This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, Q. Articles by Nesland, J. M. Search for Related Content PubMed PubMed Citation Articles by Peng, Q. Articles by Nesland, J. M.

Antitumor Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy Can Be Enhanced by the Use of a Low Dose of Photofrin in Human Tumor Xenografts

Departments of Pathology [Q. P., F. A., J. M. N.], Surgical Oncology [T. W., K-E. G.], and Biophysics [J. M.], The Norwegian Radium Hospital, Institute for Cancer Research, University of Oslo, Montebello, 0310 Oslo, and PhotoCure ASA, 0377 Oslo, [A. G.] Norway

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

 
Practically all of the exogenous photosensitizers used for clinical photodynamic therapy (PDT) target mainly vasculature. Although effective in tumor destruction, they also, unavoidably, induce phototoxicity of normal tissues. Porphyrins synthesized endogenously from 5-aminolevulinic acid (ALA) accumulate within cells. Tumor eradication would be more efficient if both cellular components and vascular stroma of a tumor could be targeted. Thus, PDT with a mixture of ALA and Photofrin (Pf, a vessel-targeted sensitizer) may simultaneously destroy the two elements. Using chemical extraction assays, pharmacokinetics of ALA and ALA-induced porphyrins were studied in the plasma and tumors of nude mice bearing human WiDr and KM20L2 colonic carcinomas after an i.p. injection of 250 mg/kg body weight of ALA. Subsequently, PDT efficacy of the two tumor models with ALA, Pf, or with the two drugs in combination was evaluated. The phototoxic effects on tumor cells in vitro with the combined drugs was also determined. Moreover, histological and ultrastructural alterations of the treated tumors were investigated, and tumor cell clonogenicity was assessed as a function of time after in vivo PDT using an in vitro colony formation assay. Finally, the photosensitivity of normal skin tissue treated according to various protocols was compared. The amounts of ALA peaked at 0.5 h after administration in both plasma and WiDr tumor. The rates of ALA clearance seemed to follow a one-compartment model with half-lives of 18 and 58 min in the plasma and tumor, respectively. About 100 and 60 times higher concentrations of ALA were needed to induce a given concentration of porphyrins in the plasma and tumor, respectively, although the plasma porphyrins may not only be released from blood cells but also from other organs. Similar kinetics of distribution patterns of ALA- and ALA methylester-induced porphyrins were found in the plasma and tumors, and the elimination rates were consistent with a two-compartment model. ALA induced much more porphyrins than ALA methylester in both plasma and tumors. Tumors PDT-treated with ALA plus Pf at a low dose (1 mg/kg) grew significantly more slowly than those treated with either of the drugs in both WiDr and KM20L2 models. However, the enhanced antitumor effect was not found in the tumor cells under in vitro conditions. Morphological studies demonstrated that PDT with the combined regimen resulted in necrosis of neoplastic cells and severe disruption of tumor microvasculature. This was supported by the findings obtained from the studies of in vivo PDT and in vitro clonogenic assay that showed a progressive reduction in tumor cell viability with times following PDT. Such a combined PDT protocol did not induce any phototoxicity in normal skin tissue. These data indicate that targeting both neoplastic cells and stroma with ALA and Pf (a low dose) can potentiate antitumor PDT effect with no risk of prolonged skin photosensitivity.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

 
PDT² has developed to become an important new cancer treatment (1) . The treatment typically involves systemic administration of a tumor-localizing photosensitizer and its subsequent activation by light of an appropriate wavelength to create a photochemical reaction causing photodamage to the tumor (1) . Pf was studied worldwide in clinical PDT during the past two decades and has been approved by governments of many countries for several medical indications. However, the major side effects associated with Pf-based PDT are the risk of skin photosensitization (2) and damage to surrounding normal tissue potentially leading to stenosis of the esophagus (3) and shrinkage of the bladder (4) . To a considerable extent these side effects restrict the use of clinical Pf PDT. PDT with ALA-derived endogenous porphyrins has already shown promising clinical results in the treatment of several superficial disorders of the skin and internal hollow organs (5) . The principle of this new approach is that ALA is a precursor of several porphyrin intermediates, particularly PpIX, a potent photosensitizer, in the heme biosynthetic pathway. The synthesis of heme is largely controlled by a heme-mediated feedback inhibition of the enzyme ALA synthase (6) . Such an ALA/heme feedback control can, however, be bypassed by administering excess amounts of exogenous ALA. As a result, endogenous porphyrins accumulate in cells because of the limited capacity of enzymes (e.g., ferrochelatase) to convert porphyrins into heme (6) . When light activated, the accumulated porphyrins produce a photodynamic reaction (6) . One of the main advantages of using ALA PDT is that ALA-derived porphyrins are cleared from the body within 24–48 h after systemic ALA administration. This would reduce or avoid the risk of prolonged skin phototoxicity.

Our earlier studies have shown that porphyrins synthesized endogenously from ALA localize within neoplastic cells of CaD2 tumors. Subsequent light activation leads to inactivation of the tumor cells (7) . Pf is known to distribute mainly in the vascular stroma of the tumor (8 , 9) , thereby destroying the vascular system of the tumor during light exposure (10 , 11) . Our data suggest that efficient eradication of a tumor by PDT requires destruction of both cellular components and vascular stroma of the tumor (12 , 13) . Therefore, we have hypothesized that the antitumor effect of ALA PDT may be enhanced by the use of vessel-localizing sensitizers like Pf, particularly at a low dose of the sensitizer that does not induce any risk of prolonged photodamage of normal tissues. In the present study, we have combined ALA with Pf in the photodynamic treatment of human colon carcinoma WiDr and KM20L2 xenografts in nude mice and demonstrate that the combined PDT regimens did not only enhance significantly PDT efficiency of the tumors but also avoided skin phototoxicity of the mice.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

 
Chemicals.
ALA and its methylester were supplied by PhotoCure ASA (Oslo, Norway). The drugs were dissolved in Dulbecco’s PBS on the same day as the experiments started, and the pH was adjusted to 7.0 by addition of 5 M of NaOH. Pf (porfimer sodium) was a kind gift from QLT PhotoTherapeutics Inc. (Vancouver, British Columbia, Canada). The stock solution of Pf was made up in isotonic solution containing 5% dextrose and stored at -70°C. Because the exact molecular weight of Pf is not known, the molarity units of Pf, ALA, and ALA methylester were not applied in the present study. All of the other chemicals used were of the highest purity commercially available.

Animals and Tumor Line.
Approval for protocols of this study was obtained from The Norwegian National Animal Research Authority, and all experiments of animals were conducted according to The National Ethical Committee’s Guidelines on Animal Welfare. Female BALB/c athymic nude mice were bred at the nude rodent facility of The Norwegian Radium Hospital, housed five/cage, and kept under specific pathogen-free conditions. The mice were 6 weeks old and weighed 20–25 g when the experiments started.

Two established human colon adenocarcinoma lines, WiDr (14) and KM20L2 (15) , were used in the present study. WiDr cells (5 x 10⁶) suspended in 0.04 ml of PBS were s.c. implanted in the flank of the mice. Nonnecrotic tumor material obtained by sterile dissection of large flank tumors was gently minced with a Kai sterile surgical blade (Kai Surgical Instrument Co., Ltd, Tokyo, Japan) to make a tumor-tissue suspension, 0.02 ml of which was then injected into the dorsal side of the right hind foot of each syngeneic mouse. At this site the tumor was easily accessible to treatment and to assessment of response. The tumor volume was calculated using the following formula:

where D₁, D₂ and D₃ are three orthogonal diameters of the tumors that were measured every 2nd day using a caliper. No spontaneous necrosis was observed in the tumors that grew to reach 130–220 mm³ on the day of treatment. The same procedure was also used for xenografts of the KM20L2 tumor.

Determination of ALA in Plasma and Tumor.
After WiDr-bearing mice had been anesthetized by i.p. injection of 50 µl/25 g of a mixture (1:1 by volume) of Dormicum (Roche, Basel, Switzerland) and Hypnorm (Janssen, Beerse, Belgium), the blood was drawn directly from the left ventricle of the heart, and the animals were immediately killed by cervical dislocation. The total volume of the blood obtained from each mouse was 0.8 ml and collected in EDTA-treated tubes. The plasma was separated from blood cells by centrifugation at 5700 x g for 10 min. In the case of the tumor, the tissue was brought into suspension in saline (0.25 g/ml) by a Potter Elvehjem homogenizer, and the tissue suspension was sonicated twice (1 min for each) before being centrifuged as described previously. The resulting supernatant was collected. ALA concentrations in the plasma and supernatants were measured by a method of Tomokuni et al. (16) with slight modifications. This assay is based on derivatization of ALA with acetylacetone followed by HPLC analysis on the fluorescent derivative. The HPLC system was composed of a pump (Spectra Physics 8800, Hempstead, United Kingdom), a reverse-phase column [Hypersil H50DS (4.6 x 250 mm); Supelco, Gland, Switzerland], a fluorescence detector (Spectra Physics FL2000) with excitation/emission wavelengths of 370/460 nm, and an integrator (Spectra Physics Data-jet) coupled to a computer. The mobile phase was methanol/water/acetic acid (430/570/10 by volume), flow rate was 0.7 ml/min, and column temperature was room temperature. The concentrations of ALA in the samples were calculated from standard curves made by addition of known amounts of ALA to respective extracts from untreated mice and expressed as µg ALA/ml plasma or g wet tumor tissue.

Determination of ALA- or ALA Methylester-derived Porphyrins in Plasma and Tumor.
Samples of plasma and tumor tissue taken from WiDr- and KM20L2-bearing mice were prepared by the same ways as described for ALA measurements except that the solvent was a mixture of acetonitrile and ethanol (1:7 by volume). The plasma or tumor tissue samples (0.1 ml) were mixed with 0.9 ml of the mixture solvent and sonicated for 1 min before centrifugation (5700 x g for 10 min). Porphyrins in the supernatants were spectrofluorometrically measured using a Perkin-Elmer LS50B spectrofluorometer. The samples were excited at 400 nm, and emission was scanned from 550 to 750 nm with a slit width of 15 nm on both sides. A cutoff filter was used on the emission side to remove scattered light of wavelength <530 nm from the light reaching the detection system. With this procedure it was possible to extract and detect porphyrins (>95%) induced by ALA or its methylester in the plasma and tumor tissue. The concentrations of porphyrins in the samples were calculated from standard curves made by addition of known amounts of PpIX to respective extracts from untreated mice and expressed as ng porphyrins/ml plasma or g wet tumor tissue.

PDT Protocol.
Mice with WiDr tumors of the appropriate size were randomly divided into groups (a minimum of 5 animals/group) as follows: group 1, neither drug nor light; group 2, light only on the tumors; group 3, mice given an i.p. injection of ALA at a dose of 250 mg/kg body weight followed by light irradiation of the tumor; group 4, mice given an i.v. administration of Pf at a dose of 1 mg/kg before light exposure of the tumor; group 5, irradiated tumors after mice had been given both i.p. ALA (250 mg/kg) and i.v. Pf (1 mg/kg). Also, tumors of mice were treated with PDT using ALA plus an increased dose of Pf to see if the increased doses of Pf could additionally enhance ALA-PDT efficiency. Group 6 was PDT of tumor with Pf (2.5 mg/kg) alone; group 7, PDT of tumors with ALA (250 mg/kg) and Pf (2.5 mg/kg); group 8, PDT of tumors with Pf (5 mg/kg) alone; and group 9, PDT of tumors with ALA (250 mg/kg) plus Pf (5 mg/kg). ALA methylester was also studied in combination with Pf in PDT of the tumors. Group 10 was PDT with ALA methylester (250 mg/kg) alone, and group 11 was PDT using ALA methylester (250 mg/kg) and Pf (1 mg/kg). The tumor model KM20L2 was applied in experiments parallel to those as described previously. The time intervals between drug administration and light irradiation were chosen to be 3 h for ALA and Pf and 1 h for ALA methylester. This was on the basis of the peak levels of ALA- and ALA methylester-induced porphyrins in the tumor tissues and on a previous report that showed a peak effect on the vascular system at 3 h after hematoporphyrin ester-mediated photosensitization (17) . Responses of the treated tumors were evaluated by tumor regression/regrowth time and Kaplan-Meier method. The size of the tumors was measured every 2nd day, and when the treated tumors reached a volume 5 times that of the volume on the day before light irradiation, the mice were sacrificed. The data obtained from the measurements on tumor volumes in each group were pooled to represent mean tumor regrowth curves. The fraction of tumors that failed to reach 4 times the initial volume was also estimated by the Kaplan-Meier method.

The laser light irradiation of the tumors was performed by fixing unanesthetized mice in specially designed plastic holders so that the mice were immobile during illumination. The tumor area was exposed to red light (632 nm) from a 0.3 W diode laser (CeramOptec GmbH, Bonn, Germany) at a fluence rate of 150 mW/cm² for 15-min exposure in all cases. The fluence rate of the light on the tumor area was regularly checked by a calibrated integrating sphere with a photodiode coupled to a digital multimeter (Keithley Instruments, Munich, Germany) before and immediately after light illumination.

Influence of Pf on Production of ALA-induced Porphyrins in WiDr Cells in Vitro.
Monolayer cultures of the WiDr cells were grown in RPMI 1640 supplemented with 10% FCS, penicillin (100 IU/ml), and streptomycin (0.1 mg/ml) at 37°C in an incubator flushed with 5% carbon dioxide in air. The cells were subcultured twice/week. WiDr cells (5 x 10⁵) were seeded into six-well dishes (Nunclon) and left for 2 days for proper attachment to the substratum. After washing with RPMI 1640, the cells had been incubated with the serum-free medium containing ALA, Pf, or ALA plus Pf at various concentrations for 4 h before they were rinsed twice with PBS and brought into a solution containing 1 M perchloric acid in 50% methanol by scraping with a Costar cell scraper. After 5 min of incubation, the cell debris was removed by centrifugation. Practically all of the ALA-induced porphyrins and Pf could be extracted from the cells with this procedure. Furthermore, aggregated porphyrins could be dissolved into fluorescent monomers in the extracting solution so that there was a linear relationship between the amount of porphyrin monomers and fluorescence intensity. The intensity of fluorescence of ALA-induced porphyrins, Pf, and porphyrins plus Pf in the samples, was recorded as described previously. The excitation wavelengths were 411, 407, and 411 for the induced porphyrins, Pf, and porphyrins plus Pf, respectively, and the emission wavelength for all of the cases was scanned from 550 to 700 nm. In this way the influence of Pf on the production of ALA-induced porphyrins in the WiDr cells can be determined.

PDT of WiDr Cells with the Combined Regimens in Vitro.
WiDr cells (400) were inoculated in 25 cm² of plastic tissue culture flasks. After incubation of the cells with ALA (168 µg/ml), Pf (0.02 µg/ml or 0.1 µg/ml), or ALA (168 µg/ml) plus Pf (0.02 µg/ml or 0.1 µg/ml) for 4 h as described previously, the cells were exposed to light (for 30 s) from a bank of four fluorescent tubes (Model 3026; Applied Photophysics, London, United Kingdom) emitting light mainly 405 nm. The fluence rate of the light reaching the cells was 36 W/m². Immediately after irradiation, the medium was replaced with drug-free RPMI 1640 containing 10% FCS, and the cells were then left for 10 days to allow for the formation of colonies. The cells were finally fixed in ethanol, stained with methylene blue, and the colonies counted.

Histological and Ultrastructural Studies.
Before and 1, 3, 24, and 48 h after PDT with ALA, Pf, or ALA plus Pf, the WiDr tumor tissues (three tumors/time point) were excised and fixed in 10% buffered formalin solution. The specimens were then dehydrated and embedded in paraffin. Sections were cut and stained with H&E for light microscopy. After excision, tumor specimens were also immediately fixed in a 0.1 M cacodylate-buffered mixture of 1% glutaraldehyde and 4% formaldehyde (18) , followed by postfixation for 1 h in cacodylate-buffered 1% osmium tetroxide. The samples were then dehydrated in progressive ethanol-water in 10-min steps (70, 90, 96, and 100%) and in propylene oxide (3 times each for 10 min) before being embedded in an Epon/Araldite mixture. Semisections were cut with glass knives, mounted on glass slides, and stained with toluidine blue for light microscopic orientation. Ultrathin sections were cut with diamond knives, floated on 200 mesh copper grids, stained with uranyl acetate as well as lead citrate, and finally examined with a Philips CM-10 transmission electron microscope.

In Vivo PDT of WiDr Tumor and in Vitro Clonogenic Assay.
Zero, 1, 3, and 24 h after PDT with ALA, Pf, or ALA plus Pf, mice were killed, and the WiDr tumors (five tumors/time point) were excised and weighed. The tumors were then minced with a scalpel, and a single-cell suspension was prepared in 5 ml of an enzyme mixture (Sigma Chemical Co.) of collagenase (0.2%), protease (0.05%), and DNase (0.02%) for 1 h at 37°C with stirring. Single cells were washed 3 times with RPMI 1640 and finally resuspended in 10 ml of the medium containing 10% FCS. Fifty and 100 µl of cell suspensions after appropriate dilutions (10–100 times depending on the number of cells) were seeded in six-well plates (six wells/tumor with 2 different volumes of cell suspension) and left for 2 weeks with medium changed once/week. All manipulations of the experiments were carried out in subdued light. Cell colonies were fixed with 96% ethanol for 10 min and stained with methylene blue for 15 min. The total number of macroscopic cell colonies was counted and calculated as per gram of wet tumor tissue. Data represent the survival fraction relative to the control tumors (light only) that are expressed as 100% survival.

PDT of Normal Skin of Mice.
To investigate the risk of prolonged skin phototoxicity with the combined PDT regimen, normal foot response of nude mice bearing no tumor (three mice/group) to PDT was examined. The feet of the mice were treated in the same manner (including light dose) as that used for tumors. Because the antitumor efficiency of PDT with 250 mg/kg ALA plus 1 mg/kg Pf is comparable with that with 5 mg/kg Pf alone, the two PDT protocols and 4 time intervals between drug administration and light irradiation (1, 2, 4, and 10 days) were used. PDT-mediated response of the right hind foot of the mice was compared with that of the unexposed left hind foot of the same mice. The average thickness (PDT-induced edema) of the treated foot (Tt) and of the untreated foot (Tu) was measured every 2nd day for 20 days, and the response was calculated as (Tt/Tu)-1.

Statistical Analysis.
Data on tumor responses to various PDT protocols were analyzed using Log-rank test and others were performed using Student’s t test.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

 
Uptake and Clearance of ALA in Plasma and Tumor.
The kinetics of uptake and clearance of ALA in the plasma and tumors of WiDr-bearing mice is shown in Fig. 1 . ALA methylester is not included, because the method applied for ALA measurement has been found to be unsuitable for the determination of ALA methylester. ALA had a similar kinetic pattern of uptake and clearance in the plasma and tumor after i.p. administration. The amounts of ALA peaked at 0.5 h after administration in both plasma and tumor, but the level of ALA in the plasma was only one-third of that in the tumor. The rates of clearance of ALA seem to follow a one-compartment model with half-lives of 18 and 58 min in the plasma and tumor, respectively.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Concentrations of ALA in the plasma of nude mice and WiDr tumors at different times after an i.p. injection of 250 mg/kg ALA. Bars, ± SE based on five individual animals.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Concentrations of porphyrins in the plasma of WiDr-bearing nude mice and WiDr tumors at different times after an i.p. injection of 250 mg/kg ALA or its methylester. Bars, ± SE based on five individual animals.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Concentrations of porphyrins in the plasma of KM20L2-bearing nude mice and KM20L2 tumors at different times after an i.p. injection of 250 mg/kg ALA or its methylester. Bars, ± SE based on five individual animals.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Kaplan-Meier (a) and regrowth (b) curves of WiDr tumors after PDT using various regimens as indicated. The tumors were exposed to laser light 3 h after drug injection (632 nm, 150 mW/cm2, and 135 J/cm2). Bars, ± SE based on a minimum of five animals (n = 5–11).

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Kaplan-Meier curves of WiDr tumors after PDT using various regimens as indicated. Others are the same as described in Fig. 4 .

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Regrowth curves of WiDr tumors after PDT using various regimens as indicated. Others are the same as described in Fig. 4 .

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Kaplan-Meier curves of WiDr tumors after PDT using various regimens as indicated. Others are the same as described in Fig. 4 .

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Kaplan-Meier curves of WiDr and KM20L2 tumors after PDT using various regimens as indicated. Others are the same as described in Fig. 4 except that tumors were exposed to light 1 h after ALA methylester administration.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. Fluorescence spectra of intracellular porphyrins in WiDr cells incubated in vitro with ALA, Pf, or ALA + Pf as indicated. Because the fluorescence intensity of Pf at a concentration used for phototoxicity was so low, the inset with a higher dose of the drug illustrates no effect of Pf on production of ALA-induced porphyrins more clearly.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 10. Phototoxicity of WiDr cells in vitro with ALA, Pf, or ALA + Pf as indicated. For details, see "Materials and Methods." Bars,± SD based on three experiments.

View larger version (147K): [in this window] [in a new window] [Download PPT slide]   Fig. 11. Transmission microphotographs of WiDr tumors taken after treatment with light alone as a control (a); 3 h after ALA-PDT (b); 3 h after Pf-PDT (c); 1 (d), 3 (e), and 48 (f) h after PDT with ALA + Pf (1 mg/kg). Bar, 300 µm for all photographs. a, unchanged tumor tissue; b, degeneration of neoplastic cells together with some edema; c, evident edema and congestion; d and e, severe edema, congestion, and some hemorrhage with some necrosis of tumor cells; and f, severe congestion and hemorrhage with extensive necrosis of tumor cells.

View larger version (112K): [in this window] [in a new window] [Download PPT slide]   Fig. 12. Electron microphotographs of WiDr tumors taken after treatment with light alone as a control (a); 1 (b) and 48 (c) h after PDT with ALA and Pf (1 mg/kg). x8,500 for all photographs. E, endothelium; P, platelet; R, RBCs. a, an unchanged vessel; b, congestion, aggregation of platelets against the wall of a vessel, and edema of subendothelial zone but endothelium undamaged; c, congestion, necrosis of endothelium, and considerable edema and disruption of subendothelial zone.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 13. WiDr tumor cell survival in vitro as a function of time for which the tumors remained in situ following in vivo PDT with various regimens as indicated. PDT treatments were the same as described in Fig. 4 . Bars, ± SE based on at least five individual animals.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 14. Normal mouse skin was treated with PDT (as indicated) in the same manner as that for PDT of tumors. The photo-induced skin edema was evaluated as described in "Materials and Methods." SE was <10% of given values for all points.

This is the first report of pharmacokinetics of ALA in both plasma and tumor tissue of nude mice. A similar kinetic pattern of ALA in the plasma and tumor was seen with a peak at 0.5 h after systemic administration (Fig. 1) . Interestingly, the maximal amount of ALA in the plasma was only about one-third of that in the WiDr tumors. This is at least partly attributable to the fact that the large fraction of injected ALA that bound to cellular components of the blood was not included. It is also likely that ALA was degraded faster in the plasma than in the tumor. The concentration ratios of ALA to induced porphyrins at peak times are 100 in the plasma and 60 in the tumor (Figs. 1 and 2 ) suggesting that 100 and 60 times higher concentrations of ALA are needed to induce a given concentration of porphyrins in the plasma and tumor, respectively. It should, however, be pointed out that ALA-induced porphyrins in the plasma are not only released from blood cells but also from other organs. Similar patterns of accumulation and clearance of porphyrins induced by ALA and its methylester were found in the plasma of the WiDr- and KM20L2-bearing mice as well as in the two models of human tumor xenografts (Figs. 2 and 3 ). The short half-lives of ALA and ALA-induced porphyrins in the body indicate the reduced risk of prolonged skin phototoxicity. The amounts of ALA-induced porphyrins were higher in the WiDr tumors than in the KM20L2 tumors, suggesting that the capacity of ALA-induced porphyrins is tumor model-dependent. Among these induced porphyrins are PpIX (>90%) and several other porphyrin species such as heptaporphyrin, coproporphyrin, and uroporphyrin (19) .

Both WiDr and KM20L2 tumors showed a significantly stronger response to PDT with ALA and Pf (at a low dose of 1 mg/kg) than the treatments with either of the drugs (Fig. 4) . The antitumor effect of the combined PDT regimen was similar to that of PDT with Pf alone at a high dose of 5 mg/kg (Fig. 5) . Moreover, the efficiency of ALA PDT could be additionally enhanced by the use of Pf at a higher concentration of 2.5 mg/kg (Figs. 6 and 7 ). However, it appeared that increasing the dose of Pf to 5 mg/kg did not continue to significantly potentiate the effect of ALA PDT, suggesting that the efficacy of ALA PDT can be enhanced by the use of Pf within a limit of certain concentrations. The antitumor efficiency of ALA methylester-based PDT was slightly increased by Pf (1 mg/kg) in both tumor models, but the effect was significantly lower than that of PDT using ALA and Pf (Fig. 8) . This may be attributable to lower amounts of porphyrins induced by ALA methylester in the tumor tissues (Figs. 2 and 3 ). However, the efficacy of ALA methylester-PDT could be significantly potentiated by disulfonated aluminum phthalocyanine (a vessel-targeted sensitizer) in the WiDr tumor model (data not shown).

Studies in vitro showed that Pf did not interfere in production of ALA-induced porphyrins in the WiDr cells (Fig. 9) . Consistently, phototoxicity of the cells with ALA was not increased by use of Pf at a low concentration. This demonstrates that no enhanced effect was achieved at a cellular level when ALA-PDT was applied in combination with Pf. Our data are in agreement with the results of Messmann et al. (20) that showed no potentiation of phototoxicity when HT-29 cells were simultaneously incubated in vitro with ALA and Photosan-3. Histological studies indicated that the combined PDT regimen led to a much earlier and stronger effect on tumor microvasculature than the treatments with ALA or Pf alone (Fig. 11) . Ultrastructural investigations showed that PDT with ALA alone gave rise to a moderate damage to the endothelial cells, whereas a treatment with a mixture of ALA and Pf destroyed both the endothelium and subendothelial layer (a sensitive target; Ref. 21 ). This may result in a lethal collapse of the whole microvascular system of the tumor that, in turn, additionally affects the viability of tumor cells that have already been attacked by the action of ALA-mediated PDT. This explanation is strongly supported by the results obtained from the studies of in vivo PDT and in vitro clonogenic assay. A progressive decrease in clonogenic cells was found with times following in vivo PDT using ALA and Pf (Fig. 13) . This reduction is known to be a consequence of the destruction of tumor microvasculature (22) . The present results are consistent with a study of Nelson et al. (23) . They found that PDT with two vessel-targeted dyes (Pf II and TPPS₄) achieved a 40% higher cure rate of EMT-6 mammary tumors than PDT with either of the drugs. The increased tumor kill was attributable to an enhanced damage to tumor microvasculature. Furthermore, two recent reports have shown that PDT with antiangiogenic agents could significantly improve the antitumor efficiency (both of which used a vessel-localizing photosensitizer for PDT; Refs. 24 , 25 ). These studies indicate that an increased PDT effect on tumor vasculature by either a vessel-targeted sensitizer or an antiangiogenic agent can potentiate tumor eradication.

Pf, like most of photosensitizers used for clinical PDT, can cause prolonged skin phototoxicity because it localizes mainly in the vasculature of the skin with a long retention. A low dose of Pf associated with only a minor skin phototoxicity will significantly improve the clinical outcome and acceptance. The present investigation shows that the effect of ALA PDT can be enhanced by the use of a low dose of Pf that does not induce any significant skin photosensitivity. Thus, this combined PDT regimen may have an important clinical implication. At present, our clinical trials with the combined protocol for the treatment of early esophageal cancer are being performed. Preliminary results indicate that PDT with the combined regimen produces a stronger response of the treated esophageal cancer than PDT with ALA alone. Although it is too early to draw any conclusion on the antitumor efficacy, none of treated patients have suffered any prolonged skin phototoxicity nor any PDT-induced perforation and stricture of the esophagus.

	ACKNOWLEDGMENTS

 
We thank Dr. Kristian Berg for valuable discussion on HPLC analysis; Vladimir Iani, Helle Anholt, Tudor Stanescu, Øystein B. Gadmar, Ellen Hellesylt, and Inger-Liv Nordli for expert technical assistance; PhotoCure ASA (Oslo, Norway) for kindly providing ALA and ALA methylester; and QLT PhotoTherapeutics, Inc. (Vancouver, British Columbia, Canada) for the generous gift of Pf.

	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 To whom requests for reprints should be addressed, at Department of Pathology, The Norwegian Radium Hospital, University of Oslo, Montebello, 0310 Oslo, Norway. Phone: 47 22935553; Fax: 47 22934832; E-mail: qian.peng{at}labmed.uio.no

² The abbreviations used are: PDT, photodynamic therapy; Pf, Photofrin; ALA, 5-aminolevulinic acid; PpIX, protoporphyrin IX; HPLC, high-performance liquid chromatography; Tt, treated foot; Tu, untreated foot.

Received 2/28/01. Accepted 5/24/01.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

 

1. Dougherty J., Gomer C. J., Henderson B. W., Jori G., Kessel D., Korbelik M., Moan J., Peng Q. Review. Photodynamic therapy. J. Natl. Cancer Inst., 90: 889-905, 1998.[Abstract/Free Full Text]
2. Dougherty T. J., Cooper M. T., Mang T. S. Cutaneous phototoxic occurrences in patients receiving Photofrin. Lasers Surg. Med., 10: 485-488, 1990.[Medline]
3. Overholt B. F., Panjehpour M. Photodynamic therapy in Barrett’s esophagus. J. Clin. Laser Med. Surg., 14: 245-249, 1996.[Medline]
4. Nseyo U. O., Shumaker B., Klein E. A., Sutherland K. Photodynamic therapy using porfimer sodium as an alternative to cystectomy in patients with refractory transitional cell carcinoma in situ of the bladder. Bladder Photofrin Study Group. J. Urol., 160: 39-44, 1998.[Medline]
5. Peng Q., Warloe T., Berg K., Moan J., Kongshaug M., Giercksky K. E., Nesland J. M. 5-aminolevulinic acid-based photodynamic therapy: clinical research and future challenges. Cancer (Phila.), 79: 2282-2308, 1997.[Medline]
6. Peng Q., Berg K., Moan J., Kongshaug M., Nesland J. M. 5-aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem. Photobiol., 65: 235-251, 1997.[Medline]
7. Peng Q., Moan J., Warloe T., Nesland J. M., Rimington C. Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma. Int. J. Cancer, 52: 433-443, 1992.[Medline]
8. Peng Q., Moan J., Farrants G., Danielsen H. E., Rimington C. Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy. Cancer Lett., 53: 129-139, 1990.[Medline]
9. Peng Q., Nesland J. M., Moan J., Evensen J. F., Kongshaug M., Rimington C. Localization of fluorescent Photofrin II and aluminum phthalocyanine tetrasulfonate in transplanted human malignant tumor LOX and normal tissues of nude mice using highly light-sensitive video intensification microscopy. Int. J. Cancer, 45: 972-979, 1990.[Medline]
10. Peng Q., Moan J., Nesland J. M. Correlation of subcellular and intratumoral photosensitizer localization with ultrastructural features after photodynamic therapy. Ultrastruct. Pathol., 20: 109-129, 1996.[Medline]
11. Moan J., Peng Q., Rrensen R., Iani V., Nesland J. M. The biological foundations of photodynamic therapy. Endoscopy, 30: 387-391, 1998.[Medline]
12. Peng Q., Moan J. Correlation of distribution of sulphonated aluminum phthalocyanines with their photodynamic effect in tumour and skin of mice bearing CaD2 mammary carcinoma. Br. J. Cancer, 72: 565-574, 1995.[Medline]
13. Peng Q., Moan J., Ma L. W., Nesland J. M. Uptake, localization, and photodynamic effect of meso-tetra(hydroxyphenyl)porphine and its corresponding chlorin in normal and tumor tissues of mice bearing mammary carcinoma. Cancer Res., 55: 2620-2626, 1995.[Abstract/Free Full Text]
14. Noguchi P., Wallace R., Johnson J., Earley E. M., O’Brien S., Ferrone S., Pellegrino M. A., Milstein J., Needy C., Browne W., Petricciani J. Characterization of the WiDr: a human colon carcinoma cell line. In Vitro (Rockv.), 15: 401-408, 1979.
15. Dykes D. J., Bissery M. C., Harrison S. D., Waud W. R. Response of human tumor xenografts in athymic nude mice to docetaxel (RP 56976. Taxotere). Investig. New Drugs, 13: 1-11, 1995.[Medline]
16. Tomokuni K., Ichiba M., Hirai Y. HPLC method for determining -aminolevulinic acid in plasma. Clin. Chem., 39: 169-170, 1993.[Medline]
17. Menezes da Silva F. A., Newman E. L. Time-dependent photodynamic damage to blood vessels: correlation with serum photosensitizer levels. Photochem. Photobiol., 61: 414-416, 1995.[Medline]
18. McDowell E. M., Trump B. F. Histological fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100: 405-414, 1976.[Medline]
19. Gederaas O. A., Berg K., Romslo I. A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolevulinic acid treatment of colon adenocarcinoma cells. Cancer Lett., 150: 205-213, 2000.[Medline]
20. Messmann H., Geisler M., Grob U., Abels C., Szeimies R. M., Steinbach P., Knuchel R., Doss M., Scholmerich J., Holstege A. Influence of a haematoporphyrin derivative on the protoporphyrin IX synthesis and photodynamic effect after 5-aminolaevulinic acid sensitization in human colon carcinoma cells. Br. J. Cancer, 76: 878-883, 1997.[Medline]
21. Nelson J. S., Roberts W. G., Liaw L. H., Berns M. W. Cellular and tumor model studies using several PDT sensitizers Kessel D. eds. . Photodynamic Therapy of Neoplastic Disease, (Vol. I): 169-188, CRC Press Boca Raton, FL 1990.
22. Henderson B. W. Probing the effects of photodynamic therapy through in vivo-in vitro methods Kessel D. eds. . Photodynamic Therapy of Neoplastic Disease, (Vol. I): 147-168, CRC Press Boca Raton, FL 1990.
23. Nelson J. S., Liaw L. H. L., Lahlum R. A., Cooper P. L., Berns M. W. Use of multiple photosensitizers and wavelengths during photodynamic therapy: a new approach to enhance tumor eradication. J. Natl. Cancer Inst., 82: 868-873, 1990.[Abstract/Free Full Text]
24. Dimitroff C. J., Klohs W., Sharma A., Pera P., Driscoll D., Veith J., Steinkamp R., Schroeder M., Klutchko S., Sumlin A., Henderson B., Dougherty T. J., Bernacki R. J. Anti-angiogenic activity of selected receptor tyrosine kinase inhibitors, PD166285 and PD173074: implication for combination treatment with photodynamic therapy. Investig. New Drugs, 17: 121-135, 1999.[Medline]
25. Ferrario A., von Tiehl K. F., Rucker N., Schwarz M. A., Gill P. S., Gomer C. J. Antiangiogenic treatment enhances photodynamic therapy responsiveness in a mouse mammary carcinoma. Cancer Res., 60: 4066-4069, 2000.[Abstract/Free Full Text]

This article has been cited by other articles:

				I. E. Furre, S. Shahzidi, Z. Luksiene, M. T.N. Moller, E. Borgen, J. Morgan, K. Tkacz-Stachowska, J. M. Nesland, and Q. Peng Targeting PBR by Hexaminolevulinate-Mediated Photodynamic Therapy Induces Apoptosis through Translocation of Apoptosis-Inducing Factor in Human Leukemia Cells Cancer Res., December 1, 2005; 65(23): 11051 - 11060. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, Q. Articles by Nesland, J. M. Search for Related Content PubMed PubMed Citation Articles by Peng, Q. Articles by Nesland, J. M.