Proc. Natl. Acad. Sci. USAVol. 92, pp. 12126-12130, December 1995Medical Sciences

Selective tumor kill of cerebral glioma by photodynamic therapyusing a boronated porphyrin photosensitizer

(brain tumor/hematoporphyrin/boron/neutron capture/phototherapy)

JOHN S. HILL*t, STEPHEN B. KAHLt, STANLEY S. STYLLI*§, YUICHI NAKAMURA*, MYOUNG-SEO Koot,AND ANDREW H. KAYE*§*Neuroscience Laboratory, Department of Surgery, and §Department of Neurosurgery, The Melbourne Neuroscience Centre, Royal Melbourne Hospital,University of Melbourne, Victoria, Australia, 3050; and tDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco,CA 94143-0446

Communicated by M. Frederick Hawthorne, University of California, Los Angeles, CA, July 24, 1995

ABSTRACT The prognosis for patients with the high-grade cerebral glioma glioblastoma multiforme is poor. Themedian survival for primary tumors is < 12 months, with mostrecurring at the site of the original tumor, indicating that amore aggressive local therapy is required to eradicate theunresectable "nests" of tumor cells invading into adjacentbrain. Two adjuvant therapies with the potential to destroythese cells are porphyrin-sensitized photodynamic therapy(PDT) and boron-sensitized boron neutron capture therapy(BNCT). The ability of a boronated porphyrin, 2,4-(a,j3-dihydroxyethyl)deuteroporphyrin IX tetrakiscarborane car-boxylate ester (BOPP), to act as a photosensitizing agent wasinvestigated in vitro with the C6 rat glioma cell line and in vivowith C6 cells grown as an intracerebral tumor after implan-tation into Wistar rats. These studies determined the doses ofBOPP And light required to achieve maximal cell kill in vitroand selective tumor kill in vivo. The data show that BOPP ismore dose effective in vivo by a factor of 10 than the currentclinically used photosensitizer hematoporphyrin derivativeand suggest that BOPP may have potential as a dualPDT/BNCT sensitizer.

Cerebral tumors are responsible for -2% of all cancer deaths,although they are the fourth most important tumor in terms oflife years lost, since they disproportionately affect youngerpeople (1). The majority of these deaths are due to high-gradegliomas-anaplastic astrocytoma and glioblastoma multi-forme. At present there is no curative treatment for thesetumors. Surgery provides a definitive histological diagnosisand relief of raised intracranial pressure, and radiotherapy andadjuvant chemotherapy are of limited benefit. Most studiesutilizing these treatments report median survival times of <1year (1-3). Treatment failures are due to recurrence of thetumor at the original site, suggesting that more aggressive localtherapies may be beneficial in controlling the tumor. Manylocally targeted experimental therapies have been investigated,but none have demonstrated definite clinical benefit. Theseinclude interstitial brachytherapy (4), retroviral gene therapy(5), locally directed immunotherapy (6), and hyperosmolardisruption of the blood-brain barrier prior to chemotherapy(7).Two therapies with the potential to control local recurrence

are photodynamic therapy (PDT) and boron neutron capturetherapy (BNCT). Both are bimodal therapies, the individualcomponents of which are nontoxic in isolation, but which aretumoricidal in combination. PDT relies on the selective uptakeof a photosensitizing chemical in tumor relative to the sur-rounding normal tissue, followed by irradiation with light of anappropriate wavelength to activate the photosensitizer (8). The

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photoactivation of the sensitizer causes oxidative damage to avariety of cellular targets and subsequent tumor necrosis (8).To date, the clinical trials of PDT as an adjuvant treatment forhuman gliomas have used the poorly defined heterogeneousporphyrin mixture hematoporphyrin derivative (HpD) or itsmore enriched commercial preparation Photofrin (for review,see ref. 9).These sensitizers have been shown to localize in glioma

relative to normal brain (9-11), and while the reports of PDTas a treatment of animal (9, 11-15) and human (9, 13, 16, 17)glioma have been encouraging, the use of a purified sensitizerthat is more tumor selective than HpD or Photofrin would havegreat clinical benefit.BNCT, like PDT, is a bimodal therapy that relies on selective

localization in tumors of a compound containing the '0Bisotope of boron followed by activation by epithermal neu-trons, resulting in production of the high linear energy particles4He and 7Li, which can cause extremely localized tumor celldeath (18). Currently, few of the potential BNCT sensitizersexhibit highly selective tumor uptake, and the neutron sourcesthat are available are suboptimal as they do not produce anepithermal beam free from a contaminating fast neutroncomponent that can cause severe normal brain toxicity. Nev-ertheless, the capacity for epithermal neutrons to deeplypenetrate brain and reach the invasive nests of tumor cellssuggests that if the two components of the therapy are opti-mized, then BNCT may be of great benefit.We have previously investigated the uptake of 2,4-(a43-

dihydroxyethyl)deuteroporphyrin IX tetrakiscarborane car-boxylate ester (BOPP) (19) into the intracerebral C6 gliomaxenograft grown in CBA mice (20). BOPP exhibits spectralabsorbance properties similar to HpD (19, 20) and is apotential sensitizer for PDT and/or BNCT. Pharmacologicalstudies have shown that BOPP is extremely selectively taken upby the glioma relative to the surrounding normal brain, withtumor/brain ratios as high as 400:1 (20), which are muchgreater than those observed for HpD in the same animal modelor in human gliomas, where ratios between 10:1 and 50:1 havebeen noted (10). Importantly, there was also retention ofBOPP in the tumor, with high selective ratios being maintainedfor a number of days postsensitization (20). In addition thetumor/blood ratios were high, indicating that the BOPPprobably partitioned into the lipid-rich membranes within thetumor and was not present in the more aqueous environmentssuch as the tumor stroma or blood vessels (20). However, themost critical finding of that study was that BOPP was detectedby confocal laser scanning microscopy in the isolated "nests"

Abbreviations: HpD, hematoporphyrin derivative; PDT, photody-namic therapy; BNCT, boron neutron capture therapy; BOPP, 2,4(a,,-dihydroxyethyl)deuteroporphyrin IX tetrakiscarborane carboxylateester.tTo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 92 (1995) 12127

of tumor cells invading into the edematous brain adjacent tothe tumor (20, 21). It is these cells that are responsible fortumor recurrence (1, 2, 9) and that must be the target of anyadjuvant therapy aiming to improve the treatment of cerebralglioma (9). We report in this paper on the use of BOPP as aphotosensitizer of rat C6 glioma cells in vitro and of thecomparison of BOPP vs. HpD as PDT photosensitizers in theC6 intracranial glioma model in Wistar rats.

MATERIALS AND METHODSMaterials. All reagents used were of analytical grade or

better. Dulbecco's PBS, RPMI 1640 liquid medium, fetal calfserum (FCS), and trypsin/versene were purchased from Com-monwealth Serum Laboratories (Parkville, Australia); HpDwas purchased and prepared as described (11).The C6 rat glioma cell line was obtained from the American

Type Culture Collection.Methods. In vitro cytotoxicity ofC6 cells exposed to BOPP or

HpD. C6 cells were cultured and harvested as described (22)and a single cell suspension (200 cells per 3 ml) in RPMI 1640medium supplemented with 10% FCS was prepared. Three-milliliter aliquots were then seeded into 25-cm2 flasks, whichwere wrapped in foil, and incubated at 37°C for 6 hr, where-upon the medium was removed and replaced with 3 ml ofRPMI 1640 medium containing either BOPP or HpD atconcentrations of 0-200 ,gg/ml. The flasks were incubated fora further 18 hr to allow for sensitizer uptake, at which time themedium and exogenous sensitizer were removed and theadherent cells were washed twice with 5 ml of fresh sensitizer-free medium, prior to addition of 3 ml of fresh medium. Theflasks were incubated for a further 7 days and the resultantcolonies were fixed, stained, and counted as described (22). Atall times before fixation, the flasks were wrapped in aluminumfoil and all procedures were carried out in dim light.

In vitro phototoxicity of C6 cells exposed to BOPP or HpD.Three-milliliter aliquots of a suspension of C6 cells (200 cells)were added to foil-wrapped 25-cm2 flasks as described above.After allowing 6 hr for adherence, the cells were washed asdescribed above and incubated for 18 hr in RPMI 1640 mediumcontaining either BOPP at 0-80 ,ug/ml or HpD at 6 ,tg/ml.The foil was then removed from the flasks in a dark room, andthe flasks were placed on a light-box and exposed to red-filtered light from a fluorescent source (NEC 15-W standardcool white; Nippon Electrical, Tokyo) for between 0 and 120min. The red light output was achieved by placing filtersbetween the flasks and the opaque Perspex top of the light box.This filter combination (Roscoe Supergel nos. 15 Deep strawand 25 Orange red; Masson Photographics, Bulleen, Australia)allowed transmission only of light > 600 nm at an energy doseof 0.2 J-min-.cm-2. After light exposure, the flasks wererewrapped in foil and incubated for a further 7 days, when theadherent colonies were fixed, stained, and counted as de-scribed above.

Cell culture and intracranial tumor implantation. The C6glioma cells were cultured in RPMI 1640 medium supple-mented with 10% FCS as described (11, 22). The cells wereharvested with trypsin/versene while in the logarithmic phaseof growth; before implantation a single cell suspension (4 x106 cells per ml) was prepared, and tumor implantation wasperformed as described (11, 22). Rats bearing the intracerebralC6 tumor used in this study were typically treated 10-12 dayspostimplantation of the tumor cells. The rats designated as thenon-tumor-bearing group were given the same operative pro-cedure as detailed above, except that no tumor cells wereinjected.

Preparation and administration of BOPP. The photosensi-tizer BOPP was prepared as described as a dried powder of thepotassium salt (19, 20) and was stored under desiccation invacuo at -20°C. Typically, solutions of 10 mg/ml in 0.9%

isotonic NaCl were prepared and were stored in darkness at4°C for no more than 24 hr before administration via i.v. tailvein injection into rats.PDT of tumor-bearing and non-tumor-bearing rats. PDT was

administered to tumor-bearing and non-tumor-bearing rats 24hr postsensitization at doses of 5, 10, 25, or 50 mg of BOPP perkg of body weight. The protocol for administration ofPDT wasas described (11). Briefly, light of 628 nm from a gold metalvapor laser (Quentron, Adelaide, Australia) was administeredvia a 600-,tm (inner core diameter) quartz optical fiber, whichwas fixed in a vertical position 6 mm over the exposed brain sothat the entire red light spot completely covered the exposedbrain or tumor and was within the limits of the craniotomy site.The power output at the fiber tip was determined with a powermeter (model 361; Scientech, Boulder, CO) and was typicallybetween 800 and 1000 mW. The dose of light administered wasbetween 0 and 500 J/cm2 of the irradiation area. The surfaceof the brain was continuously irrigated with 0.9% isotonicsaline at room temperature during the procedure, as this hasbeen previously shown to prevent any temperature increase inthe tissue caused by the administration of laser light at thepower densities used in these studies (11). After the admin-istration of PDT the craniotomy site was covered with a singlelayer of Surgicel (Johnson and Johnson, North Ryde, Austra-lia), and the scalp incision was closed with wound clips. Theanimals were then returned to cages and kept in normal roomlight, with no evidence of cutaneous photosensitivity untilsacrifice, and fed water and food ad libitum.

Quantitation of PDT necrosis mediated by BOPP. The ratswere sacrificed 5 days postadministration of PDT, as previousstudies have shown this to be the optimal time to determinephototoxic necrosis in normal brain or tumor (11). The brainswere then removed, fixed, and sectioned, and the extent ofcerebral edema and the depth and extent of brain or tumornecrosis were determined as described (11).Regrowth of intracranial C6 tumor after PDT mediated by

BOPP or HpD. Tumor-bearing rats were sensitized via i.v.administration of either 25 mg of BOPP per kg of body weight24 hr pre-PDT or 20 mg of HpD per kg of body weight 6 hrpre-PDT, drug dose regimes that have been shown to result inmaximal selective tumor destruction (ref. 11; see Fig. 4). PDTwas administered as described above, except that the laser usedwas a potassium titanium phosphate/neodymium/yttrium/aluminum-garnet pumped dye laser (Laserscope Surgical Sys-tems, San Jose, CA) producing 1000mW of 628-nm light at thefiber tip. Light was then administered at dosages of 25 J/cm2and 250 J/cm2 for the BOPP-sensitized and HpD-sensitizedanimals, respectively. These light and sensitizer dosages hadpreviously been shown to mediate the maximum depth ofselective tumor necrosis using BOPP (see Fig. 4) or HpD (11).After PDT, the animals were returned to cages, fed water andfood ad libitum, and continually monitored for any signs ofneurological deficit. If any deficit or distress was apparent, theanimal was sacrificed. Death of the animals was not used as anendpoint of the study, and at 21 days post-PDT all survivinganimals were sacrificed. The brains of all animals were re-moved, fixed, sectioned, and stained, and the PDT effects werequantitated as described above.

RESULTSThe cytotoxicity of BOPP or HpD on C6 glioma cells in vitrois shown in Fig. 1. BOPP is less toxic to glioma cells than HpDin the absence of light, with 50% colony survival (CS50) '120,ug/ml and 90% colony survival (CS0) '20 ,ug/ml, comparedto the corresponding values for HpD of 10 and 6 ,tg/ml,respectively. The in vitro phototoxicity studies shown in Fig. 2demonstrate that BOPP can mediate significant phototoxicityin glioma cells. The data from the survival curves for thephototoxicity of cells exposed to 20 ,ug of BOPP per ml or to

Medical Sciences: Hill et al.

Proc. Natl. Acad. Sci. USA 92 (1995)

60

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FIG. 1. In vitro cytotoxicity of BOPP and HpD. Rat C6 glioma cellswere incubated in the dark in RPMI 1640 medium/10% FCS con-taining either BOPP (m) or HpD (0) for 16 hr at the concentrationsindicated, washed twice in sensitizer-free medium, resuspended infresh medium, and incubated in the dark for a further 7 days to allowcolony formation. Colony survival was determined as percentagesurvival at 0 j,g of sensitizer per ml. Data represent mean colonysurvival in three flasks at each concentration, and error bars represent1 SEIS6 ,tgthesefor e,kinetan in]sublethe rs

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.t,, ;,, >,t :t. Si ffi sg ti¢*t,j ,.E ii vii e:'i.tiir3 ;i .>,g9.'2w. r> . ........... }* 2R E ,35-7. < j; - l2D * . f *B-tra fl sii5R^- ; : ;, -- ' . .w . .* yw , w ; 5 ,s5 j!R. 9.S RG ,. !:. 't ' ': .<. 5 .; | S *N @, 0lR*ffi>w 5SH - Q-

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FIG. 3. Effect on tumor or normal brain of BOPP-sensitized PDT.(a) Non-tumor-bearing normal brain sensitized with 25 mg of BOPPper kg of body weight i.v. and exposed to 628-nm light (25 J/cm2) 24hr postsensitization. (b) Non-tumor-bearing normal brain sensitized asin a and exposed to light at 200 J/cm2. (c) Unsensitized tumor innormal brain exposed to light at 200 J/cm2. (d) Tumor in normal brainsensitized with 25 mg of BOPP per kg of body weight i.v. and exposedto light at 25 J/cm2. Arrow, point of maximum depth of tumor kill.(Bars = 5 mm.)

4. - of 25 mg of BOPP per kg of body weight, in combination withlight at 25 J/cm2, mediated significant phototoxic tumor

of HpD per ml are the most directly comparable, since necrosis (Fig. 3d). Administration of the same doses of BOPPconcentrations correspond to the respective CS90 values and light to non-tumor-bearing animals resulted in no appar-ach sensitizer determined in the absence of light. The ent edema or toxicity in normal brain (Fig. 3a), althoughics of cell kill are similar for both curves, and both show higher light doses were able to induce phototoxic necrosis initial lag phase, characteristic of the cells' ability to repair the brain of sensitized non-tumor-bearing rats, even though.hal phototoxic damage, followed by a rapid ireair the levels of BOPP in brain were extremely low (Fig. 3b).thal phototoxlc damage, followed by a rapid increase in Significant macrophage infiltration and an inflammatory re-ate of cellular death. sponse were evident at the margins of the lesion (Fig. 3b). Then days post intracranial implantation into Wistar rats, the administration ofBOPP alone in doses of 50 mg per kg of bodyrs were of an appropriate size to determine the depths of weight or laser light alone, in combination with saline irriga-r kill able to be achieved after BOPP-mediated PDT. The tion, in doses up to 200 J/cm2 (Fig. 3c) caused no detectableof BOPP administered to the rats (5, 10, 25, or 50 mg per tumor or normal brain necrosis or edema.body weight) have been shown in a mouse model to result The BOPP-mediated PDT of the C6 glioma 5 days post-remely selective uptake of the drug into tumor, with very therapy is shown in Fig. 4. At all doses of BOPP, phototoxicvels in surrounding normal brain and blood (20). A dose necrosis of tumor was apparent, although normal brain dam-age could be induced at high doses of light. The light-dose

threshold for brain necrosis was dependent on the dose of................. BOPP. Normal brain damage was apparent at 25 J/cm2 after

.E;.........a BOPP dose of 50 mg per kg of body weight but was absentat lower BOPP doses. The maximum depth of tumor necrosisof 4.5 mm was induced by the combination of 25 mg of BOPPper kg of body weight and 25 J/cm2 of laser light. Arrowsindicate light doses that mediated maximum tumor kill withsparing of normal brain.The results of the study of tumor regrowth in rats post-PDT

is shown in Table 1. While the number of animals in each groupis small, the data suggest that there is a difference in the timeto occurrence of neurological symptoms in the BOPP-sensitized animals compared to those sensitized with HpD. Allanimals that were sacrificed with symptoms of neurologicaldeficit before day 21 post-PDT exhibited obvious histologicaltumors. However, the brains from the BOPP-treated animals

0 20 40 60 80 100 120 that survived for 3 weeks before sacrifice at day 21 wereTime red light exposure ( min) substantially tumor-free, whereas all of the HpD-treated an-imals had obvious tumors (data not shown). All animals in

In vitro phototoxicity of BOPP and HpD. Rat C6 glioma both groups showed inflammatory responses in the brain.r2. Incuaedi the dark in RPP and meD. FCS similar to those previously noted in PDT-treated rats, whichiere incubated in the dark in RPMI 1640 medium/10% FCS ....ining BOPP at 0 (oo...), 20 (.... ), 40 (0-0), 60 (A-A), or may be indicative of a small degree of treatment-induced-*) ,ug/ml, or HpD at 6 tLg/ml (A-A) for 16 hr. The cells were edema or phagocytic and macrophage infiltration and removal'ashed twice with fresh medium, resuspended in fresh medium, of tumor debris from the treatment site (11).

and then exposed to red light for the times indicated and incubated inthe dark for a further 7 days to allow colony formation. The colonysurvival was determined as percentage mean survival in the flasks with0 ,ug/ml and 0 min exposure. Data represent mean colony survival inthree flasks at each point, and error bars represent 1 SEM.

DISCUSSIONThe treatment of cerebral glioma remains a major challengebecause of the infiltrative nature of these tumors. They gen-

12128 Medical Sciences: Hill et aL

Proc. Natl. Acad. Sci. USA 92 (1995) 12129

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FIG. 4. Depth of tumor kill and normal brain necrosis after PDTof BOPP-sensitized rats. Depth of intracranial C6 tumor kill (-) ornormal brain necrosis (0) in Wistar rats sensitized i.v. with BOPP atthe doses indicated and exposed to various doses of 628-nm light froma gold metal vapor laser. Values shown represent mean depth of killin a minimum of three animals at each data point and error barsrepresent 1 SEM. Arrows, dose of light that results in selective tumornecrosis with sparing of normal brain.

erally do not metastasize throughout the body but rather killby local regrowth, making use of locally targeted binarytherapies, such as PDT and BNCT, very attractive (9). In thisreport, we have studied the action of a sensitizer, BOPP, which,because of its porphyrin component, is a photosensitizer forPDT and, because it consists of '30% (wt/wt) boron (19), hasthe property of also being a sensitizer for BNCT. In compar-ison to HpD, BOPP is significantly less toxic to glioma cells invitro in the absence of light (Fig. 1), although our studies haveshown that both have approximately equal toxicity whenadministered to animals in vivo (J.S.H., S.B.K., S.S.S., andA.H.K., unpublished observations).The data presented in Figs. 2-4 and Table 1 show that BOPP

can act as a potent photosensitizer of cerebral glioma in vitroand in vivo, and with the appropriate dosimetry selectivedestruction of tumor with sparing of normal brain can beachieved. We have previously shown that administration ofBOPP to mice bearing the same C6 tumor used in this studyresults in extremely selective sensitizer uptake into the tumor(20). The selectivity of uptake into the tumor was nearly 10times that observed for HpD, the sensitizer used in mosthuman clinical trials of PDT of glioma (9, 13, 16, 17). Inaddition, fluorescence microscopy with a confocal microscopehas demonstrated that BOPP is present in the tumor cellsinvading normal brain (20). These findings are critical since itis these cells that are the primary target of adjuvant therapies.The data in Fig. 4 show that BOPP is a potent photosensi-

tizer, which on a weight basis is -10 times more phototoxic invitro than HpD. We have previously reported that an optimumtumor necrosis depth of -5 mm, with sparing of normal brain,could be achieved with a dose of 20 mg ofHpD per kg of body

Table 1. Tumor regrowth in rats post-PDT

Sensitizer Rat CommentsBOPP*t 1 Sacrificed 8 days post-PDT; neurological

symptoms, histological tumor present.2 Sacrificed 17 days post-PDT, neurological

symptoms, histological tumor present.3 Sacrificed 21 days post-PDT, neurological

symptoms, small histological tumorpresent.

4 Sacrificed 21 days post-PDT, noneurological symptoms, no histologicaltumor apparent.

5 Sacrificed 21 days post-PDT, noneurological symptoms, no histologicaltumor apparent.

HpDU§ 6 Sacrificed 8 days post-PDT, neurologicalsymptoms, histological tumor present.

7 Sacrificed 8 days post-PDT, neurologicalsymptoms, histological tumor present.

8 Sacrificed 8 days post-PDT, neurologicalsymptoms, histological tumor present.

9 Sacrificed 21 days post-PDT, neurologicalsymptoms, histological tumor present.

Significance in difference in time to sacrifice atP < 0.05 by Student'spaired t test.*Dosimetry, 25 mg of BOPP per kg of body weight; 628-nm light at 25J/cm2.

tDosimetry, 20 mg of HpD per kg of body weight; 628-nm light at 200J/cm2.iMean time to sacrifice, 17.6 days; median time to sacrifice, 21 days.§Mean time to sacrifice, 11.3 days; median time to sacrifice, 8 days.

weight and 200 J/cm2 of 628-nm light (11). In this study, theoptimum dosimetry to achieve the same depth of tumor killwas 25 mg of BOPP per kg of body weight and only 25 J/cm2of light administered 24 hr postsensitization. It is difficult todirectly compare the two sensitizer doses since the molecularweights of the two drugs are so different. BOPP, which is asingular chemical entity has a molecular weight of -1300 (19),whereas HpD is a complex mixture of porphyrins, which existin varying aggregation states (for review, see ref. 9). Themolecular weight of the monomeric hematoporphyrin is "600,so it may be considered that on a molar basis the dose of BOPPrequired to achieve maximal tumor kill is half that required forHpD. In this study, PDT was administered 24 hr postsensiti-zation, the time point we have previously shown correspondsto the maximal levels of BOPP in tumor (20). While themaximum tumor/normal brain uptake ratio was evident 48 hrpost administration ofBOPP (20), PDT of animals at this timepoint (data not shown) did not result in the same depth oftumor kill as at 24 hr (Fig. 4). This suggests that the limitingfactor in tumor phototoxicity was the sensitizer level in thetumor, rather than the differential concentration betweentumor and normal brain. However, the data in Fig. 4 also showthat the level of BOPP in tumor must be below an upperthreshold, since the depth of tumor kill decreased after a doseof 50 mg/kg. This may be due to higher tumor sensitizer levels24 hr after a dose of 50 compared to 25 mg/kg. Our previousstudies in an identical mouse model have shown a BOPP levelof 140 jig per g of tumor after the former dose, compared to60 ,zg/g after the latter dose (20). High levels of BOPP mayresult in increased absorbance of the activating light near thesuperficial margin, resulting in an attenuation of the light doseto the deeper tumor regions, with consequent reduced necro-sis. This attenuation of light may be a critical factor in someclinical situations where high sensitizer levels may be presentin some tumors.The reason for the more potent tumor kill with BOPP

compared to HpD is probably related to their respective

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12130 Medical Sciences: Hill et al.

subcellular localization. Using confocal laser scanning micros-copy, in conjunction with centrifugal organelle fractionation,we have shown that BOPP is almost exclusively localized in themitochondria of glioma cells (20, 21). HpD localized in avariety of subcellular sites, including the cytoplasm, lysosomes,and mitochondria (9, 21, 23), probably reflecting the hetero-geneity of the HpD mixture (9). It has previously been shownthat the mitochondria of tumor cells may be critical subcellulartargets for PDT (24). The data in Fig. 4 and Table 1 suggestthat specific localization of high concentrations of BOPP tomitochondria may result in an increased efficacy of BOPP-sensitized PDT compared to HpD PDT. It may also explain thegreater degree of tumor eradication and an increased timeinterval between therapy and tumor regrowth in BOPP-sensitized animals. This preliminary finding indicates therequirement for a more complete study of long-term survivalafter BOPP-mediated PDT. It may have great significancesince it provides a rational basis for future drug design basedon the site of localization of the sensitizer.These findings demonstrate that with appropriate dosime-

try, BOPP is a more potent photosensitizer of glioma thanHpD. This may have direct clinical implications where the timeconstraints of surgery require the adjuvant PDT procedure tobe completed in the minimum possible time. In many HpD-sensitized treatments of cerebral glioma, or larger systemictumors such as mesothelioma, the residual tumor bed is solarge that the delivered light dose is lower than that requiredto achieve maximum tumor eradication. The use of a morepotent photosensitizing drug such as BOPP may overcome thisproblem. In addition, the lower activating light threshold ofBOPP may allow destruction of deeper nests of tumor com-pared to, HpD.

In addition, the attraction of using BOPP as a BNCTsensitizer allows the possibility of combination PDT/BNCTtreatments, which may substantially improve the degree oflocal control of tumors such as cerebral glioma.

We thank Ms. Janna Stickland, Mr. Pierre Smith, and Mr. LindsayCox for photography and preparation of figures and Ms. KallyVasilopoulos and Ms. Val Feakes for sectioning and staining of alltissue. This work was supported by grants from the National Healthand Medical Research Council (Australia), Anti-Cancer Council ofVictoria, Royal Australasian College of Surgeons, The Slezak Foun-dation, Stroke Research Foundation, Victor Hurley Foundation, andthe U.S. Department of Health and Human Services, National Insti-tutes of Health Grant CA 37961.

1. Walker, M. D., Green, S. B., Byar, D. P., Alexander, F., Batzdorf,

U., Brooks, W. H., Hunt, E. E., MacCarty, C. S., Mahaley, M. S.,

Mealey, J., Owens, G., Ransohoff, J., Robertson, J. T., Shapiro,W. R., Smith, K. R., Wilson, C. B. & Strike, T. A. (1980) N. Engl.J. Med. 303, 1323-1329.

2. Walker, M. D., Alexander, E., Hunt, W. C., MacCarty, C., Ma-haley, M. S., Mealey, J., Norrell, H. A., Owens, G., Ransohoff, J.,Wilson, C. B., Gehan, E. A. & Strike, T. (1978) J. Neurosurg. 49,333-343.

3. Wilson, C. B. (1967) N. Engl. J. Med. 300, 1469-1471.4. Gutin, P. H., Liebel, S. A., Wara, W., Choucair, A., Levin, V.,

Phillips, T., Silver, P., Da Silva, V., Edwards, M., Davis, R.,Weaver, K. A. & Lamb, S. (1987) J. Neurosurg 67, 864-873.

5. Oldfield, E. H., Ram, Z., Culver, K. W., Blaese, R. M., DeV-room, H. L. & Anderson, W. F. (1993) Hum. Gene Ther. 4,39-69.

6. Barba, D., Saris, S. C., Holdere, C., Rosenberg, S. A. & Oldfield,E. H. (1989) J. Neurosurg. 70, 175-182.

7. Gumerlock, M. K., Belshe, B. D., Madsen, R. & Watts, C. (1992).J. Neuro-Oncol. 12, 33-46.

8. Henderson, B. W. & Doughterty, T. J. (1992) Photochem. Pho-tobiol. 55, 145-157.

9. Kaye, A. H. & Hill, J. S. (1992) Neurosurg. Q. 1, 233-258.10. Hill, J. S., Kaye, A. H., Sawyer, W. H., Morstyn, G., Megison,

P. D. & Stylli, S. S. (1990) Neurosurgery 26, 248-254.11. Kaye, A. H. & Morstyn, G. (1987) Neurosurgery 20, 408-414.12. Sandeman, D. R., Bradford, R., Buston, P., Bown S. G. &

Thomas, D. G. T. (1987) Br. J. Cancer 55, 647-649.13. Kaye, A. H., Morstyn, G. & Apuzzo, M. L. J. (1988) J. Neurosurg.

69, 1-14.14. Powers, S. K., Beckman, W. C., Brown, T. & Kolpack, L. C.

(1987) J. Neurosurg. 67, 889-894.15. Boggan, J. E., Bolger, C. & Edwards, M. S. B. (1985). J. Neuro-

surg. 63, 917-921.16. Laws, E. R., Cortese, D. A., Kinsey, J. H., Eagen, R. T. & Ander-

son, R. E. (1981) Neurosurgery 9, 672-678.17. Muller, P. J. & Wilson, B. C. (1990) Can. J. Neurol. Sci. 17,

193-198.18. Gabel, D., Foster, S. & Fairchild, R. G. (1987) Radiat. Res. 111,

14-25.19. Kahl, S. B. & Koo, M.-S. (1990) J. Chem. Soc. Chem. Commun.

1769-1771.20. Hill, J. S., Kahl, S. B., Kaye, A. H., Stylli, S. S., Koo, M.-S.,

Gonzales, M. F., Vardaxis, N. J. & Johnson, C. I. (1992) Proc.Natl. Acad. Sci. USA 89, 1785-1789.

21. Hill, J. S., Kaye, A. H., Kahl, S. B., Stylli, S. S., Gonzales, M. F.,Ward, A. D. & Vardaxis, N. J. (1992) in Photodynamic Therapyand Biomedical Lasers, eds. Spinelli, P., Dal Fante, M. &Marchesini, R. (Elsevier, New York), pp. 370-374.

22. Kaye, A. H., Morstyn, G. & Ashcroft, R. G. (1985) Neurosurgery17, 883-890.

23. Woodburn, K. W., Vardaxis, N. J., Hill, J. S., Kaye, A. H. &Phillips, D. R. (1991) Photochem. Photobiol. 54, 725-732.

24. Hanzlik, C., Knox, R. S., Gibson, S. L. & Hilf, R. (1989) Photo-chem. Photobiol. 50, 45-53.

Proc. Natl. Acad. Sci. USA 92 (1995)