Original Articles

Nanoparticle delivery of an SN38 conjugate is more effective thanirinotecan in a mouse model of neuroblastomaRadhika Iyer a1 Jamie L Croucher a1 Michael Chorny bc1 Jennifer L Mangino aIvan S Alferiev bc Robert J Levy bc Venkatadri Kolla a Garrett M Brodeur aca Division of Oncology The Childrenrsquos Hospital of Philadelphia 3400 Civic Center Blvd Philadelphia PA 19104 USAb Division of Cardiology The Childrenrsquos Hospital of Philadelphia 3400 Civic Center Blvd Philadelphia PA 19104 USAc Department of Pediatrics Perelman School of Medicine Philadelphia PA 19104 USA

A R T I C L E I N F O

Article historyReceived 10 December 2014Received in revised form 3 February 2015Accepted 7 February 2015

KeywordsIrinotecanNanoparticlesNeuroblastomaSN38Tocopherol succinate

A B S T R A C T

Neuroblastoma (NB) is the most common and deadly solid tumor in children The majority of NB pa-tients have advanced stage disease with poor prognosis so more effective less toxic therapy is neededWe developed a novel nanocarrier-based strategy for tumor-targeted delivery of a prodrug of SN38 theactive metabolite of irinotecan We formulated ultrasmall-sized (lt100 nm) biodegradable poly(lactide)-poly(ethylene glycol) based nanoparticles (NPs) containing SN38 conjugated to tocopherol succinate (SN38-TS) Alternative dosing schedules of SN38-TS NPs were compared to irinotecan Comparison of SN38-TSNPs (2 doses) with irinotecan (20 doses) showed equivalent efficacy but no cures Comparison of SN38-TS NPs (8 8 and 16 doses respectively) to irinotecan (40 doses) showed that all SN38-TS NP regimenswere far superior to irinotecan and ldquocuresrdquo were obtained in all NP arms SN38-TS NP delivery resultedin 200times the amount of SN38 in NB tumors at 4 hr post-treatment compared to SN38 detected for theirinotecan arm no toxicity was seen with NPs We conclude that this SN38-TS NP formulation im-proved delivery retention and efficacy without causing systemic toxicity

copy 2015 Elsevier Ireland Ltd All rights reserved

Introduction

Neuroblastoma (NB) is the most common and deadly solid tumorof childhood NB a tumor of the sympathetic nervous system ac-counts for 8ndash10 of all childhood cancers and 15 of deaths fromcancer in children [1] Some infants experience spontaneous re-gression whereas other patients have maturation of their tumor intobenign ganglioneuromas Unfortunately the majority of patients havemetastatic disease and many progress relentlessly despite inten-sive multimodality treatment So despite dramatic improvementsin the cure rate for other pediatric neoplasms the survival rate forpatients with NB has lagged behind Recent advances in under-standing the molecular pathogenesis of NB have providedconsiderable insight into the genetic and biochemical mecha-nisms underlying these seemingly disparate behaviors [23] Thesein turn have identified the genes proteins and pathways that shouldbe effective targets for biologically based therapy [4]

The development of targeted agents for NBs and other tumorsis being actively pursued and they hold great promise for futuretreatment strategies We are also investigating a targeted deliveryapproach for chemotherapeutic and biological agents that are suit-able for encapsulation in nanoparticles (NPs) Nanoparticles canpreferentially deliver drugs to tumors by taking advantage of theenhanced permeability and retention (EPR) effect [5] An addition-al advantage of using NP formulations is in providing a biocompatiblevehicle for water-insoluble therapeutics and stabilizing labile mol-ecules thus enabling delivery of drugs otherwise unsuitable fortherapeutic use This approach permits enhanced drug delivery totumor tissue while simultaneously reducing systemic exposure andtoxicity There are a variety of different types of NPs [6] but we haveformulated poly(lactide)-poly(ethylene glycol) (PLA-PEG)-based poly-meric NPs containing a conjugate of SN38 the active metabolite ofirinotecan SN38 is 1000 times more active than irinotecan but ithas toxicity and solubility issues that make it unsuitable for patientadministration However NP encapsulation overcomes these prob-lems and allows for safe IV administration

We have compared the efficacy and toxicity of conventionallyadministered irinotecan to NP delivery of SN38 formulated as aprodrug by conjugation to tocopherol succinate in a mouse xeno-graft model of NB This approach allows for greater drug retentionin circulating NPs This in turn results in dramatically increasedtumor drug delivery and retention as well as greater antitumor ef-ficacy at reduced doses in this xenograft model

Abbreviations NPs nanoparticles NB neuroblastoma PLA-PEG poly(lactide)-poly(ethylene glycol) SN38-TS SN-38 tocopherol succinate SRB sulforhodamineB

Corresponding author Tel +1 215 590 2817 fax +1 215 590 3770E-mail address Brodeuremailchopedu (GM Brodeur)

1 These authors contributed equally

httpdxdoiorg101016jcanlet2015020110304-3835copy 2015 Elsevier Ireland Ltd All rights reserved

Cancer Letters 360 (2015) 205ndash212

Contents lists available at ScienceDirect

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journal homepage wwwelseviercom locate canlet

Materials and methods

Compounds

SN-38 was developed and evaluated as a chemotherapeutic agent but it wasboth toxic and not water soluble so irinotecan was later introduced as its water-soluble prodrug Irinotecan (Camptosar Pfizer) is a commercially available orallybioavailable topoisomerase I inhibitor that is used clinically for a variety of tumortypes It is converted by carboxylesterase converting enzyme into SN38 its activemetabolite Irinotecan (20 mgml) was diluted in 09 normal saline and adminis-tered by oral gavage once daily at 10 mgkg Monday through Friday for either 4weeks or 8 weeks Saline was used as the control The SN38-Tocopherol Succinate(SN38-TS) NPs were administered at an effective dose of 10 mgkg SN38 The SN38-TS NPs were given via tail vein injection either once every other week once per weekor twice per week (MondayWednesday) for 4 or 8 weeks Irinotecan was ob-tained from the pharmacy at The Childrenrsquos Hospital of Philadelphia The doses usedin this study were based on prior published studies [7] Human brain-derived neu-rotrophic factor (BDNF PeproTech Rocky Hill NJ) was reconstituted in distilled waterat 2 μgml For long-term storage in minus20 degC the reconstituted BDNF was further dilutedto 1 μgml and used at a final concentration of 100 ngml

Nanoparticle formulation

The SN38-TS conjugate was synthesized from SN-38 (AK Scientific Union CityCA) and D-α-tocopherol hemisuccinate (Sigma-Aldrich St Louis MO USA) by directcoupling in the presence of N-(3-dimethylaminopropyl)-Nprime-ethylcarbodiimide hy-drochloride with 4-dimethylaminopyridinium 4-toluenesulfonate as a catalyst Thestructure and purity were confirmed by 1H NMR SN38-TS loaded NPs were formu-lated by nanoprecipitation using poly(DL-lactide)-block-poly(ethylene glycol)(Advanced Polymer Materials Dorval Canada) and Pluronic F-68 (Sigma-Aldrich StLouis MO) as the particle-forming polymer and stabilizer respectively The parti-cle size was analyzed by dynamic light scattering and the drug loading wasdetermined by UVndashVis spectrophotometry after SN38-TS extraction in sec-butanolFor more information about the formulation optimization release kinetics and otherinformation related to these nanoparticles please see the recently accepted manu-script by Alferiev et al in Biomaterials [8]

Cell lines

Trk-null SH-SY5Y cells were stably transfected with TrkB (SY5Y-TrkB) and thesecells were used for all in vitro and in vivo studies The cells were grown in RPMI-1640 containing 10 fetal bovine serum and 03 mgml G418 Cells were maintainedin culture flasks at 37 degC in a humidified atmosphere of 95 air and 5 carbon dioxideCells were harvested using 02 tetrasodium EDTA in PBS

In vitro experiments

Sulforhodamine B (SRB) assays were performed to determine the effect of irinotecanand SN38-TS NPs on the survival and growth of the TrkB-expressing neuroblastomacells 5 times 103 cells per well were plated in 96 well plates and exposed to the drug atdifferent concentrations (1 nM 3 nM 5 nM 10 nM) for one hour followed by addi-tion of 100 ngml of BDNF Plates were harvested at 24 48 and 72 hours followingaddition of drug The plates were processed via standard SRB assay protocol All invitro experiments were performed in triplicate and repeated at least 3 times

Animals

Six-week-old athymic nunu mice were obtained from Jackson Laboratories Micewere maintained at five per cage under humidity- and temperature-controlled con-ditions in a lightdark cycle that was set at 12-hour intervals The Institutional AnimalCare Committee of the Joseph Stokes Jr Research Institute at CHOP approved theanimal studies described herein

In vivo experiments

For the xenograft studies animals were injected subcutaneously in the flank with1 times 107 SY5Y-TrkB cells in 01 ml of Matrigel (BD Bioscience Palo Alto CA) Tumorswere measured 2 times per week in 3 dimensions and the volume calculated asfollows [(0523 times L times W times W)1000] For the image analysis studies SN38-TS NPslabeled with the red fluorescent dye BODIPY650665 [8] were injected via tail vein whenthe average tumor size reached 1 cm3 Animals were imaged using the IVIS Spec-trum Pre-clinical In Vivo Imaging system at exem wavelengths of 640700 nm at4 24 48 144 and 192 hours post injection Dorsal side and supine images weretaken for each animal Fluorescence counts were normalized to a mouse not in-jected with any dye to correct for auto fluorescence from mouse tissues

For the first series of tumor inhibition studies animals were treated with thecompounds for 4 weeks Irinotecan was given as an oral gavage at 10 mgkg QD 5timesweek SN38-TS NPs were injected via tail vein either 1times2 weeks 1timesweek or 2timesweek The control group was injected with blank NPs 2timesweek For the next set ofstudies animals were treated for 8 weeks (with the exception of one group for 4

weeks) Irinotecan was given orally at 10 mgkg QD 5timesweek The control group re-ceived oral doses of saline SN38-TS NPs were injected via tail vein either 1timesweekor 2timesweek A fifth group was included with a treatment regimen of 2timesweek for 4weeks to validate the findings of the previous study and allow for study compari-son We used PO dosing of irinotecan as this is the route used clinically and thereare published data that the PO route has similar efficacy and pharmacokinetics asIV dosing [79]

Body weights were obtained once a week and the dose of compound was ad-justed accordingly Blood counts were checked regularly Mice were sacrificed whentumor volume reached 3 cm3 Retro-orbital and terminal bleeds were obtained forblood counts and pharmacokinetic studies Animals were given a single dose ofirinotecan or SN38-TS NPs and tumor spleen and liver were harvested post-sacrifice (at 4 24 and 72 hours) after heart perfusion with cold saline (performedto minimize organ blood content for drug concentration analysis)

Pharmacokineticspharmacodynamics analysis of mouse tissues

Tissues were homogenized using a Biologics Inc Model 3000 ultrasonic ho-mogenizer We added 2080 methanolwater with 1 formic acid to a known weightof tissue to obtain a ratio of 4 mlg sample Samples were homogenized on ice andfrozen until analysis SN38 pro-drug spiked mouse plasma and tissue homogenatewere hydrolyzed using sodium hydroxide (1 M 15 μl) and incubated for 15 minutesat 37 degC in a Thermo electron incubator The reaction was stopped by adding 98formic acid (10 μl) Analysis confirmed complete hydrolysis of SN38 pro-drug underthese conditions Standards were prepared in CD-1 mouse plasma containing sodiumheparin as an anticoagulant A nine-point calibration curve was prepared at differ-ent concentrations by spiking a working stock Plasma and tissue homogenate sampleswere extracted via acetonitrile precipitation in a 96-well format Electrospray ion-ization in the positive ion mode was utilized for the tandem mass spectrometricdetection of SN38 (mz 3932 3490) and irinotecan (mz 5873 1239) using AB Sciex4000 mass spectrometer Separation was accomplished utilizing Kinetex PFP(50 times 41 mm id 26 μm) column with Shimadzu LC 20AD HPLC system with a runtime of 45 min Assay was linear over the range of 1 ngml to 1000 ngml for bothSN38 and irinotecan in mouse plasma The matrix factors of the mouse tissues (tumorkidney spleen and liver) obtained using tissue homogenates spiked with 100 ng ofSN38 pro-drug per ml (n = 3) against mouse plasma calibration curves were appliedin the drug assay calculations

Statistical analysis

A linear mixed effects model was used to test the difference in the rate oftumor volume change over time between groups The model included groupday and group-by-day interaction as fixed effects and included a random inter-cept and a random slope for each mouse Separate models and tests were constructedfor the on-treatment period and off-treatment period Event-free survival (EFS)curves were estimated using the KaplanndashMeier method and compared using a log-rank test

Results

Effect of irinotecan and SN38-TS NPs on growth of cells in vitro

An SRB assay was performed to assess the growth inhibitionand toxicity of irinotecan and SN38 on cell growth A range ofirinotecan concentrations (1 3 5 10 nM and 3 μM) was used Therewas no apparent inhibition of cell growth observed at concentra-tions from 1 to 10 nM At 3 μM of Irinotecan the cell survivalpattern looked similar to that of control cells grown without ligand(Fig 1) SN38-TS NPs showed an increased inhibition of growth withan increase in NP drug concentration To rule out the role of TS incell growth inhibition SRB assays were performed with SN38 (freedrug) alone TS alone and the combination of SN38 (free drug)and TS at the same concentrations as the NP formulations No sig-nificant effect was seen on cell growth with TS alone at anyconcentration whereas SN38 alone exhibited complete inhibitionof cell growth at all of the concentrations studied with or withoutTS

Biodistribution of SN38-TS NPs in mice

SN38-TS NPs labeled with a red fluorophore BODIPY630650 wereinjected into mice via tail veins The maximum amount of fluores-cence in tumors was seen at 4 hours with a steady decline influorescent intensity in NB tumors at later time points (Fig 2A) Side

206 R Iyer et alCancer Letters 360 (2015) 205ndash212

and supine views of the mice revealed that there was also accu-mulation of NPs on the dorsal side of the body due to concentrationof particles in the cervical and brachial lymph nodes as well as inthe liver Similar high fluorescence is seen in the inguinal and lumbarlymph nodes as seen in the supine view (Fig 2B)

Effect of irinotecan and SN38-TS NPs on in vivo xenografts

The ability of irinotecan and SN38-TS NPs to inhibit the growthof SY5Y-TrkB cells in vivo was tested using xenograft models Forthe first study treatment was carried out for 4 weeks All the animalsin the control group were removed once they reached 1 cm3 for asubsequent study described below Tumor growth curves formice treated with irinotecan 5times per week for four weeks and mice

treated with the SN38-TS NPs 1times every other week for four weeksoverlapped demonstrating equivalent efficacy in tumor inhibitiondespite a 10-fold dose reduction with SN38-TS NPs The group treatedwith SN38-TS NPs 2timesweek maintained stable tumor growth controlup to 66 days post-cessation of treatment and had the greatest in-hibition of tumor growth when compared to the other treatmentgroups (Fig 3A) Mice treated with NPs 2timesweek exhibited a sig-nificant survival advantage compared to those with a 1timesweektreatment regimen with a 100 survival through day 130 (100 dayspost-cessation of treatment) (Fig 3B)

There was no significant difference between the group treatedwith irinotecan 5timeswk versus those treated with SN38-TS NPs 1times2weeks (Log-rank test p = 03105) However there was a significantdifference for SN38-TS NP 1timesweek versus irinotecan 5timesweek(p = 00014) for SN38-TS NP 1times2 weeks versus SN38-TS NP 1timesweek (p = 00034) for SN38-TS NP 2timesweek versus irinotecan 5timesweek (p lt 00001) for SN38-TS NP 2timesweek versus SN38-TS NP 1times2weeks (p lt 00001) and for SN38-TS NP 2timesweek versus SN38-TSNP 1timesweek (p lt 00001)

Because our SN38-TS NPs exhibited promising control over tumorgrowth we next investigated whether NP treatment for 8 weekswould provide a more protracted survival advantage Mice treatedwith oral irinotecan displayed inhibited tumor growth and de-creased tumor size over the course of treatment However theirtumors regrew within 4 weeks of treatment cessation with somemice reaching a tumor volume of over 3 cm3 by day 37 post-treatment All of the mice treated with SN38-TS NPs had negligibletumor volumes (below 01 cm3) for at least 60 days following ces-sation of therapy Furthermore the tumor regrowth patterns of theNP-treated mice were significantly slower than untreated oririnotecan-treated mice (Fig 4A) A significant survival advantagewas observed in the groups treated with SN38-TS NPs comparedto the irinotecan group treated 5timesweek8 weeks Mice treatedwith NPs 2timesweek8 weeks exhibited significant survival advan-tage over the 1timesweek8 weeks and 2timesweek4 weeks groups withalmost 100 survival through day 180 (120 days post cessation of

Fig 1 Effect of Irinotecan SN38-TS NP SN38 FD and TS FD on cell growth bySulforhodamine B analysis Cells were exposed to 1 nM 3 nM (data not shown) 5 nM(data not shown) and 10 nM of the respective compounds in the presence of BDNFPlates were harvested at 24 48 and 72 hr post-drug treatment Cell viability wasassayed using SRB dye

Fig 2 Biodistribution of SN38-TS NPs in mice at different time points post IV injection (A) Prone view at different time points after injection (B) Side and supine views revealthat thoracic fluorescence is primarily due to lymphatic and hepatic NP accumulation Animals were imaged using the IVIS Spectrum Pre-clinical In Vivo Imaging system

207R Iyer et alCancer Letters 360 (2015) 205ndash212

treatment Fig 4B) Log-rank tests revealed p lt 00001 for SN38-TSNP 1timesweek8 weeks group versus irinotecan 5timesweek8 weeksp = 00007 for SN38-TS NP 2timesweek4 weeks versus irinotecan 5timesweek8 weeks p = 00753 for SN38-TS NP 2timesweek4 weeks versusSN38-TS NP 1timesweek8 weeks p lt 00001 for SN38-TS NP 2timesweek8 weeks versus irinotecan 5timesweek8 weeks p = 00142 forSN38-TS NP 2timesweek8 weeks versus SN38-TS NP 1timesweek8 weeksand p = 00001 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP2timesweek4 weeks

Effect of SN38-TS NP treatment on large tumors

Because this NP formulation provided significant control overmoderately-sized NB xenografts (average 02 cm3) we wanted to de-termine whether SN38-TS NPs could effectively control larger NB

tumors which mimic more advanced-stage disease NB xeno-grafts were allowed to grow untreated until they reached an averageof 1 cm3 Then tumor-bearing mice were treated intravenously witha total of 16 doses of SN38-TS NPs at 2times per week Over the courseof treatment all tumors regressed to approximately 02 cm3 and re-mained in stable remission for an average of 60 days similar to thesmaller NB tumors (data not shown) Interestingly when thesetumors began to recur (at 60ndash90 days after treatment cessation)their growth was consistently slow and protracted (Fig 5A) Thisfinding led us to investigate the histology of the tumors at thetime of sacrifice Hematoxylin and Eosin staining of the SN38-TSNP-treated tumors harvested at about 18 weeks (~120 days)post last treatment showed dramatic maturation towarda ganglioneuroma phenotype in all of the treated tumorsexamined (Fig 5B) Interestingly these tumors also expressed

Fig 3 Treatment of xenografts with SN38-TS NPs for 4 weeks prolongs tumor regrowth (A) Tumor volume of xenografts after treatment with irinotecan (5timesweek4 weeks10 mgkg) or SN38-TS NP (1times2weeks4 weeks 1timesweek4 weeks 2timesweek4 weeks 10 mgkg) Data are shown as means (B) Survival curves of tumor-bearing animals

indicates last day of treatment Animals were followed for tumor regrowth and survival until the tumors reached 3 cm3 in volume

208 R Iyer et alCancer Letters 360 (2015) 205ndash212

significantly higher levels of neuronal differentiation markers suchas tyrosine hydroxylase consistent with neuronal maturation(Fig 5B)

Pharmacokinetics of NP distribution in mouse tissues

We also performed pharmacokinetic analyses of mice treated withirinotecan or SN38-TS NPs to determine the biodistribution of SN38at different time points post-treatment Blood liver spleen andtumor samples taken from mice at 4 24 and 72 hours after oralirinotecan administration or NP injection were analyzed for drugcontent via LCndashMSMS Average irinotecan and SN38 levels inirinotecan-treated mice were 164 plusmn 47 ngg (SD) and lt10 ngg oftissue respectively at 4 hours post-treatment whereas SN38 levelswere 200-fold higher in NP-treated samples (Table 1) Further-more although mice treated with irinotecan had undetectable levelsof irinotecan and SN38 in tumors at 24 or more hours after treat-

ment (lt10 ngg) NP-treated tumors retained very high levels of SN38at 24 hours post-treatment (1482 plusmn 3546 ngg) This level de-creased at 72 hours (5583 plusmn 1907 ngg) but remained significantlyelevated compared to irinotecan-treated mice at 72 hours post-treatment (Table 1)

Discussion

NBs are characterized by heterogeneous clinical behavior in-cluding spontaneous regression or differentiation into benignganglioneuromas [1] Additionally NB patients under 12ndash18 monthsof age tend to have a better outcome than older patients Unfortu-nately over half of all NBs are older with unresectable or metastaticdisease at the time of diagnosis and are considered high-risk Evenwith very intensive multimodality therapy including chemother-apy radiation therapy stem cell transplantation and immunotherapyover half of these patients do not survive [10] Furthermore we have

Fig 4 Treatment of xenografts with SN38-TS NPs for 8 weeks prolongs tumor regrowth (A) Tumor volume of xenografts after treatment with irinotecan (5timesweek8 weeks10 mgkg) or SN38-TS NP (2timesweek4 weeks 1timesweek8 weeks 2timesweek8 weeks 10 mgkg) Data are shown as means (B) Survival curves of tumor-bearing animals Log-rank tests revealed p lt 00001 for SN38-TS NP 1timesweek8 weeks group versus irinotecan 5timesweek8 weeks p = 00007 for SN38-TS NP 2timesweek4 weeks versus irinotecan5timesweek8 weeks p = 00753 for SN38-TS NP 2timesweek4 weeks versus SN38-TS NP 1timesweek8 weeks p lt 00001 for SN38-TS NP 2timesweek8 weeks versus irinotecan 5timesweek8 weeks p = 00142 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 1timesweek8 weeks p = 00001 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 2timesweek4 weeks indicates last day of treatment Animals were followed for tumor regrowth and survival until the tumors reached 3 cm3 in volume indicates a treatmentperiod of 4 weeks indicates a treatment period of 8 weeks

209R Iyer et alCancer Letters 360 (2015) 205ndash212

treatment Fig 4B) Log-rank tests revealed p lt 00001 for SN38-TSNP 1timesweek8 weeks group versus irinotecan 5timesweek8 weeksp = 00007 for SN38-TS NP 2timesweek4 weeks versus irinotecan 5timesweek8 weeks p = 00753 for SN38-TS NP 2timesweek4 weeks versusSN38-TS NP 1timesweek8 weeks p lt 00001 for SN38-TS NP 2timesweek8 weeks versus irinotecan 5timesweek8 weeks p = 00142 forSN38-TS NP 2timesweek8 weeks versus SN38-TS NP 1timesweek8 weeksand p = 00001 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP2timesweek4 weeks

Effect of SN38-TS NP treatment on large tumors

Because this NP formulation provided significant control overmoderately-sized NB xenografts (average 02 cm3) we wanted to de-termine whether SN38-TS NPs could effectively control larger NB

tumors which mimic more advanced-stage disease NB xeno-grafts were allowed to grow untreated until they reached an averageof 1 cm3 Then tumor-bearing mice were treated intravenously witha total of 16 doses of SN38-TS NPs at 2times per week Over the courseof treatment all tumors regressed to approximately 02 cm3 and re-mained in stable remission for an average of 60 days similar to thesmaller NB tumors (data not shown) Interestingly when thesetumors began to recur (at 60ndash90 days after treatment cessation)their growth was consistently slow and protracted (Fig 5A) Thisfinding led us to investigate the histology of the tumors at thetime of sacrifice Hematoxylin and Eosin staining of the SN38-TSNP-treated tumors harvested at about 18 weeks (~120 days)post last treatment showed dramatic maturation towarda ganglioneuroma phenotype in all of the treated tumorsexamined (Fig 5B) Interestingly these tumors also expressed

Fig 3 Treatment of xenografts with SN38-TS NPs for 4 weeks prolongs tumor regrowth (A) Tumor volume of xenografts after treatment with irinotecan (5timesweek4 weeks10 mgkg) or SN38-TS NP (1times2weeks4 weeks 1timesweek4 weeks 2timesweek4 weeks 10 mgkg) Data are shown as means (B) Survival curves of tumor-bearing animals

indicates last day of treatment Animals were followed for tumor regrowth and survival until the tumors reached 3 cm3 in volume

208 R Iyer et alCancer Letters 360 (2015) 205ndash212

significantly higher levels of neuronal differentiation markers suchas tyrosine hydroxylase consistent with neuronal maturation(Fig 5B)

Pharmacokinetics of NP distribution in mouse tissues

We also performed pharmacokinetic analyses of mice treated withirinotecan or SN38-TS NPs to determine the biodistribution of SN38at different time points post-treatment Blood liver spleen andtumor samples taken from mice at 4 24 and 72 hours after oralirinotecan administration or NP injection were analyzed for drugcontent via LCndashMSMS Average irinotecan and SN38 levels inirinotecan-treated mice were 164 plusmn 47 ngg (SD) and lt10 ngg oftissue respectively at 4 hours post-treatment whereas SN38 levelswere 200-fold higher in NP-treated samples (Table 1) Further-more although mice treated with irinotecan had undetectable levelsof irinotecan and SN38 in tumors at 24 or more hours after treat-

ment (lt10 ngg) NP-treated tumors retained very high levels of SN38at 24 hours post-treatment (1482 plusmn 3546 ngg) This level de-creased at 72 hours (5583 plusmn 1907 ngg) but remained significantlyelevated compared to irinotecan-treated mice at 72 hours post-treatment (Table 1)

Discussion

NBs are characterized by heterogeneous clinical behavior in-cluding spontaneous regression or differentiation into benignganglioneuromas [1] Additionally NB patients under 12ndash18 monthsof age tend to have a better outcome than older patients Unfortu-nately over half of all NBs are older with unresectable or metastaticdisease at the time of diagnosis and are considered high-risk Evenwith very intensive multimodality therapy including chemother-apy radiation therapy stem cell transplantation and immunotherapyover half of these patients do not survive [10] Furthermore we have

Fig 4 Treatment of xenografts with SN38-TS NPs for 8 weeks prolongs tumor regrowth (A) Tumor volume of xenografts after treatment with irinotecan (5timesweek8 weeks10 mgkg) or SN38-TS NP (2timesweek4 weeks 1timesweek8 weeks 2timesweek8 weeks 10 mgkg) Data are shown as means (B) Survival curves of tumor-bearing animals Log-rank tests revealed p lt 00001 for SN38-TS NP 1timesweek8 weeks group versus irinotecan 5timesweek8 weeks p = 00007 for SN38-TS NP 2timesweek4 weeks versus irinotecan5timesweek8 weeks p = 00753 for SN38-TS NP 2timesweek4 weeks versus SN38-TS NP 1timesweek8 weeks p lt 00001 for SN38-TS NP 2timesweek8 weeks versus irinotecan 5timesweek8 weeks p = 00142 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 1timesweek8 weeks p = 00001 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 2timesweek4 weeks indicates last day of treatment Animals were followed for tumor regrowth and survival until the tumors reached 3 cm3 in volume indicates a treatmentperiod of 4 weeks indicates a treatment period of 8 weeks

209R Iyer et alCancer Letters 360 (2015) 205ndash212

significantly higher levels of neuronal differentiation markers suchas tyrosine hydroxylase consistent with neuronal maturation(Fig 5B)

Pharmacokinetics of NP distribution in mouse tissues

We also performed pharmacokinetic analyses of mice treated withirinotecan or SN38-TS NPs to determine the biodistribution of SN38at different time points post-treatment Blood liver spleen andtumor samples taken from mice at 4 24 and 72 hours after oralirinotecan administration or NP injection were analyzed for drugcontent via LCndashMSMS Average irinotecan and SN38 levels inirinotecan-treated mice were 164 plusmn 47 ngg (SD) and lt10 ngg oftissue respectively at 4 hours post-treatment whereas SN38 levelswere 200-fold higher in NP-treated samples (Table 1) Further-more although mice treated with irinotecan had undetectable levelsof irinotecan and SN38 in tumors at 24 or more hours after treat-

ment (lt10 ngg) NP-treated tumors retained very high levels of SN38at 24 hours post-treatment (1482 plusmn 3546 ngg) This level de-creased at 72 hours (5583 plusmn 1907 ngg) but remained significantlyelevated compared to irinotecan-treated mice at 72 hours post-treatment (Table 1)

Discussion

NBs are characterized by heterogeneous clinical behavior in-cluding spontaneous regression or differentiation into benignganglioneuromas [1] Additionally NB patients under 12ndash18 monthsof age tend to have a better outcome than older patients Unfortu-nately over half of all NBs are older with unresectable or metastaticdisease at the time of diagnosis and are considered high-risk Evenwith very intensive multimodality therapy including chemother-apy radiation therapy stem cell transplantation and immunotherapyover half of these patients do not survive [10] Furthermore we have

Fig 4 Treatment of xenografts with SN38-TS NPs for 8 weeks prolongs tumor regrowth (A) Tumor volume of xenografts after treatment with irinotecan (5timesweek8 weeks10 mgkg) or SN38-TS NP (2timesweek4 weeks 1timesweek8 weeks 2timesweek8 weeks 10 mgkg) Data are shown as means (B) Survival curves of tumor-bearing animals Log-rank tests revealed p lt 00001 for SN38-TS NP 1timesweek8 weeks group versus irinotecan 5timesweek8 weeks p = 00007 for SN38-TS NP 2timesweek4 weeks versus irinotecan5timesweek8 weeks p = 00753 for SN38-TS NP 2timesweek4 weeks versus SN38-TS NP 1timesweek8 weeks p lt 00001 for SN38-TS NP 2timesweek8 weeks versus irinotecan 5timesweek8 weeks p = 00142 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 1timesweek8 weeks p = 00001 for SN38-TS NP 2timesweek8 weeks versus SN38-TS NP 2timesweek4 weeks indicates last day of treatment Animals were followed for tumor regrowth and survival until the tumors reached 3 cm3 in volume indicates a treatmentperiod of 4 weeks indicates a treatment period of 8 weeks

209R Iyer et alCancer Letters 360 (2015) 205ndash212

reached the limits of acute and long-term toxicity with this inten-sive approach so more effective less toxic approaches are greatlyneeded Targeted agents show promise in select subsets ofpatients that express the target protein but responses maybe short-lived and they do not work for all high-risk patients[41112]

Given the current limitations of intensive multimodality therapyfor high-risk NBs we have taken the approach of more targeted drugdelivery that could treat these tumors more effectively while main-taining reduced toxicity NP encapsulation of chemotherapeuticagents takes advantage of the EPR effect to deliver more drug totumors than conventional administration which should increase the

Fig 5 SN38-TS NP treatment of large NB tumors A Survival curve of the tumor-bearing animals Indicates last day of treatment Animals were followed for tumor re-growth and survival until the tumors reached 3 cm3 in volume B Treatment of large tumors with SN38-TS NP promotes maturation of xenografts into a ganglioneuroblastomaphenotype Tumors were followed for regrowth patterns and harvested when they reached 1 cm3 Increased Tyrosine Hydroxlyase staining is seen in the SN38-TS NP-treated tumors

210 R Iyer et alCancer Letters 360 (2015) 205ndash212

efficacy of these agents while simultaneously reducing systemic ex-posure [513ndash15] NP formulations also provide a biocompatiblevehicle for water-insoluble agents as well as stability for labile mol-ecules Irinotecan a commonly used topoisomerase I inhibitor isa weak or inactive pro-drug that is metabolized to SN38 its activeagent [16] The conversion of irinotecan to SN38 is inefficient andsubject to significant interpatient variability [1718] However 40ndash60 of administered irinotecan was in the form of SN38 in bloodand tissues at 4 hr in our animal model (Table 1) SN38 itself is 1000times more potent than irinotecan but it has toxicity and solubil-ity issues that make it unsuitable for systemic administration [1920]However SN38 is an attractive agent for NP drug delivery becausethis approach obviates the inherent disadvantages of SN38 as a freedrug In this study we examined the efficacy of NP delivery of SN38versus oral administration of irinotecan in a mouse NB xenograftmodel

Others have encapsulated SN38 in NPs [21ndash23] because of itspoor solubility Pal and coworkers [22] tested liposome entrappedSN38 had antitumor efficacy and low toxicity in mouse and dogtumor models Atyabi and colleagues [21] used pegylated lipo-somes of 150ndash200 nm containing SN38 to test their biodistributionin mice and found that the distribution in liver spleen kidney andlung was less with pegylated liposomes and they persisted longerin the circulation Zhang and coworkers [23] developed anoligoethylene glycol SN38 codrug that formed micelles of 25ndash30 nm and required esterase activation This formulation exhibitedfavorable antitumor efficacy against human xenografts Preclinicalas well as phase I studies have been conducted of an SN38-incorporating 20 nm polymeric micelles (NK012) that show improvedefficacy over irinotecan [24ndash26] and Marier et al showed that anSN38 pro-drug formulated as emulsion (SN2310) had a better safetyprofile than irinotecan [27] In the present studies we employed abiodegradable NP formulation where TS-derivatized SN38 was in-corporated in pegylated polymeric PLA nanoparticles of 70ndash80 nm designed to provide improved encapsulation efficiency andNPndashdrug association stability

Nanoencapsulated SN38 conjugated to TS acted as a potent in-hibitor of cell growth in SH-SY5Y-TrkB cells whereas TS proved tobe ineffective at inhibiting cell growth suggesting that only the SN38component of the NP formulation contributes to cell death in vitro(Fig 2) We also observed no toxicity when NB tumor-bearing micewere treated with tocopherol succinate alone (data not shown)However the levels of TS in the tumor that were achieved with NPdelivery were likely far greater than those achieved by oral admin-istration so it is possible that the resultant high local levels couldbe sufficient to exert an anti-tumor effect Furthermore even if TShad little or no effect alone it may have enhanced the efficacy ofSN38 when delivered as a conjugate

Next to determine whether these NPs could safely and effec-tively reach tumor tissue we analyzed the biodistribution of

fluorescently conjugated SN38-TS NPs Over the first 4ndash24 hourspost-injection NPs were visualized throughout the circulatorysystem but after 24 hours post-injection NPs preferentially accu-mulated in the tumor as well as in the liver spleen and lymph nodesNP-treated mice showed no evidence of toxicity as demonstratedby normal mouse weights blood counts and behavior throughoutthe course of treatment Furthermore pharmacokinetic analysis oftumor tissue showed that NP delivery of SN38 had a ~200-fold ad-vantage at 4 hr over oral administration of irinotecan and sustainedlevels in tumors for at least 72 hr post-treatment (Table 1) This sug-gests that compared to conventional irinotecan NP administrationof SN38-TS NPs can significantly increase the exposure of tumortissue to the cytotoxic effects of SN38 while preventing system ex-posure to its inherent toxicities

We next tested the ability of SN38-TS NPs to control NB tumorgrowth over time We observed significantly greater tumor controlafter cessation of treatment as well as protracted long-term sur-vival in NP-treated mice when compared to oral irinotecan treatmentwith substantially more total drug delivered Furthermore we de-termined that mice treated with the NP formulation just once everytwo weeks (2 doses) had survival curves equivalent to mice treatedwith oral irinotecan 5timesweek for 4 weeks (20 doses) Together theseresults show that the SN38-TS NP formulation is safe as well as sig-nificantly more effective at controlling NB tumor growth andrecurrence than conventional irinotecan therapy

SN38-TS NPs were also effective at controlling larger more pro-gressive NB tumors which mimic more advanced stage disease Ina pilot study we showed that SN38-TS NPs induced tumor regres-sion from an average of 1 cm3ndash01 cm3 when administered twice aweek for 8 weeks (data not shown) These mice remained in re-mission for an average of 60 days post-cessation of treatmentanalogous to our previous mouse studies Interestingly when tumorsrecurred after a period of remission they grew at a slower pace thanrecurrences in irinotecan-treated mice and they exhibited a dra-matically altered morphology All recurrent tumors examined in theSN38-TS NP treated group resembled ganglioneuromas (Fig 5A andB)

Santos and colleagues treated neuroblastoma xenografts withCPT-11 (irinotecan) and they found that the tumors differentiatedduring treatment to ganglioneuroblastomas but then reverted toan immature phenotype when treatment was discontinued [28] Al-though the mechanism for this differentiation is unknown it wasassociated with a decrease in MYCN expression Our findings of dif-ferentiation were similar but unlike the Santos study with irinotecanthe differentiation persisted months after treatment was discon-tinued SN38 is the major active metabolite of irinotecan andinactivates topoisomerase 1 [29] so the effects in both the Santosstudy and ours are presumably mediated by the same mecha-nism However the durability of the differentiation in our study ispresumably related to the higher intratumoral concentrations of

Table 1Levels of irinotecan andor SN38 in blood and tumor tissue

Blood Blood Tumor Tumor

Irinotecanngml plusmn SD

SN38ngml plusmn SD

Irinotecanngg tissue plusmn SD

SN38ngg tissue plusmn SD

Irinotecan 4 hr 513 plusmn 3 801 plusmn 17 1644 plusmn 47 1071Irinotecan 24 hr lt1 lt1 lt10 lt10Irinotecan 72 hr lt1 lt1 lt10 lt10SN38-TS 4 hr ndash 39371 plusmn 7485 ndash 19746 plusmn 4652SN38-TS 24 hr ndash 99 plusmn 257 ndash 14824 plusmn 3546SN38-TS 72 hr ndash 293 plusmn 186 ndash 5583 plusmn 1907

LLOQ for SN-38 and Irinotecan was 10 ngml (blood) and 10 ngg of tissueHLOQ for SN-38 and Irinotecan was 1000 ngml (blood) and 10000 ngg of tissueTissue distribution of irinotecan and SN38-TS NP Animals were given a single dose of either irinotecan PO (10 mgkg) or SN-38 TS NP IV (10 mgkg) and sacrificed at giventime points post-treatment Drug concentrations were analyzed by LCMSMS as described in Materials and methods Values are shown plusmn the standard deviation (SD)

211R Iyer et alCancer Letters 360 (2015) 205ndash212

SN38 we achieved (Table 1) andor the protracted duration of ex-posure afforded by NP delivery It is possible that the sustained SN38exposure killed proliferating NB cells and left a more differenti-ated population of cells that promoted Schwann cell invasion TheSchwann cells could then have provided the neurotrophic factorsthat led to neuronal differentiation [30]

Other groups have synthesized PEGylated nanoparticulate ornanoprecipitate formulations of SN38 to overcome the issues of poorsolubility and high toxicity [233132] These approaches showed su-periority over conventionally delivered irinotecan but most weredesigned primarily to address the poor solubility of SN38 and didnot take full advantage of the EPR effect Others have used lipid orchitosan nanocapsules for oral or parental administration [33ndash35]Finally another study developed polymeric NPs encapsulating SN38using poly lactic-co-glycolic acid [36] In the present study we uti-lized biodegradable PEGylated polymeric nanoparticles incombination with a pro-drug derivatization approach for deliveryof SN38 Our SN38-TS NPs were also optimized for size and releasekinetics [8] and we demonstrated dramatically superior effective-ness compared to orally administered irinotecan in our model system

Taken together our preclinical studies suggest that our SN38-TS NP formulation is an attractive new therapeutic approach for NBand other solid tumors Our results show that this formulation issafe as well as significantly more effective than oral irinotecan attargeting NB tumors and controlling tumor regrowth This formu-lation could be used to treat any tumor currently treated withirinotecan and possibly tumors previously thought resistant to thisdrug due to the dramatically increased drug delivery Further-more this approach could potentially be applied to other therapeuticagents

Acknowledgements

This work was supported in part by Alexrsquos Lemonade Stand Foun-dation for Childhood Cancer the V Foundation for Cancer ResearchNIH grant CA094194 and the Audrey E Evans Endowed Chair (GMB)

Conflict of interest

None

Appendix Supplementary material

Supplementary data to this article can be found online atdoi101016jcanlet201502011

References

[1] GM Brodeur JM Maris Neuroblastoma in PA Pizzo DG Poplack (Eds)Principles and Practice of Pediatric Oncology sixth ed Lippincott Williamsand Wilkins Philadelphia 2011 pp 886ndash922

[2] GM Brodeur Neuroblastoma biological insights into a clinical enigma NatRev Cancer 3 (3) (2003) 203ndash216

[3] JM Maris MD Hogarty R Bagatell SL Cohn Neuroblastoma Lancet 369(9579) (2007) 2106ndash2120

[4] GM Brodeur R Iyer JL Croucher T Zhuang M Higashi V Kolla Therapeutictargets in neuroblastomas Expert Opin Ther Targets 18 (2014) 277ndash292

[5] H Maeda J Wu T Sawa Y Matsumura K Hori Tumor vascular permeabilityand the EPR effect in macromolecular therapeutics a review J Control Release65 (1ndash2) (2000) 271ndash284

[6] K Cho X Wang S Nie ZG Chen DM Shin Therapeutic nanoparticles for drugdelivery in cancer Clin Cancer Res 14 (5) (2008) 1310ndash1316

[7] J Thompson WC Zamboni PJ Cheshire L Richmond X Luo JA Houghtonet al Efficacy of oral irinotecan against neuroblastoma xenografts AnticancerDrugs 8 (4) (1997) 313ndash322

[8] IS Alferiev R Iyer JL Croucher RF Adamo K Zhang JL Mangino et alNanoparticle-mediated delivery of a rapidly activatable prodrug of SN-38 forneuroblastoma therapy Biomaterials 51 (2015) 22ndash29

[9] J Thompson WC Zamboni PJ Cheshire L Lutz X Luo Y Li et al Efficacy ofsystemic administration of irinotecan against neuroblastoma xenografts ClinCancer Res 3 (3) (1997) 423ndash431

[10] KK Matthay CP Reynolds RC Seeger H Shimada ES Adkins D Haas-Koganet al Long-term results for children with high-risk neuroblastoma treated ona randomized trial of myeloablative therapy followed by 13-cis-retinoic acida childrenrsquos oncology group study J Clin Oncol 27 (7) (2009) 1007ndash1013

[11] JE Minturn AE Evans JG Villablanca GA Yanik JR Park S Shustermanet al Phase I trial of lestaurtinib for children with refractory neuroblastomaa new approaches to neuroblastoma therapy consortium study CancerChemother Pharmacol 68 (4) (2011) 1057ndash1065

[12] YP Mosse FM Balis MS Lim J Laliberte SD Voss E Fox et al Efficacy ofcrizotinib in children with relapsedrefractory ALK-driven tumors includinganaplastic large cell lymphoma and neuroblastoma a Childrenrsquos Oncology Groupphase I consortium study J Clin Oncol 30 (Suppl) (2012) abstr 9500

[13] D Di Paolo M Loi F Pastorino C Brignole D Marimpietri P Becherini et alLiposome-mediated therapy of neuroblastoma Methods Enzymol 465 (2009)225ndash249

[14] D Di Paolo F Pastorino C Brignole D Marimpietri M Loi M Ponzoni et alDrug delivery systems application of liposomal anti-tumor agents toneuroectodermal cancer treatment Tumori 94 (2) (2008) 246ndash253

[15] N Federman CT Denny Targeting liposomes toward novel pediatric anticancertherapeutics Pediatr Res 67 (5) (2010) 514ndash519

[16] LM Wagner JG Villablanca CF Stewart KR Crews S Groshen CP Reynoldset al Phase I trial of oral irinotecan and temozolomide for children withrelapsed high-risk neuroblastoma a new approach to neuroblastoma therapyconsortium study J Clin Oncol 27 (8) (2009) 1290ndash1296

[17] GG Chabot Clinical pharmacokinetics of irinotecan Clin Pharmacokinet 33(4) (1997) 245ndash259

[18] JG Slatter LJ Schaaf JP Sams KL Feenstra MG Johnson PA Bombardt et alPharmacokinetics metabolism and excretion of irinotecan (CPT-11) followingIV infusion of [(14)C]CPT-11 in cancer patients Drug Metab Dispos 28 (4)(2000) 423ndash433

[19] AM Abang The clinical pharmacology of topoisomerase I inhibitors SeminHematol 35 (3 Suppl 4) (1998) 13ndash21

[20] J OrsquoLeary FM Muggia Camptothecins a review of their development andschedules of administration Eur J Cancer 34 (10) (1998) 1500ndash1508

[21] F Atyabi A Farkhondehfai F Esmaeili R Dinarvand Preparation of pegylatednano-liposomal formulation containing SN-38 in vitro characterization andin vivo biodistribution in mice Acta Pharm 59 (2) (2009) 133ndash144

[22] A Pal S Khan YF Wang N Kamath AK Sarkar A Ahmad et al Preclinicalsafety pharmacokinetics and antitumor efficacy profile of liposome-entrappedSN-38 formulation Anticancer Res 25 (1A) (2005) 331ndash341

[23] H Zhang J Wang W Mao J Huang X Wu Y Shen et al Novel SN38conjugate-forming nanoparticles as anticancer prodrug in vitro and in vivostudies J Control Release 166 (2) (2013) 147ndash158

[24] T Hamaguchi T Doi T Eguchi-Nakajima K Kato Y Yamada Y Shimada et alPhase I study of NK012 a novel SN-38-incorporating micellar nanoparticle inadult patients with solid tumors Clin Cancer Res 16 (20) (2010) 5058ndash5066

[25] F Koizumi M Kitagawa T Negishi T Onda S Matsumoto T Hamaguchi et alNovel SN-38-incorporating polymeric micelles NK012 eradicate vascularendothelial growth factor-secreting bulky tumors Cancer Res 66 (20) (2006)10048ndash10056

[26] Y Matsumura Preclinical and clinical studies of NK012 an SN-38-incorporatingpolymeric micelles which is designed based on EPR effect Adv Drug Deliv Rev63 (3) (2011) 184ndash192

[27] JF Marier L Pheng MM Trinh HA Burris 3rd S Jones K Anderson et alPharmacokinetics of SN2310 an injectable emulsion that incorporates a newderivative of SN-38 in patients with advanced solid tumors J Pharm Sci 100(2011) 4536ndash4545

[28] A Santos L Calvet MJ Terrier-Lacombe A Larsen J Benard C Pondarre et alIn vivo treatment with CPT-11 leads to differentiation of neuroblastomaxenografts and topoisomerase I alterations Cancer Res 64 (9) (2004) 3223ndash3229

[29] CL Kline WS El-Deiry Personalizing colon cancer therapeutics targeting oldand new mechanisms of action Pharmaceuticals 6 (8) (2013) 988ndash1038

[30] SP Frostick Q Yin GJ Kemp Schwann cells neurotrophic factors andperipheral nerve regeneration Microsurgery 18 (7) (1998) 397ndash405

[31] MF Al-Kasspooles SK Williamson D Henry J Howell F Niu CJ Decedueet al Preclinical antitumor activity of a nanoparticulate SN38 Invest New Drugs31 (4) (2013) 871ndash880

[32] F Pastorino M Loi P Sapra P Becherini M Cilli L Emionite et al Tumorregression and curability of preclinical neuroblastoma models by PEGylatedSN38 (EZN-2208) a novel topoisomerase I inhibitor Clin Cancer Res 16 (19)(2010) 4809ndash4821

[33] H Liu H Lu L Liao X Zhang T Gong Z Zhang Lipid nanoparticles loadedwith 7-ethyl-10-hydroxycamptothecin-phospholipid complex in vitro and invivo studies Drug Deliv (2014) PMID 24625262

[34] E Roger F Lagarce JP Benoit Development and characterization of a novellipid nanocapsule formulation of Sn38 for oral administration Eur J PharmBiopharm 79 (1) (2011) 181ndash188

[35] E Sayari M Dinarvand M Amini M Azhdarzadeh E Mollarazi Z Ghasemiet al MUC1 aptamer conjugated to chitosan nanoparticles an efficient targetedcarrier designed for anticancer SN38 delivery Int J Pharm 473 (1ndash2) (2014)304ndash315

[36] N Sepehri H Rouhani F Tavassolian H Montazeri MR Khoshayand MHGhahremani et al SN38 polymeric nanoparticles in vitro cytotoxicity and invivo antitumor efficacy in xenograft balbc model with breast cancer versusirinotecan Int J Pharm 471 (1ndash2) (2014) 485ndash497

212 R Iyer et alCancer Letters 360 (2015) 205ndash212