Pharmacokinetic Characteristics, Pharmacodynamic Effect and In Vivo Antiviral Efficacy of Liver

19 Pages·2015·2.81 MB·English

RESEARCHARTICLE
Pharmacokinetic Characteristics,
In Vivo
Pharmacodynamic Effect and Antiviral
Efficacy of Liver-Targeted Interferon Alpha
DanielRycroft1,JaneSosabowski2,EdwardCoulstock1,MarieDavies1,JohnMorrey3,
SarahFriel1,FionaKelly4,RobertHamatake5,MilanOvečka1,RobPrince6,LauraGoodall1,
ArminSepp1,AdamWalker1*¤
1 BiopharmInnovationUnit,BiopharmResearchandDevelopment,GlaxoSmithKline,Cambridge,United
Kingdom,2 CentreforMolecularOncology,BartsCancerInstitute,QueenMaryUniversityofLondon,
London,UnitedKingdom,3 InstituteforAntiviralResearch,UtahStateUniversity,Logan,Utah,United
StatesofAmerica,4 BiopharmTranslationalMedicine,BiopharmR&D,GlaxoSmithKline,Stevenage,United
Kingdom,5 GlaxoSmithKline,ResearchTrianglePark,NorthCarolina,UnitedStatesofAmerica,6
BiopharmDiscovery,BiopharmR&D,GlaxoSmithKline,Stevenage,UnitedKingdom
¤ Currentaddress:ClinicalUnitCambridge,GlaxoSmithKline,Cambridge,UnitedKingdom
* [email protected]
OPENACCESS
Abstract
Citation:RycroftD,SosabowskiJ,CoulstockE,
DaviesM,MorreyJ,FrielS,etal.(2015)
PharmacokineticCharacteristics,Pharmacodynamic Interferonalpha(IFNα)isusedforthetreatmentofhepatitisBvirusinfection,andwhilsteffica-
EffectandInVivoAntiviralEfficacyofLiver-Targeted cious,itisassociatedwithmultipleadverseeventscausedbysystemicexposuretointerferon.
InterferonAlpha.PLoSONE10(2):e0117847.
WethereforehypothesisethattargetingIFNdirectlytotheintendedsiteofactionintheliver
doi:10.1371/journal.pone.0117847
wouldreduceexposureinbloodandperipheraltissueandhenceimprovethesafetyandtoler-
AcademicEditor:RafaelAldabe,Centrode abilityofIFNαtherapy.FurthermoreweinvestigatedwhetherdirectingIFNtothereservoirof
InvestigaciónenMedicinaAplicada(CIMA),SPAIN
infectioninthelivermayimproveantiviralefficacybyincreasinglocalconcentrationintargetor-
Received:October14,2014 gansandtissues.OurpreviousresultsshowthatthemIFNα2fusedtoanASGPRspecificliver
Accepted:January2,2015 targetingantibody,DOM26h-196-61,resultsinafusionproteinwhichretainstheactivityof
Published:February17,2015 bothfusionpartnerswhenmeasuredinvitro.InvivotargetingoftheliverbymIFNα2-DOM26h-
196-61,hereafterreferredtoastargetedmIFNα2,wasobservedinmicroSPECTimagingstud-
Copyright:©2015Rycroftetal.Thisisanopen
accessarticledistributedunderthetermsofthe iesinmice.Inthisstudyweshowbypharmacokineticanalysisthatantibodymediatedliver-tar-
CreativeCommonsAttributionLicense,whichpermits getingresultsinincreaseduptakeandexposureoftargetedmIFNα2intargettissues,and
unrestricteduse,distribution,andreproductioninany
correspondinglyreduceduptakeandexposureinsystemiccirculation,clearanceorgansand
medium,providedtheoriginalauthorandsourceare
non-targettissues.Wealsoshowthatcytokineactivityandantiviralactivityofliver-targeted
credited.
IFNisobservedinvivo,butthat,contrarytoexpectations,liver-targetingofmIFNα2using
DataAvailabilityStatement:Allrelevantdataare
ASGPRspecificdAbsactuallyleadstoareducedpharmacodynamiceffectintargetorgans
withinthepaper.
andlowerantiviralactivityinvivowhencomparedtonon-targetedmIFNα2-dAbfusions.
Funding:WorkcarriedoutbyJMwasfundedbythe
NationalInstitutesofHealth(NIH).NIHcontract
numberisHHSN272201000039I/HHSN27200001/
A19.Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript.
Introduction
CompetingInterests:Allauthors(exceptJSand
ThecurrentstandardofcareforhepatitisBvirus(HBV)infectionistreatmentwithpegylated
JM)wereemployeesofGlaxoSmithKlineatthetime
thisworkwascarriedout.JSisaresearcherat IFNalpha[1,2].Thepotentanti-viral,anti-proliferativeandimmunomodulatorymechanisms
PLOSONE|DOI:10.1371/journal.pone.0117847 February17,2015 1/19
Liver-TargetedIFN;PK,PDandEfficacy
QueenMaryUniversityofLondon,whoseworkon ofthetypeIinterferons,aclassofcytokinestowhichIFNαbelongs,arewelldocumented[3].
thisprojectwasfundedbyGlaxoSmithKline.JMisa Whilstclearlyefficacious,thesystemicdeliveryofIFNαnotonlygeneratesananti-viralre-
researcheratUtahStateUniversity.Allrelevant
sponseintheliver,butalsoresultsinleukocyteactivationinthebloodleadingtoadversere-
patentshavebeendeclared,andthereareno
sponsestothetherapyincludingcytokinerelease,flu-likesymptomsanddepression.These
productsindevelopmentormarketedproductsto
declare.Thisdoesnotaltertheauthors’adherenceto side-effectscanbeseverewhichleadstoasignificantproportionofpatientsdiscontinuing
allthePLOSONEpoliciesonsharingdataand treatment[4,5,6].
materials,asdetailedonlineintheguideforauthors. Thetargetingofbioactivemoleculestotissuesisanattractiveconceptandinparticularmay
offermultiplebenefitsinthetreatmentofHBVwithIFNα.Theperceivedbenefitsaretwo-
fold,namelyincreasingthelocalconcentrationofatherapeuticcompoundattherequiredsite
ofaction,potentiallyretainingefficacywithareduceddose,andreducingundesiredactivityof
atherapeuticinnon-targettissues,potentiallyimprovingsafetyandtolerability.Theapplica-
tionofthisconceptinmultiplediseaseindicationshasbeeninvestigatedusingawiderangeof
methodologies,forexamplesite-specificdeliveryofcytotoxicdrugsforcancertherapy[7,8],li-
posomaldeliveryofantigensinvaccinedevelopment[9]andthetargetingofblood-brainbarri-
er(BBB)receptorstofacilitatetransferofbiopharmaceuticalsfromthebloodintothebrain
parenchyma[10].
ViralreplicationinHBVinfectionoccurspredominantlyintheliver.Asialoglycoproteinre-
ceptor(ASGPR)isacellsurfacereceptorexpressedexclusivelyinhepaticparenchymalcells
[11].ASGPRisaC-type(calciumdependent)lectincomposedoftwotransmembraneglyco-
proteinsubunits,termedH1andH2.TheaglycosylH1andH2subunitsareapproximately35
and33kDainsizerespectively,thoughpurifiedASGPRproteinsubunitsaresignificantlylarg-
erduetopost-translationalmodification.ASGPRmediatesendocytosisofplasmaglycopro-
teinsthathaveexposedterminalgalactoseresiduesfromwhichterminalsialicresidueshave
beenremoved[12].Inaddition,ASGPRhasalsobeenlinkedtotheentryofHBVintohepato-
cytes[13].Despitereportsofpotentialextrahepaticexpressioninhumankidney[14],thyroid
[15]andactivatedTcells[16],ASGPRhasbeenexploitedinthetargetingoftherapeuticmole-
culestotheliver.Forexample,ASGPR-targetednanoparticlesloadedwithcytotoxicagents
suchaspaclitaxelresultinenhancedcellkillingactivityagainstASGPR-positivecelllineswhen
comparedwithfreepaclitaxel[17].ASGPR-directednanoparticleshavealsobeenusedtodeliv-
ertransgenesandantisenseoligonucleotidestoASGPR-expressingprimaryhepatocytesand
celllines[18,19].InvivoradioiodinatedcopolymerswithASGPRbindingactivityaccumulate
intheliverfollowingintravenousadministrationinrats[20].InastudyconductedbyPeng
etal.,systemicdeliveryoftheapoptingene,whichselectivelyinducesapoptosisinmalignant
cells,linkedtoasialoglycoproteinresultedinspecificdeliverytoASGPR-positiveHepG2de-
rivedtumorsxenograftedinSCIDmiceandsignificanttumourregression.Bycontrast
ASGPR-apoptintransgeneconjugateswerenotabletoinducetumourregressioninnon-hepa-
tocytederivedA549xenograftedanimals[21].
CompellingevidenceforthepotentialapplicationofASGPR-mediatedhepaticdeliveryin
improvingantiviralefficacyoftypeIinterferonsisprovidedinastudybyEtoandTakahashi.
FollowingenzymaticremovalofterminalsialicacidresiduesfromtheN-linkedoligosaccharide
chainofhumaninterferonbeta(IFNβ),theinvestigatorswereabletodemonstrateenhanced
interferonsignalingactivityandinhibitionofviralreplicationinHBVtransfectedHepG2cells
comparedtotheunmodifiedformoftheprotein[22].Thisenhancedantiviralactivitywaspre-
sumablyduetoASGPRbinding,asitcouldbepartiallyinhibitedbynaturalASGPRligands
suchasasialofetuin.Significantlyenhancedinvivoantiviralefficacyofmurineasialo-IFNβ,
comparedwiththatoftheunmodifiedprotein,wasalsoshowninHBVtransfectedBALB/c
athymicnudemice.
ThesmallsizeofdAbs(11–15kDa)coupledwiththeirhighaffinityfortheirrespectiveanti-
gencanhelppreservetheactivityoffusionpartners,whichmakestheiruseattractive[23,24,
PLOSONE|DOI:10.1371/journal.pone.0117847 February17,2015 2/19
Liver-TargetedIFN;PK,PDandEfficacy
25].WehavepreviouslyshownthatIFNα-ASGPRdAbfusionproteinscanbeexpressedin
mammaliancellswhilstretainingtheinvitroactivityofbothfusionpartners.Furthermore,
usingSPECTimagingwehaveshownthatthefusionproteintargetedmIFNα2specificallytar-
getstheliverinvivo[26].InthisstudyweshowthattargetedmIFNα2exposureisincreasedin
targetorgansandreducedinsystemiccirculationandnon-targettissues.Whilsttargeted
mIFNα2inducesIFN-responsivegeneexpressionandelicitsantiviraleffectsinvivo,theseef-
fectsarelowerincomparisontonon-targetedmIFNα-dAbfusionproteins,suggestingthatthe
targetingmethodhasmoreutilityinreducingexposureinnon-targettissuesandorgansthan
increasingefficacyasadirectconsequenceofincreasedlocalconcentrationintargettissues
andorgans.
MaterialsandMethods
ProductionandcharacterisationofmIFNα2-dAbfusionproteinsand
PEGylatedmIFNα2
FusionproteinsandPEGylatedmIFNα2wereproducedandcharacterisedasdescribedprevi-
ously[26]withthefollowingadditionalstepstoproducedPEGylatedmIFNα2;16.75fold
molarexcessof40kDaBranchedPEGNHS-ester(TOFSunbright)wasaddedtomIFNα2in
PBS(SigmaAldrich).Reactionwasincubatedatroomtemperaturefor2hrsbeforepurification
usingionexchangechromatographyusinga1mlResourceScolumn(GEHealthcare).
ConjugationofmIFNα2-dAbfusionproteinswithNHS-DOTA
Fusionproteinsweredialysedinto25mMNaAcetatesolution,pH8(SigmaAldrich)using
MaxiGeBAflexdialysistubeswitha3.5kDamolecularweightcutoff(GeneBio-Application
Ltd.).NHS-DOTA(MacrocyclicsInc.)wasthenaddedina4-foldmolarexcessandreacted
overnightatroomtemperature.ConjugationsolutionswerethenappliedtoProteinAcolumns
(GEHealthcare)equilibratedinChelex100(Bio-RadLtd.)treatedPBS,pH7.4(PAALaborato-
riesGmbH),washedwithChelextreatedPBSandelutedin0.5mlfractionsof0.1Mglycine/
HCl,pH2(SigmaAldrich),intotubescontainingammoniumacetate(finalconcentration/pH
offractionswas0.46Mammoniumacetate,pH5).Conjugationefficiencyandactivitywerede-
terminedasdescribedpreviously[26].
RadiolabellingandradiochemicalanalysisofDOTAconjugated
mIFNα2-dAbfusionproteins
Allradiolabellingwascarriedout4,3or2dayspriortothe111InCl referencedatewhenthera-
3
dioactivityconcentrationwasapproximately1,0.83or0.65MBq/μlrespectively.Thegeneral
radiolabellingprotocolwasasfollows;toalowproteinbinding1.5mlpolypropylenetube
(Nunc)wasadded40–60μl(26–50MBq)of111InCl (Covidien)in0.05MHCl(SigmaAl-
3
drich),8–12μl(1/5ththevolume)of1Mammoniumacetate(SigmaAldrich),pH4.5–5.5
(orinthecaseofmIFNα2-DOM26h-196-61,120μl0.1MMES,pH5.1(SigmaAldrich))and
12.5–92.5μgofprotein.Thesolutionwasheatedto40°Cfor1.5–2.5handquenchedwith
0.1MEDTAsolution(NorwichNHSTrust)using1/20threactionvolume.Radiochemicalpu-
ritywasdeterminedusingsizeexclusionHPLCandthinlayerchromatography(TLC)analysis
afterwhichthereactionmixturewasdilutedwithPBSorPBS/0.1%BSA(SigmaAldrich)fol-
lowedbyfiltrationthrougha0.22μmfilter.
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Liver-TargetedIFN;PK,PDandEfficacy
PharmacokineticstudiesinCD-1mice
AllanimalstudieswereethicallyreviewedandcarriedoutinaccordancewithAnimals(Scien-
tificProcedures)Act1986andtheGlaxoSmithKlinePolicyontheCare,WelfareandTreat-
mentofAnimals.
AllstudieswereconductedinaccordancewiththeGlaxoSmithKlinePolicyontheCare,
WelfareandTreatmentofLaboratoryAnimalsandwerereviewedbytheInstitutionalAnimal
CareandUseCommitteeeitheratGlaxoSmithKlineorbytheethicalreviewprocessatthein-
stitutionwheretheworkwasperformed.TheInstitutionalAnimalCareandUseCommittee
specificallyapprovedthisstudy.
Fusionproteinswereadministeredasasingleintravenousdoseat5mg/kgintoagroupof
maleCD-1miceviathecaudalvein,orasasinglesubcutaneousdoseat5mg/kgintoagroupof
maleCD-1mice.Atarangeoftimepointsupto120hourspostdose.Bloodfrom3miceof
eachgroupwastakenaseithersmallvolumetailbleedsorasterminalbleedsfollowingsacrifice.
Bloodsampleswereallowedtoclotatambienttemperature,beforesampleswerecentrifugedat
approximately2000gfor10minutestoprepareserum.Serumwasstoredfrozenuntilrequired.
AnalysisofmIFNα2-dAbfusionproteinsusingantibodycaptureand
detection
TheconcentrationsofanymIFNα2-V dAbfusioninmouseserumsampleswasdetermined
H
usinganMSDassay.Briefly,96-wellstandardbindMSDplates(MesoscaleDiscovery)were
coatedovernightwitharatanti-mouseIFNαmAb(R&DSystems).Thefollowingday,plates
werewashedwithPBS/0.1%Tween-20.Wellswerethenblockedwithassaybuffer(5%BSAin
PBScontaining1%tween-20).
Standardsamplesandstudysampleswereaddedatarangeofdilutionsinduplicate.Sam-
plesweredilutedinassaybuffercontaininganappropriateamountofcontrolmatrixtomatch
matrixconcentrationsacrosstheplate.TheappropriatemIFNα2-V dAbfusionwasaddedto
H
eachplateastriplicatestandardcurvesatarangeofknownconcentrationsinassaybuffercon-
taininganappropriateamountofcontrolmatrix.
Afterwashing,boundmIFNα2-V dAbfusionwasdetectedwithMSDsulfo-taggedmouse
H
anti-V mAb(in-housereagent,cloneM2.3G10.1G06,preparedaccordingtoMSDprotocols).
H
PlateswerereadonaSECTOR6000MSDimager.
PharmacokineticstudiesinBALB/cmice
RadiolabelledmIFNα2-dAbfusionswereadministeredasasingleintravenousdoseat20μg/kg
intothetailveinofagroupofmaleBALB/Cmice.Atarangeoftimepointsupto96hours
postdoseterminalbloodsamplesfrom3miceofeachgroupweretaken.Followingsacrifice,
thekidneys,liverandasuitableamountofmusclewasextracted.Radioactivitylevelswerethen
quantifiedinallsampletypesusinggammacounting.
Analysisofpharmacokineticdata
FinalassayresultswerefittedinWinNonLinbyNCAaccordingtostandardmethods.The
meanPKwasplottedusingGraphpadPrismversion6.DerivedPKparameterswereobtained
fromtheNCAfit.
QuantitativePCRAnalysisofInterferonInducibleGeneExpression
PurificationoftotalRNA>200nucleotides(excludingmiRNA)frombloodwascarriedout
usingRNeasyprotectanimalbloodkit(Qiagen).200ngofRNAwasarrayedintriplicatein96
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Liver-TargetedIFN;PK,PDandEfficacy
wellplatesonice.Reversetranscriptionreactionsweresetupintriplicateconvertingusinga
highcapacitycDNAconversionkit(AppliedBiosystems)followingmanufacturer'sinstruc-
tions.OncompletionofcDNAconversionsamplesweredilutedto5ng/ulofinputRNAand
arrayedin384wellplateformatsat10ng/wellandstoredatfrozenuntilrequired.
LivertissuesampleswereplacedinRNALater(Qiagen)aspermanufacturer’sinstructions.
LiverswerethenhomogenisedinTrizol(LifeTechnologies)ataratioof100mgtissueperml
liquid.A750μlaliquotwasmadeofeachsampleandstoredfrozenuntilprocessed.
150μlofchloroformwasthenaddedtothawedhomogenates,beforemixingfor5minutes
atroomtemperature.Homogenateswerethencentrifugedfor15minutesbeforetransferof
aqueousphaseofthesampletoethanoltoprovideappropriatebindingconditions.Samples
werethenappliedtoRNeasy96wellplates(Qiagen)toallowtotalRNAbinding.Contaminants
werewashedawayusingsuppliedbuffersbeforeapplicationofDNasetoallowdigestionofre-
mainingDNA.DNasewasthenremovedbywashingplateswithsuppliedbuffers.TheRNeasy
RNAmembranewasthendriedtoremoveanyethanol.High-qualityRNAwasthenelutedin
70μlwater.
2.5μgofRNAwasarrayedintriplicatein96wellplatesonice.Reversetranscriptionreac-
tionsweresetupintriplicateusingahighcapacitycDNAconversionkit(AppliedBiosytems)
followingmanufacturer'sinstructions.OncompletionofcDNAconversionsamplesdilutedto
10ng/ulofinputRNAandarrayedin384wellplateformatat20ng/wellandstoredatfrozen
untilrequired.
TaqManreactionplateswerethensetupbyadditionofuniversalPCRmastermix(Applied
Biosystems),adding8μlofmastermixtothe2ulofcDNAtemplatepreviouslyplatedintothe
384-wellplates.1μleachprimerand2μlofwaterwasaddedperwell,givingfinalconcentration
of900nMforwardprimer,900nMreverseprimerand100nMprobe.Plateswerecycledonan
ABI7900HTTaqManmachine(AppliedBiosystems)usingthefollowingcyclingconditions;
50°Cfor2min,95°Cfor10min,followedby40cyclesof95°Cfor15secand60°Cfor1min.All
dataanalysiswasperformedinArrayStudiov3.5.DatapointswereexcludedifCt'sweregreat-
erthan35orlessthan10.Technicaltriplicateswereassessedforvariabilityusingthefollowing
method;iftherangeofCt'sforasetoftriplicatesisgreaterthan1Ct,therawSDStracesareex-
aminedforevidenceofaninefficientreaction.Ifareplicateisanoutliergreaterthan1Ctand
showsevidenceofaninefficientreactionthenitwasexcludedfromthestudy.Ctvalueswere
convertedintoabundances(copies/50ngRNAforliverand/25ngRNAforblood)usinggeno-
micstandardsrunonthesameplate.
Abundancesforeachgenewerenormalisedusingthescoresfromfirstprincipalcomponent
fromaprincipalcomponentanalysisonselectedinvariantgenesGAPDH,ACTBandPPIB.
Thenormalisingconstantwasfittedinthefinalmodelasacovariatesothatthedatawasnor-
malisedandanalysedsimultaneously.
DeterminationofantiviralefficacyinHBVtransgenicmousemodel
TransgenicHBVmiceoriginallyobtainedfromDr.FrankChisari(ScrippsResearchInstitute,
LaJolla,CA)wereusedinthisstudy.HomozygousanimalswereraisedintheBiosafetyLevel3
areaoftheUSULaboratoryAnimalResearchCenter(LARC).Theanimalswerederivedfrom
founder1.3.32.Femaleandmalemice12–16weeksoldwereassignedrandomlytotreatment
groups.Animalsweretreatedonceintravenouslywithdosesof2,20,and200μg/kgoftargeted
mIFNα2ornon-targetedmIFNα2,with1.2,12,or120μg/kgofPEGylatedmIFNα2,orwitha
sterilesodiumacetate(pH5.5)vehicle.Tenanimalswereincludedineachgroup.At24hrafter
injection,themicewerenecropsiedtoobtainsamplesforliverHBVDNAusingqPCRand
Southernblothybridization.
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Liver-TargetedIFN;PK,PDandEfficacy
LiverHBVDNAwasanalysedbySouthernblothybridizationandbyreal-timePCR.The
proceduresforpreparationoflivertissue,SouthernblothybridizationandPCRaredescribed
previously[27].
Results
PharmacokineticanalysisofmIFNα2-dAbfusionproteinsfollowing
intravenousadministration
FollowingasingleintravenousbolusadministrationofmIFNα2-V D2(hereafterreferredto
H
asnon-targetedmIFNα2)andtargetedmIFNα2fusionproteinsinmaleCD-1mice,serumlev-
elsweredeterminedusinganMSDbasedassay.Thisassayformatutilisescaptureviamurine
IFNαspecificrabbitpolyclonalantiseraanddetectionviasulfo-taggedanti-V mousemono-
H
clonalantibodyandis,therefore,designedtomeasurethelevelofintactfusionproteinin
biologicalmatrices.
Fig.1showsthepharmacokineticprofilesofnon-targetedmIFNα2andtargetedmIFNα2in
theserumofmaleCD-1miceaftera5mg/kgintravenousdose.Table1showsasummaryof
thederivedkeyparametersfromnon-compartmentalanalysisofthedata.Someindividual
Fig1.PharmacokineticanalysisofmIFNα2-dAbfusionsinserumfollowingintravenousadministration.TargetedmIFNα2(solidline)andnon-
targetedmIFNα2isotypecontroldAb(dottedline)wereadministeredat5mg/kgviaintravenousinjection.Compoundlevelswereanalysedinserumby
mIFNα2specificantibodycaptureanddAbspecificdetection.Datashownaremeann=3animals.Errorbarsrepresents.e.m.Pharmacokineticparameters
areshowninTable1.
doi:10.1371/journal.pone.0117847.g001
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Liver-TargetedIFN;PK,PDandEfficacy
Table1.Pharmacokineticsofnon-targetedmIFNα2andtargetedmIFNα2inmouseserumafterasingle5mg/kgintravenousdose.
Molecule T½(hr) Tmax(hr) Cmax(ng/mL) SEofCmax AUC(0-1)(hr*ng/mL) Vz(mL/kg) Cl(mL/kg/hr) MRT(hr)
non-targetedmIFNα2 16.2 0.17 17,743.0 1,104.9 11,659.7 9,996.1 428.8 0.5
targetedmIFNα2 17.1 0.08 22,740.0 50.9 6,028.0 20,432.5 829.5 0.7
Non-targetedmIFNα2hadaterminalhalf-lifeof16.2hours,clearanceof428mL/kg/hr,avolumeofdistributionof9,996mL/kgandanAUC(0-1)of
11,660hr*ng/mL.TargetedmIFNα2hadaterminalhalf-lifeof17.1hours,clearanceof829mL/kg/hr,avolumeofdistributionof20,432mL/kgandan
AUC(0-1)of6,028hr*ng/mL.
doi:10.1371/journal.pone.0117847.t001
animalswereexcludedfromtheplotshownandwerenotincludedintheanalysisastheywere
suspectedoutliers.
PharmacokineticanalysisofmIFNα2-dAbfusionproteinsfollowing
subcutaneousadministration
Followingasinglesubcutaneousadministrationofnon-targetedmIFNα2andtargeted
mIFNα2fusionproteinsinmaleCD-1mice,serumlevelsweredeterminedusingtheMSD
assaydescribedabove,inordertodeterminewhetherdifferencesinthepharmacokineticpa-
rametersoftargetedandnon-targetedmIFNα2-dAbfusionproteinswouldbeobservedwhen
usingarouteofadministrationotherthanintravenousinjection.
Fig.2showsthepharmacokineticprofilesofnon-targetedmIFNα2andtargetedmIFNα2in
theserumofmaleCD-1miceaftera5mg/kgsubcutaneousdose.Table2showsasummaryof
thederivedkeyPKparametersfromthenon-compartmentalanalysisofthedata.
Pharmacokineticanalysisof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2inbloodfollowingintravenousadministration
Thestudiesdescribedabovetodeterminepharmacokineticparametersofintactfusionproteins
inserumwerecarriedoutatdosesmuchhigherthantheanticipatedclinicaldoseforIFNther-
apyinhumans.Thiswasarequirementduetothesensitivityoftheassayformat.Thereforein
ordertoinvestigatethekineticsoftargetedmIFNα2andnon-targetedmIFNα2atamoreclini-
callyrelevantdose,astudywascarriedouttomeasuretheconcentrationsofDOTAconjugated
non-targetedmIFNα2andtargetedmIFNα2,labelledwith111In,inwholeblood.Thiswouldin
alllikelihoodovercometheincompatibilitybetweenlowdoseadministrationofcompound
andtheknownsensitivityoftheassaydescribedinFigs.1and2.111In-DOTA-non-targeted
mIFNα2and111In-DOTA-targetedmIFNα2wereadministeredviaasingleintravenousinjec-
tionat20μg/kgintothetailveinofmaleBALB/cmice.Atarangeoftimepoints,terminal
bloodsamplesweretakenandradioactivitylevelsquantifiedinagammacounter.
Fig.3showsthepharmacokineticprofilesof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2inmiceaftera20μg/kgintravenousdose.Table3showsasummary
ofthederivedkeyparametersfromthenon-compartmentalanalysisofthedata.
Pharmacokineticanalysisof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2inliverfollowingintravenousadministration
InordertodeterminethepharmacokineticparametersoftargetedmIFNα2andnon-targeted
mIFNα2inthetargetorgan,liversofmiceinFig.3werecollectedandradioactivitylevels
quantifiedinagammacounter.Thismethod,inadditiontoovercomingassaysensitivityissues
describedabove,wouldalsoovercomepotentialvariabilityintroducedbytherequirementto
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Fig2.PharmacokineticanalysisofmIFNα2-dAbfusionsinserumfollowingsubcutaneousadministration.TargetedmIFNα2(redline)andnon-
targetedmIFNα2isotypecontroldAb(greyline)wereadministeredat5mg/kgviasubcutaneousinjection.Compoundlevelswereanalysedinserumby
mIFNα2specificantibodycaptureanddAbspecificdetection.Datashownaremeann=3animals.Errorbarsrepresents.e.m.Pharmacokineticparameters
areshowninTable2.
doi:10.1371/journal.pone.0117847.g002
extractcompoundpriortodetectionusingantibodybasedmethods.Theresultsobtainedintis-
sueusing111Inlabelledcompoundswouldbeindependentoftheextractionefficiencyofthe
processesusedduringthepreparationofhomogenatesupernatants,andthereforewouldper-
hapsbeamoreappropriatewayofinvestigatingtissuedistributionovertime.
Table2.Pharmacokineticsofnon-targetedmIFNα2andtargetedmIFNα2inmouseserumafterasingle5mg/kgsubcutaneousdose.
Molecule T½(hr) Tmax(hr) Cmax SEofCmax AUC(0-1)(hr*ng/mL) Vz_F(mL/kg) Cl_F(mL/kg/hr) MRT(hr) F(%)
(ng/mL)
non-targetedmIFNα2 15.0 1 1,026.4 207.6 3,103.9 34,775.9 1,610.9 3.1 26.6
targetedmIFNα2 8.5 0.16 75.5 10.9 440.8 138,786.6 11,342.8 14.6 7.3
InserumtargetedmIFNα2hadaterminalhalf-lifeof8.5hours,Tmaxof0.17hours,clearance(offractionabsorbed)of11,343mL/hr/kg,avolumeof
distribution(offractionabsorbed)of138,787mL/kgandanAUC(0-1)of440hr*ng/mL.non-targetedmIFNα2hadaterminalhalf-lifeof15.0hours,Tmax
of1hour,clearance(offractionabsorbed)of1,611mL/hr/kg,avolumeofdistribution(offractionabsorbed)of34,776mL/kgandanAUC(0-1)of
3,104hr*ng/mL.UsingtheanAUC(0-1)calculatedhereandfromthosedeterminedinFig.1(seeTable1)itwaspossibletodeterminethesystemic
bioavailabilityofeachmolecule.Thiswasdeterminedtobe7.3%fortargetedmIFNα2and26.6%fornon-targetedmIFNα2.
doi:10.1371/journal.pone.0117847.t002
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Liver-TargetedIFN;PK,PDandEfficacy
Fig3.Pharmacokineticanalysisof111In-DOTA-mIFNα2-dAbfusionsinbloodfollowingintravenousadministration.TargetedmIFNα2(blueline)and
non-targetedmIFNα2isotypecontroldAb(redline)wereadministeredat20μg/kgviaintravenousinjection.Radioactivitylevelswereanalysedinbloodby
gammacounting.Datashownaremeann=3animals.Errorbarsrepresents.e.m.PharmacokineticparametersareshowninTable3.
doi:10.1371/journal.pone.0117847.g003
Fig.4showsthepharmacokineticprofilesof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2intheliverofmaleBALB/cmiceaftera20μg/kgintravenousdose.
Table4showsasummaryofthederivedkeyparametersfromthenon-compartmentalanalysis
ofthedata.
Pharmacokineticanalysisof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2inkidneyfollowingintravenousadministration
InordertodeterminethepharmacokineticparametersoftargetedmIFNα2andnon-targeted
mIFNα2innon-targetorgans,kidneysofmiceinFig.3wereremovedandradioactivitylevels
quantifiedinagammacounter.Astheprimaryrouteofclearanceof111In-DOTA-mIFNα2-
Table3.Pharmacokineticsof111In-DOTA-non-targetedmIFNα2and111In-DOTA-targetedmIFNα2inmousebloodafterasingle20μg/kg
intravenousdose.
Molecule T½(hr) Tmax Cmax SEof AUC(0-1) Vz Cl MRT AUC(0–12) AUC(0–24)
(hr) (ng/mL) Cmax (hr*ng/mL) (mL/kg) (mL/kg/hr) (hr) (hr*ng/mL) (hr*ng/mL)
111In-DOTA-non-targetedmIFNα2 80.4 0.083 50.2 3.6 42.7 54,331.0 468.7 61.9 22.4 24.6
111In-DOTA-targetedmIFNα2 171.7 0.083 18.6 1.6 52.4 94,650.2 382.0 204.3 11.4 13.3
Inblood,111In-DOTA-targetedmIFNα2hadaterminalhalf-lifeof171hours,clearanceof382mL/hr/kg,avolumeofdistributionof94,650mL/kg,an
AUC(0-1)of52.4hr*ng/mL,anAUC(0–12)of11.4hr*ng/mLandanAUC(0–24)of13.3hr*ng/mL.111In-DOTA-non-targetedmIFNα2hadaterminalhalf-life
of80.4hours,clearanceof468mL/hr/kg,avolumeofdistributionof54,331mL/kgandanAUC(0-1)of42.7hr*ng/mL,anAUC(0–12)of22.4hr*ng/mLand
anAUC(0–24)of24.6hr*ng/mL.
doi:10.1371/journal.pone.0117847.t003
PLOSONE|DOI:10.1371/journal.pone.0117847 February17,2015 9/19
Liver-TargetedIFN;PK,PDandEfficacy
Fig4.Pharmacokineticanalysisof111In-DOTA-mIFNα2-dAbfusionsinliverfollowingintravenousadministration.TargetedmIFNα2(blueline)and
non-targetedmIFNα2isotypecontroldAb(redline)wereadministeredat20μg/kgviaintravenousinjection.Radioactivitylevelswereanalysedinliverby
gammacounting.Datashownaremeann=3animals.Errorbarsrepresents.e.m.PharmacokineticparametersareshowninTable4.
doi:10.1371/journal.pone.0117847.g004
dAbfusionproteinsislikelytobeviathekidney,analysingthistissueasanexampleofnon-tar-
getorgan/tissuewouldalsoallowustofurtherinvestigatedifferencesintheclearanceofthese
twomoleculesinvivo.
Fig.5showsthepharmacokineticprofilesof111In-DOTA-non-targetedmIFNα2and111In-
DOTA-targetedmIFNα2inthekidneyofmaleBALB/cmiceaftera20μg/kgintravenousdose.
Table5showsasummaryofthederivedkeyparametersfromthenon-compartmentalanalysis
ofthedata.
SystemicpharmacodynamiceffectofmIFNα2-dAbfusionproteins
followingintravenousadministration
InordertodeterminetheinvivopharmacodynamiceffectoftargetedmIFNα2andnon-tar-
getedmIFNα2inthesystemiccirculationwemeasuredbyquantitativePCRanalysisthe
Table4.Pharmacokineticsof111In-DOTA-non-targetedmIFNα2and111In-DOTA-targetedmIFNα2inmouseliverafterasingle20μg/kg
intravenousdose.
Molecule T½(hr) Tmax(hr) Cmax SEofCmax AUC(0-1) AUC(0–12) AUC(0–24)
(ng/g) (hr*ng/g) (hr*ng/g) (hr*ng/g)
111In-DOTA-non-targetedmIFNα2 92.9 12 259.1 3.3 31,945.3 2,735.8 5,333.8
111In-DOTA-targetedmIFNα2 67.9 2 397.6 30.1 32,474.0 4,159.0 7,592.6
Inliver,111In-DOTA-targetedmIFNα2hadaterminalhalf-lifeof67.9hours,Cmaxof397ng/g,Tmaxof2hours,anAUC(0-1)of32,474hr*ng/g,an
AUC(0–12)of4,159hr*ng/mLandanAUC(0–24)of7,592hr*ng/mL.111In-DOTA-non-targetedmIFNα2hadaterminalhalf-lifeof92.9hours,Cmaxof
259ng/g,Tmaxof12hours,anAUC(0-1)of31,945hr*ng/g,anAUC(0–12)of2,735hr*ng/mLandanAUC(0–24)of5,333hr*ng/mL.
doi:10.1371/journal.pone.0117847.t004
PLOSONE|DOI:10.1371/journal.pone.0117847 February17,2015 10/19