British Journal of Cancer 2005 92 12401246
`0 2005 Cancer Research UK
`rights reserved 0007 092005
`$3000
`
`All
`
`wwwbjcancercom
`
`NKI 05 a paclitaxelincorporating micellar nanoparticle
`formulation can extend in vivo antitumour activity and reduce the
`neurotoxicity of paclitaxel
`
`I
`
`T Hamaguchi I Y Matsumura2 M Suzuki3 K Shimizu3 R Goda3 I Nakamura3 I Nakatomi4 M Yokoyamas
`K Kataoka6 and T Kakizoe7
`Department of Medicine President of National Cancer Center 511 Tsukiji Chuoku Tokyo 1040045 Japan 2Investigative Treatment Division
`National Cancer Center Research Institute East 651 Kashiwanoha
`Kashiwa Chiba 2778577 Japan 3Pharmaceuticals Group Research
`Division Nippon Kayaku Co Ltd 33112 Shimo Kitaku Tokyo 1158588 Japan 4NanoCarrier Co Ltd Tokatsu Techno Plaza 546
`KSP Bldg East 404 321 Sakado Takatsuku
`Kashiwa Chiba 2770882 Japan 5Kanagawa Academy of Science and Technology
`Kashiwanoha
`The University of Tokyo 731 Hongo
`Kawasaki Kanagawa 2130012 Japan 6Departrnent
`of Materials Engineering Graduate School of Engineering
`Bunkyoku Tokyo 1138656 Japan 7President of National Cancer Center 5I I Tsukiji Chuoku Tokyo 1040045 Japan
`
`Development
`
`Paclitaxel PTX is one of the most effective anticancer agents In clinical practice however high incidences of adverse reactions of
`the drug for example neurotoxicity myelosuppression and allergic reactions have been reported NKI05 a micellar nanoparticle
`to overcome these problems and to enhance the antitumour activity of PTX Via the self association
`formulation was developed
`process PTX was incorporated
`into the inner core of the micelle system by physical entrapment through hydrophobic interactions
`between the drug and the well designed block copolymers for PTX NKI05 was compared with free PTX with respect
`to their in
`vitro cytotoxicity in vivo antitumour activity pharmacokinetics
`and neurotoxicity Consequently the plasma area
`pharmacodynamics
`the curve AUC values were approximately 90 fold higher
`for NK I 05 than for free PTX because the leakage of PTX from
`under
`normal blood vessels was minimal and its capture by the reticuloendothelial system minimised Thus the tumour AUC value was 25
`fold higher for NK105 than for free PTX NKI05 showed
`significantly potent antitumour activity on a human colorectal
`line HT 29 xenograft as compared with PTX P <0001 because the enhanced accumulation of the drug in the tumour has occurred
`and sustained release from micellar nanoparticles Neurotoxicity was significantly weaker with
`probably followed by its effective
`NKI05 than with free PTX The neurotoxicity of PTX was attenuated
`by NKI05 which was demonstrated by both histopathological
`P <0001 and physiological P <005 methods for the first
`time The present study suggests that NKI05 warrants a clinical
`solid tumours
`patients with metastatic
`British Journal of Cancer 2005 92 1240 I 246 do i101038sjbjc6602479
`Published online 22 March 2005
`D 2005 Cancer Research UK
`
`cancer cell
`
`trial
`
`for
`
`wwwbjcancercom
`
`Keywords NKI 05 pachtaxel polymer micelles DDS ERR effect
`
`Paclitaxel PTX is one of the most useful anticancer
`agents known
`including ovarian breast and lung cancers
`for various cancers
`Carney 1996 Khayat et al 2000 However PTX has serious
`adverse effects for example neutropenia and peripheral sensory
`neuropathy In addition anaphylaxis and other severe hypersensi
`
`tive reactions have been reported to develop in 24 of patients
`
`receiving the drug even after premedication with antiallergic
`agents these adverse reactions have been attributed to the mixture
`of Cremophor EL and ethanol which was used to solubilise PTX
`Weiss et al 1990 Rowinsky and Donehower 1995 Of the adverse
`reactions neutropenia can be prevented or managed effectively by
`
`Correspondence Dr Y Matsumura
`Treatment Division
`Institute East 651 Kashiwanoha
`National Cancer Center Research
`Kashiwa Chiba 2778577 Japan Email yhmatsumeastnccgojp
`27 October 2004 revised 26 January 2005
`Received
`January 2005 published online 22 March 2005
`
`accepted
`
`31
`
`Investigative
`
`a granulocyte colony stimulating factor On the
`administering
`other hand there are no effective
`therapies to prevent or reduce
`nerve damage which is associated with peripheral neuropathy
`caused by PTX therefore neurotoxicity
`constitutes a significant
`dose limiting toxicity of
`drug Rowinsky
`et al
`1993
`the
`Wasserheit et al 1996
`The above problems of PTX have been attributed to its low
`and limited efficacy due to the nonselective
`therapeutic indices
`nature of its therapeutic targets
`and its inability to accumulate
`in cancer
`tissue Therefore there is an urgent need to
`selectively
`develop modalities by which cytotoxic drugs can selectively
`target
`tumour tissue and effectively act on cancer cells in the scene The
`roles of drug delivery systems DDSs have drawn attention in this
`context Drug delivery systems are based on two main principles
`active and passive targetings The former refers to the development
`tumour related mole
`of monoclonal antibodies directed against
`cules that allow targeting of the tumour because of specific binding
`between the antibody and its antigen However
`the application of
`
`Abraxis EX2009
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`antibodies
`
`is
`
`restricted to tumours
`
`Several
`
`DDSs using monoclonal
`expressing high levels of related antigens
`Passive targeting is based on the socalled enhanced perme
`ability and retention EPR effect Matsumura and Maeda 1986
`Maeda et al 2000 The EPR effect consists
`in the pathophysio
`logical characteristics of solid tumour
`tissue hypervascularity
`secretion of vascular perme
`incomplete vascular
`architecture
`ability factors stimulating extravasation within cancer
`tissue and
`absence
`drainage from tumours
`of effective
`that
`lymphatic
`impedes the efficient clearance of macromolecules accumulated
`in solid tumour
`tissues
`to maximally use the EPR effect have been
`techniques
`is modification
`of drug structures and develop
`developed that
`ment of drug carriers The first micelle forming polymeric drug
`glycol PEGpolyaspartate block
`was polyethylene
`developed
`copolymer conjugated with doxorubicin DXR Yokoyama et al
`et al 1993 PEG constituted
`1990 Yokoyama et al 1991 Kataoka
`the outer shell of the micelle which conferred a stealth property on
`the drug that allowed
`the micellar drug preparations to be less
`avidly taken up by the reticuloendothelial system RES and to be
`retained in the circulation for a longer time Prolonged circulation
`time and the ability of polymeric micelles to extravasate
`through
`tumour
`were
`vasculature
`to result
`the leaky
`in the
`expected
`accumulation of DXR in tumour
`tissue due to the EPR effect
`Kwon et al 1994 Yokoyama et al 1999 A clinical
`trail of
`micellar DXR NK911 is now underway Nakanishi
`et al 2001
`Hamaguchi et al 2003 Recently we succeeded in constructing
`system for PTX which
`NK105
`a polymeric micelle carrier
`conferred on PTX a passive targeting ability based on the EPR
`we
`details and
`effect
`In the
`describe
`the
`paper
`present
`of NK105 We also discuss
`NK105 and other DDS formulations containing PTX
`
`characteristics
`
`differences
`
`between
`
`MATERIALS AND METHODS
`
`Materials
`
`PTX was purchased
`from Mercian Corp Tokyo Japan All other
`lines MKN45
`chemicals were of reagent grade Following cell
`MKN28 HT 29 DLD1 HCT116 TE1 TE8 PC 14 PC14TXT
`H460 MCAS OVCAR3 AsPC1 PAN 9 PAN 3 and MCF7 cells
`were purchased from American Type Culture Collection Colon 26
`from the Japan Foundation for Cancer
`cells were
`dispensed
`Japan Female BALBc
`Research Tokyo
`nunu mice were
`from SLC Shizuoka Japan Female CDF1 mice and
`purchased
`IGS rats were purchased
`from Charles River Japan Inc Kanagawa
`Japan
`All animal procedures were performed in compliance with the
`guidelines for the care and use of experimental animals which had
`been drawn up by the Committee for Animal Experimentation of
`the National Cancer Center
`these guidelines meet
`the ethical
`standards
`required by law and also comply with the guidelines for
`the use of experimental animals in Japan
`
`NK105 a PTXincorporating micellar nanoparticle
`formulation
`
`NK105 is a PTXincorporating
`core shell type polymeric micellar
`formulation Polymeric micellar
`particles were
`nanoparticle
`the selfassociation
`formed by facilitating
`block
`of amphiphilic
`copolymers in an aqueous medium Novel
`block
`amphiphilic
`namely NK105 polymers were
`for PTX
`copolymers
`designed
`entrapment NK105 polymers were constructed
`using PEG as the
`and modified polyaspartate as the hydro
`hydrophilic
`segment
`phobic segment Carboxylic groups of polyaspartate block were
`modified with 4phenyl1butanol
`by esterification
`reaction
`the half of the groups were converted
`to 4 phenyl
`
`consequently
`
`A paclitaxelincorporating micellar nanoparticle
`T Hamaguchi et al
`
`1butanolate Via the selfassociation process PTX was incorpo
`the micelle system by physical
`rated into the inner
`core of
`through hydrophobic interactions between
`the drug
`entrapment
`and specifically well designed block copolymers for PTX
`
`1 24 1
`
`Pharmacolcinetics
`NK105
`
`and pharmacodynamics of PTX and
`
`Colon 26 tumour bearing CDF1 mice aged 8 weeks were given
`intravenously iv via the tail vein PTX 50 and 100 mg kg or
`NK105 at corresponding PTXequivalent doses Mice were killed at
`5 and 30 min as well as 2 6 24 and 72 h after injection Blood was
`removed
`and
`plasma and
`tumours were
`tumours
`collected
`obtained were then stored at 20°C until
`the analysis Each time
`for collection represented three samples from three different
`point
`mice PTX was extracted from plasma obtained by deproteinisation
`followed by liquid liquid extraction with
`using acetonitrile
`tbutylmethylether Tumours
`obtained were
`homogenised in
`05 acetic acid and the resultant homogenate was deproteinised
`to the same method as that used for
`and extracted
`according
`plasma The blood and tumour extracts were analysed for PTX by
`mass
`spectrometry Reversed
`liquid chromatographytandem
`phase column switching
`chromatography was conducted
`using
`an ODS column and
`was
`enabled
`by electrospray
`detection
`of positive mode
`The mean plasma and
`tumour
`ionisation
`concentrations of PTX at each sampling point were calculated
`for both PTX
`and NK105 Pharmacokinetic modelling was
`completed using a WinNonlin
`Standard software
`version 31
`Pharsight Corp California USA
`
`In vitro cytotoxicity
`
`Various human cancer cell
`lines were evaluated
`in the present
`study The cell
`lines were maintained in monolayer cultures in
`
`Dulbeccos modified Eagles medium containing 10 vv foetal
`calf serum and 600 mg11 glutamine WST8 Cell Counting Kit8
`Dojindo Kumamoto
`Japan was used for
`the cell proliferation
`line in 90 tl of culture medium
`assay In all 2000 cells of each cell
`were plated in 96 well plates and were then incubated for 24 h at
`37°C Serial dilutions of PTX or NK105 in a volume of 10 u1 were
`added and the cells were incubated for 48 or 72 h All data were
`expressed as mean+ se of triplicate cultures The data were then
`plotted as a percentage of the data from the control cultures which
`were treated identically to the experimental cultures except
`that
`no drug was added
`
`Evaluation of the antitumour activity of PTX and NK105
`
`cells
`
`inoculated
`
`at
`
`a
`
`The antitumour activity of PTX and NK105 was evaluated using
`nude mice implanted with a human colonic cancer cell line HT 29
`of HT 29 were
`One million tumour
`subcutaneous sc site on the back skin of BALBc female nude
`mice aged 6 weeks When
`tumour
`size reached
`approximately
`58 mm in diameter mice were randomly allocated
`to the PTX
`group NK105 administration group and control
`administration
`administration group each of which was made up of five animals
`free PTX group
`Each
`treatment was carried out
`follows
`as
`100 mg kg NK105
`was administered at a dose of 25 50 or
`group was with same PTXequivalent doses and in control group
`animals were given saline Mice were administered a single iv
`injection of PTX or NK105 weekly for 3 weeks The antitumour
`activity of PTX and NK105 was evaluated by measuring tumour
`size a x b where a is the major diameter and b is the minor
`diameter at various time points after injection Changes in body
`weight were also monitored for mice which were used in the
`
`present study
`
`© 2005 Cancer Research UK
`
`BUtish Journal of Cancer 2005 927 1240 1246
`
`

`

`N
`
`Modified polyaspartate
`
`(hydrophilic segment)
`
`(hydrophobic segment) Polyethylene glycol
`
`Micelle formation
`and
`
`PTX entrapment oO
`
`o
`
`A paclitaxel-incorporating micellar nanoparticle
`e T Hamaguchi et af
`
`1242
`
`Evaluation of neurotoxicity
`
`The severity of neurotoxicity was assessed both electrophysiolo-
`gically and histologically. Under intraperitoneal ketamine anaes-
`thesia (40 mgkg™'), rats were given a single iv. injection of PTX
`(7.5mgkg~'), NK105 (a PTX-equivalent dose of 7.5mgkg~'), or
`5% glucose weekly for 6 weeks. All the solutions were administered
`through the jugular vein exposed via a small incision in the neck.
`Electrophysiological measurements were conducted 1 day before
`the first dosing and on day 6 after
`the final dosing. For
`electrophysiological
`recording,
`rats were anaesthetised by the
`intraperitoneal
`injection of pentobarbital 40mgkg'. Electrical
`stimuli were given peripherally, and caudal sensory nerve action
`potentials (caudal SNAPs) were recorded centrally from the tail.
`The amplitude of each waveform wascalculated by measuring the
`caudal SNAP from the top peak to the bottom peak. Variations in
`the amplitude after the 6th weekly administration of the solutions
`were determined.
`Forlight microscopy, rats were killed after electrophysiological
`recordings. Subsequently, a segment of the sciatic nerve was
`carefully removed, and embedded in paraffin. Sections (2 um
`thick) were stained with haematoxylin and eosin (H & E) before
`examination under light microscopy to evaluate the degenerative
`changes of myelinated nerve fibres.
`
`Statistical analysis
`
`The data of therapeutic efficacy was expressed as mean+s.e.m.
`The statistical significance of differences in therapeutic efficacy
`between two administration groups was calculated by means of
`repeated measures (analysis of variance). The statistical signifi-
`cance of the differences in neurotoxic activity between two
`administration groups was calculated using the Student’s t-test
`on the closed testing procedure. The histopathological impairment
`was scored in five grades. The statistical significance of the
`differences in histopathological impairment between two admin-
`istration groups was calculated using the Wilcoxon’s rank-sum test
`on the closed testing procedure. All data were calculated with
`software StatView, version 5 (ABACUS Concepts, Berkeley, CA,
`USA). A value of P<0.05 was consideredstatistically significant.
`
`RESULTS
`
`Preparation and characterisation of NK105
`
`
`
`Polyethylene glycol
`Inner core
`with high hydrophobicity
`
`B
`
`3
`
`10
`
`32
`
`101
`
`320
`
`1013
`
`Particle size (nm)
`
`Preparation and characterisation of NKI05. (A) The micellar
`Figure |
`structure of NKI95 PTX was incorporated into the inner core of the
`micelle. (B) The size distribution of NK105 measured by the dynamiclight
`scattering method. The mean diameter of an NKIO5 micelle was 85 nm.
`
`To construct NK105 micellar nanoparticles (Figure 1A), block
`copolymers consisting of PEG and polyaspartate, the so-called PEG
`polyaspartate described previously (9, 11, 13, 14), were used. PTX
`was incorporated into polymeric micelles formed by physical
`entrapmentutilising hydrophobic interactions between PTX and
`the block copolymer polyaspartate chain. After screening of many
`candidate substances, 4-phenyl-1-butanol was employed for the
`chemical modification of the polyaspartate block to increase its
`hydrophobicity. Treating with a condensing agent, 1,3-diisopro-
`pylcarbodiimide, the half of carboxyl groups on the polyaspartate,
`Colon 26-bearing CDF1 mice were given a single iv. injection of
`was esterified with 4-phenyl-1-butanol. Molecular weight of the
`PTX 50 or 100 mg kg", or of NK105at an equivalent dose of PTX.
`polymers was determined to be approximately 20000 (PEG block:
`Subsequently, the time-course changes in the plasma and tumour
`12000; modified polyaspartate block: 8000). NK105 was prepared
`levels of PTX were determined in the PTX and NK105 adminis-
`by facilitating the self-association of NK105 polymers and PTX.
`NK105 was obtained as a freeze-dried formulation and contained
`tration groups
`(Figure 2);
`furthermore,
`the pharmacokinetic
`ca. 23% (ww') of PTX, as determined by reversed-phase liquid
`parameters of each group were also determined (Table 1).
`chromatography using an ODS column with mobile phase
`NK105 exhibited slower clearance from the plasma than PTX,
`consisting of acetonitrile and water (9:11, vv) and detection
`while NK105 was present
`in the plasma for up to 72h after
`injection; PTX was not detected after 24h or later of injection. The
`of ultraviolet absorbance at 227nm. Finally, NK105, a PTX-
`incorporating polymeric micellar nanoparticle formulation with a
`plasma concentration at 5min (Cs min) and the area under the
`
`single and narrow size distribution, was obtained. The weight- curve (AUC) of NK105 were 11-20-fold and 50-86-fold higher for
`average diameter of the nanoparticles was approximately 85nm
`NK105 than for PTX, respectively. Furthermore, the half-life at the
`ranging from 20 to 430 nm (Figure 1B).
`terminal phase (t)2z) was 4-6 times longer for NK105 than for
`
`Pharmacokinetics and pharmacodynamics of NK105
`
`British Journal of Cancer (2005) 92(7), 1240-1246
`
`© 2005 Cancer Research UK
`
`

`

`A paclitaxelincorporating micellar nanoparticle
`T Hamaguchi et el
`
`1243
`
`superior
`
`In vivo antitumour activity
`BALBc mice bearing sc HT 29 colon
`cancer
`tumours showed
`of PTX
`decreased
`tumour growth rates after the administration
`and NK105 However NK105
`antitumour
`exhibited
`activity as compared with PTX P< 0001 The antitumour activity
`dose of 25 mgkg was
`of NK105 administered at a PTXequivalent
`comparable to that obtained after the administration of free PTX
`100 mg kg Tumour suppression by NK105 increased in a dose
`dependent manner Tumours disappeared after the first dosing to
`dose of 100 mg kg
`mice treated with NK105 at a PTXequivalent
`and all mice remained tumourfree thereafter Figure 3A In
`loss was induced in mice which were given
`addition less weight
`NK105 100 mg kg than in those that were given the same dose of
`free PTX Figure 3B
`
`Table 2
`
`IC50 values ttm of PTX and NK I 05 in various cell
`
`lines
`
`48h
`
`72h
`
`Cancer
`
`Cell line
`
`NKI 05
`
`PTX
`
`NKI 05
`
`PTX
`
`Oesophageal
`
`cancer
`
`Lung cancer
`
`Breast cancer
`
`Stomach cancer
`
`Colon cancer
`
`TE1
`TE8
`
`PCI4
`PC14TXT
`H460
`
`MCF7
`MKN28
`MKN45
`
`DLD I
`HT 29
`HCT I 16
`
`>10
`002
`
`001
`015
`ND
`
`>10
`003
`002
`
`095
`
`001
`ND
`
`>10
`002
`
`001
`009
`ND
`
`>10
`003
`007
`
`026
`
`001
`ND
`
`001
`
`001
`
`001
`008
`003
`
`001
`
`001
`
`001
`
`029
`
`001
`003
`
`002
`
`001
`
`001
`006
`
`001
`
`001
`
`021
`002
`
`020
`
`001
`
`001
`
`PTX The maximum concentration Cm and AUG of NK105 in
`Colon 26 tumours were approximately 3 and 25 times higher for
`NK105 than for PTX respectively NK105 continued to accumulate
`tumour PTX
`tumours until
`72 h after
`injection The
`in the
`concentration was higher than 10 itg g1 even at 72 h after the iv
`50 and 100 mgkg1 On the contrary
`injection of NK105
`the
`tumour PTX concentrations at 72 h after the iv administration of
`free PTX 50 and 100 mgkg1 were below detection
`limits and less
`than 01 itgg1 respectively
`
`In vitro cytotoxicity
`
`NK105 was tested on 12 human tumour
`lines derived from
`colon breast and ovarian tumours
`lung gastric
`oesophagus
`Similar dose response curves were noted for PTX and NK105
`data not shown Furthermore the IC50 values of NK105 were
`similar to those of PTX at 48 and 72 h indicating that both NK105
`cytotoxic activity in vitro Table 2
`and PTX showed equivalent
`
`cell
`
`10 000
`
`1000
`
`100
`
`10
`
`1
`
`01
`
`001
`
`0001
`
`A
`
`10000
`
`E woo
`L 100
`7
`
`10
`
`o
`
`1
`
`01 l
`
`X
`
`001
`
`0001
`
`ca
`
`a 8
`
`0
`
`0
`
`12 24 36 48 60 72
`
`0
`
`12 24 36 48 60 72
`
`Time after administration h
`Plasma and tumour concentrations of PTX after
`single iv
`Figure 2
`administration of NKI05 or PTX to Colon 26 bearing CDF I mice Plasma
`A and tumour B concentrations of PTX after NK I 05 administration at
`NK I 05 at a PTXequivalent
`dose of I 00 mg kg A PTX 50 mg kg I 0 and PTX I 00 mg kg I A
`dose of 50 mg kr I
`
`a PTXequivalent
`
`Ovarian cancer
`
`Pancreatic cancer
`
`MCAS
`OVCAR3
`
`AsPC1
`PAN 9
`PAN 3
`
`001
`>10
`
`001
`>10
`
`001
`>10
`
`001
`>10
`
`ND
`ND
`ND
`
`ND
`ND
`ND
`
`002
`003
`0010
`
`002
`002
`0004
`
`PTX= paclitaxel ND = not done
`
`Pharmacokinetic parameters for the plasma and tumour concentrations of paclitaxel
`Table I
`26 bearing CDF I mice
`Dose mg kg C51 g
`
`Treatment
`
`i2z h AUC0 Gig h m11
`
`t
`
`AUCoinf µg h m11
`
`CLtht ml h kg 1
`
`c ml kg I
`
`after
`
`single iv administration of NKI05 and PTX =o Colon
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`5932
`15767
`
`115703
`
`181237
`
`098
`184
`599
`682
`
`C
`
`Gig m11
`
`T h
`
`902
`3090b
`
`78609c
`
`155657c
`
`ti h
`
`913
`3090
`78623
`155736
`
`5476
`3236
`64
`64
`
`6846
`8122
`464
`548
`
`AUC0gh m11
`
`AUCoinf µg h m11
`
`1250
`
`20
`
`702
`
`1208b
`
`1330
`
`2857
`4245
`7109
`
`05
`240
`60
`
`806
`3507
`7366
`
`3304c
`23601
`38849
`
`3310
`31920
`79645
`
`the terminal phase AUC= area under the curve CLtot =total body clearance V volume of
`at steady state T =time of maximum concentration FIX= paclitaxel Parameters were calculated
`
`Plasma
`
`NKI 05
`NKI 05
`
`Tumour
`
`NKI 05
`NKI05
`
`distribution
`
`iv= intravenous
`
`C5 rnIn = plasma concentration
`
`at 5 min t112z= halflife at
`
`noncompartmental
`
`analysis AUC061 bAUCo24h AUCo721
`
`© 2005 Cancer Research UK
`
`British Journal of Cancer 2005 927 2401 1246
`
`from the mean value of
`
`three or two mice by
`
`

`

`which indicated markedly more numerous degenerative myeli
`nated fibres P<0001 Figure 4B and C and Table 3
`
`DISCUSSION
`
`it
`
`is still
`
`is sufficient
`
`the plasma AUC of NK105
`A pharmacokinetic study revealed that
`than that of free PTX in the
`was approximately 90 fold higher
`present rodent models Prolonged circulation
`of NK105 in the
`blood due to the EPR effect was associated with a significant
`increase in the tumour AUG In fact
`the tumour AUC of NK105
`PTX
`was
`25 fold higher
`than that
`approximately
`of
`free
`Figure 2B In mice accordingly
`NK105
`exhibited stronger
`antitumour activity than free PTX Figure 3A However
`debatable whether or not
`enhanced
`accumulation of an
`the
`anticancer drug into a tumour
`in leading the drug to
`exert its antitumour activity in vivo
`Jain etahave reported that the convective
`passage of large drug
`molecules into the core of solid tumours could be impeded by
`in solid tumours However
`abnormally high interstitial
`pressures
`they also admitted that
`low molecular weight anticancer
`agents
`might be harmful
`to normal organs because they can leak out of
`normal blood vessels freely they finally concluded
`that one useful
`strategy for evading the barriers to drug dispersion would be to
`inject patients with drug carriers such as liposomes filled with
`low molecular weight drugs Jain 1994 In this case liposomes
`should have sufficient
`time to exit from the site of tumour blood
`leakage and to accumulate
`at reasonably high dose levels in
`vessel
`the surrounding interstitium Subsequently low molecular weight
`drugs packed within liposomes should be released gradually so
`the tumour However
`they can be dispersed throughout
`that
`Unezaki et al have used fluorescence labelled PEGliposomes and
`described that the area of highest fluorescence was located outside
`tumour vessels almost all around the vessel wall even 2 days after
`drug injection Unezaki et al 1996 Therefore the study suggested
`that although PEGliposomes can be delivered effectively
`to a solid
`tumour via the EPR effect
`formulation would not
`be
`the
`distributed sufficiently to cancer cells distant from tumour vessels
`because liposomes are too large to scamper about
`in the tumour
`interstitium Liposomes have been suggested to be too stable to
`easily Therefore PEG
`allow the drug therein to be released
`to be not so effective
`liposomes have been speculated
`against
`cancers in which the tumour vessel network is irregular and loose
`because of an abundant
`collagen rich matrix Such cancers include
`scirrhous cancer of the stomach
`cancer
`and pancreatic
`In fact
`Doxilg a PEGliposomal DXR is known to be effective clinically
`and breast cancer both of which are
`against ovarian cancer
`by a high density of tumour microvessels however
`characterised
`stomach
`cancer and pancreatic
`the drug is not effective
`cancer Muggia 2001
`There are several possible reasons why NK105 exhibited higher
`antitumour activity in the present study as compared with free
`PTX 1 since NK105 is very stable in the circulation and exhibits
`a markedly higher plasma AUC than free PTX it accumulates
`better in tumour tissue than does free PTX due to the EPR effect
`2 NK105
`in size 85 nm as compared with
`Doxil 100 nm thus explaining its more uniform distribution in
`tumour tissue
`in cancer
`its greater accumulation
`and
`tissue Savic et al 2003 have recently reported
`throughout cancer
`that polymeric micelles could internalise into cells to localise in
`several cytoplasmic organelles and 3 a polymeric micelle carrier
`system for a drug has the potential
`to allow the effective sustained
`release of the drug inside a tumour following the accumulation of
`tissue Regarding NK105 in particular this
`micelles into tumour
`dose of <1 yg m11
`sustained
`release begins at a PTXequivalent
`data not shown Consequently released PTX becomes distributed
`tumour tissue and internalises into cancer cells to kill
`throughout
`them
`
`against
`
`is relatively small
`
`cells
`
`A paclitaxelincorporating micellar nanoparticle
`T Hamagechi et at
`
`1244
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Days after
`
`initial
`
`treatment
`
`Relativetumoursize
`
`B 16
`
`cy
`
`13
`
`10
`
`07
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Days after
`
`initial
`
`treatment
`
`>o
`
`o a
`
`cr
`
`c0
`
`Figure 3
`
`Relative changes
`
`in HT 29 tumour growth rates in nude mice
`A Effects of PTX open symbols and NKI 05 closed symbols PTX and
`NK 105 were injected iv once weekly for 3 weeks at PTXequivalent
`100 mg kg I 0
`50 mg kgI A A and
`injected to control animals 0 B Changes
`of 25 mg kg I El
`respectively Saline was
`relative body weight Data were derived from the same mice as those used
`for the present study
`
`doses
`
`in
`
`Neurotoxicity of PTX and NK105
`
`Treatment with PTX has resulted in cumulative sensorydominant
`in humans
`clinically by
`characterised
`peripheral neurotoxicity
`numbness andor paraesthesia
`of the extremities Pathologically
`axonal swelling vesicular degeneration and demyelination were
`observed We therefore examined the effects of
`free PTX and
`NK105
`and morphological
`using both
`methods
`
`electrophysiological
`
`Prior
`
`differences
`
`action
`
`to drug administration there
`were
`no
`significant
`of caudal
`sensory nerve
`in the amplitude
`potential caudal SNAP between two drug administration groups
`On day 6 after the last dosing at week 6 the amplitude of the
`caudal SNAP in the control group increased in association with rat
`maturation The amplitude was significantly smaller in the PTX
`group than in the control group P<001 while the amplitude was
`in the NK105 group than in the PTX group
`P<005 and was comparable between the NK105 group and the
`group Figure 4A Histopathological
`examination
`paraffin embedded sections
`longitudinal
`days after
`the sixth weekly injection revealed degenerative
`changes
`The NK105 administration group showed only a few degenerative
`to the PTX administration group
`myelinated fibres in contrast
`
`significantly larger
`
`control
`
`of
`
`the sciatic
`
`nerve 5
`
`of
`
`British Journal of Cancer 2005 927 12401246
`
`© 2005 Cancer Research UK
`
`

`

`eraeutic
`
`ransationa
`
`A paclitaxelincorporating micellar nanoparticle
`T Hamaguchi et at
`
`1245
`
`8
`
`5
`4 11
`
`1
`
`1 0
`
`0
`
`+ ++
`
`Control
`
`n=13
`
`1
`
`01
`
`I
`
`+ ++
`
`PTXa
`n=10
`
`+I + ++
`NK105b
`n=14
`
`C 15 r
`
`12
`
`10
`
`5
`
`Incidencenumber
`
`t
`
`Control
`
`PTX NK105
`
`Rif
`
`R
`
`k
`
`=a
`
`A
`
`40
`
`30
`
`> 20
`
`10
`
`0
`
`a
`
`14
`
`kgMagnification
`
`nerve H
`
`Incorporation of PTX into polymeric micelles diminishes neurotoxicity A Effects of PTX or NKI05 on the amplitude of rat caudal sensory
`Figure 4
`Glucose 5 was also injected
`action potentials as examined 5 days after weekly injections for 6 weeks Rats n= 14 were injected with NKI 05
`or PTX El at a PTX
`nerve
`P <005 P<001 B
`in the same manner to animals
`equivalent dose of 75 mg kgT
`in the control group
`in the sciatic nerve of rats Degenerating myelinated nerve fibres arrow were examined in the longitudinal section of the sciatic
`E 5 days after weekly injections for 6 weeks with 5 glucose a PTX b and NK I 05 c at a PTXequivalent
`Histopathological changes
`dose of 75 mg
`x 100 upper and x 400 lower C Incidences of degenerating myelinated nerve fibres in rats treated with PTX or NK I 05 N K I 05 or PTX
`was administered iv at a weekly dose of 75 mg kg for 6 consecutive
`weeks to female rats The degenerating myelinated fibre score was defined as follows
`fibres affected + slight degree of degenerative
`no degenerative changes +
`very slight degree of the degenerative changes
`changes scattered small groups of degenerative myelinated fibres + + moderate degree of degenerative changes disseminated degenerative myelinated
`fibres + + + marked degree of degenerative changes confluent
`vs PTX
`vs vehicle treated animals bP<0001
`fibres aP <0001
`groups of affected
`
`scattered
`
`single
`
`treated animals
`
`to DDSs have
`
`To date PTX preparations that are categorised
`been developed Among them clinical
`trials are currently ongoing
`for the following drugs CT 2103 polyglutamateconjugated
`PTX
`Singer et al 2003 ABI007 PTX coated with albumin Ibrahim
`et al 2002 and GenexolPM PTX micelle in which PTX is simply
`solubilised Kim et al 2004 The advantage
`commonly shared
`with these dosage forms is that they are injectable iv without
`the
`mixture of Cremophor EL and ethanol which potentially provoke
`serious allergic reactions The block copolymer used for forming
`NK105 micellar nanoparticles is nonimmunogenic and is injectable
`iv without Cremorphor EL and ethanol Therefore this dosage
`form is expected to possess a clinical advantage which is similar to
`that of the above PTX dosage forms Now what is the difference
`
`forms ABI007
`and other PTX dosage
`between NK105
`and
`the AUG and tumour AUG
`GenexolPM were
`found to have
`which are nearly comparable or rather slightly lower than those of
`free PTX Furthermore
`the plasma AUG and tumour AUG are
`115 and 118 fold higher respectively for CT 2103 than for free
`PTX but they are markedly low as compared with those of NK105
`have employed proper
`tumours and proper
`studies
`Respective
`rodent models However NK105 was forecasted
`to have markedly
`high plasma and tumour AUG as compared with those of other
`PTX dosage forms
`Regarding the toxicity profiles the repeated administration
`of
`NK105 to rats at 7 day intervals produced less toxic effects on
`peripheral nerves than free PTX This reduced the neurotoxicity of
`
`0 2005 Cancer Research UK
`
`BUtish Journal of Cancer 2005 927 2401 1246
`
`

`

`I
`
`1246
`
`A paclitaxelincorporating micellar nanoparticle
`T Hamaguchi et at
`
`Table 3
`Incidence of degenerating myelinated fibres in rats treated with
`PTX or NK105
`
`Degenerating myelinated nerve fibre scoreb
`
`Treatment
`
`Control vehicle
`PTX`
`NKI05d
`
`na
`
`13
`
`10
`
`14
`
`12
`
`8
`
`+1
`
`2
`
`++
`
`+++
`
`4
`
`4
`
`5
`
`PTX= pahlitaxel Vehicle NKI05 or PTX was administered iv at a weedy dose of
`75 mg kg I
`for 6 consecutive weeks to female rats Total number of animals
`accounted for that experimental
`defined as follows
`
`condition bDegenerating myelinated fibre score was
`
`no degenerative changes + very slight degree of the
`
`degenerative
`
`changes
`
`scattered single fibres affected + slight
`degree of
`small groups of degenerative myelinated fibers
`degenerative changes
`scattered
`++ moderate
`degree of
`degenerative
`changes
`disseminated
`myelinated fibers +++ marked degree of degenerative changes
`fibres P< 0001 vs vehicle treated animals dP<0001
`of affected
`
`degenerative
`
`groups
`confluent
`vs PTXtreated
`
`animals
`
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`
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`
`doxor
`
`for drug delivery
`
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`Hamaguchi T Matsumura Y Shirao Y Shimada Y Yamada Y Muro Y
`Okusaka T Ueno H Ikeda M Watanabe N 2003 Phase I study of novel
`drug delivery system NK911 a polymer micelle encapsulated
`ubicin Proc Am Soc Clin Oncol 22 571
`Ibrahim NK Desai N Legha S SoonShiong P Theriault RL Rivera E
`Esmaeli B Ring SE Bedikian A Hortobagyi GN Ellerhorst
`JA 2002
`study of ABI007 a Cremophorfree
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`Res 8 1038
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`Jain RK 1994 Barriers to drug delivery in solid tumours Sci Am 271
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`Kataoka K Kwon GS Yokoyama M Okano T Sakurai Y 1993 Block
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`Kim TY Kim DW Chung JY Shin SG Kim SC Heo DS Kim NK Bang YJ
`2004 Phase I and pharmacokinetic
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