tumor regression and curability of preclinical ......aug 11, 2010 · and characterized by cell...

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research

Tumor regression and curability of preclinical neuroblastoma models by PEGylated SN38

(EZN-2208), a novel topoisomerase I inhibitor

Fabio Pastorino1, Monica Loi1, Puja Sapra2,a, Pamela Becherini1, Michele Cilli3, Laura Emionite3,

Domenico Ribatti4, Lee M. Greenberger2, Ivan D. Horak2, and Mirco Ponzoni1

1Experimental Therapies Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Genoa,

Italy; 2Enzon Pharmaceuticals, Piscataway, NJ, USA; 3Animal Research Facility, National Cancer

Institute, Genoa, Italy; 4Department of Human Anatomy, University of Bari, Italy.

a current address: Pfizer Biotherapeutics, Pearl River, NY, USA

Running title: PEGylated SN38 therapeutic efficacy in neuroblastoma

Corresponding author: Fabio Pastorino, Ph.D, Experimental Therapy Unit, Laboratory of

Oncology, G. Gaslini Children’s Hospital, 16148-Genoa, Italy. Phone: +39-010-5636342; Fax: +39-

010-3779820. email: [email protected]

Published OnlineFirst on August 11, 2010 as 10.1158/1078-0432.CCR-10-1354

Research. on December 21, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 11, 2010; DOI: 10.1158/1078-0432.CCR-10-1354

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Translational relevance

Human neuroblastoma represents a major therapeutic challenge due to the lack of drug effective at

achieving disease eradication. Approximately a half of neuroblastoma patients present with

metastatic disease at diagnosis, and only 25% of them survive at five years in spite of high dose

chemotherapy and autologous hematopoietic stem cells rescue.

This study describes for the first time an innovative therapeutic treatment for neuroblastoma, based

on the use of the novel DNA topoisomerase-I inhibitor, EZN-2208, that, due to the extraordinary

results obtained in various biologically and clinically relevant preclinical settings, led to the rapid

translation to clinical trials in patients with various pediatric cancers, including neuroblastoma.





ABSTRACT

Purpose

Treatment of neuroblastoma (NB) is successful in less than half of patients with high-risk disease.

The anti-tumor activity of a water soluble pegylated SN38 drug conjugate (EZN-2208), was

compared with CPT-11 (a pro-drug for SN38) in preclinical models of human NB.

Experimental Design

The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye- and annexin-V-

positive cells, while its therapeutic efficacy evaluated, in terms of survival, anti-tumor, and anti-

angiogenic activities, in subcutaneous luciferase-transfected, pseudometastatic and orthotopic NB

animal models.

Results

EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis

and p53 expression and by reducing HIF-1α/ HIF-2α expression. EZN-2208 gave superior

antitumor effects compared to CPT-11 in NB xenografts. EZN-2208 treatment always resulted in

lack of tumor detection at the end of trials whereas only small therapeutic effects were observed

with CPT-11, as assessed by luciferase assay, tumor size, or even staining histological sections of

tumors with antibodies recognizing NB cells and cell proliferation. In NB model resistant to

Doxorubicin (D), Cisplatin, Vincristine (V), Fenretinide and Topotecan (T), EZN-2208 induced

100% curability. It also blocked tumor relapsed after TVD-combined treatment. Mechanistic

experiments showed statistically significant enhanced TUNEL and Histone H2ax staining and

decreased VEGF, CD31, MMP-2 and MMP-9 expression in tumors removed from EZN-2208-

treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic

membranes.





Conclusions

EZN-2208 should be considered a most promising novel anti-NB agent and ongoing phase I study

in pediatric patients should identify the optimal dose for Phase II study.

INTRODUCTION

Treatment of neuroblastoma (NB), the second most common solid tumor in childhood, is successful

in less than half of patients with high-risk disease (1). The effective treatment of NB, either at

advanced stages or at minimal residual disease, remains indeed one of the major challenges in

pediatric oncology. The use of intensive therapeutic interventions have marginally prolonged the

overall long-term disease-free survival rates, mainly due to the dose-limiting toxicity associated

with systemic delivery of cytotoxic drugs in vivo (2). Moreover, the incidence of fatal relapses is

still high and long-term survival remains very low. Innovative therapies are thus required to

eradicate residual disease after chemotherapy and surgery.

Camptothecin and its analogs irinotecan (CPT-11) and topotecan, have hold great promise in the

treatment of NB, for several reasons (3-6). First, they have impressive activity against NB cell lines

resistant to standard anti-neuroblastoma agents. These findings imply that they might provide a

novel non-cross-resistant chemotherapeutic addition to the standard drug armamentarium for NB.

Second, their non-hematological toxicities are manageable and transient. Such limitations are major

consideration in NB patients who often undergo nephrectomy, are treated with cardiotoxic

(doxorubicin) and nephrotoxic (platinum compounds) agents, and who receive irradiation. Third,

cytotoxicity occurs at relatively nonmyelosuppressive dosages, suggesting that they can be safely

given with other anticancer drugs (5, 6).

On the other hand, CPT-11 has several limitations, such as the limited and variable conversion by

endogenous enzymes to convert the pro-drug (CPT-11) to the active form (SN38) (no better than

approximately 4% of the total drug), an inter-individual variability in drug safety profile, the





induction of diarrehea (associated, in part, with the parent molecule), and the limited anti-tumor

activity as a single agent (7, 8) including in the treatment of NB (6). These limitation suggest that

direct delivery of the prodrug, SN-38 may improve the efficacy and/or safety of this compound.

However, SN-38 is insoluble in pharmaceutical acceptable vehicles and therefore cannot be given

by itself. This problem has been overcome with the development of a pegylated SN38, designated

EZN-2208.

EZN-2208 is a water soluble pegylated SN38 drug conjugate, composed of a four-arm 40 KDa

polyethylene glycol (PEG) linked via glycine residue to SN38 (9). This highly soluble molecule

shows favorable prolonged circulation time leading to a preferential accumulation in the solid

tumors and has shown considerably more efficacy than CPT-11 in various preclinical models of

solid and hematologic tumors (10, 11). Moreover, in Phase I trials in adult patients, EZN-2208 was

well tolerated, with dose-limiting toxicity (DLT) of isolated, short-lived neutropenia with or

without fever. This is in distinction to irinotecan where grade ¾ diarrhea is frequently encountered

at therapeutic doses (12, 13). In on-going Phase II trials with EZN-2208, prolonged stable disease,

sometimes associated with tumor shrinkage, was also observed.

In this report, the activity of EZN-2208 has been evaluated in experimental models of pediatric NB.

However, unlike previous compounds that have been evaluated in experimental model of pediatric

NB tumor where the tumor is grown as subcutaneous xenograft in nude mice, we have rigorously

evaluated EZN-2208 in this tumor model as well as pseudometastatic and orthotopic models of

human NB. Collectively, these models better reflect the growth of advanced NB in children (i.e.

large adrenal gland tumors and multiple small metastatic lesions), which have been difficult to

control.

EZN-2208 demonstrated excellent and clearly superior antitumor activity compared with CPT-11 as

well as with many others clinically used anti-neoplastic drugs. In all tested models, EZN-2208-





treated tumor-bearing mice survived, with complete regression of implanted tumors. Therefore,

EZN-2208 is a most promising, novel anti-tumor agent for the treatment of NB.

METHODS

Materials, cell lines and animals

EZN-2208 was prepared as described previously (9, 10). Irinotecan (CPT-11, CAMPTOSAR®) was

purchased from Pfizer (Pfizer Italia S.r.l.; Borgo S. Michele, LT, Italy); Topotecan (HYCAMTIN®)

from Glaxo (GlaxoSmithkline S.p.A.; Verona, Italy); Cisplatin and Doxorubicin from Ebewe Italia

S.r.l., Roma, Italy; Vincristine from Sigma Chemical Co. (St. Louis, MO). Fenretinide (N-4-

hydroxyphenyl retinamide, HPR) was kindly provided by Dompè (Dompè Farmaceutici S.p.A.,

Milan, Italy). Deferoxamine (Desferal, DFX) was purchased from Novartis Pharma (Stein,

Swizerland).

To broadly cover the phenotypes exhibited by neuroblastoma (NB) cells in vitro and in vivo, five

human (GI-LI-N, HTLA-230, IMR-32, SK-N-BE2c and SH-SY5Y) and one murine (NXS2) NB

cell lines were used (14, 15). In some experiments, the SH-SY5Y cell line was infected with

retrovirus expressing the firefly luciferase gene and luciferase activity of retrovirally-transduced

cells confirmed by bioluminescence imaging (IVIS Caliper Life Sciences, Hopkinton, MA) after a

10 min. incubation with 150 μg/mL of D-luciferin (Caliper Life Sciences), as described (16). All

cell lines were grown in complete DMEM or RPMI-1640 medium, supplemented with 10% heat-

inactivated FCS, as described (14, 15). All cell lines were tested for mycoplasma contamination,

and characterized by cell proliferation morphology evaluation and multiplex short tandem repeat

profiling test, both after thawing and within four passages in culture.

All animals were purchased from Harlan Laboratories (Harlan Italy, S.Pietro al Natisone, Italy) and

housed under specific pathogen-free conditions. Experiments involving animals were reviewed and





approved by the licensing and ethical committee of the National Cancer Research Institute, Genoa,

Italy, and by the Italian Ministry of Health. All experiments in vivo were performed with 5-8 mice

per group and were repeated twice.

In vitro cytotoxicity and apoptosis

The in vitro cytotoxicity of EZN-2208 was evaluated in a panel of NB cell lines by counting trypan

blue dye-stained cells. Briefly, adherent cells were plated in 75-cm3 flasks and incubated overnight

at 37°C. The day after cells were treated with serial dilutions of EZN-2208 and further incubated for

24 hours. At the end of the incubation period, cells were detached with trypsin, stained with trypan

blue dye, and counted microscopically. Apoptosis analysis was performed using the annexin V-

FITC/propidium iodide assay, as described (11).

Immunoblotting analysis

Western blotting analysis from whole cell lysates was performed as described (17). Briefly, cultured

NB cells were treated for 24 and 48 hours with the same concentration of either CPT-11 or EZN-

2208. In some experiments, as positive control for HIF-1α induction, cells were pre-incubated with

0.15 mM of Desferal (DFX) for 6 hours, washed and treated with CPT-11 and EZN-2208 for a total

of 24 hours-incubation. Monoclonal anti-p53 (clone PAb 1801) and anti-HIF-1α (clone 54) were

purchased from BD Biosciences (Buccinasco, MI, Italy), anti-HIF-2α (clone ep190b) an anti-

GAPDH (clone 14c10) antibodies were from Novus Biologicals, Inc (Cambridge, UK) and Cell

Signaling Technology (Danvers, MA, US), respectively.

Animal models





For the subcutaneous (s.c.) animal model, 2.0 x 107, luciferase-transfected, SH-SY5Y cells were

inoculated in the mid-dorsal region of five-week-old female SCID mice (5 mice/group). Tumor

expansion over time, as well as the response to treatment have been readily visualized by

bioluminescence imaging (BLI) and quantified by highly sensitive, cooled CCD camera mounted in

a light-tight specimen box (IVIS™), as described (15, 16).

For the pseudometastatic animal model, four-week-old female, athymic (nu/nu) mice (8

mice/group) were injected intravenously (i.v.) in the tail vein with 4 x 106 HTLA-230 tumor cells, as

described (18). Body weight and general physical status of the animals were recorded daily, and

mice were sacrificed by cervical dislocation after the administration of xilezine (Xilor 2%, Bio98

Srl, Milan, Italy), when they showed signs of poor health, such as abdominal dilatation, dehydration,

or paraplegia.

In the orthotopic animal model, five-week-old female, athymic (8 mice/group) or

immunocompetent (A/J mice, 5/group) animals were anesthetized with ketamine (Imalgene 1000,

Merial Italia SpA., Milan, Italy), subjected to laparotomy, and injected with either 1 x 106 GI-LI-N

or 5 x 104 NXS2 cells, respectively, in the capsule of the left adrenal gland, as previously described

(14, 19). No mice died as a result of this treatment. Mice were monitored at least two times weekly

for evidence of tumor development and quantification of tumor size, and were sacrificed by cervical

dislocation after being anesthetized with xilezine, when they showed signs of poor health, such as

abdominal dilation, dehydration, or paraplegia.

In vivo therapeutic efficacy

Mice were treated i.v. every other day for 5 total doses with 10 mg/kg of CPT-11 (CAMPTOSAR)

or with the SN38 equivalents of EZN-2208 (for details, see reference number 10), in the first set of

in vivo experiments, or with MTD doses, for multiple-dose regimen therapies (10) of both





CAMPTOSAR (40 mg/kg) and EZN-2208 in a second set of in vivo experiments. In every

experiments, a group of control mice received HEPES-buffered saline.

For the s.c. animal model, tumors were allowed to grow for 7 (first set of experiments) and 13

(second set of experiments) days, reaching a size of about 100 and 200 mm3 before i.v (tail vein)

treatment commenced. For the pseudometastatic model, mice were treated i.v. either 24 or 72 hours

post-inoculation of tumor cells. For the orthotopic animal models, tumors were allowed to grow

from the injected cells for 21 and 3 days, in immunodeficient and immunocompetent mice,

respectively, before treatments started. For the tumor regression studies, mice were sacrificed and

tumors were measured with calipers. Tumor volumes were calculated by the formula π/6 [w1 ×

(w2)2], where w1 represented the largest tumor diameter and w2 represented the smallest tumor

diameter, as described (19). The anti-tumor EZN-2208 activity was also evaluated either in

comparison with MTD doses of different, clinically used, anti-tumor compounds (Topotecan, CPT-

11, Cisplatin, Doxorubicin, Vincristine and Fenretinide), or as a second-line treatment in

Topotecan-Vincristine-Doxorubicin (TVD)-resistant, orthotopic NB-bearing mice. For these

experiments, tumors were allowed to grow from the injected cells for 21 days in nude mice (6

mice/group), before randomly treated with: EZN-2208, i.v., 10 mg/kg every other day for 5 total

doses; CAMPTOSAR, i.v., 40 mg/kg, every other day for 5 total doses; Topotecan, intraperitoneally

(i.p.), 10 mg/kg, every 4 days x 3 times; Cisplatin, i.v., 5 mg/kg, once per week, 3 weeks total;

Doxorubicin, i.v., 5 mg/kg, once per week, 5 weeks total; Vincristine, i.v., 1 mg/kg, once per week,

7 weeks total; fenretinide, i.v., 1.5 mg/kg, twice per week, 6 weeks total, as previously reported (10,

17, 19-22).

Another group of animals (7/group) was treated 21 days after tumor challenge with a combination

of Topotecan (3 mg/kg, i.p., daily from day 21 to day 25), Vincristine (50 μg/kg, i.v., at day 25) and

Doxorubicin (1.5 mg/kg, i.v., at day 25), mimicking schedule and treatment doses performed in

patients suffering from high-risk NB (23). When signs of poor health, such as abdominal dilation,





became evident (day 80), half of TVD-treated mice, randomly chosen, were then inoculated with

EZN-2208, 10 mg/kg every other day for 5 total doses.

Chorioallantoic membrane assay

Chorioallantoic membrane (CAM) experiments were performed according to the method of Ribatti

et al. (24). NB (GI-LI-N and HTLA-230 tumor cell lines) biopsy fragments (1-2 mm3) taken from

untreated mice were grafted onto the CAM and then treated with either PBS (control), EZN-2208,

or CPT-11 (SN38 equivalents and at MTD). CAMs were examined daily until day 12 and

photographed in ovo with a stereomicroscope equipped with a camera and an image analyzer

system (Olympus Italia, Rozzano Italy). On day 12, the angiogenic response, as determined by the

number of vessels converging toward the grafts, was evaluated with an image analyzer. CAMs

were then processed for light microscopy, as reported (17). Microvessels density was expressed as

the percentage of the total number of intersection points occupied by CD31-positive vessels cut

transversely (diameter, 3-10 μm). Mean values ± SD were determined for each analysis.

Histological analysis

In the first set of experiments, histological evaluation of primary tumors was performed 50 days

after GI-LI-N cell inoculation. Briefly, tumors were orthotopically implanted in athymic mice. After

21 days, animals were treated with 10 mg/kg of CPT-11 (CAMPTOSAR) or with the SN38

equivalents of EZN-2208 every other day for 5 total doses. In every experiments, a group of control

mice received HEPES-buffered saline.

Three weeks after the end of the treatment, mice were anaesthetized with xylezine and killed by

cervical dislocation. Tumors were collected and then embedded in optimum cutting temperature





(O.C.T.) (Miles Chemical Co., Elkhart, IN) compound. Tissue sections (5 μm thick) were examined

after staining with Mayer’s Hematoxylin-Eosin (H&E) (Sigma).

Tumor frozen sections were then washed twice in phosphate buffered saline (PBS) and saturated

with 2% bovine serum albumin (BSA) in PBS before staining with primary antibodies against anti-

human NB (NB84a, Dako) and Ki-67 proliferation antigen (mouse anti-human Ki-67, clone Ki-55,

Dako), as described (17).

In the second set of experiments, histological evaluation of primary tumors was performed at 44

days after GI-LI-N cell inoculation. Briefly, orthotopically implanted athymic mice were implanted.

After Thirty five days, animals were treated with 10 mg/kg of CPT-11 (CAMPTOSAR) or with the

SN38 equivalents of EZN-2208 or with MTD doses of CAMPTOSAR (40 mg/kg)., every other day

for 5 total doses. In every experiments, a group of control mice received HEPES-buffered saline.

One day after the end of the treatment, mice were anaesthetized with xylezine and killed by cervical

dislocation. Tumors were collected, divided in two, and processed as stated above.

Tumor frozen sections were stained with primary antibodies against Histone H2ax (H2AFX, clone

EP854(2)Y, Lifespan Biosciences, Prodotti Gianni, Milan, Italy) to detect DNA-damage dependent

histone phosphorylation (Huang X Cell Cycle 2003); VEGF (Thermo Fisher Scientific, Fremont,

CA, USA) and CD31 (clone SC-1506, Santa Cruz Biotechnology, D. B. A. Italia S. R. L., Segrate,

Milan, Italy) to detect inhibition of angiogenesis. TUNEL (i.e., terminal deoxynucleotidyl

transferase-mediated end labeling) staining was performed using a commercially available

apoptosis detection kit (In situ Cell Death Detection, POD; Roche Molecular Biochemicals,

Mannheim, Germany) according to manufacturer’s instructions, as described (17).

Paraffin-embedded tissue sections were de-paraffinized by a xylene-ethanol sequence, re-hydrated

in a graded ethanol solutions, and TRIS-buffered saline (TBS, pH 7.6), and then processed for

antigen retrieval by boiling tissue sections for 10 min in 1 mM EDTA, pH 8.0, in a microwave

oven. The sections were then washed twice in PBS and saturated with 2% BSA in PBS before





staining with primary antibodies against MMP-2 (clone 36006, R & D System, Abingdon, UK) and

MMP-9 (clone 443, R & D System) to detect inhibition of tumor invasiveness-related markers.

Morphometric analysis was performed on 9 randomly selected fields every 3 sections, observed at

200x magnification with an Olympus photomicroscope, using Image Analysis software (Olympus

Italia). VEGF and MMP-2/MMP-9 labeled areas were evaluated.

Statistical analysis

Results are expressed as mean ± 95% Confidence Intervals (95% CI). The statistical significance of

differential findings between experimental groups and controls was determined by ANOVA, with

the Tukey’s multiple comparison test (GraphPad Software, Inc., El Camino Real, San Diego, CA).

The significance of the differences between experimental groups in the survival experiments was

determined by Kaplan-Meier curves by the use of Chi square log-rank test (Graph-Pad Prism 3.0).

In the morphometric analysis, the mean value in each image from the section, the final mean value

for all the images and the SEM were calculated. The statistical significance of the differences

between the mean values of the different experimental conditions was determined by Student’s t test

(GraphPad software). Findings were considered significant at P values < 0.05 for all statistical

evaluations.

RESULTS

In vitro studies

EZN-2208 showed potent in vitro cytotoxicity against NB cells, with IC50 values ranging from

0.002 to 0.15 μmol/L (Table 1, supplementary). Consistent with previous results (11), EZN-2208

induced a rapid induction of annexin-V-positive NB cells, followed by massive necrosis (data not





shown). This cell death were accompanied by a rapid and strong induction of p53 and a down-

modulation of HIF-2α proteins expression (Figure 1S, A-B). Noteworthy, compared with

CAMPTOSAR, EZN-2208 led also to a strong down-modulation of both constitutive (Figure 1S, C)

and DFX-induced (Figure 1S, D) HIF-1α protein levels.

Therapeutic effects of CPT-11 and SN38 equivalents of EZN-2208

In the subcutaneously injected NB-bearing mice, the effects of EZN-2208 and CAMPTOSAR were

compared against a luciferase-transfected human NB cell line (SH-SY5Y) inoculated in the right

flank of animals. Treatment started seven days after cell injection and the anti-tumor efficacy

evaluated by bioluminescence (BLI). As showed in Figure 1,A, while CAMPTOSAR led to a

partial anti-tumor response, compared to control mice that were sacrificed at day 40 for excessive

tumor mass, a complete regression of primary tumor growth in NB-bearing mice were observed

after treatment with EZN-2208.

In the pseudometastatic model, mice were treated either 24 or 72 hours after intravenous tumor cells

(HTLA-230) inoculation. As showed in Figure 1,B, animals treated with EZN-2208 displayed

significant increased life span compared to control mice or those treated with CAMPTOSAR. After

150 days post cell implantation, all EZN-2208-treated mice were still disease-free, while control

and CAMPTOSAR-treated animals had already died with metastatic disease.

In the orthotopic NB model, the anti-tumor efficacy of EZN-2208 was evaluated in both

immunocompetent (A/J mice) and immunodeficient (nude mice) NB-bearing animals. A/J mice

were inoculated with the very aggressive NB cell line NXS2 and treated after three days post cell

inoculation, while nude mice were inoculated with GI-LI-N cells and treated after 21 days post cell

inoculation. In both models, mice treated with EZN-2208 showed a dramatic arrest in primary

tumor growth compared to control mice. Moreover, while CAMPTOSAR-treated mice died with

widespread tumor masses within 50 (CAMPTOSAR-resistant animal model) and 80





(CAMPTOSAR-sensitive animal model) days, respectively, long term survival was seen in 100% of

EZN-2208-treated animals (Figure 1,C and Figure 2,A). Furthermore, GI-LI-N-bearing, EZN-2208-

treated mice showed a dramatic regression of the tumor mass, as showed in Figure 2,B.

To assess the impact of EZN-2208 on tumor cell proliferation, viability, and apoptosis, we stained

tumor cryosections taken from NB xenografts (50 days after cells inoculation), and examined them

at low and medium magnification for tumor changes. Histopathological analysis of tumors showed

that both CAMPTOSAR and EZN-2208 inhibited tumor cell proliferation, as assessed by the drastic

decrease in NB84- and Ki-67-positive cells. However, EZN-2208 resulted in a statistically

significant increased anti-proliferative effect compared to CAMPTOSAR (p<0.001), with GI-LI-N

tumors almost disappeared, as assessed by staining histological sections of the tumors with

antibodies recognizing NB cells (NB84) and the cell proliferation marker, Ki-67 (Figure 2,C-D).

In the previous IHC analysis it has been not possible to perform tumor parenchyma- and

angiogenesis-related evaluations because, at the time examined, EZN-2208-treated tumors had

almost disappeared. Thus, we decided to study the anti-tumor, the anti-angiogenic and the anti-

invasive capabilities of EZN-2208 by performing IHC analysis on tumors derived from mice in

which treatments have started at later time point.

GI-LI-N tumors were thus allowed to orthotopically grow for 35 days before treatments started.

Mechanistic experiments performed one day after the end of the treatment showed statistically

significant (P<0.001) enhanced TUNEL and Histone H2AX staining in tumors removed from mice

treated with EZN-2208, indicating its much potent effect on tumor cell apoptosis, compared with

CAMPTOSAR (Figure 3).

Therapeutic effects of MTD doses of EZN-2208 and CPT-11

In the s.c. NB model, luciferase-transfected SH-SY5Y cells were allowed to grow in the right flank

of mice for 13 days before the treatment begun and the anti-tumor efficacy evaluated at different





time points by BLI. As showed in Figure 4,A, while CAMPTOSAR treatment showed a partial and

temporary arrest of tumor growth, EZN-2208 led to a complete regression of primary tumors.

In the very aggressive, orthotopically implanted, syngeneic animal model of NB (NXS2 cells),

CAMPTOSAR did not exert any anti-tumor effect, while EZN-2208 led to 100% of long term

survivors (Figure 4,B). In immunodeficient orthotopic NB (GI-LI-N cells) animal model,

CAMPTOSAR at MTD dose led to a partial increased in long term survival. In contrast, EZN-2208-

treated, GI-LI-N-bearing mice were 100% cured after 180 days post cells implantation (Figure 4,C).

Concordantly with previous results 8.9, EZN-2208 was well-tolerated with neither obvious toxicities

(i.e. no weight loss and no skin rush) nor acute and chronic, liver and renal toxicities (Table 2,

supplementary).

Effect of EZN-2208 on angiogenic responses in the CAM assay

The differences in the anti-angiogenic activity between EZN-2208 and CPT-11 (CAMPTOSAR) in

vivo, using the CAM assay, is shown in Figure 5, A-B. Tumor xenografts derived from NB-

bearing, untreated mice, were grafted onto CAMs. CAMs incubated with CAMPTOSAR showed a

decrease in the number of allantoic vessels radiating in a “spoked wheel” pattern towards the

xenografts, when compared to those incubated with PBS. However, incubation of the CAMs with

EZN-2208 significantly reduced the number of radiating vessels that invaded the implant compared

to either specimens alone or CAMs incubated with CAMPTOSAR, as shown by morphometric

assessment of microvessel density (P<0.01) (Figure 5,A-B).

Effect of EZN-2208 on tumor cell angiogenesis in vivo





The impact of EZN-2208 on angiogenesis was also evaluated by IHC on tumors derived from either

untreated or CAMPTOSAR- and EZN-2208-treated, orthotopically-implanted (GI-LI-N cells),

mice. As showed in Figure 5,C-D, both treatments exerted their anti-angiogenic activities, which

resulted in decreased VEGF and CD31 expression in primary NB tumors. However, in terms of

CD31-positive endothelial cell staining, EZN-2208, resulted in a statistically significant increased

anti-angiogenic effect compared to CAMPTOSAR (P<0.05).

Finally, studies on the tumor invasiveness-related markers, performed in parallel sections,

demonstrated a pronounced, statistically significant inhibition of MMP-2 and MMP-9 expression in

tumors derived from EZN-2208-treated mice (P<0.05), when compared to tissue sections from

CAMPTOSAR-treated animals (Figure 5,E-F).

Taken together, the IHC results obtained clearly indicate a pivotal role of EZN-2208 in inhibiting

tumor angiogenesis and, potentially, the systemically tumor spreading.

Clinical impact of EZN-2208

Once demonstrated the enhanced anti-tumor effect of EZN-2208 compared with Irinotecan in many

biologically relevant xenograft models of NB, we next evaluated the efficacy of EZN-2208

compared to other anti-tumor compounds, either in Phase II/III or clinically used in NB therapy.

These drugs were administered as single-agent therapy or in a combined schedule treatment. Mice

were orthotopically inoculated in the adrenal gland with GI-LI-N cells and then treated 21 days

after tumor challenge. The results are shown in Figure 6. Mice treated with Doxorubicin and

Fenretinide as single agents, behaved as control mice, dying from progression of the primary tumor

and metastatic disease within 75 days post cell inoculation. Cisplatin, Topotecan and Vincristine, as

single agents, led to a partial increased anti-tumor effect compared with control, while Irinotecan,

used at MTD dose, led to a partial increased long term survival. In comparison, EZN-2208-treated

GI-LI-N-bearing mice were 100% cured after 150 days post cells implantation (Figure 6,A).





Furthermore, enhanced life span was also observed in TVD-treated mice, but without any long term

survivors. However, when TVD-treated mice, showing signs of poor health, were subsequently

treated with EZN-2208 (see arrow), they showed a significant increased life span compared with

animals treated only with TVD (P<0.001) (Figure 6,B). From these results, we conclude that EZN-

2208 may be an excellent primary and second line therapy for patients with NB.

DISCUSSION

This work demonstrates the extraordinary therapeutic efficacy of an SN38 drug conjugate (EZN-

2208) in NB xenografts. This novel drug led to a complete tumor regression in all the NB animal

models tested and significantly outperformed CPT-11. Based on these data, exploration of EZN-

2208 for the treatment of NB is warranted.

Better therapies to treat advanced cases of NB have been sought for many years. Currently, high-

risk NB patients treated with radiotherapy, intensive chemotherapy, autologous stem-cell rescue and

with 13-cis-retinoic acid or with monoclonal antibodies targeting NB-specific proteins (25) have a

3-year event-free survival rate of 34% (2, 26). Moreover, due to limiting factors for intensive

induction therapy such as acute and chronic toxicity and the development of secondary neoplasias

(27), the overall outcome for high-risk refractory or recurrent NB patients remains very poor.

The DNA topoisomerase-I inhibitor, Irinotecan (CPT-11; Camptosar) is approved for the treatment

of metastatic colorectal cancer (28), and for some other solid tumors including NB (29). However,

particularly when administered as a single agent in NB, patients given CPT-11 have demonstrated

limited partial responses, disease stabilization (29), or no objective response(6), and to the

Camptothecin analogue Topotecan (5). When given in combination with other agents,

camptothecin analogs have been demonstrated efficacy in preclinical NB models (30, 31) and in





Phase I clinical trials (32, 33). More interestingly, an orally available formulation of Irinotecan, in

combination with Temozolomide, led to partial response in a small number of relapsed high-risk

NB patients and was well tolerated (34).

The lack of remarkable efficacy of CPT-11 is associated with many limitations including limited

conversion of the CPT-11 prodrug to the highly active moiety, SN38 (7, 8), deactivation of SN38 or

CPT-11 by opening of the lactone “E” ring (7, 8), and induction of gastrointestinal side-effects

mediated by the bispiperizane moiety unique to CPT-11, intact CPT-11, or SN38 (35). Pegylated

SN38 overcomes some or all of these limitations since only the highly active moiety, SN38, is

released from the drug conjugate, the pegylation at the C20 position preserves the lactone ring, and

only SN38 is available to possibly induce GI side-effects (9). In addition, it has been experimental

determined in tumor-bearing animals that the pegylated molecule accumulates at the tumor

providing sustained release of SN38, consistent with the proposed “enhanced permeation retention”

(EPR) effect observed with other pegylated molecules (36, 37). However, the basis of the superior

efficacy achieved with PEG-SN38 compared to CPT-11 is incompletely understood. In our

previous studies in mice we have demonstrated that the conversion of CPT-11 to its more active

metabolite, SN38 is low (1.5%) and SN38 derived from PEG-SN38 will be approximately 50-fold

higher and for a longer duration that that derived from CPT-11 (10). Therefore, PEG-SN38 delivers

a higher and sustained level of SN38 in the plasma and the tumor compared with CPT-11 in our

mouse models. This should be compared with others where the conversion of CPT-11 to SN38 in

rodents after a single dose has been reported to vary between 3 to 45% (38, 39) as well as the 2-5%

conversion rate of CPT-11 to SN38 in cancer patients. The basis for this difference in animal

models may be related to methodological details, routes of administration, the exact species and

strains of animals used, and variable carboxyesterase activity that would produce SN38 from CPT-

11 (8).





We have observed that EZN-2208 was highly efficacious and cured not only subcutaneous-

implanted NB tumors-bearing mice, but exerted its anti-tumor effect also against both

pseudometastatic and orthotopic NB xenografts. These models are particularly relevant since they

closely model advanced cases of NB. In the case of the pseudometastatic model, with NB cells

injected intravenously in mice, this xenograft model mimics the metastatic spread observed in

advanced stage NB patients (18). Circulating NB cells in the blood and micrometastases in the

bone marrow at the time of primary surgery of NB patients is a strong predictor of relapse (40).

Since bone marrow micrometastases are a direct measurement of the ability of tumor cells to spread

systemically, the establishment of a model that closely mimics the clinical situation allows a more

realistic evaluation of anti-tumor therapies. Thus, the pseudometastatic model employed in these

studies provides a consistent test for the potential use of EZN-2208 in human metastatic disease.

The schedule of treatment performed, beginning either 24 or 72 hours after NB cells injection, was

deliberately chosen to allow evaluation of the effects of EZN-2208 during the metastatic cascade

(18). While CPT-11 exerted a partial anti-tumor response, EZN-2208, at both schedule treatments

used, displayed 100% of tumor-free mice for more then five months, suggesting a complete

inhibition of both circulating cells and residual disease.

We have also compared the efficacy of EZN-2208 and CPT-11 in orthotopic tumor models of NB,

since such models closely mimic tumor progression, angiogenesis, invasion, and metastasis (41). In

the case of NB, orthotopic models mimic the large adrenal gland tumors and multiple small

metastatic lesions observed in patients (19). Interestingly, EZN-2208 led to a dramatic tumor

regression and to a complete curability of both early-stage, CPT-11-resistant, and more established,

CPT-11-sensitive, NB models (Figure 4), highlighting the overall enhanced anti-tumor activity of

this pegylated SN38 formulation, compared with the pro-drug CPT-11, independently from tumor

stage, sensitivity and schedule of treatment.





EZN-2208 also had superior anti-tumor effect with respect to several clinically used anti-neoplastic

drugs, such as Topotecan, Cisplatin, Doxorubicin and Vincristine. More clinically relevant, EZN-

2208 led to a statistically significant enhanced anti-tumor effects (P<0.001) also in mice that

resulted sensitive, but not cured, to a Topotecan-Vincristine-Doxorubicin (TVD) combination

treatment, which is clinically used as first line treatment for stage IV NB patients (23). These mice,

which were treated with EZN-2208 when signs of poor health became evident as a consequence of

TVD treatment failure (day 80 post cell implantation), displayed a dramatic arrest and regression of

tumor growth, and a long-term survival of about 85% of mice. From these excellent results we

conclude that EZN-2208 could be also used as second-line therapy for refractory NB tumors.

Several molecular mechanisms of action seem to be involved in the EZN-2208-based treatment.

Compared to CPT-11, EZN-2208 was able to induce more DNA damage, apoptosis/necrosis and

p53 expression. Moreover, EZN-2208 exerted its superior potential in inhibiting tumor

angiogenesis. Several recent studies implicate angiogenesis as an essential mechanism regulating

NB growth and anti-angiogenic therapies have been reviewed (42). Sprouting of new blood vessels

from pre-existing capillaries under the influence of pro-angiogenic growth factor expression, such

as VEGF, has been reported (43). HIF-1α mediates angiogenesis by induction of VEGF(44) and

regulates tumor angiogenesis and invasion (45). Moreover, hypoxia-induced decrease in p53

protein level is known to result in cancer cell resistance to DNA damage-induced apoptosis (46).

Thus, the inhibition of HIF-1α could decrease HIF-1α-mediated VEGF production, leading to

apoptosis-mediated cell death and a subsequent block of tumor neo-angiogenesis.

In this work we demonstrated that EZN-2208 was extremely potent in down modulating HIF-1α

and HIF-2a expression, whereas little effect was observed with Irinotecan (Figure 1S, and Sapra P.

et al, manuscript in preparation). It is likely that the reduction in HIF-1α led to an increase of p53

protein, and to a statistically significant decrease in CD31, VEGF, MMP-2, and MMP-9, thus

validating the anti-angiogenesis and the anti-invasion effect of EZN-2208 on NB tumors.





Previously, other topoisomerase I and II inhibitors have been shown to inhibit HIF-1a expression

(47, 48); it is likely that SN38 is a more potent inducer of HIF-1a degradation. In addition, since

HIF-2α is also strongly correlated with high tumor vascularization (49) and it has been

demonstrated promoting aggressive NB phenotypes (50), it is striking that EZN-2208 dramatically

decrease HIF-2α protein level compared with CPT-11. Hence, the anti-angiogenic potential of this

novel drug may further explaining in more details its superior efficacy with respect to CPT-11.

Thus, this anti-angiogenic response could be also wished for the inhibition of secondary neoplasias,

after anti-NB therapies (27).

Collectively, the results obtained provided experimental evidence to strongly support the beginning

of Phase-I for EZN-2208 in pediatric patients with relapsed or refractory high-risk NB patients.

There is ongoing Phase I study of EZN-2208 in a pediatric patients with relapsed solid tumors.

AUTHOR’S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

P. Sapra, L.M. Greenberger and I.D. Horak own stock in Enzon Pharmaceuticals, Inc

Acknowledgements

We thank A. Daga A., H. Zhao, G. Taverniti, and A. Rapisarda for expert technical assistance.

Work supported by Enzon Phamaceuticals, Associazione Italiana per la Ricerca sul Cancro (AIRC,

MFAG and IG), and the Fondazione Italiana per la Lotta al Neuroblastoma.

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LEGENDS

Figure 1. Therapeutic effects of EZN-2208 and CPT-11, at SN38 equivalents, against NB-

bearing mice. Mice were treated every other day for 5 total doses with 10 mg/kg of CPT-11

(CAMPTOSAR) or EZN-2208. Control mice received HEPES buffered saline. A) SCID mice were

subcutaneously (s.c.) injected in the right flank with luciferase (luc)-transfected, human NB cells,

SH-SY5Y, and treated one week after tumor cell implantation. The anti-tumor response was

evaluated for BLI intensity over time (12-50 days). Histograms quantified BLI imaging showing

complete regression of primary tumor growth in mice treated with EZN-2208. Control mice were

sacrificed for excessive tumor mass. Columns depicts the mean of expression of bioluminescent and

errors bars show 95% CI. B) Mice were intravenously (i.v.) inoculated with human NB cells,

HTLA-230, and treated after either 24 or 72 hours from cell inoculation. C) A/J mice

orthotopically implanted in the left adrenal gland with murine NB cells (NXS2) were allowed to

form tumors of approximately 200 mm3 in size and treated 3 days after cells implantation.

Figure 2. Therapeutic effects of EZN-2208 and CPT-11, at SN38 equivalents, against NB-

bearing mice and impact on tumor cell proliferation. Mice were treated every other day for 5

total doses with 10 mg/kg of CPT-11 (CAMPTOSAR) or EZN-2208. Control mice received





HEPES buffered saline. A) Nude mice orthotopically implanted in the left adrenal gland with

human NB cells, GI-LI-N, were allowed to form tumors of approximately 200 mm3 in size and

treated 21 days after cells implantation. B) Dramatic arrest and regression of tumor growth in EZN-

2208-treated mice. Each point represents the mean ± 95% CI. of eight replicates. C) IHC analysis of

NB primary tumors removed from untreated mice (control) and mice treated either with

CAMPTOSAR or EZN-2208. Tumors were harvested on day 50 and tissue sections were

immunostained for NB84, to show NB cells and Ki-67, to show tumor proliferating cells. Cell

nuclei were stained with DAPI. Scale bar 100 μm. D) Columns, mean of NB84 and Ki-67

expressions; errors bars show 95% CI. *, p<0.05; **, p<0.01;***, p<0.001.

Figure 3. Impact of EZN-2208 on in vivo tumor cell apoptosis. Nude mice orthotopically

implanted in the left adrenal gland with human NB cells, GI-LI-N, were allowed to form tumors of

approximately 400 mm3 in size (day 35) and then injected with 10 mg/kg CPT-11 (CAMPTOSAR)

or EZN-2208, every other days for 5 total doses. Control mice received HEPES buffered saline

Histological analysis have been performed on primary NB tumors removed from untreated (control)

and CAMPTOSAR- or EZN-2208-treated mice (44 days after cell inoculation), 24 h after the last

treatment (day 43). Tissue sections were immunostained with TUNEL, to detect apoptosis, and with

primary antibody against Histone H2a.x (H2AFX) to detect DNA-damage depending Histone

phosphorylation. Scale bars 150 μm. Pictures are representative images. Columns, mean of TUNEL

and H2AFX expressions; errors bars show 95% CI. *, p<0.05; **, p<0.01;***, p<0.001.

Figure 4. Therapeutic effects of EZN-2208 and CPT-11, at MTD doses, against NB-bearing

mice. Mice were treated every other day for 5 total doses with 40 mg/kg of CPT-11

(CAMPTOSAR) or with the 10 mg/kg of EZN-2208. Control mice received HEPES buffered saline

A) SCID mice were subcutaneously (s.c.) injected in the right flank with luciferase (luc)-





transfected, human NB cells, SH-SY5Y, and treated 13 days after tumor cell implantation. The

anti-tumor response was evaluated for BLI intensity before (12 day) and after treatment over time

(20-75 days). Histograms quantified BLI imaging showing complete regression of primary tumor

growth in mice treated with EZN-2208. Control mice were sacrificed for excessive tumor mass.

Columns depicts the mean of expression of bioluminescent and errors bars show 95% CI. A/J (B)

and nude (C) mice, orthotopically implanted in the left adrenal gland with NXS2 or GI-LI-N NB

cells, respectively, were allowed to form tumors of approximately 200 mm3 in size and then treated

as in Figures 1-2.

Figure 5. Impact of EZN-2208 on angiogenesis and tumor invasiveness-related markers. A-B)

Chorioallantoic membrane (CAM) assay. A) Biopsy fragments, 1-2 mm3, from xenografts derived

from NB (GI-LI-N and HTLA-230) cells injected in athymic mice were then grafted onto the CAM

either alone (specimens control) or together with CAMPTOSAR or EZN-2208. CAMs were

examined daily until day 12 and photographed in ovo with a stereomicroscope equipped with a

camera and image analyzer system (Olympus Italia, Italy). Original magnification, 50X. B)

Morphometric assessment of microvessel area. Columns depicts the mean of suppression of vessel

density with respect to control (% area = 100%) and errors bars show SEM. C-F) IHC evaluation

of primary NB tumors. Nude mice orthotopically implanted in the left adrenal gland with human

NB cells, GI-LI-N, were allowed to form tumors of approximately 400 mm3 in size (day 35) and

then injected with 40 mg/kg CPT-11 (CAMPTOSAR) or EZN-2208, every other days for 5 total

doses. Tumors were harvested one day after the end of the treatment and tissue sections were

immunostained for VEGF and CD31 (C), to show tumor angiogenesis and for MMP-2 and MMP-9

(E) to show tumor invasion capability. Cell nuclei were stained with DAPI. Scale bar 200 μm.

Pictures are representative images. D, F) Columns, mean value of VEGF and CD31 and MMP-2





and 9 expressions; errors bars show 95% CI. n.s., not significant; *, p<0.05; **, p<0.01;***,

p<0.001.

Figure 6. Clinical relevance of the therapeutic effects of EZN-2208. Nude mice orthotopically

implanted in the left adrenal gland with the human NB cells, GI-LI-N, were allowed to form tumors

of approximately 200 mm3 in size. Treatments started 21 days after cells implantation. A) Mice

were treated every other day for 5 total doses with 40 mg/kg of CPT-11 (CAMPTOSAR) or with

the 10 mg/kg of EZN-2208. Other groups of mice were treated with Topotecan, Cisplatin,

Doxorubicin, Vincristine, Fenretinide at the doses and schedules of treatment showed in M&M

section. B) Mice were treated with a combination of Topotecan (T, daily from day 21 to day 25),

Vincristine (V, at day 25) and Doxorubicin (D, at day 25) with schedule and doses showed in M&M

section. When signs of poor health became evident (arrow), half of TVD-treated mice, randomly

choosen, were then inoculated with EZN-2208, 10 mg/kg every other day for 5 total doses.




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Published OnlineFirst August 11, 2010.Clin Cancer Res Fabio Pastorino, Monica Loi, Puja Sapra, et al. inhibitor

Imodels by PEGylated SN38 (EZN-2208), a novel topoisomerase Tumor regression and curability of preclinical neuroblastoma

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