Nonclinical Evaluation of Novel Cationically Modified Polysaccharide Antidotes for Unfractionated

21 Pages·2015·1.18 MB·English

RESEARCHARTICLE
Nonclinical Evaluation of Novel Cationically
Modified Polysaccharide Antidotes for
Unfractionated Heparin
BartlomiejKalaska1,KamilKaminski2,EmiliaSokolowska1,DominikCzaplicki3,
MonikaKujdowicz2,KrystynaStalinska3,JoannaBereta3,KrzysztofSzczubialka2,
DariuszPawlak1,MariaNowakowska2,AndrzejMogielnicki1*
1 DepartmentofPharmacodynamics,MedicalUniversityofBialystok,Bialystok,Poland,2 Facultyof
Chemistry,JagiellonianUniversity,Krakow,Poland,3 DepartmentofCellBiochemistry,Facultyof
a11111
Biochemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Poland
* [email protected]
Abstract
OPENACCESS
Protamine,theonlyregisteredantidoteofunfractionatedheparin(UFH),mayproducea
Citation:KalaskaB,KaminskiK,SokolowskaE, numberofadverseeffects,suchasanaphylacticshockorserioushypotension.Weaimed
CzaplickiD,KujdowiczM,StalinskaK,etal.(2015) todevelopanalternativeUFHantidoteasefficientasprotamine,butsaferandeasiertopro-
NonclinicalEvaluationofNovelCationicallyModified
duce.Asastartingmaterial,wehavechosengenerallynon-toxic,biocompatible,widely
PolysaccharideAntidotesforUnfractionatedHeparin.
PLoSONE10(3):e0119486.doi:10.1371/journal. available,inexpensive,andeasytofunctionalizepolysaccharides.Ourapproachwasto
pone.0119486 synthesize,purifyandcharacterizecationicderivativesofdextran,hydroxypropylcellulose,
AcademicEditor:PauloLeeHo,InstitutoButantan, pullulanandγ-cyclodextrin,thentoscreenthemforpotentialheparin-reversalactivityusing
BRAZIL aninvitroassayandfinallyexamineefficacyandsafetyofthemostactivepolymersinWis-
Received:October16,2014 tarratandBALB/cmousemodelsofexperimentallyinducedarterialandvenousthrombo-
sis.Efficacystudiesincludedthemeasurementofthrombusformation,activatedpartial
Accepted:January13,2015
thromboplastintime,bleedingtime,andanti-factorXaactivity;safetystudiesincludedthe
Published:March17,2015
measurementofhemodynamic,hematologicandimmunologicparameters.Linear,high
Copyright:©2015Kalaskaetal.Thisisanopen molecularweightdextransubstitutedwithglycidyltrimethylammoniumchloridegroupsata
accessarticledistributedunderthetermsofthe
ratioof0.65perglucoseunit(Dex40-GTMAC3)isthemostpotentandthesafestUFHinhib-
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany itorshowingactivitycomparabletothatofprotaminewhilepossessinglowerimmunogenici-
medium,providedtheoriginalauthorandsourceare ty.CationicpolysaccharidesofvariousstructuresneutralizeUFH.Dex40-GTMAC3isa
credited.
promisingandpotentiallybetterUFHantidotethanprotamine.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:ThestudywassupportedbyNational
ScienceCentreGrantNo.DEC-2011/03/B/NZ7/
00755.BKwassupportedbyfundsfromLeading
Introduction
NationalResearchCenterinBialystok(31/KNOW/
2013).KKwassupportedbyNationalScienceCentre Althoughmanynewantithromboticagentswereintroducedinthelastfewyears,unfractio-
GrantNo.UMO-2013/09/D/ST5/03864.KK,KS,and
natedheparin(UFH),ananionicpolysaccharide,remainsakeydruginhibitingbloodcoagula-
MNacknowledgethefinancialsupportofthe
tionincaseofemergency.Itenablesopen-heartsurgerybypreventingbloodclottinginthe
FoundationforPolishScienceTeamProgrammeco-
heart-lungmachine(cardiopulmonarybypass)oxygenatingandsupplyingbloodtothemain
financedbytheEUEuropeanRegionalDevelopment
Fund,PolyMed,TEAM/2008-2/6.Thefundershadno bodyorgans.Aftersurgeryalmostallpatientshavetoreceiveprotamine:acationicprotein
PLOSONE|DOI:10.1371/journal.pone.0119486 March17,2015 1/21
PolysaccharideAntidotesforHeparin
roleinstudydesign,datacollectionandanalysis, inactivatingheparinandrestoringcoagulation;mostwouldprobablybleedtodeathwithout
decisiontopublish,orpreparationofthemanuscript. thisantidote[1].
CompetingInterests:Theauthorshavedeclared Kimmeletal.[2]predictedthat7%ofthesepatientsmaydieanywaybecauseofseriousside
thatnocompetinginterestsexist. effectsofprotamine,suchasanaphylacticshock,pulmonaryvasoconstrictionorsystemichy-
potension[3–6].Accordingtoreportstheincidenceofprotamine-relatedanaphylaxisvaries
from0.2to10%[4,6].In2003666,000open-heartsurgeriesand470,000CoronaryArteryBy-
passGraft(CABG)procedureswereperformedwithintheUSA[7].Althoughin2010these
numbersdecreasedby15%duetomorefrequentpercutaneousinterventions[8],protamine
usageincreasedinvascularsurgery,suchasfistulaplacement,abdominalaorticaneurysmre-
pair,andotheropenabdominalsurgeries,ortranscatheteraorticvalveimplantations[9].
Thus,intheUSAalonemorethanonethousandlife-threateningcomplicationsayearcouldbe
attributedtothetoxicityofprotamine[8].Allergytofishmaypredisposetothisacutere-
sponse,becausecommercialprotamineoriginatesfromspermofsalmonfishedaroundJapan
[10].Sinceprotamineisalsousedtoslowdownabsorptionandtoprolongactionofsomeinsu-
lins,diabeticpatientsmightbepronetofatalallergicreactions[10,11].EuropeanMedicines
AgencyalertedaboutpotentialsupplyshortageofprotamineafterFukushimanuclearplantdi-
saster.Obviously,becauseoftheproblematicsupplysourceofprotamineandsafetyreasons,a
newheparinantidoteisstronglydesired.Thequestforasaferalternativetoprotaminehasa
longhistoryandhasbeenexhaustivelydescribedinarecentreview[12].Sofarthepharmaceu-
ticalcompanieshavesuspendedthedevelopmentofanumberofUFHantidotes,forexample
L-lysine[13],duetoexpensivelarge-scalesynthesis.Thus,asastartingmaterialforthesynthe-
sisofcationicpolymers,wechosegenerallynon-toxic,biocompatible,widelyavailable,inex-
pensive,andeasytofunctionalizepolysaccharides.Wehadpreviouslyexploredthemechanism
ofUFHbindingbymodifiedpolysaccharidessuchaschitosan[14,15],dextran[16,17],and
hydroxypropylcellulose[16].Theircationicgroupsattachedtothepolysaccharidechainbind
totheanionicgroupsattachedtotheUFHchainandforminactivepolycation-UFHcomplex.
InthepresentstudyweaimedtodevelopanewUFHantidotewithfullinhibitorypotency,
butsaferandeasiertoproducethanprotamine.First,ourteam—consistingofchemists,phar-
macologistsandimmunologists—synthesizedandcharacterizedseveralpositively-charged
polymers,whosestructure,likethatofheparin,isbasedonapolysaccharidemainchain.The
novelmoleculesoriginatedfromdextran,hydroxypropylcellulose,pullulanorγ-cyclodextrin
anddifferedinthemolecularweight,architectureandcationicchargedensity.Then,basedon
invitroUFHbindingassayweselectedthemostpromisingpolymersand,finally,wecompared
theefficacyandsafetyofthemostactiveagentsintheanimalmodelsofthrombosis.Weselect-
edthemostpotentandsafeheparinantidoteusingratmodelofelectrically-inducedarterial
thrombosis[18],whichwefoundpreviouslytobesuitablefortestingantithromboticandantic-
oagulativeeffectsofvariousagents[17,19–24].Wealsoevaluatedimmunogenicpropertiesof
selectednovelpolymersandcomparedthemwithprotamineinarepeated-doseanimalstudy.
MaterialsandMethods
Animals
AnimalswerepurchasedfromandhousedintheCentreofExperimentalMedicineofMedical
UniversityofBialystokinspecificpathogenfreeconditionsaccordingtoGoodLaboratory
Practicerules.166maleWistarratsand45BALB/cmicewereusedinallexperiments.Animals
werehousedwitha12hlight/darkcycleintemperature(22±2°C)andhumidity(55±5%)
controlledroom,groupedcagesasappropriate,andallowedtohaveadlibitumaccesstosteril-
izedtapwaterandastandardchow(SsniffR-ZV1324).Theanimals’healthstatuswasmoni-
toredthroughouttheexperimentsbyahealthsurveillanceprogrammeaccordingtoFederation
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PolysaccharideAntidotesforHeparin
ofEuropeanLaboratoryAnimalScienceAssociations(FELASA)guidelines.Theratsandmice
werefreeofallviral,bacterial,andparasiticpathogenslistedintheFELASArecommendations.
AlltheproceduresinvolvinganimalsandtheircarewereapprovedbyLocalEthicalCommittee
onAnimalTestingattheMedicalUniversityofBialystok(PermitNumbers28/2012and15/
2013)andbyFirstLocalEthicalCommitteeonAnimalTestingattheJagiellonianUniversity
inKrakow(PermitNumber92/2012)andconductedinaccordancewithARRIVEguidelines
[25],directive2010/63/EUoftheEuropeanParliamentandoftheCouncilontheprotectionof
animalsusedforscientificpurposesandthenationallaws.Procedureswereconductedinthe
lightphaseofcycleinthesurgicalroomofourlaboratory.Allanimalswereeuthanizedbyex-
sanguinationattheendofexperiments.
Chemicalsanddrugs
Dextran(Dex40,Mw=40kDafromLeuconostocspp.;Dex6,Mw=6kDafromLeuconostoc
spp.),hydroxypropylcellulose(HPC,Mn10kDa,Mw80kDa),pullulan(Pul,200kDa,from
Aureobasidiumpullulans),γ-cyclodextrin(GCD,(cid:1)90.0%cyclodextrinbasis(HPLC),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimidehydrochloride(EDC,(cid:1)99.0%),N-hydro-
xysuccinimide(NHS,98%),L-arginine(reagentgrade,(cid:1)98%TLC),allylamine((cid:1)99%),sper-
mine(Spm,>97%),2,20-azobis(2-methylpropionamidine)dihydrochloride(AAPH,97%),N,
N'-carbonyldiimidazole(CDI,reagentgrade),N-acrylamidopropyl-N,N,N-trimethylammo-
niumchloride(APTMAC,75wt%solutioninwater,stabilizedwith3000ppmMEHQ),glyci-
dyltrimethylammoniumchloride(GTMAC,technical(cid:1)90%),benzoylperoxide(BPO,75%
remainderwaterLuperoxA75),heparinsodiumsaltfrombovineintestinalmucosa(UFH),
protamine(protaminesulfatesaltfromsalmon,gradeX),N,N-dimethylformamide(DMF,an-
alyticalgrade),dimethylsulfoxide(DMSO,analyticalgrade),trisodiumcitrate((cid:1)99%)were
purchasedfromSigma-Aldrich(Germany).Dex1(Mw=1kDafromLeuconostocspp.)was
purchasedfromPharmacosmos(Denmark).AzureAchloride(Flukastandard)waspurchased
fromFluka(Switzerland).Acetone,ethanol96%,methanol,potassiumchloride,potassium
dihydrogenphosphate,disodiumhydrogenphosphate,sodiumchloride,NaOH,wereallana-
lyticalgradeandpurchasedfromPOCh(Poland).Routinelaboratoryreagentstodetermineac-
tivatedpartialthromboplastintime(aPTT)inplasmawerepurchasedfromBio-Ksel(Poland).
Anti-XaassaykitswerepurchasedfromSekisuiDiagnostics(USA).Pentobarbital,ketamine,
andxylazinewerepurchasedfromBiovet(Poland).
Polymersynthesisandcharacteristics
PolysaccharidessubstitutedwithGTMACweresynthesizedusingthegeneralprocedurede-
scribedpreviously[16].AllthedetailsofpolymerssynthesisandsolubilityarepresentedinS1
File.UV-VisabsorptionspectrawererecordedusinganHP8452Adiode-arrayspectrophotom-
eterin1-cmopticalpathquartzcells.Thedimensionsoftheaggregatesinaqueoussuspensions
weredeterminedusingMalvernInstrumentsLtdNanoZetaSizer.FTIRspectrawereobtained
usingaBrukerIFS48spectrometer.NMRspectraweremeasuredinD OusingaBrukerAMX
2
500spectrometer.GPCanalyseswereperformedusingaWatersGPCsystemequippedwitha
bankofthreecolumns(PLAquagel-OH30,40,and60)andtandemPDA/RIdetectors.Theel-
uentwas0.1MNaCl,flowratewas0.6ml(cid:3)min-1,samplevolumewas150μl,andtheconcen-
trationofpolymerswas5g(cid:3)l-1.
BindingofUFHbythecationicpolymers
TheabilityofthestudiedsubstancestobindUFHwasassessedwithacolorimetricmethod
usingAzureA,acationicdye,asdescribedpreviously[14,15].Briefly,UFHchains,when
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PolysaccharideAntidotesforHeparin
Fig1.BindingofUFHbythecationicpolymers.DependenceoffreeUFHconcentrationontheconcentrationofDex6-GTMACexpressedastheratioof
theDex6-GTMACmassandtotalUFHmass.Dataforprotamineareshownforcomparison.
doi:10.1371/journal.pone.0119486.g001
presentinthesolutionofAzureA,complexthedyemolecules,whichthenformaggregatesab-
sorbingat513nm,whilethemonomericformofthedye(intheabsenceofUFH)absorbsat
630nm.Addedpolymerdisruptsdyeaggregatespartially(orfullyinthecaseofcompletebind-
ingofUFH)andincreases630nmanddecreases513nmabsorptionbandintensity.Arepre-
sentativeexampleoftheplotshowingthedependenceoffree(i.e.,uncomplexedandtherefore
possessinganticoagulantactivity)UFHconcentrationonthecationicpolymerconcentration,
expressedastheratioofthecationicpolymermassandtotalUFHmass,isshowninFig.1.
Experimentalarterialthrombosisinrats
84maleWistarratsweighing216±10.8g(nosignificantdifferencebetweengroups)wereran-
domlydividedinto10groups(8–10perexperimentalgroup),anesthetizedbyanintraperitone-
alinjectionofpentobarbital(45mg(cid:3)kg-1b.w.)andplacedinasupinepositiononaheated
operationtable.UFHwasadministeredintorightfemoralveininavolumenotexceeding0.5
ml(cid:3)kg-1b.w.10minbeforetheinductionofthrombosisindoseof300U(cid:3)kg-1b.w.aloneorfol-
lowed(3min)byintravenous(iv)administrationofDex40-GTMAC2(4.2mg(cid:3)kg-1b.w.),
Dex40-GTMAC3(2.5and7.5mg(cid:3)kg-1b.w.),Dex6-GTMAC(9.6mg(cid:3)kg-1b.w.),
GCD-GTMAC2(10.8mg(cid:3)kg-1b.w.),orprotamine(3mg(cid:3)kg-1b.w.).Vehicle(PBS)treatedani-
malsservedasacontrolgroup.Additionally,UFHwasadministeredintorightfemoralveinof
16maleWistarrats,weighing209.7±9.9gindoseof900U(cid:3)kg-1b.w.aloneorfollowedbyiv
administrationofDex40-GTMAC3(22.5mg(cid:3)kg-1b.w.).Dex40-GTMAC3wasalsoadminis-
teredaloneindoseof7.5mg(cid:3)kg-1b.w.Arterialthrombosiswasinducedbyelectrical
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PolysaccharideAntidotesforHeparin
stimulationofthecommoncarotidartery,accordingtomethodspreviouslydescribed[18,26].
Briefly,theanode,astainlesssteelL-shapedwire,wasinsertedunderthearteryandconnected
toacircuitwithaconstantcurrentgenerator.Thecathodewasasubcutaneousmetalneedleat-
tachedtothehindlimb.Thestimulation(1mA)took10min.45minafterstimulationtheseg-
mentofthecommoncarotidarterywiththeformedthrombuswasclippedatbothsides,
dissectedopenedlengthwiseandthethrombuswascompletelyremoved,air-driedin37°Cand
weighed24haftertheendofexperiment.Thrombusweightwasourprimaryendpoint,whereas
coagulationmarkersmeasuredinplasmaservedassecondaryendpoints.Afterremovalof
formedthrombusbloodsamplesweretakenfromtheheart.Thebloodsamplesweredrawn
into3.13%trisodiumcitrateinavolumeratioof9:1,thencentrifugedat3500xgat4°Cfor20
minandplasmawasdeep-frozen(-70°C)inaliquotsof1mluntilfurtherassayscouldbeper-
formed.Atime-linediagramofthisexperimentwaspresentedinourpreviouspublication[17].
Experimentalvenousthrombosisinmice
25maleBALB/cmiceweighing24.2±0.2g(nosignificantdifferencebetweengroups)and
aged8–10weekswererandomlydividedinto4groups(5–7perexperimentalgroup).Animals
wereanesthetizedwithamixtureofketamine(125mg(cid:3)kg-1b.w.)andxylazine(12.5mg(cid:3)kg-1b.
w.).Aftertheanesthesiaanimalswereheparinizedwith300U(cid:3)kg-1b.w.(50μlivintotailvein).
Threeminutesafterinjection,UFHwasneutralizedwithprotamine(3mg(cid:3)kg-1b.w.)or
Dex40-GTMAC3(7.5mg(cid:3)kg-1b.w.).TheElectrolyticInferiorVenaCavaModelwasperformed
aspreviouslydescribed[27].Briefly,theabdomenwasopened,andallinferiorvenacavaside
brancheswerecarefullyligated.The25Gstainless-steelneedlewasinsertedintotheexposed
caudalvenacavaandconnectedtoacurrentsourceof25μAover15minutes(anode).After15
minutestheneedlewasremovedandabdomenwasclosedinatwo-layerfashion.Aftersix
hourstheanimalswerereanesthetizedandtheabdomenwasreopened,thevenacavawascare-
fullydissectedandinspectedforthepresenceofthrombus.Thethrombuswaskeptat37°Cand
after24hoursitsdryweightwasmeasured.
Bleedingtimeinrats
Thetailbleedingtimewasmeasuredafterremovalofformedthrombususingastandardized
Simplatedevice(SimplateII,OrganonTeknikaCorp.,USA).Thedevicemakestwoincisions5
mmlongand1mmdeeponthedorsalpartofthetailbetween9and11cmfromthetip,taking
caretoavoidlargeveins.Immediatelyafterinjury,thetailwasplacedintothecylinderwithiso-
tonicsalineat37°C.Bleedingtimewasmeasuredfromthemomentthetailwassurgicallycut
untilbleedingstoppedcompletely(norebleedingwithin30s).Ifbleedingwasstillpresentat
theendofthe300sobservationperiod,avalueof300swasascribedforthesakeofstatistical
analysis.Bleedingtimewasexpressedinseconds.
aPTTmeasurementinratsandmice
TheaPTTwasmeasuredinplasmacollectedfromthromboticratsormiceasabasiccoagula-
tionparameterusedformonitoringofUFHaction.Additionally,Dex40-GTMAC3wasas-
sayedforpotentialanticoagulantactivitybymeasuringofaPTTaccordingtoamodified
procedureofByunetal.[28].Thebloodsamplesweretakenfromtheheartof5maleWistar
rats,andweredrawninto3.13%trisodiumcitrateinavolumeratioof9:1,thencentrifugedat
3500×gat4°Cfor20minand200μlofpooledplasmawasmixedwith20μlofasolutioncon-
tainingincreasingconcentrationsofDex40-GTMAC3.TheaPTTvalueswereautomatically
determinedwithanopticalmethod(Coag-Chrom3003;Bio-ksel,Poland)addingroutinelabo-
ratoryreagentstocollectedanimalplasma.
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PolysaccharideAntidotesforHeparin
Anti-factorXaactivityinrats
Anti-factorXaactivitywasanalyzedwithELISAtechniqueat25°Cusingamicroplatereader
(DynexTech.,USA)accordingtothekitmanufacturerinstructions.
Bloodcellcountinrats
BloodcellcountwasassessedwithanAnimalBloodCounter(ABCVet,Horiba,Germany)ac-
cordingtothemanufacturerdirections.
Bloodpressuremeasurementinrats
Themeanbloodpressure(MBP)wasmeasureddirectlythroughacannulafilledwithUFHso-
lution(150U(cid:3)ml-1),placedintheleftcommoncarotidarteryandconnectedtothepressure
transducer(Plugsys,TransonicsSystem,USA)inanaesthetizedWistarratsweighing
239.7±7.2g(nosignificantdifferencebetweengroups),asdescribedpreviously[29].74Wistar
ratswererandomlydividedinto16groups(4–6perexperimentalgroup).Thescheduleofdrug
administrationwasthesameasinarterialthrombosisexperiment.Additionally,polymerswere
administeredalonein3timeshigherdoses.
Immunizationexperiments
20femaleBALB/cmiceweighing26.3±0.5g(nosignificantdifferencebetweengroups)and
aged8–10weekswererandomlydividedinto4groups(5perexperimentalgroup).Animals
wereanesthetizedwithamixtureofketamine(125mg(cid:3)kg-1b.w.)andxylazine(12.5mg(cid:3)kg-1b.
w.)andwereheparinizedwith150U(cid:3)kg-1b.w.(50μlivintotailvein).Threeminutesafterin-
jection,UFHwasneutralizedwithprotamine(1.5mg(cid:3)kg-1b.w.),orDex40-GTMAC2(6.25
mg(cid:3)kg-1b.w.),orDex40-GTMAC3(3.75mg(cid:3)kg-1b.w.).Allantidoteswereinjectedivintotail
veinin50μlofsaline,whilethecontrolgroup(UFH)wasnotadministeredanyantidote.The
heparinization/neutralizationregimenwasrepeated5times,onceeveryweek(days1,8,15,22,
and29).BloodsampleswerecollectedfromthetailveinofeachmouseonedaypriorUFHad-
ministrationandserumwasisolatedbycentrifugation.Oneweekafterthelastinjection(day
36)allmiceweresacrificed;finalbloodsampleswerecollectedandspleensoftheanimalswere
isolatedforevaluation.
Immuneresponseevaluation
Thelevelsofantibodiesspecifictoprotamine,Dex40-GTMAC2,andDex40-GTMAC3were
evaluatedusingstandardindirectELISA.Briefly,wellsofa96-wellplate(NuncMaxiSorp)
werecoatedO/NatRTwith50μlofantigensolution(25μg(cid:3)ml-1inPBS).Thewellswere
blockedO/Nwith200μlof1%BSAinPBSat4°C.SerumsamplesweredilutedinPBS,added
tothewellswashedwithPBS,andincubatedO/Nat4°C.Murineantibodiesboundtotheanti-
genweredetectedwithhorseradishperoxydase-conjugatedsecondaryantibodiesspecificto
mouseIgM(SigmaA8786,1:5000)ormouseIgG(SigmaA3673,1:10000).Colorimetricdetec-
tionwasbasedonTMBsubstratesolution(BDBiosciences)andtheenzymaticreactionwas
stoppedbyadding50μlof0.18MH SO .ELISAsignalsweremeasuredat450nmusingVER-
2 4
SAmaxmicroplatereader(MolecularDevices).Allseraweretestedoneachantigen(prot-
amine,Dex40-GTMAC2,andDex40-GTMAC3)todetectpotentialcross-reactivity.
Statisticalanalysis
Inthestudy,nreferstonumberofanimalsineachexperimentalgroup.Foreachtest,theex-
perimentalunitwasanindividualanimal.Wechoosetheminimalnumberofanimalstodetect
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PolysaccharideAntidotesforHeparin
differencesbetweeneachgroupbasingonourandothersexperienceusingtheseprocedures.
Thedataareshownasmean±SDandanalyzedusingthenon-parametricMann-Whitneytest.
Pvalueslessthan0.05wereconsideredsignificant,lessthan0.01highlysignificantandless
than0.001extremelysignificant.
Results
Characteristicsofthesynthesizedpolymers
Table1showscharacteristicsofobtainedpolymers.Theirstructurevariedinthetypeof:modi-
fiedpolysaccharide(Dex1,6or40kDA,Pul,HPCorGCD),typeofcationicgroup(GTMAC,
APTMAC,Spm,PAHorPAH-ARG),degreeofsubstitution,andchargeexpressedaszetapo-
tential.Allthedetailsofelementalanalysis,1Hnuclearmagneticresonance(NMR)andFourier
transforminfrared(FT-IR)spectroscopymeasurementsarepresentedinS2File.
InvitroUFHbindingbythecationicpolymers
TheconcentrationoffreeUFHinthesolutiondecreasedwithincreasingconcentrationofall
thesynthesizedpolymers,exceptforDex1-GTMACandGCD-GTMAC1.Generally,thepoly-
merswithlowerdegreeofsubstitutionwithcationicgroupsboundUFHweakerthanprot-
aminei.e.,theirhigherconcentrationwasrequiredtoachievethesamedecreaseinfreeUFH
concentration.Theamountofpolymersnecessaryforbinding90%of1mgofUFHwasused
Table1. Characteristicsofthepolymers.
Polymer MW(kDa)a ξb(mV) Degreeofsubstitutionc UFHbindingd
Dex1-GTMAC 1 - 0.45 nobinding
Dex6-GTMAC 6 3.0±0.6 0.59 2.7
Dex40-GTMAC1 40 ~0 0.10 insufficientbindinge
Dex40-GTMAC2 40 1.2±0.4 0.50 2.7
Dex40-GTMAC3 40 3.2±0.6 0.65 1.6
Dex40-APTMAC 40 1.0±0.7 0.42 insufficientbindinge
Dex40-Spm 40 1.9±0.3 0.71 1.0
Dex40-PAH 40 0–2f 1.17 insoluble
Dex40-PAH-Arg 40 1.2±2.8 0.10g 2.2
Pul-GTMAC 200 2.6±0.1 0.71 1.9
GCD-GTMAC1 1.297 - 0.66 nobinding
GCD-GTMAC2 1.297 - 0.99 2.8
HPC-APTMAC1 80 2.8±0.2 0.22 insufficientbindinge
HPC-APTMAC2 80 8.5±0.4 4.11 1.0
Dex—dextran,Pul—pullulan,GCD—γ-cyclodextrin,HPC—hydroxypropylcellulose,GTMAC—ammoniumgroupofglycidyltrimethyl-ammoniumchloride,
APTMAC—ammoniumgroupofN-acrylamidopropyl-N,N,N-trimethylammoniumchloride,Spm—primaryandsecondaryaminegroupsofN,N0-Bis
(3-aminopropyl)-1,4-diaminobutane(Spermine),PAH—primaryaminegroupofpoly(allylaminehydrochloride),PAH-Arg—primaryaminegroupofpoly
(allylaminehydrochloride),guanidylgroupofarginine,
aMW—weightaveragemolecularweight,forGCDexactmolecularweightisgiven
bξ—zetapotential
cdegreeofsubstitutiondefinedasthenumberofthecationicgroupsperaglucoseunit,asfoundfromelementalanalysis
dUFHbinding—ratioofpolymermasstoUFHmassrequiredforbinding90%ofUFH
ethemassofthepolymerrequiredtobindUFHwasatleast8timeshigherthanthatofprotamine
fdependingonconcentration
gdegreeofsubstitutiondefinedasthenumberofargininegroupsperPAHrepeatingunit
doi:10.1371/journal.pone.0119486.t001
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PolysaccharideAntidotesforHeparin
Fig2.ReversaloftheeffectsofUFHonthearterialthrombosisinrats.DrythrombusweightinWistar
ratstreatedwithvehicle(PBS),UFHindoseof300or900U(cid:3)kg-1)aloneorfollowedbyDex40-GTMAC2(4.2
mg(cid:3)kg-1),Dex40-GTMAC3(2.5,7.5or22.5mg(cid:3)kg-1),Dex6-GTMAC(9.6mg(cid:3)kg-1),GCD-GTMAC2(10.8
mg(cid:3)kg-1),andprotamine(3.0mg(cid:3)kg-1),c-P<0.001vs.vehicle;d-P<0.05,e-P<0.01vs.UFH300U(cid:3)kg-1;l-
P<0.001vs.UFH900U(cid:3)kg-1,Mann-Whitneytest.Resultsareshownasmean±SD,n=8–10.
doi:10.1371/journal.pone.0119486.g002
asameasureofthebindingefficiency.Ifthisvalueexceededtheanalogousvalueforprotamine
morethaneighttimesthecompoundwasdisqualifiedfromfurtherbiologicalstudies.This
valuewasalsousedforthepreliminaryassessmentofthedoseofacationicpolymerrequired
inbiologicaltests.
ReversaloftheeffectsofUFHonthearterialthrombosisinrats
UFHdosedependentlydecreasedtheweightofarterialthrombusinrats.Studiedagentsre-
versedantithromboticeffectofUFH(Fig.2).Thepolymerswereadministeredindosescorre-
spondingwiththeratioofpolymer/UFHestimatedintheUFHbindingassay.
Dex40-GTMAC3wasthemostpotentanditseffectwascomparabletotheeffectofprotamine.
ItalsoreversedtheantithromboticeffectofUFHadministeredindoseof900U(cid:3)kg-1b.w.
(Fig.2).Dex40-GTMAC2administeredindoseof4.2mg(cid:3)kg-1b.w.wascompletelyineffective.
PLOSONE|DOI:10.1371/journal.pone.0119486 March17,2015 8/21
PolysaccharideAntidotesforHeparin
Fig3.ReversaloftheeffectsofUFHonthevenousthrombosisandaPTTinmice.Drythrombusweight
(A)andtheaPTT(B)inmicetreatedwithvehicle(PBS),UFH(300U(cid:3)kg-1)aloneorfollowedby
Dex40-GTMAC3(7.5mg(cid:3)kg-1),andprotamine(3.0mg(cid:3)kg-1),b-P<0.01,c-P<0.001vs.vehicle;e-P<0.01vs.
UFH300U(cid:3)kg-1;h-P<0.01vs.UFH300U(cid:3)kg-1+Protamine3.0mg(cid:3)kg-1,Mann-Whitneytest.Resultsare
shownasmean±SDn=5–7.
doi:10.1371/journal.pone.0119486.g003
WhenadministeredaloneDex40-GTMAC3didnotinfluencethrombusweight(0.98±0.25
mg)incomparisontocontrolgroup(0.92±0.17mg).
ReversaloftheeffectsofUFHonthevenousthrombosisinmice
Similarlytotheresultsinthemodelofthearterialthrombosisinrats,UFHsignificantlyde-
creasedthrombusweightandprolongedaPTTinmicedevelopingelectricallyinducedvenous
thrombosis.WeconfirmedtheabilityofDex40-GTMAC3toreverseantithromboticandanti-
coagulanteffectsofUFHinmice(Fig.3).
InfluenceofthecationicpolymersonaPTT
Invivo,Dex40-GTMAC3,Dex6-GTMAC,GCD-GTMAC1,andprotaminesignificantlyshort-
enedaPTTwhichwasprolongedbyearlieradministrationofUFH.Wefoundthattheeffectof
GCD-GTMAC2wastheweakest,whereasDex40-GTMAC3andprotaminecompletelyre-
versedtheeffectsofUFHtothecontrolvalue(Fig.4).Dex40-GTMAC3reversedalsoaPTT
prolongedby900U(cid:3)kg-1b.w.ofUFH,andwhenadministeredaloneslightly(by30%),butsig-
nificantlyprolongedaPTTto26.28±6.94secondsincomparisonto20.28±1.08secondsin
controlgroup(P<0.01).
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PolysaccharideAntidotesforHeparin
Fig4.InfluenceofthecationicpolymersonaPTTinrats.aPTTinWistarratstreatedwithvehicle(PBS),UFH(300U(cid:3)kg-1)aloneorfollowedby
Dex40-GTMAC2(4.2mg(cid:3)kg-1),Dex40-GTMAC3(2.5and7.5mg(cid:3)kg-1),Dex6-GTMAC(9.6mg(cid:3)kg-1),GCD-GTMAC2(10.8mg(cid:3)kg-1),andprotamine(3.0
mg(cid:3)kg-1),c-P<0.001vs.vehicle;f-P<0.001vs.UFH300U(cid:3)kg-1;i-P<0.001vs.UFH300U(cid:3)kg-1+Protamine3.0mg(cid:3)kg-1;l-P<0.001vs.UFH900U(cid:3)kg-1,
Mann-Whitneytest.Resultsareshownasmean±SD,n=8–10.
doi:10.1371/journal.pone.0119486.g004
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