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[Cancer Research 59, 6164-6170, December 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 6164-6170, December 15, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

In Vivo Fluence Rate and Fractionation Effects on Tumor Response and Photobleaching

Photodynamic Therapy with Two Photosensitizers in an Orthotopic Rat Tumor Model1

Seiichi Iinuma, Kevin T. Schomacker, Georges Wagnieres, Milind Rajadhyaksha, Michael Bamberg, Tetsuo Momma and Tayyaba Hasan2

Wellman Laboratories of Photomedicine, Department of Dermatology [K. T. S., G. W., M. M., M. B., T. M., T. H.] and Department of Urology [S. I.], Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Appendix 1
 REFERENCES
 
The effect of fluence rate and light fractionation on phototoxicity was investigated in vivo in an orthotopic rat bladder tumor model. Two photosensitizers, benzoporphyrin derivative monoacid ring A and 5-aminolevulinic acid-induced protoporphyrin IX, were studied. For a given cumulative light dose of 30 J/cm2, enhanced tumor destruction was observed from both photosensitizers when using either lower fluence rates or fractionated light delivery. Photobleaching experiments in vivo demonstrated that the photobleaching rate, however, was not fluence rate dependent. The fluence rate and light fractionation effects on tumor phototoxicity lead to rapid local depletion in oxygen concentration that inhibited subsequent photochemical reactions necessary for efficient photodestruction of tumor cells. Nicotinamide did not enhance photodynamic therapy efficacy, suggesting that the added increase of oxygen within the tumor was not sufficient to enhance photodestruction of hypoxic cell fractions. The independence of the photobleaching rate with fluence rate suggests distinct mechanisms, at least in part, of photodestruction of the tumor and the photosensitizer and that the rate of photosensitizer photobleaching may not always be an appropriate monitor for singlet oxygen availability and photodynamic therapy dosimetry.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Appendix 1
 REFERENCES
 
PDT3 is emerging as an alternative modality for cancer therapy. It offers improved selectivity for targeting tumor compared with conventional chemotherapy and radiotherapy because of preferential accumulation of photoactivatable compounds (PSs) to tumor tissue and due to controlled light delivery directed toward the tumor that leads to spatially confined photodynamic effects. Hematoporphyrin derivative and its somewhat purified version, PFs, are the most frequently used PSs in experimental clinical PDT (1, 2, 3) . Currently, PF is the only PS that is approved in the United States for treating cancers in humans for the treatment of early- and late-stage non-small cell lung cancers and for palliation of patients with completely obstructing esophageal cancer. Because of the poor selectivity of PF to cancerous tissue and prolonged skin photosensitivity, other PSs with better selectivity (4, 5, 6) and alternative methods of improving the localization of PSs are being investigated (7, 8, 9) .

The efficacy of PDT using PF and other sensitizers depends on the partial pressure of oxygen in tissue that can be depleted during periods of irradiation, leading to therapeutic inefficiencies. PDT-induced hypoxia can limit direct photodynamic cytotoxicity in vivo (10) , as suggested by numerous studies that measured an enhanced photodynamic response (11 , 12) or a lower rate of decrease in the partial pressure of interstitial oxygen (13, 14, 15) with decreasing fluence rate or fractionated light doses. Prior to these observations, clinical applications using the highest subthermal fluence rates were thought to be optimal because this led to the shortest irradiation time for a given light dose. However, using s.c. implanted tumor models, Gibson et al. (11) reported less tumor destruction with higher fluence rates and attributed the decrease to more rapid consumption of oxygen during the time of irradiation. More detailed and elegant studies by Foster et al. (16) have since quantified this observation using a theoretical model of oxygen diffusion and consumption in tumors during PDT. The distribution of oxygen in tumor tissues is not homogeneous and can lead to nodules of hypoxic cells when located some distance away from blood vessels. It is especially in such hypoxic regions where PDT may be less effective because of the limited availability of oxygen (10) . Even for tumor cells located near blood vessels, interstitial oxygen levels will become depleted during PDT if oxygen consumption is greater than oxygen replacement from circulating blood and the rate of replacement is further reduced by photodynamic destruction of the microvasculature. We hypothesize that the effect of oxygen diffusion into hypoxic tumor cells is less important if the mechanism of tumor destruction is attributable entirely to PDT-induced thrombosis of feeding vessels and that oxygen replacement is crucial if the destructive photochemistry occurs via singlet oxygen-mediated destruction of tumor cells.

The above hypothesis was tested in the present study with two different PSs. The first, ALA, is often considered a cellular PS, being a precursor in the pathway of heme biosynthesis that is transformed to the PS (PpIX) in situ intracellularly (17, 18, 19, 20, 21, 22, 23, 24, 25) . ALA-PpIX derived in situ has been used in clinical investigations with reasonable success for the treatment of primary and secondary cutaneous (21 , 22) and gastrointestinal tract (25) lesions. The second PS, BPD-MA, has been suggested to derive its photocytotoxic effect indirectly via vascular destruction (5 , 26) . Blood flow and oxygenation levels within tumors are strongly influenced by the host organ. Hence, the effects of fluence rate and light fractionation on PDT destruction of tumor were investigated in an orthotopic rat bladder carcinoma model. Fluorescence images of the tumor in situ were obtained to study the localization of the PSs within the tumors at times of irradiation. The emergence of photoproducts and the photobleaching of both PSs and their photoproducts were also investigated during irradiation in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Appendix 1
 REFERENCES
 
Tumor Cells.
NBT-II cells (a rat bladder carcinoma cell line) was obtained from American Type Culture Collection and grown in Eagle’s MEM with L-glutamine (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS (Life Technologies, Inc.), 0.1 mM nonessential amino acid (Life Technologies, Inc.), 1 mM sodium pyruvate (Whittaker Bioproduct, Walkersville, MD), and penicillin-streptomycin (100 units/ml penicillin G and 100 µg/ml streptomycin; Sigma, St. Louis, MO). Cells were incubated at 37°C in an atmosphere of 5% CO2 and 95% air and passaged every 4–5 days.

Chemicals and PS.
Collagenase, proteinase, and DNase were obtained from Sigma. Nicotinamide was purchased from Aldrich (Milwaukee, WI). ALA was a gift from DUSA Pharmaceutical, Inc. (Tarrytown, NY) and was dissolved in normal saline. The pH was adjusted to 6.8 with 7 N sodium hydroxide, and the final concentration was 100 mg/ml of liposomal BPD-MA (72.5 mg of liposomal BPD-MA is equivalent to 1 mg of BPD-MA) was a gift from QLT (Vancouver, British Columbia, Canada). BPD-MA solution was freshly made by dissolving it in normal saline immediately before use. A 1 mg/ml BPD-MA equivalent solution was used for all experiments.

Animal Tumor Model.
All animal procedures were approved by the Subcommittee on Research Animal Care at the Massachusetts General Hospital. Experiments were carried out on female Fisher CDF rats (Charles River, Wilmington, MA). The tumor transplantation technique was performed as described in previous work (27 , 28) . Briefly, after general anesthesia, rats were placed in a supine position, and the urinary bladder was exposed through a midline incision. Cell suspensions of 2 x 106 NBT-II cells in 0.1 ml of culture medium without FCS were injected directly into the bladder wall, and the abdominal wall was closed. Experiments were carried out 6 days after the implantation of tumor cells, where the average tumor size in the bladder wall was 5 mm in diameter and 3 mm in thickness. Histologically, the tumor mass was generally located in the bladder muscle layer, and an intact urothelium was present.

Fiber Assembly for PDT and Light Dosimetry.
A 320-µm core fiber was used. After removing the nylon jacket, the distal tip was grounded to an optically diffusive conical shape using a lathe and a grinder. Aluminum oxide was mixed (10% w/w) with an epoxy that is cured with UV light (Loctite; Loctite Corp., Newington, CT). Light from a nitrogen laser (Laser Science, Inc., Franklin, MA) was coupled into the proximal end of the fiber while the distal conical end was immersed into the glue. The laser emitted 10 pulses of 337 nm of light/s at 100 µJ/pulse. After 5–7 min, a small sphere of aluminum oxide-impregnated, cured epoxy formed at the tip of the fiber. The cure time was varied to achieve optimal light distribution without drastically reducing the power emitted from the tip. The sphere is easily passed through a 18-gauge needle. Light emitted from this fiber was shown to be isotropic at 630 and 690 nm, as determined from a goniometric measurement of output power versus angle. The determination of total power emitted from the fiber was obtained using an integrating sphere (Labsphere Inc., North Sutton, NH) prior to PDT. The incident fluence rate at the inner surface of the bladder was determined from the output power divided by the calculated urothelial surface area, assuming the bladder to be spherical.

Photodynamic Treatment.
ALA (500 mg/kg body weight) or liposomal BPD-MA (1 mg/kg body weight BPD-MA equivalent) were injected i.v. through the tail vein. Four h after administration of ALA or 1 h after administration of BPD-MA, the animals were irradiated with an argon laser-pumped dye laser (Coherent, Inc., Palo Alto, CA). The excitation wavelength was 630 and 690 nm for ALA-induced PpIX and BPD-MA, respectively. For irradiation of the bladder, an 18-gauge Angiocath, minus the needle, was placed into the bladder through the urethra, and the fiber was inserted through this catheter. Laparotomy was performed to measure the diameter of the bladder and to position the fiber in the center of the bladder. The bladder was distended with normal saline until it reached 8 mm in diameter. The incident fluence rate at the inner surface of the bladder wall was adjusted to either 30 or 100 mW/cm2. Fluences of 30, 50, and 100 J/cm2 were used for ALA-PpIX-treated animals, whereas 30 J/cm2 were used for BPD-MA-treated animals.

In addition to continuous irradiation, fractionated light doses were performed at 100 mW/cm2 by opening and closing a mechanical shutter. During irradiation, the light was turned on for a fixed period and then turned off for the same equivalent period. Periods of 15, 30, and 60 s were used. The on-off cycle was repeated until a fluence of 30 J/cm2 was delivered. The periods were chosen to follow the protocol of Foster et al. (16) .

In another set of experiments, ALA was administered i.v. 4 h prior to PDT, and nicotinamide (0.5 mg/kg body weight) was injected i.p. 1 h prior to irradiation to see if nicotinamide enhances tumor destruction at high fluence rates. Tumor survival was measured 24 h after PDT for fluences of 30 and 50 J/cm2 at a fluence rate of 100 mW/cm2.

For all PDT experiments, animals were sacrificed 24 h after irradiation, and the bladder tumor was removed aseptically. A single tumor cell suspension was prepared and processed for "in vivo/in vitro" assay (29) . Tumor tissue was minced with scissors and then enzymatically digested (15 mg of collagenase, 92 units of proteinase, and 0.5 mg of DNase in 5 ml of HBSS) for 1 h at 37°C. The cell suspension was filtered with a fine wire mesh to remove stromal components. The live cells were counted in a hemocytometer using a trypan blue dye exclusion assay. Five hundred live cells were plated in a 60-mm diameter culture dish, and forming colonies were counted 8 days later after methanol fixation and staining with 0.1% crystal violet. The clonogenic cells in the tissue were calculated by multiplying the number of live cells by the plating efficacy (number of colonies per number of cells plated) of the cells divided by the mass of the tumor and was expressed as number of clonogenic cells per gram of tissue. At least five animals were used for each experimental group.

Photobleaching Experiment.
Changes in the concentration of PS during irradiation were predominantly attributed to photodegradation (photobleaching) because the time delay between light treatment and PS administration was chosen to be when the pharmacokinetics of the PS was essentially in steady state during the light treatment period. Laser-induced fluorescence spectroscopy is a useful tool for estimating static and dynamic PS concentration in tissue (30) . Photobleaching decay constants were measured by following the fluorescence decay of the PS in the tumor during irradiation. In these experiments, the bladder was irradiated from both sides because simple calculation showed that the ratio defined by dividing the space irradiances at the top and bottom surfaces of the tumor was 4.5 when irradiating the top surface only (see "Appendix" ). When irradiating from both sides, the homogeneity of the space irradiance was calculated to be within ± 30%. ALA and BPD-MA were given 4 and 1 h prior to the irradiation, respectively, as described above. The fluence rate at the tissue was either 26 or 8 mW/cm2 and corresponds to a space irradiance in the tumor at the site of the collecting fiber of 95 and 30 mW/cm2, respectively.

A minimum of five animals were used for these experiments. After general anesthesia and laparotomy, the rat bladder with tumor was exposed, opened, and held extraperitoneally with stay sutures. The two major blood vessels feeding the bladder were spared to minimize disruption of the blood circulation system. For both PSs, a 630-nm beam was split into two equal intensity beams with a 50/50 beamsplitter (Fig. 1)Citation . Each beam was focused into a 1-mm core diameter fiber. The output of the fibers was expanded to 25-mm-diameter homogeneous spots using objective lenses and used to irradiate the tumor from both sides. A third 600-µm core diameter fiber was positioned on the mucosal side of the tumor for fluorescence detection. A 694-nm bandpass filter with a 10-nm full width at half maximum (694BP10; Corion Corp., Holliston, MA) was used to eliminate backscattered excitation light. After filtering, the fluorescence was launched into a spectrograph (Jarrell Ash, Monospec 27; Anaspec, Acton, MA) and wavelength dispersed onto an intensified 1024 photodiode array (1421BR-1024-HQ; PAR/EG&G, Inc., Princeton, NJ). The spectrum was digitized via an OMA (OMA III; PAR/EG&G, Inc.), and the total fluorescence signal integrated between 675 and 720 nm was found. During the 15-min irradiation period, the total fluorescence signal was measured every 1 s by accumulating the signal for 500 ms. An adequately filtered photomultiplier-based system could have been used in place of the spectrograph/diode array system; however, the latter was used here because it was readily available with code for making automatic measurements every 1 s.



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  		Fig. 1. Schematic diagram of photobleaching experiments. After administration of the PSs, bladder tumor was irradiated from both sides of the bladder wall, and the fluorescence was collected and delivered by the third fiber to an OMA. B.S., 50/50 beam splitter; 694-10, 694-nm center band pass filter having a 10-nm full width at half maximum.

 
The photobleaching data of ALA-induced PpIX were fitted with the following equation:

where I(D) is the fluorescence signal after a given light dose D [space irradiance x irradiation time, (J/cm2)], Io is the fluorescence signal prior to irradiating (D = 0), k is the photobleaching decay constant (cm2/J), and IPh is a dose-independent background fluorescence term which is attributable to the fluorescence of the photoproducts, as we will see later. The photobleaching data for BPD-MA were fitted with a monoexponential decay curve:


The total fluorescence signal obtained from control animals lacking PS was 2–3% of that obtained with the injected ones, and this background fluorescence was subtracted from the photobleaching decay curves. Each photobleaching decay curve was normalized to its respective signal measured at D = 0.

Measurement of Fluorescence Spectra during Irradiation.
To see whether there was any emergence of fluorescent photoproducts during PDT in vivo, the fluorescence emission of ALA-induced PpIX and BPD-MA were measured just prior to and immediately after light irradiation using the laser-induced fluorescence spectroscopy system modified to measure the entire porphyrin fluorescence band, not a narrow band between 675 and 720 nm as described above. The instrument has been described in detail elsewhere (30) . Briefly, a pulsed nitrogen laser (VSL-337ND; Laser Science, Inc., Cambridge, MA) was used to pump a dye laser (DLM220; Laser Science) tuned to 420 nm. The energy per pulse on the sample was ~1 µJ. This light was coupled into a 600-µm core diameter fiber, and the distal end was gently apposed with the mucosal side of the tumor. The fluorescence light from the tissue was collected by a second fiber held parallel to the first and was filtered through a 550-nm long pass filter (LL550; Corion Corp., Holliston, MA) before it reached the OMA. A total of 50 scans were integrated per measurement. Innate tissue fluorescence was recorded prior to administering the PS and subtracted from the collected signal for analysis.

In Vivo Fluorescence Imaging of BPD-MA and ALA-induced PpIX.
To understand the tumor response data, PS localization in vivo was established. In vivo fluorescence imaging of normal and tumoral tissue samples was performed on a microscope-based fluorescence system illustrated schematically in Fig. 2Citation . BPD-MA or PpIX fluorescence in the tumor vasculature and in tumor tissues surrounding the blood vessels was observed. Rats were injected i.v. with either 500 mg/kg of ALA or 1 mg/kg of BPD-MA, and the fluorescence observations were performed 4 and 1 h after injection, respectively. Fluorescence images of BPD-MA and PpIX in tissue were measured with illumination from the mercury lamp filtered to pass 420 ± 10 nm light. The fluorescence emission was measured between 590 and 750 nm. The imaging system is based on a Zeiss fluorescence microscope, a Gen-II image intensifier (Model M942; Litton Electron Devices, Tempe, AZ), and a monochrome CCD camera (TM-745E; Pulnix, Inc., Sunnyvale, CA). Images from the CCD camera were transferred to an IBM-PC clone computer via a frame grabber board (IP8AT; Matrox Electronic Systems, Ltd., Dorval, Quebec, Canada).



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  		Fig. 2. Schematic diagram of the microspectrofluorometer. The system obtains fluorescence images and spectra from aperture-defined regions within the image. The details are discussed in the text. DICH., dichroic mirror.

 
Statistics.
The Wilcoxon rank sum test was used to compare the number of clonogenic cells among different modalities of irradiation in the PDT experiments and to compare the photobleaching rate constant between high and low fluence rate methods.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Appendix 1
 REFERENCES
 
Fluence Rate-dependent Phototoxicity.
The fluence rate dependence of tumor survival after PDT with BPD-MA and ALA-PpIX was evaluated by an in vivo/in vitro assay and the data are presented as survival fraction (Fig. 3)Citation . In the case of ALA-PpIX, fluence-dependent phototoxicity was also examined. For a fluence rate of 100 mW/cm2, there was a shoulder, followed by an exponential decay of the clonogenic cells, suggesting the existence of a threshold fluence of 30 J/cm2 for obtaining tumoricidal effects as discussed by Henderson (31) . No such shoulder was observed for a fluence rate of 30 mW/cm2. For a fluence of 30 J/cm2, there were fewer clonogenic cells when the tumors were irradiated with 30 mW/cm2 as compared with 100 mW/cm2 (P < 0.05). A fluence rate effect was not observed at the higher light dose (100J/cm2). In the case of BPD-MA, a similar fluence rate effect was observed for a fluence of 30 J/cm2. The number of surviving clonogenic cells were much less when a low fluence rate was used (P < 0.01). Higher fluence experiments were not performed on BPD-MA-treated animals because of the limited fluence rate information gained for the ALA-treated animals at 100 J/cm2.



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  		Fig. 3. Surviving fraction of cells, comparing PDT of two PSs at two fluence rates. Survival fraction was calculated as number of clonogenic cells PDT treated to number of clonogenic cells not treated (no light, no drug). All controls were measured (light, no drug; drug, no light; and no drug, no light), and no statistically significant differences were noted. For clarity, only the data for ALA-PpIX are shown for fluence rates equal to zero. Two fluence rates were used with the two PSs: {circ}, ALA-PpIX 30 mW/cm2; •, 100 mW/cm2 ALA-PpIX; {square}, 30 mW/cm2 BPD-MA; {blacksquare}, 100 mW/cm2 BPD-MA. Points, means; bars, SE. *, P < 0.05, and #, P < 0.01, when compared with the same PS at 30 cm2.

 
Light Fractionation.
Three different fractionation modes were used: repetitive 15 s light on and 15 s light off, 30 s light on and 30 s light off, or 60 s light on and 60 s light off. A fluence of 30 J/cm2 was delivered in all three fractionation modes at the highest fluence rate of 100 mW/cm2. For PDT with BPD-MA, the in vivo/in vitro assay revealed that tumor destruction was enhanced using 30- and 60-s fractionated doses (P < 0.05 and P < 0.01, respectively; Fig. 4Citation ). With ALA-PpIX, a significant decrease in the number of clonogenic cells in the tumor was noted for the 60-s light fractionation (P < 0.01) when compared with continuous irradiation at the same fluence rate. The 60-s fractionation caused the highest enhancement of tumor destruction compared with other light fractionation modalities for both PSs.



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  		Fig. 4. Enhancement of tumor destruction by light fractionation for BPD-MA and PpIX. The bladder was irradiated with 30 J/cm2 with either continuous or fractionated 630-nm light at 100 mW/cm2. Tumors were removed 24 h later, and phototoxicity was evaluated by in vivo/in vitro assay to determine the number of clonogenic cells/gram of tissue. 15s/15s, 15 s light on, 15 s light off; 30s/30s, 30 s light on, 30 s light off; 60s/60s, 60 s light on, 60 s light off; cont, continuous irradiation. Points, means; bars, SE. *, P < 0.05, and #, P < 0.01, when compared with continuous irradiation.

 
Effects of Nicotinamide.
Nicotinamide is believed to circumvent microheterogeneities in tumor oxygenation, which should enhance PDT of tumors at high fluence rates. Nicotinamide was given prior to PDT with ALA-PpIX, and tumor clonogenicity was assessed 24 h later after fluences of 30 and 50 J/cm2 at a fluence rate of 100 mW/cm2. At both doses, the number of clonogenic cells observed was equivalent comparing when nicotinamide was given to the animal 1 h prior to irradiation (data not shown) to when nicotinamide was not given. The plating efficacy of the cells at each fluence in the in vivo/in vitro assay was not significantly different from that of the control group. The mean plating efficacy was 8.15 ± 0.81% (mean ± SE).

Photobleaching Experiments.
Typical photobleaching curves are shown in Figs. 5Citation and 6Citation . The photobleaching decay constant (k) and light dose-independent background fluorescence (IPh) were measured for two-sided irradiation of tumors at 630 nm using space irradiances of 30 and 100 mW/cm2 (Table 1)Citation . No statistically significant difference in the photobleaching decay constant was noted between high and low space irradiances within the same PS. When the two PSs are compared, the photobleaching decay constant of ALA-PpIX is higher than that of BPD-MA by almost one order of magnitude (P < 0.01). Even when corrected for the peak absorption (i.e., a factor of 3.3, the 690–630 nm absorption ratio for BPD-MA), the photobleaching rate of PpIX is still higher by a factor of 2–2.5.



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  		Fig. 5. A typical photobleaching curve of ALA-PpIX in tumor in situ. Tumor was irradiated from both sides with 630-nm laser light and a space irradiance of 95 mw/cm2. Fluorescence was detected with the OMA system. The fluorescence decay values were obtained by integrating the fluorescence signals from 675 to 720 nm. For comparison, typical spectra before (A) and after (B) 42 J/cm2 of irradiation are shown as insets in the photobleaching curve. The emergence of a new peak at 667 nm was observed in the fluorescence spectra after irradiation.

 


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  		Fig. 6. A typical photobleaching curve of BPD-MA in tumor in situ. The experimental set-up was the same as for ALA-PpIX. A typical spectrum before (A) and after (B) 90 J/cm2 of irradiation was inserted in the photobleaching curve showing fluorescence from a new photoproduct at 650 nm.

 

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  		Table 1 Photobleaching decay constants and background fluorescence for ALA-induced PpIXa and BPD-MAb

 
Measurement of Fluorescence Spectra during Irradiation.
Typical fluorescence spectra of tumors with ALA-PpIX or BPD-MA using 420 nm excitation were obtained in vivo before and after irradiation with 630-nm laser light and are presented as insets in Figs. 5Citation and 6Citation . A marked decrease of the 635- and 702-nm peaks was noted for ALA-PpIX during irradiation, with the appearance of a new fluorescence band at 667 nm. For BPD-MA, a decrease of the fluorescence band at 697 nm was noted, with the appearance of a new peak at 650 nm.

In Vivo Fluorescence Imaging of BPD-MA and ALA-induced PpIX.
A marked fluorescence signal attributable to PpIX in the tumor was observed 4 h after ALA injection in extravascular tissue (Fig. 7a)Citation . There was no detectable fluorescence from the blood vessels. A similar PS distribution was obtained with BPD-MA (Fig. 7b)Citation ; however, in this case, the PS appears to also localize in the vessel wall perhaps because of binding to endothelial cells. An image taken 5 min after injection of BPD-MA is also shown for comparison (Fig. 7c)Citation . In this case, the drug is predominantly limited to intravascular and perivascular spaces.



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  		Fig. 7. In vivo fluorescence images of bladder tumor obtained 4 h after given 500 mg/kg of ALA i.v. (a) or 1 h after 1 mg/kg i.v. administration of BPD-MA (b). In both cases, strong extravascular fluorescence was noted with no detectable fluorescence signal in the major blood vessels. For BPD-MA, enhanced fluorescence is noted in perivascular tissue, perhaps attributable to binding to vascular endothelium. An image taken at 5 min after injection of BPD-MA (c) shows relatively little fluorescence outside of the vessels. Bars, 100 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Appendix 1
 REFERENCES
 
Fluence rate effects in PDT attributed to oxygen consumption have now been demonstrated in a number of elegant studies in in vitro and in vivo s.c. animal models (11 , 16 , 32) and in human studies of cutaneous lesions (14) . Our motivations for the present study were to investigate these effects in an orthotopic tumor model and to do so in relation to the intratumoral localization of the PS in vivo. The site of implantation can be an extremely important determinant of the biological behavior of tumors. Properties as diverse as blood flow (1) and gene expression (33) are affected by whether or not the tumor is in its natural anatomical site. Because all of the existing in vivo reports on the topic of fluence rate dependence have used ectopic tumor models, we have studied these effects in an orthotopic tumor model in the current investigation. We were also interested in determining whether the extent of photobleaching of the PS could be used as a monitor for singlet oxygen generation (34 , 35) and the efficiency of tumor destruction. Fluence rate effects reported for hematoporphyrin derivative during PDT of multicell tumor spheroids in vitro (32) and a s.c. tumor model in vivo (11 , 16) have been reported for PpIX and BPD-MA. In these reports, PDT was noted to be less effective with higher fluence rates, and this effect has traditionally been attributed to oxygen depletion. On the basis of these theoretical models, tumor cells relatively far from the tumor vasculature or within large tumor spheroids are thought to be more hypoxic compared with cells adjacent to blood vessels or on the surface of the spheres because these regions are far more dependent on oxygen diffusion. If the fluence rate effect is entirely attributable to oxygen depletion, then the influence of oxygen depletion should be more pronounced during PDT with ALA-PpIX because the critical photochemistry, with this PS is presumed to be cellular where access to oxygen may be limited by diffusion. On the other hand, oxygen effects might not be as important during PDT with BPD-MA because the presumed primary target for this drug, the vascular endothelium, is well exposed to oxygen in the blood stream.

However, enhanced tumor destruction was seen at lower fluence rates for both PSs with, to our surprise, a larger fluence rate effect seen during PDT with BPD-MA. PpIX exhibited a 10-fold reduction in phototoxicity in contrast to a 40-fold reduction for BPD-MA. For PpIX, this result might reasonably be explained by an oxygen depletion hypothesis (16) ; however, for the vascular PS BPD-MA, the observation was contrary to our expectation. A possible explanation for this discrepancy may be that, although vascular occlusion plays a role in tumor destruction, direct tumor cell destruction is the critical event, and as long as blood flow was intact, destruction of tumor cells will be dependent on the diffusion of oxygen from the blood vessel. Consistent with this explanation, in vivo fluorescence imaging 1 h after i.v. administration of BPD-MA demonstrated that almost all of the BPD-MA fluorescence was extravascular, with some perivascular fluorescence in what may be the vascular endothelium. In addition, we observed that all of the tumor cells survived during PDT with BPD-MA when irradiated at the higher fluence rate, suggesting early vascular occlusion with little oxygen support for photodestruction of tumor cells. Fewer tumor cells survived at the lower fluence rate, consistent with incomplete vascular shutdown with oxygen available for photodestruction of tumor cells. These observations suggest that BPD-MA may not be exclusively a vascular PS and are consistent with a report by Korbelik and Frosi (36) , demonstrating that BPD-MA phototoxicity occurs more by direct tumor cell killings rather than secondary vascular effects.

The importance of local oxygen concentration on phototoxicity was further confirmed by light fractionation experiments where multiple dark intervals were given during the irradiation period to allow time for oxygen to diffuse into the target cells. For a dark interval of 60 s, enhanced tumor destruction was noted for both PpIX and BPD-MA. Once again, the effect was more pronounced with BPD-MA (Fig. 4Citation ; ~1000-fold for BPD-MA and ~100-fold for ALA-PpIX). In addition, the effect of light fractionation was less dramatic for 15- and 30-s intervals, supporting the fact that oxygen depletion had not recovered within this time frame. Because the total irradiation time in our experimental set-up was 5 or 10 min for continuous or fractionated irradiation, respectively, and little enhancement was observed comparing continuous and 30-s irradiation intervals, the enhanced tumor destruction was not caused by an increase in the operation time. Tromberg et al. (13) have suggested that the tissue oxygen recovery time is about twice the time of irradiation, which suggests that more efficient photodestruction may be obtained in our experiment with a longer dark interval. However, the optimal light interval where oxygen is depleted during irradiation is also important for optimizing the toxicity of fractionated light irradiation. From a theoretical study, it was shown that the optimum fractionation period depends only on the intercapillary spacing and not on the intensity of irradiation or PS concentration (37) . Intercapillary distances of 1 mm are needed to achieve optimal fractionation time of 60 s, suggesting that microscopic pockets of cells are present that are immune to PDT because they exist within hypoxic regions created by heterogeneities in tumor vasculature and PDT-induced oxygen depletion.

More recently, Gibson et al. (38) using PF as the PS reported that different amounts of tumor photodestruction occurred comparing tumor xenografts and isografts. They found that the xenograft of R3230 AC rat mammary carcinoma was fluence rate independent, whereas the isograft of the same carcinoma was fluence rate dependent, as reported previously (11) . Therefore, the effect of PDT requires optimizing fluence rate and light fractionation, which can vary depending on tumor structure (vascularization) and type of PS.

Nicotinamide is a well-known enhancer of ionizing irradiation therapy in experimental tumor models, and it is reported to have several effects on tumor blood flow circulation. In certain cases, it decreases the tumor interstitial pressure (39) and homogenizes tumor blood flow (40 , 41) . Kelleher and Vaupel (40) reported no increment of tumor oxygenation by direct measurement of oxygen tension in the tumor and attributed the nicotinamide-enhanced toxicity of ionizing radiation to more effective circulation of blood in the tumor. However, Lee et al. (39) demonstrated decreased tissue interstitial pressure and estimated the increase in tumor oxygenation. In either case, nicotinamide increased the oxygen availability in the tumor tissue. In our study, nicotinamide was administered 1 h prior to PDT, based on previous reports that the maximum effect of nicotinamide was obtained 1 h after administration (40 , 41) . Because oxygen has an important role for ionizing radiation therapy and appears to be important for PDT based on fluence rate and fractionation effects, nicotinamide was expected to enhance the PDT effect. However, our results demonstrated no enhancement to PDT and suggest that oxygen consumption by PDT was too rapid to be compensated by the nicotinamide effect. A similar test of increased tumor oxygenation was performed by Fingar et al. (42) , where they used an artificial oxygen carrier, Fluosol-DA (20%), to increase oxygen content in the blood but also failed to show enhanced tumor destruction. These data suggest that oxygen depletion occurs quite rapidly, and that although nicotinamide has been shown to influence either tumor blood flow or blood oxygen concentration, effective PDT is also limited by the rate of oxygen delivery to tissue (37) , which is not influenced by nicotinamide.

An alternative to the oxygen depletion mechanism is the direct photodestruction of the PS at high fluence rates, leading to a lower production of reactive singlet oxygen (43) . However, the rate of PS fluorescence decay was followed during irradiation for both PSs and was found to be independent of the fluence rate. The values of space irradiance and irradiation time were set to correspond to the effective fluence used for the PDT experiments. The change of fluorescence emission spectra during irradiation supports the findings that long wavelength-emitting hydroxyaldehyde porphyrin adducts were formed from PpIX and BPD-MA (44 , 45) . In the case of PpIX, the emission wavelength of the photoproduct lies in the same fluorescence region used to monitor PpIX photobleaching during PDT. Therefore to correct for this, the PpIX photobleaching curve should be fitted by the following equation:

where Ip and IPh are preexponential fluorescence factors corresponding to the PpIX and its photoproduct fluorescence, respectively, D is the light dose, and k1 and k2 are the photobleaching decay constants of PpIX and its photoproduct, respectively. In vivo and ex vivo evaluations of these photobleaching decay constants have shown that k2 << k1 when red light is used. Because the photobleaching of the photoproducts was not measurable in the context of our "in vivo" experiments, we have assumed that e-k2D = 1, and the above equation may be rewritten as:

which is identical to the original equation used to fit the data (Eq. A), where Io = IP - IPh. The steady-state fluorescence signal seen in the PpIX photobleaching curve (Fig. 6)Citation at high doses is mainly attributable to the slowly photobleaching fluorescent PpIX photoproducts, IPh. It is possible that the circulating PpIX could reequilibrate with the tissue and contribute to the fluorescence signal seen at high doses. However, no abrupt change of photobleaching curve was noted after clamping the blood vessels, which suggests that contribution from circulating PpIX was negligible.

It has been suggested that PS photobleaching may be a useful monitor for 1O2 production and by extension of PDT dosimetry (32 , 46 , 47) . Our data suggest that the two processes may, at least in part, be unrelated. Although photodegradation of PpIX requires oxygen and is reported to produce different porphynoid photoproducts (48) , a study of BPD-MA photobleaching in methanol or PBS with different oxygen concentrations demonstrated that BPD-MA photodegradation was virtually oxygen independent, with a breakdown of the porphyrin macrocycle (45) . However, the process of photobleaching is complicated, because oxygen-dependent porphyrin-like photoproducts were seen when BPD-MA was irradiated in the presence of FCS or human bladder carcinoma cells (44) . In our experiments, the rate of photobleaching in vivo appears to be oxygen independent because the photobleaching decay constants were comparable for both high and low fluence rates. Consistent with our data, the rate of photobleaching of PpIX in normal skin of BALB/c nude mice was shown to be minimally dependent on fluence rate (49) , whereas others have shown fluence rate effects in mouse skin and UV-induced skin tumors (46 , 47) . This is not surprising because it is well established that some molecules that photodegrade by oxygen-dependent processes in the presence of oxygen can easily switch to oxygen-independent photodegradation mechanisms in the absence of oxygen (50 , 51) . In a situation such as this, the photoproducts of oxygen-dependent and oxygen-independent mechanisms are different. Because our experiments in vivo measured the decrease in fluorescence of the PSs and not the photoproduct in the tumor, it is difficult to sort the mechanistic details of the process during photodegradation. It is possible that there exist two different photobleaching processes for both PpIX and BPD-MA in vivo: one is oxygen dependent, and the other is oxygen independent, and that the measured decay constant is a composite of the two processes.

In summary, this investigation demonstrates, in an orthotopic tumor model, the fluence rate effects on PDT response using ALA-PpIX and BPD-MA as PSs. Despite similarities in photophysical characteristics of PpIX and BPD-MA in a homogeneous solution, the two sensitizers exhibited markedly different fluence rate dependencies. BPD-MA photosensitization appeared four times more sensitive to fluence rate changes than PpIX PDT. The study also demonstrated that photobleaching rates in vivo and 1O2 generation (the presumed critical species for tumor destruction) are not directly related and suggest that the use of photobleaching rates for PDT dosimetry is more complex than may have been anticipated (32 , 34 , 35) .


   Appendix 1
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Appendix 1
REFERENCES
 
The space irradiance distribution {Phi}(z), in the context of the diffusion approximation theory, is given by the following expression if a semi-infinite medium is illuminated with an homogeneous, infinite, and collimated light beam (52 , 53) :

(A1)
where {Phi}0, is the incident fluence rate (mW/cm2), {Phi}(z), is the space irradiance (mW/cm2) at the depth of z (mm), K is a pre-exponential constant, and µeff, is the effective attenuation coefficient (mm-1).

If K is 3, µeff is 0.5 mm-1 (54) , and Z = d (tumor thickness = 3 mm), and if the irradiation is performed only from the inside of the bladder with 100 mW/cm2, the space irradiance just underneath the inner surface is 300 mW/cm2 and the space irradiance at the outer surface of the tumor {Phi} (3 mm) is equal to about 67 mW/cm2, i.e., ~4.5 times less than on the inner surface.

If the irradiation is performed from both sides of the tumor with 26 mW/cm2 each, the space irradiance at each surface of the tumor is equal to 95 mW/cm2, according to the diffusion approximation theory. The space irradiance at the center of the tumor (d = 1.5 mm) is equal to 73 mW/cm2. Therefore, the ratio of space irradiance between the surface and the center of the tumor is only 1.3. In this rough evaluation, we have neglected the variation of K because of the different refractive index matching conditions on both sides of the bladder wall (54 , 55) .


   ACKNOWLEDGMENTS
 
We thank Drs. S. P. Dretler and H. Tazaki for support and encouragement; Dr. R. Webb for helpful suggestions on in vivo imaging; QLT Phototherapeutics, Inc. and DUSA Pharmaceutical, Inc. for providing BPD-MA and the ALA, respectively; and Coherent, Inc. for loan of the argon ion laser.


   FOOTNOTES
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Department of Defense Medical Free Electron Laser Program, Contract N00014-91-C-0084, and NIH R01-AR40352. S. I. is a recipient of Research Fellowship DE-FG02-91ER61228 funded by the Department of Energy. G. W. a recipient of a fellowship from the Swiss Research Council. Back

2 To whom requests for reprints should be addressed, at Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Boston, MA 02114. Back

3 The abbreviations used: PDT, photodynamic therapy; PS, photosensitizer; ALA, 5-aminolevulinic acid; ALA-PpIX, ALA-induced protoporphyrin IX; BPD-MA, benzoporphyrin derivative monoacid ring A; PF, Photofrin; OMA, optical multichannel analyzer. Back

Received 5/24/99. Accepted 10/15/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Appendix 1
 REFERENCES
 

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