The antiparasitic agent ivermectin induces chloride - Blood

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MYELOIDNEOPLASIA
The antiparasitic agent ivermectin induces chloride-dependent membrane
hyperpolarization and cell death in leukemia cells
*SumaiyaSharmeen,1*MarkoSkrtic,1*MahadeoA.Sukhai,1RoseHurren,1MarcelaGronda,1XiaomingWang,1
SonaliB.Fonseca,2HongSun,1TabithaE.Wood,1,3RichardWard,1MarkD.Minden,1RobertA.Batey,3AlessandroDatti,4,5
JeffWrana,4ShanaO.Kelley,2andAaronD.Schimmer1
1PrincessMargaretHospital,OntarioCancerInstitute,Toronto,ON;2DepartmentofPharmaceuticalSciences,FacultyofPharmacy,UniversityofToronto,
Toronto,ON;3DepartmentofChemistry,UniversityofToronto,Toronto,ON;4SamuelLunenfeldResearchInstitute,MountSinaiHospital,Toronto,ON;and
5DepartmentofExperimentalMedicineandBiochemicalSciences,UniversityofPerugia,Perugia,Italy
Toidentifyknowndrugswithpreviously miacelllinesandprimarypatientsamples plasmamembranehyperpolarization,but
unrecognizedanticanceractivity,wecom- preferentiallyovernormalhematopoietic didnotchangemitochondrialmembrane
piledandscreenedalibraryofsuchcom- cells. Ivermectin also delayed tumor potential.Ivermectinalsoincreasedreac-
pounds to identify agents cytotoxic to growth in 3 independent mouse models tiveoxygenspeciesgenerationthatwas
leukemia cells. From these screens, we ofleukemiaatconcentrationsthatappear functionally important for ivermectin-
identifiedivermectin,aderivativeofaver- pharmacologicallyachievable.Asananti- inducedcelldeath.Finally,ivermectinsyn-
mectin B1 that is licensed for the treat- parasitic,ivermectinbindsandactivates ergizedwithcytarabineanddaunorubicin
mentoftheparasiticinfections,strongy- chloride ion channels in nematodes, so that also increase reactive oxygen spe-
loidiasisandonchocerciasis,butisalso we tested the effects of ivermectin on cies production. Thus, given its known
effectiveagainstotherworminfestations. chloridefluxinleukemiacells.Ivermectin toxicologyandpharmacology,ivermectin
As a potential antileukemic agent, iver- increased intracellular chloride ion con- could be rapidly advanced into clinical
mectininducedcelldeathatlowmicromo- centrationsandcellsizeinleukemiacells. trial for leukemia. (Blood. 2010;116(18):
larconcentrationsinacutemyeloidleuke- Chloride influx was accompanied by 3593-3603)
Introduction
Antimicrobials with previously unrecognized anticancer activity patients with relapsed or refractory hematologic malignancies
can be rapidly repositioned for this new indication given their (ClinicalTrials.govIDNCT00963495).
extensivepriorpharmacologyandtoxicologytesting.Forexample, Here,weusedachemicalscreentoidentifyknowndrugswith
the broad spectrum antiviral ribavirin was found to suppress previously unrecognized activity against leukemia. From this
oncogenictransformationbydisruptingthefunctionandsubcellu- screen,weidentifiedtheantiparasiticagent,ivermectin.Ivermectin
lar localization of the eukaryotic translation initiation factor isaderivativeofavermectinB1andlicensedforthetreatmentof
eIF4E.1,2Assuch,ribavirinwasrecentlyevaluatedinaphaseIdose theparasiticinfections,strongyloidiasisandonchocerciasis,aswell
escalation study in patients with relapsed or refractory M4/M5 asotherworminfestations(eg,ascariasis,trichuriasis,andentero-
acutemyeloidleukemia(AML).Inthisstudyof13patientstreated biasis),buthasnotbeenpreviouslytestedasananticanceragent.
with ribavirin, there was 1 complete remission and 2 partial Aspartofthedevelopmentofthisagentasanantiparasiticagent,
remissions.Thus,ribavirinmaybeefficaciousforthetreatmentof ivermectinwasextensivelyevaluatedforitspharmacology,safety,
AML.3 Likewise, the antifungal agent ketoconazole inhibits the andtoxicityinhumansandanimals.Forexample,theLD oforal
50
productionofandrogensfromthetestesandadrenalsinrats.Given ivermectininmice,rats,andrabbitsrangesfrom10to50mg/kg.7
thisfinding,ketoconazolewasrapidlyadvancedintoclinicaltrials In humans, when used to treat onchocerciasis, 100-200(cid:1)g/kg
forpatientswithprostatecancerwhereitdisplayedclinicalefficacy ivermectin is administered as a single dose.8 This brief and
inearlystudies.4,5 low-dose treatment is sufficient to achieve an antiparasitic effect,
Recently, we demonstrated that the antiparasitic agent clio- but higher doses and treatment beyond 1 day have been safely
quinol inhibits the proteosome and induces cell death in administered for other conditions. For example, in patients with
leukemia and myeloma cells through copper-dependent and spinal injury and resultant muscle spasticity, up to 1.6mg/kg
-independent mechanisms.6 Thus, our preclinical data suggest ivermectinwasadministeredsubcutaneouslytwiceweeklyforup
that this antiparasitic could be repurposed for the treatment to 12 weeks. In this study, no significant adverse effects were
of hematologic malignancies. Therefore, we initiated a phase reported.9 Likewise, to evaluate the safety of oral ivermectin,
1 study to evaluate the dose-limiting toxicity, maximum toler- healthyvolunteersreceived30-120mgondays1,4,and7andthen
ated dose, and recommended phase 2 dose of clioquinol in afurtherdoseinweek3.10Evenatadoseof120mg((cid:2)2mg/kg),
SubmittedJanuary5,2010;acceptedJuly11,2010.Prepublishedonlineas The publication costs of this article were defrayed in part by page charge
BloodFirstEditionpaper,July19,2010;DOI10.1182/blood-2010-01-262675. payment. Therefore, and solely to indicate this fact, this article is hereby
marked‘‘advertisement’’inaccordancewith18USCsection1734.
*S.S.,M.S.,andM.A.S.contributedequallytothiswork.
Theonlineversionofthisarticlecontainsadatasupplement. ©2010byTheAmericanSocietyofHematology
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3594 SHARMEENetal BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18
noseriousadverseeffectswerenoted.Finally,reportsofivermectin sublethally irradiated (3.5 Gy) nonobese diabetic/severe combined
overdosesalsosupporttheevaluationofhighdosesofivermectin immunodeficiency (NOD/SCID) mice (Ontario Cancer Institute, To-
in humans, as in the majority of these cases no serious adverse ronto,ON).Four(OCI-AML2),5(MDAY-D2),or7(K562)daysafter
eventswerereported.11 injection,oncetumorswerepalpable,micewerethentreateddailyfor
10days(K562)ortreatedwith8dosesover10days(OCI-AML2)with
Inourcurrentstudy,wedemonstratedthativermectindisplayed
ivermectin (3mg/kg) by oral gavage in water or vehicle control
preclinicalactivityagainsthematologicmalignanciesinvitroand
(n(cid:4)10pergroup).MDAY-D2miceweretreatedsimilarly,butdosage
delayed tumor growth in vivo at concentrations that appear
escalatedfrom3(4days)to5(3days)and6mg/kg(3days)asthedrug
pharmacologically achievable. Mechanistically, ivermectin in- was well tolerated. Tumor volume (tumor length(cid:5)width2(cid:5)0.5236)
ducedchlorideinflux,membranehyperpolarization,andgenerated was measured 3(cid:5) a week using calipers. Fourteen (MDAY-D2),
reactive oxygen species (ROS). Furthermore, ivermectin syner- 15(OCI-AML2),or17(K562)daysafterinjectionofcells,micewere
gizedwithcytarabineanddaunorubicin.Thus,givenitspriorsafety killed, tumors were excised, and the volume and mass of the tumors
and toxicity testing, ivermectin could be rapidly advanced into weremeasured.
clinicaltrialforpatientswithleukemia. To measure gene expression changes in vivo, OCI-AML2 cells
(2.5(cid:5)105) were injected subcutaneously into the flanks of sublethally
irradiated NOD/SCID mice. Once tumors were established, mice were
Methods treatedwithivermectin(7mg/kg)orvehiclecontrolintraperitoneallyfor
5 days. After treatment, mice were killed and tumors were harvested.
Reagents mRNAwasextractedandchangesinSTATexpressionweremeasuredby
quantitative reverse-transcription polymerase chain reaction (qRT-PCR).
The compounds in the chemical library were purchased from Sigma- EvidenceofapoptosiswasmeasuredbyTunelstainingandimmunohisto-
Aldrich. AnnexinV–fluorescein isothiocyanate (FITC) and propidium chemistry (Pathology Research Program, University Health Network,
iodide(PI)werepurchasedfromBiovision.Indo-1AM,6-methoxy-N-(3- Toronto,ON).
sulfopropyl) quinolinium (SPQ), carboxydichlorofluorescein diacetate Allanimalstudieswerecarriedoutaccordingtotheregulationsofthe
(CarboxyH2DCF-DA),5,5(cid:3),6,6(cid:3)-tetrachloro-1,1(cid:3),3,3(cid:3)-tetraethylbenzimida- Canadian Council onAnimal Care and with the approval of the Ontario
zlycarbocyanine iodide (JC1), and bis-(1,3-dibutylbarbituric acid)trime- CancerInstituteAnimalEthicsReviewBoard.
thineoxonol(DiBAC(3))wereallpurchasedfromInvitrogenCanada.
4
Intracellularionmeasurements
Celllines
Intracellular chloride concentration was measured using a fluorescent
Human leukemia (OCI-AML2, HL60, U937, KG1a) and prostate cancer
indicatorforchloride,SPQaspreviouslydescribed.12Uponbindinghalide
(DU145andPPC-1)celllinesandmurineleukemia(MDAY-D2)cellswere
ions such as chloride, SPQ is quenched, resulting in a decrease in
maintainedinRPMI1640medium.Mediumwassupplementedwith10%
fluorescence without a shift in wavelength. After treating OCI-AML2
fetalbovineserum(FBS),100(cid:1)g/mLpenicillin,and100U/mLstreptomy- (5(cid:5)105)andDU145(4(cid:5)105)cellsovernightwithivermectin(3-10(cid:1)M),
cin (all from Hyclone). TEX human leukemia cells were maintained in
cells were incubated for 15 minutes with SPQ (5mM) at 37°C in a
Iscove modified Dulbecco medium (IMDM), 15% FBS, 1%, penicillin-
hypotonic solution of Hanks balanced salt solution (HBSS/HO; 1:1) to
streptomycin,20ng/mLSCF,and2ng/mLIL-3.Allcellswereincubatedat 2
promote the intracellular uptake of SPQ.After 15 minutes of incubation
37°Cinahumidifiedairatmospheresupplementedwith5%CO.
2 with SPQ, cells were diluted (15:1) in HBSS and centrifuged. The
Primarycells supernatantwasremoved,andcellswereresuspendedin200(cid:1)Loffresh
HBSSandincubatedfor15minutesat37°Ctoallowrecoveryfromthe
PrimaryhumanAMLsampleswereisolatedfromfreshbonemarrowand hypotonicshock.CellswerethenstainedwithPI,andSPQfluorescence
peripheral blood samples of consenting patients and mononuclear cells wasdeterminedinthePInegativecellsusinganLSR-IIflowcytometer
fractionatedbyFicollseparation.Similarly,primarynormalhematopoietic (Becton Dickinson; excitation 351nm, emission 485nm). In parallel,
mononuclear cells were obtained from healthy consenting volunteers changesincellsizeweredeterminedbymeasuringforwardlightscatterby
donatingperipheralbloodstemcells(PBSCs)forstemcelltransplantation. flowcytometry.ResultswereanalyzedwithFlowJo,version8.8(TreeStar).
Primary cells were cultured at 37°C in IMDM, supplemented with 20% Changes in cytosolic calcium concentration were detected with the
FBS,andappropriateantibiotics.Thecollectionanduseofhumantissuefor fluorescent dye, Indo-1 AM (final concentration 6(cid:1)M) as previously
thisstudywereapprovedbytheUniversityHealthNetworkinstitutional described.13
reviewboard.
Determinationofplasmaandmitochondrialmembrane
Chemicalscreenforcytotoxiccompounds
potentialandROSgeneration
HL60, KG1a, and OCI-AML2 leukemia cells were seeded into 96-well
Plasmaandmitochondrialmembranepotentialweremeasuredbystaining
polystyrenetissuecultureplates(Nunc).Afterseeding,cellsweretreated
cells with DiBAC(3) and JC-1 (Invitrogen), respectively, as described
withaliquotsofthechemicallibrary(n(cid:4)100)atincreasingconcentrations 4
above. Intracellular ROS were detected by staining cells with Carboxy-
(3-50(cid:1)M),withafinaldimethylsulfoxide(DMSO)concentrationof0.5%.
HDCFDA(finalconcentration10(cid:1)M)andanalyzingwithflowcytometry
Seventy-two hours after incubation, cell proliferation and viability were 2
aspreviouslydescribed14andasdescribedinthesupplementalMethods.
measuredbytheMTSassay.LiquidhandlingwasperformedbyaBiomek
FXLaboratoryAutomatedWorkstation(BeckmanCoulter).
Geneexpressionstudies
Cellviabilityassays
OCI-AML2leukemiacellsweretreatedwithbuffercontrolorivermectin
Cellgrowth,viability,andclonogenicgrowthofprimarycellsweremeasuredas (3(cid:1)M) for 30 and 40 hours.After treatment, cells were harvested, total
describedinthesupplementalMethods(availableontheBloodWebsite; RNAwasisolated,andgeneexpressionwasmeasuredasdescribedinthe
seetheSupplementalMaterialslinkatthetopoftheonlinearticle). supplementalMethods.
Assessmentofivermectin’santicanceractivityinmouse Drugcombinationstudies
modelsofleukemia
Thecombinationindex(CI)wasusedtoevaluatetheinteractionbetween
MDAY-D2murineleukemiacells,K562,andOCI-AML2humanleukemia ivermectin and cytarabine or daunorubicin as previously described.15
cells (2.5(cid:5)105) were injected subcutaneously into the flanks of OCI-AML2andU937cellsweretreatedwithincreasingconcentrationsof
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ivermectin,cytarabine,anddaunorubicin.Seventy-twohoursafterincuba-
tion,cellviabilitywasmeasuredbytheMTS[3-(4,5dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay.The
CalcuSyn median effect model was used to calculate the CI values and
evaluatewhetherthecombinationofivermectinwithcytarabineordaunoru-
bicinwassynergistic,antagonistic,oradditive.CIvaluesof(cid:6)1indicate
synergism,CI(cid:4)1indicateadditivity,andCI(cid:7)1indicateantagonism.16
Results
Achemicalscreenidentifiesivermectinwithpotential
anticanceractivity
Off-andon-patentdrugswithpreviouslyunrecognizedanticancer
activity can be rapidly repurposed for this new indication given
theirpriortoxicologyandpharmacologytesting.Toidentifysuch
compounds, we compiled a small chemical library (n(cid:4)100)
focused on antimicrobials and metabolic regulators with wide
therapeutic windows and well-understood pharmacokinetics. We
treated OCI-AML2, HL60, and KG1a leukemia cell lines with
aliquotsofthischemicallibraryat5concentrations(rangingfrom
3-50(cid:1)M). Seventy-two hours after incubation, cell growth, and
viability were measured by the MTS assay. From this screen, we
identifiedivermectinthatreducedcellviabilityinallcelllinesin
the screen with an EC (cid:6)10(cid:1)M. The results for the screen of
50
OCI-AML2cellswithcompoundsaddedatafinalconcentrationof
6(cid:1)MareshowninFigure1A.
Figure 1. A screen of off-patent drugs identifies the antiparasitic agent,
Ivermectiniscytotoxictomalignantcelllinesandprimary ivermectin,thatreducesviabilityofleukemiacells.(A)OCI-AML2cellswere
patientsamples treatedwithaliquotsofasmallchemicallibrary(n(cid:4)100)focusedonantimicrobials
and metabolic regulators. Seventy-two hours after incubation, cell growth and
Havingidentifiedivermectininourchemicalscreens,wetestedthe viabilityweremeasuredbytheMTSassay.Datarepresentthepercentageofviable
OCI-AML2cellstreatedwiththecompounds(6(cid:1)M)sortedinorderofincreasing
effects of ivermectin on cell growth and viability in a panel of
activity. (B) Leukemia cell lines were treated with increasing concentrations of
5leukemiacelllines.Cellsweretreatedwithincreasingconcentra- ivermectin. Seventy-two hours after incubation, cell growth and viability were
tionsofivermectin,and72hoursafterincubation,cellgrowthand measured by the MTS assay. Data represent the mean EC50 and 95% CI from
3independentexperiments.(C)Primarynormalhematopoieticcells(PBSC;n(cid:4)3),
viabilitywereassessedbytheMTSassay.Ivermectindecreasedthe
primaryAMLpatientsamples(AML;n(cid:4)3),andU937leukemiacellsweretreated
viability of the tested leukemia cell lines with an EC50 of with increasing concentrations of ivermectin for 48 hours. After incubation, cell
approximately5(cid:1)M(Figure1B).Thelossofviabilitywasdetected viabilitywasmeasuredbyannexinVandPIstaining.Datarepresentthemean(cid:9)SD
at 24 hours after treatment and increased in a time-dependent percentviablecellsfromexperimentsperformedintriplicate.(D)PrimaryAMLcell
samples(AML;n(cid:4)6)andnormalhematopoieticperipheralbloodstemcellsamples
manner.CelldeathandapoptosiswereconfirmedbyannexinVand (PBSC;n(cid:4)3)weretreatedwithivermectin(6(cid:1)M)for24hoursandthenplatedina
PI staining (Figure 1C). Cell death was caspase-dependent, as methylcellulosecolonyformingassay.Seven(AML)or14days(PBSCs)daysafter
cotreatmentwiththepan-caspaseinhibitorz-VAD-fmkabrogated plating,thenumberofcolonieswascounted.Datarepresentthemean(cid:9)SDpercent
colonyformationcomparedwithcontroltreatedcells.
cell death (supplementary Figure 1A). Furthermore, times and
concentrations of ivermectin that preceded cell death induced G
2
cell-cyclearrest(supplementalFigure1Banddatanotshown). cells (n(cid:4)3) with(cid:6)15% reduction in clonogenic growth. In
Giventhecytotoxicityofivermectintowardleukemiacelllines, contrast,ivermectinreducedclonogenicgrowthby(cid:1)40%in3of
we compared its cytotoxicity to primary normal hematopoietic
6 primaryAMLsamples (Figure 1D). Similar effects were noted
cellsandAMLpatientsamples(n(cid:4)4intermediateriskcytogenet-
when primary cells were directly plated into clonogenic assays
ics,n(cid:4)1goodriskcytogenetics,andn(cid:4)1unknowncytogenet-
withivermectin(supplementalFigure2).
ics). Normal hematopoietic cells and patient sample cells were
treatedfor48hourswithincreasingconcentrationsofivermectin.
Afterincubation,cellviabilitywasmeasuredbyannexinVandPI Ivermectindelaystumorgrowthinmousemodelsofleukemia
staining.IvermectinwascytotoxictoAMLpatientsamplesatlow
Giventheeffectsofivermectinasapotentialantileukemicagent,
micromolarconcentrations.Incontrast,itdidnotinducecelldeath
inthePBSCsatconcentrationsupto20(cid:1)M(Figure1C).However, we evaluated ivermectin in mouse models of leukemia. Human
whengatingontheCD34(cid:8)cellsfromonePBSCsample,ivermec- leukemia (OCI-AML2 and K562) and murine leukemia (MDAY-
tin induced cell death with an IC of 10.5(cid:9)0.6(cid:1)M. Thus, D2) cells were injected subcutaneously into the flank of NOD/
50
ivermectininducedcelldeathinprimaryAMLcellspreferentially SCIDmice.Four(OCI-AML2),5(MDAY-D2),or7(K562)days
over normal cells, but the therapeutic window over normal stem afterinjection,oncetumorswerepalpable,miceweretreatedwith
cellsmaybenarrow. ivermectin (3mg/kg) by oral gavage in water or vehicle control
Ivermectinwasalsoevaluatedinclonogenicassaysinprimary (n(cid:4)10 per group) for 10 days (K562) or 8 doses over 10 days
normal hematopoietic and AML cells. Ivermectin (6(cid:1)M) had (OCI-AML2). MDAY-D2 mice (n(cid:4)10 per group) were treated
minimaleffectsontheclonogenicgrowthofnormalhematopoetic similarly,butwithescalatingdoses(3mg/kgfor4days,5mg/kg
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3596 SHARMEENetal BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18
measured by staining cells with the fluorescent dye SPQ that is
quenchedathighchlorideionconcentrations.InOCI-AML2cells,
ivermectin decreased SPQ fluorescence, consistent with an in-
crease in levels of intracellular chloride at concentrations that
inducedcelldeath,butattimesthatprecededcelldeath(Figure3B
anddatanotshown).Incontrast,chlorideinfluxwasnotobserved
inDU145cellsthatwereresistantto10(cid:1)Mivermectin(Figure3C
anddatanotshown).
Chlorideinfluxcanincreasecellsize.Therefore,wemeasured
changes in cell size in parallel to measuring changes in chloride
flux.As measured by flow cytometry, after 2 hours of treatment,
ivermectincausedanincreaseincellsizeinOCI-AML2butnotin
the resistant DU145 cells, consistent with its effects on chloride
influx(Figure3D-E).
Innematodes,increasesinintracellularchlorideafterivermec-
tin treatment cause membrane hyperpolarization. Therefore, we
evaluated the effects of ivermectin on plasma and mitochondrial
membranepolarizationinleukemiacells.OCI-AML2,U937,and
TEX leukemia cells sensitive to ivermectin-induced death, a
primaryAMLpatient sample, DU145 and PPC-1 prostate cancer
cells, and primary normal hematopoietic cells were treated with
increasingconcentrationsofivermectin.Atincreasingtimesafter
incubation,plasmamembranepotentialwasmeasuredbystaining
cellswithDiBAC4(3)andflowcytometricanalysis.InOCI-AML2
cells,treatmentwithivermectininducedmembranehyperpolariza-
tioninadose-dependentmanner(Figure4A)andasearlyasafter1
Figure2.Ivermectindelaystumorgrowthandreducestumormassinleukemia
mousexenografts.Humanleukemia(OCI-AML2andK562)andmurineleukemia
(MDAY-D2)cellswereinjectedsubcutaneouslyintotheflankofsublethallyirradiated
NOD/SCID mice. Four (OCI-AML2), 5 (MDAY-D2), or 7 (K562) days after
injection,oncetumorswerepalpable,miceweretreatedwithivermectin(IVM;
3mg/kg)byoralgavageinwaterorvehiclecontrol(n(cid:4)10pergroup)for10days
(K562)or8dosesover10days(OCI-AML2).MDAY-D2mice(n(cid:4)10pergroup)
weretreatedwithescalatingdosesofivermectin(3mg/kgfor4days,5mg/kgfor
3days,andthen6mg/kgfor3days).Fourteen(OCI-AML2),15(MDAY-D2),or
17(K562)daysafterinjectionofcells,micewerekilled,tumorswereexcised,and
thevolumeandmassofthetumorsweremeasured.Thetumorweightandthe
meanvolume(cid:9)SEMareshown.Differencesintumorvolumeandweightwere
analyzedbyanunpairedttest.***P(cid:6).0001.
for 3 days, and then 6mg/kg for 3 days) as the drug was well
tolerated. Tumor volume and mass were measured over time.
Compared with buffer control, oral ivermectin significantly
(P(cid:6).05) decreased tumor mass and volume in all 3 models
(Figure 2) by up to 70% without any gross organ toxicity. In an
OCI-AML2xenograft,weshowedthativermectinincreasedapo-
ptosis in the subcutaneous tumor as measured by Tunel staining
(supplemental Figure 3). Of note, a dose of 3mg/kg in mice
translatestoadoseof0.24mg/kginhumans,basedonscalingof
bodyweightandsurfacearea,andappearsreadilyachievablebased
onpriorstudies.9,11Thus,theactivityinthexenograftstudiesand
theinvitrostudiesabovesuggeststhatatherapeuticwindowmay
beachievable.
Ivermectininducesintracellularchlorideflux,increaseincell
Figure3.Ivermectininduceschlorideinfluxandincreasescellsizeinleukemia
size,andhyperpolarizationoftheplasmamembrane cells.(A)OCI-AML2leukemiaandDU145prostatecancercellsweretreatedwith
increasingconcentrationsofivermectin.After24hoursofincubation,cellgrowthand
Asanantiparasiticagent,ivermectinactivateschloridechannelsin viability were measured by MTS assay. Data represent the mean(cid:9)SD percent
nematodes,causinganinfluxofchlorideionsintothenematode’s viablecellsfromrepresentativeexperiments.(B)OCI-AML2and(C)DU145cells
cells.17Thus,weinvestigatedchloridefluxafterivermectintreat- weretreatedwith10(cid:1)Mivermectinfor1hour,andlevelsofintracellularchloride
were measured after staining cells with the fluorescent dye, SPQ, that is
mentinOCI-AML2leukemiacellswhereivermectininducedcell
quenchedbyhighchlorideionconcentrations.Histogramsfromrepresentative
deathafter24hoursoftreatmentandDU145prostatecancercells experimentsareshown.(D)OCI-AML2and(E)DU145cellsweretreatedwith
that were more resistant to ivermectin-induced cell death (Figure 6and10(cid:1)Mivermectinfor1hour.Aftertreatment,cellsizewasmeasuredby
3A). OCI-AML2 and DU145 cells were treated with 10(cid:1)M forwardlightscatterandflowcytometry.Datarepresentmean(cid:9)SDfoldchange
incellsizecomparedwithcontrolfromrepresentativeexperimentsperformedin
ivermectin for 2 hours, and levels of intracellular chloride were triplicate.**P(cid:6).01,byunpairedttest.
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BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18 PRECLINICALACTIVITYOFIVERMECTININLEUKEMIA 3597
Figure 4. Ivermectin induces plasma membrane
hyperpolarization dependent on chloride influx.
OCI-AML2cellsweretreatedwithincreasingconcentra-
tionsofivermectinfor24hours(A)or6(cid:1)Mivermectin
forincreasingtimesofincubation(B).Aftertreatment,
plasmamembranepotentialwasmeasuredbystaining
cellswithDiBAC4(3)andflowcytometricanalysis.Data
representthemean(cid:9)SDfoldchangeinplasmamem-
brane potential compared with control treated cells.
Representativeexperimentsperformedintriplicateare
shown.Differencesinchangeofmembranepotential
comparedwithcontrolwereanalyzedbyanunpaired
ttest.***P(cid:6).001;*P(cid:6).05.U937andTEXleukemia
cells,aprimaryAMLsample(AML),(C)DU145and
PPC-1 prostate cancer, and 2 samples of normal
hematopoieticcells(D)weretreatedwith6(cid:1)Mivermec-
tinforincreasingtimes.Aftertreatment,plasmamem-
brane potential was measured as described above.
Datarepresentthemean(cid:9)SDfoldchangeinplasma
membrane potential compared with control treated
cells.Representativeexperimentsperformedintripli-
cateareshown.Differencesinchangeofmembrane
potential,comparedwithcontrol,wereanalyzedbyan
unpairedttest.***P(cid:6).001;*P(cid:6).05.(E)OCI-AML2
cells were treated with 6(cid:1)M ivermectin in chloride-
replete and chloride-free media for 5 hours. After
incubation,plasmamembranepotentialwasmeasured
asdescribedabove.Datarepresentthemean(cid:9)SD
changeinplasmamembranepotential,comparedwith
untreatedcellsinchloride-repletemedia.Representa-
tive experiments performed in triplicates are shown.
Differences in change of membrane potential, com-
paredwithcontrol,wereanalyzedbyanunpairedttest.
***P(cid:6).001;*P(cid:6).05.
hour of treatment (Figure 4B), consistent with the influx of depolarization (Figure 4E). Thus, the effects of ivermectin on
intracellular chloride and the effects observed in nematodes. plasma membrane polarization appear to be related to increased
Likewise,U937andTEXleukemiacells,aswellasprimaryAML chlorideflux.
cells sensitive to ivermectin-induced death, also demonstrated
plasma membrane hyperpolarization after ivermectin treatment
Ivermectinincreasesintracellularcalciumbutisnot
(Figure 4C). In contrast, DU145 and PPC-1 cells, as well as
functionallyrelevantinleukemiacells
primary normal hematopoietic cells that were more resistant to
ivermectin, did not show changes in their plasma membrane Plasma membrane hyperpolarization can lead to calcium in-
potential when treated with up to 6(cid:1)M ivermectin for up to flux.18Therefore,wetestedtheeffectsofivermectinoncalcium
24hours(Figure4D). influx in leukemia cells. OCI-AML2 cells were treated with
Todeterminewhethertheplasmamembranehyperpolarization ivermectin and the concentration of intracellular calcium was
observed after ivermectin treatment was related to increased measuredbystainingcellswiththeratiometricdye,Indo-1AM.
chlorideionflux,wemeasuredplasmamembranepolarizationafter Asapositivecontrol,cellsweretreatedwithdigoxin,whichis
treatingcellswithivermectininbufferswithandwithoutchloride. known to increase intracellular calcium.19,20 Similar to the
OCI-AML2 cells were treated for 5 hours with ivermectin in a effects of digoxin, ivermectin increased intracellular calcium
chloride-repletebufferorachloride-freebuffer,wheresodiumand (supplementalFigure4A-B).However,theincreaseinintracel-
potassiumchloridewerereplacedwithequimolargluconatesaltsof lularcalciumdidnotappearsufficienttoexplainthecytotoxic-
sodiumandpotassium.Whenaddedtocellsinthechloride-replete ity of ivermectin, because chelation of intra- and extracellular
buffer, ivermectin induced plasma membrane hyperpolarization calciumwithBAPTA-AMandEDTA(ethylenediaminetetraace-
similartocellstreatedinRPMImedium.However,whenaddedto tic acid), respectively, did not inhibit ivermectin-induced cell
cellsinchloride-freebuffer,ivermectincausedplasmamembrane death(datanotshown).
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3598 SHARMEENetal BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18
Figure 5. Ivermectin induces generation of ROS.
OCI-AML2leukemiacellsweretreatedwithincreasing
concentrations of ivermectin overnight (A) or 6(cid:1)M
ivermectin for increasing incubation times (B). After
incubation,ROSwasdetectedbystainingcellswith
Carboxy-H2DCFDA(finalconcentration10(cid:1)M)andflow
cytometric analysis. Data represent the mean(cid:9)SD
foldchangeinROSproduction,comparedwithcontrol.
Representativeexperimentsperformedintriplicateare
shown.DifferencesinchangeofROScomparedwith
controlwereanalyzedbyanunpairedttest.***P(cid:6).001;
**P(cid:6).005. (C) U937 and TEX leukemia cells and
DU145 and PPC-1 prostate cells were treated with
ivermectinat6(cid:1)Mfor2hours.Aftertreatment,ROS
generationwasmeasuredasdescribedabove.Data
representthemean(cid:9)SDfoldchangeinROSproduc-
tioncomparedwitheachoftheirbuffertreatedcontrols.
Representativeexperimentsperformedintriplicateare
shown.DifferencesinchangeofROScomparedwith
controlwereanalyzedbyanunpairedttest.***P(cid:6).001.
PrimaryAMLcells(n(cid:4)3)andnormalhematopoietic
stemcells(PBSCs,n(cid:4)3)weretreatedwithivermectin
(6(cid:1)M) for 6 hours.After treatment, ROS generation
wasmeasuredasdescribedabove.Datarepresentthe
mean(cid:9)SDfoldchangeinROSproductioncompared
with each of their buffer treated controls for experi-
ments performed in triplicate. Differences in ROS
productioncomparedwithcontrolwereanalyzedbyan
unpairedttest.***P(cid:6).001.(D)OCI-AML2cellswere
treatedsimultaneouslywithivermectin(3(cid:1)M),theROS
scavenger,NAC(5(cid:1)M),orthecombinationofNACand
ivermectin.After48hoursoftreatment,cellgrowthand
viability were measured by the MTS assay. Data
representthemean(cid:9)SDpercentviablecellsfroma
representativeexperimentperformedintriplicate.Differ-
encesinchangeofcellviability,comparedwithcontrol,
wereanalyzedbyanunpairedttest.***P(cid:6).001.
IvermectinincreasesintracellularROS production and in keeping with its effects on plasma membrane
hyperpolarizationandchlorideinflux(Figure5D).
Manganese chloride, cobalt chloride, and mercuric chloride can
Changes in ROS production are indicative of a biologic
lead to generation of ROS.21-23 Therefore, we tested whether
response to ivermectin, but are very difficult to measure in the
ivermectin increased ROS production in leukemia cells due to
contextofaclinicaltrial.Therefore,toidentifyalterationsingene
theobservedchlorideinflux.OCI-AML2cellsweretreatedwith
expressionthatarearesultofROSproductionandcouldbeusedas
ivermectinatincreasingconcentrationsandtimesofincubation.
biomarkers in the context of a clinical trial, we undertook gene
After treatment, levels of intracellular ROS were measured by
expressionprofilinganalysis(usingAffymetrixHGU133Plus2.0
staining cells with Carboxy-H DCFDA and flow cytometry.
2 arrays) of RNA derived from OCI-AML2 cells treated with
Treatment with ivermectin increased ROS production at times ivermectin for 30 and 40 hours (supplemental Table 1). One
andconcentrationsthatcoincidedwithplasmamembranehyper- hundredfiftygeneswerederegulated(cid:7)4-foldatbothtimepoints
polarization(Figure5A-B).Likewise,U937andTEXleukemia (33 underexpressed; 117 overexpressed), compared with control.
cells that were sensitive to ivermectin-induced death demon- Among these genes dysregulated were STAT1, which has been
strated increased ROS generation 2 hours after ivermectin associated with increased ROS generation24-26 and the STAT1
treatment(Figure5C).Incontrast,DU145andPPC-1cellsthat downstream targets, IFIT3, OAS1, and TRIM22. We validated
weremoreresistanttoivermectindidnotshowchangesinROS the up-regulation of STAT1 and target genes IFIT3, OAS1, and
generation. Likewise, primary AML cells, but not normal TRIM22 after ivermectin treatment by qRT-PCR, (Figure 6A).
hematopoietic cells, demonstrated increased ROS generation Likewise,U937andHL60leukemiacellsthatweresensitiveto
afterivermectintreatment(Figure5C). ivermectin-induced death also demonstrated increased STAT1
TodeterminewhethertheincreasedROSproductionwasfunction- mRNA.Incontrast,DU145andPPC-1cellsthatweremoreresistantto
ally important for ivermectin-induced cell death, cells were treated ivermectindidnotshowchangesinSTAT1expression(Figure6B).We
simultaneouslywithivermectinalongwiththefreeradicalscavenger alsoevaluatedchangesinSTAT1expressionintumorsfromaleukemia
N-acetyl-L-cysteine (NAC). NAC abrogated ivermectin-induced cell xenograftmodel.MicewithOCI-AML2subcutaneousxenograftswere
death consistent with a mechanism of cell death related to ROS treated with ivermectin for 5 days. After treatment, tumors were
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BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18 PRECLINICALACTIVITYOFIVERMECTININLEUKEMIA 3599
Figure6.IvermectinincreasesexpressionofSTAT1
and its target genes through a ROS-dependent
mechanism. (A) OCI-AML2 cells were treated with
3(cid:1)M ivermectin (IVM) for 30 hours.After treatment,
RNAwasisolated,reversetranscribed,andsubjected
toquantitativePCRusingspecificprimersforSTAT1A,
STAT1B,andSTAT1targetgenesOAS1,TRIM22,and
IFIT3.Datarepresentmean(cid:9)SDfoldincreaseingene
expression normalized to 18S expression and com-
pared with control cells. (B) OCIAML2, U937, and
HL60leukemiaandDU145andPPC-1prostatecancer
cellsweretreatedwith6(cid:1)Mivermectinfor24hours,
andmRNAlevelsofSTAT1AandSTAT1Bweremea-
suredusingqPCRandnormalizedto18Sexpression
as (A). Data represent mean(cid:9)SD fold increase in
gene expression, compared with control cells.
(C)OCI-AML2cells(2.5(cid:5)105)wereinjectedsubcuta-
neouslyintotheflanksofsublethallyirradiatedNOD/
SCIDmice.Oncetumorswereestablished,micewere
treated with ivermectin (7mg/kg) intraperitoneally or
vehicle control for 5 days (n(cid:4)3 per group). After
treatment, mice were killed, and tumors were har-
vested.mRNAwasextracted,andchangesinSTAT1A
and1BexpressionweremeasuredbyqRT-PCR.Data
representmean(cid:9)SDfoldincreaseingeneexpression
normalizedto18Sexpression,comparedwithtumors
from controltreated mice.(D) OCI-AML2 cells were
treatedsimultaneouslywithivermectin(3(cid:1)M),theROS
scavenger, NAC (5(cid:1)M), or both for 30 hours, and
STAT1A and STAT1B expression was assessed as
described for panel A. Relative expression values
normalizedto18sarereportedasfold-change(cid:9)SD
comparedwiththeuntreatedcontrolforeachgene.
harvested,mRNAwasextracted,andSTAT1expressionwasmeasured treated with increasing concentrations of ivermectin alone and in
byqRT-PCR.STAT1mRNAwasincreasedin2of3testedtumorsfrom combination with cytarabine and daunorubicin. Cell growth and
micetreatedwithivermectin,comparedwithSTAT1mRNAexpression viabilityweremeasured72hoursafterincubationusingtheMTS
fromtumorsharvestedfrommicetreatedwithvehiclecontrol(Figure assay.DatawereanalyzedbytheCalcuSynmedianeffectmodel,
6C).WealsodemonstratedthatchangesinSTAT1genesweresecondary wheretheCIindicatessynergism(CI(cid:6)0.9),additivity(CI(cid:4)0.9-
to ROS production as pretreatment with NAC blocked their up- 1.1) or antagonism (CI(cid:7)1.1). In both OCI-AML2 and U937
regulation(Figure6D). leukemia cells, the combination of ivermectin and cytarabine
Of note, we also compared our array dataset to a ROS gene demonstratedstrongsynergism,withCIvaluesattheED ,ED ,
25 50
signature reported by Tothova et al.27 Of the 55 genes in the andED of0.51,0.58,and0.65,respectively,inOCIAML2cells
75
Tothovasignature,two-thirdswerepresentinourdataset.Ofthese and ED , ED , and ED of 0.55, 0.71, and 0.91 in U937 cells
25 50 75
36 genes, 55% (20 genes) were found to be differentially (Figure 7C). Likewise, in OCI-AML2 cells, the combination of
regulated on ivermectin treatment (fold-change of 1.25 up or ivermectinanddaunorubicinwasalsosynergistic,withCIvaluesat
down, compared with the untreated control sample). Thus,
theED ,ED ,andED of0.48,0.51,and0.54,respectively.In
25 50 75
ivermectin appears to induce genetic changes consistent with
contrast, the combination of ivermectin and daunorubicin was
ROSinduction.
closertotheadditiveinU937,withCIvaluesattheED ,ED ,and
25 50
ED of1.1,0.98,and0.85,respectively(Figure7D).
Ivermectinsynergizeswithcytarabineanddaunorubicin 75
Wealsotestedthecombinationofivermectinandcytarabinein
Cytarabine and daunorubicin increase ROS production through normal hematopoeitic cells. In contrast to the effects observed in
mechanisms related to DNAdamage (Figure 7A-B) and are used theleukemiacelllines,ivermectindidnotenhancethecytotoxicity
clinicallyinthetreatmentofAML.28,29Therefore,weevaluatedthe ofcytarabineinnormalcells(Figure7E).
effects of the combination of ivermectin with cytarabine and Drugsequencingcanaffecttheactivityofdrugcombinations.
daunorubicin on cell viability. OCI-AML2 and U937 cells were Therefore,wetestedtheeffectofdrugsequencingonthesynergism
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3600 SHARMEENetal BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18
Figure 7. Ivermectin synergizes with cytarabine and
daunorubicin to induce cell death in leukemia cells.
OCI-AML2 cells were treated with increasing concentra-
tionsofdaunorubicin(A)andcytarabine(B)overnight.After
treatment,ROSwasmeasuredbystainingcellswithCar-
boxy-H2DCFDA(finalconcentration10(cid:1)M)andflowcyto-
metricanalysis.Datarepresentthemean(cid:9)SDfoldchange
inROSproductioncomparedwithcontrol.Representative
experimentsperformedintriplicateareshown.Theeffects
ofdifferentconcentrationsofivermectinincombinationwith
cytarabineanddaunorubicinontheviabilityofOCI-AML2
and U937 cells were measured by MTS assay after
72hoursofincubation.DatawereanalyzedwithCalcUSyn
software as described in Methods. CI versus Fractional
effect(Fa)plotshowingtheeffectofthecombinationof
ivermectinwithcytarabine(C)andivermectinwithdaunoru-
bicin (D) in OCI AML2 and U937 are illustrated in the
isobolograms.CI(cid:6)0.9indicatessynergism.Representa-
tiveisobologramsofexperimentsperformedintriplicateare
shown. (E) Normal hematopoietic cells (PBSCs; n(cid:4)2)
weretreatedwithTincreasingconcentrationsofivermectin
and cytarabine (0, 2.5, and 5(cid:1)M). After 48 hours, cell
viability was measured by annexinV–PI staining. Data
represent the mean(cid:9)SD percent of viable cells from
experimentsperformedintriplicate.
betweenivermectinandcytarabineordaunorubicn.InOCI-AML2and for compounds cytotoxic to leukemia cells. From this screen, we
U937 cells, the combination of ivermectin and cytarabine remained identifiedtheantiparasiticagent,ivermectin,whichinducedcelldeathin
synergistic,regardlessofwhethertheivermectinwasgivenwith,before, leukemiacelllinesatlowmicromolarconcentrationsanddelayedtumor
or after the addition of cytarabine (Figure 7F). In contrast, in OCI- growthinmousemodelsofleukemia.
AML2cells,thecombinationofivermectinwassynergisticwhengiven As part of its development as an antiparasitic agent, the
beforeorsimultaneouslywithdaunorubicin.However,theeffectsofthe pharmacology and toxicology of ivermectin have been studied
combination were additive when the ivermectin was given after the extensivelyinhumansandanimals.Humanstreatedforonchocer-
additionofdaunorubicin.(Figure7F) ciasistypicallyreceiveasingledoseof100-200(cid:1)g/kgivermectin
We also evaluated the combination of ivermectin with the toeradicatetheparasite.Insuchpatients,plasmaconcentrationof
anthelmintic agent, albendazole, as this agent synergized with 52.0ng/mLhavebeenachievedin5.2hours,withanareaunderthe
ivermectin in the treatment of nematodes.30,31 In contrast to the curveover48hoursof2852ng•h/mL.32Similarpharmacokinetics
synergy observed with cytarabine and daunorubicin, albendazole havebeenreportedinhealthymalevolunteersreceivinga14-mg
antagonizedtheantileukemiceffectsofivermectin,withCIvalues capsuleofradiolabeledivermectin.Inthesesubjects,themeanT
max
at the ED , ED , and ED of 1.59, 1.09, and 0.89, respectively was 6 hours, with a half-life of 11.8 hours.10 These doses of
25 50 75
(datanotshown). ivermectin produce plasma levels that are likely lower than the
concentrationsrequiredtoinduceanantileukemiceffectandmay
explainwhyantitumoreffectsofivermectinhavenotbeenpreviously
Discussion reported in patients receiving standard doses of this drug for the
treatmentofonchocerciasis.However,higherconcentrationsofivermec-
To identify known drugs with previously unrecognized antileukemia tinthatmaypossessantitumoractivityhavebeenwelltoleratedinboth
activity,wecompiledandscreenedalibraryofoff-andon-patentdrugs humans and animals. For example, the LD of oral ivermectin is
50
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BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18 PRECLINICALACTIVITYOFIVERMECTININLEUKEMIA 3601
Figure7(continued).(F)OCI-AML2(i)andU937(ii)cells
weretreatedwithivermectin,cytarabine,orthecombination
of the 2 drugs at varying concentrations for 72 hours.
Ivermectin3cytarabinedenotesthativermectinwasadded
initially,andcytarabinewasaddedforthelast48hoursofthe
72-hour experiment. Cytarabine3ivermectin denotes that
cytarabinewasaddedinitially,andivermectinwasaddedfor
thelast48hoursofthe72-hourexperiment.OCI-AML2(iiii)
cells were treated with ivermectin, daunorubicin, or the
combination of the 2 drugs at varying concentrations for
72hours.Ivermectin3daunorubicindenotesthativermectin
was added initially, and the daunorubicin was added
for the last 48 hours of the 72-hour experiment.
Daunorubicin3ivermectin denotes that daunorubicin
wasaddedinitially,andtheivermectinwasaddedforthelast
48 hours of the 72-hour experiment. After treatment, cell
growth and viability was measured by the MTS assay.
Representativeexperimentsperformedintriplicateareshown.
Datarepresentmean(cid:9)SDfractionaleffect(celldeath).
approximately 28-30mg/kg in mice, 80mg/kg in dogs, and above and this rash responded to ivermectin. Moreover, the leukemia
24mg/kg in monkeys.7,32 Humans with spinal injury and muscle cells in the peripheral blood were controlled while receiving
spasticity have been treated with up to 1.6mg/kg of ivermectin ivermectin
subcutaneouslytwiceweeklyforupto12weekswithouttoxicity.9In Ourstudiessuggestthativermectininducescelldeaththrougha
addition,reportsofivermectinoverdosesalsosupportthepotentialwide mechanismrelatedtoitsknownfunctionasanactivatorofchloride
therapeutic window of this drug. For example, an individual who channels.Asanantiparasiticagent,ivermectinactivatesglutamate-
self-administered 6g of veterinary ivermectin 30-50 times over the gatedchloridechannelsuniquetoinvertebrates.However,athigher
course of 1 year had no evidence of toxicity from the ivermectin.11 concentrations, ivermectin also activates mammalian chloride
Multipleotheringestioneventshavealsobeenreported,particularlyin channels.7 Mammalian chloride channels broadly fall into
pediatric subjects who accidentally consumed veterinary ivermectin 5classes,basedontheirregulation:cysticfibrosistransmembrane
keptinthehouseholdforthefamilydog.Inthemajorityofthese,no conductance regulator, which is activated by cyclicAMPdepen-
seriousadverseeventswerereported.9,11 dentphosphorylation;calcium-activatedchloridechannels;voltage-
Although no prior clinical studies have directly evaluated gated chloride channels (ClCs); ligand-gated chloride channels
ivermectin as an anticancer agent, a case report suggests that ((cid:10)-aminobutyricacidandglycine-activated);andvolume-regulated
ivermectinmayhaveactivityinthetreatmentofleukemia.33An chloride channels. These channels act in heteromeric complexes
adult male with T-cell leukemia/lymphoma presented with a dependent upon cell type, with many possible permutations and
generalizedpruritic,erythrodermicrashwithareasofhyperker- combinations of the subunits.34 Currently, it is unclear which
atosis and was diagnosed with scabies. He received 200(cid:1)g/kg mammalian chloride channels are being activated by ivermectin,
ivermectin on days 1 and 10 with complete resolution of the but the complexity of their organization makes it difficult to
rash. While not a focus of the paper, it is possible that a identify single “target” channels for ivermectin activity using
componentofthepatient’srashmayhavebeenduetoleukemia standardgeneticexperiments.
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(cid:1)
3602 SHARMEENetal BLOOD,4NOVEMBER2010 VOLUME116,NUMBER18
Theshort-termcytotoxicitystudiesandtheinvivoexperiments tinanddemonstratedsynergywithbothofthesedrugs.Therefore,
support a therapeutic window for ivermectin as an antileukemia ivermectincouldbeevaluatedincombinationwiththeseagentsto
agent,butthedifferencebetweennormalCD34(cid:8)cellsandmalig- enhancetheefficacyofstandardtherapyforAML.
nantcellswasnarrower,aswerethedifferencesintheclonogenic In summary, we have shown that ivermectin induces cell
growthassays.However,itisimportanttonotethatresultsofthese deathinleukemiacellsviachlorideinflux,membranehyperpo-
assays do not always predict clinical toxicity. For example, larization,andincreasinglevelsofintracellularROS.Givenits
cytarabine and amsacrine are chemotherapeutic agents routinely prior safety record in humans and animals coupled with its
usedinthetreatmentofAML,butshowlittleornoselectivityfor preclinicalefficacyinleukemia,aphase1clinicaltrialcouldbe
malignantcellsovernormalcellsincolonyformationassays.35,36In conducted to determine the tolerance and biologic activity of
addition, we demonstrated that oral ivermectin delayed tumor oralivermectininthesepatients.
growthin3mousemodelsofleukemiawithoutuntowardtoxicity,
supportingatherapeuticwindow.Finally,toxicologystudieswith
ivermectin in animals and humans did not report hematologic Acknowledgments
toxicity. Nonetheless, the small differential sensitivity between
primary AML and normal hematopoietic cells raises concerns A.D.S. is a Leukemia & Lymphoma Society Scholar in Clinical
aboutthepotentialhematologictoxicity,anditssafetywillhaveto Research. M.A.S. is a Canadian Institutes of Health Research
becarefullyevaluatedinphase1clinicaltrials. postdoctoral fellow, and holds a postdoctoral research fellowship
The basis for the therapeutic window after invermectin treat- fromtheMultipleMyelomaResearchFoundationandMillennium
mentislikelymultifactorial.Chloridechannelsareoftenincreased Pharmaceuticals.M.S.isaCanadianInstitutesofHealthResearch
on the surface of malignant cells, compared with normal cells, BantingandBestCanadaGraduateScholar.
potentially making them more sensitive to alterations in chloride
flux by ivermectin. For example, compared with normal neutro-
phils, HL60 cells overexpress the ClC-5 chloride channel that is Authorship
normallyexpressedinrenalcells.37Insupportofthismechanism,
weobservedlesschloridefluxincellsmoreresistanttoivermectin. Contribution: S.S. designed research, analyzed data, performed
Alterations in intracellular chloride concentrations also affect research, and wrote the paper; M.S. designed research, analyzed
basichomeostaticparameters,suchasintracellularCa2(cid:8)levels, data,andperformedresearch;M.A.S.designedresearch,analyzed
pH, and cell volume,38 and alteration of these parameters can data, and performed research; R.H. performed research and ana-
induceapoptosis.39Finally,thetherapeuticwindowwithivermec- lyzed data; M.G. performed research and analyzed data; X.W.
tin treatment may reflect differences in sensitivity to ROS performedresearchandanalyzeddata;S.B.F.performedresearch
generation.IvermectinincreasedROSgenerationthatappeared and analyzed data; H.S. performed research and analyzed data;
functionallyimportantforitscytotoxicity,andpreviousstudies T.E.W.designedresearch,analyzeddata,andperformedresearch;
support a mechanism of ROS generation related to increased R.W. performed research and analyzed data; M.D.M. contributed
chloride influx.22,40-42 Previous studies have also demonstrated criticalreagentsandanalyzeddata;R.A.B.designedresearchand
thatmalignantcellshavehigherbasallevelsofROSandareless supervisedresearch,;A.D.designedresearch,preformedresearch,
tolerantofROS-inducingagents,comparedwithnormalcells.43,44 and analyzed data; J.W. and S.O.K. supervised research; A.D.S.
Future studies will help clarify the basis of the therapeutic designed research, analyzed data, supervised research, and wrote
windowaswellasidentifysubgroupsofpatientsmostlikelyto themanuscript;andallauthorsreviewedandeditedthepaper.
respondtothistherapy. Conflict-of-interestdisclosure:Theauthorsdeclarenocompet-
Cytarabineanddaunorubicin,whichareusedinthetreatmentof ingfinancialinterests.
AML,induceROSgenerationthroughamechanismlinkedtoDNA Correspondence:AaronD.Schimmer,PrincessMargaretHospi-
damage and thus a mechanism distinct from ivermectin. Conse- tal, Ontario Cancer Institute, 610 University Ave, Toronto, ON,
quently,weevaluatedthecombinationofthesedrugswithivermec- Canada,M5G2M9;e-mail:[email protected].
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