Gemcitabine and glioblastoma: challenges and current perspectives

Gemcitabine and glioblastoma:

challenges and current perspectives

Chiara Bastiancich

, Guillaume Bastiat

and Frederic Lagarce

1MINT,UNIVAngers,INSERM1066,CNRS6021,UniversitéBretagneLoire,Angers,France

2PharmacyDepartment,CHUAngers,AngersUniversityHospital,France

3UniversitéCatholiquedeLouvain,LouvainDrugResearchInstitute,AdvancedDrugDeliveryandBiomaterials,Brussels,Belgium

Gemcitabine is a nucleoside analog currently used for the treatment of various solid tumors as a single agent or in combination with other chemotherapeutic drugs. Its use against highly aggressive brain tumors (glioblastoma) has been evaluated in preclinical and clinical trials leading to controversial results. Gemcitabine can inhibit DNA chain elongation, is a potent radiosensitizer and it can enhance antitumor immune activity, but it also presents some drawbacks (e.g., short half-life, side effects, chemoresistance). The aim of this review is to discuss the challenges related to the use of gemcitabine for glioblastoma and to report recent studies that suggest overcoming these obstacles opening new perspectives for its use in the field (e.g., gemcitabine derivatives and/or nanomedicines).

Introduction

Glioblastoma(GBM)isoneofthegreatestchallengesinoncology.

Itisanaggressivemalignantbraintumorcharacterizedbyrapid proliferation,propensitytoinfiltrateinhealthybraintissuesand strongchemoresistance. Its standard-of-care treatmentincludes surgicalresectionfollowedbyradiotherapy(RT)andconcomitant plusadjuvantchemotherapywithtemozolomide(TMZ)[1].After oral administration, TMZ is spontaneously converted into its activemetabolitemethyltriazeno-imidazole-carboximide(MTIC) atphysiologicalconditions.MTICshowsexcellentbioavailability, iscan crossthe blood–brainbarrier (BBB)and quicklydegrades intomethyldiazoniumion,whichisapotentmethylatingagent [2].TheDNAmethylationleadstomismatchrepairsystemfailure (owingto theimpossibilityoffindingcomplementarybasesfor methylatedadducts),inhibitionofcellreplicationandapoptosis (Fig. 1a) [3]. The use of TMZ as standard therapy for GBM in combination with RT is the result of a successful clinical trial publishedby Stuppetal.thatproved modestsurvival improve- mentcomparedtoRTaloneandledtoFDA approvalonnewly diagnosedGBMin2005.However,despitetheaggressive thera- peuticregimen,mostGBMpatientsquicklydeveloptumorrecur- rencesthatinevitablyleadtodeath(mediansurvival14.6months;

5-year survival rate <10%)[4].Some intrinsiccharacteristicsof GBMlimittheeffectivenessofchemotherapeutics.Theseinclude GBM anatomic location (BBB) and unique microenvironment (extracellularmatrix,nutrition,oxygenation,pHvalue),GBMcell heterogeneity(cancerstemcells,tumormicrotubes),highprolif- erationrate(variationincell-cycledistribution,cell–cellcontact), angiogenesisand chemoresistance[5,6].Resistancetoalkylating agents can be caused by intrinsic resistance as a result of O⁶- methylguanine-DNAmethyltransferase (MGMT) genepromoter methylationand/oracquired resistancecausedby mutationsin DNAmismatchrepairenzymes[7].MGMTisaDNArepairenzyme thatcantransferthealkylgroupattheO⁶positionofguanineto theactivesiteoftheenzymethereforereversingtheDNAalkyl- ation producedbyTMZ. Acorrelationhasbeen foundbetween MGMT promoter methylation status (leading to MGMT gene silencing and lower MGMT expression) and increased survival inGBMpatientstreatedwithTMZ[8,9].

As an attempt to ameliorate the management of GBM patients, increasingtheir survivalrate andqualityoflife,many researchers have tried to explore different strategies(e.g., local delivery of chemotherapeutics, nanomedicines, gene therapy, etc.)[10].Choosingthecorrectdrugorcombination(e.g.,single ormultimodalchemotherapy,combinationofchemotherapyand RT),theproperdoses,timingofadministrationanddeliveryroute

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Correspondingauthor:Lagarce,F. ([email protected])

1359-6446/ã2017ElsevierLtd.Allrightsreserved.

are crucialfor GBM investigators. Historically, the tangible in- creaseinGBMpatientsurvivalwasobservedwhenRTwasincluded as the standard treatment following surgical resection in the 1970s, shifting the median survival from 3–6 to 9–12 months [11].Thestudyofradiosensitizingmoleculesthatcouldenhance RTefficacyleadingtoareductionofrecurrenceshassincebeenof greatinterest.Thisreviewfocusesontheuseofgemcitabine(Gem) – a potent chemotherapeutic and radiosensitizing agent acting through a MGMT-independent mechanism-of-action – for the treatmentofGBM,defining itshistoricalbackground,potential, challengesandcurrentapplications.

Gemcitabine

Gem(2⁰,2⁰-difluoro-deoxycytidine)isanucleosideanalogcurrent- lyapprovedforthetreatmentofvarioussolidtumors(pancreatic, non-small-celllung,breastand ovarian),asa singleagentor in combinationwithotherchemotherapeuticdrugs.Gemisgeneral- lyadministeredonceaweekbyintravenousinfusionover30min, at a maximumdosageof1250mg/m² for21-daycycles (longer administration cyclesfor pancreatic cancer).Gem isa prodrug, becauseitneedstobetransportedintothecellsthroughnucleo- side transporters(mainly hENT1, hCNT1 and hCNT3) whereit undergoes sequential phosphorylation by deoxycytidine kinase (DCK)tobeactive(Fig.1b).Gemtriphosphateactsasadeoxycy- tidine-triphosphate-competitive substrateand itis incorporated intoDNAduringreplication,inhibitingDNAchainelongationby

‘mask chaintermination’.The formationofGem-inducedDNA fragmentsleadstocelldeathbyapoptosis[12].Atthesametime, Gem diphosphateinhibitsribonucleotidereductase, anenzyme ofDNAsynthesis,depletingthebiosynthesisofthedeoxyribonu- cleoside triphosphate precursors and avoiding the intracellular

inactivationofGem monophosphatethus‘self-potentiating’ its ownconcentrationandcytotoxicactivity[13].Ithasbeenrecently hypothesizedthatGemanditsmetabolitescanpassivelydiffusein goodcommunicatingcellsthroughgapjunctions(composedof connexin proteins).Even thoughthe connexin expressionand functioninGBMisnotwellknown[14],ithasbeendemonstrated invitroondifferentGBMcelllinesthatGem-mediatedtoxicitycan diffuseand canbetransferred betweencellsby a phenomenon calledthebystandereffect[15].

Gemisapowerfulradiationsensitizeratnoncytotoxicconcen- trationsevenafterabriefexposuretime[16,17].Moreover,difluor- odeoxyuridine(dFdU),oneoftheGemmetabolites,canactasa radiosensitizerandshowscytotoxicactivityatconcentrationsthat are easily reached in plasma [18,19]. Even if the mechanism involved remains unclear, it is believed that the main factors contributing to Gem-radioenhancing activity are depletion of phosphorylated deoxynucleotides (especially deoxyadenosine triphosphate) and Gem-induced cell-cycle redistribution into theS-phase[20–24].

Gempresentssignificantimmunomodulatoryactivityindiffer- entanimaltumormodels,independentlyofitscytotoxic effect.

Indeed,GemhasbeenshowntoselectivelydepleteBlymphocytes, myeloid-derivedsuppressorcellsandregulatoryTcellsintumor- bearinganimals[25–28].Gemisalsoanattractivemoleculeforthe treatmentof GBM (Fig. 2a).Indeed, aspreviously cited, itis a powerful chemotherapeutic and radiosensitizing agent acting througha MGMT-independent mechanism,which could avoid crossed-linkedresistancewithTMZ.Itstoxicityisprobablymedi- atedthroughgapjunctionssuggesting thatitcould beauseful agentintumorsdisplayinggapjunctionsandexpressingdifferent typesofconnexins,suchasGBM.EveniftheabilityofGemtopass (a) Alkylating agents: Temozolomide (b)

Temozolomide CONH₂

CONH₂

CONH₂

CH₃ CH₃

NH₂ NH CH₃

+H₂O

– CO₂ N

N N N

Methyldiazonium cation

Gemcitabine (Gem)

Gem Difluorodeoxyuridine

Inactive compound

-Gem P-Gem

P P

P P P-Gem

Nucleoside transporters (hENTs or hCNTs)

MTIC

AIC

+ +

N

HO N N O O

OH F

F NH₂

NH N

H N N N

N N N

O

Non phase specific (cell-cycle independent) Cell-cycle dependent (S-phase specific)

Deoxycytidine kinase

Cytidien deaminase

Cytidylate deaminase

5′-nucleotidase

5′-nucleotidase Nucleoside

monophosphate kinase

Diphosphate kinase

Nucleoside analogs: Gemcitabine

Nu (h

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FIGURE1

Schematicrepresentationofmechanism-of-actionoftemozolomideandgemcitabine;adapted,withpermission,fromRefs[7]and[64].(a)Temozolomideis spontaneouslyhydrolyzedintotheactivemetabolitemethyltriazeno-imidazole-carboximide(MTIC)atphysiologicalconditions.MTICdegradesinto methyldiazoniumcation(methylatingagent)andAIC(degradationproduct).MethyldiazoniumcationinteractswithDNAproducingO⁶-methylguanine,N⁷- methylguanineandO³-methyladenineadducts.AlkylatedazotateleadstoDNAmismatchrepairevents,DNA-strandbreakandapoptosis.(b)Gemcitabine (Gem)uptakeismediatedbynucleosidetransportersandfollowedbyaseriesofthreephosphorylationevents.Gemdiphosphateinhibitsribonucleotide reductasereducingtheconcentrationofdeoxycytidinetriphosphate(self-potentiation).GemtriphosphateincorporatesintoDNAduringreplicationactingasa competitivesubstrateofdeoxycytidinetriphosphate,leadingtoirreversibleinhibitionofDNApolymerasesandblockingofDNAchainelongation(maskedchain termination).

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theBBB ismodest, ithasbeen shownto passthe blood–tumor barrierinGBMpatientsatconcentrationshighenoughtoenable radiosensitization.Moreover,theclinicaluseofGemhasshownits abilityto act in synergy notonly with RTbut also with other chemotherapeuticagents(e.g.,carboplatin,cisplatin,paclitaxel).

Finally,theimmunomodulatingcapacitiesofGemmightbeuseful foritsuse againstGBM incombinationwith immunotherapies [29].Owingtotheaggressivenessandtheuniquecharacteristicsof GBM,therationalefortheuseofsuchaversatilemoleculeandthe combinationofmultipletherapeuticstrategiesisconvincing.

Gemcitabine for the treatment of glioblastoma

ThefirststudysuggestingtheuseofGemforthetreatmentofGBM waspublishedbyRiegeretal.in1999[30](Table1),thesameyear that TMZ received accelerated approval for use in anaplastic astrocytoma. In this work, the authors studied the effect of Gem on12human malignant GBMcell lines invitro,showing themtobesusceptibletothecytotoxicandanticlonogenicaction ofGem. Gem was100-foldmore potent than its related agent cytarabine.Interestingly,itwasdemonstratedthatpre-exposureof thecellstodexamethasone(asteroiddrugcommonlyusedforthe controlofcerebraledemainGBMpatients)moderatelyreduced the cytotoxic effect of Gem, as previously shown with other anticancerdrugs.Subsequently,thesamegroupperformedaPhase IIclinicaltrialenrolling21patientswithnewlydiagnosedGBMto evaluatethetherapeuticefficacyofpre-irradiation Gemchemo- therapyfollowedbystandardRT[31].Patientswereadministered

1000mg/m²intravenouslyondays1,8and15ofeach1-month cycleandforamaximumoffourcycles.Radiotherapywasthen administered2 weeksafterthefinaldoseofGem. Theregimen usedinthestudywassafebutdidnotimprovesurvivalcompared toRTalone.Inthesameperiod,anothergroupperformedaPhase IIclinicaltrialonpatientswithanaplasticastrocytomaorGBMat first relapse showing similar results [32]. Indeed, no objective response was obtained fromthis study. Authors suggested that selection bias might have confounded results because patients couldstart thetreatmentonly2monthsafterpriorRT,leaving timeforthediseasetoprogress.Moreover,theconcomitantuseof anticonvulsants and steroids might have reduced the effect of Gem. AnotherPhaseIIclinicalstudy reportedthe combination ofGemandtreosulfanasapre-irradiatingchemotherapyregimen beforestandard RTin newlydiagnosedGBM patients [33].The doses used (days1 and 8: 3500mg/m² treosulfan, 1000mg/m² Gempercycle,intravenousadministration)hadbeenestablished basedonapreviousPhaseItrialinvarioussolidtumors(notGBM), whichshowed beneficialpalliativeeffectsand minimaltoxicity owingtothechemotherapycombination[34].However,inGBM patientsthisregimenproducedsomedeepvenousthrombosisand hematological toxicities,and no survival increase wasreported comparedtoRTalone[33].

Despite thepromising invitroresults,theseclinicaltrials un- equivocallyshowedthatpretreatmentwithGemfollowedbyRT weeks after the completion of the chemotherapy was not an efficient strategy for the management of GBM. Several factors canexplainthislackofefficacy.Forexample,thecytotoxicaction

Rationale for the use of gemcitabine against GBM

Challenges

Plasma half-life Combination therapy

Local administration in the brain Active targeting

Use of gem derivatives Use of nanocarriers Dose-related toxicity

Chemoresistance BBB penetration Efficacy

Drug at target site

Strategies

Potent chemotherapeutic agent

Radiation sensitizer agent at low concentration

Mechanism of action different from TMZ and MGMT-indepentent Toxin activity mediated by gap junctions (bystander effect) Passes blood-tumor barrier in GBM patients

Good for combination therapy (RT and other chemotherapeutic agents) Immunomodulatory activity

(a)

(b)

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FIGURE2

Listoftheadvantages(a)andchallenges(b)relatedtotheuseofgemcitabine(Gem)forthetreatmentofglioblastoma(GBM)andsomestrategiesthathave beendevelopedtoovercomethem.

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ofGemisS-phase-dependent.Indeed,itislikelythatGemoptimal cytotoxic activity is achieved in rapidly growing tumors with concomitantRT(e.g.,recurrenttumorsdevelopingaftersurgery) instead of established GBM tumors with a largepopulation of nonproliferatingcellsandweeksbeforeRT[35,36].Moreover,Gem ishydrophilic,meaning thatitspenetrationthroughthe BBBis

low[37–39].Itsconcentrationinthe brainismaximal2hafter intravenousadministrationandthenrapidlydecreasesinhealthy animals[37].Intumor-bearingmodelsaswellasinGBMpatients theBBBispartiallydisruptedandGemuptakecanincreasereach- ing concentrations high enough to enable radiosensitization [39,40]. Because radiosensitization depends on the length of TABLE1

Preclinicalstudiesandclinicaltrialsusinggemcitabineastherapeuticstrategyforglioblastomatreatment

Therapeuticstrategy Characteristicsofthestudy Mainresults Refs

Preclinicalstudies

Gem Cytotoxicitystudies(LN-18,U138MG,U87MG, LN-428,D247MG,T98G,LN-319,LN-229,A172, U251MG,U373MG,LN-308cells)

GBMcellssensitivetocytotoxicand anticlonogenicactionofGem

[30]

C GemRT Cytotoxicityandradiosensitivitystudies(U251, D54GBMcells)

ProgressionofcellsinS-phaseafterGem treatmentinfluencesradiosensitization

[23]

C GemRT CytotoxicityandradiosensitivitystudiesonGli-6 monolayersandspheroids

Effectdependsonthestateofthecellsandthe sizeofthespheroids

[35]

Gem Cytotoxicityinvitroandefficacyinvivo(C6 model)

Reductionintumorvolume,alterationofcell- cycleprogressionandincreaseofapoptosis

[22]

C Gem+RT CytotoxicandradiosensitivityonGBMspheroids (GaMG,U87MG)andpatient-derivedOMS

Goodefficacyincell-linespheroids, heterogeneousresponseinOMS

[36]

Gem Gemmechanismofactioninvitro(U87MG,SKI-1 cells)

Gemtoxicitycandiffusethroughgapjunctions inducingbystandereffect

[15]

N Gem-PBCA-NP Cytotoxicityinvitroandefficacyinvivo(C6 model)

Inhibitcellgrowthinvitro,reducetumorgrowth invivo

[53]

L Gem SafetyandefficacyofCEDdelivery(9Lmodel) Increasedanimalsurvival [51]

T PBR–Gemconjugate Efficacyofactivetargeting(pharmacokinetics, distribution)

IncreasedtumortargetselectivityagainstPBR [50]

C Gem+RT EfficacyinproneuralPDGFGBMsubtypemouse

model

Reductionoftumorburdenandincreased survival

[46]

C Gem+DCK+RT Cytotoxicandradiosensitivitystudiesinvitro andinvivo(GI261,U373,C6)

Increasedefficacywiththetriplecombination [52]

C,N PEG-lipo-CD133-GemBV EfficacyinGBMstemcellsinvitroandscGBM modelinvivo

Enhancedantitumoreffect,increasedsurvival, synergybetweenthedrugs

[54,55]

N,T IONP-Gem-CTX Stability,cellularuptake(SF-763,U-118MG cells),activetargeting

Goodcellularuptake,prolongedblood circulation,abilitytocrossBBB

[56]

C,L,D,N PEG-SQ-Gem-NPRT Cytotoxicityinvitroandefficacyinvivo(GR2 model)

Gooddistributioninthebrainandincreased animalssurvival

[57]

L,D,N GemC12-LNChydrogel Cytotoxicityinvitroandefficacyinvivo(U87 model)

Hydrogelwelltoleratedinmousebrain, effectiveinreducingtumorsizeordelay recurrenceformation

[62,63]

Clinicalstudies

Gem,followedbyRT PhaseIIonNDGBM Safebutnoteffective [31]

Gem,followedbyRT PhaseIIonRGBM Safebutnoteffective [32]

C Gem+treosulfan, followedbyRT

PhaseIIonNDGBM Noteffective,somehematologicaltoxicitiesand thrombosis

[33]

Surgery+Gem Phase0 GemanditsmetabolitedFdUcanpassblood–

tumorbarrierandbetakenupbytumorcellsin GBMpatientsreachingradiosensitizing concentrations

[40]

C Gem+RT,followedbyTMZ PhaseIandIIonNDGBM Safeandeffectiveregimen [43,44]

C Gem+RT PhaseIonNDHGG Safeandpromisingregimen,especiallyforpoor

prognosispatientsubgroups

[45]

Abbreviations:C,combinationtherapy(e.g.,withRT,chemotherapeuticagents,etc.);L,localadministration;D,useofGemderivatives;N,useofnanocarriers;GBM,glioblastoma;Gem, gemcitabine;OMS,organotypicmulticellularspheroids;PBR,peripheralbenzodiazepinereceptor;PBCA,polybutylcyanoacrylate;NP,nanoparticle;PEG,polyethyleneglycol;lipo, liposomes;BV,bevacizumab;IONP,ironoxidenanoparticle;CTX,chlorotoxin;SQ-Gem,squalenoyl-gemcitabine;GemC12-LNC,4-lauroyl-gemcitabinelipidnanocapsules;ND,newly diagnosedpatients;R,recurrentpatients;HGG,high-gradegliomapatients.

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intervalbetweendrugtreatmentandradiation[41],choosingthe correcttimingbetweenGemadministrationandRTiscrucial.

Combinationofgemcitabineandconcomitantradiation therapy

OnewaytoexploitGempotentialinthetreatmentofGBMwasto studytheradiosensitizingactivityofGeminGBM(invitroandin vivo)tooptimizethecombinationregimenbetweenGemandRT (Table 1). Using GBM cell lines, it has been shown that Gem radiosensitization is highly dependent on the cell line and its cell-cycle progression after Gem+RT treatment [23]. Ostruzka and Shewachreportedthat U251cells(presenting mutantp53) accumulatein S-phaseafterincubation withGem and ionizing radiationand S-phase-specificcelldeathwasinduced.Withthe sametreatmentconditions,D54cells(presentingwild-typep53) showedG1 block withfewercells inS-phaseand absence ofS- phase-specificcelldeathinduction[23].Anotherstudy,performed byGencetal.onGli-6cells,showedthatGemcaninduceradio- sensitizationinexponentiallygrowingcellsand smallspheroids (Ø 250–400

m

spheroids(Ø400–500

m

in cell-cycle distribution, cell–cell contact, nutrient and drug diffusionormetabolisminthedifferentconditions[35].Fehlauer etal.evaluatedthecytotoxicandradiosensitizingeffectofGemin vitroonGaMGandU87MGspheroidsandonorganotypicmulti- cellularspheroids(OMS)derivedfromGBMpatients [36].Their resultsshowedmigrationinhibitionandproliferationinhibition inthe twoestablishedcelllinesfollowingcombinationtherapy (Gem+RT).TheresponseinOMSwasmoreheterogeneous,with no obvious changes in volume or histological damage but a decreaseinproliferatingcellsandalterationsofproteinexpression levels(MIB-1,p21andp53).Carpinellietal.testedGemactivityin C6ratmalignantgliomastoevaluateitseffectsoncell-cyclephase distribution,apoptosisanditsefficacyinvivo[22].They showed thatGeminducesaccumulationinS-phaseandapoptosisinC6 cellline.Moreover,asignificantreductionintumorvolumewas observedinratsafterintraperitonealGemadministration,aswell asperturbationincell-cycleprogressionandincreaseinapoptosis.

Thesestudiesconfirmed the potentialinterestofusing Gemin combinationwithradiotherapyforthetreatmentofGBM.

Gemradiosensitizationinvitrocanbeachievedeitherbylong exposure to low drug concentrations or brief treatment with higherbutclinically relevant concentrations(radiosensitization isdetectable4haftertreatmentandcanlastfor2days),therefore definingtheGemadministrationscheduleisessentialforitseffect [41,42]. Maraveyaset al. [42] thought that in humans a twice- weeklydosingoraslowerrateofinfusionwouldbepreferableas theradiosensitizationstrategyandtheyevaluatedthemaximum tolerateddosefortheconcomitantuseofGemandRTincarcino- ma patients with brain metastasis in a clinical Phase I study.

Subsequently,aPhaseIstudywasdesignedbyFabietal.totest GemwithconcomitantRTinnewlydiagnosedGBMpatients[43].

Asa differencecomparedwiththe previously describedclinical studies,patientswereenrolledinthestudywithina40-dayperiod aftersurgery.From24to72hbeforethefirstRTsessionandthen onceweekly,patientsreceivedGemintravenouslyatafixed-dose rateof10mg/m²/min.RT(involvedfieldirradiation,2.0Gys)was givendaily,5daysperweekover6weeks.Theaimofthestudywas

to identify the dose-limiting toxicity and maximum tolerated dose, which werefound at 175mg/m²/weekly. Subsequently, a PhaseIIstudywasconductedtoevaluatetheactivityofGemasa radiosensitizer fornewlydiagnosedGBM[44].Patientsreceived standard cranialirradiationandconcomitantfixed-doserate in- travenous Gem(175mg/m²weeklyfor6 weeks).Irrespectiveof tumorresponse,nolaterthan6weeksafterchemo-RT, patients weretreatedwithTMZ.BecauseGemhasadifferentmechanism- of-actioncomparedwithTMZ(Fig.1),itcanbeusefulforpatients withunmethylatedMGMTstatuswhoareexpectedtorespondina lesser extentto alkylatingagents.This studyshowed that Gem administeredconcurrentlywithRTissafeandclinicallyactiveasa radiosensitizerinGBMpatients,anditseffectisachievedirrespec- tiveofthemethylationstatusoftheMGMTpromoter.

Kimetal.recentlypublishedthelong-termresultsofaPhaseI dose-escalation study on Gem+RT for newly diagnosed high- grade glioma(grade3or 4 supratentorialglioma) patients[45].

The maximumtolerated dose was750mg/m²/weekduring the final4weeksofradiation.Thisregimenwaswelltoleratedandthe survivalresultswerepromisingforfurtherstudies,particularlyon poorprognosispatientsubgroups.TheefficacyofGemincombi- nationwithRTwasalsoreportedinapreclinicalstudyperformed byGalba`netal.,showingreductionoftumorburdenandincreased survival after treatment in a proneural platelet-derived growth factor(PDGF)GBMsubtypemousemodel[46].

Alternative deliverystrategiesforgemcitabine

Gemisastrongchemotherapeuticandradiosensitizingagentbut presentssomedrawbacks.First,ithasashort-termplasmahalf-life owingtoextensivedegradationbycytidinedeaminaseintheliver [13].Second,sideeffectscanbeobservedowingtohighdrugdoses, frequent administration schedules or combination with other drugs(e.g.,myelosuppression,thrombocytopenia,edema,cutane- oustoxicity)[13,47].Moreover,resistancetoGemcanoccurandis mainlyattributedtoalteredexpressionofnucleosidetransporters, kinases,enzymesresponsibleforDNApolymerizationandrepair orisimpliedinGeminactivationandaberrantexpressionofgenes associatedwithcellularsurvivalandapoptosis[18].Also,limited GempenetrationthroughsolidtumorssuchasGBMcanresultin reducedefficacyandincreasedresistance[48,49].ToincreaseGem deliverytothetargetsiteandenhancethechemotherapeuticand/

or radiosensitizing properties ofGem against GBM,researchers havestudieddifferentdeliverystrategiesincludingactivetargeting to pass the BBB or target GBM cells, local delivery of Gem, encapsulation of Gem or its derivatives in nanomedicines or combinationalstrategies(Fig.2b,Table1).

Guoetal.,whodevelopedaperipheralbenzodiazepinereceptor (PBR)ligand–GemconjugatesystemtotargetselectivelythePBR receptors(overexpressedinbraintumors),werethefirsttochange the deliverystrategytoincreaseGemefficacyinGBM[50].The tumortargetselectivitywassignificantlyincreased afterintrave- nous administration of PBR–Gem conjugate in an orthotopic SF188/VEGF⁺ model in rats compared to nativeGem. This ap- proachwouldenableanincreasedconcentrationatthetargetsite, enhancingdrugefficacyandreducingsideeffects.

Bycontrast,Diegenetal.deliveredGemdirectlyinthecentral nervous system (CNS) by convection-enhanced delivery (CED) in rats [51].CEDefficiently distributesinfusate throughoutthe

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interstitial spaces ofneural parenchymaby bulk-flow, enabling drugdeliveryacrosstheBBB.Intheaggressive9Lgliomamodel, the CED delivery of Gem showed reduction in tumor volume comparedwithintraperitonealadministrationofGemandlong- termsurvivalofsomeanimals.Aratherdifferentstrategywasused bySzatma´rietal.,whousedagene-therapyapproachtoincrease thetoxicandradiosensitizingeffectofGemindifferentinvitroand invivogliomamodels[52].Theyintroducedanadenovirusvector encodingforthe humanDCKgeneintogliomacellsandtrans- plantedtheminrodentbrains.Theirresultsshowthatthecombi- nation of DCK overexpression, Gem treatment and irradiation significantly increased the toxic and radiosensitizing effects of Gem invitro,aswell asanimal survivalinvivo(evenifthis was toadifferentextentforthetwomodels).Wangetal.werethefirst onestousenanoparticles(NPs)forthedeliveryofGeminGBM [53].Polybutylcyanoacrylate(PBCA)NPswereloadedwithGem, coatedwithpolysorbate-80toincreasetheirabilitytopasstheBBB andtestedinC6gliomacells.TheresultsshowedthatGem-PBCA- NPs can effectively inhibit the growth of C6 cells in vitro and enhanceantitumoractivityonbraintumorsinvivoafterintrave- nousadministration.Shinetal.encapsulatedGeminPEGylated liposomesconjugatedtoanti-CD133monoclonalantibodyasan attempt to increase drug penetration into the tumor, reduce systemic toxicity and target GBM stem cells overexpressing CD133surfacemarkertoincreaseitstherapeuticefficacy.Intheir firststudy,theauthorsdemonstratedthatPEG-lipo-CD133-Gem wasstable,withalongcirculatingtimeinvivoafterintravenous administration and they wereableto reduce toxicity and exert

significantantitumorefficacyinasubcutaneoustumormodel[54].

Inasecondstudy,asynergisticeffectwasobservedbetweenPEG- lipo-CD133-Gem and the antiangiogenic drug bevacizumab, achieving good antitumoral response reducing the drug doses andthesideeffects[55].

ToincreaseGemcirculationtimeandovercometheBBB,Mu etal.developedaGem-loadedironoxideNPandconjugateditvia hyaluronicacidtochlorotoxin(CTX)–apeptideabletocrossthe BBB and target braintumor cells[56].The IONP-HA-Gem-CTX formulationshowedcellularuptakeinSF-763andU-118MGcells, prolongedbloodcirculationand the abilityto crossthe BBBin healthymiceafterintravenousadministration.Gaudinetal.bio- conjugatedGemwithliquidsqualeneproducingsqualenoyl-gem- citabine(SQ-Gem),aprodrugthatspontaneouslyformsNPs[57].

Squalenoylation of nucleoside analogs has been extensively reported in the literature as a way to protect the drug from degradation,bypassresistancemechanismsandimproveantican- ceractivity[58].InGBM,theCEDadministrationofPEGylatedSQ- Gemallowedwidespreaddistributioninthebrainandincreased animalsurvivalinratsbearingintracranialRG2tumorscompared withfreeGemwhenusedaloneorincombinationwithRT[57].

Recently,auniquenanomedicinehydrogelplatformformedof N-4-lauroyl-gemcitabine(GemC12)andlipidnanocapsules(LNCs) wasdevelopedbyMoysanetal.[59].GemC12(Fig.3a)isalipo- philicderivativeofGemthatshowsimprovedstabilityinplasma owingtotheprotectionoftheaminegroupbyanamidelinkage stable at pH 4–9, and improved cytotoxicity in different cell lines [59]. LNCs are biocompatible nanocarriers obtained by a

(a)

(b)

4-N-lauroyl gemcitabine (GemC₁₂)

4-N-lauroyl gemcitabine lipid nanocapsule hydrogel (GemC₁₂-LNC)

100

50

0 0

Drug GemC₁₂

Non ionic surfactant (Span80^®) PEG (Kolliphor HS15^®) Triglycerides (Labrafac^®)

Water phase immobilized in get structure

15 Percent survival Resection and treatment administration

Time (days)

30 45 60

No treatment Unloaded LNC GemHCL GemC₁₂ GemC₁₂-LNC

75 90

O C CH₂

OH NH N

N

F H H H

F O O

HOCHO 2

CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ CH₃

Drug Discovery Today

FIGURE3

4-N-Lauroyl-gemcitabine(GemC12)chemicalformula(a)andschematicrepresentationofGemC12–lipidnanocapsule(GemC12–LNC)nanomedicinehydrogel(b).

GemC12islocatedattheoil–waterinterfaceoftheLNCandtheGemmoietiesorientedoutsidetheLNCcanformH-bondcross-linkagesresultingina3Dpearl necklacethatcanimmobilizethewaterphaseformingahydrogel.Imageadapted,withpermission,fromRef.[62].

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phase-inversionprocess,formedofanoilycore(Labrafac^TMWL 1349;medium-chaintryglicerides)surroundedbyahighly orga- nizedmembraneoflowmolecularweightsurfactants(Span¹80 and Kolliphor¹ HS15) [60]. When GemC12 is encapsulated in LNCs,theformulationspontaneouslyformsahydrogelwithout theadditionofpolymers,gellingagentsorexternalstimuli.The gelationtimeandgelmechanicalpropertiesstronglydependon GemC12andLNCconcentration.Thegelisformedasaresultofan inter-NPassociationofGemC12–LNCinwhichGemC12behavesas anamphiphilicmoleculelocatingitselfattheLabrafac^TM–water interfaceoftheLNC,andtheGemmoietiesoutsidetheLNCsform H-bondcross-linkagesentrappingthewaterphaseandforminga gel(Fig.3b).GemC₁₂–LNCs cantarget the monocyticmyeloid- derived suppressor cells in lymphoma and melanoma-bearing miceandhumanbloodsamplesfromhealthydonorsandmela- nomapatients[61].Recently,ourgrouptestedGemC₁₂–LNCson GBMcelllinesinvitroshowingincreasedcytotoxicactivitycom- paredwiththeparenthydrophilicdrugGem.TheGemC12–LNC hydrogelwaswelltoleratedinmousebrainintheshort-,mid-and long-term(1week,2and6months)andshowedgoodantitumor efficacyinasubcutaneousandinanorthotopicGBMtumormodel after intratumoral administration, as well as in an orthotopic tumormodelfollowingresectionandlocaldeliveryinthetumor resectioncavity[62,63].Theuseofananomedicinehydrogelin the treatment of GBM could combine the advantages of local delivery (bypassingthe BBB, highlocal concentration and low systemictoxicity,increasedtherapeuticbenefitatthetumorsite), hydrogels(adaptationto the resectioncavity,injectability)and nanomedicines(sustaineddrugrelease,combinationofmultiple anticancerdrugs),resultinginincreasedantitumorefficacy.

Concluding remarks

Toconclude,Gemisadrugusedintheclinicforthemanagement of different solid tumors, alone or in combination with other chemotherapeutic agents. Its cytotoxic activity, mediated by MGMT-independentmechanisms,combinedwiththeradiosensi- tizing properties and its abilityto pass the GBM blood–tumor barrierallmakeitaveryinterestingmoleculeforGBMtreatment (Fig.2,Table1).Severalchallengesneedtobeovercometoincrease GemefficacyagainstGBM,suchasincreasingplasmahalf-life,BBB penetration,drugconcentrationattargetsiteandreducingdose- related toxicity and chemoresistance. To achieve these goals, several strategies have already been tested (e.g., local delivery, useofGemderivatives,useofnanocarriers),whereasotherswill surelybeexperiencedinthefuture.Inthissense,webelievethat notenoughattentionhasyetbeenpaidtoGemimmunomodulat- ing capacities and its synergisticactivity with othermolecules.

Indeed,combiningdifferentdeliverystrategiesandattackingthe GBMmicroenvironmentfromdifferentsidescouldreallymakea difference in the management of these highly malignant and incurablebraintumors.

Conflicts of interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbytheEducation,Audiovisualand CultureExecutiveAgency(EACEA)oftheEuropeanCommission inthefieldoftheErasmusMundusJointProgrammeNanoFar (C.Bastiancich).TheauthorswouldliketothankDrJohnBianco forhislinguisticcontribution.

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