1996;56:1075-1082. Published online March 1, 1996.Cancer Res Kelly L. Molpus, Daniel Kato, Michael R. Hamblin, et al. Ovarian Carcinomatosis in a Xenograft Murine ModelIntraperitoneal Photodynamic Therapy of Human Epithelial  

  

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(CANCERRESEARCH56. 1075-1082, March1. 19961

ABSTRACT

The objective of this investigation was to determine the efficacy of i.p.photodynamic therapy (PDT) against solid, multifocal ovarian carcinomausing a newly described NIH:OVCAR-5 induced murine modeL PDT wasinitiated when diffuse microscopic disease and small multifocal tumornodules were present, similar to the extent of residual carcinoma that maypersist clinically after laparotomy and tumor debulking. The photosensitizer, benzoporphyrin derivative monoacid ring A (BPD.MA), was administered in a dose of 0.25 mg/kg body weight i.p. 90 mm prior to lightexposure. An argon-pumped dye laser was used to deliver low intensitylight (20 J) i.p. through a cylindrically diffusing fiberoptic tip. Treatmentregimens consisted of a series of three to five treatments at 3-7-day

intervals, with the extent of macroscopic disease or death from diseasebeing the evaluable outcome parameters for tumoricidal and survival

studies, respectively. The mean tumor burden at necropsy for treatedanimals was 0.034 ±0.014 g compared to 0.379 ±0.065 g in untreatedcontrols (P < 0.001). Survival studies were initiated in two groups at day7 and day 14 following cell inoculation. The first group received eitherthree or five treatments at 5-day intervals, and both had a significantincrease in median survival compared to untreated controls (57 and 53days, respectively, compared to 43 days, P < 0.05). The second group wastreated every 7 days until death and also had a significant survival

advantage over controls (57 days compared to 47 days, P < 0.05). Thesestudies suggest that benzoporphyrin derivative mono acid ring A-mediated PDT is a feasible, well-tolerated, experimental treatment approachthat elicits a tumoricidal response against diffuse, solid i.p. disease intumor-bearing mice, with concomitant prolongation of survival and needscareful optimization.

INTRODUCTION

Epithelial ovarian carcinoma is the leading cause of death fromgynecological malignancy, and is the fourth most frequent cause ofcancer death among women in the United States. In this country,approximately 1 in 70 women will develop ovarian cancer in theirlifetime and 1 in 100 will die of the disease. Overall 5-year survivalis 37% and depends on the extent of disease at the time of diagnosis.Unfortunately, more than one half of patients present with advancedstages of disease, with an associated 5-year survival of 21% (1).Chemotherapy is the most frequently utilized adjunctive treatment forresidual carcinoma that persists after surgical debulking. Despite

advances in chemotherapeutic regimens, the prognosis of patientswith advanced disease at this time remains uncertain. The best hopefor improved survival lies in early detection and development of moreeffective treatment modalities (2). One approach currently underinvestigation for the treatment of ovarian carcinoma is PDT3.

Received 9/8/95; accepted 1/2/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by NIH Grant ROl AR40352, Office of Naval Research

Contract N 00014-94-1-0927, the Massachusetts General Hospital Laser Center (partialfellowship to D. K.).

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: PDT, photodynamic therapy; BPD-MA, benzoporphyrin

derivative monoacid ring A; HPD, hematoporphyrin derivative; PF, Photofrmn; TNF-a,tumor necrosis factor a.

PDT utilizes a combination of low-intensity illumination and tissue-localized photoactivatable chemicals (photosensitizers) to produce photochemical effects leading to tumoricidal effects (3). Thephotosensitizer is generally administered systematically and has somedegree of preferential accumulation in malignant tissues. After aspecified time for optimal biodistribution (increased contrast betweentumor and surrounding normal tissue), the target tissues are irradiatedat the absorbance maximum of the photosensitizer (4, 5). Phototoxiceffects occur due to the formation of reactive intermediates such assinglet oxygen and free radicals from the activated photosensitizer (6).This results in the disruption of cellular and vascular structures, celldeath, and tissue necrosis.

PDT is well suited for the treatment of ovarian cancer due to therelatively easy access throughout the peritoneal cavity via fiberoptictechniques during laparoscopy or laparotomy. However, the advantages of the ready accessibility of the peritoneal cavity are mitigatedby its geometric complexity, which makes light dosimetry a chal

lenge. PDT circumvents the lack of specificity of conventional therapies to some extent due to its inherent dual selectivity: (a) increasedselectivity is obtained by localization of the photosensitizer to thetumor, and (b) toxicity is confined due to spatial control of illuminatedareas. Prior applications of PDT have most often used the administration of a free photosensitizer. In the case of ovarian cancer, wherethe disease is disseminated throughout the peritoneal cavity, it isimportant to have improved methods of photosensitizer localization.This may be achieved by binding photosensitizers to carrier systemssuch as liposomes or antibodies (4). Reasonably promising resultsusing antibody-conjugated photosensitizers have been reported byGoff et a!. (7—9)in a murine xenograft model, and the present studyinvestigates i.p. PDT with liposomally encapsulated BPD-MA.

BPD-MA is in Phase I—Illclinical trials for cutaneous diseases (10).Initial laboratory and clinical studies suggest that BPD-MA is anefficient long wavelength-absorbing photosensitizer which lacks theside effects (prolonged cutaneous photosensitivity) of PF, the onlyPDT agent with regulatory approval in some countries. Almost all ofthe studies using BPD-MA-mediated PDT in oncology have beenfocused on either s.c. tumor models in the laboratory (1 1, 12) orcutaneous diseases clinically (10, 13). The purpose of this investigation was to explore the tumoricidal effect of PDT with liposomalBPD-MA in diffuse multifocal i.p. disease, which presents additionalchallenges for both uptake of photosensitizer and light delivery. Anew xenograft model that more closely parallels the human diseaseprocess was developed4 and utilized in these experiments. The specific aim of the study was to evaluate the destruction of this smallvolume disease to demonstrate the efficacy and feasibility of PDTwith liposomal BPD-MA as a potential adjunctive therapy in thetreatment of ovarian carcinoma after debulking surgery.

4 K. L. Molpus, D. Koelliker, L. Atkins, D. Kato, 3. Buczak-Thomas, A. F. Fuller, Jr.,

and T. Hasan. Characterization of a xenograft model of human ovarian carcinoma whichproduces intraperitoneal carcinomatosis and metastases in mice, manuscript submitted forpublication.

1075

Intraperitoneal Photodynamic Therapy of Human Epithelial OvarianCarcinomatosis in a Xenograft Murine Model'

Kelly L. Molpus, Daniel Kato, Michael R. Hamblin, Lothar Lilge, Michael Bamberg, and Tayyaba Hasan2

Weliman Laboratories of Photomedicine [K. L M., D. K., M. R. H., M. B., T. H.], WLM 222B and Vincent Gynecologic Oncology Service (K. L M., D. K.], VBK-I, MassachusettsGeneral Hospital. Boston, Massachusetts 02114 and Ontario Cancer Institute/Princess Margaret Hospital. Department of Clinical Physics, 610 University Ave., Toronto, Ontario.M5G 2M6, Canada (L LI

American Association for Cancer Research Copyright © 1996 on July 13, 2011cancerres.aacrjournals.orgDownloaded from

PHOTODYNAMICTHERAPY

Tumoricidal Response. Treatmentwas initiatedon postinoculumday 14and administered as described above. Based on the toxicology studies described above, all treatment studies used 0.25 mg/kg body weight of BPD-MA

and 20 J of 690 nm light administered i.p. Low-intensity light (20 J total, —5J/quadrant) was delivered in equivalent fractions to each i.p. quadrant. Minoradjustments in the duration of illumination were made based on energy outputof the fiberoptic tip to keep the total fluence constant during each treatmentsession. Typical illumination times per quadrant were in the range of 70—80s;total time of treatment was in the range of 280—320s.

For tumoncidal studies, this sequence of steps was repeated every 72 h untilthree treatments were completed. Multiple treatments were given based onprevious work in our laboratory in which there was no significant tumoricidalresponse to a single i.p. treatment using the aforementioned regimen. All micewere sacrificed for necropsy 72 h after the third treatment (approximately day26 postinoculation). Prior to sacrifice, the treatment and control mice weregrouped together and randomly selected in a blinded fashion for necropsy.Initial tumor resection was performed by the same investigator (K. M.), andcomplete resection of macroscopic disease was confirmed by a second investigator (M. B.). Tumors ex vivowere grouped according to anatomical location,and the wet tissue weights were obtained (Mettler AE 163; Mettler InstrumentCorp., Highstown, NJ). Tumoricidal response was assessed by comparing theextent of gross residual disease in treated animals to the extent of disease inuntreated controls. Tumoricidal PDT experiments were performed in duplicate.The differences in the means of resected tumor weights were tested forsignificance using the two-tailed Student t test.

A subset of resected tissue was fixed in 10% phosphate-buffered formalin(Mallinckrodt Inc., Paris, KY) and embedded in paraffin. Sections cut 5-smthick were stained with hematoxylin and eosin for microscopic evaluation toconfirm carcinoma as the predominant cell type in resected tissue specimens.

Survival Studies. For survival studies modeling treatment of advanced

disease, mice were divided into two groups (treatment and controls). Treatment

mice underwent initial PDT on postinoculum day 14 and subsequent treat

ments at 7-day intervals. Treatments were continued throughout the life spanof the mice, for a total of five to nine treatments. For survival studies modelingtreatment of minimal residual disease, the mice were divided into three groupsaccording to the number of treatments scheduled (3, 5, and untreated controls).Treatment was initiated on postinoculation day 7, and the sequence ofBPD-MA injection followed by 690 nm illumination was repeated every 5days until the appropriate number of treatments were completed. The end pointwas defined as death due to disease, which was confirmed in all mice atnecropsy. Survival analysis was performed using the Kaplan-Meier method.Survival curves were compared, and differences in survival were tested forsignificance using the Wilcoxon signed rank test. A P < 0.05 was consideredsignificant.

Blodistribution. The presentstudies used an irradiationtime of 90 mmfollowing BPD-MA administration. For future work we were interested inestablishing times for irradiation following photosensitizer administrationwhich would give the optimum tumoncidal response. Preliminary biodistribu

Lionstudies were therefore initiated.Standard curves for the estimation of the concentration of BPD-MA in

tissue extracts were obtained as follows. Serial dilutions of a 1.25 @g/mlsolution BPD-MA in PBS containing 2% FCS were prepared in methanol, andfluorescence emission spectra were recorded (Fluorolog 2; Spex Industries,Edison, NJ) with an excitation wavelength of 430 nan (excitation maximum ofBPD-MA), and the emission spectra were monitored between 600 and 800 nm(emission maximum of BPD-MA, 695 nm). A linear equation was determined

from each standard curve and used to calculate the concentration of BPD-MAfrom the fluorescence spectrum of a sample.

BPD-MA was injected i.p. at a dose of 2.0 mg/kg body weight in 1.0 mlsolution. At time points of I, 2, 4, 8, 12, 24, and 48 h after injection (n = 5—7mice/time point) animals were sacrificed by CO2 inhalation. At necropsy theskin, peritoneum, liver, spleen, kidney, small intestine, heart, lung, adipose

tissue, and tumor nodules were harvested. Wet tissue samples were weighedimmediately upon resection (Mettler AE 163; Mettler Instrument Corp.) andfrozen at —70°C.For extraction of the photosensitizer, the tissues werehomogenized (homogenizer model PT 10/35; Brinkman Instruments, Westbury, NY) in 3.0 ml 100% MeOH (Fisher Scientific, Fair Lawn, NJ) and

centrifuged at 17,000 rpm (Sorvall RC-SB, refrigerated superspeed centrifuge;Dupont Instruments) at 22°Cfor 10 mm. The supernatant was collected by

1076

MATERIALS AND METHODS

Tumor Cells. NIH:OVCAR-5cells were obtained from the Fox ChaseCancer Institute (Philadelphia, PA). Cells were grown in RPMI 1640 mediumsupplemented with 5% heat-inactivated fetal bovine serum and maintained inan incubator at 37°Cin an atmosphere of 5% carbon dioxide. Cells wereharvested for injection during the log growth phase, when they were 80—85%

confluent. For tumor harvest and transplantation, cells were trypsinized (tiypsin-EDTA: GIBCO, Grand Island, NY), centrifuged at 1000 rpm for 10 mm(model 6000B: Sorvall Centrifuges, Dupont, Wilmington, DE), and resuspended in PBS without Ca2@or Mg2@(GIBCO) and counted on a hemocytometer plate. The suspension volume was adjusted to a concentration of17.5 X 106 cellslml. In previous studies,4 it was shown that a 2.0-ml suspension containing 35 x 106 cells resulted in reproducible disease progression

in vivo.Animal Model. A xenograft model for human epithelial ovarian carcinoma

was developed in our laboratory4 and utilized in these experiments. In brief,6—8-week-old Swiss athymic nude mice (Cox Breeding Laboratories, Cambridge, MA) were given injections i.p. with 35 X 106 cells from the establishedNIH:OVCAR-5 cell line. Within 7 days, diffuse granular carcinomatosis isapparent, and within 14 days the animals develop small multifocal tumornodules throughout the peritoneal cavity. Within 28—35days after inoculation,animals develop significant macroscopic tumor burden in the abdomen andpelvis.

Mice received proper care and maintenance in accordance with institutionalguidelines. They had continual access to food and water, which was taken adlibitum. Throughout the experiments, mice were housed in laminar flow racksunder specific pathogen-free conditions and were monitored daily for generalhealth status. Cancer growth was made evident by the presence of abdominaldistention and palpable tumor masses in the peritoneal cavity. Animals weresacrificed when they became moribund or had an evident excessive tumor

burden.Photosensitizer. Liposomal BPD-MA was a generous gift from Quadra

Logic Technologies, Inc. (Vancouver, British Columbia, Canada) and was

stored in powder form and kept refrigerated. Samples were prepared immediately prior to use by dissolution of BPD-MA in D5W solution and diluted withsterile PBS for injection. All work involving the use of photosensitizer wasperformed in subdued lighting.

Phototoxicology. PDT was performed 28—35days after i.p. injection of35 X l0@NIH:OVCAR-5 cells. This time point was chosen to provide a moresevere criterion for determining dose-limiting toxicity than earlier time pointswhen toxicity was found to be similar. Mice (n 5—7/group)were given i.p.injections of the photosensitizer (0.25—2.0mg/kg BPD-MA) 90 mm prior tolight exposure. Two ml of a 0.1% w/v intralipid solution (Intralipid, soybeanoil emulsion for iv. use; Kabi Pharmacia, Inc., Clayton, NC) was injected i.p.just prior to treatment to enhance light scattering (14). Animals were anesthetized by i.p. administration of 0.02—0.04ml ketamine cocktail (ketamine, 30mg/mi; xylazine, 5 mg/mI; and atropine, 0.5 mg/mI). An argon-pumped dyelaser (Coherent, Palo Alto, CA). focused through a X 10 microscope objectiveonto the proximal end of an optical fiber, was used to deliver 690 nm light i.p.via a 8.0 mm X 0.4-mm cylindrically diffusing fiberoptic tip (built in-house).This fiber was introduced into the peritoneal cavity of a supine anesthetized

mouse through a centrally placed 22-gauge catheter that traversed the abdominal wall. One fourth of the total light energy was delivered to each i.p.quadrant over equivalent time periods. At the conclusion of treatment, micerecovered in an animal warmer until they awoke and resumed normal activity.No mice died during treatment. Toxicity thresholds were established, andtreatment parameters were defined based on the percentage of animals alive 72

h after treatment. Pilot experiments established that any deaths related to theeffects of PDT occurred within 48 h of light administration.

Photodynamic Therapy in Vivo. Mice were inoculated i.p. with 35 X 106NIH:OVCAR-5 cells, given a numeric ear tag for identification, and thenrandomly divided into two groups (treatment and control groups). Controlgroups consisted of animals that received no treatment, those that received ani.p. injection of intralipid and i.p. light delivery, and those that received i.p.

BPD-MA and intralipid but no light. No significant difference in tumor weightor survival was seen between any of these control groups (data not shown). i.p.disease was confirmed in all mice (treatment and control) prior to therapy by

the presence of positive pentoneal cytology.

American Association for Cancer Research Copyright © 1996 on July 13, 2011cancerres.aacrjournals.orgDownloaded from

p <0.01

Table I Phototoxicology: tumor-bearing mice survivingPDrTotal

photosensitizerdoseFluence

(J) 2.0 mg/kg 1.0 mg/kg 0.5 mg/kg 0.25mg/kg40

0% 0%71%300% 29%100%20

0% 29% 57%100%1514% 43% 71%100%1029% 43% 100%100%a

Animals were given injections i.p. of 35 X 106 @4ffl:OVCAR5 cells. On postinoculum day 21, —90mm after i.p. injection of the photosensitizer (BPD-MA), i.p. 690nmlight

were administered. Numbers represent percentage of mice which survived 72 haftertreatment.Each group contains seven mice.

a) IIn

04.'I.,a)C

Fig. 2. Comparison of site-specific tumor weights in treated (U) and control ( 0) mice.The mice that had been treated in Fig. 1 had their tumors weighed separately byanatomical site. Mean values of tumor are given for treated and control mice and the Pvalues compare treatment and control groups for each site.

(Fig. 3). Using gross dissection, the lower limit of visibly detectabletumor implants was —1 mm in diameter. The frequency data shown inFig. 3, therefore, represent any animal with at least one implant of >1mm, at any specific anatomical location. All untreated mice hadevidence of disease on gross inspection at necropsy. In comparison, 6(27%) treated mice had no detectable macroscopic disease. The pelvicand subgastric regions most often demonstrated tumor growth and hadthe highest volumes of disease as well. A single mouse (6%) in thecontrol group had disease limited to these two areas. In contrast, 10mice (45%) treated with PDT had disease limited to the pelvis andsubgastric regions, suggesting destruction of macroscopic disease atall other sites. In cases without overt evidence of carcinoma, somedegree of subclinical disease most likely persisted. This was suggestedby survival studies in which treated mice eventually succumbed toadvanced disease.

There was only one prolonged PDT-related untoward effect on themice. In mice that consistently survived PDT, significant weight lossoccurred when treatments were administered at 72-h intervals. Themean weights of the two groups were not dissimilar prior to treatment,

1077

PHOTODYNAMICTHERAPY

aspiration, and the pellet was resuspended (Thermolyne Maxi Mix II; Thermolyne Corp., subsidiary of Sybron Corp., Dubuque, IA) in 2.0 ml MeOH.This suspension was refrigerated for 4 h, centrifuged, and the supernatant fluidwas aspirated and combined with that previously collected. The fluorescenceof the supernatant fluid was measured with a spectrofluonmeter as describedfor the standard solutions, and concentrations of BPD-MA were determinedfrom the equations of the standard curves. The fluorescence values of tissuesamples from untreated animals were used for background correction.

RESULTS

Phototoxicology

Results of phototoxicology experiments are shown in Table 1.Survival was determined by the percentage of animals in each groupthat survived 72 h after PDT. More severe phototoxicity was observedwith both increasing light exposure and photosensitizer dose. Toxicity, however, more closely paralleled incremental changes in thedosage of BPD-MA, as compared to changes in the total light energydelivered. Necropsy of animals that died due to phototoxicity revealeddiffuse inflammation, hepatic congestion, and tissue edema, most

notably in the bowel and pentoneum. Clinically, these animals hadincreasing abdominal distention and progressive lethargy. They became dehydrated, hypothermic, and appeared to develop shock, withdeath generally occurring 12—48h following high-intensity PDT.Intestinal perforation and/or ischemic bowel was noted in severalmice treated with higher doses of photosensitizer. Mice survivingtoxicity testing suffered no prolonged untoward effects.

Photodynamic Therapy

Tumoricidal Response. i.p. disease was confirmed in all mice(treatment and control) before therapy by the presence of positiveperitoneal cytology. A significant reduction in the overall solid tumorburden (Fig. 1) was noted. For treated animals, the mean weight ofresidual carcinoma at necropsy was 0.034 ±0.014 g as compared to0.379 ±0.065 g in controls (P < 0.001). This overall tumor reductioncollectively reflects the significant decrease in disease noted at allanatomical sites (Fig. 2). The greatest bulk of tumor in this xenograftmodel is in the pelvis, which was reduced to 0.015 +1—0.007 g inmice given PDT as compared to 0.187 +1—.038 g in those not treated(P < 0.001). PDT effectively reduced the subgastric tumor volume to0.009 +1—0.004 g in comparison to 0.075 +1—0.01 g (P < 0.001)in untreated controls. A comparable tumoncidal effect on i.p. carcinoma was also noted in the hepatic, splenic, and mesenteric areas.Small implants on the inferior surface of the diaphragm were notconsistently present and accounted for the least volume of disease.Diaphragmatic studding was noted in six control mice (35%) with amean weight of 0.012 +1—0.007 g, while only one treated mouse (5%)had any visible implants, which weighed 0.002 g (not significant).

In the mice treated with PDT, there was a marked reduction in thefrequency of macroscopic tumor implants at multiple anatomical sites

p (control v treatment) < 0.001

C —C) QIa)@ @a 0U)

control treatment

(I)E 0.5

C)

@0.4E

.@ 0.1

Fig. 1. Comparison of total tumor weights in treated (U) and control (0) mice. Swissnude mice (n = 17 or 22) with human ovarian carcinoma were given injections at day 14postinoculation with BPD-MA (2 mg/kg) i.p. After 90 mm, 20 J of 690 nm light weredelivered in equal fractions to the peritoneal quadrants. This sequence was repeated threetimes at 72-h intervals. At necropsy, the total i.p. tumors from treated and control micewere completely resected and weighed.

Cl)ECa

01

.C0)a)

5-

0E

C(8C)E

<0.001

p <0.001

0.25

0.2

0.15

0.1

0.05

0

p <0.001p <0.01@ p <o.ooi

U

U 4J.- Cl)> Ca.@; 0)0. .@

U)

4.'

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American Association for Cancer Research Copyright © 1996 on July 13, 2011cancerres.aacrjournals.orgDownloaded from

20

100

80

60

40

20

0

Table 2 Meanconcentrations (p.g/g tissue) ofBPD-MA in peritoneal organs of tumor-ladenmice―Time(h)IntestinesSpleenLiverKidney

Tumor

PHOTODYNAMICTHERAPY

Survival. Because of the marked weight loss observed with treatments administered at 72-h intervals, survival analysis was performedon mice treated at 5- and 7-day intervals. Sustained weight loss wasnot observed using these regimens, suggesting adequate time forrecovery was allowed between treatments. In the assessment of PDTadministered every 7 days and commencing 14 days after tumor cellinoculation (advanced disease model), the median survival for controlanimals (n = 9) was 47 days, and all controls died between 38 and 61days. For the treated mice (n 11), 45% were still alive after 61 days,and the median survival for this group was 57 days. Kaplan-Meiersurvival curves are shown in Fig. 4. Evaluation using the Wilcoxonsigned rank test confirmed a significant survival advantage with i.p.PDT (P < 0.05). For the treatment group commencing at 7 days aftertumor cell inoculation (minimal residual disease model) PDT wasrepeated either three or five times at 5-day intervals. Kaplan-Meiersurvival curves are shown in Fig. 5. Both of the courses consisting of

three treatments and five treatments gave significant survival advantages (median survivals of 57 and 53 days, respectively) over untreated controls (median survival of 43 days, P < 0.05), but did notsignificantly differ from each other. The two survival studies havesignificantly different survivals for control mice (P < 0.05). Thereason for these differences is not fully understood. Possibilitiesinclude variations in the immune status of the nude mice betweenbatches, and increase in aggressiveness in the OVCAR-5 cell line withrepeated in vitro passaging. Nevertheless, the treatment mice and thecontrol mice in each survival experiment were directly comparable.

Mice from the same batch each received the same number of cellsfrom the same passage number.

Biodistribution. The tissue concentrationsof BPD-MA in resectedi.p. organs and tumor are shown in Table 2. The maximum concen

tration of BPD-MA in each tissue is reached within 2 h after i.p.injection of 2.0 mg/kg. The highest levels are present in the tumor

0E

C).0CaUC)C,@0

.C

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E

0C)01Ca

CC)UC)0.

C)4-'

C —

a) a)

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a)C

U4-'

U

@ 0)0.a) .C

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@0

UI.

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> (a.; I@@)

0. .@

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Fig. 3. Comparison of frequency of tumor in anatomical sites between treated (U) andcontrol (i'l) mice. The mice that had been treated in Fig. I were classified for thepercentage of treated mice that showed macroscopic tumor (> I mm) at each anatomicalsite.

C0

.05-

Cl)@0

Ca>>

U)

32 40 48 56 64 72 80 88

days surviving post inoculation 1

Fig. 4. Kaplan-Meier survival curves for treated (—) and control ( ) mice(advanced disease model). Treatment mice underwent initial PDT 14 days postinoculumand were treated every 7 days until death.

yet there was an average weight loss of 1.9 g after three treatments ascompared to a mean weight gain of 0.7 g in controls (P < 0.05). Theextent of weight loss is several times greater than can be explained byreduction in tumor burden alone, suggesting some manifestation ofsystemic stress. The extent of weight gain in control mice is notunexpected in the context of normal growth of immature mice andtumor progression.

Four mice died during treatment: two from excess anesthesia sedation, one from i.p. hemorrhage due to a liver laceration, and onefrom undetermined causes. Two control mice also died during thesame time interval of undetermined causes.

0 50 60

C0

.05-

Cl)@0

Ca>

U)

days surviving post-inoculation

Fig. 5. Kaplan-Meier survival curves for treated (three treatments, ; five treatments, —) and control ( ) mice (minimal residual disease model). Treatmentmice underwent initial PDT 7 days postinoculation and received a total of either three orfive treatments at 5-day intervals.

I 3.35 ±0.85― 1.94 ±0.56― 2.92 ±042b 1.02 ±0.16― 4.51 ±118b2 2.18±0.36 0.94±0.14 2.59±0.75 0.73±0.2 1.76±0.454 1.17 ±0.28 0.92 ±0.27 1.79 ±0.31 0.54 ±0.07 1.01 ±0.228 1.02 ±0.39 0.65 ±0.33 2.1 1 ±0.89 0.83 ±0.29 0.71 ±0.18

12 0.88 ±0.26 0.05 ±0.02 0.35 ±0.07 0.19 ±0.1 0.38 ±0.1724 0.32 ±0. 15 0.07 ±0.03 0.03 ±0. 1 0.2 1 ±0.08 0. 15 ±0.0348 0.09 ±0.05 0.01 ±0.01 0.06 ±0.01 0.05 ±0.02 0.03 ±0.01

a Swiss nude mice (n = 5—7) with human ovarian carcinoma were given liposomal BPD-MA (2 mg/kg i.p.) and sacrificed after varying times. Samples of organs were dissected,

weiEhed. homogenized, and extracted with methanol. The fluorescence signals were compared with standard curves to generate tissue concentrations of BPD-MA.Mean ±SE.

1078

American Association for Cancer Research Copyright © 1996 on July 13, 2011cancerres.aacrjournals.orgDownloaded from

the relatively poor survival benefits can be explained by the fast rateof regrowth of tumors that had been substantially reduced but noteliminated. We believe the findings are an important addition to thepresent body of information for several reasons. First, the xenograftmodel utilized in these experiments manifests tumor derived fromhuman ovarian carcinoma cells and has been described in detail.4 Incontrast to models expressing primary murine malignancies, thesetumors possess many of the inherent biological properties of humandisease. Advanced disease is characterized by extensive solid tumorburden and ascites, with parenchymal invasion, lymphatic metastases,and vascular dissemination. Individual tumor nodules exhibit developing neovasculature characterized by the absence of mature basement membrane. Second, the anatomical site of carcinoma is withinthe pentoneal cavity and presents a more realistic set of challengesthan the treatment of s.c. tumors. Third, the focus of treatment isagainst solid tumors, in contrast to the more frequently studied ascitictumor models for ovarian cancer. Fourth, a tumoricidal effect isdemonstrated against diffuse i.p. carcinomatous implants at a timewhen the extent of disease is similar to the amount of residual diseasethat may persist following laparotomy and tumor debulking. Light at690 nm may penetrate tissues 5—10mm; because of the limitedpenetration, a substantial reduction in massive tumor burden is notexpected using PDT. This modality, however, is an appealing adjunctive, therapeutic option to follow aggressive surgical debulking, forthe treatment of microscopic implants and small volume residualdisease, especially since it has been shown to be phototoxic to humanovarian cancer cells that are platinum resistant (7, 17). Delaney et al.(18), andSindelaret al. (19)havedemonstratedthe feasibilityofdelivering PDT to the peritoneal cavity at the time of staging laparotomy in patients with disseminated intraperitoneal tumors.

Benzoporphyrin derivatives belong to the group of compounds

often referred to as second-generation photosensitizers (20) whichhave higher absorption coefficients associated with their activationwavelengths in the 650—800nm range and enhanced target specificity(14). These relatively new molecules are proposed as possible alternatives to the first-generation porphyrin mixtures, PF and HPD.BPD-MA is a synthetic chlorin (17) with an absorbance peak at 690nm [e = 33,000 M@ cm ‘in MeOH (21)]. BPD-MA has been shownto be a potent photosensitizer both in vitro (22) and in vivo (23).Treatment effect varies with the wavelength of light that activates aspecific sensitizer, and second-generation sensitizers are activated atlonger wavelengths, allowing for a 30% increase in treatment depths(17). The BPD-MA used in the present experiment was encapsulatedin liposomes, which has been reported to increase the tumor specificity when administered i.v (4, 24). A clinical advantage is offered by

the lack of significant cutaneous photosensitization associated withBPD-MA.

Phototoxicology studies, following treatment of the entire pentoneal surface, have demonstrated significant toxicity associated witheither high light or photosensitizer doses (18, l9).@ Results from ouranimal studies concur with these findings. PDT-induced injuries of thepentoneal surface would be expected to cause a burn-like reaction thatcould lead to major fluid and electrolyte disturbances, prolongedparalytic ileus, and elevated liver enzymes (15). In Phase 1111clinical

trials using i.p. PDT with dihematoporphyrin ethers, major morbidity

was seen in 9 (23%) of 39 patients (18) and 5 (22%) of 23 patients(19) who underwent surgery plus PDT. Serious complications included gastric and small bowel perforations, intestinal fistulas, intraabdominal abscesses, ureteral leak and urinoma, necrotizing pancre

5 B. A. Goff. J. Blake. M. P. Bamberg. and T. Hasan. Treatment of ovarian cancer with

photodynamic therapy and immunoconjugates in a murine ovarian cancer model, manuscript submitted for publication.

100a.

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PHOTODYNAMICTHERAPY

5- Ca) a, a)

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C @—.— 4-' C.@ U) 0

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Fig. 6. Tumor:normal tissue ratios in mice given i.p. BPD-MA measured after I h (U),4 h (0), and 24 h (n). Tumor-bearing mice were given liposomal BPD-MA (2 mg/kg i.p.)and sacrificed after varying times. Samples of organs were dissected, weighed, homogenized, and extracted with methanol. The fluorescence signals were compared withstandard curves to generate tissue concentrations of BPD-MA. Values given are @gBPD-MA/g tumor divided by @.tgBPD-MAIg tissue.

(4.51 +1— 1.18 @tg/g),followed by comparable levels in the liver(2.92 +1—0.42 p.g/g) and small intestine (3.35 +1—0.85 jtg/g). Thetumor:tissue ratios for these organs are shown in Fig. 6. Initially, at1-h postinjection, the tumor:tissue ratios are > 1 for intestine, spleen,liver, and kidney. At 2 h postinjection, the tumor:tissue ratios become< 1 for the liver and intestine. These higher concentrations, relative to

that of tumor, persist throughout the 72-h period of investigation. Thetissue concentrations of BPD-MA in adipose tissue, the abdominalwall (skin and peritoneum), and thoracic cavity (heart and lungs) areshown in Table 3. The maximum concentration of BPD-MA in eachof these organs, except the skin, is also reached within 2 h after i.p.administration. The maximum concentrations of photosensitizer in theskin are found at the 2-h time point. The highest concentrations arenoted in the peritoneum (6.88 +1—0.73 @tg/g)and adipose tissue(3.70 +1—0.88 p@g/g).Initially, at 1 h postinjection, the tumor:tissueratios are > 1 for skin, adipose, lung, and heart. The concentration ofBPD-MA in tumor is consistently higher than those in the heart andlungs throughout the 72-h period of investigation. At 24 h postinjection, the tumor and tissue concentrations are approximately equivalentin the skin and adipose tissue, with tumor:tissue ratios of 1.08: 1 and1.29: 1, respectively. The photosensitizer is consistently more concentrated in the peritoneum during the first 12 h, but at 24 h a tumor:tissueratio of 1.20: 1 is observed.

DISCUSSION

This initial study demonstrates the feasibility, tumoricidal activity,and survival benefit of BPD-MA-mediated PDT directed againstdiffuse i.p. ovarian carcinoma. Previous in vivo investigations of PDTfor ovarian carcinoma have primarily focused on the treatment ofascitic or s.c. tumors. Tochner et a!. (15) effectively demonstratedascitic tumoricidal response and prolonged survival with i.p. PDTwith PF in a primary murine tumor model. Using the 0C125 antibody,conjugated to the photosensitizer chlorine ethylene diamine monoamide, Goff et a!. (7—9)demonstrated selective destruction of ascitictumor cells and enhanced survival. Tumors derived from s.c. implantation of NIH:OVCAR-3 cells into athymic mice have been completely eradicated using PF-based PDT (16).

Although there was an almost 10-fold decrease in tumor burdenfollowing PDT therapy, the survival advantages seen in the presentstudy were not dramatic. It can be seen from Fig. 3 that the number ofmice which had no detectable tumor was very small. We believe that

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Table 3 Mean concentrations (pg/g tissue) of BPD-MA in tumor-ladenmice―Time(h)

Skin Lung Heart AdiposePeritoneumI

0.58 ±012b 0.98 ±023b 0.45 ±0.09― 3.70 ±0.88― 6.88 ±0.73―20.71 ±0.13 0.41 ±0.09 0.28 ±0.08 2.35 ±1.07 1.88 ±0.3840.39 ±0.1 1 0.34 ±0.1 1 0.14 ±0.02 2.80 ±0.63 2.36 ±0.5580.35 ±0.05 0.42 ±0.22 0.19 ±0.09 1.32 ±0.4 1.63 ±0.54120.20 ±0.08 0.1 ±0.06 0.07 ±0.06 0.19 ±0.1 1 0.17 ±0.08240.22 ±0.07 0.04 ±0.02 0.01 ±0.01 0.12 ±0.06 0.12 ±0.06480.17±0.08 0 0 00a

Swiss nude mice (n 5—7) with human ovarian carcinoma were given liposomal BPD-MA (2 mg/kg i.p.) and sacrificed after varying times. Samples of organs weredissected,weighed,

homogenized, and extracted with methanol. The fluorescence signals were compared with standard curves to generate tissue concentrations of BPD-MA.Mean ±SE.

PHOTODYNAMIC THERAPY

atitis, and postoperative hemorrhage. Additionally, third spacing offluids, greater than expected during normal postoperative course, wasnoted in the majority of patients. Delaney et a!. (18) showed that 59%of patients developed pleural effusions and 15% required thoracentesis or prolonged intubation. Sindelar et a!. (19) reported postoperativehypoalbuminemia in all patients, with varying degrees of fluid sequestration, transient edema, and ileus. One patient developed apericardial effusion.

Animal studies using PF-mediated PDT have suggested that acutePDT-induced lethality is consistent with a traumatic shock syndrome(25).Veenhuizeneta!. (26)havestudiedlethaltoxicityafteri.p.PDTand draw a distinction between death due to PDT-mediated intestinaldamage over a period of 2 weeks and death due to a shock syndromewithin 20 h. Lethal shock can be caused by sepsis (27), hemorrhage(28), severe burns (29), or trauma (28), and is thought to involve acommon cascade of cytokines, eicosanoids, and other mediators (30).Central to this cascade is the production of TNF-cx by macrophages(31), especially peritoneal macrophages. TNF-a then causes release ofvasoactive substances which mediate the drop in blood pressure anddisseminated intravascular coagulation typical of multiorgan failure(32). Peritoneal macrophages have been shown (33) to accumulatelarge amounts of photosensitizers, and PDT causes them to release1'NF-a (34). Other authors have suggested that macrophages play arole in the tumoricidal effect of PDT either by becoming photodynamically activated (35) or by being more cytotoxic toward cellswhich have mild PDT injuries (36). Two factors apply in the presentcase which make this mechanism for systemic toxicity likely. First,the BPD-MA was administered in liposomal form. It is well known(37,38) thatpentonealmacrophagesphagocytosei.p.administeredliposomes. Hunt et a!. (39) have shown that mouse peritoneal macrophages take up liposomal BPD-MA in vitro and, on photoactivation,release nitric oxide. Macrophages activated with IFN--y were moresensitive to this effect than those activated with lipopolysaccharide. Ithas also been shown (40) that after i.v. administration of PF to s.c.tumor-bearing C3H mice, pentoneal macrophages took up the photosensitizer nine times more than the malignant cells. Second, athymicnu/nu mice have been shown (41) to have a 25-fold enhanced phagocytosis capacity of peritoneal macrophages over their nu/+ and +1+litter mates. This is thought (41) to be one way in which they

compensate for their immune defect. It would seem therefore that inthis model, pentoneal macrophages may be significantly involved inboth tumor destruction at modest light doses and in lethal shock athigh light doses.

A major obstacle in the i.p. treatment of tumors with PDT isuniform light delivery to all visceral and parietal peritoneal surfaces,without producing significant toxicity. The confinement of i.p. organswith vastly different optical properties [i.e., liver, absorption coefficient at 630 nm, pa —0.23—0.65mm ‘,aorta pa —0.056—0.23mm@‘,and bladder @ta—0.14 mm â€(̃42)] presents a substantialchallenge for uniform light delivery and accurate dosimetry (43).

Therefore, despite a reasonable selectivity of the photosensitizer fortumor, damage of normal tissues at high fluence regions proximal to

the source, and insufficient treatment of ascites, or carcinoma distantfrom the source are observed (44). Cancer cells implanted along thedorsal pentoneum and around the liver or diaphragm are difficult totreat due to being shielded from direct light exposure by intraabdominal organs. To reduce this problem, multiple treatments have beengiven in some studies (9, 45) at 48—72-hintervals, allowing redistribution of the ascites in the peritoneal cavity between treatments. Lightdelivery and dosimetry must be optimized to all peritoneal surfaces,however, to reach microscopic and macroscopic foci of carcinomathat are not redistributed by peritoneal circulation (14).

In the NIH:OVCAR-5-induced ovarian cancer model, the majorityof macroscopic disease develops in the pelvic and subgastric regions.These areas are readily accessible by direct illumination during PDT,as administered in the experiments reported in the present study.Accordingly, a significant tumor reduction occurred in both areas asa result of treatment. Tumors along the bowel serosa may be shieldedsomewhat from direct light due to the extensive length of the bowel,with numerous folds and layers within the peritoneal cavity. Similarly,direct illumination of the splenic tumors is hindered by the dorsallocation of the spleen. Carcinomatous implants along the inferiorsurface of the liver and diaphragm are obscured by the stomach andproximal small bowel, and liver, respectively. The unexpected resultfrom this study was the significant degree of tumor reduction in theseanatomical locations that, in all probability, were suboptimally illuminated as was reported by Lilge et a!. (14), who measured the lightdistribution with implanted optical fiber fluence rate detectors.Among the possible reasons for this are the high therapeutic efficacyof BPD-MA-mediated PDT, or that the combination of the cylindrically diffusing fiberoptic tip and i.p. intralipid solution may contributesubstantially to enhanced uniform light delivery. Other effects such asenhanced oxygen supply to these areas may well play a role. Our

study suggests that for optimal tumoricidal effect, PDT may need tobe administered in multiple treatments. Similar results were noted byTochner et al. (15) who used HPD for PDT to treat a murine ovariancancer model.

Although mechanisms for the preferential accumulation of liposomally encapsulated photosensitizers administered i.v. have been pro

posed (46, 47), little is known about the routes of uptake into thetumor or other tissues after i.p. administration. No comparison between liposomal and nonliposomal BPD-MA has yet been made for

i.p. tumors. The pharmacokinetics and biodistribution of liposomallyencapsulated drugs are generally thought to depend mainly on the sizeof the liposomes, but also on the constituents of the phospholipidbilayer which affect parameters such as surface charge and membranerigidity. In a comparison of free and liposomally encapsulated cefoxitin administered i.p., it was found (48) that the liposomal formulationpersisted longer in the peritoneum and gave higher concentrations inthe liver and spleen, while the free formulation was more quickly

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PHOTODYNAMIC THERAPY

taken up into the systemic circulation. Large liposomes however were

found (49) to have higher uptake in the liver and spleen after iv.administration than i.p. Intact liposomes are known (50) to be takenup from the pentoneum by subdiaphragmatic lymphatics from wherethey reach the circulation with an absorption half life of 3—4h and awhole-body half-life of 10—13h. Zinc phthalocyanine encapsulated inliposomes was injected i.p. into mice bearing s.c. tumors (5 1). Theserum concentration reached a peak in 3 h, and tumor localization was

at a peak at 18 h.In the biodistribution study, the dose of BPD-MA administered was

8-fold higher than in the treatment study. This was in order to obtainsufficient fluorescent signal from the tissue homogenates. It should benoted that the dose of photosensitizer may effect tumor selectivity[low doses may give greater tumor:normal ratios (52)]. With theexception of the skin, the maximum concentration of BPD-MA ineach tissue was reached approximately 1 h after i.p. injection. Thehighest levels were present in the pentoneum and tumor followed bycomparable levels in the liver and small intestine. The liver is theprimary organ of metabolism of BPD-MA, and, accordingly, persistently higher levels have been demonstrated in previous biodistnbution studies (53). The present study confirms these findings. Initially,at 1 h postinjection, the tumor:liver ratio was > 1, but this ratio fell to<1 at 2 h postinjection and was persistently < 1 during the 72-hperiod of investigation. The i.p. route of administration, with subsequent circulation of i.p. fluid, may have enhanced the uptake andconcentration of BPD-MA within the peritoneum. The tumor:normaltissue ratios are relatively modest, especially tumor:peritoneum ratioswhich however could be affected by the presence of diffuse microscopic tumor foci in the peritoneal wall.

The splenic concentration of BPD-MA was consistently less thanthat observed in tumor at all time points studied. This is in contrast tothe relatively higher concentrations of BPD-MA maintained in thespleen following i.v. administration (53). This may be explained, inpart, by the high capture rate of photosensitizer by the reticuloendothelial system following i.v. injection. The maximum concentrationsof photosensitizer in the skin were detected 2—8h after administration.Although detectable levels persisted in the skin for 72 h, the concentrations were low in comparison to other organs. This relatively low

uptake of BPD-MA by the skin is consistent with the clinicallyobserved lack of cutaneous photosensitivity associated with use of thiscompound. Most previous studies of i.p. PDT have used PF or HPDas sensitizers, and although the disadvantages of these compounds arewell known, it would be interesting to carry out a study comparing PFwith BPD-MA in this model.

These preliminary results of PDT treatment are encouraging, specifically the selective phototoxicity of human ovarian cancer cells,reduction of diffuse, solid i.p. tumor implants, and improved survivalafter fractionated i.p. PDT therapy for both models of minimal residual and advanced disease. The parameters used in this study have notbeen optimized. Optimal drug and light dose and the optimal time forirradiation after injection remain to be determined following carefulbiodistribution studies.

ACKNOWLEDGMENTS

We thank Quadra Logic Technologies, Inc. (Vancouver, Britich Columbia,Canada) for the gift of BPD-MA and Coherent Inc. (Palo Alto, CA) for theloan of the argon-pumped dye laser. We thank Imran Rizvi and RiniChakraborty for technical assistance.

REFERENCES

1. Wingo, P. A., Tong, T., and Bolden, S. Cancer Statistics. CA Cancer J. Clin., 45:1—128.1995.

2. McNeil,C.Ovariancancer:latestgainsestablishnewbattlefronts.J. NatI.CancerInst., 87: 871—873, 1995.

3. Spikes, J. D. Photobiology of porphyrins. In: D. R. Dorian and C. L. Gomer (eds.),Porphyrin Localization and Treatment of Tumors. pp. 19—39.New York: Alan R.Liss, 1984.

4. Hasan, T. Photosensitizer delivery mediated by macromolecular carrier systems. In:B. Henderson and T. Dougherty (eds.). Photodynamic Therapy: Basic Principles andClinical Applications, pp. 187—200.New York: Marcel Dekker, 1992.

5. Henderson, B. W., and Dougherty. T. J. How does photodynamic therapy work?Photochem. Photobiol., 55: 145—157,1992.

6. Delaney, T. F., and Glatstein, E. Photodynamic therapy of cancer. Comprend. Ther..14: 43—55,1988.

7. Goff, B. A., Bamberg, M., and Hasan, T. Photoimmunotherapy of human ovariancarcinoma cells ex vivo. Cancer Res.. 51: 4762—4767, 1991.

8. Goff, B. A., Bamberg, M., and Hasan, T. Experimental photodynamic treatment ofovarian carcinoma cells with immunoconjugates. Antibody, Immunoconj. Radiopharm., 5: 191—199,1992.

9. Goff, B. A., Hermanto. U., Rumbaugh, J., Blake, J., Bamberg. M.. and Hasan. 1.Photoimmunotherapy and biodistribution with an 0C125-chlorin immunoconjugatein an in vivo murine ovarian cancer model. Br. J. Cancer, 70: 474—480, 1994.

10. Lui, H. Photodynamic therapy in dermatology with porfimer sodium and benzoporphyrin derivative: an update. Semin. Oncol., 21: 11—14,1994.

11. Richter, A. M.. Cerruti-Sola, S., Sternberg. E. D.. Dolphin. D., and Levy, J. G.Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA). a new potentphotosensitizer, in normal and tumor-bearing mice. J. Photochem. Photobiol. B.. 5:231—244,1990.

12. Waterfield, E. M., Renke, M. E, Smits, C. B., Gervais, M. D., Bower, R. D., Stonetield,M. S., and Levy, J. G. Wavelength-dependent effects of benzoporphyrin derivativemonoacid ring A in vito and in vitro. Photochem. Photobiol., 60: 383—387.1994.

13. Levy, J. G. Photosensitizers in photodynamic therapy. Semin. Oncol.. 21: 4—10.1994.

14. Lilge, L., Dabrowski, W.. Holdsworth, D., Blake, J., Kato, D., Wilson, B.. and Hasan,T. Lightdeliveryand dosimetryfor photodynamictherapyin an ovarian-cancermouse model.SPIE Proc..2133:150—161.1994.

15. Tochner, Z., Mitchell, J. B., Harrington, F. S.. Smith. P., Russo, D. 1., and Russo, A.Treatment of murine intraperitoneal ovarian ascitic tumor with hematoporphyrinderivative and laser light. Cancer Res., 45: 2983—2987, 1985.

16. Peterson,C.M.,Reed,R..Jolles,C.J.,Jones,K.P.,Straight,R.C.,andPoulson,A.M.Photodynamic therapy of human ovarian epithelial carcinoma. OVCAR-3. heterotransplanted in the nude mouse. Am. J. Obstet. Gynecol., 167: 1852—1855, 1992.

17. Pass, H. I. Photodynamic therapy in oncology: mechanisms and clinical use. J. NatI.Cancer Inst., 85: 443—456,1993.

18. DeLaney, T. F., Sindelar, W. F.. Tochner. Z., Smith, P. D., Friauf, W. S., Thomas, G.,Dachowski, L., Cole, J. W., Steinberg, S. M., and Glatstein, E. Phase I study ofdebulking surgery and photodynamic therapy for disseminated intraperitoneal tumors.Int. J. Radiat. Oncol. Biol. Phys., 25: 445—457, 1993.

19. Sindelar, W. F., DeLaney, T. F., Tochner, Z., Thomas, G. F., Dachoswki. L. J., Smith.P. D., Friauf, W. S., Cole, J. W., and Glatstein, E. Technique of photodynamic therapyfor disseminated intraperitoneal malignant neoplasms. Phase I study. Arch. Surg..126: 318—324.1991.

20. Richter, A. M., Kelly. B., Chow, J., Liu, D. J., Towers, G. H.. Dolphin, D., and Levy,J. G. Preliminary studies on a more effective phototoxic agent than hematoporphyrin.J. NatI.CancerInst..79: 1327—1332,1987.

21. Aveline, B., Hasan, T., and Redmond, R. W. Photophysical and photosensitizingproperties of benzoporphyrin derivative monoacid ring A (BPD-MA). Photochem.Photobiol., 59: 328—335, 1994.

22. Richter, A. M., Waterfield, E., Jam, A. K., Sternberg, E. D., Dolphin. 0., and Levy.J. G. In vitroevaluationof phototoxicpropertiesof fourstructurallyrelatedbenzoporphyrin derivatives. Photochem. Photobiol., 52: 495—500.1990.

23. Richter, A. M., Waterfield, E., Jam, A. K., Allison. B., Sternberg, E. D., Dolphin, D.,and Levy, J. G. Photosensitising potency of structural analogues of benzoporphyrinderivative (BPD) in a mouse tumour model. Br. J. Cancer, 63: 87—93,1991.

24. Jiang, F. N., Jiang, S., Liu, D., Richter. A., and Levy, J. G. Development oftechnology for linking photosensitizers to a model monoclonal antibody. J. Immunol.Methods, 134: 139—149,1990.

25. Ferrario, A., and Gomer, C. J. Systemic toxicity in mice induced by localizedporphyrin photodynamic therapy. Cancer Res., 50: 539—543,1990.

26. Veenhuizen, R. B., Ruevekamp-Helmers, M. C., Helmerhorst, T. J., Kenemans. P.,Mooi, W. J., Marijnissen, J. P., and Stewart, F. A. Intraperitoneal photodynamictherapy in the rat: comparison of toxicity profiles for photofrin and MTHPC. Int. J.Cancer, 59: 830—836. 1994.

27. Galanos, C., and Freudenberg, M. A. Mechanisms of endotoxin shock and endotoxinhypersensitivity. Immunobiology, 187: 346—356, 1993.

28. Thu. X. L., Ayala, A., Zellweger, R., Morrison, M. H., and Chaudry, I. H. Peritonealmacrophages show increased cytokine gene expression following haemorrhagicshock. Immunology, 83: 378—383, 1994.

29. Marano, M. A., Fong, Y., Moldawer, L. L., Wei, H., Calvano, S. E., Tracey, K. J.,Bade, P. S., Manogue, K., Cerami, A., Shires, G. 1., and Lowry, S. F. Serumcachectin/tumor necrosis factor in critically ill patients with bums correlates withinfection and mortality. Surg. Gynecol. Obstet., 170: 32—38,1990.

30. Rothstein, J. L., and Schreiber, H. Relationship of tumour necrosis factor andendotoxin to macrophage cytotoxicity. haemorrhagic necrosis and lethal shock. CibaFound.Symp.,131:124—139,1987.

31. Dofferhoff, A. S., Vellenga, E., Limburg, P. C., van Zanten, A., Mulder, P. 0., andWeits, J. Tumour necrosis factor (cachectin) and other cytokines in septic shock: areview of the literature. Neth J. Med., 39: 45—62,1991.

1081

American Association for Cancer Research Copyright © 1996 on July 13, 2011cancerres.aacrjournals.orgDownloaded from

PHOTODYNAMICTHERAPY

32. Schlag, 0., RedI, H., and Hallstrom, S. The cell in shock: the origin of multiple organfailure. Resuscitation, 21: 137—180,1991.

33. Korbelik, M., Krosl, 0., and Chaplin, D. J. Photofrin uptake by murine macrophages.Cancer Res., 51: 2251—2255,1991.

34. Evans, S., Matthews, W., Perry, R., Fraker. D., Norton, J., and Pass, H. I. Effect ofphotodynamic therapy on tumor necrosis factor production by murine macrophages.J. Natl. Cancer Inst., 82: 34—39,1990.

35. Steubing, R. W., Yeturu, S., Tuccillo, A., Sun, C. H., and Berns, M. W. Activation ofmacrophagesbyPhotofrinIIduringphotodynamictherapy.J. Photochem.Photobiol.B, 10:133—145,1991.

36. Korbelik,M.,and Krosl,G. Enhancedmacrophagecytotoxicityagainsttumorcellstreated with photodynamic therapy. Photochem. Photobiol., 60: 497—502,1994.

37. Biewenga, J., van der Ende, M. B., Krist, L. F., Borst, A., Ghufron, M., and vanRooijen, N. Macrophage depletion in the rat after intrapentoneal administration ofliposome-encapsulated clodronate: depletion kinetics and accelerated repopulation ofperitoneal and omental macrophages by administration of Freund's adjuvant. CellTissue Res., 280: 189—196,1995.

38. Malik, S. 1., Martin, D., Hart, I., and Balkwill, F. Therapy of human ovarian cancerxenografts with intraperitonealliposome encapsulated muramyl-tripeptidephosphoethanolamine (MTP-PE) and recombinant GM-CSF. Br. J. Cancer, 63: 399-403, 1991.

39. Hunt, D. C. W., Jiang, H-J., Levy, J. G., and Chan, A. H. Sensitivity of activatedmurine peritoneal macrophages to photodynamic killing with benzoporphyrin denyative. Photochem. Photobiol., 61: 417—421, 1995.

40. Korbelik, M., and Krosl, G. Photofrin accumulation in malignant and host cellpopulations of a murine fibrosancoma. Photochem. Photobiol., 62: 162—168,1995.

41. Vetvicka, V., Fornusek, L., Holub, M., Zidkova, J., and Kopecek, J. Macrophages ofathymic nude mice: Fc receptors, C receptors, phagocytic and pinocytic activities.Eur. J. Cell Biol., 35: 35—40,1984.

42. Cheong, W. F., Prahi, S. A., and Welch, A. J. A review of the optical properties ofbiological tissue. IEEE J. Quantum Electronics. 26: 2166—2185, 1990.

43. Wilson, B. C. Photodynamic therapy: light delivery and dosage for second-generationphotosensitizers. Ciba Found. Symp., 146: 60—73,1989.

44. Evnard, S., Aprahamian, M., and Marescaux, J. Intra-abdominal photodynamic thenapy: from theory to feasibility. Br. J. Sung., 80: 298—303, 1993.

45. Tochner, Z., Mitchell, J. B., Smith, P., Hannington, F., Glatstein, E., Russo, D., andRusso, A. Photodynamic therapy of ascites tumounswithin the penitonealcavity. Br.J. Cancer, 53: 733—736,1986.

46. Allison, B. A., Pritchard, P. H., and Levy, J. 0. Evidence for low-density lipoproteinreceptor-mediateduptake of benzoporphyninderivative. Br. J. Cancer, 69: 833—839,1994.

47. Hamblin, M. R., and Newman, E. L. On the mechanism of the tumour-localisingeffect in photodynamic therapy. J. Photochem. Photobiol. B, 23: 3—8,1994.

48. Kresta, A., Shek, P. N., Odumenu, J., and Bohnen, J. M. Distribution of free andliposome-encapsulated cefoxitin in experimental intra-abdominal sepsis in rats. J.Pharm. Pharmacol., 45: 779—783,1993.

49. Parker,R. J., Pniester,E. R., and Sieben,S. M. Effectof routeadministrationandliposome entrapment on the metabolism and disposition of Adniamycin in the rat.Drug. Metab. Dispos., 10: 499—504, 1982.

50. Hwang, K. J., and Mauk, M. R. Fate of lipid vesicles in vivo: a gamma-nay perturbedangular correlation study. Proc. Natl. Acad. Sci. USA, 74: 4991—4995,1977.

51. Reddi, E., La Castro, G., Biolo, R., and Jori, 0. Pharmacokinetic studies withzinc(ll)-phthalocyanine in tumour-bearing mice. Br. J. Cancen, 56: 597—600, 1987.

52. Baert, L., Berg, R., Van Damme, B., D'Hallewin, M. A., Johansson, J., Svanbeng, K.,and Svanbeng, S. Clinical fluorescence diagnosis of human bladder carcinoma following low-dose Photofrmn injection. Urology, 41: 322—330,1993.

53. Richter, A. M., Wateffield, E., Jam, A. K., Canaan, A. J., Allison, B. A., and Levy,J. G. Liposomal delivery of a photosensitizen, benzoponphyninderivative monoacidring A (BPD), to tumor tissue in a mouse tumor model. Photochem. Photobiol., 57:1000—1006,1993.

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