Brit.J.Pharmacol.(1964),22,246-253.THEUSEOFCHANGESINCAPILLARYPERMEABIL - PDF document

Brit.J.Pharmacol.(1964),22,246-253.THEUSEOFCHANGESINCAPILLARYPERMEABILITYINMICETODISTINGUISHBETWEENNARCOTICANDNONNARCOTICANALGESICSBYB.A.WHITTLEFromImperialChemicalIndustriesLtd.,PharmaceuticalsDivision,AlderleyPark,Macclesfield,Cheshire(ReceivedSeptember9,1963)Anextensionofthe"squirmingtest"isdescribedwhichmakesthemethodspecificfornonnarcoticanalgesics.Theintraperitonealinjectionofaceticacidcausessquirmingandanincreaseincapillarypermeabilitythatismeasuredbydirectestimationofplasma-bounddye(PontamineSkyBlue)whichhasleakedintotheperitonealcavity.Nonnarcoticanalgesicsinhibitsquirmingandleakageofdye.ValuesfortheoralED50sforbotheffectsaregivenforanumberoftypicalcom-pounds.Narcoticanalgesics,indosesthatproduceanalgesia,inhibitsquirmingbutdonotsignificantlyaffectleakageofdye.Drugsthatstimulatethecentralnervoussystemandalsoinhibitsquirminghavenosignificanteffectonleakageofdyeovertherangeofdoseswhichinhibitsquirming.Corticosteroidsdonotsignificantlyinhibiteithersquirmingorleakageofdye.Theuseofthesquirmingsyndromeinmiceandratsisnowwellestablishedasamethodoftestinganalgesiccompounds.Squirmingishereusedtodenoteasyndromewhichappearsaftertheinjectionofmildlyirritantsubstancesintotheperitonealcavity.Itisvariouslydescribedas"stretching"byKoster,Anderson&deBeer(1959),"cramping"byMurray&Miller(1960)and"writhing"byHendershot&Forsaith(1959).Squirmingisunderstoodtoindicateoneormoreofthefollowingresponses:inwardrotationofoneormorefeet,twistingorturningofthetrunk,drawing-inoftheabdominalwall,lordosis,andarchingoftheback.Althoughthistestissufficientlysensitivetodetecttheeffectofanalgesicswhichareclinicallylessactivethanacetylsalicylicacid,itsuffersfromthedisadvantageofnotbeingspecific.Thetestdoesnotdistinguishbetweennarcoticandnon-narcoticdrugsanditmayevengiveapositiveresultwithcompoundswhicharenotregardedasclinicallyusefulanalgesics(Seigmund,Cadmus&Lu,1957;Eckhardt,Cheplovitz,Lipo&Govier,1958;Hendershot&Forsaith,1959).Variousauthorshavediscussedtheconditionswhichinfluencetheefficiencyofthetest(Murray&Miller,1960;Brown&Hughes,1962;Okun,Liddon&Lasagna,1963).Theobjectofthepresentworkwastogainmoreinformationfromthetestbymeasuringtheconcurrentchangesintheperitonealcapillarypermeabilityduringsquirming.Byextendingthetestinthiswayitwaspossibletoincreasethespecificity ANALGESICSANDCAPILLARYPERMEABILITYofthetestandsotodistinguishbetweentheeffectsofnarcoticandnonnarcoticanalgesics.METHODSMaleandfemalespecificpathogen-freemiceoftheAlderleyParkstrainI,weighing20±1g,weregiventest-compoundsbystomachtube.After20to25mineachanimalwasgivenanintravenousinjectionof0.1ml.ofa4%solutionofPontamineSkyBlue.At30minafteradministrationoftheanalgesic,1mgofaceticacidwasinjectedintraperitoneallyas0.4ml.ofa0.25%(v/v)solution.Eachdosegroupconsistedoftwelvemice.Thesewereplacedinatwelve-compartmentPerspexobservationbox,andthenumberofsquirmsforeachanimalwasrecordedonatwelve-unittallycounter.After20minthemicewerekilledbydislocationoftheneckandtheviscerawereexposed,afterallowing1minforbloodtodrainawayfromtheabdominalwall.TheanimalwasheldbyaflapofabdominalwallandtheviscerawereirrigatedwithdistilledwateroveraPetridish.Thecombinedwashingswerefilteredthroughglasswoolandmadeupto10ml.inagraduatedtesttube.0.1ml.of0.1N-sodiumhydroxidesolutionwasaddedtoeachtubeinordertoclearanyturbidityduetoprotein,andtheabsorptionwasreadat590my.Theamountofdyewasexpressedasug/20gofmouse.Controlanimalsweretreatedsimilarlyexceptthattheyreceivedanoraldoseofvehiclealone.Theeffectsintermsofpermeabilityandanalgesiaareexpressedaspercentagereductionsofcontrolvalues.Comparisonsofpotencyweremadeatdoseswhichgavea50%reduction.Alogdose/effectcurvewasobtainedforeachofthecompounds.Theslopeandpositionoftheregressionlinewerecalculatedbythemethodofleastsquares,andtheED50andfiduciallimitsbystandardstatisticalmethods.Thetestswereperformedatroomtemperature(21±1°C).Animalswereprimedbyinjecting0.05mgofstilboestrolintramuscularly48hrbeforethetest.Thestilboestrolwasadministeredasasolutioninethyloleatecontaining1mg/ml.Drugs.TheanalgesicdrugsusedwereofB.P.qualityandwereadministeredassolutionsorball-milledsuspensionsinadispersingagentcontaining(per1.):LissapolNX1ml.,LissapolC1g,andDispersolOG30%3.3ml.(eachfromI.C.I.),adjustedtopH7.Acetylsalicylicacidwasadministeredasasolutionofthesodiumsaltpreparedimmediatelybeforeusebyneutralizationoftheacidwithaslightexcessofsodiumbicarbonate.Dosesofamphetaminesulphate,ephedrinehydrochloride,andmebanazineoxalate(a-methylbenzylhydrazineoxalate,Actomol,I.C.I.)refertothesalts.PontamineSkyBlue6BX,batchnumber14530,wasobtainedfromG.T.GurrLtd.Variationsintheabsorptionmaximaandconcentrationofdyewerenotedindifferentbatchesofthismaterial.Thepresentworkwascarriedoutusinga4%solution(w/v)indistilledwaterwhichwasstoredat40Cuntilrequiredforuse.Phenyl-quinone(2-phenyl-1,4-benzoquinone)wasobtainedfromKodakLtd.RESULTSDifference,insensitivityofdifferentstrainsofmiceBrown&Hughes(1962)haveexaminedthesensitivityofmiceofvariousstrainstosquirmingproducedbyphenylquinone.Themiceusedinthepresentstudygavemeannumbersofsquirmsof36.2and49.5after20and30minrespectively,inresponsetoanintraperitonealdoseof1mg/kgofphenylquinone.ThisrateofsquirmingcompareswiththatofthemoreactivestrainsofBrown&Hughes(1962)whousedanintraperitonealdoseof2mg/kg.Comparativestudiesusingotheragentshavenotsofarbeenreported,butsimilarvariationsinsensitivitybetweenstrainsarelikelytoexist.Changesinpermeabilityafterphenylquinonearesimilarintimecoursetothoseproducedbyaceticacid,buttheyarelessconspicuousandhencelessusefulasthebasisforanassaymethod.247 B.A.WHITTLEEffectsofenvironmentaltemperatureParkes&Pickens(personalcommunication,1960)haveshownthattherateofsquirmingincreaseswithenvironmentaltemperature,andausefulincreaseinsensitivitycanbeobtainedbycarryingoutthetestat35"C.Thechangesinpermeabilityarealsosensitivetotemperature,buttheoptimumtemperatureappearstobeabout200C.Anyincreasedpermeabilityathighertemperaturesispresumablyoffsetbymorerapidreabsorptionofdye.Miles&Miles(1952)foundthatanenvironmentaltemperatureof370Creducedtheleakageofcirculatingdyeinducedbyhistamineinguinea-pigs.EffectsofprimingwithstilboestrolFig.1showstheeffectofprimingwithstilboestrolonmaleandfemalemice.Theincreaseinsensitivitywasseenineachsexoverarangeofdosesofaceticacid.AtthedoseusedinthemethoddescribedaboveitamountedtoatwofoldincreaseFemalesMales40.30/'5206z0I0-0.06250.1250.250.50.06250.1250.250.5Concentrationofaceticacid(%)Fig.1.Theeffectofprimingonthenumberofsquirmsofmaleandfemalemiceinducedbyintra-peritonealinjectionofaceticacid.Theprimedanimalsreceived0.05mgofstilboestrolinethyloleate48hrbeforetesting.Abscissa:theconcentrationofaceticacidinjected;ordinate:thenumberofsquirmsperanimalin20min.Eachpointisthemeanfortwelvemicewithstandarderror.Thecontinuouslinerepresentsprimedanimalsandthebrokenlineunprimedanimals.insensitivity.Theeffectdidnotappeartobedirectlyrelatedtothestageofoestrus.Primingdoesnotmateriallyaffecttheestimatesofpotency,butprovidesausefulmeansofincreasingsensitivity.EffectsofpHandconcentrationofaceticacidSquirmingandincreasedpermeabilitywereproducedbyalloftheacidstested(acetic,citric,hydrochloric,lacticandphosphoricacids)andtheresponseappearedtoberelatedtopHandtoconcentration(Fig.2).Inthisexperimentsolutionsofaceticacidwereneutralizedwithsodiumhydroxidesolutiontogivesolutionscontaining0.25and0.5%ofacetate,witharangeofpH'sfrom3.1to5.4SquirmingandleakageofdyeweremaximalatapHoflessthan3.8,andthechangeinbothparameterswasgreatestintherange4.0to5.0.ThisistherangeofpHwherethe248 ANALGESICSANDCAPILLARYPERMEABILITY80~-25070*~^~~~}~-~~*0.5%Aceticacid0.25%Aceticacid60i"200O~~~.A3.05Er1504015,bpZ30'-1000a20id:'5010I0I.03.03'.54.04551.0PHFig.2.TheeffectofpHandconcentrationofaceticacid/acetatesolutiononsquirmingandcapillarypermeabilityinmice.Ordinate:thenumberofsquirmsperanimalin20min(solidline)andtheamountofdyerecoveredin,ug(brokenline);abscissa:pHofinjectedsolution.Solutionsofaceticacidwerepartiallyneutralizedwiththecalculatedamountofsodiumhydroxidesolutionandweredilutedtogivefinalconcentrationsof0.25%(*andx)and0.5%(eandA)ofaceticacid.concentrationofun-ionizedacidischangingmostrapidly(thepKaofaceticacidis4.75).Otheracidsweretestedbutfoundtobelesssatisfactorythanaceticacidoverthesamerangeofconcentrations.Citricacidgaveaflatdose/responsecurveandathigherconcentrationsproducedconvulsions.Lacticacidandhydrochloricacidgaveirregulardose/responsecurves.EffectsofnarcoticandnonnarcoticanalgesicsNarcoticanalgesicsdidnotcauseasignificanteffectonthepermeabilityresponse,whenthecriterionofactivitywasreductionoftheresponseintreatedanimalsto50%ofthevalueincontrolanimals.Table1recordsvaluesfortheED50'sofTABLE1COMPARISONOFANALGESICACTIVITIESOFNARCOTICANALGESICSAGAINSTSQUIRMINGINDUCEDBYACETICACIDTheanalgesicsweregivenorally30minbeforethesquirmingtestED5095%Compound(mg/kg)confidencelimitsMorphine130-9-18Methadone4112-7-60Pethidine6-65-0-9-0Codeine7-25.5-9.324A9 B.A.WHITTLEMethadoneCodeine3C-r~~~~~~~~~~r200ED20-9o1~r'-5001I0I-z100LL002.551020024816Dose(mg/kg)Fig.3.Analgesiceffectsofmethadoneandcodeineinmice.Ordinate:thenumberofsquirm,peranimalin20min(solidline)andtheamountofdyerecoveredin,tg(brokenline);abscissa:oraldoseofdruginmg/kg.Treatmenttimebeforetestingwas30min.Verticallinesgivestandarderrorsofthemeans.sometypicalnarcoticanalgesics,butsincereductionofthepermeabilityresponsedidnotapproach50%overtherangeofdoseswhichinhibitedsquirming,therearenocorrespondingED5O'sforthepermeabilityeffect.Thecompoundsweregivenorally30minbeforeinjectingaceticacid.Typicaldose/responsecurvesforcodeineandmethadoneareshowninFig.3.Nonnarcoticanalgesicsantagonizedsquirmingandalsoreducedthepermeabilityresponse.ValuesoftheED5O'sforbotheffectsaregiveninTable2,andthedose/effectcurvesforsodiumacetylsalicylateandcinchophenareshowninFig.4.TABLE2COMPARISONOFTHEACTIVITIESOFNONNARCOTICANALGESICSAGAINSTSQUIRMINGANDINCREASEINPERITONEALPERMEABILITYInhibitionofInhibitionofpermeabilitysquirmingresponseOral95%conidencc95%confidenceCompoundED50limitsED50limitsGiven05hrbeforetestsAmidopyrine25-118-5-34200Cinchophen46-432-6613083-205Sodiumacetylsalicylate48-835-6710461-176Phenazone78-559-105445Paracetamol137-0102-182415Sodiumsalicylate190.0134-268270155-470Salicylamide211-0150-298519298-930Given1hrbeforetestsPhenylbutazone61-5200EflectsofnonanalgesiccompoundsCentralnervoussystemstimulantsgivealargeproportionofthe"falsepositives"whicharedetectedbytheconventionalsquirmingtest.Althoughthecharacteristics250 ANALGESICSANDCAPILLARYPERMEABILITYSodiumacetylsalicylate2001.00-150t,0SL100aI,50L002550100200012.52550100200Dose(mg/kg)Fig.4.Theeffectsofcinchophenandofsodiumacetylsalicylateonsquirmingandcapillaryper-meability.Ordinate:thenumberofsquirmsperanimalin20min(solidline)andtheamountofdyerecoveredin,tg(brokenline).Treatmenttimebeforetestingwas30min.Verticallinesgivestandarderrorsofthemeans.ofthistypeofcompoundcanbedistinguishedinothertests,theinhibitionofsquirmingbythesenonanalgesiccompoundscanbeanembarrassmentinascreeningprogramme.Table3showstheeffectofamphetamine,ephedrineandmebanazine,amonoamineoxidaseinhibitor:allinhibitedsquirmingbutdidnotaffectthepermeabilityresponsesignificantlyandhencecanbedistinguishedfromnonnarcoticanalgesics.TABLE3EFFECTOFCENTRALNERVOUSSYSTEMSTIMULANTSONSQUIRMINGANDCAPILLARYPERMEABILITYPercentagesarecalculatedfromthemeansofgroupsofmicecomparedwithcontrols(12to24mice)CompoundAmphetamineEphedrineMebanazineOraldose(mg/kg)4851020102040No.ofReductionofmicesquirming1046-51092-11216-22453-11288-2105-91038-310645Changeinpermeability(%)+14+16+16+1X3+16-21X4-13-5-23-8Corticosteroids,suchascortisoneandparamethasoneacetate(Metilar,SyntexCorp.),hadlittleeffectwhengivenparenterally,2hrbeforechallengingwithaceticacid(Table4).Animalstreatedwithcortisone24and2hrbeforechallengingwithaceticacidshowedasmalldecreaseinthenumberofsquirmsandanincreaseinpermeability,neitherofwhichissignificant(P=0.05).Similarresultswereobtainedwithparamethasonewhenanimalsweretreatedwith2mg/kg,administeredorally18and1hrbeforetheinjectionofaceticacid.Cincophen30.;200~00z100F.I-251 B.A.WHITTLETABLE4EFFECTOFANTI-INFLAMMATORYSTEROIDSONSQUIRMINGANDCAPILLARYPERMEABILITYPercentagesarecalculatedfromthemeansofgroupsofmicecomparedwithcontrols(12to24mice).I.v.==intravenous;s.c.==subcutaneousTimeNo.ofChangeinChangeinCompoundDosebeforetestsRoutemicesquirmingpermeability(mg/kg)(hr)(%)(%)Paramethasone22I.v.6+12-1acetate218andlOral12-14-5+12Cortisone2502S.c.6-19-2+19610024and2S.c.12-15+25d1DISCUSSIONMethodsbasedonthesquirmingresponseinrodentsareamongstthemostsensitivemethodsofdetectinganalgesicactivity.Theconventionalsquirmingtestissensitivebutnotspecific,andattemptstoimprovespecificitybythecontrolofexperimentalconditionshavemetwithlittlesuccess.Themeasurementofconcurrentchangesinperitonealcapillarypermeabilitythereforeappearstoofferarelativelysimplewayoftestingnonnarcoticanalgesics.Theeffectoncapillarypermeabilityandsquirminghavesimilartime-courses,andithasbeenproposedthatboththeseeffectsareconsequencesoftheactivationofaproteasesystem(Whittle,1963).Nonnarcoticanalgesicscouldinhibitsquirmingbyinterferingwiththelocalreactiontoperitonealirritationandsoreducetheintensityofafferentnervousstimulation.Squirmingcouldalsobeinhibitedbynarcoticanalgesicswhichactbyblockingcentralnervoussynaptictransmissioninthepathwayforpain.Thesensitivityofthetestdependsontheconditionsunderwhichthetestisperformedandmaythereforevaryfromonelaboratorytoanother.Forthisreasonpreliminaryexperimentsmaybeneededtofindthedoseofaceticacidwhichwillproduceasatisfactoryresponseundertheconditionsofthetest.Ingeneral,satisfactoryassayshavebeenobtainedwhencontrolanimalshaveexhibitedmorethanfifteensquirmspermousein20min.Atthislevelofresponseitisusuallypossibletorecoveratleast140jugofdyefromtheperitonealcavity.Whenthevaluesforrateofsquirminganddyeconcentrationarelower,thedose/responsecurvesfornonnarcoticanalgesicsbecomeflatandsothetestgiveserraticresults.Intestingforanalgesiaitisdesirable,thoughrarelypossible,toestimatethetissueresponsetotheappliedstimulus.Whenthemethodisbasedontheuseofthermal,electricalormechanicalstimuliitisusuallyonlypossibletomeasuretheamountofenergyapplied.Thepresentmethodmakesitpossibletomeasurethelocalresponsetoperitonealirritationasanincreaseincapillarypermeability,andtocorrelatethisresponsewiththatarisingfromthesimultaneousstimulationofsensorynerveendings.ItisapleasuretoacknowledgetheskilledtechnicalassistanceofMissS.Bentley,Mrs.S.Brocklehurst,Mrs.S.Diggle,MissB.Field,MissG.TurnerandMissS.Wallbanks.IamindebtedtomycolleagueMr.C.J.Clarkforvaluablediscussionsaboutthestatistics,andtoMr.N.Harrisonforthepreparationoftheillustrations.252 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