Cancer Therapy: Preclinical

In vivo Evaluation of Mucoadhesive Nanoparticulate Docetaxel forIntravesical Treatment of Non–Muscle-Invasive Bladder Cancer

Clement Mugabe1, Yoshiyuki Matsui5, Alan I. So2,5, Martin E. Gleave2,5, Jennifer H. E. Baker6,Andrew I. Minchinton6, Irina Manisali7, Richard Liggins7, Donald E. Brooks3,4, and Helen M. Burt1

AbstractPurpose: The present work describes the development and in vitro and in vivo evaluation of a

mucoadhesive nanoparticulate docetaxel (DTX) formulation for intravesical bladder cancer therapy.

Experimental Design: Mucoadhesive formulations based on hyperbranched polyglycerols (HPG),

hydrophobically derivatized with C8/C10 alkyl chains in the core andmodified withmethoxy-polyethylene

glycol (MePEG) and amine groups in the shell (HPG-C8/10-MePEG-NH2) were synthesized and DTX was

loaded into these by a solvent evaporation method. Both low-grade (RT4, MGHU3) and high-grade

(UMUC3) human urothelial carcinoma cell lines were treated with various concentrations of DTX

formulations in vitro. KU7 cells that stably express firefly luciferase (KU7-luc) were inoculated in female

nude mice by intravesical instillation and quantified using bioluminescence imaging. Mice with estab-

lished KU7-luc tumors were given a single intravesical instillation with PBS, Taxotere (DTX from Sanofi-

aventis), andDTX-loadedHPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2. Drug uptake was conducted

using LC/MS-MS (liquid chromatography/tandem mass spectrometry) and tumor microenvironment and

uptake of rhodamine labeled HPGs was assessed.

Results: In vitro, all DTX formulations potently inhibited bladder cancer proliferation. However, in vivo,

DTX-loaded HPG-C8/10-MePEG-NH2 (mucoadhesive DTX) was the most effective formulation to inhibit

tumor growth in an orthotopic model of bladder cancer. Furthermore, mucoadhesive DTX significantly

increased drug uptake in mouse bladder tissues. In addition, rhodamine labeled HPG-C8/10-MePEG-NH2

showed enhanced uptake of these nanoparticles in bladder tumor tissues.

Conclusions: Our data show promising in vivo antitumor efficacy and provide preclinical proof of

principle for the intravesical application of mucoadhesive nanoparticulate DTX formulation in the

treatment of bladder cancer. Clin Cancer Res; 17(9); 2788–98. �2011 AACR.

Introduction

Bladder cancer is a significant public health problemresponsible for more than 130,000 deaths annually world-wide (1). In the United States, bladder cancer is the fourthmost common cancer diagnosed in men, with an estimated70,530 new cases and 14,680 deaths from the disease in2010 (2). Approximately 70% to 80% of patients withbladder cancer initially present with superficial disease that

does not invade the muscularis propria (3). Transurethralresection (TUR) is the primary treatment method for non–muscle-invasive or "superficial" carcinoma, which consistsof the surgical removal of tumor nodules from the bladderwall (4, 5). However, there is a high rate of tumor recur-rence and high chance of disease progression after TURalone (6). This is probably due to incomplete removal ofthe tumors and/or implantation of cancer cells in normaltissues of the bladder. Intravesical therapy, which involvesinstillation of one or more chemo- and/or immunother-apeutic agents into the bladder following resection, hasbecome the standard of care for the treatment of non–muscle-invasive bladder cancers (7). Systemic administra-tion of anticancer drugs offers little therapeutic benefit inthese patients, as the urothelial layer of the bladder is notvascularized (8). In contrast, intravesical chemotherapy hasthe potential to selectively deliver high drug concentrationsto tumor-bearing bladder tissues while minimizing thesystemic exposure.

Immunotherapy with BCG (bacille Calmette–Gu�erin) isthe most effective form of intravesical therapy for prophy-laxis of recurrence and progression in non–muscle-invasivebladder cancer patients (9). However, it is associated with

Authors' Affiliations: 1Faculty of Pharmaceutical Sciences, Departmentsof 2Urologic Sciences and 3Pathology & Laboratory Medicine and Chem-istry, 4Centre for Blood Research, University of British Columbia; 5TheVancouver Prostate Centre; 6Department of Medical Biophysics, BritishColumbia Cancer Research Centre; and 7Centre for Drug Research andDevelopment, Vancouver, British Columbia, Canada

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Helen M Burt, Faculty of PharmaceuticalSciences, University of British Columbia, Vancouver, British Columbia,Canada V6T 1Z3. Phone: 604-822-6354; Fax: 1-604-822-3035; E-mail:burt@interchange.ubc.ca

doi: 10.1158/1078-0432.CCR-10-2981

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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frequent local and/or systemic adverse effects includingBCG sepsis (10, 11). The incidence of local and systemicadverse effects is significantly lower with chemotherapeuticagents. However, the response to intravesical chemother-apy is incomplete and tumor recurrence is still high (up to80%) in patients with non–muscle-invasive bladder cancer(12).Treatment failure is thought to be, in part, due to the

short dwell time of intravesical drugs and the low drugpermeability of urothelium. Conventional formulationsare typically maintained in the bladder for 2 hours andurothelial drug exposure rarely lasts beyond the first void-ing of urine after instillation (13). Our approach to increasethe dwell time and uptake of intravesical drugs is to targetthe mucin glycoproteins on the surface of urothelium byexploiting the concept of mucoadhesion, which is anadhesive phenomenon occurring between certain type ofpolymers known as "mucoadhesive" polymers and themucin gel layer covering mucosal membranes.The systems we are currently developing are based on

hydrophobically derivatized hyperbranched polyglycerols(HPG) which are dendritic-like macromolecules, with ahydrophobic core and a hydrophilic shell connected bycovalent bonds in a single molecule (14). The hydrophobiccore is based on a mixture of alkyl (C8/C10) chains which isimportant for the binding of hydrophobic molecules,including drugs, whereas the hydrophilic shell based onmethoxy-polyethylene glycol [MePEG; molecular weight

(MW) ¼ 350] keeps the system soluble in water (15). Inaddition, HPGs possess many hydroxyl groups on the outersurface that can be further derivatized to increase theirmucoadhesiveness.

Mucoadhesive systems possess numerous advantageswhen compared with conventional dosage forms. Locali-zation at a specific region of the body helps improve druguptake at the site of interest (16). Mucoadhesion alsoincreases the intimacy of contact between a drug-contain-ing polymer and mucous surface, which can enhance thepermeability of the drug (17). Mucoadhesive polymers canalso prolong residence time of the incorporated drugs,leading to less frequent dosing and improved patientcompliance (18).

Recently, we have reported the synthesis and character-ization of HPG-C8/10-MePEG nanoparticles with surface-derivatized amine groups (19). The surface amine groupson HPG-C8/10-MePEG resulted in positively charged nano-particles (HPG-C8/10-MePEG-NH2) with improvedmucoadhesive properties. We conducted a dose rangefinding and tolerability study of intravesical docetaxel(DTX) in HPG formulations in an orthotopic model ofbladder cancer and showed that intravesical DTX in eithercommercial or in HPG formulations (up to 1.0 mg/mL)was well tolerated with no apparent signs of toxicity inmice(19).

The objective of the present work was to evaluate theeffectiveness of intravesical mucoadhesive DTX loaded inHPG nanoparticles in an orthotopic model of bladdercancer and to investigate bladder tissue and tumor uptakeof these nanoparticulate formulations.

Materials and Methods

ChemicalsAll chemicals were purchased from Sigma-Aldrich and all

solvents were high-performance liquid chromatography(HPLC) grade from Fisher Scientific. MePEG epoxide(a-epoxy, w-methoxy-PEG 350) was synthesized from areaction of MePEG 350, sodium hydroxide, and epichlor-ohydrin. Potassium methylate, trimethyloylpropane(TMP), and octyl/decyl glycidyl ether were obtained fromSigma-Aldrich and used without further purification. N-(2,3-epoxypropyl)-phthalimide (EPP) was obtained fromSigma-Aldrich. Tetramethylrhodamine-5-carbonylazide(TMRCA) was purchased from Invitrogen. DTX powderwas obtained from Natural Pharmaceuticals, Inc. and Tax-otere (DTX in Tween 80) was purchased from Sanofi-aventis Canada Inc.

Cell linesThe human bladder cancer cell lines RT4 and UMUC3

were purchased from the American Type Culture Collec-tion. Cells were maintained in McCoy’s medium (Invitro-gen) containing 10% heat-inactivated FBS and kept at 37�Cin a humidified 5% CO2 atmosphere. MGHU3 cells wereobtained as a generous gift from Dr. Y. Fradet (L’Hotel-Dieu de Quebec, Quebec, Canada) and maintained in

Translational Relevance

Development of novel therapeutics for patients withhigh-risk non–muscle-invasive bladder cancer is animportant topic considering the rather limited optionscurrently available. Microtubules are one of the mostsuccessful targets in cancer therapy to date and therecent phase I trial of intravesical docetaxel (DTX) forthe treatment of non–muscle-invasive bladder cancer,refractory to BCG (bacille Calmette–Gu�erin) therapy,has shown this to be a promising intravesical agent.Treatment failure is thought to be, in part, due to theshort dwell time of intravesical drugs and low druguptake in the bladder wall. Conventional formulationsare typically maintained in the bladder for 2 hours andurothelial drug exposure rarely lasts beyond the firstvoiding of urine after instillation. In this study, wedeveloped a novel, mucoadhesive, controlled-releaseformulation of DTX. Mucoadhesive polymers have sev-eral advantages when compared with conventional drugcarriers including localization at the specific target site,prolonged residence time, and increased drug uptake.Overall, our data show promising antitumor efficacyand safety in a recently validated orthotopic model ofbladder cancer and provide a clear rationale for theintravesical use of this nanoparticulate formulation overthe commercial formulation of DTX in the treatment ofnon–muscle-invasive bladder cancer.

Mucoadhesive Nanoparticulate Docetaxel for Intravesical Therapy

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MEM (minimum essential media) supplemented with 10%FBS and 2 mmol/L L-glutamine (Invitrogen). KU7 waskindly provided by Dr. C. Dinney (MD Anderson CancerCenter, Houston, TX) and maintained in DMEM(Dulbecco’s modified Eagle’s media) containing 5% FBS.Although controversial, KU7 cells were initially thought tobe of bladder origin, but recent studies in our laboratorysuggest they may be of HeLa origin (data not shown).However, this cell line provides for an excellent in vivorepresentation of an aggressive epithelial carcinoma thatcan be modeled intravesically. For visualization purposes,KU7 cells were infected with a lentivirus containing thefirefly luciferase gene by Dr. Graig Logsdon (MD AndersonCancer Center), and these subclones were named KU7-lucas described previously (20).

Synthesis and characterization of HPG-C8/10-MePEGand HPG-C8/10-MePEG-NH2

The synthesis and characterization of HPG-C8/10-MePEGhas been previously reported by our group (15). Recently,we have described the surface derivatization of HPG-C8/10-MePEG with amine groups using EPP followed by cleavageof the phthalimide functional groups by hydrazinolysis(19). The details on the synthesis and characterization ofHPG-C8/10-MePEG andHPG-C8/10-MePEG-NH2 have beenprovided in the Data Supplements section.

Rhodamine labeling of HPGsHPG-C8/10-MePEG and HPG-C8/10-MePEG-NH2 poly-

mers were covalently labeled with TMRCA as previouslyreported (21, 22).

DTX loading into HPGsDTX (0.2 mg) and HPGs (100 mg; HPG-C8/10-MePEG

and HPG-C8/10-MePEG-NH2) were dissolved in 1 mL acet-onitrile solution in 4-mL vials and dried in an oven at 60�Cfor 1 hour and flashed with nitrogen stream to eliminatetraces of the organic solvent. The resulting HPG/DTXmatrix was hydrated with 1 mL of 10 mmol/L PBS (pH6) and vortexed for 2 minutes. The amounts of DTXincorporated in HPGs were determined by reversed phaseHPLC. Drug content analysis was conducted using a sym-metry C18 column (Nova-Pak; Waters) with a mobilephase containing a mixture of acetonitrile, water, andmethanol (58:37:5, v/v/v) at a flow rate of 1 mL/min.Sample injection volumes were 20 mL and detection wasconducted using UV detection at a wavelength of 232 nm.Total run time was set to 5minutes and DTX retention timewas 2.9 minutes. Our HPLC method for quantification ofDTX has been previously validated. This method wasevaluated over a linear range of 0.5 to 100 mg/mL. Overthis range, the accuracy (percent deviation from theoreticalvalue) was found to be within 3% and the precision (%relative SD) was less than 2% using clean DTX standards.

In vitro cytotoxicity studiesCells were plated at 5,000 cells per well in 96-well plates

in a 100 mL volume of McCoy’s medium supplemented

with 10% FBS and allowed to equilibrate for 24 hoursbefore freshly prepared solutions of Taxotere, or DTX inHPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2 (dis-solved in PBS, pH 7.4), were added. Cells were exposedto the drugs for 2 hours and cell viability was determinedafter 72 hours using the CellTiter 96 AQueous Non-Radio-active Cell Proliferation Assay (Promega) as describedpreviously (23). Each experiment was repeated 3 timesand MTS values fell within a linear absorbance range forall cell lines.

In vivo studiesEfficacy of intravesical DTX in orthotopic murine model of

bladder cancer. The orthotopic mouse model used in thepresent study has been described in earlier publications (20,23–26). Animal studies were carried out in accordance withthe Canadian Council on Animal Care. Briefly, 11-week-oldfemale nude mice (Harlan) were anaesthetized with iso-flurane. A superficial 6-0 polypropylene purse-string suturewas placed around the urethral meatus before a lubricated24 G Jelco angiocatheter (Medex Medical Ltd.) was passedthrough the urethra into the bladder. After a single irrigationof the bladder with PBS, 2 million KU7-luc cells wereinstilled as a single cell suspension in 50 mL and thepurse-string suture was tied down for 2.5 hours. To quantifyin vivo tumor burden, animals were imaged in supine posi-tion 15minutes after intraperitoneal injection of 150mg/kgluciferin on days 4, 11, 18, and 25 with an IVIS200 ImagingSystem(Xenogen/Caliper Life Sciences).Datawere acquiredand analyzed using Living Image software (Xenogen).

On day 5 post–tumor inoculation, mice were rando-mized to receive a single 50 mL intravesical treatment withPBS (control); Taxotere (0.2 mg/mL); DTX (0.2 mg/mL)-loaded HPG-C8/10-MePEG; and DTX (0.2 mg/mL)-loadedHPG-C8/10-MePEG-NH2. Levels of bioluminescence wereequivalent among the groups; however, as tumors variedbetween individual mice, for statistical analyses, tumorbioluminescence after treatment was normalized againstthe initial flux on day four in each mouse.

Necropsy was conducted on day 25 after tumor inocula-tion. The whole bladders were removed, fixed in 10%buffered formalin and embedded in paraffin. Sections of5 mm were prepared and stained with H&E (hematoxylinand eosin) using standard techniques. All slides werereviewed by a pathologist (Dr. L. Fazli, The VancouverProstate Centre) and scanned on a BLISS microscopeimaging workstation (Bacus Laboratories Inc.).

Drug uptake studies. Drug uptake studies were con-ducted in 15-week-old female nude mice with establishedKU7-luc tumors (33 days post–tumor inoculation). Tailblood samples were taken at 0, 30, and 60 minutes post–intravesical instillation. During this period, mice were stillanaesthetized with isoflurane. After 2 hours, all mice wereeuthanized using CO2 asphyxiation and additional bloodwas removed by cardiac puncture. Blood samples werecentrifuged in microhematocrit tubes (Fisher Scientific)or serum separator tubes (Becton Dickinson) and theserum was snap frozen in liquid nitrogen. Urine and

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bladder of each mouse were also harvested, and beforefreezing, the bladders were cut, opened to expose thelumen, and were vigorously washed in 5 sequential 10-mL PBS washes. All samples were stored at �80�C.The UPLC/MS-MS (ultraperformance liquid chromato-

graphy/tandem mass spectrometry) system used for ana-lysis consisted of an integrated Waters Acquity UPLCseparation system (Acquity BEH C18, 1.7 mm, 2.1 �50 mm column) coupled to a mass spectrometry analysisusing Waters TQD mass spectrometer. The system wasoperated at an electrospray ion source block temperatureof 150�C, a desolvation temperature of 350�C, a conevoltage of 14 V, a capillary voltage of 0.70 kV, extractorvoltage of 3 kV, RF voltage of 0.1 kV, a cone gas flow at 25 L/h, a desolvation gas flow at 600 L/h, and a collision gas flowat 0.2 mL/min. The molecules undergo electronspray ioni-zation in the positive ion mode. DTX was quantified inmultiple reaction monitoring with the transition of m/z808.5 ! 527.2 as previously established (22).DTX was extracted from the mouse serum by solvent/

solvent extraction method. Briefly, 50 mL aliquots of themouse plasma and standards were mixed with 150 mL of0.1% formic acid in acetonitrile in a 96-well plate andvortexed for 1 minute at room temperature. The sampleswere centrifuged at 5,500 rpm (Allegra 25 R centrifuge;Beckman Coulter) for 10 minutes at 4�C. Then, 100 mL ofthe supernatant was mixed with 50 mL of distilled water,remixed, and vortexed for 30 seconds.Bladder tissues were weighed and homogenized in 0.1%

formic acid/methanol using zirconia beads (Biospec Pro-ducts) and Mini-beadbeater equipped with microvialholder (Biospec Products) for 60 seconds. The sampleswere centrifuged at 14,000 rpm (Allegra 25 R centrifuge;Beckman Coulter) for 2minutes at 4�C. A total of 150 mL of0.1% trifluoroacetic acid in methanol was added to thesamples, mixed, and vortexed at 14,000 rpm (Allegra 25 Rcentrifuge; Beckman Coulter) for 15 minutes at 4�C. Allsample analysis was conducted using UPLC/MS-MS. Thelimit of quantification for DTX was 10 ng/mL with arecovery of 97% from spiked control samples. Withinrun precision (%relative SD) was less than 15% in all cases.Assessing tumor microenvironment and uptake of rhoda-

mine labeled HPGs. Fifteen-week-old female nude micewith orthotopic bladder tumors (33 days post–tumorinoculation) were anaesthetized with isoflurane. A super-ficial 6-0 polypropylene purse-string suture was placedaround the urethral meatus and the bladder was emptiedbymanual compression. A lubricated 24-gauge Jelco angio-catheter was passed through the urethra into the bladderand then 50 mL of either PBS, free rhodamine (TMRCA),HPG-C8/10-MePEG-TMRCA, and/or HPG-C8/10-MePEG-NH2-TMRCA was instilled and the purse-string suturewas tied down for a 2-hour period, during which the micewere kept anesthetized. After the 2-hour period, the purse-string suture was removed, the bladder was emptied bymanual compression, and washed twice with 150 mL of PBS(pH 6.0). The mice were euthanized and the bladders wereexcised and frozen on an aluminum block, then embedded

in OCT (optimal cutting temperature) for cryosectioning.Cryosections of 10 mm were cut at distances of 1, 2, and3 mm from the bladder edge. Sections were dried at roomtemperature and imaged for rhodamine fluorescence using10� objective (0.75 mm/pixel resolution). Slides were fixedin 1:1 acetone: methanol solution for 10 minutes andstained using a custom capillary action staining apparatusfor CD31 (1:50 hamster anti-CD31 with an anti-hamsterAlexa 647 secondary) and Hoechst 33342 (nuclear dye).Following fluorescent imaging of CD31 and Hoechst33342, sections were counterstained lightly with hematox-ylin, mounted, and imaged in bright field.

Image analyses. Images were reduced to 1.5 mm/pixelresolution to improve manageability in Image J software.With user-supplied algorithms, image stacks were thencreated, aligned, and cropped to tumor tissue boundarieswith artifacts removed; necrosis was further cropped on thebasis of the hematoxylin image. The bladder lumen wasartificially traced along the tumor tissue boundary onHoechst 33342 images. User-supplied analysismacros wererun to generate the following types of data: (a) thresholdwas manually determined to include positive stain but thatdoes not pick up background outside of necrosis areas; themacro determines the number of positive pixels meeting orexceeding this threshold and was reported as an average forthe whole tumor section and (b) intensity was reported asthe average intensity of staining for a whole tumor section,or the average intensity of pixels sorted on the basis of theirdistance from a secondary stain (i.e., CD31) or artificiallytraced boundary (bladder lumen). Calculations to deter-mine averages � standard error (SE) were conducted andgraphic displays created using Microsoft Excel; nonpara-metric ANOVA (Kruskal–Wallis tests) statistical analyseswere conducted using Prism v5 for Mac software.

Results

In vitro cytotoxicityWe evaluated the cytotoxic effects of the commercial

formulation of Taxotere and DTX-loaded HPG formula-tions against the KU7-luc cell line, and both low-grade(RT4, MGHU3) and high-grade (UMUC3) human urothe-lial carcinoma cell lines. Cells were exposed to the drugformulations for 2 hours to simulate the current clinicalstandard for instillation therapy, and cell viability wasdetermined after 72 hours by MTS assay. All formulationsresulted in concentration-dependent inhibition of the pro-liferation of cells. DTX-loaded HPG-C8/10-MePEG or HPG-C8/10-MePEG-NH2 were found to be as cytotoxic as thecommercial formulation of Taxotere (Fig. 1). The IC50 ofDTX formulations were in the low nanomolar range (4–12nmol/L) for all cell lines tested. Control HPGs nanoparti-cles (no drug) showed no cytotoxicity across the testedconcentration range (15–1,500 nmol/L; data not shown).

In vivo efficacy of Taxotere and DTX-loaded HPGsIn vivo studies were done in a total of 42 nude mice to

evaluate the efficacy of a single intravesical treatment with

Mucoadhesive Nanoparticulate Docetaxel for Intravesical Therapy

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Taxotere (0.2 mg/mL) and DTX (0.2 mg/mL)-loaded HPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2. The KU7-luc tumor cell line, initially thought to be of humanbladder origin (i.e., high-grade urothelial carcinoma),was recently found to be of HeLa origin using arrayCGH (comparative genomic hybridization) in our labora-tory (data not shown). Nonetheless, KU7-luc is the onlycell line to date that reproducibly provides reliable tumor"take" rates as orthotopic xenografts without the use ofsecondary agents such as trypsin or electrocautery thattraumatize the urothelium to promote tumor cell adhe-sion. KU7-luc xenografts represent an epithelial "intrave-sical carcinoma model" that clinically resembles humanbladder cancer and the key characteristic of this model isthe lack of deeper invasion at early time points, whichmakes these tumors amenable to intravesical therapy. Thus,this bladder cancer "model", we believe, is still appropriateto test pharmacokinetic and pharmacodynamic propertiesof intravesical agents. After intravesical inoculation of KU7-luc cancer cells, all mice developed bladder tumors. How-ever, one mouse in DTX-loaded HPG-C8/10-MePEG-NH2

group died unexpectedly on day 4 posttreatment. Overall,intravesical DTX administered as either the commercialTaxotere or the HPGs formulations were well toleratedby mice and no major toxicities were observed. Comparedwith control mice, DTX-loaded HPGs inhibited tumorgrowth. However, DTX-loaded HPG-C8/10-MePEG-NH2

was the most effective formulation to inhibit tumor growthin KU7-luc orthotopic bladder cancer xenografts andreached statistical significance compared with either thePBS control or Taxotere groups (Fig. 2, P < 0.01, post hocBonferroni analysis after 2-way ANOVA). At the end of thestudy, a single intravesical instillation of DTX-loaded HPG-C8/10-MePEG-NH2 nanoparticles inhibited tumor growthby 88% compared with the PBS control groups. DTX-loaded HPG-C8/10-MePEG nanoparticles exhibited a 54%tumor inhibition in this treatment arm. On the other hand,the commercial formulation of Taxotere failed to inhibittumor growth in this orthotopic xenograft model. Repre-sentative bioluminescence images of mice over time ineach treatment group are shown in Figure 2. Histologicexamination of bladder tissues show that KU7-luc tumorsexhibited an aggressive growth pattern and frequent multi-focality, but after 25 days post–tumor inoculation, theywere generally confined to the lamina propria and corre-lated with high-grade T1 stage disease (Fig. 3). AlthoughDTX (0.2 mg/mL) did not cause any remarkable histologicchange in KU7-luc xenograft compared with the PBStreatment, DTX (0.2 mg/mL)-loaded HPG-C8/10-MePEGand/or HPG-C8/10-MePEG-NH2 inhibited tumor growth.Tumors treated by DTX loaded in HPG-C8/10-MePEG-NH2 decreased significantly in size with heterogeneouscellular size, nuclear shape, and infiltrating inflammatorycells.

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Figure 1. In vitro cytotoxicity of DTX formulations against the KU7-luc cell line, and both low-grade (RT4, MGHU3) and high-grade (UMUC3) humanurothelial carcinoma cell lines. Cells were exposed to either Taxotere or DTX-loaded HPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2 for 2 hours and cellviability was determined after 72 hours using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay. Data shown are representative of3 independent experiments.

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Drug uptake studiesTo evaluate the bladder tissue and serum uptake follow-

ing intravesical DTX formulations, mice with orthotopicbladder tumors were instilled with either Taxotere (0.2mg/mL, n ¼ 3) or DTX (0.2 mg/mL)-loaded HPG-C8/10-MePEG (n ¼ 4) and/or HPG-C8/10-MePEG-NH2 (n ¼ 4).The amount of DTX in urine, bladder tissue, and serumwere measured 2 hours postinstillation. Several serumsamples had nonquantifiable or no detectable signals.Mice instilled with Taxotere had no detectable DTX inserum at all time points. DTX-loaded HPG-C8/10-MePEG-NH2 exhibited the highest serum levels at the 2-hour timepoint (150.87� 34.98 vs. 23.97 � 16.71 ng/mL, P < 0.01,2-way ANOVA, Bonferroni posttest). However, serumconcentrations of DTX were several orders of magnitudelower than the concentrations in urine and bladder tissue(Table 1). DTX-loaded HPG-C8/10-MePEG-NH2 resultedin significantly higher amounts in bladder tissue accumu-lation compared with Taxotere or DTX-loaded HPG-C8/10-MePEG (P < 0.001, 1-way ANOVA, Bonferroni’s multiplecomparison test). There was no significant difference(P > 0.05, 1-way ANOVA) in bladder tissue accumulationbetween Taxotere and DTX-loaded HPG-C8/10-MePEGtreatment groups. The final urine concentrations wereabout 5- to 7-fold lower than the initial dosing solution.This was due to the urine dilution during the 2-hour periodof intravesical instillation. However, there was no signifi-cant difference (P > 0.05, 1-way ANOVA) in the final urineconcentrations of DTX between different treatmentgroups.

Tumormicroenvironment and uptake of rhodamine labeledHPGs. Bladder tumor microenvironment and distributionof rhodamine labeled HPGs into tumor tissue was assessed.Bladder tumor tissues were highly vascularized with anaverage distance of 40 to 60 mm to the nearest blood vessel(Fig. 4A). No significant difference was seen between differ-ent groups (P ¼ 0.8). The amount of fluorescence insidewhole bladder tumors was measured. Rhodamine labeledHPG-C8/10-MePEG-NH2 (HPG-C8/10-MePEG-NH2-TMRCA)exhibited the highest tumor uptake comparedwith the othergroups (P ¼ 0.037). There was no significant difference (P >0.05) in tumor uptake of the bladders instilled with freerhodamine (TMRCA) and rhodamine labeled HPG-C8/10-MePEG (Fig. 4B). The depth profile of rhodamine uptakeinto the tumor tissues was assessed as a function of distancefrom the bladder lumen. HPG-C8/10-MePEG-NH2-TMRCAnanoparticles showed enhanced tumor uptake at all dis-tances from lumen, showing a 5- to 6-fold increase overHPG-C8/10-MePEG-TMRCAnanoparticles (Fig. 4C). Figure 5shows sectionsof anorthotopic bladder tumor that hadbeeninstilled with either PBS, TMRCA, HPG-C8/10-MePEG-TMRCA, and/or HPG-C8/10-MePEG-NH2-TMRCA, wherethe red fluorescence shows the location of the rhodaminelabeled HPG nanoparticles.

Discussion

The objective of the present work was to evaluate theeffectiveness of intravesical mucoadhesive DTX formula-tions in an orthotopic model of bladder cancer and to

Figure 2. Treatment effectsof single intravesical DTXformulationson orthotopic bladdercancer xenografts. Female nudemice (n ¼ 9–13/group) wereintravesically inoculated with 2 �106 KU7-luc tumor cells on day 0and randomized on day 5 toreceive 50 mL instillation volumesof formulations; Taxotere (0.2 mg/mL; Sanofi-aventis) or DTX (0.2mg/mL)-loaded HPG-C8/10-MePEG and/or HPG-C8/10-MePEG-NH2 (mucoadhesiveDTX). Tumor growth wasdetermined in an IVIS 200 ImagingSystem (Xenogen; Caliper LifeScience). Bioluminescenceimaging of mice is shown on theleft.

4 11 18 254 11 18 25

TaxotereTaxotere

Days post–tumor inoculation

PBSControl

HPG-C8/10-MePEG/DTX

HPG-C8/10-MePEG-NH /DTX2

PBS (n = 13)

Taxotere (0.2 mg/mL, (r = 10)

HPG-C8/10-MePEG/DTX (0.2 mg/mL, (n = 9)HPG-C8/10-MePEG-NH2/DTX (0.2 mg/mL, (n = 9)

600

500

400

300

200

100

00 5 10 15 20 25

Time (d post–tumor inoculation)

Treatment

Tum

or b

iolu

min

esce

nce

(fol

d-ch

ange

of i

nitia

l flu

x)

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Table 1. Drug uptake of intravesical DTX formulations in orthotopic xenografts

DTX formulations (No. of mice) Curine,a mg/mL Cbladder,

b mg/g Cserum,c ng/mL

0.5 h 1 h 2 h

Taxotere (3) 31.4 � 15.5 1.24 � 0.54 BLOQ BLOQ BLOQDTX/HPG-C8/10-MePEG (4) 53.8 � 8.1 1.09 � 0.70 55.87 27.88 23.97 � 16.71DTX/HPG-C8/10-MePEG- NH2 (4) 27.6 � 4.0 13.07 � 4.32 81.47 � 23.76 88.21 � 39.42 150.87 � 34.98

NOTE: Data shown are the mean � SD. BLOQ, below the limit of quantification (lowest limit of quantification was 10 ng/mL).aFinal concentration of DTX in mouse urine after 2 hours of intravesical instillation measured by HPLC.bConcentration of DTX in mouse bladder tissue following a 2-hour intravesical instillation measured by LC/MS-MS.cConcentration of DTX in mouse serum taken at 0.5, 1, and 2 hours post–intravesical instillation measured by LC/MS-MS.

A1 A2 A3

2X 6X 20X

B1 B2 B3

2X 6X 20X

A

B

CC1 C2 C3

2X 6X 20X

Figure 3. Representative histologic sections of bladders harvested at the end of study, from mice receiving various formulations of 0.2 mg/mL DTX:(A) HPG-C8/10-MePEG-NH2, (B) HPG-C8/10-MePEG, and (C) Taxotere. After H&E staining of whole bladder section, the low magnification (2� on a BLISSmicroscope imaging workstation) show multifocal and aggressive pT1 disease. KU7-luc tumors grow partially below the urothelial lining (magnification6�) after 25 days post–tumor inoculation, there was no evidence of muscularis propria invasion despite some extremely large tumors observed in Taxotereand PBS control groups. High-power view (magnification 20�) showing evidence of hyperchromasia and mitotic figures within KU7-luc tumors. Withintreatment groups, A, B, and C, numbers 1 to 3 designate different magnifications.

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investigate bladder tissue and tumor uptake of these nano-particulate formulations.HPGs were synthesized via anionic ring opening multi-

branching polymerization of glycidol using trimethylol-propane (TMP) as an initiator. The HPG core was

derivatized with C8/C10 alkyl chains to create a hydropho-bic core, to allow the loading of DTX, whereas the MePEGchains linked to hydroxyl groups on HPGs create a hydro-philic shell intended to increase the aqueous solubility ofthese molecules. To increase their mucoadhesiveness,amine groups were conjugated to some of the hydroxylgroups on HPG-C8/10-MePEG polymer in a 2-step reactionprocedure (Supplementary Fig. S1 and Data Supplements).HPGs are small nanoparticles with hydrodynamic radii lessthan 10 nm. The surface modification of HPG-C8/10-MePEG with amine groups resulted in highly positivecharged nanoparticles with improved mucoadhesive prop-erties (Supplementary Table S1 and Data Supplements).DTX was loaded into these HPG nanoparticles due tohydrophobic interactions between the hydrophobic drugand the hydrophobic core of theHPGs. Themaximumdrugloading of DTX in HPGs was about 5%, which corre-sponded to about 5 to 6 DTXmolecules per HPGmolecule.Previously, we have shown that paclitaxel (PTX) can beloaded in HPGs; however, the loading capacity was lower(2% vs. 5%w/w) and this may be due to the fact that PTX isslightly larger (MW ¼ 854 vs. 808 Da) and more hydro-phobic than DTX (22). The release profiles of DTX fromHPGs were characterized by a continuous controlledrelease with little or no burst phase of release. Approxi-mately 55% of initially encapsulated drug was releasedwithin the first 24 hours of incubation in artificial urine.However, the presence of amine groups on the surface ofHPGs had no effect on drug release (19).

We evaluated the cytotoxic effects of the standard Tax-otere and DTX-loaded HPGs against the KU7-luc cell line,and both low-grade and high-grade human urothelialcarcinoma cell lines. All DTX formulations resulted inconcentration-dependent inhibition of proliferation inall cell lines tested. In agreement with our previous resultswith PTX, themore aggressive and fast growing KU7-luc cellline was the most sensitive to DTX formulations (23, 24).Loading of DTX in HPGs had no effect on its cytotoxicity.Indeed, DTX-loaded HPG-C8/10-MePEG or HPG-C8/10-MePEG-NH2 were found to be as cytotoxic as the commer-cial formulation of Taxotere (Fig. 1).

Next, we evaluated the effectiveness of a single intrave-sical therapy with DTX formulations in a mouse xenograftmodel of bladder cancer. Orthotopic implantation ofhuman cancer cells into immunodeficient mice is one ofthe best models currently available to assess the effectsof intravesical anticancer therapy in vivo. Using both his-tologic evaluation and magnetic resonance imaging, ourgroup has previously validated an orthotopic mousemodelof bladder cancer in which luciferase expressing KU7-luccells are transurethrally inoculated in nudemice and tumorburden is longitudinally quantified using bioluminescenceimaging (20). However, the fact that KU7 may not be of ahuman bladder origin has to be acknowledged and ourgroup is actively seeking new human urothelial carcinomacell models for evaluation of intravesical therapy. Themajor issue with most human urothelial carcinoma celllines is the low and nonreproducible tumor take in nude

80

A

B

C

50

12

9

6

3

0

80

60

40

20

00 50 100 150 200 250

Distance from lumen (µm)

30

0Ave

rage

dis

tace

to

near

est v

esss

el (

µm)

Ave

rage

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SE

)A

vera

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tens

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)

PBSHPG-C

8/10 -MePEG-TMRCA

HPG-C8/10 -MePEG-NH

2 -TMRCA

TMRCA

PBSHPG-C

8/10 -MePEG-TMRCA

HPG-C8/10 -MePEG-NH

2 -TMRCA

TMRCA

PBS

HPG-C8/10-MePEG-TMRCA

HPG-C8/10-MePEG-NH2-TMRCA

TMRCA

Figure 4.Orthotopic bladder carcinoma instilled with PBS, free rhodamine(TMRCA), rhodamine labeled HPG-C8/10-MePEG (HPG-C8/10-MePEG-TMRCA), and rhodamine labeled HPG-C8/10-MePEG-NH2 (HPG-C8/10-MePEG-NH2-TMRCA). Bladders were rinsed twice with PBS, excisedwhole, and frozen immediately. cryosections of 10 mm were subsequentlyimaged for fluorescent signal of rhodamine, then stained and imaged forvasculature CD 31 (A). B, amount of fluorescence inside the bladdertumors. C, observed rhodamine fluorescence in tumor tissues as afunction of distance from bladder lumen.

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mice which makes intravesical evaluation challenging. Ourobjectives in this study, however, were neither to developnor to establish a "true" non–muscle-invasive bladdercancer model but rather to evaluate the drug uptake andthe efficacy of our newly developed mucoadhesive DTXformulations. Compared with control mice, DTX-loadedHPGs inhibited tumor growth. However, DTX-loadedHPG-C8/10-MePEG-NH2 was the most effective formula-tion to inhibit tumor growth in KU7-luc orthotopic bladdercancer xenografts and reached statistical significancecompared with either the PBS control or Taxotere groups(Fig. 2, P < 0.01, post hoc Bonferroni analysis after 2-wayANOVA). This increase in efficacy likely resulted fromenhanced drug uptake in bladder and tumor tissues of micetreatedwithDTX-loadedHPG-C8/10-MePEG-NH2 nanopar-ticles. Our uptake studies have showed significantlyincreased drug uptake in bladder tissues and enhancedtumor uptake of rhodamine labeled HPG-C8/10-MePEG-NH2 nanoparticles (Table 1 and Fig. 4). These results arein agreement with our recent studies in which DTX-loadedHPG-C8/10-MePEG-NH2 nanoparticles significantlyincreased the urinary bladder wall permeability and uptakeofDTX in both isolated pig bladder aswell as in a livemousebladder tissues without tumors. Our preliminary studies on

the effects of HPGs on the urothelium have showed thatHPG-C8/10-MePEG-NH2 nanoparticles increase the perme-ability of the urinary bladder wall by causing changes to theurothelial barrier function and morphology through open-ing of tight junctions and exfoliation of the urothelium.These effects are similar to the effects on bladder mucosacaused by chitosan dispersions, a well-established, cationic,mucoadhesive polymer, which increased the permeabilityof isolated pig bladder to a model drug by causing desqua-mation of the urothelium (27–30).

Systemic absorption of chemotherapeutic drugs throughthe bladder wall is thought to be unlikely for agents with amolecular weight over 300 Da (31). Thus, DTX (808 Da)theoretically has little risk of systemic uptake. However,similar to our previous studies with PTX (854 Da) inhealthy mice (24), and in this model (23), we detectedsome systemic uptake especially in DTX-loadedHPG-C8/10-MePEG-NH2 treatment group. Systemic levels of DTX in theTaxotere group were below our limit of quantificationwhich was 10 ng/mL. This may be due to DTX sequestra-tion in micelles as has been reported for Taxol (32). Thepresence of Cremophol EL in the commercial formulationof PTX (Taxol) has been shown to reduce its penetrationinto the bladder tissues in dogs. This was due to PTX

TMRCAPBS

HPG-C8/10-MePEG-TMRCAHPG-C8/10-MePEG-NH2-TMRCA

Figure 5. Orthotopic bladdercarcinoma instilled with PBS (topleft), free rhodamine (TMRCA, topright), rhodamine labeled HPG-C8/

10-MePEG (bottom left), andrhodamine labeled HPG-C8/10-MePEG-NH2 (bottom right).Bladders were rinsed twice withPBS, excised whole, and frozenimmediately. Cryosections of 10mm were subsequently imaged forfluorescent signal of rhodamine(red), then stained, and imaged forcell nuclei (Hoechst 33342, gray)and vasculature (CD31, blue).Scale bar, 250 mm.

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sequestration into micelles which reduced the free fractionof PTX and consequently, lowed the drug penetration intothe bladder tissues (32). DTX is commercially formulatedin Tween 80 and mixed with 13% v/v ethanol and furtherdiluted prior to administration and is also entrapped inmicelles. The highest serum levels were obtained in theDTX-loaded HPG-C8/10-MePEG-NH2 arm at the 2-hourtime point [150.87 � 34.98 vs. 23.97 � 16.71 ng/mL(DTX-loadedHPG-C8/10-MePEG), P < 0.01, 2-way ANOVA,Bonferroni posttest]. This was not surprising as the levels inbladder tissues weremore than 10-fold higher than those ofDTX-loadedHPG-C8/10-MePEG (Table 1). Nevertheless, wedid not observe any local or systemic toxicity in eithergroup. Compared with humans, the mouse bladder wall isvery thin and with an assumed logarithmic decrease in drugpenetration across the tissue (33), lower serum levelswould be expected in humans. In the recent phase I trialof intravesical DTX for the treatment of non–muscle-inva-sive bladder cancer refractory to BCG therapy, DTX wasadministered as in 6 weekly instillations with a dose-escalation up to 75mg (34). The authors reported minimaltoxicity with dysuria being the most common; however, nosystemic absorption was reported for intravesical DTX.BCG therapy is the most effective intravesical therapy todate; unfortunately, head-to-head comparison with ourDTX formulations was not possible because our orthotopicmurine model lacks an immune response, which is impor-tant for the effectiveness of BCG therapy.In conclusion, the surface modification of HPG-C8/10-

MePEG with amine groups resulted in highly positivecharged nanoparticles (HPG-C8/10-MePEG-NH2) withimproved mucoadhesive properties. DTX-loaded HPGswere found to have equivalent cytotoxicities as the com-mercial Taxotere formulation in vitro. However, in vivo,DTX-loaded HPG-C8/10-MePEG-NH2 was the mosteffective formulation to inhibit tumor growth in anorthotopic model of bladder cancer. Furthermore, thisformulation significantly increased drug uptake inmouse bladder tissues. In addition, rhodamine labeled

HPG-C8/10-MePEG-NH2 showed enhanced tumor uptakeof these nanoparticles. Our data show promising in vivoantitumor efficacy and provide preclinical proof-of-principle for the intravesical application of mucoadhesiveDTX in the treatment of bladder cancer. We are in theprocess of understanding the effects of these mucoadhe-sive nanoparticles on the bladder urothelium and itsintegrity, which will give us valuable information abouttheir biocompatibility, toxicity, and mode of action. Oncethese studies have been completed, we hope to moveforward with early phase clinical trials to evaluate thesafety and efficacy of our mucoadhesive nanoparticulateDTX formulation in patients refractory to BCG.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Irina Chafeeva, Markus Heller, and Lucy Ye for theirexcellent technical assistance.

Grant Support

The above work was supported by an operating grant from theCanadian Institutes of Health Research (H.M. Burt and D.E. Brooks), theNatural Sciences and Engineering Research Council of Canada and theMichael Smith Foundation for Health Research (C. Mugabe and D.E.Brooks), Canadian Blood Services (D.E. Brooks). Research carried out withthe assistance of the LMB Macromolecular Hub at the Centre for BloodResearch was supported in part by grants from the Canada Foundation forInnovation and the Michael Smith Foundation for Health Research. Thiswork was also provided with support from the Centre for Drug Research andDevelopment and a grant from the Canada Foundation for Innovation.Research carried out by Y. Matsui and A.I. So were supported by NCIC/CCSRI research grant 018075.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 8, 2010; revised February 3, 2011; accepted February21, 2011; published OnlineFirst February 28, 2011.

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2011;17:2788-2798. Published OnlineFirst February 28, 2011.Clin Cancer Res   Clement Mugabe, Yoshiyuki Matsui, Alan I. So, et al.   Bladder Cancer

Muscle-Invasive−Docetaxel for Intravesical Treatment of Non Evaluation of Mucoadhesive Nanoparticulate In vivo

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