Epiisopilosine alkaloid has activity against Schistosoma mansoni in mice without acute toxicity

19 Pages·2017·14.33 MB·English

RESEARCHARTICLE
Epiisopilosine alkaloid has activity against
Schistosoma mansoni in mice without acute
toxicity
MariaA.Guimarães1,2,3☯,RosimeireN.deOliveira4☯¤,RebecaL.deAlmeida1☯,Ana
C.Mafud5,6☯,AnaL.V.Sarkis7☯,RayaneGanassin8☯,MarcosP.daSilva9,Daniel
B.Roquini9,LeizM.Veras1☯,TaˆniaC.H.Sawada2‡,CristinaD.Ropke3‡,Luis
A.Muehlmann8,10‡,GraziellaA.Joanitti8,10,SelmaA.S.Kuckelhaus7‡,Silmara
M.Allegretti4‡,YvonneP.Mascarenhas5‡,Josue´deMoraes9‡,Jose´R.S.A.Leite3,7‡*
a1111111111
1 Nu´cleodePesquisaemBiodiversidadeeBiotecnologia,BIOTEC,UniversidadeFederaldoPiau´ı,
a1111111111
Parna´ıba,Piau´ı,Brazil,2 PhytobiosPesquisaDesenvolvimentoeInovac¸ãoLTDA,Parna´ıba,Piau´ı,Brasil,
a1111111111
3 ProgramadePo´s-graduac¸ãoemBiotecnologia,RENORBIO,PontoFocalUniversidadeFederaldoPiau´ı,
a1111111111
Teresina,Piau´ı,Brazil,4 DepartamentodeBiologiaAnimal,InstitutodeBiologia,UniversidadeEstadualde
a1111111111 Campinas,Campinas,SãoPaulo,Brazil,5 InstitutodeF´ısicadeSãoCarlos,UniversidadedeSãoPaulo,
SãoCarlos,SãoPaulo,Brazil,6 DeptMedicalParasitologyandInfectionBiology,SwissTropicalandPublic
HealthInstitute,Basel,Switzerland,7 FaculdadedeMedicina,UniversidadedeBras´ılia,UNB,Campus
DacyRibeiro,Bras´ılia,DistritoFederal,Brazil,8 Laborato´riodeNanobiotecnologia,InstitutodeBiologia,
UniversidadedeBras´ılia,UNB,CampusDacyRibeiro,Brasilia,DistritoFederal,Brazil,9 Nu´cleodePesquisa
emDoenc¸asNegligenciadas,UniversidadeGuarulhos,Guarulhos,SãoPaulo,Brazil,10 Faculdadede
OPENACCESS Ceilandia,UniversidadedeBras´ılia,UNB,Bras´ılia,DistritoFederal,Brazil
Citation:GuimarãesMA,deOliveiraRN,de
☯Theseauthorscontributedequallytothiswork.
AlmeidaRL,MafudAC,SarkisALV,GanassinR,et
¤ Currentaddress:UniversidadeEstadualdePontaGrossa–UEPG,PontaGrossa-Parana´,Brazil
al.(2018)Epiisopilosinealkaloidhasactivity
‡Theseauthorsalsocontributedequallytothiswork.
againstSchistosomamansoniinmicewithout
*[email protected]
acutetoxicity.PLoSONE13(5):e0196667.https://
doi.org/10.1371/journal.pone.0196667
Editor:SalahA.Sheweita,AlexandriaUniversity, Abstract
EGYPT
Received:November24,2017 SchistosomiasisisadiseasecausedbyparasitesofthegenusSchistosoma,currently
affectingmorethan200millionpeople.Amongthevariousspeciesofthisparasitethatinfect
Accepted:April17,2018
humans,S.mansoniisthemostcommon.Pharmacologicaltreatmentislimitedtotheuseof
Published:May11,2018
asingledrug,praziquantel(PZQ),despitereportsofparasiteresistanceandlowefficacy.It
Copyright:©2018Guimarãesetal.Thisisanopen isthereforenecessarytoinvestigatenewpotentialschistosomicidalcompounds.Inthis
accessarticledistributedunderthetermsofthe
study,wetestedtheefficacyofepiisopilosine(EPIIS)inamurinemodelofschistosomiasis.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and AsingledoseofEPIIS(100or400mg/kg)administeredorallytomiceinfectedwithadultS.
reproductioninanymedium,providedtheoriginal mansoniresultedinreducedwormburdenandeggproduction.Thetreatmentwiththelower
authorandsourcearecredited.
doseofEPIIS(100mg/kg)significantlyreducedtotalwormburdenby60.61%(P<0.001),
DataAvailabilityStatement:Allrelevantdataare aswellasdecreasinghepatosplenomegalyandeggexcretion.Scanningelectronmicros-
withinthemanuscript.
copyrevealedmorphologicalchangesinthewormtegumentaftertreatment.Despitegood
Funding:TheauthorsaregratefultoPhytobios activityofEPIISinadultS.mansoni,oraltreatmentwithsingledoseofEPIIS100mg/kghad
PesquisaDesenvolvimentoeInovac¸ãoLTDA., onlymoderateeffectsinmiceinfectedwithjuvenileS.mansoni.Inaddition,weperformed
companyoftheCentrofloraGroup,foritssupport
cytotoxicityandtoxicologicalstudieswithEPIISandfoundnoinvitrocytotoxicity(inHaCaT,
duringtherealizationofthisresearch.SMAis
gratefultoConselhoNacionaldeDesenvolvimento andNIH-3T3cells)ataconcentrationof512μg/mL.Wealsoperformedinsilicoanalysisof
Cient´ıficoeTecnolo´gico(CNPq)(MCTI/Cnpq/MS- toxicologicalpropertiesandshowedthatEPIIShadlowpredictedtoxicity.Toconfirmthis,
SCTIEDECITN.404134/2012-2).ACMisgrateful
weinvestigatedsystemicacutetoxicityinvivobyorallyadministeringa2000mg/kgdoseto
toFoundationforResearchSupportofSãoPaulo
Swissmice.Treatedmiceshowednosignificantchangesinhematological,biochemical,or
State(FAPESP)(Grants2014/02282-76and2016/
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Epiisopilosineforschistosomiasiswithouttoxicity
18023-5).JMisgratefultoFAPESP(Grant2016/ histologicalparameterscomparedtonon-treatedanimals.Epiisopilosineshowedpotential
22488-3).Phytobioscompanyplayedaroleinthe asaschistosomicidaldrug:itdidnotcauseacutetoxicityanditdisplayedanacceptable
studydesign,dataanalysis,decisiontopublishand
safetyprofileintheanimalmodel.
preparationofthemanuscript.Thespecificrolesof
theseauthorsarearticulatedinthe‘author
contributions’section.Theotherfundershadno
roleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthe
manuscript.
Competinginterests:Thecommercialaffiliation Introduction
PhytobiosdoesnotalterouradherencetoallPLOS
Brazilianbiodiversityhasbeenstudiedextensivelyandincreasingnumbersofstudiesrelated
ONEpoliciesonsharingdataandmaterials.
toplantspeciesandnaturalresourceshavecontributedtonewtherapeuticalternatives.An
exampleofthisdevelopmentconcernsthePilocarpusmicrophyllusStapfexWardlewspecies,
popularlyknownasjaborandi,originatingfromNorthandNortheastBrazil[1,2].Pilocarpine
alkaloidisproducedcommerciallybyextractionfromjaborandileaves;thealkaloidisused
commerciallyforeyeproceduresandtreatmentofglaucoma,andisthereforeofgreateco-
nomicinterest[3,2].
Jaborandileaves,likeotherplantspecies,containseveralbioactivemetaboliteswhosephar-
macologicalandphysiologicalpropertieshavenotbeenfullyelucidatedorarestillbeingstud-
ied;theyalsocontaintheepiisopiloturine(EPI),analkaloidwithantischistosomalactivity[4,
5,6,7,8].
EPIIS,or(3R,4S)3[(S)hydroxy(phenyl)methyl]4[(3methyl3H1λ2imidazolidin4yl)methyl]
oxolan2one(Fig1),isanotheralkaloidfromjaborandithathasbeenreportedtoactasa
peripheralparasympatheticnervoussystemstimulant[5].Despitefewstudieshavingfocused
onEPIISbiologicalactivities,itsinvitroactivityagainstS.mansonihasbeenpreviously
reported[2].TheseresultshighlightedtheneedtoperforminvivostudiesofEPIIS,primarily
regardingitspossibleuseinthetreatmentofschistosomiasis.Moreover,itisimportanttoeval-
uatepossibletoxicity,asacutetoxicitystudiesinanimalsareusedtofulfillvariousrequire-
mentsrelatedtothecontrolofriskstohumanhealthandtheenvironment[9,10].
Inthisstudy,weevaluatedtheinvivoantischistosomalactivityofEPIISagainstadultand
juvenileS.mansoniworms.Theeffectsofthisalkaloidontegumentmorphologywereinvesti-
gatedviascanningelectronmicroscopy(SEM),andeffectsonegglayingandhepatosplenome-
galyreductionwerealsorecorded.Furthermore,weevaluatedinvitrocytotoxicityindifferent
celllinesandinsilicoandinvivoacutetoxicity.
Materialsandmethods
EPIISpurification
EPIISalkaloidwaspurifiedfrombiomassgeneratedbyproductionofpilocarpinesalts.The
biomassassessmentprocesswasaspreviouslydescribed[11].Alkaloidswereextractedby
aprocessbasedonacidificationandfiltration,followedalkalization[11].EPIISisolation
wascarriedoutusinghighperformanceliquidchromatography(HPLC)accordingtopre-
viouslydescribedmethods[2].Massspectrometrywasusedtoevaluatethepurityandmono-
isotopicmolecularmassofEPIIS(AmaZonSL,BrukerDaltonics,Bremen,Germany),
acquiredinamassrangeofm/z100to400Da,andthepositiveelectrospraymodewasused.
MS/MSwasperformedinmanualmodewithfragmentationoftheprecursorionbycollision
induceddissociation(CID)usingHeasthecollisiongas.Precursorionswereselectedwithin
anisolationwidthof2DaandscanswereaccumulatedwithvariableRFsignalamplitudes
[11].
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Epiisopilosineforschistosomiasiswithouttoxicity
Fig1.ChemicalstructureofEPIISalkaloidwithitsheteroatomsandstereoisomerannotated.EPIISpossesses39atoms(C H ON).
16 18 3 2
https://doi.org/10.1371/journal.pone.0196667.g001
InvivoassayagainstS.mansoni
Schistosomastrainandhosts. WeusedS.mansoniBHstrainBeloHorizonte,Minas
Gerais,Brazil,maintainedintheplanorbidmolluscBiomphalariaglabrataastheintermediate
host.Fordefinitivehosts,MusmusculusfemaleBALB/cSPF,weighing20gat4weeksofage,
werepreviouslyinfectedusingexposuretoasuspensioncontaining70cercariaebythetail
immersiontechnique,asdescribed[12].Afterinfection,theanimals(8pergroup)weremain-
tainedinventedracksystem,VentilifeAlescomini-isolatorswith32cmlengthperfloorarea
(451cm2),temperature24˚C,autoclavedshavings,rationfeedandadlibitumwater.For
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Epiisopilosineforschistosomiasiswithouttoxicity
managementandmonitoringofanimals,twoweeklyshavingswereperformedandbehavior
wasobserveduntiltheendoftheexperiment.AllstudieswereperformedattheBiologyInsti-
tute—HelminthologyLaboratoryofNeglectedDiseasesattheDepartmentofAnimalBiology,
IB,Unicamp,SãoPaulo,Brazil.
Animalgroupsandinvivotreatments. WeanalyzedtheeffectofEPIISinmicewith
adultS.mansoniinfections(patentinfections).Inthefirststep,animalsweredividedintotwo
groups(n=8)andweretreated60dayspost-infection(dpi).GroupIanimalsweretreated
withasingledoseof400mg/kgEPIIS,andgroupIIanimals(controlgroup)weregiven0.3
mLphosphatebufferedsaline(PBS).Inthesecondstep,animalsweredividedintotwogroups
(n=8)andwerealsotreated60daysdpi.GroupIIIanimalsweretreatedwithasingledoseof
100mg/kgEPIIS,andgroupIVanimals(controlgroup)weregiven0.3mLPBS.Inallgroups,
EPIISsolubilizedwithPBSwasadministeredbytheoralroute.
Subsequently,onthebasisoftheirinvivoactivityagainstadultschistosomes,EPIISwas
testedinmiceharboringjuvenileS.mansoni(pre-patentinfection).Inthiscase,21dayspost
infection,agroupof8miceweretreatedwithasingle100mg/kgoraldoseofEPIIS,andthe
othergroupof8untreatedmiceservedascontrols.
Wormrecoveryandtreatmentanalysis. Theanalyseswereperformedtwoweeksafter
treatment.Micewereeuthanizedviacervicaldislocationandadultwormswereretrieved
throughperfusionofthehepaticportalsystemandmesentericveinsasdescribed[13].The
percentageofwormreduction(WR)wascalculatedasdescribed[14].Thecountingofthe
eggseliminatedthroughthefeces(OPG)wasperformedusingtheKato-Katzquantitative
method[15].Thepercentagesofthevariouseggdevelopmentalstages:immature,mature,and
deadeggs(theoogrammethod)werecalculatedfromthesmallintestinalwallofinfectedmice
asdescribed[16].Noanimalsdiedbeforethefinaltrialperiod(75days).
Scanningelectronmicroscopy(SEM). Formicroscopicanalysis,anewgroupofanimals
wastreatedseparatelyfollowingthetreatmentschemereportedabove,butwiththedifference
thatmicewereeuthanizedviacervicaldislocation48haftertreatment.AdultS.mansoni
wormswereretrievedasdescribedabove.Forpreparationandanalysis,thewormswere
washedwithsodiumcacodylatebuffer(0.1M),fixedin2.5%glutaraldehyde(pH7.4,Merck)
for24h,andthenfixedin1%osmiumtetroxidefor1h.Specimensweredehydratedwith
increasingconcentrationsofethanol(70%,80%,90%,and100%)for30mineach,driedina
criticalpointdryer,mountedonstubs,metalizedwithgoldparticlesusingasputtercoater,
andfinallyanalyzedandphotographedusinganelectronmicroscope(Jeol-JSM-820).
Insilicoanalysisoftoxicologicalproperties
Toanalyzethetoxicologicalparameters,wefirstneededtocalculateLogP(lipophilicity),total
polarsurfacearea,numberofhydrogenbonddonorsandacceptors,molecularweightand
numberofrotatablebondsforcomparisonwithmoleculesthathavesimilarcharacteristics.All
thesedataandinsilicotoxicologicalpropertieswerecalculatedusingpkCSMsoftware,aninsil-
icotoolwebservermaintainedbyVLS3D(CambridgeUniversity)thatgraph-basedsignatures
todeveloppredictivemodelsofcentraltoxicologicalpropertiesfordrugdevelopment[17].
Cellcultureandinvitrocytotoxicityassays
Murinefibroblasts(NIH-3T3)andhumankeratinocytes(HaCaT)cellsweregrowninDulbec-
co’smodifiedEagle’smediumsupplementedwith10%fetalbovineserumand1%antibiotic
solution.Thecellsweremaintainedinanincubatorunderhumidifiedatmospherewith5%
CO at37˚C.Forreal-timecellanalysis(RTCA,xCelligence,Roche,Switzerland),theNIH-
2
3T3andHaCaTcellswereseededincultureplatescontainingelectronicbiosensors.After24h
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Epiisopilosineforschistosomiasiswithouttoxicity
ofculture,cellsweretreatedwithEPIISornodrug(control).TheconcentrationsofEPIIS
were32and512μg/mL(equivalentto111.8and1790.2μM,respectively),andtheadheredcell
ratesweremeasuredevery30minfor137h.
Acutetoxicityevaluation
TheinvivotoxicologicalevaluationofEPIISwasperformedusingtheacutetoxicclasstest[18],
guidenumber423[9].FemaleSwissmiceweredividedintotwogroupsofthreeanimalseach:a
controlgrouptreatedorallywithasolutionof5%DMSOinsalineandagrouptreatedorally
withEPIIS.Thedrugwassolubilizedatthehighestrecommendeddose(2000mg/kgbody
weight)inasolutionof5%DMSOinsaline.Clinicalandbehavioralparameterswereevaluated
accordingtotheOECD’sGuideforRecognition,EvaluationandUseofClinicalSignals[19].
Evaluationofhematologicalandbiochemicalparameters. Afterthe14thdayofobser-
vation,theanimalswereeuthanizedbyasphyxiationinaCO chamberandbloodsamples
2
werecollectedfromtheanimalsviatheorbitalplexus.Forbiochemicalanalyses,thematerial
wascentrifugedat1200xgfor5minbeforedeterminingglucose,urea,creatinine,aspartate
aminotransferase(AST),alanineaminotransferase(ALT),totalcholesterol,HDL,LDL,alka-
linephosphatase,totalprotein,albumin,andglobulinlevels.Thevaluesforredbloodcells,leu-
kocytes,platelets,hemoglobin,meancorpuscularvolume,andmeancorpuscularhemoglobin
weredeterminedimmediatelyaftercollectionbymeansoftheautomatichematologyAdvia
120/hematology(Siemens)analyzer.Differentialleukocytecountswereperformedonsmears
stainedwithMay-Gru¨nwald-Giemsa[20,21].
Histopathologicalanalysis. Immediatelyaftereuthanasia,fragmentsofliver,spleen,kid-
ney,brain,lung,stomach,smallintestine,andlargeintestinewerecollectedandfixedin10%
bufferedformaldehyde.Afterfixation,theorgansweredehydratedinserialdilutionsofethyl
alcoholfor1hineachdilution,diaphanizedinxylenefor30min,andsoakedinparaffinfor5
min.Sectionsoftheseorgans(4μm)werestainedwithhematoxylinandeosinandexamined
underalightmicroscope[22].
Ethicalconductofresearch
TheanimalsusedintheinvivostudieswereprovidedbyMultidisciplinaryCenterforBiologi-
calResearch(CEMIB)oftheStateUniversityofCampinas.Allexperimentswereapprovedby
theEthicsCommissionfortheUseofAnimals(CEUA/UNICAMP,protocoln˚2170–1),
accordingtoBrazilianSocietyofScienceinLaboratoryAnimals(SBCAL)andEthicalPrinci-
plesfortheUseofAnimals.
Statisticalanalysis
GraphPadPrism(version5.0)softwarewasusedforanalysesofdatasignificance.Fortherapeu-
ticeffectassays(invivoassays)ANOVAandDunnett’stestwereusedtoanalyzethestatistical
significanceofdifferencesbetweenmeanexperimentalandcontrolvalues.FortheRTCAassay
weanalyzedmeanandstandarddeviation.APvalueof<0.05wasconsideredsignificant.
Results
EPIISpurification
Themassspectrometryanalysisshowedthattheseparationmethodsusedweresuccessful.
MS/MSanalysisshowedapseudomolecularionwithm/z286.9Da[M+H]+andaMS2frag-
mentswithm/z268.8Da[M–H O+H]+and180.7Da.This‘‘molecularfingerprint”con-
2
firmedEPIIS(Fig2).
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Epiisopilosineforschistosomiasiswithouttoxicity
Fig2.EPIISmassspectrum.(A)pseudomolecularionwithm/z286.9Da[M+H]+.(B)MS2fragmentswithm/z268.8Da[M—HO+H]+e180,7Da.
2
https://doi.org/10.1371/journal.pone.0196667.g002
EPIISshowedactivityagainstadultS.mansoni-infectedmice(patent
infections)
InmiceinfectedbyadultS.mansoni,therewasasignificantreductioninwormburdenwith
oraltreatmentbyEPIIS(Fig3).AsingledoseofEPIISof400mg/kgreducedwormburdenby
57.78%(P<0.001).Atadoseof100mg/kg,thetotalwormburdenreductionwas60.61%
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Epiisopilosineforschistosomiasiswithouttoxicity
Fig3.EffectonwormburdenofasingleoraldoseofEPIISadministeredtomiceharboringa60-day-oldadultS.
mansoniinfection,stratifiedbysex.PointsrepresentdatafromindividualmicethatwereinfectedandtreatedwithEPIIS,
orinfectedanduntreated(control).Eachtreatedgroupwascomparedtoitscorrespondinguntreatedgroup.Thehorizontal
barsrepresentmedianvalues.(cid:3)P<0.05,(cid:3)(cid:3)P<0.01,(cid:3)(cid:3)(cid:3)P<0.001comparedwithuntreatedgroups.
https://doi.org/10.1371/journal.pone.0196667.g003
(P<0.001)thatoftheinfecteduntreatedcontrols.Thetotal,maleandfemalewormburdens
followingtreatmentofS.mansoniinfectionswithEPIISareshowninFig3.
Becausethesingledoseof400mg/kgshowednodifferenceintermsofwormburdencom-
paredwith100mg/kgdose,weshowedtheresultsforeggsproduction,liverandspleen
weights,andscanningelectronmicroscopyinvestigationsonlyforthelowestdoseinvestigated
(100mg/kg).
EPIISdisruptedeggdevelopmentaswellasliverandspleenweightsin
miceharboringpatentinfections
TheeffectsofEPIISoneggdevelopmentstages(oogrampattern)at15daysaftertreatmentare
showninFig4A.Administrationofsingledoseof100mg/kgreducedthenumberofimmature
eggsby45.84%.Theoogrammethodrevealedasignificantincreaseinthepercentageofdead
eggsintreatedmice(19.16%)comparedwiththecontrolgroup(2.25%).
Infecessamplescollectedfrommice,theKato-KatzmethodrevealedthatEPIISsignifi-
cantlyreducedthenumberofeggscomparedwiththecontrolgroup(untreated),witha
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Fig4.EffectoneggdevelopmentandonStolleggcountofasingledose100mg/kgofEPIISadministeredtomice
harboringa60-day-oldadultS.mansoniinfection.(A)EPIISeffectoneggdevelopmentstages(oogram)(B)Effect
onStolleggcount.PointsrepresentdatafromindividualmicethatwereinfectedandtreatedwithEPIIS,orinfected
anduntreated(control).Thehorizontalbarsrepresentmedianvalues.(cid:3)(cid:3)P<0.01and(cid:3)(cid:3)(cid:3)P<0.001comparedwith
untreatedgroups.
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Epiisopilosineforschistosomiasiswithouttoxicity
Fig5.EffectofEPIISonrelativeliverandspleenweights.Pointsrepresentdatafromindividualmicethatwere
infectedandtreatedwithEPIISorwereinfectedanduntreated(control).Thehorizontalbarsrepresentmedianvalues.
(cid:3)P<0.05and(cid:3)(cid:3)P<0.01comparedwithuntreatedgroups.
https://doi.org/10.1371/journal.pone.0196667.g005
reductionof58%(P<0.001)atdoseof100mg/kg(Fig4B).Furthermore,oraltreatmentwith
EPIISsignificantlydecreasedhepatosplenomegalyasmeasuredbyliverandspleenweights,
P<0.01andP<0.05,respectively(Fig5)comparedtothecontrolgroup.
EPIIScausedchangesinthetegumentmorphologyofadultS.mansoni
Inthecontrol(untreated)group,theoralandventralsucker,tubercles,andspinesofmaleand
femalewormsshowednormalintegrity(Fig6A–6D).EPIIStreatmentcausedchangestothe
tegumentinbothmalesandfemales(Fig6E–6L),buttegumentaldamagewasparticularlyevi-
dentinmaleworms.Themalewormsshowedextensivepeelingoftubercles,absenceofspines,
andtheformationofprotuberancesinthedorsalregion(Fig6I–6L).
EPIISshowsmoderateactivityagainstjuvenileS.mansoni-infectedmice
(pre-patentinfections)
BecauseEPIISatdoseof100mg/kgwasthemostpotentagainstS.mansoniadults,wealso
analyzedtheeffectsofEPIISonjuvenileS.mansoniworms.ThetreatmentofjuvenileS.man-
soni-infectedmicewithsingle100mg/kgoraldoseofEPIISshowedamoderatereductionin
wormburdenof58.06%comparedtothecontrolgroup(Fig7A).Analysisoftheoogrampat-
ternshowedreductionsinthenumberofimmatureeggsof58.60%(Fig7B).Infecessamples,
theKato-Katztechniquerevealedeggreductionof64.18%comparedtothecontrolgroup
(Fig7C).
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Fig6.ScanningelectronmicroscopyofS.mansoniadultsretrieved48haftertreatmentwithEPIIS(100mg/kg).A-D:Controlgroup—untreated;E–L:
Maleandfemaleworms.Thearrowsshowdamagetotegumentwithreductioninthesizeofthespines,anddamagetotheoralandventralsuckersinmale
worms,followedbybodycorrugationinfemaleworms.Imagebarscale:(A)100X___10kv;(B)120X___10kv;(C-D)180X___10kv;(E-F)127X___10kv;
(G-H)250X___10kv;(I-L)350X___10kv.
https://doi.org/10.1371/journal.pone.0196667.g006
EPIISgavelowprobabilitytoxicitypredictions
InsilicoanalysisofLogP,totalpolarsurfacearea,numberofhydrogenbonddonorsandaccep-
tors,molecularweightandnumberofrotatablebondsareshowninTable1.Weanalyzedthe
predictedtoxicityonhumanether-à-go-go-relatedgene(hERG)inhibitorIandII,toevaluate
whetherEPIIScausedeffectsoncardiacrepolarization.Inaddition,weanalyzedwhetherEPIIS
causedhepatotoxicity,becausetheliverissubstantiallyaffectedinschistosomiasis;inaddition,
liverisprimarilyresponsibleforthefirst-passmetabolism.Thehumanmaximumtolerated
doseandskinsensitizationwerealsoanalyzed,becauseitisimportanttoknowwhetherthe
dosecausesallergicresponses.AllresultsfortoxicitypropertiesareshowninTable2.
EffectofEPIISoncellviability
CytotoxicactivityofEPIISwasdeterminedviareal-timecellindexanalysis(RTCA)ontwo
normalcelllines:NIH-3T3andHaCaT.RTCAshowedthatadhesionindicesinbothNIH-
3T3andHaCaTcellsweredecreasedbyEPIISataconcentrationof32μg/mL.However,no
significantdecreaseincelladhesionindiceswerefoundincomparisontothecontrolgroups
whenEPIISwasusedat512μg/mL(Fig8).
EPIISdidnotcauseacutetoxicityinmice
Inacutetoxicitytestsinmice,noanimalshowedsignsoftoxicity(cyanosis,piloerection,con-
tortion,eyelidptosis,tremors,seizures,ataxia,ordiarrhea)andallmicesurvivedthe14days
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