Correction

MEDICAL SCIENCES ENGINEERINGCorrection for ldquoIntracranial microcapsule chemotherapy delivery forthe localized treatment of rodent metastatic breast adenocarcinomain the brainrdquo by Urvashi M Upadhyay Betty Tyler Yoda PattaRobert Wicks Kevin Spencer Alexander Scott Byron Masi LeeHwang Rachel Grossman Michael Cima Henry Brem and RobertLanger which appeared in issue 45 November 11 2014 of Proc Natl

Acad Sci USA (11116071ndash16076 first published October 272014 101073pnas1313420110)The authors note that because Fig 2 did not publish in color

the legend for Fig 2 does not accurately describe the figure Thelegend has been corrected to describe a black and white figureThe figure and its corrected legend appear below

wwwpnasorgcgidoi101073pnas1600771113

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Release Devices

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Fig 2 (A) In vitro release of DOX from one- (triangle) three- (square) and five-hole (diamond) microcapsule devices loaded with 5 mg DOX (B) DOX LCPsingle-orifice device in vitro release compared with leak devices

wwwpnasorg PNAS | March 1 2016 | vol 113 | no 9 | E1327

CORR

ECTION

Intracranial microcapsule chemotherapy delivery forthe localized treatment of rodent metastatic breastadenocarcinoma in the brainUrvashi M Upadhyaya1 Betty Tylerb Yoda Pattac Robert Wicksb Kevin Spencerc Alexander Scottc Byron MasidLee Hwangb Rachel Grossmanb Michael Cimace Henry Brembfg and Robert Langerde1

aDepartment of Neurosurgery Brigham and Womenrsquos Hospital Boston MA 02115 bDepartment of Neurosurgery Johns Hopkins University HospitalBaltimore MD 21231 Departments of cMaterials Science and Engineering and dChemical Engineering and eKoch Institute for Integrative CancerResearch Massachusetts Institute of Technology Cambridge MA 02139 and Departments of fOncology and gBiomedical Engineering Johns HopkinsUniversity Hospital Baltimore MD 21205

Contributed by Robert Langer July 17 2013 (sent for review August 14 2012)

Metastases represent the most common brain tumors in adultsSurgical resection alone results in 45 recurrence and is usuallyaccompanied by radiation and chemotherapy Adequate chemo-therapy delivery to the CNS is hindered by the bloodndashbrain barrierEfforts at delivering chemotherapy locally to gliomas have shownmodest increases in survival likely limited by the infiltrative natureof the tumor Temozolomide (TMZ) is first-line treatment for glio-mas and recurrent brain metastases Doxorubicin (DOX) is used intreating many types of breast cancer although its use is limited bysevere cardiac toxicity Intracranially implanted DOX and TMZmicrocapsules are compared with systemic administration of thesame treatments in a rodent model of breast adenocarcinoma brainmetastases Outcomes were animal survival quantified drug expo-sure and distribution of cleaved caspase 3 Intracranial delivery ofTMZ and systemic DOX administration prolong survival more thanintracranial DOX or systemic TMZ Intracranial TMZ generates themore robust induction of apoptotic pathways We postulate thatthese differences may be explained by distribution profiles of eachdrug when administered intracranially TMZ displays a broader dis-tribution profile than DOX These microcapsule devices providea safe reliable vehicle for intracranial chemotherapy delivery andhave the capacity to be efficacious and superior to systemic deliveryof chemotherapy Future work should include strategies to improvethe distribution profile These findings also have broader implica-tions in localized drug delivery to all tissue because the efficacy ofa drug will always be limited by its ability to diffuse into surround-ing tissue past its delivery source

microcapsule | intracranial | chemotherapy | brain | metastases

The rationale behind localized drug delivery is that high con-centrations of drug may be reached in target tissue while

minimizing systemic exposure to the large quantities of drug thatmight otherwise be necessary to achieve the desired biologic effectLocalized drug delivery has been used such as in drug-elutingstents for coronary artery disease metered dose inhalers for thetreatment of asthma and intracranial chemotherapy wafers forbrain tumors This premise is particularly applicable to conditionsaffecting tissue that is sequestered from systemic circulation suchas conditions affecting the CNSAdequate and efficacious drug delivery to the CNS has proved

challenging Systemic delivery methods are hindered by lowpermeability at the bloodndashbrain barrier (BBB) to nonlipophilicdrugs which requires high systemic doses to be administeredthereby increasing systemic toxicity (1 2) One method of cir-cumventing the BBB is to place polymers that can release drug ina time-controlled manner at the site of tumor resection (3ndash6)Localized drug delivery devices provide high drug concentrationsto the tumor site while preventing drug degradation and clearanceuntil its release (7 8) The efficacy of current intracranial drugdelivery such as Carmustine wafers has promise with a doublingof median survival from 9 to 20 mo in the case of Carmustine

wafers (6 9 10) Phase III trial data indicated a 2-to 3-mo survivaladvantage in Carmustine wafers-treated patients (11) and Car-mustine wafers continue to have a role combined with radiationand oral temozolomide (TMZ) However nearly all patients stillsuccumb to tumor progressionThe clinical focus of CNS drug delivery has been high-grade

glioma (12) The modest increases in survival seen with suchstrategies have been attributed in part to the limited efficacy ofthe chemotherapies used and the disease target itself becausethese tumors are among the most infiltrative and aggressive tumorsstudied (6 8) It has also been postulated that the chemotherapythat has been used (Carmustine) does not diffuse well throughbrain tissue to produce tissue effects away from the implantationsite Such strategies have not been widely tested in more focalnoninfiltrative lesions of the brain such as brain metastases Sev-eral experiments have been conducted with polymer-based vehi-cles which locally delivery chemotherapy that suggest theirefficacy in brain metastases (13) The microcapsules described herewere studied in a rodent glioma model and showed tunable releasekinetics and efficacy (12) These vehicles have however not beentested in brain metastasesMetastases to the brain are the most commonly encountered

tumors in the brain (14ndash17) It is estimated that 100000ndash200000new cases are identified in the United States each year Fur-thermore 20 of patients with disseminated cancer have evi-dence of CNS involvement at autopsy (14) Certain tumors have

Significance

Brainmetastases represent themost common intracranial tumorsin adultsAdequate chemotherapydelivery to theCNS is hinderedby the bloodndashbrain barrier Efforts in intracranial chemotherapydelivery aim to maximize CNS levels while minimizing systemictoxicity however the success has been limited thus far In thiswork intracranial implanted doxorubicin and temozolomidemicrocapsules are compared with systemic administration in anovel rodent model of breast adenocarcinoma brain metastasesThese microcapsules are versatile and efficacious but that effi-cacymay depend on the ability of the chemotherapy to diffuse inbrain tissue These insights apply to other non-CNS applicationsof local drug delivery and drugs should be evaluated based ontheir ability to penetrate the targeted tissue as well as their in-herent efficacy

Author contributions UMU BT MC HB and RL designed research UMU BTYP RW KS LH and RG performed research UMU BT AS BM and HBcontributed new reagentsanalytic tools UMU BT MC and RL analyzed data andUMU wrote the paper

The authors declare no conflict of interest1To whom correspondence may be addressed Email urvashimupadhyaygmailcom orrlangermitedu

This article contains supporting information online at wwwpnasorglookupsuppldoi101073pnas1313420110-DCSupplemental

wwwpnasorgcgidoi101073pnas1313420110 PNAS | November 11 2014 | vol 111 | no 45 | 16071ndash16076

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a predilection to metastasize to the brain with nonsmall cell lungcancer breast carcinoma small cell lung carcinoma malignantmelanoma renal cell carcinoma gastrointestinal carcinoma uter-ine carcinoma and unknown primary carcinoma in decreasingfrequency (14 15) Cell surface markers seem to predict metastaticpotential for example human epidermal growth factor receptor2-positive breast carcinoma is more likely to present with CNSlesions (16) Metastases are focal in nature with no true invasionof brain tissue in contrast to high-grade gliomasThe treatment of brain metastases is often multimodal with

surgery whole-brain radiotherapy (WBRT) stereotactic radio-surgery and chemotherapy all playing a role (18 19) The selec-tion of specific modalities of treatment is dependent on patient-specific factors such as number size and location of metastasespatientrsquos overall health and prognosis and degree of symptomsattributable to these metastases Surgery remains a standard treat-ment for metastatic lesions particularly those that are symptomaticfor patients with good functional status and well-controlled extra-cranial disease (19) Metastases tend to be noninfiltrative histo-logically suggesting that surgical treatment should reduce oreliminate tumor burden and be beneficial Multiple retrospectiveanalyses have shown survival benefit from resection of singlebrain metastases particularly combined with WBRT (19ndash22)More recent data from 2005 suggest that patients with two orthree metastases also show increases in overall survival when thedominant symptomatic lesion is surgically resected (23) Themost promising trial of combination chemotherapy showed re-sponse rates of 40ndash59 in breast cancer metastases to the brain(24) although more recent studies show a wide range of responsefrom 10 to 45 with variability based on the primary tumor(19) Systemic chemotherapy delivery has played a limited role inthe treatment of brain metastases in part because of the poorpenetration through the BBB (19) More recent studies suggestthat chemotherapeutic agents that have better penetration of theBBB such as TMZ may have a role in newly diagnosed metas-tases not just as salvage therapy (25 26) The agents that haveshown promise include cyclophosphamide 5-fluorouracil metho-trextate and vincristine combined and TMZ although the use ofTMZ has been restricted to recurrent breast metastases (19 2728) The use of TMZ in recurrent breast cancer brain metastaseshas shown modest increases in survival although this patientpopulation represents a selection of the most aggressive and re-fractory cancers that have withstood surgery WBRT and multipleother systemic chemotherapiesDoxorubicin (DOX) has been used to treat early-stage node-

positive breast cancer particularly HER2-positive tumors al-though it is more commonly used for treatment of non-Hodgkinslymphoma and certain leukemias (29) Its clinical use in breastmetastases to the brain has largely been limited by the resistanceof the tumor to the chemotherapy as well as its poor penetrationof the BBB and its dose-related adverse effects The risk of de-veloping potentially lethal cardiotoxicity increased significantlywhen doses of DOX reach 500 mgm2 (30)Microcapsules loaded with DOX or TMZ were administered

intracranially in an experimental model of metastases to the brainin the following set of experiments The CRL1666 cell line hasbeen used in a metastatic spine model This body of work rep-resents the first time that it has been successfully used in a brainmetastasis model to the best of our knowledge (31) Their efficacywas compared with systemic administration of DOX and TMZTissue harvested from these animals was then processed for evi-dence of induction of apoptosis and the distribution of drug re-leased from the devices was also examined to test the relationshipbetween drug distribution and biologic activity and efficacy

ResultsTMZ Liquid Crystal Polymer Devices Liquid crystal polymer (LCP)is nonbiodegradable but biocompatible and inert and its liquidcrystal structure along with polymer chain rigidity do not allowdrug partitioning through its membrane which has been an issuewith other polymer compounds used (such as polylactic acid)

Fickrsquos Law of Diffusion was used to estimate the rate of drugdelivery from microcapsule devices for the device prototypeRelease kinetics were controlled by the diameter of device ori-fices the number of orifices and the solubility of the drug Theinner volume of the reservoir measured 15 mL which allowed forhigher drug loading per device volume compared with previousdrug delivery devices (10 32) One important innovation in thiswork was the use of the injection molding process for high-pre-cision device production which gave reliable and consistentresults including very small tolerances in volume These devicescan be engineered to achieve desired release kinetics Thesedevices are not biodegradable and were intact on retrieval frombrain tissue at the time of animal euthanasia without evidence ofbiofouling Multiple-orifice microcapsules were fabricated to in-crease the release rate of TMZ These devices were manufacturedwith a total of five orifices for drug release one 889-μm-diameterorifice in the cap and four 403-μm-diameter orifices around thecircumference of the microcapsule reservoir The theoretical re-lease rate of TMZ from a single 889-μm-diameter orifice micro-capsule was calculated by calculation of the flux (J) and the area(A) over which drug release occurs The release rate of TMZ fromthese devices was found to be 25 times 10minus5 mgs or 216 mgd (Fig 1)

In Vitro TMZ Drug Release Kinetics The in vitro release of TMZfrom the LCP microcapsules into HPLC-grade water was assayedby HPLC Control devices with sealed orifices were fabricated totest the hermeticity of the UV-curable epoxy and polymer housingA theoretical release curve based on Fickrsquos Law was constructedfor comparison with experimental drug release data The calcu-lated mass flow rate was 90 mgh The experiment was completedin triplicate Results were reported as average values with SDsThe experimental release curves were in agreement with the the-oretical release curve The experimental TMZ release rate duringthe first 100 h of release was 88 mgh plusmn 675 μg At this time 67of the total 12-mg payload was released The release rate de-creased after 100 h with 71 after 190 h The devices fabri-cated for drug leak tests showed that the epoxy sealed thedevices preventing leakage No drug partitioning through thepolymer housing was detected suggesting that LCP is capable ofstoring TMZ throughout the length of therapy

In Vitro DOX Drug Release Kinetics The in vitro release of DOXfrom LCP microcapsules was assayed using spectroscopy A the-oretical release curve was again constructed for comparison withexperimental release data The calculated mass flow rate for sin-gle-orifice devices was 121 mgd The calculated rate for multiple-hole devices was 216 mgd (Fig 2) The experiment was com-pleted in triplicate Results were reported as average values withSDs At 300 h nearly 70 of the 1-mg payload was released fromsingle-orifice devices Multiple-hole devices released 70 of thepayload from multiple-hole devices over a shorter time period(Fig 2) The experimental and theoretical release curves werein agreement

0102030405060708090

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LCP Devices

Theorecal Release

Fig 1 TMZ LCP device in vitro release

16072 | wwwpnasorgcgidoi101073pnas1313420110 Upadhyay et al

In Vivo Efficacy ExperimentsEfficacy of locally delivered DOX and TMZ The primary efficacy out-come was animal survival (Fig 3) The median survival of un-treated control animals was 11 d with mean of 10 d Animals withunfilled (blank) LCP-implanted devices had mean and mediansurvival of 12 d Animals with DOX-loaded LCP devices (payload =5 mg) had mean and median survival of 14 d Rats from thesystemic DOX and intracranial TMZ microcapsule groupsexhibited longer survival rates TMZ LCP device-treated animalshad median and mean survival of 19 and 18 d respectively withall dying at 21 d DOX-treated animals had mean and mediansurvival of 19 d with all animals dying by 25 d Statistical anal-yses of the survival data were performed and significant differ-ences were observed between the controlDOX (P lt 005)controlTMZ (P lt 005) blankDOX (P lt 005) and blankTMZ(P lt 005) groups There was no statistical significance shownbetween the control and DOX microcapsule groupsThe second in vivo experiment tested the efficacy of DOX

microcapsules loaded with lower payload (35 and 5 mg) againstother groups Animal survival was the primary outcome measure(Fig 4) Untreated control animals experienced a median sur-vival of 13 d Those animals treated with DOX LCP micro-capsules (payload = 5 mg) had median survival of 13 d and meansurvival of 15 d Animals treated with DOX LCP devices (pay-load = 35 mg) had median and mean survival of 12 and 13 drespectively DOX animals had median survival of 18 d withmean survival of 13 d Animals treated with oral TMZ had meanand median survival of 19 d with all animals dying by 21 dAnimals with TMZ microcapsules had median and mean survivalof 21 and 25 d respectively with all dying by 56 d Significantdifferences were observed between controloral TMZ (P lt 005)controlTMZ LCP (P = 001) DOX LCP (5 mg)TMZ LCP(P = 002) DOX LCP (35 mg)TMZ LCP (P = 002) and DOXTMZ LCP (P = 001) Increased survival in TMZ LCP micro-capsules was not statistically significant compared with oral TMZadministration (P = 009)Cleaved caspase 3 expression on immunohistochemistry Brain tissueharvested from the in vivo efficacy studies was formalin-fixed andparaffin-embedded for immunohistochemistry Coronal sectionswere obtained from each treatment group Cells expressing thecleaved caspase 3 protein were counted per high-power field(hpf) in all sections and compared between treatment groups(Fig 5) Untreated control animals had an average of 12 cellshpf DOX animal tissue had on average 10 cellshpf DOX LCP(5 mg) animal tissue had 81 cellshpf Blank LCP tissue had anaverage of 2 cellshpf TMZ LCP animal tissue had 103 cellshpfUsing the unpaired t test significant differences were observedbetween controlDOX microcapsule (P lt 005) controlTMZmicrocapsule (P lt 005) controloral TMZ (P = 0001) DOXDOX microcapsule (P lt 005) and oral TMZTMZ microcap-sule (P = 0003)Drug distribution after release from microcapsules Healthy rodentbrain tissues exposed to microcapsules filled with DOX or TMZwere examined for the spatial distribution of drug released overtime (Fig 6) Microcapsules loaded with DOX achieved narrowdistribution with a large peak seen at the sight of implantationand a steep drop out of drug level within 025 cm Rodent tissueexposed systemically to DOX showed low but uniform levelsof DOX Microcapsules loaded with TMZ achieved broader

distribution with more uniform levels of drug seen within 05 cmand a dropout was seen around 07 cm Systemic TMZ achieveda similar distribution profile but with lower exposure levels thanTMZ delivered through microcapsule

DiscussionA growing trend in pharmacology is designing targeted therapiesthat minimize indiscriminate delivery and maximize delivery tothe desired end organ Multiple strategies have been used in-cluding nanoparticle-driven delivery liposomal formulation andlocal delivery Each strategy aims to minimize systemic toxicityIn these experiments intracranial drug delivery was explored inmetastatic breast tumors to the brain The findings suggest thatlocal chemotherapy delivery may be more efficacious than itssystemic delivery and this efficacy may hinge on drug diffusionThe broader implication for delivery outside the CNS is thatsuccessful local delivery depends on the efficacy and distributionof the drug For those drugs that show strong biologic efficacybut with narrow distribution strategies to improve distributionsuch as chemical modification should be deployed to improvetheir tissue penetration

In Vitro Drug Release The rates of microcapsule devices exhibitedclose agreement with theoretical rates for the first 100 h of re-lease The release from single- and multiple-hole microcapsulesplateaus after this time as chemical driving force decreases fordiffusion This plateau results in slower drug diffusion thantheoretically predicted The stability of TMZ also has a profoundimpact on the in vitro release kinetics TMZ is a prodrug thatquickly degrades into the bioactive MTIC (3-methyl-(triazen-1-yl)imidazole-4-carboxamide) molecule under basic conditionsand in high-ionic strength solvents (32) This instability does notseem to be the case for DOX because it is not a prodrug In vitrorelease into water differs from in vivo conditions The half-life ofTMZ in water is considerably greater than its half-life in PBS inFBS and in vivo (32) This difference accounts for the closeagreement with theory that is seen during the first 100 h of theTMZ water release profiles Degradation of TMZ because of itsinstability in solution may account for the observed plateau andmay explain why 100 drug is not released experimentally

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A B

Fig 2 (A) In vitro release of DOX from one- (green)three- (red) and five-hole (blue) microcapsule devicesloaded with 5 mg DOX (B) DOX LCP single-orificedevice in vitro release compared with leak devices

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CRL1666 Control (n=8)

Blank Chip Day 0 (n=8)

Doxorubicin Chip 5mg Day 0 (n=8)

TMZ Chip 5mg Day 0 (n=8)

Doxil Injecon 567 mgkg (n=8)

Fig 3 Efficacy of locally delivered DOX and TMZ in breast metastasis model

Upadhyay et al PNAS | November 11 2014 | vol 111 | no 45 | 16073

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The microcapsule was designed for delivery of a large payloadfrom a device small enough for rodent intracranial implantationThe tight dimensional tolerances between the rodent skull and themicrocapsule device along with the introduction of a growing tu-mor could cause brain damage because of increased intracranialpressure (mass effect) This mass effect is pronounced in metas-tases patients and the primary indication for tumor resection sur-gery The consequence of mass effect is shown in the short survivalof blank microcapsule groupsThe microcapsule represents an improvement over prior in-

tracranial delivery As a reservoir-based vehicle it maximizespayload delivered per unit volume compared with other local de-livery strategies such as polymer-composite wafers Microcapsulesare capable of delivering solid or liquid drug formulations anability that polymer-composite wafers do not currently possessSuch devices may house multiple classes of therapeutics not justchemotherapy but macromolecules or tumor-directed viral par-ticles Release kinetics may be reliably tuned without the need forpolymer degradation to occur for drug release because the mi-crocapsule is made from LCP which does not permit drug parti-tioning These properties allow for highly versatile and tun-able drug delivery with the possibility of delivering a wide rangeof therapeutic compounds to brain tumors as well as otherhuman diseases

In Vivo Experiments The in vivo efficacy of localized TMZ andDOX therapy was investigated in a rodent breast metastasismodel TMZ microcapsules most effectively prolonged animalsurvival rates and these results reached statistical significance inboth experiments Systemic DOX also showed prolonged survivalcompared with controls Interestingly DOX microcapsules didnot show prolonged survival regardless of payload chosen sug-gesting that intracranial delivery is not always the most efficaciousdelivery route Locally delivered TMZ is equivalent or superior tosystemic TMZ However locally delivered DOX does not per-form as well as systemic DOX Systemic DOX is delivered in aliposomal formulation which aides in its ability to evade therecticuloendothelial system circulating longer The differ-ences in survival may be explained by distribution profiles seenin these experiments Intracranial TMZ and systemic DOX doachieve more uniform distributions compared with intracranialDOX delivery which seems to be quite narrow Oral TMZ wasadministered 5 d after tumor implantation to mimic the peakrelease of TMZ from our deviceIntracranial DOX had narrow distribution with levels drop-

ping significantly at 25 mm compared with the broader profileof intracranial TMZ with drop off first seen at 5ndash7 mm Strasseret al (33) found that drug delivered from a biodegradablepolymer implant was seen to diffuse only 1ndash2 mm from the site ofimplantation (33 34) The reasons for the differences observedcould be twofold the polymer composite delivery vehicle mayimpact drug distribution itself or the chemical properties of the

agent delivered [BCNU (bis-chloroethylnitrosourea) DOX orTMZ] are the larger determinant of the ability to diffuse in tissueGiven our experiments that used the same delivery vehicle forDOX and TMZ but yielded different profiles results suggest thatthe chemical properties are likely the larger factor in determiningdepth of drug distribution We suspect that prolonged survival inthe systemic DOX is because of a low uniform level of DOX whichmay be more ideal in treating cancer cells beyond the site of releaseOne important caveat to our findings is that the distribution

profiles were conducted in healthy rodents and not diseasedanimals The relatively hypoxic environment of cancer coupledwith the effect of tumor edema on diffusion may have a bearinghere These studies were a proof of principle of chemotherapydiffusion in brain tissue they highlight the stark differences inintracranial distribution for different agents These differencesmay explain limitations in efficacy of intracranial delivery evenin circumstances where the chemotherapy is potent and inducesa robust apoptotic response (such as in intracranial DOX) Cer-tainly additional studies in diseased animals where 3D models ofdrug distribution may be compared with the spatial distribution ofbiologic activity (such as cleaved caspase 3 expression) wouldbe enlighteningThe histological analysis of each experimental group was also

interesting Intracranial delivery of chemotherapy led to morerobust expression of apoptotic markers for both TMZ and DOXHowever the tissue evidence of cleaved caspase 3 did not ex-plain or follow survival data that is intracranial (IC) DOXanimals actually displayed poor survival compared with systemicDOX animals The local activity of a drug may not adequatelyincrease survival because the drug must also act at some dis-tance away from the implantation site The ability of micro-capsular TMZ to induce apoptosis more robustly than any othermodality tested coupled with its wide distribution profile supportthe improved survival observed in the intracranial TMZ groupThe CRL1666 cell line has been used in a model of breast

metastases to the spine This body of work represents its first usein a brain metastasis model Metastases are noninfiltrative tumorsand this cell line represents an appropriate histological model Insharp contrast primary brain cancer models should ideally modelthe infiltrative nature of these tumors We believe that this body ofwork represents the potential to use local intracranial drug de-livery for a different indication to treat brain metastases Theirhistologic nature may render them a better suited target by less-ening the burden of broad drug distribution because chemo-therapies may only need to diffuse centimeters away from theirdelivery site to be efficaciousTMZ was chosen based on the experience of certain groups with

its use as a salvage treatment in breast metastases to the brain Invitro cytotoxicity experiments against this cell line supported thechoice to test its efficacy in vivo Our results suggest that TMZmayplay a clinical role in treatment of breast metastases to the brainWe feel that these results bear additional investigation Currentlyit is only used in a rescue setting but may have a role in first-linetreatment of breast metastases to the brain particularly when

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CRL 1666 Control (n=8)

Dox Chip 5 (n=8)

Dox Chip 35 (n=16)

Doxil (n=16)

TMZ Chip (n=8)

TMZ Oral (n=8)

Days

Fig 4 Efficacy of locally delivered DOX and TMZ in second breast metas-tasis experiment

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Doxil (n=3) DOX IC (n=3) TMZ IC (n=4) Oral TMZ (n=4)

No treatment (n=4)

Blank LCP Device (n=3)

Posi

ve

cells

hpf

Treatment Group

Cleaved Casp 3 Expression

Fig 5 Cleaved caspase 3 (cleaved casp 3) expression IC intracranial

16074 | wwwpnasorgcgidoi101073pnas1313420110 Upadhyay et al

The microcapsule was designed for delivery of a large payloadfrom a device small enough for rodent intracranial implantationThe tight dimensional tolerances between the rodent skull and themicrocapsule device along with the introduction of a growing tu-mor could cause brain damage because of increased intracranialpressure (mass effect) This mass effect is pronounced in metas-tases patients and the primary indication for tumor resection sur-gery The consequence of mass effect is shown in the short survivalof blank microcapsule groupsThe microcapsule represents an improvement over prior in-

tracranial delivery As a reservoir-based vehicle it maximizespayload delivered per unit volume compared with other local de-livery strategies such as polymer-composite wafers Microcapsulesare capable of delivering solid or liquid drug formulations anability that polymer-composite wafers do not currently possessSuch devices may house multiple classes of therapeutics not justchemotherapy but macromolecules or tumor-directed viral par-ticles Release kinetics may be reliably tuned without the need forpolymer degradation to occur for drug release because the mi-crocapsule is made from LCP which does not permit drug parti-tioning These properties allow for highly versatile and tun-able drug delivery with the possibility of delivering a wide rangeof therapeutic compounds to brain tumors as well as otherhuman diseases

In Vivo Experiments The in vivo efficacy of localized TMZ andDOX therapy was investigated in a rodent breast metastasismodel TMZ microcapsules most effectively prolonged animalsurvival rates and these results reached statistical significance inboth experiments Systemic DOX also showed prolonged survivalcompared with controls Interestingly DOX microcapsules didnot show prolonged survival regardless of payload chosen sug-gesting that intracranial delivery is not always the most efficaciousdelivery route Locally delivered TMZ is equivalent or superior tosystemic TMZ However locally delivered DOX does not per-form as well as systemic DOX Systemic DOX is delivered in aliposomal formulation which aides in its ability to evade therecticuloendothelial system circulating longer The differ-ences in survival may be explained by distribution profiles seenin these experiments Intracranial TMZ and systemic DOX doachieve more uniform distributions compared with intracranialDOX delivery which seems to be quite narrow Oral TMZ wasadministered 5 d after tumor implantation to mimic the peakrelease of TMZ from our deviceIntracranial DOX had narrow distribution with levels drop-

ping significantly at 25 mm compared with the broader profileof intracranial TMZ with drop off first seen at 5ndash7 mm Strasseret al (33) found that drug delivered from a biodegradablepolymer implant was seen to diffuse only 1ndash2 mm from the site ofimplantation (33 34) The reasons for the differences observedcould be twofold the polymer composite delivery vehicle mayimpact drug distribution itself or the chemical properties of the

agent delivered [BCNU (bis-chloroethylnitrosourea) DOX orTMZ] are the larger determinant of the ability to diffuse in tissueGiven our experiments that used the same delivery vehicle forDOX and TMZ but yielded different profiles results suggest thatthe chemical properties are likely the larger factor in determiningdepth of drug distribution We suspect that prolonged survival inthe systemic DOX is because of a low uniform level of DOX whichmay be more ideal in treating cancer cells beyond the site of releaseOne important caveat to our findings is that the distribution

profiles were conducted in healthy rodents and not diseasedanimals The relatively hypoxic environment of cancer coupledwith the effect of tumor edema on diffusion may have a bearinghere These studies were a proof of principle of chemotherapydiffusion in brain tissue they highlight the stark differences inintracranial distribution for different agents These differencesmay explain limitations in efficacy of intracranial delivery evenin circumstances where the chemotherapy is potent and inducesa robust apoptotic response (such as in intracranial DOX) Cer-tainly additional studies in diseased animals where 3D models ofdrug distribution may be compared with the spatial distribution ofbiologic activity (such as cleaved caspase 3 expression) wouldbe enlighteningThe histological analysis of each experimental group was also

interesting Intracranial delivery of chemotherapy led to morerobust expression of apoptotic markers for both TMZ and DOXHowever the tissue evidence of cleaved caspase 3 did not ex-plain or follow survival data that is intracranial (IC) DOXanimals actually displayed poor survival compared with systemicDOX animals The local activity of a drug may not adequatelyincrease survival because the drug must also act at some dis-tance away from the implantation site The ability of micro-capsular TMZ to induce apoptosis more robustly than any othermodality tested coupled with its wide distribution profile supportthe improved survival observed in the intracranial TMZ groupThe CRL1666 cell line has been used in a model of breast

metastases to the spine This body of work represents its first usein a brain metastasis model Metastases are noninfiltrative tumorsand this cell line represents an appropriate histological model Insharp contrast primary brain cancer models should ideally modelthe infiltrative nature of these tumors We believe that this body ofwork represents the potential to use local intracranial drug de-livery for a different indication to treat brain metastases Theirhistologic nature may render them a better suited target by less-ening the burden of broad drug distribution because chemo-therapies may only need to diffuse centimeters away from theirdelivery site to be efficaciousTMZ was chosen based on the experience of certain groups with

its use as a salvage treatment in breast metastases to the brain Invitro cytotoxicity experiments against this cell line supported thechoice to test its efficacy in vivo Our results suggest that TMZmayplay a clinical role in treatment of breast metastases to the brainWe feel that these results bear additional investigation Currentlyit is only used in a rescue setting but may have a role in first-linetreatment of breast metastases to the brain particularly when

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Dox Chip 5 (n=8)

Dox Chip 35 (n=16)

Doxil (n=16)

TMZ Chip (n=8)

TMZ Oral (n=8)

Days

Fig 4 Efficacy of locally delivered DOX and TMZ in second breast metas-tasis experiment

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Posi

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cells

hpf

Treatment Group

Cleaved Casp 3 Expression

Fig 5 Cleaved caspase 3 (cleaved casp 3) expression IC intracranial

16074 | wwwpnasorgcgidoi101073pnas1313420110 Upadhyay et al

whole-brain radiation therapy may not be attractive given its long-term effects on cognition This body of work is not meant to sug-gest that intracranial TMZ therapy alone may cure metastaticbreast cancer however these studies represent a proof of conceptthat a chemotherapy-like TMZ which until now has been usedwith limited success may be more beneficial when delivered in-tracranially in a device with tunable release kineticsIntracranial chemotherapy delivery has largely focused on

high-grade gliomas which are infiltrative by nature and quitedifferent from metastatic lesions (13 35) The limited efficacy ofcurrent intracranial delivery schema including Carmustine wafersmay be attributed to the inability of chemotherapy to penetratebrain tissue well Our experiments support the notion that dif-ferences in chemotherapeutic drug distribution seem to correlateto tissue response to chemotherapy and length of survival There-fore efforts in exploring intracranial drug delivery should focus inpart on strategies to improve and broaden the distribution of thedrug through tissue

ConclusionThis work has shown that polymer microcapsules are capable ofachieving tunable rates of release of various chemotherapiesTMZ microcapsules and systemic DOX seem to show promisingefficacy against breast metastases to the brain We have alsoshown that the tissues harvested from these experiments indicatethat TMZ microcapsules induce apoptosis at higher levels thanDOX microcapsules or systemic DOX Finally we have shownthat the drug distribution profiles for intracranial and systemicDOX are quite different from that of intracranial TMZ and mayexplain the differences observed in survival Microcapsules offerversatile targeted delivery without the requirement for intensivedrug formulation necessary for liposomal nanoparticle or sol-vent drug-loading methods These devices also have the uniqueability to simultaneously deliver multiple therapies and may beused to deliver a variety of treatments beyond chemotherapy in-cluding nanoparticles and small molecules The ability of a drug topenetrate tissue requires additional investigation drugs that show

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Fig 6 (A) DOX distribution in rodent tissue when released from the microcapsule (B) DOX distribution in rodent tissue when delivered systemically (C) TMZdistribution in rodent tissue when released from the microcapsule (D) TMZ distribution in rodent tissue when delivered systemically (E) Comparison of drugdistribution profiles when TMZ and DOX were delivered through the microcapsule

Upadhyay et al PNAS | November 11 2014 | vol 111 | no 45 | 16075

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improved distribution profiles are likely to show more promise intreating lesions of the brain Finally the limitations of prior effortsat intracranial chemotherapy delivery may be overcome with focuson new therapeutic targets and strategies to improve the ability ofa given chemotherapy to diffuse through CNS tissue These insightsapply to non-CNS applications of local drug delivery where drugdiffusion and inherent efficacy should be analyzed in target tissueIn cases where tissue penetration is poor drug formulation strat-egies although cumbersome should improve the drugrsquos overalleffect When this two-pronged strategy is used many new appli-cations of localized drug delivery may be identified andfurther studied

Materials and MethodsIn Vitro Drug Release Kinetics of TMZ Twelve milligrams TMZ was loaded intoLCP single- and multiple-hole devices TMZ quantification was done usingHPLC with acetonitrile (928) mobile phase and UV absorption at 316 nmInterpolation of the readings with a standard curve was used to quantify theamount of drug released

In Vitro Drug Release Kinetics of DOX DOX (35 and 5 mg) was loaded into LCPsingle- and multiple-hole polymer microcapsules for in vitro drug releasecharacterization DOX was quantified using the UV spectrophotometer at481 nm Interpolation of readings with a standard curve was used to quantifythe amount of drug released

In Vitro Cytotoxicity CRL1666 mammary adenocarcinoma cells were exposedto TMZ and DOX at varying concentrations to determine cytotoxicity Cellproliferation was quantified using the MTT assay (Invitrogen)

In Vivo ExperimentsTumor and device implantation Briefly animals were anesthetized and the durawas exposed Stereotactic injection consisted of 75000 tumor cells in 25 μL thatwere placed into the brain at a depth of 35 mm and delivered over 2 minDevices were implanted at the time of tumor injection

Efficacy of locally delivered TMZ and DOX Two experiments were conducted todetermine the efficacy of locally delivered TMZ and DOX in tumor-bearingrats All TMZ microcapsules used in this experiment had a total TMZ payloadof 12 mg with a single 889-μm-diameter orifice cap All DOX microcapsuledevices used in this experiment had a total payload of 5 mg with a single180-μm-diameter orifice cap and one 430-μm-diameter orifice on the reser-voir The first experiment included five experimental groups and 40 F344rats (i) no treatment (n = 8) (ii) ip DOX (Doxil Johnson amp Johnson) on days0 6 and 13 after tumor implantation (n = 8) (iii) empty devices (n = 8) (iv)TMZ-filled devices (n = 8) and (v) DOX-filled devices (n = 8) The secondstudy included 80 F344 rats divided into seven experimental groups (i) notreatment control (n = 16) (ii) oral TMZ by gavage on days 5ndash9 after tumorimplantation (n = 8) (iii) TMZ-filled LCP microcapsule (n = 8) (iv) DOX-filledLCP microcapsule with 5 mg DOX payload (n = 8) (v) DOX-filled LCPmicrocapsule with 35 mg DOX payload (n = 16) (vi) ip DOX ondays 0 6 and 13 (n = 16) and (vii) empty device controls (n = 8) Overallanimal survival was compared with that of untreated controls Death wasthe primary end point for efficacy studies KaplanndashMeier survival curveswere constructedImmunohistochemical staining Cleaved caspase 3 stainingwas developed by CellSignaling Technology The whole brain was sectioned and counted in ag-gregate including tumor as well as peritumoral tissueSpatial drug distribution Nondiseased F344 rats underwent implantation of LCPdevices with DOX or C13-radiolabeled TMZ DOX LCP device animals were killedon days 2 4 6 8 and 10 (n = 4) Radiolabeled TMZ animals were killed on days2 4 6 8 and 10 (n = 5) Rodent brain tissue was homogenized and processedfor quantification of radiolabeled TMZ or DOXWestern blotting Anti-cleaved caspase 3 Western blotting was done on ratbrain tissue homogenates obtained from efficacy trials Cleaved caspase 3rabbit mAb was obtained from Cell Signaling Technology

ACKNOWLEDGMENTS The authors thank Dr Nancy Lin a breast canceroncologist at DanandashFarber Cancer Institute who advised us on the clinicalaspects of metastatic breast cancer treatment This work was funded par-tially by National Institutes of Health Grant R01 EB006365-06 and BrainScience Foundation Private Grant 106708

1 DeAngelis LM (2001) Brain tumors N Engl J Med 344(2)114ndash1232 Raghavan R et al (2006) Convection-enhanced delivery of therapeutics for brain

disease and its optimization Neurosurg Focus 20(4)E123 Brem H Langer R (1996) Polymer-based drug delivery to the brain Sci Med (Phila) 3

52ndash614 Lesniak MS Brem H (2004) Targeted therapy for brain tumours Nat Rev Drug Discov

3(6)499ndash5085 Sawyer AJ Piepmeier JM Saltzman WM (2006) New methods for direct delivery of

chemotherapy for treating brain tumors Yale J Biol Med 79(3-4)141ndash1526 Brem H et al The Polymer-brain Tumor Treatment Group (1995) Placebo-controlled

trial of safety and efficacy of intraoperative controlled delivery by biodegradablepolymers of chemotherapy for recurrent gliomas Lancet 3451008ndash1012

7 Santini JT Jr Cima MJ Langer R (1999) A controlled-release microchip Nature397(6717)335ndash338

8 Santini J Jr Richards AC Scheidt R Cima MJ Langer R (2000) Microchips as controlleddrug-delivery devices Angew Chem Int Ed Engl 39(14)2396ndash2407

9 McGirt MJ et al (2009) Gliadel (BCNU) wafer plus concomitant temozolomide ther-apy after primary resection of glioblastoma multiforme J Neurosurg 110(3)583ndash588

10 Lechapt-Zalcman E et al (2012) O(6) -methylguanine-DNA methyltransferase (MGMT)promoter methylation and low MGMT-encoded protein expression as prognosticmarkers in glioblastoma patients treated with biodegradable carmustine wafer im-plants after initial surgery followed by radiotherapy with concomitant and adjuvanttemozolomide Cancer 118(18)4545ndash4554

11 Westphal M et al (2003) A phase 3 trial of local chemotherapy with biodegradablecarmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant gliomaNeuro-oncol 5(2)79ndash88

12 Scott AW et al (2011) Intracranial microcapsule drug delivery device for the treat-ment of an experimental gliosarcoma model Biomaterials 32(10)2532ndash2539

13 Ewend MG Sampath P Williams JA Tyler BM Brem H (1998) Local delivery of che-motherapy prolongs survival in experimental brain metastases from breast carci-noma Neurosurgery 43(5)1185ndash1193

14 Nussbaum ES Djalilian HR Cho KH Hall WA (1996) Brain metastases Histologymultiplicity surgery and survival Cancer 78(8)1781ndash1788

15 Rusciano D Burger MM (1993) Mechanisms of metastases Molecular Genetics ofNervous System Tumors eds Levine AJ Schmidek HH (Wiley New York)

16 Debinski W Tatter SB (2009) Convection-enhanced delivery for the treatment of braintumors Expert Rev Neurother 9(10)1519ndash1527

17 Bindal RK Sawaya R Leavens ME Lee JJ (1993) Surgical treatment of multiple brainmetastases J Neurosurg 79(2)210ndash216

18 Bindal AK et al (1996) Surgery versus radiosurgery in the treatment of brain me-tastasis J Neurosurg 84(5)748ndash754

19 Eichler AF Loeffler JS (2007) Multidisciplinary management of brain metastasesOncologist 12(7)884ndash898

20 Patchell RA et al (2006) Radiosurgery plus whole-brain radiation therapy for brainmetastases JAMA 296(17)2089ndash2090

21 Patchell RA et al (1990) A randomized trial of surgery in the treatment of singlemetastases to the brain N Engl J Med 322(8)494ndash500

22 Pirzkall A et al (1998) Radiosurgery alone or in combination with whole-brain ra-diotherapy for brain metastases J Clin Oncol 16(11)3563ndash3569

23 Paek SH Audu PB Sperling MR Cho J Andrews DW (2005) Reevaluation of surgeryfor the treatment of brain metastases Review of 208 patients with single or multiplebrain metastases treated at one institution with modern neurosurgical techniquesNeurosurgery 56(5)1021ndash1034

24 Rosner D Nemoto T Lane WW (1986) Chemotherapy induces regression of brainmetastases in breast carcinoma Cancer 58(4)832ndash839

25 Stark AM (2010) Neurosurgical treatment of breast cancer metastases to the neuro-cranium Patholog Res Int 2011(2010)549847

26 Chang JE Robins HI Mehta MP (2007) Therapeutic advances in the treatment of brainmetastases Clin Adv Hematol Oncol 5(1)54ndash64

27 Abrey LE et al (2001) A phase II trial of temozolomide for patients with recurrent orprogressive brain metastases J Neurooncol 53(3)259ndash265

28 Christodoulou C et al Hellenic Cooperative Oncology Group (2001) Phase II study oftemozolomide in heavily pretreated cancer patients with brain metastases AnnOncol 12(2)249ndash254

29 Laginha KM Verwoert S Charrois GJ Allen TM (2005) Determination of doxorubicinlevels in whole tumor and tumor nuclei in murine breast cancer tumors Clin CancerRes 11(19 Pt 1)6944ndash6949

30 Ortho Biotech Products LP (2007) DOXIL Product Information Available at stagedoxilcomsharedproductdoxilprescribing-informationpdf Accessed April 19 2007

31 Mantha A et al (2005) A novel rat model for the study of intraosseous metastaticspine cancer J Neurosurg Spine 2(3)303ndash307

32 Newlands ES Stevens MF Wedge SR Wheelhouse RT Brock C (1997) TemozolomideA review of its discovery chemical properties pre-clinical development and clinicaltrials Cancer Treat Rev 23(1)35ndash61

33 Strasser JF Fung LK Eller S Grossman SA Saltzman WM (1995) Distribution of 13-bis(2-chloroethyl)-1-nitrosourea and tracers in the rabbit brain after interstitial deliveryby biodegradable polymer implants J Pharmacol Exp Ther 275(3)1647ndash1655

34 Fung LK et al (1998) Pharmacokinetics of interstitial delivery of carmustine4-hydroperoxycyclophosphamide and paclitaxel from a biodegradable polymerimplant in the monkey brain Cancer Res 58(4)672ndash684

35 Ewend MG et al (1996) Local delivery of chemotherapy and concurrent externalbeam radiotherapy prolongs survival in metastatic brain tumor models Cancer Res56(22)5217ndash5223

16076 | wwwpnasorgcgidoi101073pnas1313420110 Upadhyay et al

improved distribution profiles are likely to show more promise intreating lesions of the brain Finally the limitations of prior effortsat intracranial chemotherapy delivery may be overcome with focuson new therapeutic targets and strategies to improve the ability ofa given chemotherapy to diffuse through CNS tissue These insightsapply to non-CNS applications of local drug delivery where drugdiffusion and inherent efficacy should be analyzed in target tissueIn cases where tissue penetration is poor drug formulation strat-egies although cumbersome should improve the drugrsquos overalleffect When this two-pronged strategy is used many new appli-cations of localized drug delivery may be identified andfurther studied

Materials and MethodsIn Vitro Drug Release Kinetics of TMZ Twelve milligrams TMZ was loaded intoLCP single- and multiple-hole devices TMZ quantification was done usingHPLC with acetonitrile (928) mobile phase and UV absorption at 316 nmInterpolation of the readings with a standard curve was used to quantify theamount of drug released

In Vitro Drug Release Kinetics of DOX DOX (35 and 5 mg) was loaded into LCPsingle- and multiple-hole polymer microcapsules for in vitro drug releasecharacterization DOX was quantified using the UV spectrophotometer at481 nm Interpolation of readings with a standard curve was used to quantifythe amount of drug released

In Vitro Cytotoxicity CRL1666 mammary adenocarcinoma cells were exposedto TMZ and DOX at varying concentrations to determine cytotoxicity Cellproliferation was quantified using the MTT assay (Invitrogen)

In Vivo ExperimentsTumor and device implantation Briefly animals were anesthetized and the durawas exposed Stereotactic injection consisted of 75000 tumor cells in 25 μL thatwere placed into the brain at a depth of 35 mm and delivered over 2 minDevices were implanted at the time of tumor injection

Efficacy of locally delivered TMZ and DOX Two experiments were conducted todetermine the efficacy of locally delivered TMZ and DOX in tumor-bearingrats All TMZ microcapsules used in this experiment had a total TMZ payloadof 12 mg with a single 889-μm-diameter orifice cap All DOX microcapsuledevices used in this experiment had a total payload of 5 mg with a single180-μm-diameter orifice cap and one 430-μm-diameter orifice on the reser-voir The first experiment included five experimental groups and 40 F344rats (i) no treatment (n = 8) (ii) ip DOX (Doxil Johnson amp Johnson) on days0 6 and 13 after tumor implantation (n = 8) (iii) empty devices (n = 8) (iv)TMZ-filled devices (n = 8) and (v) DOX-filled devices (n = 8) The secondstudy included 80 F344 rats divided into seven experimental groups (i) notreatment control (n = 16) (ii) oral TMZ by gavage on days 5ndash9 after tumorimplantation (n = 8) (iii) TMZ-filled LCP microcapsule (n = 8) (iv) DOX-filledLCP microcapsule with 5 mg DOX payload (n = 8) (v) DOX-filled LCPmicrocapsule with 35 mg DOX payload (n = 16) (vi) ip DOX ondays 0 6 and 13 (n = 16) and (vii) empty device controls (n = 8) Overallanimal survival was compared with that of untreated controls Death wasthe primary end point for efficacy studies KaplanndashMeier survival curveswere constructedImmunohistochemical staining Cleaved caspase 3 stainingwas developed by CellSignaling Technology The whole brain was sectioned and counted in ag-gregate including tumor as well as peritumoral tissueSpatial drug distribution Nondiseased F344 rats underwent implantation of LCPdevices with DOX or C13-radiolabeled TMZ DOX LCP device animals were killedon days 2 4 6 8 and 10 (n = 4) Radiolabeled TMZ animals were killed on days2 4 6 8 and 10 (n = 5) Rodent brain tissue was homogenized and processedfor quantification of radiolabeled TMZ or DOXWestern blotting Anti-cleaved caspase 3 Western blotting was done on ratbrain tissue homogenates obtained from efficacy trials Cleaved caspase 3rabbit mAb was obtained from Cell Signaling Technology

ACKNOWLEDGMENTS The authors thank Dr Nancy Lin a breast canceroncologist at DanandashFarber Cancer Institute who advised us on the clinicalaspects of metastatic breast cancer treatment This work was funded par-tially by National Institutes of Health Grant R01 EB006365-06 and BrainScience Foundation Private Grant 106708

1 DeAngelis LM (2001) Brain tumors N Engl J Med 344(2)114ndash1232 Raghavan R et al (2006) Convection-enhanced delivery of therapeutics for brain

disease and its optimization Neurosurg Focus 20(4)E123 Brem H Langer R (1996) Polymer-based drug delivery to the brain Sci Med (Phila) 3

52ndash614 Lesniak MS Brem H (2004) Targeted therapy for brain tumours Nat Rev Drug Discov

3(6)499ndash5085 Sawyer AJ Piepmeier JM Saltzman WM (2006) New methods for direct delivery of

chemotherapy for treating brain tumors Yale J Biol Med 79(3-4)141ndash1526 Brem H et al The Polymer-brain Tumor Treatment Group (1995) Placebo-controlled

trial of safety and efficacy of intraoperative controlled delivery by biodegradablepolymers of chemotherapy for recurrent gliomas Lancet 3451008ndash1012

7 Santini JT Jr Cima MJ Langer R (1999) A controlled-release microchip Nature397(6717)335ndash338

8 Santini J Jr Richards AC Scheidt R Cima MJ Langer R (2000) Microchips as controlleddrug-delivery devices Angew Chem Int Ed Engl 39(14)2396ndash2407

9 McGirt MJ et al (2009) Gliadel (BCNU) wafer plus concomitant temozolomide ther-apy after primary resection of glioblastoma multiforme J Neurosurg 110(3)583ndash588

10 Lechapt-Zalcman E et al (2012) O(6) -methylguanine-DNA methyltransferase (MGMT)promoter methylation and low MGMT-encoded protein expression as prognosticmarkers in glioblastoma patients treated with biodegradable carmustine wafer im-plants after initial surgery followed by radiotherapy with concomitant and adjuvanttemozolomide Cancer 118(18)4545ndash4554

11 Westphal M et al (2003) A phase 3 trial of local chemotherapy with biodegradablecarmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant gliomaNeuro-oncol 5(2)79ndash88

12 Scott AW et al (2011) Intracranial microcapsule drug delivery device for the treat-ment of an experimental gliosarcoma model Biomaterials 32(10)2532ndash2539

13 Ewend MG Sampath P Williams JA Tyler BM Brem H (1998) Local delivery of che-motherapy prolongs survival in experimental brain metastases from breast carci-noma Neurosurgery 43(5)1185ndash1193

14 Nussbaum ES Djalilian HR Cho KH Hall WA (1996) Brain metastases Histologymultiplicity surgery and survival Cancer 78(8)1781ndash1788

15 Rusciano D Burger MM (1993) Mechanisms of metastases Molecular Genetics ofNervous System Tumors eds Levine AJ Schmidek HH (Wiley New York)

16 Debinski W Tatter SB (2009) Convection-enhanced delivery for the treatment of braintumors Expert Rev Neurother 9(10)1519ndash1527

17 Bindal RK Sawaya R Leavens ME Lee JJ (1993) Surgical treatment of multiple brainmetastases J Neurosurg 79(2)210ndash216

18 Bindal AK et al (1996) Surgery versus radiosurgery in the treatment of brain me-tastasis J Neurosurg 84(5)748ndash754

19 Eichler AF Loeffler JS (2007) Multidisciplinary management of brain metastasesOncologist 12(7)884ndash898

20 Patchell RA et al (2006) Radiosurgery plus whole-brain radiation therapy for brainmetastases JAMA 296(17)2089ndash2090

21 Patchell RA et al (1990) A randomized trial of surgery in the treatment of singlemetastases to the brain N Engl J Med 322(8)494ndash500

22 Pirzkall A et al (1998) Radiosurgery alone or in combination with whole-brain ra-diotherapy for brain metastases J Clin Oncol 16(11)3563ndash3569

23 Paek SH Audu PB Sperling MR Cho J Andrews DW (2005) Reevaluation of surgeryfor the treatment of brain metastases Review of 208 patients with single or multiplebrain metastases treated at one institution with modern neurosurgical techniquesNeurosurgery 56(5)1021ndash1034

24 Rosner D Nemoto T Lane WW (1986) Chemotherapy induces regression of brainmetastases in breast carcinoma Cancer 58(4)832ndash839

25 Stark AM (2010) Neurosurgical treatment of breast cancer metastases to the neuro-cranium Patholog Res Int 2011(2010)549847

26 Chang JE Robins HI Mehta MP (2007) Therapeutic advances in the treatment of brainmetastases Clin Adv Hematol Oncol 5(1)54ndash64

27 Abrey LE et al (2001) A phase II trial of temozolomide for patients with recurrent orprogressive brain metastases J Neurooncol 53(3)259ndash265

28 Christodoulou C et al Hellenic Cooperative Oncology Group (2001) Phase II study oftemozolomide in heavily pretreated cancer patients with brain metastases AnnOncol 12(2)249ndash254

29 Laginha KM Verwoert S Charrois GJ Allen TM (2005) Determination of doxorubicinlevels in whole tumor and tumor nuclei in murine breast cancer tumors Clin CancerRes 11(19 Pt 1)6944ndash6949

30 Ortho Biotech Products LP (2007) DOXIL Product Information Available at stagedoxilcomsharedproductdoxilprescribing-informationpdf Accessed April 19 2007

31 Mantha A et al (2005) A novel rat model for the study of intraosseous metastaticspine cancer J Neurosurg Spine 2(3)303ndash307

32 Newlands ES Stevens MF Wedge SR Wheelhouse RT Brock C (1997) TemozolomideA review of its discovery chemical properties pre-clinical development and clinicaltrials Cancer Treat Rev 23(1)35ndash61

33 Strasser JF Fung LK Eller S Grossman SA Saltzman WM (1995) Distribution of 13-bis(2-chloroethyl)-1-nitrosourea and tracers in the rabbit brain after interstitial deliveryby biodegradable polymer implants J Pharmacol Exp Ther 275(3)1647ndash1655

34 Fung LK et al (1998) Pharmacokinetics of interstitial delivery of carmustine4-hydroperoxycyclophosphamide and paclitaxel from a biodegradable polymerimplant in the monkey brain Cancer Res 58(4)672ndash684

35 Ewend MG et al (1996) Local delivery of chemotherapy and concurrent externalbeam radiotherapy prolongs survival in metastatic brain tumor models Cancer Res56(22)5217ndash5223

16076 | wwwpnasorgcgidoi101073pnas1313420110 Upadhyay et al