Fig 2

20 Pages·2016·2.42 MB·English

RESEARCHARTICLE
Formulation, Pharmacokinetic, and Efficacy
Studies of Mannosylated Self-Emulsifying
Solid Dispersions of Noscapine
TerrickAndey1,ApurvaPatel2,SrujanMarepally3,MahavirChougule4,ShawnD.Spencer2,
ArunK.Rishi5,MandipSingh2*
1 DepartmentofPharmaceuticalSciences,SchoolofPharmacy,MassachusettsCollegeofPharmacyand
HealthSciencesUniversity,19FosterStreet,Worcester,MA,UnitedStatesofAmerica,2 Departmentof
Pharmaceutics,CollegeofPharmacyandPharmaceuticalSciences,FloridaA&MUniversity,1520South
MartinLutherKingJr.Blvd.,Tallahassee,FL,UnitedStatesofAmerica,3 InstituteforStemcellbiologyand
RegenerativeMedicine(inStem),NationalCentreforBiologicalSciences(NCBS),Bangalore,India,
4 DepartmentofPharmaceuticalSciences,DanielK.InouyeCollegeofPharmacy,UniversityofHawaiiat
Hilo,200W.St.,Hilo,HI96720,UnitedStatesofAmerica,5 DepartmentofOncology,WayneState
University,Detroit,MI,UnitedStatesofAmerica
*[email protected]
Abstract
OPENACCESS
Citation:AndeyT,PatelA,MarepallyS,Chougule
M,SpencerSD,RishiAK,etal.(2016)Formulation, Purpose
Pharmacokinetic,andEfficacyStudiesof
MannosylatedSelf-EmulsifyingSolidDispersionsof Toformulatehydroxypropylmethylcellulose-stabilizedself-emulsifyingsoliddispersiblecar-
Noscapine.PLoSONE11(1):e0146804.doi:10.1371/ riersofnoscapinetoenhanceoralbioavailability.
journal.pone.0146804
Editor:DhyanChandra,RoswellParkCancer Methods
Institute,UNITEDSTATES
Formulationofnoscapine(Nos)self-emulsifyingsoliddispersiblemicroparticles(SESDs)
Received:July10,2015
wasaffordedbyemulsificationusinganoptimizedformulaofLabrafilM1944,Tween-80,
Accepted:December22,2015
andLabrasolfollowedbyspray-dryingwithhydroxypropylmethylcellulose(HPMC),with
Published:January12,2016 andwithoutmannosamine(Mann-Nos_SESDsandNos_SESDsrespectively);self-microe-
Copyright:©2016Andeyetal.Thisisanopen mulsifyingliquiddispersions(SMEDDs)withandwithoutmannosamine(Mann-Nos_S-
accessarticledistributedunderthetermsofthe MEDDsandNos_SMEDDsrespectively)werealsoprepared.SMEDDsandSESDswere
CreativeCommonsAttributionLicense,whichpermits
characterizedforsize,polydispersity,surfacecharge,entrapmentefficiency,invitroperme-
unrestricteduse,distribution,andreproductioninany
ability,invitroreleasekinetics,andoralpharmacokineticsinSprague-Dawleyrats(10mg/
medium,providedtheoriginalauthorandsourceare
credited. kgp.o).TheantitumorefficacyofMann-Nos_SESDsonthebasisofchemosensitizationto
cisplatin(2.0mg/kg,IV)wasinvestigatedinachemorefractorylungtumorNu/Numouse
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. modeluptoamaximaloraldoseof300mg/kg.
Funding:ThisstudywasfundedbyaP20MD006738
grantfromtheNationalInstituteonMinorityHealth Results
andHealthDisparities(NIMHD).Thefundershadno
Theoil/surfactant/co-surfactantmixtureofLabrafilM1944,Tween-80,andLabrasoloptimized
roleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript. atweightratiosof62.8:9.30:27.90%producedstableself-microemulsifyingdispersions
(SMEDDs)ataSMEDDtowaterratioof1–3:7–9partsbyweight.SMEDDshadhydrody-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. namicdiametersbetween231and246nm;surfacechargesrangedfrom-16.50to-18.7mV;
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OralNoscapineFormulationforEnhancedBioavailability
andentrapmentefficiencieswerebetween32and35%.SESDsrangedinsizebetween5.84
and6.60μmwithsurfacechargesfrom-10.62to-12.40mVandentrapmentefficienciesof
30.96±4.66and32.05±3.72%(Nos_SESDsandMann-Nos_SESDsrespectively).Mann-
Nos_SESDsexhibitedsaturatinguptakeacrossCaco-2monolayers(P =4.94±0.18×
app
10−6cm/s),withcontrolledreleaseof50%ofNosin6hratpH6.8followingHiguchikinetics.
Mann-Nos_SESDswas40%morebioavailablecomparedtoNos_SESDs;andwaseffective
insensitizingH1650SPcellstoCisplatininvitroandinanorthotopiclungtumormodelof
H1650SPorigin.
Conclusions
Mannosylatednoscapineself-emulsifyingsoliddispersions(Mann-Nos_SESDs)arebio-
availableandpotentiatetheantineoplasticeffectofcisplatin-basedchemotherapyincis-
platin-resistantNSCLC.
Introduction
Noscapine,alowtoxicity,naturally-occurringbenzylisoquinolinealkaloidisassociatedwith
anticanceractivity[1,2].Themodeofactionofnoscapine’santicanceractivityispolymeriza-
tionandstabilizationofmicrotubules[3];andwhenadministeredincombinationwithcon-
ventionalchemotherapy,itpotentiatestheinductionofcelldeath[4].However,theprospectof
noscapineasaneffectiveanticancertherapyintheclinicremainsunknown[5,6].Thatnosca-
pine,alipophiliccompound(LogP~2.6)withmoderateaqueoussolubility(solubility(cid:1)0.05
mg/mL)shouldsufferfromlimitedoralbioavailabilityisunderpinnedbyashorthalf-lifestem-
mingfromextensivehepaticmetabolism,asiscommonwithopioids[7,8].Noscapine’santi-
canceractivitytherefore,necessitatesahighoraleffectivedose(ED 300–600mg/kg),thereby,
50
limitingitstranslationalutilityduetopotentialadversereactions[9,10].Therehasconse-
quentlybeenmuchinterestinnanoparticleencapsulationofnoscapineasameansofovercom-
ingreducedplasmaexposureviaprotectionfromenzymaticdegradationandefflux[11,12,13].
Fororaladministration,functionalizingananoparticleviamannosylationhasthebenefitof
allowingforsustainedinputviaintestinallymphaticabsorption,whichmayincreasesystemic
exposure[14].Theintestinallumenhasmicrofold(M)cellsinthefollicularepithelium,cover-
ingimmuneresponsezoneswithinthePeyer’spatch.TheseMcellsexpressmannosereceptors
whichfacilitateendocytictraffickingofparticlesintothelymphatics[15,16,17].Thus,the
designofsystemswithamannosepresentingsurfacehasbecomeapotentiallyviableapproach
forenhancingthedeliveryoforaldrugcandidates[18,19,20].However,engineeringofadrug
carrierforlymphatictraffickingmustfacilitatedelayedintestinalreleasetopromotelymphatic
transit.Hydroxypropylmethylcellulose(HPMC),asemi-syntheticpolymervariouslyusedasa
thickening,suspending,andemulsifyingagenteffectivelystabilizesemulsionsandfacilitates
controlledreleaseofdrugs[21,22,23].Formulationoflowbioavailabilitynoscapineinaself-
emulsifyingdrugdeliverycarrierstabilizedbyspray-dryingwithHPMCwastherefore,pre-
dictedtodelaysystemicinputandpredisposetolymphatictransitandpotentialuptake.
Inthisstudytherefore,wehypothesizedthatnoscapine,orallyadministeredviaamannosy-
latedHPMC-coatedself-emulsifyingcarrierwillbebioavailableandenhancetumorrespon-
sivenesstocisplatin.Thisweexpectedtoresultfroma)slownoscapinerelease,b)increased
lymphaticsystemtransit,c)delayed/sustainedsystemicinput,andd)increasedplasma
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OralNoscapineFormulationforEnhancedBioavailability
exposure,allpotentiallycontributingtoenhancedtumorsensitization.Totestourhypothesis,
weutilizedaspray-driedmannosamine-HPMCmatrixoveranoscapine-loadedself-microe-
mulsifyingdrugdeliverysystem(SMEDD)toevaluate(a)thereleaserate,(b)transportacross
acoloncancercarcinomacell(Caco-2)model,(c)pharmacokineticplasmaexposureinorally
administeredrats,and(d)tumorresponsetoacombinationregimenofnoscapineandcisplatin
inanorthotopicmousemodelofchemorefractoryH1650-inducednon-smallcelllungcancer
(NSCLC).
MaterialsandMethods
Reagents
1
Noscapinehydrochloride,D-mannosaminehydrochloride,laminin,Accutase ,poly-D-lysine,
epidermalgrowthfactor,andfibroblastgrowthfactorwerefromSigmaAldrich(St.Louis,
1 1
MO);Labrafil M1944andLabrasol fromGattefosse(Paramus,NJ).Fetalbovineserum
(FBS),RPMI-1640media,DMEM:F12basemediaandnitrogensupplementwerefromLife
Technologies(GrandIsland,NY).Allothermaterialswereofcellcultureoranalyticalgrade.
Cellculture
H1650cells,originallyfromtheAmericanTypeCultureCollection(ATCC)werekindly
donatedbyDr.SrikumarChellappanattheH.LeeMoffittCancerCenter&ResearchInstitute
(Tampa,FL).SubculturesofH1650cancerstemcells(CSCs)/sidepopulation(SP)cellswere
generatedfromH1650parent/mixedpopulation(MP)cellsbyfluorescenceactivatedcellsort-
ingwithHoechst33342stainingaspreviouslydescribed[24].Caco-2andH460cellswere
fromtheAmericanTypeCellCulture(Manassas,VA).H1650SPcellsweremaintainedin
DMEM:F12supplementedwithfibroblastgrowthfactor(10μg/mL),epidermalgrowthfactor
(10μg/mL),andpenicillin-streptomycin-neomycin(PSN,2%v/v).H1650MPcellsweremain-
tainedinDMEM:F12supplementedwith10%v/vFBSand2%v/vPSN.Caco-2cellsandH460
cellswereculturedinDMEM:F12andRPMI-1640respectively,eachsupplementedwithFBS
(10%v/v),PSN(2%v/v),non-essentialaminoacidsolution(1.2%v/v)andHEPES(10mM).
Allcelllinesweremaintainedundercarbondioxide(5%)at37°C.
Preparationofsolidmicroparticles
1 1
Optimizedconcentrationsofoil(Labrafil M1944),surfactant(Tween 80)andco-surfactant
1
(Labrasol )weredeterminedfromtenaryphasediagrams.Theoptimizedcompositioncon-
1 1
sistedofanoilphase(Labrafil M1944;63.5mL,andLabrasol ;9.39mL)andanaqueous
1
phase(Tween 80;27.9mL).Noscapine(5.0g)wasdissolvedinchloroform(5.0mL)andthen
dispersedinanoilphasecomprisingLabrafilM1944andLabrasol(astheoilandco-surfactant
respectively).Thenoscapine-oildispersionwaseitheremulsifiedinanaqueousphase(consist-
1
ingofTween 80andwater)toformself-microemulsifyingdrugdeliverysystems(Nos-
SMEDDs)orinjectedintoaspray-dryer.Spray-dryingwascarriedoutbymixingtheaqueous
andoilphaseswithinaheated(110°C)nitrogenatmosphereinthedryingchamberofaPulvis
Mini-SprayGS-310(YamatoScientificAmerica,USA).Theaqueousphase(comprising
1 1
Tween 80andHPMCcolloid(10%w/v),orTween 80andHPMC-MannosamineHCl
(1.0%w/v)colloidwasinductedintoa1550AutoJetModularSpraySystem(SprayingSystems
Co.,Wheaton,IL)andtheatomizedmixturefedthroughchannel1whereastheoilphasewas
simultaneouslyfedthroughchannel2ofthedualspraynozzleandatomizedintofineoildrop-
lets.Coatingofoildropletsbycolloidalaqueousphaseoccursinthedryingchamberunder
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OralNoscapineFormulationforEnhancedBioavailability
pulseflowwithresultantnoscapineself-emulsifyingsoliddispersiblemicroparticles(Mann-
Nos_SESDsorNos_SESDs).
Validationofnanoparticlesandmicroparticles
Nos_SMEDDsandMann-Nos_SMEDDsandNos_SESDsandMann-Nos_SESDswerechar-
acterizedforsize,polydispersity(PDI),zetapotential,andentrapmentefficiency.Particle
sizeandzetapotentialofnoscapineformulationsweredeterminedusingaNicomp380ZLS
(ParticleSizingSystems,PortRichey,FL).SMEDDs(10μL),orSESDs(2.0mg)weredis-
persedindouble-distilledwaterto4.0mLinacuvetteandparticlesize,PDI,andzetapoten-
tialreadingstaken.Entrapmentefficiency(EE)ofSMEDDsandSESDswasdeterminedby
centrifugationofformulationusingavivaspincolumnwithmolecularweightcut-off
(MWCO)of10kDa.ThefiltratewascollectedandelutedoveraC18columnbyHPLC(see
BioanalysisMethod).EEwasestimatedaccordingto,EE(%)=[(W —W)/W ]×100,
n r n
where,W =weightofNosinSMEDDsorSESDs,andW =weightofNosinfiltrate.Deter-
n r
minationofEEforSESDswasdonebydissolving10mgofsamplein250μLofaceticacidin
amicrocentrifugetube.Thesamplewasvortexedandcentrifugedat12,000×gfor5min.
Theaqueousphasewascollectedand0.75mLNaOH(4M)addedtoprecipitateNos.The
samplewascentrifugedat14,000×gfor15minandthesupernatantdiscarded.Theprecipi-
tatewasdissolvedinacetonitrileandelutedoveraC18columnbyHPLC.Determinations
weredoneintriplicatesforeachformulation.
Releaseofnoscapinefrommicroparticles
Thedissolutionofspray-driednoscapinemicroparticles(i.e.Nos_SESDsandMann-Nos_
SESDs)wasinvestigatedunderUSPguidelinesfordelayedreleasedosageformsinaVanKel
7000DissolutionSystem(Varian,Cary,NC)usingtheUSPDissolutionApparatus1basket
method.Theglassvesselwasfilledwith0.75Lofdissolutionmedium(i.e.0.1NHClbuffer,
pH2.0)andallowedtoequilibrateat37°C.Nos_SESDsorMann-Nos_SESDs(0.5g)was
placedinthemeshbasketandrotatedat50rpmfor2hr.Sampling(2.0mL)wasdonefromthe
dissolutionmediumat5,10,15,20,30,45,60,90and120min.Volumecorrectionwasmain-
tainedbyreplacingthealiquotvolumewithanequalvolumeoffreshdissolutionmedium.A
volumeof0.25Lof0.2MNa PO4wasaddedtothedissolutionmediumandthepHadjusted
3
to6.8at120min.Samplingandvolumecorrectionwascontinuedat4,6,20,and24hr.The
sampleswereloadedontoaC18columnforHPLCanalysis.Cumulativeconcentrationwas
normalizedagainstinitialconcentrationandpresentedasPercentDrugReleasevsTime.
Determinationsweredone2×intriplicates.
NoscapinetransportacrossCaco-2monolayers
At80%confluence,Caco-2cellsweredetachedwithtrypsin/EDTAandwashed2×withPBS.
Cellsuspensions(105cellspermL)wereseededintheapicalcompartmentofaCorning1
Costar1Transwell1permeablesupportinsertwithpolycarbonatemembrane(0.4μmpore
size)ina12-wellplateformatusing0.5mLvolume.Cellsweremaintainedfor21dayswith
mediachangesonalternatedaysfor14daysanddailyclosetoconfluence.Formationand
integrityofCaco-2monolayerandtightjunctionsweremonitoredbytransepithelialelectri-
calresistance(TEER=200–300O(cid:3)cm2usingaMillicell1ERS-2Volt-OhmMeter(Millipore,
Billerica,MA).TEERbelow190O(cid:3)cm2wasdiscarded.Drug-freebuffer(HBSS-HEPES)was
addedtothedonor(pH6.5)andacceptor(pH7.4)compartmentsinahumidified(5%CO ),
2
pre-warmed(37°C)incubatorfor10minforpreconditioning.Samplesolutionwasaddedto
donororacceptorcompartmentforflux:apical-basolateralorefflux:basolateral-apical
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OralNoscapineFormulationforEnhancedBioavailability
estimates(i.e.A-B,B-Arespectively),anddrug-freebuffertocountercompartmentforsam-
plingat15,30,45,60,90,and120min.Transportwascarriedoutonanorbitalshaker(50
oscillations/min)tominimizetheeffectofanaqueousboundarylayer.Thedirectionaltrans-
portoftotalnoscapine(i.e.freeNos+SMEDDentrappedNos)wasestimatedbyliquid
extractionwithtert-methylbutyletherandquantificationbyHPLC.Theapparentpermeabil-
ity(P )ofNosusingthisdeliverysystemwascomputedatequilibrium(steady-stateflux)
app
accordingto:P (cm/s)=(ΔQ/Δt)×[(1/Q )×(1/A)]×V,whereΔQ/Δt=discretediffer-
app ss r
enceinreceivermasspertimeinterval(μg/s);Q =massofdrugindonorchamberatsteady
ss
state(μg);t=time(s);A=areaofmembraneinsert(cm2);V =volumeofsolutionreceiver
r
chamber(cm3).ThenetfluxdirectionoftotalNosacrossCaco-2monolayerswasestimated
fromanEffluxRatio(ER)=(P B-A)/(P A-B).Experimentswererepeated2×andPapp
app app
estimatesdoneintriplicatesforeachgroup.
Bioanalysismethod
Noscapine(Nos)stocksolution(1.0mg/mL)waspreparedbydissolvingNosinacetonitrile.
WorkingsolutionsofNoswerepreparedbydilutingtheprimarystocksolutiontoobtain
2.5,5,10,25.0,50.0,and100.0μg/mL.Plasmacalibrationstandards(0.4,0.5,1.0,2.0,10.0,
and20.0μg/mL)werepreparedindrug-naiveratplasmaobtainedfromSprague-Dawley
(SD)rats.Ratplasma(160μL)wasspikedwithNosworkingsolution(40μL)andvortexed
for1min.Noswasextractedbyadding100μLtert-methylbutylethertothemixtureand
vortexingfor1min.Theorganicphasewasseparatedandthesolventevaporated.Quality
control(QC)sampleswerepreparedsimilarlytoobtainplasma-drugconcentrationof0.4,
1.0,and10.0μg/mLcorrespondingtolow,intermediate,andhighQCrespectively.Samples
werecentrifugedat12,000×gfor15minandthesupernatantcollected.Mobilephasecon-
sistingof20mMammoniumacetate(pH4.5):water(65:35)waspreparedandfilteredusing
a0.45μmfilter.Sampleswereinjected(0.1μL)intoaSymmetry1C18columnconnectedto
aWaters1HPLCunderisocraticflowof1.0mL/minanddetectioncarriedoutatλ of
max
232nm.HPLCrunsweredone3×foreachsampleandthesamemethodwasutilizedforall
analysesinthisstudy.
Ethicsstatement
TheprotocolsforinvivoexperimentswereapprovedbytheInstitutionalAnimalCareandUse
Committee(IACUC)ofFloridaA&MUniversity.
PharmacokineticsinSprague-Dawleyrats
Sprague-Dawleyrats,withmeanweightof250g,weredividedinto4groups(n=3)andgiven
eitherasingledoseofNoscapine(Nos)asanintravenousbolusat2.0mg/kg,ororallyat10
mg/kg,aseitherNosincornoil,Nos_SESDsorMann-Nos_SESDs.TheI.V.dosewasprepared
bydissolvingNosina0.1mLvolumeofPBS(pH4.5)andthenadministeredbytailveininjec-
tionwhereasoraldosesweregivenbygavage.Bloodsampleswerecollectedintoheparinized
tubesat0.167,0.5,1,4,8,and24hr.Thesampleswerecentrifugedat12,000×gfor15min,the
plasmacollectedandanalyzedasdescribed(seeBioanalysisMethod).Aconcentration-time
plotofNoswasobtainedandthedataanalyzedbysimultaneousI.V.andOralnonlinear
regressionofpooleddataofallanimalsineachgrouptoobtainpharmacokineticparameters
foratwocompartmentmodelwithvascular(I.V.)extravascular(oral)input.Thedrug
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OralNoscapineFormulationforEnhancedBioavailability
concentrationwasdescribedbydifferentialequationsaccordingto:
dA
DOSE ¼Ka(cid:4)F(cid:4)A
dt DOSE
dA
C ¼Ka(cid:4)F(cid:4)A þ K (cid:4)A (cid:5)K (cid:4)A (cid:5)Ke(cid:4)A
dt DOSE 21 P 12 C C
dA
P ¼K (cid:4)A (cid:5) K (cid:4)A
dt 12 C 21 P
A ¼C (cid:4)V
C P C
K ¼Q=V
12 C
K ¼Q=V
21 P
Ke¼CL=V
C
A ð0Þ ¼ administereddose
DOSE
A ð0Þ ¼ 0
C
TheVsswasthemodelestimatedsumofvolumeofcentral(Vc)andtissue(Vp)compart-
ments,whereasCmaxandTmaxwerecalculatedwithstandardformulasusingthepharmaco-
kineticparameters.
H1650SPandH1650MPspheroidformation
H1650SPorH1650MPcellsuspensionsinserum-freemediawereseededat104cellsperwell
1
ina24-wellLipidure -CoatMulti-Dishplate(NOFCorporation,Japan)upto7daysfor3D
culture,changingmediaonalternatedays.Spheroidformationwasobservedondays3,5,and
7inbright-fieldusinganOlympusBX40fluorescencemicroscopeconnectedtoaDP71camera
(Olympus,Japan)at40×magnification.Thespheroidsurfaceareawasestimatedusingthe
ImageJsoftware(NationalInstitutesofHealth,Bethesda,MD).Resultswereexpressedasmean
surfacearea±standarddeviationofatleast3replicatesoftwoexperimentseach.
Cellviability
Thecellviabilityassaymethodwasaspreviouslydescribed[25,26].Briefly,H1650MP,H1650
SP,orH460cellswereseededina96-wellformat(1×104perwell)andincubatedfor16–18
hr.Treatmentwascarriedoutwithdifferentconcentrationsofcisplatinand/ornoscapineand
incubatedfor72hr.Cellviabilitystudiesforcombinationsofnoscapineandcisplatinwere
donebyvaryingtheconcentrationsofcisplatinwithfixedconcentrations(5and20μM)of
noscapineandincubatingfor72hrpost-treatment.ThecellswerewashedwithPBS2×and
fixedin0.1mLglutaraldehydesolution(0.025%w/v)andincubatedat37°Cfor30min.Glu-
taraldehydewasremovedand1μLof(0.01%w/v)crystalvioletsolutionwasaddedandincu-
batedfor15minatroomtemperature.Theplateswerewashedandair-driedfollowedbythe
additionofdisodiumhydrogenphosphatesolution.Theabsorbanceofthedissolvedcrystal
violetwasreadat540nmandthecellviabilitycalculatedasapercentageofuntreatedcontrols.
Determinationsofcellviabilityweremadeatleast3×andthedatapresentedasaplotofcell
viabilitywithlogdoseoftreatment.TheIC ofeachdrugwascomputedwhereappropriateby
50
regressionandpresentedasmeanIC ±SD.Similarly,theeffectof20μMnoscapinebasein
50
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OralNoscapineFormulationforEnhancedBioavailability
Nos_SESD,Mann-Nos_SESD,andtheircombinationwithdifferentconcentrationofcisplatin
onthecell72-hrviabilityofH1650SPcellswasinvestigated.The20μMequivalentweightof
Nos_SESD(26.70μg)andMann-Nos_SESD(25.80μg)permlofsolvent(RPMI1640)media
wereaddedtoH1650SPcellstreatedwithandwithoutdifferentconcentrationscisplatinto
obtainatotalnoscapinebaseconcentrationof20μMin200μLfinalvolume.Thecellswere
incubatedfor72hrandanalyzedforcellviabilityusingthecrystalvioletassay.IC valuesrep-
50
resentingmean±SDoftriplicateassayswereestimated.
OrthotopicH1650NSCLCtumormodelinNu/Numice
FemaleNu/Numice,sixweeksold(Harlan,Indianapolis,IN)wereplacedunderisoflurane-
inducedanesthesiaunderasepticconditions.Leftlateralchestwasdousedwithiodineand
cleanedwithanalcoholswab.Asmalllateralincision(~5mm)wasmadetotheleftchestin
1
theplaneoftheleftfore-limbjustbelowthescapula.Acellsuspension-filledB-D 1mL
1
latexfreesyringeconnectedtoa27-gaugeSurflo wingedinfusionsetwasusedtodeliveran
inoculumof1×104H1650SP(n=29)orMP(n=1)cellsin0.1mLserum-freeDMEM:
F12,throughthesixthintercostalspaceintotheleftlung.Incisionswereclosedwithsurgical
skinclipsandanimalsobservedforfullmotorandcognitiverecovery.Miceweremaintained
for30daysfordevelopmentoflungtumorverifiedbydissectionofamouse(bearingH6150
SPorMPtumorseach)foranatomicalobservationandextractionoftumorlysateforwest-
ernblot.
AntitumorevaluationofnoscapineinNu/Numice
Animalswererandomizedinto7groups(n=4)of(i)untreated,(ii)Cisplatin,(Cis)(2mg/kg/
biweeklyI.V.,),(iii)Nos_SESDs(300mg/kg/day),(iv)Mann-Nos_SESDs(150mg/kg/day),(v)
Mann-Nos_SESDs(300mg/kg/day),(vi)Mann-Nos_SESDs(150mg/kg/day)+Cis(2mg/kg/
wkI.V.),and(vii)Mann-Nos_SESDs(300mg/kg/day)+Cis(2mg/kg/wk,I.V.,).Doseswere
selectedbasedonpreviousstudiesinourlaboratory[2,4,27,28].Treatmentwasstartedonday
8post-inoculationandcontinuedfor14daysconsistingofdailyoraladministrationofNosfor-
mulationsandintravenousinjectionsofCissolution(biweeklyasasingletreatmentorweekly
incombinationwithNos_SESDs).CissolutionsweremadebydispersingCisinnormalsaline
undersonicationandpHadjustedto7.4toobtaina1.0mg/mLsuspension.Tocheckforevi-
denceoftoxicity(weightloss>25%)orunreasonabletumorburden,theanimalswereweighed
weekly.Themicewerefedwithfoodandwateradlibitum.MiceweresacrificedunderCO -
2
inducedhypoxiaandlungtumorsresected.Tumorgrowthinhibitionwasestimatedfromlung
weightandtumorweight.
Statisticalanalysis
Analysesofnanoparticlecharacterization,spheroidformation,cellviability,westernblot,lung
1
andtumorweightsweredoneusingGraphPadPrism 5.0(GraphPadSoftware,Inc.).Caco-2
permeationdataanalysiswasdoneinMicrosoftExcel.Pharmacokineticdatawereanalyzed
1
usingWinNonlin (Pharsight,Cary,NC).Differencesbetweenmeanswereanalyzedby
unpairedt-testorOne-wayANOVA,andconsideredsignificantatP<0.05.Differencesacross
groupmeanswereanalyzedbyOne-wayANOVA.Comparisonsbetweengroupswereana-
lyzedbyTukey’stest.Datawerepresentedasmean±SDexceptotherwisestated.
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OralNoscapineFormulationforEnhancedBioavailability
Table1. CharacterizationofNoscapineself-microemulsifyingdrugdelivery(SMEDD)andself-emulsifyingsoliddispersible(SESDs)formulations
fororaldelivery.
Sample ParticleSize(nm) PolydispersityIndex,PDI Zeta-Potential,ζ(mV) EntrapmentEfficiency,EE(%)
BlankSMEDD 230.91±6.33 0.26±0.002 -16.50±0.82 -
Nos-SMEDD 246.33±3.79 0.303±0.005 -18.17±0.68 32.36±4.28
Mann-Nos_SMEDD 238.52±4.88 0.361±0.002 -18.69±0.91 35.02±6.56
Sample ParticleSize(μm) PolydispersityIndex,PDI Zeta-Potential,ζ(mV) EntrapmentEfficiency,EE(%)
BlankSESDs 5.84±3.63 0.316±0.002 -12.18±1.32 -
Nos-SESDs 6.42±5.03 0.293±0.003 -10.62±0.83 30.96±4.66
Mann-Nos_SESDs 6.60±3.16 0.266±0.003 -12.40±1.61 32.05±3.72
Noscapinewasformulatedinaself-microemulsifyingdrugdelivery(SMEDD)systemwithandwithmannosamine(Mann)andspray-driedtoobtainself-
emulsifyingsoliddispersible(SESD)particles.Particleswerecharacterizedforsize,polydispersity,zetapotential,andentrapmentefficiencyasshownin
MaterialsandMethods.Dataarepresentedasmean±SDofatleast3analyses.Differencesobservedwerenotsignificant(onewayANOVA,P<0.05).
doi:10.1371/journal.pone.0146804.t001
Results
Spray-driedmannosamine-coatedself-emulsifyingcarriersproduce
stablesolidmicroparticles
Validityofinvivodatawillbedependentonaccuratecharacterizationofstableparticles.Opti-
mizationoftheternarysystemconsistingofoil(LabrafilM1944),surfactant(Tween80)and
co-surfactant(Labrasol)resultedinasuitablecompositionbyweightof62.8%and9.3%and
27.9%respectively.StableSMEDDsresultedfrommixingthecomponents(1–3parts)and
dispersingindistilledwater(7–9parts).ResultsofSMEDDandSESDscharacterizationare
presentedinTable1.BlankSMEDDS,Nos_SMEDDs,andMann-Nos_SMEDDsweremondis-
perse(PDIof0.260±0.002,0.303±0.005,and0.361±0.002respectively)withmeanhydrody-
namicdiametersof230.91±6.33,246.33±3.79,and238.52±4.88nmrespectively.Negative
surfacechargesof-16.50±0.82,-18.17±0.68,and-18.69±0.91mVwererecordedforBlank
SMEDDs,Nos_SMEDDs,andMann-Nos_SMEDDsrespectively;allofwhichwerewithinthe
rangefornanoparticleemulsionstability.EntrapmentefficiencyofnoscapineinNos_
SMEDDsandMann-Nos_SMEDDswere32.36±4.28%and35.02±6.56%(n=3)respectively,
whichenabledaccuratecalculationofdoses.ParticlesizeforBlank-SESDs,Nos_SESDs,and
Mann-Nos_SESDswere5.84±3.63,6.42±5.03,and6.60±3.16μmrespectively;theSESDs
showednarrowdistributioninparticlesizewithpolydispersityindicesof0.316±0.002,0.293
±0.003,and0.266±0.003respectively.Zetapotentialwas-12.18±1.32,-10.62±0.83,and-12.40
±1.61mVforBlankSESDs,Nos_SESDs,andMann-Nos_SESDsrespectively.Entrapmenteffi-
ciencyofnoscapineinNos_SESDsandMann-Nos_SESDswere30.96±4.66and32.05±3.72%
respectively.
Mannosylatedmicroparticleretains50%oforalnoscapinethrough6
hours
Theaqueousreleasecharacteristicsofspray-driedNos_SESDsandMann-Nos_SESDswere
investigatedusingtheUSPApparatus1methodfordelayed-releasedosageformsundergastric
andintestinalconditions(i.e.pH2.0and6.8respectively)(Fig1).AtgastricpH,amaximumof
29.4%ofNos_SESDsversus16.4%ofMann-Nos_SESDswasobservedattheendof2hr(Fig
1a).AtintestinalpH,51.3%and44.4%ofnoscapinewasreleasedfromNos_SESDsandMann-
Nos_SESDsrespectivelywithinthenext4hr(Fig1a).Noscapinereleasefrombothformula-
tionswasalmost100%at24hours.Thedatawerefittedintozero-order,first-order,and
PLOSONE|DOI:10.1371/journal.pone.0146804 January12,2016 8/20
OralNoscapineFormulationforEnhancedBioavailability
Fig1.Mannosylatedmicroparticleretaines50%oforalnoscapinein6hours.(a)ThedissolutionrateandreleaseoftotalnoscapinefromHPMC-coated
spray-driedNos_SMEDDandMann-Nos_SMEDDareshowninsimulatedgastric(pH2.0)andintestinal(pH6.8)buffersfrom5minto24hr.Resultswere
presentedasmeanpercentofNosreleasevstime.(b)KineticreleaseofNos_SMEDDandMann-Nos_SMEDDshowingHiguchirelationshipofdrugamount
releasedfromsolidmicroparticleintodissolutionbufferagainstsquarerootoftime.Straightlineequationobtainedbylinearregression.Bothformulations
werelinearwithR2rangingbetween0.9905and0.9968.Datarepresents6replicatesofexperimentsrepeated2×anddifferenceswerenotconsidered
significant.
doi:10.1371/journal.pone.0146804.g001
PLOSONE|DOI:10.1371/journal.pone.0146804 January12,2016 9/20
OralNoscapineFormulationforEnhancedBioavailability
Higuchikineticmodelsfortimepredictability,andshowedthehighestfidelitytotheHiguchi
relationshipwhereQ(Mass)=K ×t1/2.Aplotofconcentrationofdrugreleasedagainstsquare
H
rootoftimeproducedlineardiffusionprofilesforNos_SESDsandMann-Nos_SESDs
describedbytheequationsQ=2.124t1/2–0.254(R2=0.997)andQ=3.308t1/2–2.153(R2=
0.991)respectively(Fig1b).
Mannosylatedmicroparticlesaturatesnoscapinetranscytosis
Thepermeationresultsacrosscaco-2monolayersareshowninFig2.Theestimatedapparent
permeability(Papp)fortotalnoscapinefromNos_Solution,Nos_SMEDD,andMann-Nos_S-
MEDD,revealedthattheconstantfortranscytosiswashighestusingthemannosylatedformu-
lation.Theapical/donor(pH6.5)tobasolateral/acceptor(pH7.4)permeabilityoftotal
noscapinewaslowinNos_Solution(2.50±0.17×10−6cm/s)comparedtoNos_SMEDD(3.43
±0.13×10−6cm/s),andMann-Nos_SMEDD(4.94±0.18×10−6cm/s).Aplotofconcentration
vstimefortotalnoscapineinNossolutionandNos_SMEDDwereapproximatelylinearthere-
fore,estimationoftheirPappwasbasedontheassumptionofratesaturationandzero-order
kinetics.ThetimecourseoftotalnoscapinetransportfromMann-Nos_SMEDDSfollowed
first-orderkineticswithapparentreceptorsaturation(Fig2b).Thedifferencesinthesecretory
transportoftotalNoswasintheorderofNossolution(13.66±1.01×10−6cm/s)>>>Nos_S-
MEDDS(5.50±0.65×10−6cm/s)>>Mann-Nos_SMEDDS(1.03±0.15×10−6cm/s)(Fig2c).
Inparallelorder,effluxratios(ER)followedthehierarchyofNossolution(5.46)>>>Nos_S-
MEDD(1.61)>>Mann-Nos_SMEDD(0.21±0.13),indicatingthemannosylatedmicroparti-
clewasleastsubjecttoefflux(Fig2d).
Mannosylatedmicroparticleshavedelayedabsorptionandhigher
bioavailability
Plasmadrugconcentration-timeplotforeachformulationisshowninFig3.Pharmacokinetic
parameterswereobtainedbynonlinearregressionofplasmaconcentrationvstimedatapoints
usingtwo-compartmentalanalysesofintravenousandoralinputofnoscapineareshownin
Table2.IntravenousbolusinjectionofNoscapinesolutionat2mg/kgwasdistributedintothe
peripheral/tissuecompartmentatarateof3.867h-1(k12);therateofdrugtransportfromthe
peripheralcompartmentintothecentralcompartment(k21)andrateofdrugeliminationwere
4.677h-1each.Totalvolumeofdrugdistributionatsteadystate(Vss)was1856.442mL,and
thevolumeofdistributioninthecentral(Vc)was1016.209mL.Therateofsystemicclearance
(Cl)was46.524mL/hwithaneliminationhalf-life(T1/2)of15.137handareaunderthecon-
centration-timecurve(AUC )of3.925μg(cid:3)h/mL.Oralformulationofnoscapineincornoilat
0-4
adoseof10mg/kgwascharacterizedbyanabsorptionrate(ka)of5.161h-1withahalf-lifeof
drugabsorption,T1/2,absof0.134h.Therateofdrugelimination,kelwas0.301h-1withater-
minalhalf-life,T1/2eliminationof2.302h.Themaximumplasmadrugconcentration,Cmax
was0.945μg/mLandthetimetoreachmaximumconcentration,Tmaxwas0.585h.Thearea
underthecurvewas3.812μg(cid:3)h/mLandtheabsolutebioavailabilitywas19.424%.At10mg/kg
oraldose,Nos_SESDtheka,T1/2abs,kel,andT1/2eliminationwere4.729h-1,0.147h,0.160
h-1,and4.331hrespectively;theCmax,Tmax,AUC,andabsolutebioavailabilitywere
1.627μg/mL,0.742h,5.191μg(cid:3)h/mL,and26.451%respectively.Theka,T1/2abs,kel,andT1/
2eliminationoforalMann_Nos_SESDwere0.636h-1,1.090h,0.101h-1,and6.861hrespec-
tively;theCmax,Tmax,AUC,andabsolutebioavailabilitywere1.308μg/mL,3.439h,
7.274μg(cid:3)h/mL,and36.927%respectively.
PLOSONE|DOI:10.1371/journal.pone.0146804 January12,2016 10/20