactivesiteofCOX-2inthesamewayasitbindstoSer530intheactivesiteofCOX-1.However,theactivesiteofCOX-2isslightlylargerthantheactivesiteofCOX-1,soJ.R.Vane,R.M.Botting/ThrombosisResearch110(2003)255–258 thatarachidonicacidcanstill‘squeezepast’theaspirinmoleculeinactivatingCOX-2andbecomeconvertedto15-15-.ThesmalldifferenceinsizebetweentheactivesitesofCOX-1andCOX-2hasbeenexploitedbypharmaceuticalcompaniestodevelopselectiveCOX-2inhibitors,suchascelecoxibxib,rofecoxibxibandmeloxicamoxicam,whichreduceinflammationwithoutdamagingthestomachmuco-sa.Onecompanyhasalsoproducednitroaspirinrin,whichcombinesaspirinwithanitricoxide-releasingmoiety.Thenitricoxideliberatedinthestomachprotectsthestomachmucosafromdamagebygastrichydrochloricacid.Asnewanti-inflammatorydrugswithfewerseveresideeffectsthannon-selectivenon-steroidanti-inflammatorydrugs(NSAIDs)aredeveloped,theuseofaspirinforosteoarthritisandrheumatoidarthritiswilldecline.However,itsuseasapotentanti-thromboticagentforthepreventionofsecondheartattacksislikelytoincrease.ArecentreportfromChandrasekharanetal..describesathirdcyclooxygenase(COX-3)selectivelyinhibitednotonlybyparacetamolbutalsobylowconcen-trationsofsomenon-steroidanti-inflammatorydrugsin-cludingaspirin.COX-3isavariantofCOX-1whichhasretainedintron-1duringtranslationandwhichisfoundinhumantissuesinapolyadenylatedform.SelectiveinhibitionofCOX-3willdiscoverpotentandvaluablenewdrugsforcontrollingpainandfever.References[1]MaclaganTJ.Thetreatmentofacuterheumatismbysalicin.Lancet1876;i:342–83.[2]DreserH.PharmacologischesuberAspirin(Acetylsalicyl-saure).PflugersArch1899;76:306–18.[3]BeaverWT.Mildanalgesics:areviewoftheirclinicalpharmacology.AmJMedSci1965;250:577–604.[4]GuzmanF,BraunC,LimRKS,PotterGD,RodgersDW.Narcoticandnon-narcoticanalgesicswhichblockvisceralpainevokedbyintra-arterialinjectionsofbradykininandotheralgesicagents.ArchIntPharmacodyn1964;149:571–88.[5]LimRKS,GuzmanF,RodgersDW,GotoK,BraunC,DickersonGD,etal.Siteofactionofnarcoticandnon-narcoticanalgesicsdeterminedbyblockingbradykinin-evokedvisceralpain.ArchIntPharmacodyn1964;152:25–58.[6]WhitehouseMW.Somebiochemicalandpharmacologicalpropertiesofanti-inflammatorydrugs.ProgDrugRes1965;8:321–429.[7]PiperPJ,VaneJR.Releaseofadditionalfactorsinanaphylaxisanditsantagonismbyanti-inflammatorydrugs.Nature1969;223:29–35.[8]VaneJR.Theuseofisolatedorgansfordetectingactivesubstancesinthecirculatingblood.BrJPharmacolChemother1964;23:360–73.[9]HambergM,SvenssonJ,SamuelssonB.Thromboxanes:anewgroupofbiologicallyactivecompoundsderivedfromprostaglandinendo-peroxides.ProcNatlAcadSciUSA1975;72:2994–8.[10]PiperPJ,VaneJR.Thereleaseofprostaglandinsduringanaphylaxisinguinea-pigisolatedlungs.In:MantegazzaP,HortonEW,editors.Prostaglandins,peptidesandamines.London:AcademicPress;1969.p.15–9.[11]AnggardE,SamuelssonB.Biosynthesisofprostaglandinsfromarachidonicacidinguinea-piglung.JBiolChem1965;240:3518–21.[12]VaneJR.InhibitionofprostaglandinsynthesisasamechanismofactionforAspirin-likedrugs.NatNewBiol1971;231:232–5.[13]SmithJB,WillisAL.Aspirinselectivelyinhibitsprostaglandinpro-ductioninhumanplatelets.Nature1971;231:235–7.[14]FerreiraSH,MoncadaS,VaneJR.IndomethacinandAspirinabolishprostaglandinreleasefromspleen.Nature1971;231:237–9.[15]CollierJC,FlowerRJ.EffectofAspirinonhumanseminalprosta-glandins.Lancet1971;ii:852–3.[16]MiltonAS,WendlandtS.ApossibleroleforprostaglandinE1asamodulatoroftemperatureregulationinthecentralnervoussystemofthecat.JPhysiol1970;207:76–7.[17]DiRosaM,GiroudJP,WilloughbyDA.Studiesofthemediatorsoftheacuteinflammatoryresponseinducedinratsindifferentsitesbycarrageeninandturpentine.JPhysiol1971;104:15–29.[18]FlowerR,GryglewskiR,Herbaczynska-CedroK,VaneJR.Theef-fectsofanti-inflammatorydrugsonacell-freeprostaglandinsynthe-tasesystemfromdogspleen.Nature1972;238:104–6.[19]HemlerM,LandsWEM,SmithWL.Purificationofthecyclooxyge-nasethatformsprostaglandins:demonstrationoftwoformsofironintheholoenzyme.JBiolChem1976;251:5575–9.[20]SmithWL.Prostaglandinbiosynthesisanditscompartmentationinvascularsmoothmuscleandendothelialcells.AnnuRevPhysiol1986;48:251–62.[21]RothGJ,MajerusPW.ThemechanismoftheeffectofAspirinonhumanplatelets:1Acetylationofaparticulatefractionprotein.JClinInvest1975;56:624–32.[22]SmithWL.Theeicosanoidsandtheirbiochemicalmechanismsofaction.BiochemJ1989;259:315–24.[23]DeWittDL,El-HarithEA,KraemerSA,AndrewsMJ,YaoEF,ArmstrongRL,etal.TheAspirinandheme-bindingsitesofovineandmurineprostaglandinendoperoxidesynthases.JBiolChem1990;265:5192–8.[24]RazA,WycheA,SiegelN,NeedlemanP.Regulationoffibroblastcyclooxygenasesynthesisbyinterleukin-1.JBiolChem1988;263:3022–8.[25]RazA,WycheA,NeedlemanP.Temporalandpharmacologicaldivi-sionoffibroblastcyclooxygenaseexpressionintotranscriptionalandtranslationalphases.ProcNatlAcadSciUSA1989;86:1657–61.[26]XieW,ChipmanJG,RobertsonDL,EriksonRL,SimmonsDL.Ex-pressionofamitogen-responsivegeneencodingprostaglandinsyn-thaseisregulatedbymRNAsplicing.ProcNatlAcadSciUSA1991;88:2692–6.[27]VaneJR,BakhleYS,BottingRM.Cyclooxygenases1and2.AnnuRevPharmacolToxicol1998;38:97–120.[28]SilversteinFE,FaichG,GoldsteinJL,SimonLS,PincusT,WheltonA,etal.Gastrointestinaltoxicitywithcelecoxibvsnonsteroidalanti-inflammatorydrugsforosteoarthritisandrheumatoidarthritis:theCLASSstudy:arandomisedcontrolledtrial.CelecoxibLong-termArthritisSafetyStudy.JAmMedAssoc2000;284:1247–55.[29]BombardierC,LaineL,ReicinA,ShapiroD,Burgos-VargasR,DavisB,etal.Comparisonofuppergastrointestinaltoxicityofrofecoxibandnaproxeninpatientswithrheumatoidarthritis.NEnglJMed2000;343:1520–8.[30]DequekerJ,HawkeyC,KahanA,SteinbruckK,AlegreC,BaumelouE,etal.Improvementingastrointestinaltolerabilityoftheselectivecyclooxygenase(COX)-2inhibitor,meloxicam,comparedwithpirox-icam:resultsofthesafetyandefficacylarge-scaleevaluationofCOX-inhibitingtherapies(SELECT)trialinosteoarthritis.BrJRheumatol1998;37:946–51.[31]FiorucciS,SantucciL,GreseleP,FaccinoRM,DelSoldatoP,MorelliA.GastrointestinalsafetyofNO-aspirin(NCX-4016)inhealthyhu-manvolunteers:aproofofconceptendoscopicstudy.Gastroenterol-ogy2003;124:600–7.[32]ChandrasekharanNV,DaiH,RoosKL,EvansonNK,TomsikJ,EltonTS,etalCOX-3,acyclooxygenase-1variantinhibitedbyacetamino-phenandotheranalgesic/antipyreticdrugs:cloning,structureandexpressionProcNatlAcadSciUSA99:13926–31.J.R.Vane,R.M.Botting/ThrombosisResearch110(2003)255–258