Metronomic Oral Topotecan with Pazopanib Is an Active Antiangiogenic Regimen in Mouse Models of Aggressive Pediatric Solid Tumor

Published OnlineFirst July 25, 2011; DOI: 10.1158/1078-0432.CCR-11-0078

Clinical Cancer Cancer Therapy: Preclinical Research

Sushil Kumar1,3, Reza Bayat Mokhtari1,2,3, Reihaneh Sheikh2, Bing Wu1, Libo Zhang1,2, Ping Xu4, Shan Man4, Indhira Dias Oliveira1,5, Herman Yeger2, Robert S. Kerbel4, and Sylvain Baruchel1,3

Abstract Purpose: Low dose metronomic (LDM) chemotherapy, combined with VEGF signaling pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth in many adult preclinical cancer models. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, a VEGF receptor inhibitor, in three pediatric extracranial solid tumor mouse models. Experimental Design: In vitro dose–response study of topotecan and pazopanib was conducted on several neuroblastoma, osteosarcoma, and rhabdomyosarcoma cell lines. In vivo antitumor efficacies of the LDM topotecan and pazopanib as single agents and in combination were tested on 4 subcutaneous xenograft models and on 2 neuroblastoma metastatic models. Circulating angiogenic factors such as circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and microvessel densities were used as surrogate biomarker markers of antiangiogenic activity. Results: In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, combination of topotecan þ pazopanib (TP þ PZ) showed significant antitumor activity and significant enhancement in survival compared with the respective single agents in all models. Reductions in viable CEP and/or CEC levels and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. Pharmacokinetic studies of both drugs did not reveal any drug–drug interaction. Conclusion: Metronomic administration of TP þ PZ showed a statistically significant antitumor activity compared with respective single agents in pediatric tumor mouse models and represent a valid option as a maintenance therapy in aggressive pediatric solid tumors. Clin Cancer Res; 17(17); 5656–67. 2011 AACR.

Introduction regimens targeting residual disease represent an exciting opportunity to improve survival rate. Most of these recur- Neuroblastoma and sarcoma are the most common rent patients would have received dose intensitiy che- extracranial childhood solid tumors. Although the 5-year motherapy as an induction or consolidation therapy, overall survival rates in childhood cancers have reached therefore a minimally toxic regimen targeting alternate 80%, the survival rates of a subtype of these aggressive targets should be used in this context. cancers are still below 70% (1). New strategies targeting Angiogenesis is a potential therapeutic target for several alternate mechanisms of resistance and minimal residual types of cancers including pediatric cancers (2). Low dose disease are needed to improve the outcome of these aggres- metronomic (LDM) chemotherapy, which acts in part by sive cancers. Discovery of new maintenance treatment targeting the endothelial cells of the neovasculature of tumors and blocking mobilization of endothelial progenitor cells (CEP) from the bone marrow, represents an Authors' Affiliations: 1Division of Hematology and Oncology, Department attractive and effective antiangiogenic strategy (3–5). of Pediatrics; 2Department of Pediatric Laboratory Medicine, The Hospital for Sick Children; 3Institute of Medical Sciences, University of Toronto; Drawbacks to using pulse topotecan include resistance of 4Sunnybrook Health Sciences Centre, Toronto, Canada; and 5Pediatric tumor cells and the crossresistance with topoisomerase-II Oncology Institute-GRAACC, Department of Pediatrics, Federal University inhibitors in neuroblastoma cell lines (6, 7). LDM regi- of Sao~ Paulo, Sao Paulo, Brazil mens of topotecan, which may target endothelial cells and Corresponding Author: Sylvain Baruchel, New Agent and Innovative Therapy Program, The Hospital for Sick Children, 555 University Ave., tumor cells by other mechanisms, can possibly overcome Room 9418, Black Wing, Toronto, Ontario M5G 1 8, Canada. Phone: some of these limitations. 416-813-7795; Fax: 416-813-5327; E-mail: [email protected] Despite its advantages, relapses may occur in patients doi: 10.1158/1078-0432.CCR-11-0078 who have initially responded to single agent LDM che- 2011 American Association for Cancer Research. motherapy. VEGF, a survival factor for endothelial cells,

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Metronomic Topotecan and Pazopanib in Pediatric Solid Tumors

Translational Relevance line-3,14 (4H,12H)-dione monohydrochloride and pazopanib (5-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)methyl- amino]- Despite advancements in treatments for pediatric 2-pyrimidinyl]amino]-2-methylbenzolsulfonamide were cancers, high mortality is still prevalent in aggressive provided by GlaxoSmithKline. Topotecan-d6 was pur- and advanced stage diseases. Therefore, alternate chased from Toronto Research Chemicals (catalogue # strategies are needed to improve childhood cancer out- T542502). come. The impact of active and abnormal angiogenesis on tumor growth and drug resistance can be exploited to Cell lines develop new promising therapeutic regimens. In this SK-N-BE(2) (N-Myc amplified) and SH-SY5Y (non-N- study, we have tested the overall antitumor efficacy of Myc amplified neuroblastoma cell lines; ref. 16), osteosar- administration of low dose metronomic (LDM) topo- coma cell line KHOS and rhabdomyosarcoma cell lines tecan alone and its combination with a VEGF receptor RH30 and RD, human umbilical vein endothelial cells tyrosine kinase inhibitor pazopanib in 3 aggressive (HUVEC) were obtained from American Type Culture pediatric extracranial solid tumors mouse models: neu- Collection; BE(2)-c subclone of SK-N-BE(2) (17) was roblastoma, osteosarcoma, and rhabdomyosarcoma. obtained from Dr. Michelle Haber (Children’s Cancer Administration of LDM topotecan showed a significant Institute for Medical Research, Lowry Cancer Research antitumor efficacy in most tumor types, either localized Centre, Randwick, Australia); NUB-7, an I-type neuroblas- or metastatic, which was amplified in all tumor types by toma cell line (18) was obtained from Dr. Herman Yeger the adjunction of pazopanib. These results support the (The Hospital for Sick Children, Toronto, Ontario). Neu- development of phase-I clinical trials in these 3 cate- roblastoma cell lines were grown in alpha-MEM (# 310- gories of aggressive childhood solid tumors. 010-CL, Wisent Bioproducts), while sarcoma cell lines were grown in Dulbecco’s Modified Eagle’s Medium (# 319-010- CL, Wisent bioproducts), both containing 10% FBS and 1% antibiotic mixture in humidified atmosphere at 37C may be responsible, at least in part, for the loss of anti- with 5% CO2. tumor efficacy of LDM chemotherapy (8). As a result, combining metronomic therapy with agents that target In vitro cytotoxicity the VEGF-signalling pathways is associated with an overall A toatl of 50,000 cells were seeded in 48 well plates and increase in antitumor activity. We have previously shown incubated for 48 hours, after which they were treated with the therapeutic advantage of such combinations by com- topotecan and/or pazopanib for 72 hours. Cell viability bining LDM vinblastine with VEGF receptor 2 (VEGFR2)- was determined by Alamar blue assay. Alamar blue (10% of neutralizing antibody, DC101, in a human neuroblastoma total volume) was added to each well 3 hours before xenograft preclinical study (9). Such combinations have fluorometric detection. Fluorometric detection was done also been found to be effective in other preclinical and using the SPECTRAmax gemini Spectrophotometer at exci- clinical trials (10–12). Recently, 2 independent studies tation wavelength of 540 nm and emission wavelength of have reported the marked superiority of the combination 590 nm. of daily oral LDM topotecan and the small molecule receptor tyrosine kinase inhibitor (RTKi) pazopanib com- In vivo models pared with either single agent therapy in models of For subcutaneous xenograft studies, we used SK-N-BE advanced ovarian cancer (13, 14). This combination and (2), SH-SY5Y, KHOS, and RH30. 1 106 cells were their relevant pharmacokinetic and pharmacodynamic implanted subcutaneously into the inguinal fat pad of markers have never been explored in multiple preclinical each of nonobese diabetic/severe combined immune pediatric tumor models. deficient (NOD/SCID) mice. When tumors reached The main objective of this study was to compare the 0.5 cm in diameter, the animals were randomized into antitumor efficacies of LDM topotecan and its combination 4 groups and treated daily by oral gavage. The animals with pazopanib, a multikinase inhibitor (15) in 3 types of were grouped as: Control group, LDM topotecan group or pediatric solid tumors and to evaluate circulating angio- LDM TP (1.0 mg/kg topotecan), pazopanib group or PZ genic factors (CAF) such as viable CEPs and circulating (150 mg/kg pazopanib) and combination group or TP þ endothelial cells (CEC) and tumor microvessel densities as PZ (1.0 mg/kg topotecan þ 150 mg/kg pazopanib). To surrogate markers of drug activity. Pharmacokinetic (PK) compare pulse topotecan with LDM TP in KHOS osteo- analysis was conducted as well, to detect possible drug– sarcoma model, PZ was replaced by weekly oral dose of drug interaction. pulse topotecan or Pulse TP (15 mg/kg topotecan). The criteria for endpoint were tumor sizes exceeding 2.0 cm in Materials and Methods diameter or animals showing signs of morbidity. The tumor sizes were measured on a daily basis until the Drugs and reagents endpoint or sacrifice. The long (D) and short diameters Topotecan (S)-10-[(dimethylamino)methyl]-4-ethyl-4, (d) were measured with calipers. Tumor volume (cm3) 9-dihydroxy-1H-yrano [30,40:6,7] indolizino[1,2-b]quino- was calculated as V ¼ 0.5 D d2. When the endpoint

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was reached or at the end of the treatment, the animals CD13þ, while CECs were defined as CD45, VEGFR-2þ, were sacrificed by cervical dislocation. CD117, and CD13þ. 7-AAD was used to exclude the apoptotic cells. The absolute number of CEPs was calcu- Metastatic mouse model lated as the percentage of events collected in CEP enumera- A total of 1 106 BE(2)-c cells or NUB-7 cells were tion gates multiplied by the total white blood cell (WBC) injected into lateral tail veins of NOD/SCID mice to gen- count. erate experimental metastases as previously described (19). Fourteen days after injection, the mice were randomized Bone marrow progenitor assay into 4 groups and treated in same way as the inguinal The bone marrow progenitor culture was done as pre- xenograft model. The treatment was continued until death viously published by our group (21). After the sacrifice of or endpoint for BE(2)-c model and till 14 days for NUB-7 animals belonging to the RH30 model, the femurs were model. isolated and the bone marrow was flushed out with AMEM Protocol and endpoints were approved by the Sickkids containing 2% FBS. Bone marrow cells of mice belonging animal committee facility following the CACC guidelines. to each group was pooled and 200,000 cells were cultured in methyl cellulose media (Methocult #3434, Stem Cell Immunohistochemistry and histopathology Technologies) in 35 mm culture suspension dish, in Formalin-fixed tissues were paraffin embedded and sec- triplicate. Bone marrow cells of nontumor-bearing mice tions cut at 7 um. Sections were deparaffinized through (n ¼ 3) were pooled and cultured (in triplicate) concur- xylene and ethanol, rehydrated in PBS (# 311-010-CL, rently with those of each group of tumor-bearing mice and Wisent Bioproducts) and incubated overnight with primary were used as reference culture plates. After 14 days, colony antibodies for von Willebrand factor (vWF; # A0082; forming units-granulocyte, macrophages (CFU-GM) were DakoCytomation) at 4C. After the primary antibody treat- counted under the optical microscope. ment, all the slides were washed 3 times with PBS and incubated with broad spectrum poly-horse radish perox- PKs of topotecan and pazopanib idase (HRP) conjugated secondary antibody (# 87-9663, Nontumor-bearing animals were randomized into 4 Invitrogen) for 1 hour at room temperature. After washing groups (n ¼ 3): Control, PZ, LDM TP, and TPþPZ. The 3 times with PBS, slides were stained with diaminobenzi- doses of the drugs were the same as for the inguinal dine and counterstained with hematoxylin. Microscopic xenograft and metastatic models described above. images were captured by Olympus UTV1-X microscope After single drug administration, the saphenous vein mounted with Qimaging Retiga 2000R camera. blood samples (30 mL) were collected in heparinized Frozen sections from SH-SY5Y tumor model were fixed microcentrifuge tubes at 30 minutes, 1 hour, 2 hours, 4 with 4% paraformaldehyde, permeabilized with 0.05% hours, 8 hours, 12 hours, 18 hours, and 24 hours as per the Triton X-100. After blocking with 5% bovine serum albu- Laboratory Animal Services protocol. Plasma was imme- min in PBS for 1 hour, the sections were incubated over- diately isolated after blood collection by centrifugation. For night with rabbit polyclonal anti-CD31 antibody the topotecan assay, 10 mL plasma was immediately pre- (#ab28364, Abcam, dilution 1:50). The sections were cipitated with 20 mL methanol and centrifuged. The super- washed 3 times with PBS containing 0.1% tween 20 (PBST) natant and rest of the plasma were stored at 80 mC until and incubated with Alexa fluor 594 donkey anti-rabbit IgG analysis. (#A21207, Invitrogen, dilution 1:300) for 1 hour. After Assay of pazopanib. A total of 5 mL of plasma was washing with PBST, the slides were mounted with Vecta- precipitated with 40 mL methanol and centrifuged. A total shield mounting medium (#H-1200, Vector, with DAPI). of 30 mL of supernatant was injected into the high-perfor- The microscopic images of the stained sections were cap- mance liquid chromatography (HPLC) system, which con- tured by Nikon ECLIPSE Ti series fluorescence microscope, sisted of Phenomenox C18 column (Luna; 150 4.6 mm; using NIS Elements (BR 3.10) software. particle size 5 m), UV detector (267 nm). The mobile phase Microscopic images of 6 fields of high vascular density was 50:50 mixture of 10 mmol/L potassium phosphate and were digitally captured and the pixel values for stained methanol, with flow rate 1.0 mL/min. The concentrations of areas were quantified by using ImageJ software. Tumor calibration standards were 5.0, 10, 50, 100, and 200 mg/mL. angiogenesis was quantified as the number of pixels of Assay of topotecan. Before analysis, the previously pre- regions positive for von Willebrand Factor or CD31. pared 20 mL methanolic extract was mixed with 50 mLof internal standard solution (5 ng/mL d6 topotecan dis- Analysis of CAFs (CEPs and CECs) solved 0.1% formic acid in acetonitrile). The mixture Approximately 160 mL of mouse blood was collected in was then centrifuged and the supernatant was transferred K2-EDTA tubes by saphenous vein puncture in SH-SY5Y, to autosampler vials. KHOS, and RH30 model after 20, 28, and 31 days, respec- The LC/MS system consisted of an HPLC (Agilent Infinity tively. Blood was immediately stored at 4C until analysis. 1290), Column (Kinetex HILIC, 2.6 m, 100 A, 50 4.6 The CEP/CECs were measured by flow cytometry within 48 mm) and a mass spectrometer (Sciex 5500-QTrap). The hours of blood collection as previously described (20). analytes were eluted by gradient flow. Mobile phase A CEPs were defined as CD45, VEGFR-2þ, CD117þ, and was water:acetonitrile (10:90) and mobile phase B was

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Metronomic Topotecan and Pazopanib in Pediatric Solid Tumors

A CD120 140 120 Topotecan 100 Topotecan 120 100 Combination Combination 100 80 80 80 60 60 60 40 40 % Viability 40 SH-SY5Y SK-N-BE2

20 % Viability [RD] 20 20 KHOS RH30 0 0 0 % Viability [SK-N-BE(2)] 0 2,000 4,000 6,000 8,000 10,000 0.01 0.1 1 10 100 1,000 0.01 0.1 1 10 100 1,000 10,000 [Pazopanib], ng/mL [Topotecan], ng/mL [Topotecan], ng/mL B 120 120 120 Topotecan Topotecan 100 100 100 Combination Combination 80 80 80 60 60 60 40 40 40 20 20 20 % Viability [RH30] % Viability [BE(2)-c] % Viability [HUVEC] 0 0 0 0.01 0.1 1 10 100 1,000 0.01 0.1 1 10 100 1,000 0.01 0.1 1 10 100 1,000 10,000 [Topotecan], ng/mL [Topotecan], ng/mL [Topotecan], ng/mL

120 120 100 Topotecan Topotecan 100 100 Combination 80 Combination 80 80 60 60 60 40 40 40 20 20 20 % Viability [KHOS] % Viability [HUVEC] 0 % Viability [SH-SY5Y] 0 0 0.1 1 100101,000 10,000100,000 0.01 0.1 1 10 100 1,000 1 10 100 1,000 10,000 [Pazopanib], ng/mL [Topotecan], ng/mL [Topotecan], ng/mL

Figure 1. In vitro efficacy of topotecan and pazopanib. A, effect of pazopanib on neuroblastoma cell lines. B, dose–response plots of topotecan and pazopanib on HUVEC. C and D, dose–response plots of topotecan alone and in combination with 5,000 ng/mL pazopanib on neuroblastoma cell lines and sarcoma cell lines, respectively.

10 mmol/L ammonium acetate (pH 3.2). The mobile phase (Fig. 1A). Both topotecan and pazopanib caused a ratio was 5% A for 0 to 2 minutes, 20% A for 4 to 6 minutes dose-dependent reduction in viability of HUVEC with and 5% A for 8 to 10 minutes at a flow rate 0.5 mL/min. The IC50 of 4.87 ng/mL and 398.0 ng/mL, respectively samples were analyzed by positive ion electrospray ioniza- (Fig. 1B). Topotecan showed a dose-dependent reduction tion technique in multiple reaction monitoring modes. in the viability of all the tumor cell lines (Fig. 1C and D). The following mass transitions were monitored: 422.2 to Among neuroblastoma cell lines, SH-SY5Y (IC50 ¼ 377.0 m/z, (topotecan MþH) and 428.2 to 377.0 m/z 5.3 ng/mL) was more sensitive to topotecan than (topotecan d6 MþH). The concentrations of calibration BE(2)-c (IC50 ¼ 45.6 ng/mL) and SK-N-BE(2) (IC50 ¼ standards were 0.5, 1.0, 5.0, 10, and 100 ng/mL. 65.0 ng/mL; Fig. 1C). Among sarcoma cell lines, the IC50 of topotecan on RH30, Statistical analysis RD, and KHOS cell lines were 7.4 ng/mL, 7.5 ng/mL, and In vitro dose response, in vivo tumor growth curves and 4.9 ng/mL, respectively (Fig. 1D). Among all tumor cell the number of pixels for immunohistochemistry are pre- lines tested, addition of 5,000 ng/mL pazopanib only sented as mean SD. Statistical significance was assessed caused a significant reduction of IC50 in SK-N-BE(2) cells by student’s t test. Prism 5 (Version 5.04) for Windows, (IC50 ¼ 35.1 ng/mL, P ¼ 0.046; Fig. 1C). GraphPad Software was used for the calculation of IC50 and P values. LDM topotecan and pazopanib in neuroblastoma mouse models Results The criteria for effectiveness of the treatments in SK-N-BE (2) xenograft model were tumors regression and enhance- Drug-induced in vitro cytotoxities ment of survival times. Drugs were administered daily over Pazopanib did not affect the viabilities of the any of a period of 56 days. In addition, the animals belonging to the treated cell lines at any of the concentrations tested the TP þ PZ group, which remained alive, were also

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A SK-N-BE(2) xenograft model 7 100 6 n = 4 5 80 4 60 3 40 2 % survival 20

Tumor volume (cc) volume Tumor 1 0 0 Figure 2. In vivo effects of 0 25 50 75 100 125 0 20 40 60 80 100 120 140 topotecan and pazopanib Days Days treatment. A, tumor growth rate (left) and Kaplan–Meier survival B SH-SY5Y xenograft model BE(2)-c metastatic model curve (right) for SK-N-BE(2) xenograft model. Dotted line n = 5 6 100 indicates that the treatment was n = 5 stopped after 56 days, while solid 80 line indicates that the animals belonging to combination group 4 60 were retreated starting from 103 † 40 days. , One animal in combination 2 % survival group died due to gavaging error on 9th day of treatment. B, tumor

Tumor volume (cc) volume Tumor 20 growth rate in SH-SY5Y xenograft 0 0 model. C, Kaplan–Meier survival 0 4 8 12 16 20 0 20 40 60 80 100 120 140 curve for BE(2)-c metastatic Days Days model. In BE(2)-c metastatic model, all 3 treatment groups had C KHOS xenograft model RH30 xenograft model significantly higher life span than 6 control group (P < 0.005). D and E, n = 5 6 n = 5 tumor growth rate for model, 5 KHOS osteosarcoma model, and 4 RH30 rhabdomyosarcoma model, 4 respectively. 3

2 2

Tumor volume (cc) volume Tumor (cc) volume Tumor 1 0 0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 80 Days Days

Conrol PZ LDM TP Pulse TP TP + PZ

retreated from the 103rd day to evaluate the impact of the tumor endpoint was reached. Tumor growth delay and resuming treatment on reversing drug resistance (Fig. 2A). the difference in tumor weights at the end of the treatment Retreatment was continued until the 125th day, after were the criteria for assessment of treatment effectiveness. which the mice were sacrificed. In this model, no sig- The treatments caused tumor growth delay in the order PZ< nificant difference was observed between the tumor LDM TP< TPþPZ (Fig. 2B). All the 3 treatment groups growth rates of the LDM TP and PZ-treated groups. showed significant difference in tumor weights compared Compared with control, both the single agents signifi- with the control (Fig. 3A). LDM TP caused significant cantly enhanced survival of animals (P < 0.05). The tumor weight reduction compared with PZ, while TP þ survival in TP þ PZ group was significantly higher com- PZ caused significant tumor weight reduction compared pared with both control (P < 0.005) and the single agents’ with both LDM TP and PZ. groups (P < 0.005). Retreatment was associated by tran- BE(2)-c and NUB-7 are N-Myc amplified, I-type malig- sient tumor growth delay up to 120 days. All the animals nant neuroblastoma cells which have high potential to were sacrificed by 125th day. migrate and metastasize (17, 18). Survival time was used In SH-SY5Y xenograft model, animals belonging to all as the parameter to assess the efficacy of treatments in our the 4 groups were sacrificed after 20 days treatment, when BE(2)-c metastatic model. All the treatment groups showed

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Metronomic Topotecan and Pazopanib in Pediatric Solid Tumors

ABSH-SY5Y xenograft model K HOS metastatic model C NUB-7 metastatic model

Control

PZ or Pulse TP

LDMTP

TP + PZ

P < 0.0001 P < 0.0001 P = 0.0008 P = 0.002 P < 0.0001 4 P = 0.004 4 P = 0.01 P = 0.04 4 P < 0.0001 3 3 P < 0.0001 3 P = 0.04 P = 0.003 2 2 2 P = 0.007 P = 0.008 1 1 1 Weight of liver (g) of liver Weight

0 (g) KHOS tumor weight SH-SY5Y tumor weight (g) SH-SY5Y tumor weight 0 0 Control PZ LDMTP TP + PZ Control Pulse TP LDMTP TP + PZ ControlPZ LDMTP TP + PZ D Control PZ LDMTP TP + PZ

BE (2)-c

NUB-7

Figure 3. A and B, photographs and histograms depicting the tumor weight comparison in SH-SY5Y and KHOS models, respectively. C, photograph and histogram depicting the liver weight comparison in NUB-7 metastatic model. D, microscopic images of hematoxylin and eosin stained sections of livers in BE(2)-c (magnification, 40) and NUB-7 metastatic models (magnification, 10). The tumor-bearing areas in the microscopic sections can be distinguished by dark blue color, compared with pink colored nontumor-bearing areas.

a statistically significant enhanced survival (Fig. 2C). Sur- confirming the ability of TP þ PZ to control liver metastasis vival of LDM TP–treated animals was higher than PZ- (Fig. 3D). treated animals (P < 0.05). The mean survival span of animals in TP þ PZ group was approximately 2-fold Effect of LDM topotecan and pazopanib on the tumor (100.8 days) compared with the LDM TP group (52.4 growth in sarcoma models days), P < 0.005. At the time of death or endpoint, the Because PZ had shown limited efficacy in neuroblastoma animals belonging to control, PZ and LDM TP groups had models, we decided to evaluate the antitumor activity of macroscopically detectable tumors in liver. Animals Pulse TP and compare it with LDM TP in KHOS osteosar- belonging to TP þ PZ group did not reveal any evidence coma model, in which the mice were sacrificed after 28 days of liver metastasis (Fig. 3D). Animals in all the 4 groups of treatment. Here, both Pulse TP and LDM TP delayed the BE(2)-c model had evidence of tumors present in kidney, tumor growth, with significantly lower tumor weight at the adrenal gland, and bone marrow. end of the treatment (Fig. 2D, Fig. 3B). The tumor growth NUB-7 metastatic model, the animals belonging to all rate curve (Fig. 2D) reveals that the single agents caused the 4 groups were sacrificed after 14 days treatment. Com- tumor growth delay, but not tumor size reduction, while TP pared with the control, LDM TP and TP þ PZ liver weights þ PZ, induced tumor growth delay until 22 days, after were significantly lower in TP þ PZ–treated animals, com- which tumor size reduction was observed. The TP þ PZ pared with PZ (Fig. 3C). Microscopic tumors were visible in group had significantly lower tumor weights compared the livers of mice belonging to all the groups except TP þ PZ with the control, Pulse TP and LDM TP (Fig. 3B).

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In RH30 rhabdomyosarcoma RH30 xenograft model, reduced WBC level significantly, whereas LDM TP did not the animals were treated for 56 days. The animals belong- (Fig. 5B). Despite the significant lowering of WBC induced ing to control and LDM TP reached the endpoint before by the combination, compared with the control and the this period, while those in PZ and TP þ PZ–treated groups single agents, the animals belonging to this group in all the remained alive after the discontinuation of treatment models were active and showed no signs of illness during or (Fig. 2E). LDM TP was ineffective in controlling the tumor after this period until tumor sizes reached the endpoint. growth. In view of activity of PZ in soft tissue sarcoma, we decided to test PZ. PZ as a single agent and the combination Bone marrow CFU-GM assay TP þ PZ delayed the tumor growth and enhanced the To better understand the impact of LDM TP and combi- survival by 2-fold, compared with both control and nation on bone marrow function, CFU-GM were counted LDM TP. TP þ PZ group had significantly lower tumor in RH30 model, where the mice were sacrificed at different size (P ¼ 0.03), compared with those of PZ group. times, that is, day 32, day 35, day 71, and day 73 for control, LDM TP, PZ, and TP þ PZ, respectively. Percentage Effect of treatment on tumor microvessel densities CFU-GM count for each plate was calculated as the per- Comparison of the pixel counts of 6 fields of highly centage of CFU-GM number in that plate to the average vascularized regions of tumor sections stained for CD31 CFU-GM number in reference plates (Fig. 5D). LDM TP– and vWF revealed that TP þ PZ significantly reduced the treated group had significantly lower CFU-GM counts microvessel density of the tumors, compared with the compared with the control. TP þ PZ–treated group had control in SH-SY5Y, RH30, and KHOS models (Fig. 4A– signficantly lower CFU-GM number compared with the C). In SH-SY5Y model, PZ but not LDM TP caused sig- control but not compared with the single agent groups. nificant reduction in microvessel densities compared with the control. In RH30 model, none of the single agents PK did not reveal drug interaction between topotecan caused reduction in microvessel densities, compared with and pazopanib in TP PZ group the control, while in KHOS model, both Pulse TP and LDM The PK of topotecan and pazopanib was conducted to TP caused reduction in microvessel densities. detect any PK interaction between topotecan and pazopanib in TP þ PZ group. The peak plasma concentration of Effect of the treatments on CAFs (CECs/CEPs) pazopanib was reached in 2 hours in both PZ and TP þ PZ CEPs originate from the bone marrow and also the groups (Fig. 6A and B). The Cmax of pazopanib was 133.5 adipose tissue (22). VEGF stimulates the recruitment of ng/mL and 122.4 ng/mL in PZ and TP þ PZ groups, CEPs into the tumor neovasculature and thus contributes respectively, while the trough concentration was 9.46 to the endothelial lining (4). Therefore blockade of the ng/mL and 14.56 ng/mL, respectively. Peak plasma con- VEGF-signalling pathway is expected to reduce the CEP centrations of topotecan in LDM TP and TP þ PZ groups level in blood and thus inhibit angiogenesis. In our SH- were 19.75 ng/mL and 33.05 ng/mL, respectively, while the SY5Y neuroblastoma model, after 20 days treatment, TP þ trough concentration was 0.77 ng/mL and 2.79 ng/mL PZ significantly reduced both viable CEC and CEP levels (Fig. 6A and B). For both drugs, no significant difference compared with the control and single agents groups was observed between plasma concentrations of single (Fig. 5A). Though the single agents caused reduction in agent and combination treated animals at any time point both CEC and/or CEP levels, compared with the control, A significant interanimal drug concentration variability was the differences were not statistically significant. detected and larger group studies may be necessary to In RH30 rhabdomyosarcoma model, after 31 days treat- detect drug–drug interactions and changes in trough con- ment, TP þ PZ caused significant reductions in both viable centration. The previously reported optimal plasma con- CECs and CEPs levels compared with the control and LDM centration of pazopanib effectiveness (40 mmol/L or 18 TP (Fig. 5B). Compared with PZ, TP þ PZ caused significant mg/mL; ref. 15) was maintained until at least 18 hours in viable CEP reduction. PZ-treated group had significantly both PZ and TP þ PZ groups. lower viable CEP levels compared with the control. In KHOS osetosarcoma model, CEP and CEC levels were Discussion measured after 28 days treatment (Fig. 5C). TP þ PZ caused significant reduction in viable CEC and CEP levels com- Angiogenesis plays important roles in cancer growth, pared with the control and Pulse TP. Also, LDM TP caused a metastasis, and response to therapy. In pediatric tumors significant reduction in viable CEP levels compared with such as neuroblastoma, osteosarcoma, and rhabdomyosar- Pulse TP. coma, in situ tumor angiogenesis and the levels of circulat- WBC count was used as a parameter to assess bone ing angiogenic factors correlates with metastatic disease marrow toxicity. In SH-SY5Y and KHOS model model, and poor prognosis (23–25). all the 3 treatment regimens significantly reduced WBC. LDM chemotherapy alone has shown clinical benefit in TP þ PZ–treated group had significantly lower WBC levels several pediatric cancers and its maximum-tolerated dose compared with both the single agents (Fig. 5A and C). has been established in phase-I trials (26–28). The combi- Surprisingly, LDM TP had significantly lower WBC count nation of LDM chemotherapy with RTKis have been tested compared with pulse TP. In RH30 model, PZ and TP þ PZ in several preclinical studies, including metronomic

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Metronomic Topotecan and Pazopanib in Pediatric Solid Tumors

ABSH-SY5Y Model RH30 Model CD31 CD3 and DAPI Control LDMTP Control

2000 µm 2000 µm

PZ TP + PZ

µ µ PZ 2000 m 2000 m P = 0.002 P = 0.002

12 P = 0.003 10 8 TP 6 Figure 4. A, microscopic images 4 of highly vascularized areas, stained for CD31 in tumor sections 2 from SH-SY5Y xenograft (original of vVVF + areas Pixels 0 magnification, 10). The red color Control PZ LDMTP TP + PZ represents regions stained for CD31. (B) and (C) are the sections TP + PZ from RH30 and KHOS xenografts, C KHOS Model respectively, stained for vWF Control LDMTP (original magnification, 10). Arrows point toward regions stained for vWF.

2000 µm 2000 µm

PZ TP + PZ P = 0.0003

P = 0.04 P = 0.03 P = 0.0003 12.5 2000 µm 2000 µm P = 0.001 10.0 P = 0.01 7.5 10 P = 0.02 5.0 8 P = 0.002 2.5 6 P = 0.02 Pixels of CD31 + areas Pixels 0.0 Control PZ LDMTP TP + PZ 4 2

Pixels of vVVF + areas Pixels 0 Control Pulse TP LDMTP TP + PZ

topotecan and pazopanib in ovarian cancer (13, 14, 29, 30) Despite reports regarding antitumor activity of such com- and in clinical trials (31, 32). binations, their effectiveness in a particular pediatric cancer Neuroblastoma was the first preclinical tumor model to model cannot be predicted on the basis of its effects on validate the concept of combining metronomic chemother- other cancer models. In our experience combination of apy with antiangiogenic therapy (9). However, the metronomic cyclophosphamide and sunitinib did not have mechanism of increased efficacy and safety of metronomi- any advantage over sunitinib monotherapy when tested in cally administered of drug combinations and their PKs a neuroblastoma preclinical xenograft model (19). Also, have never been studied widely in pediatric cancers. in a previous study, the combination of axitinib with

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Kumar et al.

A SH-SY5Y Model BCRH30 Model KHOS Model P = 0.02 P = 0.003 P = 0.001 0.5 0.5 P = 0.006 4 P = 0.003 0.4 P = 0.002 0.4 P = 0.01 3 0.3 P = 0.009 0.3 P = 0.01 2 0.2 0.2 0.1 0.1 1 Viable CEC/ µ L blood Viable Viable CEC/ µ L blood Viable 0.0 0.0 0 Control PZ LDMTP TP + PZ Control PZ LDMTP TP + PZ CEC/10 µ L blood Viable Control Pulse TP LDMTP TP + PZ

P = 0.02 P = 0.0008 P = 0.0007 P = 0.001 P = 0.02 0.15 P = 0.02 P = 0.01 0.06 P = 0.03 P = 0.008 1.5 Figure 5. The effect of the P = 0.003 0.10 P = 0.04 P = 0.02 treatment regimens on CEP/CEC 0.04 1.0 levels in blood. A, comparison of 0.05 0.02 0.5 CAF and WBC levels in SH-SY5Y xenograft model after 20 days 0.00 CEP/ µ L blood Viable 0.00 0.0 Viable CEP/ µ L blood Viable Control PZ LDMTP TP + PZ Control PZ LDMTP TP + PZ CEP/10 µ L blood Viable Control Pulse TP LDMTP TP + PZ treatment; B, the comparison of CAF and WBC levels in RH30 P < 0.0001 P = 0.004 P = 0.007 model after 31 days treatment; C, P = 0.0005 P = 0.02 20 300 the comparison of CAF and WBC 30 P = 0.03 P < 0.0001 P = 0.002 P = 0.004 P = 0.004 P = 0.001 levels in KHOS xenograft model 15 P = 0.006 P = 0.02 20 P < 0.0001 200 after 28 days treatment; and D, the P = 0.01 10 comparison of percentage CFU- 10 100 5 GM among the 4 groups in RH30 xenograft model. WBC/100 µ L blood WBC/1000 µ L blood

WBC/100 µ L blood 0 0 0 Control PZ LDMTP TP + PZ Control PZ LDMTP TP + PZ Control Pulse TP LDMTP TP + PZ

D Bone marrow progenitor assay P = 0.0007 150 P = 0.046

100

50 % CFU-GM

0 Control PZ LDMTP TP + PZ

metronomic cyclophosphamide was less effective than nomic chemotherapy with a particular RTKi should be metronomic cyclophosphamide alone in gliosarcoma confirmed preclinically and the proper dose and preclinical model (33). Therefore, the benefit of combining metro- PK need to be established before moving to phase-I clinical trials. Here, we evaluated the effectiveness of LDM regimen of oral topotecan and its combination with one of the Single agent groups clinically approved RTKi, pazopanib, in the murine models A 150 60 AUC0–24 = 1,802 ng/mL.h AUC = 93.55 ng/mL.h 125 of 3 pediatric solid tumors, with particular emphasis on the 50 0–24 100 antiangiogenic mechanism and their potential bone mar- 40 75 30 row toxicity. 50 20 The doses of drugs were selected on the basis of previous 10 25

[Pazopanib], µ g/mL [Pazopanib], studies. The daily oral doses of 1.0 mg/kg topotecan and [Topotecan], ng/mL [Topotecan], 0 0 0 5 10 15 20 25 0 5 10 15 20 25 150 mg/kg pazopanib have been previously found to be Time (h) Time (h) B TP + PZ group effective in ovarian cancer mouse models (14). Shaked and 60 150 colleagues has previously defined the optimal biologic AUC0–24 = 1,733 ng/mL.h 50 AUC0–24 = 122.0 ng/mL.h 125 dose (OBD) of LDM chemotherapy as the dose causing 40 100 maximum reduction in CEPs with minimal or no toxicity 30 75 20 50 after daily treatment for 1 week; this dose is associated with 10 25 maximum antiangiogenic efficacy (34). In a previous dose– [Topotecan], ng/mL [Topotecan], 0 µ g/mL [Pazopanib], 0 0 5 10 15 20 25 0 5 10 15 20 25 response study, the daily dose of oral metronomic topo- Time (h) Time (h) tecan (0.5, 1.0, and 1.5 mg/kg) caused greater reduction in microvascular density compared with weekly maximum- Figure 6. Plasma concentration–time profiles of topotecan and pazopanib tolerated dose regimen (7.5 and 15 mg/kg) in an ovarian in single agents group (A) and in combination group (B). cancer model, but the mice treated with 1.5 mg/kg daily,

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Metronomic Topotecan and Pazopanib in Pediatric Solid Tumors

oral topotecan showed decreased food intake, and a lesser CEP level was showed with PZ alone after 31 days treat- antitumor effect (35). By applying the aforementioned ment, thus correlating with its tumor response. After definition of OBD, we postulated that 1.0 mg/kg oral exposure to single agent PZ, the microvessel densities topotecan administered daily, would be the OBD, or of tumor xenografts, isolated at the time of tumor pro- within the range of the OBD. The antiangiogenic efficacy gression 2 weeks after discontinuation of treatment, were of weekly pulse topotecan and daily LDM topotecan has not different from those of control group. TP þ PZ had also been compared in our osteosarcoma model. significantly low viable CEPs than PZ. By analyzing the In vitro, pazopanib neither had any effect on the viability observations from tumor growth rate, CAF levels, and of any of the cell lines, nor did it enhance the cytotoxicity of microvessel density experiment, we are postulating that in topotecan on any of the cell lines except SK-N-BE(2) but rhabdomyosarcoma model, PZ and TP þ PZ are more was active on HUVEC cell lines. In agreement with our effective than LDM TP and that the antiangiogenic effec- hypothesis, in vivo, LDM topotecan and its combination tiveness of TPþPZ is more sustained than PZ after the with pazopanib delayed the tumor growth and significantly discontinuation of the treatment. enhanced the animal survival in all the models, TP þ PZ PK interaction between 2 coadministered drugs is an showing higher antitumor efficacy compared with LDM TP important consideration. No such studies have been and PZ or Pulse TP. LDM TP was more effective than PZ in conducted so far in the context of metronomic che- neuroblastoma models, while in RH30 model, PZ was motherapy and combination with VEGF RTKi agents. more effective in delaying tumor growth than LDM TP. Pazopanib is a substrate of CYP3A4 (15), while topo- The delay of tumor growth at metastatic sites by TP þ PZ in tecan is a CYP3A4 inhibitor, which is reported to reduce NUB-7 and BE(2)-c metastatic models indicates that the the clearance of another CYP3A4 substrate (37). There- combination of LDM topotecan and pazopanib can poten- fore, we compared the plasma concentration–time pro- tially control minimal residual disease and enhance the files of each drug when administered alone and in survival in high risk neuroblastoma. combination. Our PK did not reveal any significant Numerous preclinical and clinical studies have shown differences in the plasma concentrations of LDM TP the value of CECs and CEPs as potential biomarkers of or PZ between single agent and the combination groups, antiangiogenic activity (36). Dose-dependent decreases at any of the time points examined. However, a signifi- of CEPs have been observed with LDM administration of cant interanimal variability was detected at the trough several cytotoxic agents (34). Recently, a similar combina- level of TP in the TP þ PZ group, though it did not reach tion of metronomic topotecan and pazopanib, caused a statistical significance; it was higher in the TP þ PZ significant reduction in CEP levels in an ovarian cancer group than in TP group. For pazopanib, 40 mmol/L preclinical model (14). Our data confirm these findings ( 18 mg/mL) has been reported to be the optimum in all pediatric tumor models with various degree of plasma concentration for the inhibition of VEGFR2 responses. phosphorylation in mice (15). Since the plasma con- In neuroblastoma, TP þ PZ delayed tumor growth in centration of pazopanib was above this limit until 18 SK-N-BE(2) and SH-SY5Y models, and reduced micro- hours, it can be concluded 150 mg/kg pazopanib can metastasis in BE(2)-c and NUB-7 models. The superiority inhibit VEGFR2 phosphorylation for at least 18 hours of the combination over the single agents could be after oral drug administration. partially explained by its antiangiogenic activity, as In summary, combination of LDM topotecan and pazo- observed by the significant reduction of all the 3 markers: panib has higher antitumor efficacy compared with single viable CECs, viable CEPs and tumor microvessel density, agents in neuroblastoma, rhabdomyosarcoma, and osteo- by the TPþPZ, compared with the both LDM TP and PZ in sarcoma. This combination caused significant lowering of SH-SY5Y neuroblastoma models. However, among the CAFs, compared with control and single agents. Since no single agents, only PZ shows antiangiogenic activity, as other synergism or additive effect have been observed observed by the significant reduction in the microvessel between topotecan and pazopanib in in vitro and PK density. experiments, the mechanism behind the efficacy of the In KHOS osteosarcoma model, all the regimens tested combination can be attributed to antiangiogenic activity caused significant reduction in the levels of viable CECs in all 3 solid tumor models. Direct interaction with VEGF and CEPs and microvessel densities after 28 days treatment. signaling pathway within tumor cells cannot be excluded Though there was no significant difference between the (38). Potential bone marrow toxicity may be expected with tumor weights of Pulse TP and LDM TP upon sacrifice, the the combination of LDM topotecan and pazopanib and viable CEP levels in LDM TP treated group were signifi- caution should be taken before claiming that such a com- cantly lower than those in Pulse TP treated group, indicat- bination is not myelotoxic. ing that metronomic topotecan is more antiangiogenic These results support development of this phase-I com- than the pulse dosing of topotecan. bination in pediatric solid tumors with a potential, if In RH30 rhabdomyosarcoma model, TP þ PZ caused proven to be safe to be integrated into poststem cell significant reduction in viable CEC and CEP levels and transplantation regimen. This new maintenance stategy microvessel density compared with both control and would potentially target highly resistant minimal residual LDM TP. In addition, significant reduction in viable disease.

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Disclosure of Potential Conflict of Interest Dr. Kai Du in the Development and Stem Cell Biology Program, Hospital for Sick Children for his technical help and expert assistance.

No potential conflicts of interest were disclosed. Grant Support Acknowledgments This work is supported by the James Birrell Neuroblastoma Fund; GlaxoSmithKline, Collegeville, PA, Sickkids Foundation, Toronto, and University Mass Spectrometry was done by Michael Leadley at the Analytical Facility of Toronto Fellowship (Institute of Medical Sciences). for Bioactive Molecules (AFBM) of the Centre for the Study of Complex The costs of publication of this article were defrayed in part by the payment Childhood Diseases (CSCCD) at the Hospital for Sick Children, Toronto, of page charges. This article must therefore be hereby marked advertisement in Ontario. CSCCD was supported by the Canadian Foundation for Innova- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tion (CFI). Tissue section preparation, histology, and von Willebrand Factor staining were done by Huimin Wang and Erika Takarich at Pathology Research Laboratory, Department of Laboratory Medicine and Pathobiology Received January 11, 2011; revised July 5, 2011; accepted July 6, 2011; (DPLM) at the Hospital for Sick Children, Toronto, Ontario. We thank published OnlineFirst July 25, 2011.

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Metronomic Oral Topotecan with Pazopanib Is an Active Antiangiogenic Regimen in Mouse Models of Aggressive Pediatric Solid Tumor

Sushil Kumar, Reza Bayat Mokhtari, Reihaneh Sheikh, et al.

Clin Cancer Res 2011;17:5656-5667. Published OnlineFirst July 25, 2011.

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