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A Comparison of Two Controlled-Release Delivery Systems for the Delivery of Amiloride

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MicrovascularResearch58,1–9(1999)

ArticleIDmvre.1999.2149,availableonlineathttp://www.idealibrary.comon

AComparisonofTwoControlled-ReleaseDeliverySystemsfortheDeliveryofAmiloridetoControlAngiogenesis

AndrewKnoll,StevenSchmidt,MichelleChapman,DavidWiley,JeffreyBulgrin,JamesBlank,andLorenKirchner

TheFalorCenterforVascularStudies,AkronCityHospital,SummaHealthSystem,525E.MarketStreet,Akron,Ohio,44309inconjunctionwithKentStateUniversity,SchoolofBiomedicalScience,Kent,Ohio,44224ReceivedJune1,1998

ThediureticamiloridehasbeenreportedtoinhibitbothNa؉–H؉antiportandtheurokinase-typeplasminogenac-tivator.Asaconsequenceoftheseinhibitions,neovascu-larizationmayalsobeinhibited.Wehypothesizedthatifamiloridecouldbeeffectivelydeliveredinasite-speciﬁcmanner,asystemmightbedevelopedthatcouldinhibitlocalizedangiogenesis.Inordertoevaluatethispossibil-ityweconductedastudythatcomparedtwodifferentcontrolled-releasesystemsintowhichamiloridehadbeenincorporated.Theeffectivenessofamiloridereleasefromeachdeliverysystemwasdeterminedbyquantitat-ingangiogenicpatternsinachickchorioallantoicmem-brane(CAM)systemusingafractalanalysissoftwareprogram.Thetwodeliverysystemscomparedweresu-croseacetateisobutyrate(SAIB)andcalciumalginate.InitialHPLClaboratorytestsconﬁrmedthatamiloridecouldbereleasedfrombothSAIBandcalciumalginateinvitroinasustainedmannerfor72h.TheCAMstudiesconﬁrmedthatneitherSAIBnorcalciumalginatealonepromotedorinhibitedangiogenesiswhencomparedtonontreatedcontrols.Thereleaseofamiloridefromeachdeliveryvehicleresultedinasigniﬁcant(P<0.05)inhi-bitionofangiogenesisfollowingboth24and48hofre-leasecomparedtocontrols.Therewasnodifferenceininhibitionofangiogenesis,however,whencomparingSAIB؉amiloridetreatedCAMswithcalciumalginate؉amiloridetreatedCAMs.Thesedatasuggestthatboth

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SAIBandcalciumalginatemaybeusefuldeliveryvehi-clesforthelocalizedapplicationofamiloridetocontrolangiogenesis.Suchasystemcouldpotentiallycontroltumorangiogenesiswithoutsystemiceffects.©1999AcademicPress

KeyWords:angiogenesis;inhibitor;amiloride;calciumalginate;controlled-release;sucroseacetateisobutyrate(SAIB).

INTRODUCTION

Angiogenesisistheprocessbywhichnewbloodvesselsareformed.Oneinhibitorofangiogenesisisthedrugamiloride(Harguidneyetal.,1995).Amilo-rideisapyrazinoylguanidinediureticthathasthedistinctpropertyofreducingtheexcretionofpotas-siumwhilepromotingsodiumexcretion(Gomez-SanchezandGomez-Sanchez,1994;Kooetal.,1992).Thisdrugisusedprimarilyinthetreatmentofhyper-tension.Amilorideisthoughttoinhibitangiogenesisbyoneorbothofthefollowingmechanisms:theblockageoftheNaϩ–Hϩantiporterand/ortheinhibi-tionoftheurokinaseplasminogenactivator(uPA)–urokinaseplasminogenactivatorreceptor(uPAR)complex.Bothofthesemechanismsmaybeimportantinregulatingangiogenesis.Eventhoughamiloridemaybeausefulagentinregulatingangiogenesis,

2systemicadministrationofamilorideinanefforttoregulatefocalangiogenesiscouldproducenumerousdetrimentalconsequencesphysiologically.

Thepurposeofthisresearchwastoevaluatetwonewbiodegradablepolymericdeliverysystemsthatcouldbeusefulinreleasingamiloridelocally,atafocalsitewhereangiogenesisneedstoberegulated.Clini-cally,anoptimizedformulationoftheamiloride/de-liverypolymercouldbeadministeredviapumporportinthesamemannerthatvariouschemotherapiesarecurrentlydeliveredforregionaltreatmentofcolo-rectalcancermetastatictotheliver.Alternatively,theamiloride/deliverypolymersystemoflocalregula-tionofangiogenesismightprovetherapeuticforgrowthcontrolnonresectablecarcinomas,suchasthosesurroundingthecarotidarteryortheopticnerve.

ThepolymersthatweretestedwereSAIB(sucroseacetateisobutyrate)andcalciumalginate.Theeffec-tivenessofcontrolledreleaseofamiloridefromthesepolymersininhibitingangiogenesiswasthentestedusingachickchorioallantoicmembrane(CAM)assaysystem.ThehypothesisbeingtestedwasthattherewasnosigniﬁcantdifferenceintheeffectofamilorideuponCAMangiogenesisasdeterminedbyvasculardensityandfractaldimensioncomparingthetwomethodsofdelivery.

MATERIALSANDMETHODS

PreparationofSAIBandAmiloride

Sterileﬁltered85:15sucroseacetateisobutyrate:eth-anol(SAIB85:15,EastmanKodak)wasgraciouslydo-natedbySouthernBioSystems,Inc.(Birmingham,AL).Amiloride(LotA-7410)andmethyleneblue(LotM-9140)wereobtainedfromtheSigmaChemicalCo.(St.Louis,MO).AmiloridewasincorporatedintotheSAIBbyadding1.0mgofamilorideto5.0mlofSAIBinasterilevialandvortexinguntildissolved.

PreparationofCalciumAlginateandAmiloride

Alginicacid(Lot85H1329)wasobtainedfromtheSigmaChemicalCo.Calciumchloride(Lot702982)

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waspurchasedfromtheFisherScientiﬁcCo.(FairLawn,NJ).Fourhundredmicrogramsofalginicacidwasdissolvedin30mlsterilewater.Inaseparatesterilevial,40mgofcalciumchloridewasdissolvedin20mlofsterilewater.Thealginicacidmixturewasdrippedintothecalciumchloridemixtureandwasstirreduntiltheﬂuidwasconsistentthroughout.Thesolutionwasthenwashedwithwatertoriditofchloride.Thesolutionwastestedforacidityandba-sicityandwasfoundtohaveapHofapproximately7.0.Amiloridewasthenaddedtoderivean800␮m/mlconcentration.Thesolutionwasstirredforapproximately1–1.5horuntilalltheamiloridewasdissolvedandthesolutionappearedconsistent.

High-PerformanceLiquidChromatography

High-performanceliquidchromatography(HPLC)wasperformedinordertodeterminetherateofre-leaseofamiloridefromtheSAIBandcalciumalginate.Brieﬂy,amiloridewasdissolvedinwaterandastan-dardcurvewasestablishedranginginconcentrationfrom10to400␮m.Seventeensamplesof0.75mlof85:15SAIB:ethanolϩamiloride(800␮m)alongwith0.75mlofwaterwereplacedinseparatetesttubes.Twosamplesoftheresultingsolutionweredrawnoffat1hand3sampleseachat4,12,24,48,and72h.ThesesampleswererunagainstthestandardcurveusingaprotocoldevelopedbyLeeandTannock(1996)tomeasureamiloriderelease.Anadditional17sam-plesof0.75mlofcalciumalginateϩamiloride(800␮m)alongwith0.75mlwaterwerealsoruninamannersimilartothatdescribedaboveinordertomeasureamiloridereleasefromthecalciumalginate.

CAMCulture

Chickembryosweregrownusinganestablishedwindowculturemethod.ThemethodfortheCAMassayfollowedtheprotocolofAuerbachetal.(1974).Brieﬂy,fertilizedeggs(WhiteLeghorn,DepartmentofPoultryScience,theOhioStateUniversity)wereincu-batedbluntendupinastandardeggincubator(ModelRX2,LyonElectricCo.)at37°C,95%relativehumidityfor3days.EggstobewindowedwererubbedlightlywithBetadine(providone–iodine,5%)solutionandallowedtodry.Usinganelectricengrav-

AmilorideControlofAngiogenesis3

TABLE1GroupUsed

Note.AstardenotesasigniﬁcantdifferenceatPϽ0.05betweenthetwogroups.Numbersinparenthesessignifysamplesize(n).A,amiloride.1

Fractaldimension,nonintegernumberrangingfrom1.000to2.000,withincreasingvaluesrepresentingincreasedbranchingofthevasculartree.2

Vasculardenisty,numberofgrids(0.5cmonaside)ﬁlledortraversedbyasinglevessel.

ingtool,acircularwindowapproximately1.5–2.0cmindiameterwasmadeintotheshellatday3.Thewindowwassealedwithparaﬁlmandtheeggswerethenreplacedintotheincubatoruntilsuchtimeasneeded.

ApplicationofTreatmentstoCAMSurfaces

Atday6,theshellmembranewasremovedfromeacheggandalleggsunderwenttreatmentspeciﬁctothegroupsdescribedinTable1.Inday6controls,CAMswereimagedtodetermineinitialfractaldimen-sionsandvasculardensities.Eggsinday7andday8controlgroupsweretreatedwith0.1mlofsterilewaterandthenwerereturnedtotheincubator.EggsbelongingtoSAIBonlytreatmentgroupsweretreated

onday7andday8withSAIBappliedtotheirmem-branesurfacefollowedby0.1mlofwater.Eggsincalciumalginatecontrolgroupsweretreatedonday7andday8byadding0.1mlcalciumalginateϩ0.1mlsterilewatertothesurfaceoftheCAM.Eggsinvehi-cleϩamiloridegroupsweretreatedbyadding0.1mlofSAIBorcalciumalginateincorporatedwithamilo-rideϩ0.1mlsterilewater.Afterthetreatmentwasapplied,alleggswerereturnedtotheincubator.Atapproximately12h,alltreatedeggsreceivedaseconddoseof0.1mlofsterilewater.Onday7,eggsfromday7control,day7SAIBandcalciumalginateonly,andday7SAIBϩamilorideandcalciumalginateϩamilo-ridewereimagedtodocumenttheangiogenicpat-terns.Alsoonday7,0.1mlofeitherSAIBorcalciumalginatemixedwithamiloridewasreappliedtoday8

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4control,day8SAIBonlyandcalciumalginateonly,day8SAIBϩamiloride,andday8calciumalginateϩamilorideateachspotwheretheprevioustreatmenthadbeenappliedonday6.Sterilewater(0.1ml)wasappliedtoeachCAMimmediatelyaftertherespectivetreatment.Theseeggsweresubsequentlyparaﬁlmedandreturnedtotheincubator.Atday8,allremainingeggswereimaged.

CAMImaging

CAMswereimagedontoavideoﬂoppydiskusingavideomicroscope(Keyence,VH-6100)at20ϫmag-niﬁcation(Keyence,VH-20lens).Analysisoftheim-agewasaccomplishedbyutilizinganimageanalysissystem,GlobalLab,DT2871framegrabberboardinstalledinapersonalcomputer,GlobalLabDT2879videodecoder/encoderboard,andaSonyTrinitron(PVM2030monitor)withGlobalLab-ColorVer.1.0imageanalysissoftware(Kirchneretal.,1996).InordertoimagethewindowedCAMs,portionsofeggshellswereneatlyremovedtonearlytheedgeoftheCAM,allowingawideﬁeldofview.AlightgrademineraloilwasusedtoﬁlltheresultingconcavespacesoastocompensatefortheopacityoftheCAMsurface.Whenpossible,imagesoftheCAMwereacquiredaroundandincludingtheareaoftreatmentinordertoquan-tifytheeffectsoftreatmentsonsurroundingareasoftheCAMs.Threeseparateareassurroundingthetreat-mentzoneweretypicallyacquiredandaveragedinordertoquantifyfractaldimensionandvascularden-sity.Insomeinstances,itwasnecessarytoacquiretheimageoftheentireCAMinordertogatherthesethreedistinctareas.

FractalAnalysisandDensity

Angiogen,createdbyKirchneretal.(1996),wasutilizedtoobtaintwoquantitativedescriptiveparam-etersofangiogenesisfromwhichcomparisonsbe-tweentreatmentgroupscouldbemade:fractaldimen-sionandvesseldensity.Fractaldimensionisanindicationofthedegreeofcomplexityofthevasculartree,whiledensityisreportedasthenumberofgridblocksintersectedtimesthelinearsizeofthegridblocks,whichshouldprovideareasonableestimateof

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thetotallinearlengthofthevasculature(Kirchneretal.,1996).

Fractaldimensionisanonintegernumberthat,inouranalysisoftwo-dimensionalimages,rangedfrom1.000to2.000,withincreasingvaluesrepresentativeofagreatercomplexityorincreasedbranchingofthetree.Vesseldensitywascalculatedusingthegriddataforthesmallestdivisionthatwasspeciﬁedtoestimatedensity.Thuswhenonespeciﬁedagridthatwasscaledto10ϫ10cmofactualsurfacearea,withthesmallestdivisionbeing0.5cm,thedensitywascalcu-latedasthenumberofboxesofthissize(0.5cmonaside)thatwereﬁlledortraversedbyavesselwithinthe10ϫ10cmregionofinterest.Angiogenmakestheassumptionthateachboxcountedisonlyenteredbyonevesselsegment.

StatisticalMethods

Forfractaldimensiondatafromday7SAIB,day7SAIBϩamiloride,andday7controlgroups,statisticalanalysesweredoneusingKruskal–Wallisone-wayANOVAonranksandDunn’stestatPϽ0.05.Datafromallremainingtreatmentgroupsforfractaldimen-sionwereanalyzedbyone-wayANOVAandStudent–Newman–Keulstestatthe0.05level.Forvasculardensity,allcomparisonsbetweentreatmentgroupswereevaluatedstatisticallybyone-wayANOVAandStudent–Newman–Keulstestsatthe0.05level.

RESULTS

High-PressureLiquidChromatography

High-pressureliquidchromatographywasusedtostudyreleaseofamiloridefrombothSAIBandcal-ciumalginate.ForSAIB,itwasfoundthatmeanamiloridereleaseincreasedovertimefrom19.45Ϯ0.43␮mat1hto81.45Ϯ5.84␮mat72h(Fig.1).Fromcalciumalginate,meanamiloridereleasepeakedat12hat186.27Ϯ32.68␮mbutdecreasedto85.32Ϯ28.92␮mat72h(Fig.1).

CAMFractalDimensionsandVascularDensities

Table1showsthedifferencesinmeanfractaldimen-sionsandmeanvasculardensitiesamongthecontrol,

AmilorideControlofAngiogenesis5

FIG.1.Graphofcontrolledreleaseofamiloride(␮m)fromSAIBandcalciumalginateovera72-hperiodasmeasuredbyhigh-pressureliquidchromatography.

vehiclealone(SAIBorcalciumalginate),andtheve-hicleϩamilorideCAMs.Figure2speciﬁcallyillus-tratestheCAMfractaldimensiondata.Figure3illus-tratesCAMvasculardensitydatainhistogramform.One-wayANOVAoffractaldimensiondatafromcontrolCAMsbetweendays6and8showedthattherewasastatisticallysigniﬁcantdifferenceamongthethreegroups(PϽ.0001).Testsforpairwisemultiplecomparisonprocedures(Dunn’smethod)revealeddif-ferencesbetweenday6versusday7andday6versus

day8(PϽ0.05)CAMs.However,therewasnosig-niﬁcantdifferenceinmeanfractaldimensionfromday7today8incontrolnontreatedCAMs.Inaddition,one-wayANOVArevealedthattherewerestatisti-callysigniﬁcantdifferencesamongvasculardensitiesforthecontrolnontreatedgroupscomparingdays6–8.ApairwisemultiplecomparisonprocedureusingtheStudent–Newman–Keulsmethodshowedthattherewerestatisticallysigniﬁcantdifferencesbetweenday6versusday8controlsandday7versusday8controlsinvasculardensity(PϽ0.05).Therewasnostatisticaldifferencebetweenday6andday7controlsformeanvasculardensity.

Nostatisticaldifferenceswerefoundbetweencon-trolsandvehicleonly(SAIBorcalciumalginate)foreitherfractaldimensionorvasculardensity(Figs.2and3)atanytimeperiod.ControlCAMfractaldimen-sionsandvasculardensitiesweresigniﬁcantlydiffer-entfromthoseofSAIBϩamilorideandcalciumalgi-nateϩamilorideonbothdays7and8(Table1andFigs.2and3).Oneachofthesedays,meanfractaldimensionsandvasculardensitiesofCAMstreatedwithSAIBϩamiloridedifferedfromCAMstreatedwithSAIBalone;likewise,foreachdaymeanfractaldimensionsandvasculardensitiesofCAMstreated

FIG.2.CAMfractaldimensionbarchartshowingrelationshipamongcontrols,vehicleonly,andvehicleϩamiloridetreatedCAMsfordays6–8.*Fractaldimensionisanonintegerrangingfrom1.000to2.000,withincreasingvaluesrepresentativeofagreatercomplexityorincreasedbranching.

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FIG.3.CAMfractalvasculardensitybarchartshowingrelationshipamongcontrols,vehicleonly,andvehicleϩamiloridetreatedCAMsfordays6–8.*Vasculardensityiscalculatedasthenumberofboxes(0.5cm2)ﬁlledortraversedbyavesselovera10ϫ10cmregionanalyzed.

withcalciumalginateϩamiloridedifferedfromCAMstreatedwithcalciumalginatealone.However,therewasnosigniﬁcantdifferenceoneitherdayfortheseparameterscomparingCAMstreatedwithSAIBϩamiloridewithCAMstreatedwithcalciumalginateϩamiloride.

Figure4displaysanexampleoftracedvascularimagesfromday8control,day8SAIBonly,andday8SAIBϩamiloridetreatedCAMs.AscanbeseeninFig.4,thereisreducedvascularbranchingintheSAIBϩamiloridetreatedCAMswhencomparedtocontrolandSAIBonly.

DISCUSSION

Therehavebeennumerousstudiesthathavede-scribedangiogenesisusingthechickchorioallantoicmembranemodel(Kirchneretal.,1996;DeFouwetal.,1991;MacDonaldetal.,1992).Mostofthesehavefocusedonhowtheapplicationofanangiogenicstim-ulussuchasatumorresultsinincreasedangiogenesisinaspokewheelpatternaroundtheareaofthestim-ulus(PalczakandSplawinski,1989).Thisangiogene-siswill,inturn,facilitatetumorgrowth.Itisobviousthatifangiogenesiscanberegulated,thenitmaybepossibletolimittumorgrowth.

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Therationaleforstudyingthedeliveryofamilorideviacontrolledlocaldeliveryratherthanviasystemicadministrationisrelatednotonlytothepotentialun-towardphysiologiceffectsofamiloride,butalsotoitsbioactivity.Amiloridelosesitseffectivenesswith48hfollowingsystemicabsorptionfromtheperitonealcavity.However,controlledreleaseofamiloridefrompolymerscouldtheoreticallyoccuroveralongerpe-riodoftime.Ifasystemcouldbedevelopedtodeliveramiloridelocallytoaregionalareasurroundingatumor,thenperhapsgrowthofatumorcouldbelim-itedthroughinhibitionofangiogenesis.Theresultsofthisstudy,althoughverypreliminaryinnature,sup-portthispossiblity.

Accordingtoanumberofstudies(Kooetal.,1992;Kellenetal.,1988;Tatsutaetal.,1995),amiloridemaybecapableofinhibitingtumorgrowth.AmiloridemaycompetewiththeuPAreceptororblocktheNaϩ–Hϩionantiport.ThisstudytestedwhetheramiloridewascapableofinhibitingangiogenesisintheCAMwhendeliveredtotheCAMviacontrolledreleasesystems.Asoftwarepackage(Angiogen)wasusedtoquan-titateangiogenicpatternsintheCAMs.ThisallowedanobjectivemeasureofangiogenesisineachCAM.Theuseofthecontrolledreleasesystemwasespeciallyimportantinensuringsustained,localizeddeliveryoftheamiloridetotheCAMsurface.Whenamiloride

AmilorideControlofAngiogenesisFIG.4.Tracedimagesofday8control,SAIBonly,andSAIBϩamilorideCAMs.

solutionissimplyapplieddropwisetothesurfaceoftheCAM,itdispersesovertheCAMandisnoteffec-tive.TheSAIBandcalciumalginateprovidedviscoussolutionsfromwhichtheamiloridecouldbereleasedinaslowandsustainedmannerinordertoelicitaresponse.

OurHPLCresultsshowedthatamiloridewasre-leasedfrombothofthetwotestedcontrolleddeliverysystems.FromSAIB,amiloridereleaseincreasedthroughtime.Thehighestconcentrationofamiloridereleasewasat72h,suggestingthatSAIBreleasesamilorideslowlythroughtime.Notalloftheamilo-ridewasreleasedfromtheSAIB.Wecalculatedthatonly7%oftheincorporatedamiloridewasreleasedfromtheSAIB.Releaseofamiloridefromcalciumalginatepeakedat12handthendecreased.Thiswasnotwhatwasexpected.Inpreviousstudies,releaseoftestsubstancesfromcalciumalginateoccurredveryearlyfollowingtheadditionofwater(AslaniandKennedy,1996).However,releaseofamiloridefromcalciumalginatewasdelayedinouranalysisforap-proximately12h.Approximately23%oftheincorpo-ratedamiloridewasreleasedfromthecalciumalgi-nate.Alliegroetal.(1993)showedthata54␮Mconcentrationofamiloridewasenoughtoproduceasigniﬁcantdifferencebetweencontrolsandamiloridetreatedgroupsforsuppressionofangiogenesis.There-fore,baseduponourHPLCresults,weanticipatedthattherewouldbeasufﬁcientamountofamiloridereleasedfromeachvehicletoinhibitangiogenesisinourCAMs.

Wefoundthatmeanfractaldimensionandmeanvasculardensitybothincreasedfromday6today8incontrolCAMs,whichindicatedthattherewasnewvesselgrowthandbranchingtakingplace.Thesedataaresupportedbypreviousreports(Kirchneretal.,1996;FolkmanandShing,1992).Itwasinterestingtonotethattherewasasigniﬁcantdifferenceinfractaldimensionbetweenday6andday7controlCAMsyetnosigniﬁcantdifferenceinvasculardensitybetweenday6andday7controlCAMs.ThismaybepossiblebecausetheCAMsmayhaveincreasedbranching.Ontheotherhand,densityiscountedbythenumberofboxesthatareﬁlledortraversedbyavesselwithintheregionofinterestandassumesthatonlyonevesselsegmententerseachbox.However,theremaybemorethanonevesselsegmententeringeachbox,which

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8couldaccountforasigniﬁcanceinfractaldimensionbutnosigniﬁcanceinvasculardensity.Therewasnostatisticalsigniﬁcancebetweenmeanfractaldimen-sionsofday7andday8controlCAMsbuttherewasasigniﬁcantdifferenceinday7vsday8controlCAMs.

OurdatashowedthatneitherSAIBnorcalciumalginate,alone,hadaneffectonpromotingorinhib-itingangiogenesis.TherewasnosigniﬁcantstatisticaldifferenceonanydaybetweenthecontrolgroupsandtheSAIBonlyorcalciumalginateonlygroupsforeithervasculardensityorfractaldimension.Thus,itappearsthatthesedeliveryvehiclesarebenignintheireffectsupontheCAMsfortheﬁrst48hoftreatment.Itwasnotdeterminedifthedeliveryvehiclesaffectangiogenesisbeyondthe2-daylengthofourexperi-ment.Ourstudywasfocusedontheshort-termeffectsofamiloridereleasefromthesetwodeliverysystemsfortworeasons.Kooetal.(1992)andAlliegroetal.(1993)haveshownthat48hafteramilorideaddition,vesselgrowthreturnstonormalandthatamiloridenolongerplaysanyroleintheinhibitionofvesselgrowth.ThesecondreasonisthattheCAMmodelstartstodeclineingrowthatapproximatelyday10.Thus,itwouldbedifﬁculttodetermineiftheeffectsofinhibitionofangiogenesisareduetoamilorideinhibi-tionortojusttheCAMitselfdeteriorating.Infutureexperiments,itwouldbeusefultodeterminethedu-rationthatcalciumalginateorSAIBcanreleasephys-iologicallyrelevantconcentrationsofamilorideandhowlongvesselgrowthcanbeeffectivelyinhibitedbyrepeateddosageofamiloride.Unfortunately,thismodeldidnotallowustodeterminetheseparameters.Incontrasttothedeliveryvehiclesalone,whenamiloridewasincorporatedintobothcontrolledde-liverysystemsandplacedontothesurfaceoftheCAM,meanCAMvasculardensitiesandfractaldi-mensionsdecreased.Itwasfoundthatatdays7and8,bothmeanfractaldimensionandvasculardensitydifferedsigniﬁcantlyamongSAIBϩamiloride,SAIBonly,andcontrolCAMs.Itwasalsoshownthatre-leaseofamiloridefromcalciumalginateresultedindecreasedvasculardensitiesandfractaldimensionsintheCAMs.Atdays7and8,meanvasculardensitiesandmeanfractaldimensionsfromamiloridetreatedgroupsweresigniﬁcantlydifferentthanthosefromthecontrolandcalciumalginategroupsonly.Collec-Copyright©1999byAcademicPress

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tively,theseresultssuggestthatangiogenesisonthesurfaceoftheCAMwasinhibitedbythepresentationofamiloridefrombothSAIBandcalciumalginate.ThetwovehiclesinthisstudywereequallyeffectiveintheirinhibitionofangiogenesisintheCAMassaydespitethefactthatcalciumalginatehadagreaterreleaseofamilorideaccordingtoHPLCdata.

Theremaybeseveralmechanismsthroughwhichamilorideexertsaneffect.Wangetal.(1994)showedthatamiloridewascapableofinhibitinguPAmRNAproductionat0.1–1mMconcentrations.Thisconcen-trationalsoinhibitstheNaϩ/Ca2ϩexchanger,Naϩ–KϩATPasepump,andNaϩ-coupledsolutetransport(Wangetal.,1995).TheseauthorsalsostatedthatamiloridehadnomeasurableeffectsoncellviabilityorRNAyield.Ourdatadonotsuggestamechanismofactionofamiloride—bethattheinhibitionoftheNaϩ–HϩantiporterortheuPAreceptor.However,ourdatadosupportthecontention(Kooetal.,1992;Al-liegroetal.,1993)thatamilorideiscapableofblockingangiogenesiswithintheCAM.Alliegroalsonotedthatamilorideblockedproliferationbutnotmigrationofendothelialcellsinestablishingnewbloodvessels.Theobservationsfromourstudysuggestthatthismayinfactbethecase.Inourstudy,itwaspossibletoseetheelongationofthesproutstakingplaceduringtheﬁrst24hatday7,whichsuggestedthatmigrationofendothelialcellshadoccurred.Buttherewasnore-productionofcellsbranchingofffromthesprouts,thuslimitingcapillarynetworkformation,whichsug-gestedthatproliferationwasblocked.

Controloftumorgrowthviaagentsmodulatingan-giogenesishasreceivedmuchrecentattention.TheresultsofthisstudysuggestthatamiloridecanbedeliveredviaSAIBorcalciumalginateandisasignif-icantblockerofangiogenesisintheCAMassay.Onceaformulationoftheamiloride/deliverysystemisop-timized,itcouldbepresentedtoaregionalvascularbedortumorlocalityinmuchthesamewaythatchemotherapeuticagentsaredeliveredviathehepaticarteryfromimplantableinfusionpumps,orviaports,fortreatmentofcolorectalcancermetastatictotheliver.EnsmingerandGyves(1983)describedthera-tionaleforregionalchemotherapy,particularlyinre-lationshiptometastaticcolorectalcancertotheliver.Germeretal.(1996)describedaminimallyinvasivehepaticarterycatheterfordeliveryofchemotherapeu-

AmilorideControlofAngiogenesisticagentswhichcouldalsoserveasasystemfordeliveryofourantiangiogeniccombinationofamilo-rideandvehicle.Ourstudyisonlyaﬁrststepinunderstandingthecontrolledreleaseofamiloridetocontrolangiogenesis.Ourdatasuggest,however,thatfurtherstudiesintotheuseofamiloride-inducedinhi-bitionofangiogenesisinrelationtotumorgrowtharewarranted.

ACKNOWLEDGMENTS

ThisworkwassupportedbytheSummaHealthSystemFounda-tion,theFalorFoundation,andKentStateUniversitySchoolofBiomedicalSciences.TheauthorsgratefullyacknowledgeDr.DougEvansandKimberlySloan-Stakleff,CalhounResearchCenter,Ak-ronGeneralMedicalCenter,fortheiradviceonthemechanismsofamilorideandtheirassistanceinHPLCanalysis.

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