Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
`
`cancer Therapy Preclinical
`
`Clinical
`Cancer
`Research
`
`Is an Active Drug in Preclinical Model of
`NabPaclitaxel
`Pediatric Solid Tumors
`
`Libo Zhang12 Paula Marrano2 Sushil Kunnar4 Michael Leadley2 Evelyn Elias2 Paul Thorner and
`Sylvain Baruchell
`
`Abstract
`
`an albumin stabilized nanoparticle
`
`Purpose To investigate the antitumor effect of nabpaclitaxel
`formulation of paclitaxel on pediatric solid tumor models
`Experimental Design A panel of three rhabdomyosarcoma
`one osteosarcoma and seven neuroblas
`toma cell
`in vitro Cell viability was
`to increasing concentrations of nabpaclitaxel
`lines were exposed
`evaluated using the Alamar Blue Assay Antitumor effect was further assessed in vivo in NODSCID xenograft
`and metastatic neuroblastoma mouse models Tumor sections were analyzed by immunohistochemistry for
`cleaved caspase3 and phosphohistone H3 Plasma and intratumoral paclitaxel concentrations were
`measured by liquid chromatography mass spectrometry Ratio of intratumoral and plasma concentration
`and paclitaxel
`was compared between nabpaclitaxel
`treatment groups
`displayed significant cytotoxicity against most pediatric solid tumor cell lines in
`Results Nabpaclitaxel
`vitro in a dose dependent manner In vivo nabpaclitaxel
`showed antitumor activity in both rhabdomyo
`sarcoma RH4 and RD and neuroblastoma SKNBE2 and CHLA20 xenograft models In the SKN BE
`2 metastatic model nabpaclitaxel
`extended animal survival compared with
`significantly
`control P < 001 Nabpaclitaxel
`induced tumor cell cycle arrest and apoptosis in vivo In the
`treatment 377 ± 32
`RH4 model
`increased local relapsefree intervals were observed with nabpaclitaxel
`days comparing with paclitaxel 136 ± 207 days Local relapsed tumors following paclitaxel
`treatment
`and remained responsive to nabpaclitaxel Mechanistically
`proved to be paclitaxelresistant
`was observed
`tumorplasma paclitaxel drug ratio in favor of nabpaclitaxel
`showed significant antitumor activity against all pediatric solid tumors
`Condusions Nabpaclitaxel
`associated with an enhanced drug intratumor delivery Furthermore testing of nabpaclitaxel
`in pediatric
`solidtumor patient population is under development Clin Cancer Res 1921 597283 ©2013 AACR
`
`treatment
`
`treatment
`
`a higher
`
`Introduction
`Solid tumors make up about 60 of all pediatric cancers
`1 The most common types of solid tumors in children
`
`include brain tumors neuroblastoma rhabdomyosarco
`ma Wilms tumor and osteosarcoma Although significant
`progress has been made during the last decades
`in the
`treatment and prognosis of pediatric malignancies
`high
`in patients with advanced unre
`mortality is still prevalent
`
`sectable or highgrade disease 2 Rhabdomyosarcoma is
`
`the most common pediatric soft tissue sarcoma and the
`third most common extracranial solid tumor in children
`
`Authors Affiliations
`
`New Agent and Innovative Therapy Program
`2Department of Paediatric Laboratory Medicine 3Division of Hematology
`and Oncology Department of Paediatrics The Hospital
`for Sick Children
`of Toronto Toronto Canada
`and 4Institute of Medical Sciences University
`
`Corresponding Author Sylvain Baruchel The Hospital
`for Sick Children
`Room 9418 Black Wing 555 University Avenue Toronto Ontario M5G
`1X8 Canada Phone 41681359777795
`Fax 4168135327 Email
`sylvai nbaruchelsickkidsca
`
`doi 10115810780432CCR131485
`
`©2013 American Association
`
`for Cancer Research
`
`following neuroblastoma and Wilms tumor with an annu
`the
`al incidence of 4 to 7 cases per million children under
`age of 16 years 3 4 Multimodal
`treatment has signifi
`to approximately 70 Although
`cantly improved survival
`a majority of patients achieve a complete remission with
`primary therapy a substantial number still experience
`recurrences with poor prognosis 5 In addition at
`least
`15 children with rhabdomyosarcoma
`present with met
`astatic disease The Intergroup Rhabdomyosarcoma Study
`Group IRSG and their prognosis has not improved sig
`nificantly in the last 15 years 6
`hood malignancy It accounts for 8 to 10 of all pediatric
`malignancy and is responsible for 15 of all childhood
`cancer related deaths 7 Despite intensive treatment pro
`
`Neuroblastoma is the most common extracranial child
`
`survival
`
`stem
`tocols including megatherapy with hematopoietic
`and immunotherapy the prognosis of
`cell transplantation
`patients with this malignancy remains poor Disease free
`is only about 30 for metastatic disease compared
`with 95 for localized tumors 8 9
`Paclitaxel originally isolated from Taxus brevifolia Pacific
`yew is a cytotoxic microtubule stabilizing agent
`that
`
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`ARAmerican Association
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`for CancerResearch
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`2013 American Association for Cancer Research
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`Abraxis EX2018
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`1PR201701101 1PR201701103 1PR201701104
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`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
`
`Translational Relevance
`
`Paditaxel has been used widely for the treatment of
`it has shown minimal activity in
`adult solid tumors but
`pediatric solid tumors Here we evaluated a novel
`solvent free formulation of paditaxel nabpaditaxel
`in
`pediatric solid tumor models Nabpaditaxel
`is effective
`against both rhabdomyosarcoma and neuroblastoma in
`predinical models We also showed that nabpaclitaxel
`in all tumor models tested
`more active than paditaxel
`tumors developed resistance against
`Paditaxelrelapsed
`treatment but those tumors remained respon
`elevat
`treatment Mechanistically
`sive to nabpaclitaxel
`ed intratumor and correspondingly
`lower plasma pac
`levels were observed with nabpaditaxel com
`litaxel
`pared with paditaxel resulting in higher tumorplasma
`paditaxel drug ratio for nabpaditaxel Therefore this
`predinical study supports further testing of nabpacli
`taxel in pediatric solidtumor patient population
`
`paditaxel
`
`is
`
`stabilizes microtubules and as a result interferes with the
`normal breakdown of microtubules during cell division and
`lead to mitotic arrest 10 This mitotic arrest triggers the
`mitotic spindle checkpoint and results in apoptosis 10
`shown to mediate apoptosis
`Paditaxel
`has also been
`through pathways that may be independent of gross micro
`tubule dysfunction 1113 Paditaxel
`is widely used in the
`including ovarian can
`treatment of various malignancies
`cer breast cancer non small cell lung carcinoma NSCLC
`bladder cancer head and neck cancer and a wide range of
`other malignancies 10 14 However due to its very low
`like Taxol uses a
`aqueous solubility conventional paditaxel
`Cremophor ELethanol vehicle as the solvent The Cremo
`phor EL containing paditaxel
`formulation can cause severe
`and anaphylactic reactions in ani
`allergic hypersensitivity
`mals and humans which limited its application in patients
`with pediatric cancer 1517 Although paditaxel were
`reactive to several neuroblastoma cell
`lines in vitro it dis
`played little efficacy in vivo in nude mice 18 In a phase I
`in children with refractory solid tumors severe acute
`neurologic and allergic toxicity was encountered with 3
`hour paditaxel
`infusion after premedication dexametha
`sone dexchlorpheniramine with one treatment related
`death occurred 19 For
`reasons
`unclear
`that are still
`taxanes showed minimal activity in treating solid tumors
`trials 2022
`in children in several clinical
`Nabpaditaxel also known as ABI007 Abraxane Cel
`gene Corporation is a novel solvent free 130 nm protein
`that was developed to
`stabilized formulation of paditaxel
`reduce the toxicity of solvent based paditaxel known as
`Taxol refs 23 24 In human adult
`tumor models nab
`in inducing regression of implanted
`paditaxel
`is effective
`human malignant carcinomas and prolonging survival
`in
`tumor bearing mice 25 Clinically
`nabpaclitaxel
`approved globally for the treatment of metastatic breast
`cancer MBC and is also recently approved in the United
`
`trial
`
`is
`
`Preclinical Study of Nabpaclitaxel
`
`States for the first line treatment of locally advanced
`or
`metastatic NSCLC in combination with carboplatin in
`patients who are not candidates for curative surgery or
`radiotherapy As limited pediatric data are currently avail
`able in this study we tested the antitumor activity of nab
`paditaxel against different pediatric solid tumor models to
`the development of earlyphase clinical
`support
`trials in
`
`pediatric patients
`
`Materials and Methods
`
`Materials and reagents
`Nabpaclitaxel Celgene Corporation was supplied as
`a lyophilized powder and stored at room temperature
`until reconstitution Nabpaclitaxel was reconstituted fol
`lowing the package insert with 20 mL 09 saline to 5
`mgmL stock solution The dosing solutions were pre
`pared by diluting the stock solution with 09 saline to
`
`the desired concentration Paditaxel was dissolved in
`dimethyl sulfoxide DMSO Sigma Aldrich to 25 mgmL
`stock solution The dosing solutions were prepared by
`diluting the stock solution with 09 saline to the desired
`
`concentration
`The Annexin VFITC Early Apoptosis Detection Kit was
`Inc The cleaved
`purchased from Cell Signaling Technology
`caspase3 Asp175 antibody was obtained from Cell Sig
`Inc The antihistone H3 phospho S10
`naling Technology
`antibody was purchased from Abcam Inc
`
`for
`
`Cell culture
`RH4 RH30 and RD rhabdomyosarcoma cells and KHOS
`osteosarcoma cells were gifts from Dr David Malkin The
`for Sick Children Toronto Canada LAN5 SK
`Hospital
`NBE2 BE2C and SHSY5Y neuroblastoma cells were
`kindly provided by Dr Herman Yeger The Hospital
`Sick Children CHLA15 CHLA20 and CHLA90 were
`obtained from the Childrens Oncology Group Cell Culture
`and Xenograft Repository under a signed and approved
`Material Transfer Agreement RH4 RH30 and RD rhabdo
`myosarcoma cells were cultured in Dulbeccos modified
`Eagle medium supplemented with 10 FBS CHLA15
`CHLA20 and CHLA90 neuroblastoma cells were cultured
`in Iscoves modified Dulbeccos medium supplemented with
`3 mmolL Lglutamine insulin and transferin 5 ugmL each
`and 5 ngmL selenous acid ITS Culture Supplement Col
`and 20 FBS complete
`laborative Biomedical Products
`medium LAN5 SKNBE2 BE2C and SHSY5Y neu
`roblastoma cells were cultured in a minimum essential
`medium with 10 FBS KHOS osteosarcoma cells were
`cultured in Eagles minimumessential medium supplemen
`ted with 10 FBS
`
`Test animals
`Female NO DS CID nonobese diabeticsevere combined
`immunodeficient mice 4 to 6week old were obtained
`from Charles River Laboratories Animals were housed in
`
`for Sick Children These
`the animal facility of the Hospital
`for Sick Chil
`studies were conducted under
`the Hospital
`dren Animal Care Committee approval
`
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`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
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`Zhang et al
`
`Cell viability assay
`Cells were seeded into 24 well
`tissue culture plates at a
`in culture medium and
`density of 200000 cells per well
`incubated for 24 hours at 37°C before starting drug treat
`ment Cells were exposed to increasing concentrations of
`for 72 hours The viability of proliferating
`nabpaditaxel
`cells in the control and treated media were measured with
`the Alamar Blue Assay according to manufacturers protocol
`Trek Diagnostics Systems Inc Briefly Alamar Blue was
`diluted 1 to 10 in the cell culture media and the fluorescent
`color change was monitored after 3 hours Colorimetrical
`evaluation of cell proliferation was conducted
`using a
`SpectraMax Gemini spectrophotometer with 540 nm as
`excitation wavelength and 590 nm as emission wavelength
`and values
`as relative fluorescence units Cell
`expressed
`viability was measured in triplicate and calculated relative
`to control nontreated cells
`
`Immunofluorescence analysis of apoptosis
`Annexin V was used to detect apoptosis with the Annexin
`VFITC Early Apoptosis Detection Kit Cell Signaling Tech
`nology Inc Cells were cultured 2 x 105 cells on cover
`slips overnight before the treatment with nabpaditaxel for
`48 hours For apoptosis staining with annexin Vfluores
`cein is othiocyanate FITC after incubated with annexin V
`FITC according to manufacturers protocol
`washed and fixed in 2 formaldehyde before visualization
`
`the cells were
`
`under a fluorescence microscope using a dual
`set for
`FITCAnnexin V green and DAPI nuclei staining blue
`
`filter
`
`Xenograft development
`The antitumor activity of nabpaditaxelpaditaxel was
`investigated in vivo against subcutaneous rhabdomyosarco
`ma RH4 and RH30 and neuroblastoma SKNBE2 and
`CHLA20 using NODSCID tumor xenografts Briefly
`tumor cells were washed three times with Hanks Balanced
`Salt Solution HBSS before injection Mice were given a
`tumor cells Tumor
`1 x 106
`subcutaneous injection of
`growth was measured weekly in two dimensions using a
`digital caliper and tumor volume was calculated as width2 x
`length x 05 Once the tumor diameter
`reached 05 cm
`mice were randomized into treatment groups with 7 to ten
`animals in each group Nabpaditaxel was administered
`either at low dose metronomic LDM administration three
`different doses of 2 5 or 10 mgkg iv daily consecutively
`for over 3 weeks or cytotoxic dose 50 mgkg iv weekly
`15 cm3 Pacli
`until tumor volume reached approximately
`at 20 or 30 mgkg
`taxel was administered intravenously
`weekly Control mice received saline
`Tumor volume mouse body weight and signs of animal
`distress were evaluated twice or three times a week for any
`potential drug toxicity Animals were sacrificed once the
`tumor size reached 15 cm3
`In tumor rechallenge experiments when RH4relapsed
`reached 05 cm in diam
`tumors from paditaxel
`treatment
`eter we randomized
`these animals into two treatment
`groups n = 3 nabpaditaxel and paditaxel For nab
`paditaxelrelapsed tumors we randomized
`
`animals into
`
`two groups n = 3 nabpaditaxel
`treatment and saline
`control Drugs were given with the same schedule and
`dosage as above Tumor growth and animal body weight
`were monitored
`The anti metastatic activity of nabpaditaxel was further
`investigated in SKNBE2 neuroblastoma metastatic mod
`els Tumor cells were injected intravenously into the lateral
`tail vein 26 gauge needle 1 x 106 cells in 100 iL total
`volume Mice were randomized into two groups control
`and nabpaditaxel 50 mgkg iv weekly with 10 mice in
`each group and treatments started 14 days after inoculation
`the event of endpoint The event of endpoint was
`until
`defined according to our animal committee guidelines as
`mice in severe clinical condition such as loss of 20 of
`body weight body temperature lower than 32°C or signs of
`stress The survival time of control and nabpaditaxel treat
`ment groups was compared and statistically analyzed
`
`Immunohistochemistry
`staining
`To assess the effect of nabpaditaxel on inducing cell
`cycle arrest and apoptosis in vivo SKNBE2 subcutaneous
`xenografts treated with nabpaclitaxel or DMSOpaditaxel
`were harvested at the end of study and analyzed by immu
`nohistochemistry IHC for the apoptotic marker cleaved
`and mitotic marker phosphohistone H3
`caspase3
`RH4 xenografts were harvested and analyzed by IHC for
`phosphohistone H3 Briefly formalinfixed paraffin
`embedded tissues were cut
`into 5 iimthick sections and
`immunostained with the polydonal antibodies
`against
`caspase3 150 Cell Signaling
`Technology
`cleaved
`9661 and histone H3 phospho S10 12000 Abcam
`ab5716 Heat induced epitope retrieval was conducted
`and the ImmPRESS Anti Rabbit Ig Peroxidase Reagent Kit
`Vector Laboratories MP 7401 was used for detection
`followed by DAB and hematoxylin staining
`
`LCMS plasmaintratumor concentration
`Plasma and intratumor drug concentration was studied
`after single or repeated drug administration In RH4 xeno
`graft model bloodtumor samples were collected 24 hours
`dosage of nabpaditaxel 50 mgkg or
`the first
`after
`paditaxel 30 mgkg In the SKNBE2 xenograft model
`paditaxel 20 mgkg and nabpaclitaxel 50 mgkg were
`administered on day 1 8 and 15 LDM nabpaditaxel 10
`mgkg was administered daily from day 1
`to day 15
`Twentyfour hours after the last dosage of nabpaditax
`elpaditaxel blood and tumor samples were collected and
`analyzed for paditaxel concentration by liquid chroma
`tographytandem mass spectrometry LCMSMS The
`system consisted of HPLC Agilent 1200 reverse phase
`column Phenomenex Kinetex XBC18 26 u 100A 50 x
`30 mm and mass spectrometer Sciex 4000 The ana
`lytes were eluted by gradient flow The mobile phase A was
`01 formic acid in water and mobile phase B was 01
`formic acid in acetonitrile The mobile phase ratio was
`65 A and 35 B till 25 minutes gradual
`increase to
`100 B till
`35 minutes 100 B till 55 minutes and
`finally return to initial condition ie 65 A and 35 till
`
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`Clinical Cancer Research
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`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
`
`Preclinical Study of Nabpaclitaxel
`
`10 minutes The run time was 10 minutes and the flow rate
`was 300 4min Samples were ionized by electrospray
`ionization in positive polarity mode and acquisition was
`done by multiple reaction monitoring Following mass
`transitions were monitored paclitaxel M + H 8545
`2861 mz docetaxel M + H 8085
`5272 mz Ratio of
`intratumoral versus plasma concentration was calculated
`and compared between nabpaclitaxel and DMSObased
`treatment groups
`
`paclitaxel
`
`Statistical analysis
`Data from different experiments were presented as mean
`± SD For statistical analysis Student t test for independent
`means was used A P value of < 005 was considered
`significant To compare the effects of different
`treatments
`on tumor growth in vivo oneway ANOVA with Dunnett
`multiple comparison test was used Mann Whitney U test
`was used in tumor rechallenge experiments for the statistical
`evaluation of significant
`differences in growth patterns
`
`between two study groups Survival curve comparisons were
`conducted using GraphPad Prism software for Kaplan
`Meier survival analysis
`
`Results
`
`In vitro cytotoxicity of nabpaditaxel against pediatric
`tumor cell
`lines
`against a wide
`To determine the efficacy of nabpaclitaxel
`panel of pediatric cancer cells lines three rhabdomyosar
`coma RH4 RH30 and RD seven neuroblastoma cell lines
`CHLA20 CHLA15 CHLA90 LAN5 SKNBE2 BE2
`line KHOS
`C and SHSY5Y and one osteosarcoma cell
`for viability with Alamar Blue assays after
`were tested
`exposing cells to increasing concentrations of nabpaclitaxel
`in vitro for 72 hours As shown in Fig 1A the cell viability of
`cell lines were reduced following
`three rhabdomyosarcoma
`increasing dose of nabpaclitaxel
`treatment
`IC50 values
`were calculated and ranged from 056 to 468 nmolL
`
`RH4
`R H30
`
`100
`
`>
`
`50
`
`CHLA20
`CHLA15
`SKNBE2
`LAN 5
`CHLA90
`SHSY5Y
`BE2C
`
`100
`
`50
`
`0
`
`101
`
`101
`
`100
`102
`103
`Nabpaclitaxel nmolL
`
`104
`
`101
`
`101
`
`100
`102
`103
`Nabpaclitaxel nmolL
`
`104
`
`103 102 101
`100
`102
`103
`Nabpaclitaxel nmolL
`
`101
`
`104
`
`SK N BE2 Nab paclitaxel
`SKNBE2Paclitaxel
`0SHSY5YPaclitaxel
`
`SHSY5YNabpaclitaxel
`
`100
`
`50
`
`5
`
`CH LA 20N abpaclitaxel
`CH LA20Paclitaxel
`
`100
`
`50
`
`CH LA15Nabpaclitaxel
`4 CH LA15Paclitaxel
`CH LA90Paclitaxel
`4 CH LA90Nabpaclitaxel
`
`100
`
`50
`
`100
`
`50
`
`A
`
`5 a0
`
`0
`
`o I
`103 102 101
`
`100
`
`101
`
`102
`
`103
`
`104
`
`o I
`103 102 101
`
`100
`
`101
`
`102
`
`103
`
`104
`
`103 102 101
`
`10°
`
`101
`
`102
`
`103
`
`104
`
`Nabpaclitaxelpaclitaxel nmolL
`
`Nabpaclitaxelpaclitaxel nmolL
`
`Nabpaclitaxelpaclitaxel nmolL
`
`CHM
`
`LI=2
`
`LE=
`
`100 ngmL
`
`Figure 1 Effects of nabpaclitaxel on pediatric tumor cell
`after exposing tumor cells to increasing
`lines in vitro Alamar Blue assays were conducted
`cell lines RH4
`viability was plotted
`concentrations
`in vitro for 72 hours Cell
`with GraphPad Prism software on three rhabdomyosarcoma
`of nabpaclitaxel
`cell line KHOS B and seven neuroblastoma
`RH30 and RD A the osteosarcoma
`cell lines CHLA20 CHLA15 CHLA90 LAN 5 SKNBE2 BE2C and
`SHSY5Y C D neuroblastoma
`lines were treated with the same concentration
`viability was compared between
`paclitaxel and nabpaclitaxel E cell apoptosis was analyzed by annexin VFITC fluorescence
`staining RH4 cells were treated with 12 60 or 120 nmolL
`in vitro for 48 hours followed by annexin VFITC staining Untreated cells are shown for comparison control
`nabpaclitaxel
`
`in vitro Cell
`
`cell
`
`of
`
`paclitaxel
`
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`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
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`Zhang et al
`
`Limited response was observed with osteosarcoma cell line
`KHOS Fig 1B
`For the seven neuroblastoma
`lines nabpaclitaxel
`cell
`exhibited dose dependent cytotoxicity in vitro as measured
`by cell viability Fig 1C Different
`cell
`lines displayed
`variable sensitivity for nabpaclitaxel Among all
`these cell
`IC50 36 nmolL whereas
`lines CHLA20 has the highest
`LAN 5 and SKNBE2 have the lowest EC50 Furthermore
`when neuroblastoma cell lines were treated for 72 hours in
`vitro all the tested cell lines showed more sensitivity to nab
`than to paclitaxel dissolved in the solvent DMSO
`Fig 1D suggesting that paclitaxel
`in albumin bound
`formulation in solution more readily available for tumor
`
`paclitaxel
`
`cell uptake
`
`Nabpaditaxelinduced apoptosis in
`rhabdomyosarcoma cells
`We further assessed cell apoptosis after in vitro drug
`treatment Rhabdomyosarcoma RH4 cells were incubated
`for 48
`with increasing concentrations of nabpaclitaxel
`hours and analyzed for apoptosis with annexin VFITC
`Annexin VFITCconjugated protein binds to cell surfaces
`expressing phosphatidylserine an early apoptosis marker
`as shown by annexin V
`apoptotic RH4
`Increased
`cells
`FITCpositive staining was observed following nabpacli
`taxel treatment Fig 1E With the higher concentration of
`60 or 120 nmolL most cells detached
`nabpaclitaxel
`from the coveslips but almost all
`the remaining cells
`showed annexin VFITCpositive staining
`
`In vivo antitumor activity of nabpaditaxel against
`rhabdomyosarcoma
`The in vivo antitumor activity of nabpaclitaxel was
`tumor xenografts In
`evaluated in multiple pediatric
`rhabdomyosarcoma models mice bearing RH4 and RD
`xenografts were treated intravenously with nabpaclitaxel
`50 mgkg and paclitaxel 30 mgkg The 50 mgkg
`is estimated to be compa
`weekly dose of nabpaclitaxel
`rable with human maximum tolerated dose MTD 150
`mgm2 weekly based on the calculation from US Food
`and Drug Administration dose conversion
`guidelines
`is the MTD in mice
`The 30 mgkg dose of paclitaxel
`26 and it
`is comparable with the highest
`in
`adult patients too
`and DMSOpaclitaxel
`treatments sig
`Both nabpaclitaxel
`nificantly inhibited RH4 tumor growth with tumor regres
`after the second dosage on day 8 Fig 2A
`sion observed
`animals treated with paclitaxel showed lower
`However
`and control
`body weight compared with nabpaclitaxel
`animals Fig 2B and 1 of 7 mice in paditaxel group died
`on day 10 Especially during the first week after paclitaxel
`administration all mice showed signs of
`inappetence
`hunched posture dry feces and significant body weight
`loss Necropsy of the dead animal showed that both cecum
`and colon were distended with hard stools Our results
`showed that paclitaxel
`even at a lower dose had higher
`In the RH4 model
`toxicity compared with nabpaclitaxel
`increased local
`relapse free intervals were observed with
`
`dosage
`
`treatment 377 ± 32 days comparing to
`nabpaclitaxel
`paclitaxel 136 ± 207 days
`In RD xenograft model both paclitaxel and nabpacli
`inhibited tumor growth but
`taxel treatment significantly
`tumor shrinkage was only observed
`in nabpaclitaxeltrea
`ted tumors Fig 2C
`
`Efficacy of nabpaditaxel in paditaxelresistant or
`relapsed rhabdomyosarcoma xenografts
`In RH4 xenografts either paclitaxel or nabpaclitaxel was
`administered on day 1 8 and is Complete regression was
`in paclitaxeltreated mice after day 31 Fig 2D
`observed
`showed tumor
`However
`animals
`all paclitaxeltreated
`relapse after 11 to 15 days On day 52 when paclitaxel
`relapsed tumors reached 05 cm in diameter we random
`ized animals into two treatment groups nabpaditaxel and
`paclitaxel Drugs were given on day 52 59 and 66 with the
`same schedule and dosage as above As shown in Fig 2D
`relapse RH4 xenografts were drug resistant against paclitax
`el but
`remained sensitive to nabpaclitaxel
`treatment
`Tumor
`relapsed tumors
`regression was observed
`in all
`which were treated again with nabpaclitaxel P < 005
`Mann Whitney U test
`Complete regression was observed
`in nabpaclitaxel
`treated mice after day 29 Fig 2E Six of 7 animals devel
`oped relapsed tumor after 37 to 42 days On day 75 when
`nabpaclitaxelrelapsed tumors reached 05 cm in diameter
`we randomized
`animals into two groups nabpaclitaxel
`treatment and saline control Nabpaclitaxel or saline was
`given on day 75 82 and 87 with the same schedule and
`dosage as above As seen in Fig 2E when we treated those
`relapsed RH4 tumors with nabpaclitaxel 50 mgkg week
`ly again refractory tumors remained responsive to nab
`paclitaxel We observed significant difference between nab
`tumor volumes P< 005
`paclitaxel rechallenge and control
`Mann Whitney U test
`In RD xenograft model paclitaxel 30 mgkg weekly or
`nabpaclitaxel 50 mgkg weekly was administered on day
`1 and 8 Tumor regression was observed with nabpaclitaxel
`treatment Comparing with control animals paclitaxel
`treatment was able to slow the growth of RD tumors but
`those tumors grew progressively with no signs of tumor
`regression On day 15 when we replaced the paclitaxel drug
`50 mgkg weekly those
`treatment with nabpaclitaxel
`tumors regressed rapidly after the first dosage of nabpac
`litaxel Fig 2C
`
`Antitumor effects of nabpaditaxel with different
`regimens in neuroblastoma models
`We further compared different
`and doses of
`schedules
`nabpaclitaxel LDM and standard MTD schedule in neu
`roblastoma xenograft models Subcutaneous mouse xeno
`graft tumors SKNBE2 and CHLA20 were treated with
`at 2 5 and 10 mgkg
`either vehicle alone nabpaclitaxel
`daily or 50 mgkg weekly Control mice received saline
`at 2 5 10 mgkg iv daily
`Increasing doses of nabpaclitaxel
`clearly showed greater tumor growth inhibition with SKN
`BE2 in a dose dependent manner Fig 3A The 2 mgkgd
`
`5976
`
`Clin Cancer Res 1921 November 12013
`
`Clinical Cancer Research
`
`Downloaded from clincancerresaacrjournalsorg on June 26 2017 © 2013 American Association for Cancer Research
`
`Abraxis EX2018
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`Page 5 of 13
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`

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`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
`
`Preclinical Study of Nabpaclitaxel
`
`A
`
`treatment
`Nabpaclitaxelpaclitaxel
`in RH4 xenograft model
`
`Effect of nabpaclitaxelpaclitaxel
`on mouse body weight RH4
`
`1400
`
`Control
`Nabpaclitaxel 50 mgkg
`Radix 30 mgkg
`
`> 130
`
`o 120
`
`0o T
`
`Control
`Nabpaclitaxel 50 mgkg
`Paclitaxel 30 mgkg
`4 Paclitaxel RelapsePaclitaxel
`
`Nabpaclitaxel Relapse
`
`Ot 40120
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`90
`
`100 110
`
`Days after nabpaclitaxeVpaclitaxel
`
`treatment
`
`111
`
`100
`
`a
`
`1g
`
`90
`
`0
`
`20
`
`40
`
`60
`
`80
`
`Days after nabpaclitaxelpaclitaxel
`
`treatment
`
`treatment
`Nabpaclitaxelpaclitaxel
`in RD xenograft model
`
`Paclitaxel treatment
`in RH4 xenograft model
`
`treatment
`Nabpaclitaxel
`in RH4 xenograft model
`
`_ Control
`Radix 30 mgkg
`
`RelapsePaclitaxel
`RelapseNabpaclitaxel
`
`M Control
`
`Nabpaclitaxel
`
`LI Relapse
`Relapse nab pachtaxel
`
`Control
`_ Nabpaclitaxel
`Paclitaxel
`
`Paclitaxelpaclitaxel
`
`Paclitaxelnabpaclitaxel
`
`4
`
`3
`
`2
`
`2 1
`
`2
`
`0
`
`°
`
`10
`
`20
`
`30
`
`20
`
`Ot
`
`Days after nabpaclitaxelpaclitaxel
`
`treatment
`
`40
`
`30
`
`5 f 170
`Days after paclitaxel
`
`80
`
`90
`
`100 110
`
`f0t20
`
`30
`
`40
`
`50
`
`60
`
`708Q
`
`00 110
`
`treatment
`
`Days after nabpaclitaxel
`
`treatment
`
`paclitaxel
`
`nabpaclitaxeltreated
`
`xenografts Small arrows indicate time points of drug administration A in RH4
`Figure 2 Effects of nabpaclitaxel on the growth of rhabdomyosarcoma
`xenografts when xenograft
`tumors reached above 05 cm in diameter mice were randomized into 3 groups control nabpaclitaxel
`treatment and
`on day 18 and 15 at the dose of 30 and 50 mgkg
`treatment with 7 animals in each group Paclitaxel or nabpaclitaxel was administered
`respectively Tumor volumewas measured and calculated aswidth2 x length x 05 Complete regression was observed in both paclitaxel and
`RH4 tumors B animal body weight was measured in RH4 model
`to monitor the potential drug toxicity C tumor growth was assessed in RD
`treatment group n = 10 tumorbearing mice received nabpaclitaxel
`In the nabpaclitaxel
`treatment
`xenograft model with paclitaxelnabpaclitaxel
`after 2 week paclitaxel
`50 mgkg weekly treatment
`treatment group n = 10 as tumor sizes reached the endpoint
`In the paclitaxel
`treatment
`in the
`receiving 30 mgkg
`paclitaxel group we randomized those mice into two groups with 5 animals in each group on day 15 one group of animals continued
`instead shown in orange curve D on day 52 when
`tumor growth curve shown in red and the other group received 50 mgkg of nabpaclitaxel
`of paclitaxel
`reached 05 cm in diameter we randomized these animals into two treatment groups nabpaclitaxel and
`treatment
`relapsed tumors from paclitaxel
`paclitaxel Drugs were given on day 52 59 and 66 with the same schedule and dosage as above Tumor growth was monitored in this paclitaxelrelapsed
`tumor model E in the nabpaclitaxelrelapsed tumor model on day 75 when relapsed tumors from nabpaclitaxel
`reached 05 cm in diameter we
`treatment
`treatment and saline control Drugs were given on day 75 82 and 87 with the same schedule and
`randomized animals into two groups nabpaclitaxel
`dosage as above
`
`at
`
`showed no significant
`effect on tumor growth
`dosage
`whereas the 5 and 10 mgkg daily doses significantly inhib
`ited tumor growth P < 005 oneway ANOVA The stron
`gest antitumor activity was observed with nabpaclitaxel
`50 mgkg iv weekly In CHLA20 xenograft model nab
`paclitaxel at 50 mgkg iv weekly showed similar antitumor
`activity compared with LDM therapy at 10 mgkg daily
`Fig 3B
`treatment was
`from nabpaclitaxel
`The animal survival
`in SKNBE2 metastatic models
`further investigated
`Tumor bearing mice were treated with control vehicle or
`50 mgkg iv weekly with all
`treatments
`nabpaclitaxel
`inoculation As shown
`starting 14 days after tumor cell
`in Fig 3C nabpaditaxel
`treatment significantly prolonged
`animal survival compared with the control group median
`group vs 32 days for
`survival of 59 days for nabpaclitaxel
`
`control group P < 001 Nabpaclitaxel
`treatment signifi
`in these mice Fig 3D com
`cantly increased body weight
`pared with control
`
`Plasma and tumor paditaxel concentrations following
`treatment
`nabpaclitaxel
`Plasma and intratumor drug concentration was measured
`after single or repeated drug administration details in
`Materials and Methods section Twentyfour hours after
`the last dose blood and tumor samples were collected and
`analyzed for paclitaxel concentration by LCMS In both
`tumor models nabpaclitaxel
`treatment displayed lower
`compared with DMSO
`plasma paclitaxel concentrations
`paclitaxel whereas the intratumor paclitaxel concentrations
`groups Fig 4A and B As a
`were higher with nabpaclitaxel
`had a higher
`consequence nabpaclitaxel
`
`tumorplasma
`
`wwwaacrjournalsorg
`
`Clin Cancer Res 1921 November 12013
`
`5977
`
`Downloaded from clincancerresaacrjournalsorg on June 26 2017 © 2013 American Association for Cancer Research
`
`Abraxis EX2018
`Apotex Inc. and Apotex Corp. v. Abraxis Bioscience, LLC
`IPR2018-00151; IPR2018-00152; IPR2018-00153
`Page 6 of 13
`
`

`

`Published OnlineFirst August 29 2013 DOI 10115810780432CCR131485
`
`treatment
`Nabpaclitaxel
`SKNBE2 xenograft model
`
`in
`
`treatment in
`Nabpaclitaxel
`CHLA20 xenograft model
`
`IF Control
`A 10 mgkgd
`50 mgkgwk
`
`5
`
`10
`
`15
`
`3
`
`cm3
`
`2H
`
`1
`
`0
`
`0
`
`Tumorvolume
`
`Control
`
`a 2 mgkgd
`a 5 mgkgd
`10 mgkgd
`4 50 mgkgwk
`
`10
`
`20
`
`30
`
`Days after nabpaclitaxel
`
`treatment
`
`Days after nabpaclitaxel
`
`treatment
`
`Survival curve of
`SKNBE2 metastatic model
`
`Mouse body weight in metastatic
`SKNBE2 model
`
`s Control
`0 Nabpaclitaxel 50 mgkgwk
`
`10
`
`20
`
`30
`
`40
`
`50
`
`140
`
`130
`
`120
`
`110
`
`100
`
`90
`
`0
`
`foriginalbodyweight
`
`Percentageo
`
`L
`
`0 Control
`
`+ Nabpaclitaxel
`
`50 mgkgwk
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`Zhang et al
`
`20
`
`15
`
`10
`
`05
`
`00
`
`0
`
`A
`
`cm3
`
`Tumorvolume
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Percentagesurvival
`
`Days after nabpaclitaxel
`
`treatment
`
`Days after nabpaclitaxel
`
`treatment
`
`tumors SKNBE2 were
`regimens in neuroblastoma models A subcutaneous mouse xenograft
`Figure 3 Antitumor effects of nabpaclitaxel with dif