[PDF] pharmacological properties of phenyldiguanide and other amidine

8032_2bripharmchem00051_0121.pdf

Brit.J.Pharmacol.(1959),14,527PHARMACOLOGICALPROPERTIESOFPHENYLDIGUANIDEANDOTHERAMIDINEDERIVATIVESINRELATIONTOTHOSEOF5-HYDROXYTRYPTAMINEBYF.N.FASTIER,M.A.McDOWALLANDHENDRIEKAWAALFromtheDepartmentofMedicine,OtagoUniversityMedicalSchool,Dunedin,NewZealand(RECEIVEDJuLY30,1959)Catsinwhichthecoronaryandalliedchemoreflexescouldnotbeobtainedwithsmallintravenousdosesof5-hydroxytryptaminewereinsensitivealsotophenyldiguanide.Incatswhichrespondedtophenyldiguanidewithreflexfallsofbloodpressureandheartrate,abolishedbyvagotomy,theeffectsofgradeddoses(5to150pg./kg.)ofphenyldiguanideboreastrikingresemblancetothoseproducedinitiallyby5-hydroxytryptamineinsomewhatsmallerdoses.Differencesinthecardiovascularresponsestothetwodrugsareattributedtoadditional(non-reflex)actionsof5-hydroxytryptamine.Thereflexactionsofbothdrugswereblockedreversiblyalsoby2-naphthylguanidine(500pxg.).Certainotherdrugs(bufotenine,procaine,S-decyliso-thiourea)antagonizedthedepressoractionofphenyldiguanideaswellasthereflexdepressoractionof5-hydroxytryptamine.Like5-hydroxytryptamine,phenyldiguanideandcertainotheramidinederivativescausedpainwhenappliedtothebaseofblistersinhumansubjects.Unlike5-hydroxytryptamine,phenyldiguanidedidnotconstrictperfusedratbloodvesselsorincreasethetoneoftheratfundalstrippreparationofVane(1957).Phenyldiguanidedidnotaffectthesensitivityofthesesmoothmusclepreparationsto5-hydroxytryptamine,butotheramidinederivativesprovedtobemoderatelystrongantagonistsofthevasoconstrictoractionsof5-hydroxytryptamineandofadrenaline.Unlike5-hydroxytryptamine,phenyldiguanidedidnotproducegastrichaemorrhageinthemouse.Phenyldiguanidedidnotprolongchloralhydratesleepingtimeinmicebythesamemechanismasdid5-hydroxytryptamine.Phenyl-diguanidewasnothighlytoxictomice(LD50being240mg./kg.).Itisconcludedthatphenyldiguanideandcertainotheramidinederivativesactonsensoryreceptorswhichrespondto5-hydroxytryptamine,butthattheyshowlittlepharmacologicalresemblanceto5-hydroxy-tryptamineinotherrespects.Thechemoreflexesproducedbyphenyl-diguanide(I)andotheramidinederivativeswerefirststudiedbyDawesandMott(1950),whoconcludedthatthesecompoundscausedthestimulationofcertainsensoryreceptorsinthecardio-pulmonaryarea.DawesandFastier(1950)testedabout100substancesrelatedchemicallytophenyldiguanideinthehopethatthestructuralfeaturesofcompoundsfoundtoactlikephenyldiguanidemightrecallthoseofasubstancewhichcouldbethenaturalstimulantofthereceptorsonwhichthesedrugsact.Theyfoundthatnearlyallthemorepotentcompoundswerebasedonthestructureconsistingofaromaticnucleus-shortsidechain-unsubstitutedamidinegroup(II)."Serotonin"hadnotbeenfullycharacterizedatthetimeofthisinvestigation;however,therewasevidencethatitwasatryptaminederivative.Sinceitwasknownthattheintravenousinjectionofserumintocatscancauseareflexfallofbloodpressureandheartrate(Brodie,1900),trypta-mine(III)wastestedbyDawesandFastier(1950)andwasfoundtobeinactive.However,5-hydroxytryptamine(IV)produceschemoreflexeswhichcloselyresemblethoseproducedbysuchamidinederivativesasphenyldiguanide(Page,1952;Comroe,VanLingen,StroudandRoncoroni,1953;KottegodaandMott,1955).Itwasthereforesuggested(Fastier,1955)thatsuchcompoundsasphenyldiguanidemayresemble5-hydroxytryptaminesufficientlycloselyin

F.N.FASTIER,M.A.McDOWALLandHENDRIEKAWAALONH-C-NH-C'NfIt~~~~N=/HNH'E.-C-NH2IINHCH2-CH2-NHzHOCH2-CH2-NH2N~Ichemicalstructureforthemtobeabletostimulatethesamechemoreceptors.Evidencebearingonthishypothesishasbeenobtainedintheinvestigationnowtobedescribed.METHODSCatswereanaesthetizedwithchloralose(60to70mg./kg.).Bloodpressurewasrecordedfromacarotidartery.ThemethodusedforrecordingrespiratorymovementwasessentiallythatofGaddum(1941).Drugsolutionswereinjectedintoasaphenousvein.Rabbitswereanaesthetizedwithpentobarbitonesodium,guinea-pigswithurethane,andAustralianpossums(Trichosurusvulpecula)withchloralose.EffectsoncutaneouspainreceptorsinmanwerestudiedbythemethodofArmstrong,Dry,Keele,andMarkham(1953).Thisinvolvesraisingablisterontheflexorsurfaceoftheforearm,cuttingawaytheseparatedepidermistoexposethebaseoftheblister,andrecordingthedegreeofdiscomfortestimatedbythesubjectwhenvarioussolutionsareappliedtotheexposedblisterbase.Afive-pointscalewasused,nopainbeingrated0,slightpain1,moderatepain2,fairlyseverepain3,andveryseverepain4.Thesubjectnotedthedegreeofdiscomfortat15sec.intervals.Cantharidinwasusedastheblisteringagentforalltheexperimentsdescribed.However,asoneofusdevelopedanextensivepapularrashafterthethirdapplicationofcantharidin,wenowuseapelletofC02snowinplaceofcantharidin.Drugswereappliedinneutralisotonicsolution.Thesubjectwasnottoldwhatdrugswerebeingtested.Insleepingtimeexperiments,miceinbatchesof10weregivenasubcutaneousinjectionofeitheranamidinederivativeorsaline2hr.beforeanintra-peritonealinjectionofchloralhydrate(250mg./kg.).Micewerenumberedsothatindividualsleepingtimescouldbeaccuratelyassessed.Thesleepingmicewerekeptinboxesatatemperatureof24to250.Across-overexperimentwasperformedfourdayslater,themicewhichpreviouslyhadbeengiventheamidinederivativebeinggivensaline,andviceversa.Thesleepingtimesofindividualmicewhengivenchloralhydratealoneweresubtractedfromtheirsleepingtimeswhengivenchloralhydratetogetherwiththeamidinederivative.Thelogarithmofthisdifferencewastakenasthevariateinstatisticaltests,sincewefoundthatthelog.sleepingtimesofcontrolmicewerenormallydistributedwhereasthesleepingtimeswerepositivelyskewed.Therectaltemperaturesofthemiceweremeasuredbyinsertingaglue-coveredconstantan-copperthermocoupleintotherectumtoafixeddistance.RathindquarterswereperfusedataconstantratewithRinger-LockesolutionasdescribedbyFastierandSmirk(1947).Effectsofdrugsonthefundalportionoftherat'sstomachwerestudiedfollowingthetechniqueofVane(1957).Compoundsotherthanamidinederivativeswereobtainedfromcommercialsources.Mostoftheamidinederivativesweresamplesusedinpreviousstudies.Specimensofl-naphthylguanidine,I-naphthyldiguanide,and2-naphthyldiguanideweresynthesizedforusbyMr.L.C.K.Wong.WeareindebtedtoProfessorAdrienAlbertforthe2-naphthylguanidine.RESULTSEffectsontheCirculatorySystemReflexActionsItisnowwellestablishedthatthecoronaryandalliedchemoreflexesarenotelicitedbyphenyldiguanideinvagotomizedanimals.Eveninanimalswithintactvagi,smallintravenousdosesofphenyldiguanidedonotalwayselicitsharpfallsofbloodpressureandofheartrate.Thisledustoinquirewhetherthoseanimalswhichrespondedatypicallytophenyldiguanidewouldalsodosoto5-hydroxytryptamine.Cats.-Examinationofthekymographrecordsofpreviousexperimentshadshownthattheresponsestophenyldiguanideand5-hydroxy-tryptamineareverysimilar,consideringthevariationfromcattocat.TypicalpairsofresponsesareillustratedinFig.1.Asthevariationinthecourseofasingleexperimentwasoftenconsiderable,inallthepairsofresponsesshowntheseconddrugwasgivenwithin5to8min.ofthefirst.Predominantlydepressorresponseswereobtainedwithbothphenyl-diguanideand5-hydroxytryptaminein27outof32cats,almostwhollypressorresponsesinthreeothers.IntwocatstheresponsestothetwodrugsdifferedmuchmorethanthepairedresponsesshowninFig.1;whereasasmalldoseofphenyldiguanideproducedslightfallsofbloodpressure,oneof5-hydroxytryptamineproducedaslightrise.528

ANALOGUESOFS-HYDROXYTRYPTAMINEFso.1.-Facsimilesofkymographrecordsshowingeffectsofsmalldosesof5-hydroxytryptamine(5-HT)andofphenyldiguanide(P)onthearterialbloodpressureofanaesthetizedcats.Notethattheconsiderablevariationfromonecattoanotherintheresponsetos-HTisreflectedbyasimilarvariationintheresponsetoP.Thenumeralsindicatethedoseinpg.,4iS-HTI100I1X020304050s070809cb1020100Is20so010200toolFIG.2.-Facsimilesofkymographrecordsshowingeffectsof5-hydroxytryptamineingradeddoses(indicatedinpg.bythenumerals)onthearterialbloodpressureoffouranaesthetizedcats(a,b,c,andd).v0529.2.55I020so04Soo

F.N.FASTIER,M.A.McDOWALLandHENDRIEKAWAALInafurtherseriesof18experimentsawiderangeofdoseswasgiven.In9oftheseexperi-mentsdosesfrom1to500jug.5-hydroxytrypta-mineweregiven.Therewasagradualtransitionfromasmallpressortoalargedepressorresponse.Fig.2ashowsatypicalseriesofresponses.Pressorresponsesonlywereobtainedin4outoftheother9cats(Fig.2b),depressorresponsesonlyintheremaining5(Figs.2candd).Apressorelementwasmuchlessevidentintheresponsestogradeddosesofphenyldiguanide.Differencesinthecardiovasculareffectsofthetwodrugscouldthereforebeexplainedbysupposingthat5-hydroxytryptamineexertsnotonlyreflexactionspracticallyidenticalwiththoseofphenyldiguanide,butalsoanbyphenyldiguanide.Whilethelaterphasesoftheresponseto5-hydroxytrypt-aminedifferedsubstantiallyfromthosetophenyl-diguanidein12cats,theinitialphasesoftheresponseswereusuallysimilar.Aftervagotomy,pressorresponsesweregenerallyobtainedwith5-hydroxytryptamine,butphenyldiguanidewasalmostwithoutactiononbloodpressure.OtherSpecies.-Reflexdepressorresponseswerereadilyobtainedwithbothphenyldiguanideand5-hydroxytryptamineinexperi-mentsonrabbitsbutnotonguinea-pigsorAustralianpossums.Thebloodpres-sureofthelatterwasusuallyraisedby10to100jug./kg.of5-hydroxytryptamine,sometimesbymorethan50mm.ofmercury,thoughonlyforafewminutes.Phenyl-diguanideproducedsmallrisesofbloodpressurein4outof5possums;thefifthanimalrespondedtobothphenyldiguanideand5-hydroxytryptaminewithfallsofbloodpressure.Thepressorresponsesinotherpossumsweresometimespre-cededbyslightfallsofbloodpressureandheartrate.actionnotsharedaGuinea-pigsresembledpossumsratherthancatsandrabbitsintheirresponsestobothdrugs.InteractionwithOtherDrugs.-Inexperimentsoncatsanaesthetizedwithchloralose,otherdrugsweregiventoseeiftheyselectivelyblockedthereflexdepressoractionsof5-hydroxytryptamineandphenyldiguanide.2-NaphthylguanidinewastestedinitiallybecauseofitschemicalresemblancetoS-2-(2'-naphthyl)ethylisothiourea,themostpotentoftheamidinesexaminedbyDawesandFastier(1950).Both1-and2-naphthylguanidine,aswellasthecorrespondingdiguanides,werefoundhighlyactiveinevokingchemoreflexes.Theeffectsof2-naphthylguanidine(100to200,Fg.)wereverysimilartothoseproducedbysomewhatsmallerb-~~~~~~~~~~~~~1...rk.ItIFIG.3.-a,Anaesthetizedcat,6.7kg.Recordsfromabovedownwards,respiratorymove-ments,arterialbloodpressure(initiallevel190mm.ofHg)andtime(10sec.).Notetemporaryinhibitionofreflexresponsesto5-hydroxytryptaminegivenin100,ug.dosesateacharrowafteradministrationof500pg.2-naphthylguanidine(P-NG).Injectionsweremadeat5min.intervals.b,Continuationoftheexperimentillustratedina.Notetemporaryinhibitionofreflexeffectsof5-bydroxytryptaminegivenatarrowsbyS-decylisothioureahydrobromide.(S-n-decyl)intwodosesof6mg.atwhitedots.c,Anaesthetizedcat.Noteinthisexperimentthatbufotenine(Bufo.)inlOOpug.dosesproducesreflexfallsofbloodpressurewhichcloselymatchthoseproducedby100pg.dosesofphenyldiguanideand5-hydroxytryptamine(givenatPandthearrowsrespectively),andthatalargedoseofbufoteninetemporarilyinhibitsthereflexdepressoractionofphenyldiguanide.530

ANALOGUESOFS-HYDROXYTRYPTAMINEdosesofphenyldiguanideinthesamecats.Afteralargerdoseof2-naphthylguanidine(500,ug.)theresponseofthecattosubsequentdosesofphenyldiguanideor5-hydroxytryptaminewasgreatlymodifiedforsome10to30min.Fig.3showsatypicalresult:whereasinuntreatedanimals5-hydroxytryptamineproducedtypicalfallsofbloodpressure,after2-naphthylguanidineitcausedrisesofbloodpressure,theresponsegraduallyreturningtonormalastheeffectoftheguanidineworeoff.Theresponsetophenyl-diguanidewasgreatlydiminishedorevenabolishedby2-naphthylguanidineforsome10to20min.,buttherewasnoreversaloftheactionofphenyldiguanide.Otheramidinederivatives,suchas2-naphthyl-diguanideandS-decylisothiourea,resembled2-naphthylguanidineinbeingabletoinhibitreversiblythereflexdepressoractionsof5-hydroxytryptamineandphenyldiguanide.ThisisshownforS-decylisothioureainFig.3.Blockingofthedepressoractionof5-hydroxy-tryptaminebyprecedinglargedoses(upto1mg.)ofphenyldiguanidecouldnotbeclearlydemon-strated.Whenphenyldiguanideand5-hydroxy-tryptamineweregivenatintervalsof5min.ormore,therewasnosignofinteraction.Bufoteninewastestedlike2-naphthylguanidineforitsabilitytoevokechemoreflexes.Depressoreffectsobtainedwithbufotenine(50or100ug.)incatscloselyresembledthosewith5-hydroxy-tryptamineinthesamedoses.Theseamountsofbufoteninedidnotaltertheresponseto5-hydroxytryptamineorphenyldiguanidegivenSmin.later,but500Mg.'ofbufoteninesub-stantiallydiminishedthefallsofbloodpressureobtainedwith100Mug.of5-hydroxytryptamineorphenyldiguanide,thoughonlyforSto10min.ThisisalsoshowninFig.3.Procainehadlittleornoblockingactionindosesof1to3mg./kg.;10to20mg./kg.abolishedthefallofbloodpressureobtainedwith100Mg.ofeither5-hydroxytryptamineorphenyldiguanide.Adrenalineornoradrenalinewasinfusedfor15to30min.atintervalsthroughajugularveinwhiletestdosesofphenyldiguanideand5-hydroxytryptaminewereinjectedintothesaphenousvein.Thecatecholaminesusuallyraisedarterialbloodpressureby50mm.ormoreofmercurywhengiveninsalineatarateof8to20,g./ml./min.Inthreecats,thedepressoreffectsofphenyldiguanideand5-hydroxytrypta-minewereenhancedduringthefirstinfusion,andintheremainingfourcatstheywerenotmuchaffected.Differentresultswereobtainedwhenadrenalineornoradrenalinewasinfusedforasecondorthirdtime,thedepressoractionsofphenyldiguanideand5-hydroxytryptaminebeingeithermarkedlyreduced(3cats)orentirelyabolished(4cats).Iproniazidorreserpinewasgiventosomecatsbeforeexperiments,asitseemedpossiblethattheresponsetoinjected5-hydroxytryptaminemightdependtoalargeextentupontheamountofendogenous5-hydroxytryptaminepresent.Iproniazidisbelievedtodelaythedestructionof5-hydroxytryptamineinvivobyinhibitingamineoxidase,andreserpinetodepletethebodyof5-hydroxytryptamineandcertainotheramines.In5of6catsgiventwodosesofiproniazid(50mg./kg.,thefirstdose18hr.andthesecond1hr.beforethemainexperiment),smallintravenousdosesof5-hydroxytryptamineandphenyldiguanideproducedfallsofbloodpressurewhichdidnotdifferfromthoseobtainedinuntreatedcats.Oneofthecatspre-treatedwithiproniazidrespondedwithariseofbloodpressuretosmalldosesof5-hydroxytryptamineandphenyldiguanide.Pre-treatmentwithreserpine(2mg./kg.intraperitoneally18hr.beforetheexperiment)likewisefailedtoproduceinanaesthetizedcatsreflexresponsesdifferenttothoseseeninuntreatedanimals.Otherdrugs,suchasmethylamphetamineorpecazine(Pacatal,10mg.),temporarilyinhibitedthereflexdepressoractionsof5-hydroxytrypta-mineandphenyldiguanide.Lysergicaciddiethylamidedidnotblockthereflexdepressoractionof5-hydroxytryptaminewhengiveneithershortlybeforeitoratthesametimeindosesofupto200pug.2-(Pentachlorophenoxy)ethylaminewastestedbecauseWoolley(1957)hasshownthatcompoundsofthistyperesemble5-hydroxy-tryptamineincertainrespects.Largerdoses(5or7mg.)producedsharpfallsofbloodpressureandtemporaryarrestofbreathing.Theaminedidnotantagonizethedepressoractionof5-hydroxy-tryptaminetoanynoteworthyextent,andthisactionwasonlypartlyabolishedbyvagotomy.Thisactionthereforeappearstobedueonlyinparttochemoreflexes.ActionsonPerfusedBloodVesselsInexperimentsonperfusedhind-quartersoftherat,5-hydroxytryptaminehadabout1/10ofthevasoconstrictoractivityofadrenaline.Whereas0.2Mtg.ofadrenalinewasgenerallysufficienttoincreaseperfusionpressuretemporarilybysome60to100mm.Hg,3to5jig.of5-hydroxy-tryptaminewasneededtoproduceacomparable531

F.N.FASTIER,M.A.McDOWALLandHENDRIEKAWAALeffect.Phenyldiguanidehadnoeffectonperfusionpressurewhentestedindosesoffrom10to100pug.,anditdidnotmodifytheresponseoftheperfusedvesselstoeither5-hydroxytrypta-mineoradrenaline.Likephenyldiguanide,manyoftheotheramidinederivativeshighlyeffectiveinevokingchemoreflexeshadnoeffectonvesseltoneindosesofupto100ug.;however,someofthemdifferedfromphenyldiguanideinthattheystronglyantagonizedthevasoconstrictoractionof5-hydroxytryptamine.Theactiveamidinesincluded2-naphthylguanidine,S-(3-phenylpropyl)-isothiourea,andtheo-andp-chloroderivativesofS-benzylisothiourea.Allthesecompoundsantagonizedtheactionof5-hydroxytryptamineappreciablywhengiveninadosenotmorethan20timesthatof5-hydroxytryptamine,namely50jug.ofamidineasagainst5pig.5-hydroxy-tryptamine.However,themostpotentofthesecompoundshadstilllessthan1/100oftheactivityoflysergicaciddiethylamideasanantagonistof5-hydroxytryptamineunderthesameconditions.Mostoftheamidinesantagonizedthevasoconstrictoractionofadrenalineaboutasreadilyasthatof5-hydroxy-tryptamine.Therewasnocorrelationbetweenpotencyinreflexlyloweringbloodpressureincatsandinantagon-izingthevasoconstrictoractionof5-hydroxytryptamine.EffectsontheRespiratorySystemPreviouswork,reviewedbyDawesandComroe(1954),indicatesthattheeffectsofphenyldiguanideand5-hydroxytryptamineonrespiratorymovementarechieflyduetochemo-reflexesevokedbythestimulationofsensoryendingsinthecardio-pulmonaryarea.Inthecatmoderatedoses(20to50ug./kg.)usuallycausetransientapnoeafollowedbyrapidshallowbreathing.Inotherspeciesbothagentsmayproducesomewhatdifferenteffects.Theirmostusualeffectinourexperimentsonguinea-pigsandpossumswastoinhibitbreathingforafewsecondsandthenstimulateitmarkedlyforseveralminutes.Withtheseanimals,aswithcats,afairlyclosesimilarityintherespiratoryeffectsofthetwodrugswasnoted.Insupportoftheideathattheyactonthesamesensoryendingswhencausingtem-poraryapnoeainthecatistheobservationthatthiseffectisinhibitedreversiblyby2-naphthyl-guanidine(seeFig.3)andbyS-decylisothiourea.EffectsonCutaneousPainReceptorsArmstrongetal.(1953)haveshownthat5-hydroxytryptamineexcitescutaneouspainreceptorsinmanwhenappliedindilutesolutiontotheexposedblisterbase.Wehaveusedtheirtechniquetoseeifphenyldiguanideandcertainotheramidinederivativesresemble5-hydroxy-tryptamineinthisrespect.Experimentswereperformedonfoursubjects.Theyreactedtomostdrugsinasimilarfashion,butseveraldifferenceswerenoticed.Thusonesubjectexperiencednopainwithacetylcholineevenwhentheconcentrationwasincreasedto10-3g./ml.Bygivingvariousdrugsolutionsrepeatedly,it~waspossibletogaugetheabilityofthesubjecttodiscriminatebetweendifferentsolutions.SalineoraverydilutesolutionofapotentdrugneverproducedmorethanathresholdKCI103ACh10-3iL5-HTI0-5IKCI10-3ACh10-35-HTI0-5P.B.ii5-HTI054,5-HT10-44,F.F.S-M-C110-3S-3-PhI10-34i~~~~~011012012p10-34S-n-BuI0-3III012Min.FIG.4.-Diagramsindicatingtheintensityofcutaneouspainexperiencedby3observers(H.W.,P.B.,andF.F.)whencertaindrugsolutionswereappliedtotheexposedbaseofblisters.Notethedistinctdelayintheresponseto5-hydroxytryptamine(5-HT)andphenyldiguanide(P).TheamidinederivativestestedonF.F.afterMwereS-m-chlorobenzylisothiourea(S-rn-Ca).S-3-phenyl-propylisothiourea(S-3-Ph),andS-butylisothiourea(S-n-Bu).Allcon-centrationsaregivening./ml.KCI,potassiumchloride;ACh,acetyl-choline.Verticalscalegivesthenumeralsindicatingintensityofpain(seetext).532

ANALOGUESOF5-HYDROXYTRYPTAMINEeffect.Furthermore,thetimecourseoftheeffectwasgenerallythatexpectedfromthepreviousreactionofthesubject,namelyanalmostimmediateresponsetoacetylcholine,histamine,orpotassiumchloride,butadelayedresponseto5-hydroxytryptamineorphenyldiguanide(Fig.4).However,whensomeofthedrugsweregivenrepeatedlyatshortintervals,sensitivitytothemrapidlydiminished.Forexample,whenF.N.F.wasgivenfive.successivedosesof5-hydroxy-tryptamine,heexperiencedconsiderablepainwiththefirstdose,thoughtheconcentrationof5-hydroxytryptaminewasonly10-8g./ml.,somepainwiththeseconddose,whichwas100timesstronger,andsubsequentlynopainatallwithconcentrationsof10-5,10-4,and10-7g./ml.Yetheremainedsensitivetohistamineandpotassiumchloride,thoughnottotryptamine.Similarresultswereobtainedwiththeothersubjects.Treatmentwithphenyldiguanideresultedindesensitizationtothepain-producingactionof5-hydroxytryptamine.Phenyldiguanideandotheramidinederivatives(S-benzylisothiourea,S-m-chlorobenzylisothiourea,S-(3-phenylpropyl)iso-thiourea)resembled5-hydroxytryptamineincausingdelayedpain.Amorerapidpainwascausedbystillotheramidinederivatives(guanidine,asym.-dimethylguanidine),whichdonotevokecirculatoryandrespiratoryreflexesinsmalldoses.OtherEfectsToxicityMicethathadreceived10mg./kg.ofphenyldiguanideintraperitoneallyweresedatedandshowedsomerespiratoryirregularity;breathingwasalternatelyfastandslow.Adoseof20mg./kg.producedsimilareffectswithhunchingoftheback.With50mg./kg.,therewasalmostimmediaterespiratoryirregularitybutlesssedationthanwiththesmallerdoses.100mg./kg.producedsimilareffectsatfirst;prolongedtachypnoeadeveloped5to10min.later.Wheninjectedintraperitoneallyindosesof230,250,280,and300mg./kg.,phenyldiguanidekilled3,7,9,and10micerespectivelyoutofbatchesof10.Deathwasusuallydelayedforanhourormore.Itwasprecededbyutterprostra-tion,labouredbreathing,andanoxicconvulsions.LoweringofRectalTemperatureAnintraperitonealdoseofphenyldiguanide(100mg./kg.)didnotlowertherectaltemperatureofmicetoanygreaterextentthandidacontrolinjectionofsaline.Incontrast,5-hydroxytrypta-mineloweredrectaltemperatureconsiderablyevenindosesof10or20mg./kg.When5-hydroxytryptaminewasgivenbytheintra-cerebralroute,adoseof1,ug.sufficedtolowerappreciablytherectaltemperatureoftreatedmice,muchsmallerfallsinrectaltemperaturebeingobtainedwithmicethathadbeengivencontrolinjectionsofsaline.ProlongationofSleepingTimeIthasbeenshown(Fastier,Speden,andWaal,1957)that5-hydroxytryptamineprolongstheaveragesleepingtimeofmicethathavebeengivenastandarddoseofchloralhydrateorabarbiturate,probablybyloweringbodytempera-ture.TheresultsofthreeexperimentswithphenyldiguanidearegiveninTableI.Itcanbeseenthat10and50mg./kg.ofphenyldiguanideTABLEIEFFECTOFPHENYLDIGUANIDEONCHLORALHYDRATESLEEPINGTIMEToindicatethebroadtrendoftheresults,thegeometricalmeanforthesleepingtimesofbatchesofmicearegiveninthesecondcolumn.However,"t"wascalculatedfromlog.sleepingtimes,asdescribedunder"Methods."DoseMeanSleepingTime"t10mg.!kg.21-8min.saline20-3,,04050mg./kg.18-8saline17-5,,0-28100mg./kg.35-8saline17-3,,7-2didnotaffectsleepingtimeafterchloralhydrate,but100mg./kg.producedahighlysignificantincrease.ActionsonGutGastrichaemorrhageoccursinmiceafterlargedosesof5-hydroxytryptamine(Blackman,Campion,andFastier,1959).PhenyldiguanideorS-(3-phenylpropyl)isothiourea(100mg./kg.)givensubcutaneously6hr.beforethemicewerekilledandtheirstomachsexamineddidnotproducesuchhaemorrhages.Judgingbythenumberandconsistencyofthefaecalpelletsaftertreatment,phenyldiguanidewasmuchlessactivethan5-hydroxytryptamineinstimulatingperistalsisinmice.Ontheotherhand,inspectionoftheabdominalviscerashowedthatinrabbitsphenyldiguanide,like5-hydroxytryptamine,causedanincreaseinperistalticactivitywheninjectedintravenouslyinsmalldoses(50to100ptg./kg.).Thiseffectwaslessevidentinexperimentsoncats.TheratfundalstrippreparationofVane(1957),whichwascontractedby5-hydroxytryptaminein533

F.N.FASTIER,M.A.McDOWALLandHENDRIEKAWAALdosesofaslittleas10-9g./ml.,wasnotcontractedbyphenyldiguanideinconcentrationsupto10-g./ml.,norwastheresponseto5-hydroxy-tryptamineaffectedbytheadministrationofphenyldiguanide.ActiononStripedMusclePhenyldiguanideinconcentrationsofupto50uLg./ml.didnotaffectthemaximalcontractionsoftheratdiaphragmpreparationofBulbring(1946),stimulateddirectlyorthroughthephrenicnerve.DISCUSSIONThereisaremarkablyclosesimilaritybetweensomeoftheactionsofphenyldiguanideandcorrespondingactionsof5-hydroxytryptamine.Andyetthereisnogeneralsimilaritybetweenthepropertiesofphenyldiguanideandthoseof5-hydroxytryptamine.PointsofDifferenceSomeofthecharacteristicpropertiesof5-hydroxytryptaminearelargelyorentirelylackinginphenyldiguanide.Whereasinourexperimentsonperfusedratbloodvessels5-hydroxytryptamineconstrictedbloodvesselsalmostaspowerfullyasadrenaline,phenyl-diguanidedidnotaffectvesseltone.Phenyl-diguanidedidnotraisethetoneofratfundalstrips,thoughthesewerehighlysensitiveto5-hydroxytryptamine.Phenyldiguanidedidnotproducegastrichaemorrhageinthemouse.Moderatedosesofphenyldiguanidedidnotprolongchloralhydratesleepingtime;afteralargedoseofphenyldiguanideasignificantprolongationwasseen,butthiswasnotaccompaniedbyafallofbodytemperature,suchashasbeenobservedinexperimentswith5-hydroxytryptamineandwhichmayexplaintheprolongationby5-hydroxytryptamineofsleepingtimeafterchloralhydrate(Fastier,Speden,andWaal,1957).Asnotedpreviously(FastierandWaal,1957),phenyldiguanidelacksthestrongantidiureticactionof5-hydroxytryptamine.PointsofSimilaritySeveraltypesofsensorynerveendingswhicharestimulated(notnecessarilydirectly)by5-hydroxytryptaminerespondsimilarlytophenyl-diguanide.Aresemblancebetweenthetwodrugshasbeennoticedintheireffectsonsensoryreceptorssituatedintheheartandlungs(MottandPaintal,1953;DawesandComroe,1954;KottegodaandMott,1955;Paintal,1957),ondifferenttypesofgastro-intestinalreceptor(Paintal,1954),andontheperistalticreflex(BulbringandLin,1958).Ourexperimentsinwhichdrugswereappliedtotheexposedblisterbaseprovideevidenceforyetanothertypeofsensoryreceptorwhichisacteduponbybothagents.Themerefactthatamidinederivativessuchasphenyldiguanideresemble5-hydroxytryptamineinproducinghypotension,bradycardiaandapnoeabyreflexeswithvagalpathwaysdoesnotprovidesufficientevidencethatthesecompoundsareactingatthesamesites,forthesametriadofeffectsmaybeproducedbytheexcitationofvarioussensoryreceptors,asemphasizedbyDawesandComroe(1954).Thus,thecomposite"vonBezoldreflex'sobtainedwithtypicalveratrumalkaloidsdiffersinseveralimportantrespectsfromthecoronaryandalliedchemo-reflexeselicitedbyphenyldiguanide.Unlikeveratrumalkaloids,phenyldiguanidefailstoexcitethepulmonarydepressorreflexinthedog,thoughitdoessoreadilyinthecat.Again,whentransmissionthroughvagalfibresisselectivelyinhibitedbygradualcoolingofthevagaltrunk,thereflexactionsofveratrumalkaloidsareblockedlongbeforethose*ofphenyldiguanide(Dawes,MottandWiddicombe,1951).Thereflexactionsofphenyldiguanideand5-hydroxytryptaminecannotbedifferentiatedbysuchmeans,however.Investigatorswhohavestudiedbothcompoundsfortheirreflexactionshavebeenimpressedbypointsofsimilarityratherthanbypointsofdifference.Theresultsoftheexperimentsinwhichwehavecomparedthereflexdepressoreffectsof5-hydroxytryptamineandphenyldiguanideunderavarietyofconditionsreinforcetheimpressionthatthesetwodrugsareactingatthesamesites.SomePossibleExplanationsTheverylimitedpharmacologicalresemblanceofphenyldiguanideto5-hydroxytryptamineisastrongargumentagainstthepossibilitythatthepharmacologicalactionofphenyldiguanidemightbemediatedinsomewayby5-hydroxy-tryptamine.Weretheeffectsofphenyldiguanideduetoanabilitytoliberate5-hydroxytryptamineinvivo,thenonewouldexpectphenyldiguanidetohavethedirectaswellasthereflexactionsof5-hydroxytryptamine.Forthesamereasonitseemsunlikelythatphenyldiguanidecouldactindirectlybypotentiatingtheeffectsofendogenous5-hydroxytryptamine.Someoftheamidinederivativeswhichhavepowerfulreflexactionsdohappentobepowerfulinhibitorsofamineoxidase(FastierandHawkins,1951).However,thetwoformsofactivitydonotrunparallel.Furtherevidenceagainstthisideais534

ANALOGUESOFS-HYDROXYTRYPTAMINE535providedbytheobservationthatthereflexeffectsofphenyldiguanidecomeonasrapidlyasthoseof5-hydroxytryptamineandareasevanescent.Bothdrugsmightactonthesamecellsbutinfundamentallydifferentways.Thegrossreactionsofanervecellareprobablyfew.Itisnotunlikelythatthesameend-effectscouldbeproducedbydrugswhichactedondifferentpartsofthesamecellsorwhichaffecteddifferentchemicalreactionsgoingonthere.Thealternativehypothesis,thatphenyldiguanideactsinessentiallythesamewayas5-hydroxytrypt-amineatcertainsensorynerveendings,impliesthatthedifferenttypesofcellular"receptor"for5-hydroxytryptaminediffersomarkedlyfromoneanotherthatphenyldiguanideisabletoaffectonlycertainofthem.Thereceptorsonwhich5-hydroxytryptaminecanactmaywelldiffersomewhatfromonetoanotherasregardsaccessibilityorconfiguration.Differenttypesofreceptorhavealreadybeenpostulatedfor5-hydroxytryptamine(GaddumandHameed,1954),astheyhavebeenalsoforadrenaline(Ahlquist,1948)andyetotheragents.BlockingAgents.-Perhapsthemostconvincingevidencethatphenyldiguanideand5-hydroxy-tryptamineactonthesamereceptorsisprovidedbytheexperimentswith2-naphthylguanidineandwithbufotenine.Itdidnotprovepossibletoantagonizethereflexdepressoractionof5-hydroxytryptaminebygivingoneoftheseblockingagentswithoutproducingasimilarmodificationoftheresponsetophenyldiguanide.Theblockingactionof2-naphthylguanidineisprobablyexertedonthosesensoryreceptorswhicharestimulatedbyphenyldiguanide,sincethereischemicalsimilaritybetweenthecom-poundsandsince2-naphthylguanidineactsverymuchlikephenyldiguanidewhenitisgiveninsmallerdosesthanthosehavingablockingaction.Similarly,theblockingactionofbufotenineisprobablyexertedonthosesensoryreceptorswhicharestimulatedby5-hydroxytryptamine.Wethereforethinkitsignificantthatthereflexdepressoractionsofbothphenyldiguanideand5-hydroxytryptaminecanbeblockedbygivingeither2-naphthylguanidineorbufotenine.Pre-sumably,allfourcompoundshaveacommonsiteofaction,butonly2-naphthylguanidineandbufotenineformsufficientlystablecombinationswiththereceptorstopreventfurtherexcitationforaconsiderableperiod.BlockingofreflexactionshasbeenobservedpreviouslywithS-nonylisothioureaandwith5-amino-3-ethyl-2-methylindole(DawesandComroe,1954).Itseemslikelythattheseeffectsarecloselysimilartothosedescribedinthepresentwork.ItisapleasuretoacknowledgeourindebtednesstotheMedicalResearchCouncilofNewZealandandtoWilliamR.Warner&Co.forfinancialassistance.Oneofus(F.N.F.)isindebtedalsototheWellcomeTrustforatravelgrant,whichhelpedtheresearchbypermittingtheinterchangeofideaswithotherworkersinthefield.REFERENCESAhlquist,R.P.(1948).Amer.J.Physiol.,153,586.Armstrong,D.,Dry,R.M.L.,Keele,C.A.,andMark-ham,J.W.(1953).J.Physiol.(Lond.),120,326.Blackman,J.G.,Campion,D.S.,andFastier,F.N.(1959).Brit.J.Pharmacol.,14,112.Brodie,T.G.(1900).J.Physiol.(Lond.),26,48.Biibring,E.(1946).Brit.J.Pharmacol.,1,38.-andLin,R.C.Y.(1958).J.Physiol.(Lond.),140,381.Comroe,J.H.,VanLingen,B.,Stroud,R.C.,andRoncoroni,A.(1953).Amer.J.Physio.,173,379.Dawes,G.S.,andComroe,J.H.(1954).Physiol.Rev.,34,167.-andFastier,F.N.(1950).Brit.J.Pharmacol.,5,323.-andMott,J.C.(1950).Ibid.,5,65.--andWiddicombe,J.G.(1951).J.Physio.(Lond.),115,258.Fastier,F.N.(1955).Proc.Univ.Otagomed.Sch.,33,35.-andHawkins,J.(1951).Brit.J.Pharmacol.,6,256.-andSmirk,F.H.(1947).J.Pharmaco!.exp.Ther.,89,256.--Speden,R.N.,andWaal,H.(1957).Brit.J.Pharmaco!.,12,251.-andWaal,H.(1957).Ibid.,12,484.Gaddum,J.H.(1941).J.Physiol.(Lond.),99,257.-andHameed,K.A.(1954).Brit.J.Pharmaco!.,9,240.Kottegoda,S.R.,andMott,J.C.(1955).Ibid.,10,66.Mott,J.C.,andPaintal,A.S.(1953).Ibid.,8,238.Page,I.H.(1952).J.Pharmaco!.exp.Ther.,105,58.Paintal,A.S.(1954).J.Physiol.(Lond.),126,271.-(1957).Quart.J.exp.Physiol.,42,56.Vane,J.R.(1957).Brit.J.Pharmacol.,12,344.Woolley,D.W.(1957).Nature(Lond.),180,630.