Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate.

It has been proposed that the generation of O2 during photodynamic therapy (PDT) may lead to photochemical depletion of ambient tumour oxygen, thus causing acute hypoxia and limiting treatment effectiveness. We have studied the effects of fluence rate on pO2, in the murine RIF tumour during and after PDT using 5 mg kg(-1) Photofrin and fluence rates of 30, 75 or 150 mW cm(-2). Median pO2 before PDT ranged from 2.9 to 5.2 mmHg in three treatment groups. Within the first minute of illumination, median tumour pO2 decreased with all fluence rates to values between 0.7 and 1.1 mmHg. These effects were rapidly and completely reversible if illumination was interrupted. During prolonged illumination (20-50 J cm(-2)) pO2 recovered at the 30 mW cm(-2) fluence rate to a median value of 7.4 mmHg, but remained low at the 150 mW cm(-2) fluence rate (median pO2 1.7 mmHg). Fluence rate effects were not found after PDT, and at both 30 and 150 mW cm(-2) median tumour pO2 fell from control levels to 1.0-1.8 mmHg within 1-3 h after treatment conclusion. PDT with 100 J cm(-2) at 30 mW cm(-2) caused significantly (P = 0.0004) longer median tumour regrowth times than PDT at 150 mW cm(-2), indicating that lower fluence rate can improve PDT response. Vascular perfusion studies uncovered significant fluence rate-dependent differences in the responses of the normal and tumour vasculature. These data establish a direct relationship between tumour pO2, the fluence rate applied during PDT and treatment outcome. The findings are of immediate clinical relevance.     

In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here, we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10, 25, or 50 mWcm(-2) and, (1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells, the photobleaching, and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However, in all of the cases, cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT, as well as confirming the likely role of (1)O(2) in porphyrin-based PDT.     

Relationship of tumor hypoxia and response to photodynamic treatment in an experimental mouse tumor

The relationship between tumor oxygenation and the effectiveness of photodynamic therapy (PDT) was studied in vitro and in vivo using the RIF mouse tumor model. The oxygen dependence of photodynamic inactivation of RIF cells, which had been exposed to 25 mg/kg porphyrin (dihematoporphyrin ether) in vivo, isolated and illuminated in vitro, was determined. No cell kill was achieved under anoxic conditions, full effect was reached at 5% O2, and the half value of cell inactivation was found to be at 1% O2. Tumor hypoxia was assessed after in vivo gamma-irradiation of control and PDT-treated tumors by in vitro clonogenic assay of cell radiosensitivity. In vitro control experiments established that the radio-sensitivity of PDT-surviving RIF cells was identical to that of untreated control cells. RIF tumors of treatment size (80-120 mg) contained no detectable hypoxic tumor cell fraction. PDT treatment consisting of i.p. injection of 10 mg/kg dihematoporphyrin ether 24 h prior to 45 J/cm2 of 630 nm light, rendered approximately 9% of tumor cells severely hypoxic within 10 min of treatment time. An illumination period of 30 min (135 J/cm2) induced a hypoxic tumor cell fraction of 17%, which increased to 47% within 1 h posttreatment. Despite the prompt induction of tumor hypoxia during PDT light treatment, the tumors proved highly curable (81% cures) under the present treatment conditions (depilation of tumor area, 10 mg/kg dihematoporphyrin ether i.p., 135 J/cm2). Considering the reduced effectiveness of photodynamic cell kill at low oxygen concentrations, the rapid induction of tumor hypoxia by PDT itself, and the high tumor cure rate, it has to be concluded that in the RIF tumor hypoxic tumor cells are inactivated by a mechanism other than direct photodynamic cytotoxicity, and are thus not limiting to PDT tumor response.     

Evaluation of Hypocrellin B in a human bladder tumor model in experimental photodynamic therapy: biodistribution, light dose and drug-light interval effects

Hypocrellin B (HB), a monomeric perylenequinone pigment, is a promising second-generation photosensitizer for photodynamic therapy. We have evaluated the efficacy of HB mediated PDT by experimenting with various drug-light intervals, based on the biodistribution analysis in human bladder tumor (MGH cell line) models. Tumor growth rates were assessed at 10-day post treatment followed by morphometric analysis. Biodistribution of HB was evaluated using spectrofluorophotometry analysis (Ex: 480 nm, Em: 620-630 nm). The level of HB peaked at 6 h postinjection in tumor, peritumoral skin and normal muscle followed by a decline over the next 42 h. Concurrently, the ratio of drug in tumor versus skin was relatively low at all times in comparison to tumor to muscle ratio. In serum, concentration of HB peaked at 1 h. Almost 88% of its original uptake level was cleared at 48 h. The level of PDT response revealed a strong dependence on the drug-light intervals (DLI) and light dose. For both high and low fluence/fluence rate, comparable tumor response was observed at 1 h DLI; treated tumors exhibited significant tumor regression compared to 6 and 24 h DLI. The absence of tumor response was observed at 24 h DLI even at high light dose (100 J/cm(2); 100 mW/cm(2)). Tumor response detected at low light dose (12 J/cm(2); 12 mW/cm(2)) at short DLI suggests that the tumor vasculature is a more sensitive target compared to the cellular compartment of the tumor, correlating significantly with the bioavailability of the drug in serum. Therefore, HB mediated PDT effect is characteristics of a predominantly vascular mediated effect. This study confirms that for short drug-light intervals, PDT seems to target tumor vasculature, which contributes to tumor destruction.     

Whole bladder wall photodynamic therapy of transitional cell carcinoma rat bladder tumors using intravesically administered hypericin

Whole-bladder wall photodynamic therapy (PDT) is a promising treatment for carcinoma in situ (CIS) and diffuse premalignant changes of the bladder. After the results of our clinical studies showing that intravesical hypericin selectively accumulates in superficial bladder tumors, we investigated the hypericin-PDT efficacy in an AY-27 orthotopic transitional cell carcinoma rat bladder tumor model. After the instillation of hypericin (30 microM, 2 hr) in the bladder, tumors were irradiated (25-50 mW/cm 6-48 J/cm(2)) using 595 nm laser light. Data demonstrate that light doses of 12-48 J/cm(2) resulted in selective PDT-induced urothelial tumor damage without damaging detrusor musculature. Histological assessment of bladder sections 2 days after PDT showed tumor destruction, with tumor cells shrinking and detaching from the bladder wall. There were tumor regrowth 1-3 weeks after treatment. The in vivo/in vitro clonogenic assay results revealed up to 98% of tumor cell kill by hypericin PDT. In conclusion, hypericin PDT can be used to safely induce a selective urothelial tumor damage without damaging detrusor musculature, when optimum hypericin concentration and light fluences are used. A small percentage (2-5%) of tumor cells that survive the photodynamic treatment resulting in tumor regrowth after a prolonged period of time is likely due to oxygen depletion during light irradiation.     

Photodynamic therapy in women with cervical intraepithelial neoplasia using topically applied 5-aminolevulinic acid

Photodynamic therapy (PDT) is a novel treatment modality that produces local tissue necrosis with laser light after prior administration of a photosensitizing agent. We performed a study of topically applied 5-aminolevulinic acid (5-ALA) in the photodynamic treatment of women with high-grade cervical intraepithelial neoplasia (CIN) using fixed 5-ALA doses and application protocols derived from previous in vitro and in vivo results. Three to 5 hr prior to PDT, 10 ml of a 20% solution of 5-ALA was topically applied using a cervical cap. PDT was performed with irradiation of 100 J/cm2 at an irradiance of 100-150 mW/cm2 with an argon-ion-pumped dye laser at 635 nm. For the endocervix, a specifically designed cylindrical applicator was used. Ten treatment cycles of PDT using 5-ALA were performed in 7 patients with high-grade CIN. Non-thermal laser treatment with 100-150 mW/cm2 was well tolerated. Local toxicity was minor as several patients reported burning sensations and vaginal discharge, but no necrosis, sloughing or scarring occurred. After 3 months, a significant reduction in the size of the ectocervical CIN lesions was noted in only 3 patients, who underwent a second PDT cycle. However, no significant improvement in CIN lesions was noted since cold knife conization revealed persistent CIN in all 7 cases. Therefore, PDT after topical application of 5-ALA using an irradiation of 100 J/cm2 produces only minimal side effects. However, it does not appear to be effective in treating CIN.     

Effects of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo

To improve efficacy of photodynamic therapy (PDT) with intravenously administered 5-aminolaevulinic acid (ALA) fractionating the light dose or reducing the light intensity may be a possibility. Therefore, Syrian Golden hamsters were fitted with dorsal skinfold chambers containing an amelanotic melanoma (n=26). PDT was performed (100 mW cm(-2), 100 J cm(-2), continuously or fractionated, and 25 mW cm(-2), 100 J cm(-2); continuously or fractionated) using an incoherent light source following i.v. application of ALA. Following fractionated irradiation, the light was paused after 20 J cm(-2) for 15 min. Prior to and up to 24 h after PDT tissue, pO(2) was measured using luminescence lifetime imaging. The efficacy was evaluated by measuring the tumour volume of amelanotic melanoma cells grown subcutaneously in the back of Syrian Golden hamsters (n=36). Only high-dose PDT resulted in a significant decrease of pO(2). Irrespective of the mode of irradiation only high-dose PDT induced complete remission of all tumours (13 out of 13). It could be shown that low-dose PDT failed to induce a significant decrease of pO(2). No significant effect of fractionated irradiation was shown regarding the therapeutic efficacy 28 days after PDT. Thus performing a fractionated PDT with ALA or reducing the light intensity seems not to be successful in clinical PDT according to the present data.     

Photodynamic cell killing effects and acute skin photosensitivity of aluminum-chloro-tetrasulfonated phthalocyanine and hematoporphyrin derivative

Aluminum-chloro-tetrasulfonated phthalocyanine (PC) showing an absorption peak at 678 nm was compared to hematoporphyrin derivative (HpD), a photosensitizer commonly used in the photodynamic therapy (PDT) of cancers. In vitro studies: KK-47 cells were exposed to long-wavelength ultraviolet (UVA) or red light (greater than 600 nm, greater than 640 nm and greater than 660 nm) after drug sensitization. With UVA irradiation, a higher photodynamic cell killing effect was observed in the cells treated with HpD than with PC. However, with red light irradiation (both greater than 640 nm and greater than 660 nm) PC resulted in greater cell damage. PC was less toxic to KK-47 cells in the dark. In vivo studies: Using a gold vapor laser (GVL: 627.8 nm, 200 mW/cm2, 200 J/cm2), the photodynamic tumor response was determined in C3H/He mice bearing transplantable squamous cell carcinoma. No significant difference was observed in the tumor volume between the PC and HpD groups, except that the PC group (10.0 mg/kg body weight) showed a significantly higher remission rate (3/6) than the control group (0/10, P less than 0.05). Skin photosensitivity test: Skin photosensitivity was estimated by measuring changes in back skin thickness due to photosensitization. With UVA irradiation, a stronger skin reaction was observed in the HpD group, while with visible light irradiation there was no significant difference between the HpD and PC groups. Based on the superior cell killing effect with red light, reduced toxicity to the cells in the dark and mild skin reaction with UVA, PC may be a more promising photosensitizer for PDT.     

Selectivity of the photosensitiser Tookad for photodynamic therapy evaluated in the Syrian golden hamster cheek pouch tumour model

The response to photodynamic therapy (PDT) with the photosensitiser (PS) Tookad was measured in the Syrian hamster cheek pouch model on normal mucosae and chemically induced squamous cell carcinoma. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 538 and 762 nm. The light dose, drug dose and drug injection-light irradiation times (DLI), ranging between 100 and 300 J cm(-2), 1-5 mg kg(-1) and 10-240 min respectively, were varied and the response to PDT was analysed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. A fast time decay of the tissular response with drug dose of 1-5 mg kg(-1) was observed for DLI ranging from 10 to 240 min and for light doses of 100-300 J cm(-2) delivered at a light dose rate of 150 mW cm(-2). A significantly higher level of tissular response was observed for squamous cell carcinoma compared to normal tissue. Nevertheless, the threshold level of the drug-light dose for a detectable response was not significantly different in the tumoral vs normal tissue. The highest response at the shortest DLIs and the absence of measurable response at DLI larger than 240 min at light dose of 300 J cm(-2) and drug dose of 5 mg kg(-1) reveals the predominantly vascular effect of Tookad. This observation suggests that Tookad could be effective in PDT of vascularised lesions.     

Irradiance-dependent photobleaching and pain in delta-aminolevulinic acid-photodynamic therapy of superficial basal cell carcinomas

PURPOSE: In superficial basal cell carcinomas treated with photodynamic therapy with topical delta-aminolevulinic acid, we examined effects of light irradiance on photodynamic efficiency and pain. The rate of singlet-oxygen production depends on the product of irradiance and photosensitizer and oxygen concentrations. High irradiance and/or photosensitizer levels cause inefficient treatment from oxygen depletion in preclinical models. EXPERIMENTAL DESIGN: Self-sensitized photobleaching of protoporphyrin IX (PpIX) fluorescence was used as a surrogate metric for photodynamic dose. We developed instrumentation measuring fluorescence and reflectance from lesions and margins during treatment at 633 nm with various irradiances. When PpIX was 90% bleached, irradiance was increased to 150 mW/cm(2) until 200 J/cm(2) were delivered. Pain was monitored. RESULTS: In 33 superficial basal cell carcinomas in 26 patients, photobleaching efficiency decreased with increasing irradiance above 20 mW/cm(2), consistent with oxygen depletion. Fluences bleaching PpIX fluorescence 80% (D80) were 5.7 +/- 1.6, 4.5 +/- 0.3, 7.5 +/- 0.8, 7.4 +/- 0.3, 12.4 +/- 0.3, and 28.7 +/- 7.1 J/cm(2), respectively, at 10, 20, 40, 50, 60 and 150 mW/cm(2). At 20-150 mW/cm(2), D80 doses required 2.5-3.5 min; times for the total 200 J/cm(2) were 22.2-25.3 min. No significant pain occurred up to 50 mW/cm(2); pain was not significant when irradiance then increased. Clinical responses were comparable to continuous 150 mW/cm(2) treatment. CONCLUSIONS: Photodynamic therapy with topical delta-aminolevulinic acid using approximately 40 mW/cm(2) at 633 nm is photodynamically efficient with minimum pain. Once PpIX is largely photobleached, higher irradiances allow efficient, rapid delivery of additional light. Optimal fluence at a single low irradiance is yet to be determined.     

Interstitial photodynamic therapy. Clinical experience with diffusing fibres in the treatment of cutaneous and subcutaneous tumours.

Interstitial photodynamic therapy has a number of potential advantages over superficial treatment. We have treated 50 subcutaneous and cutaneous tumours interstitially, in nine patients. An additional 22 tumours in the same patients, were treated by superficial PDT. Patients received 1.5-2.0 mg kg-1 of polyhaematoporphyrin and 72 h later underwent treatment using a copper vapour dye laser producing red light at 630 nm. All interstitial treatments were delivered using cylindrical diffusing fibres and a wide range of light doses (5-1500 J cm-3). The complete response rate for all tumours treated interstitially was 52%, rising to 81% in those patients who received 2.0 mg kg-1 PHP and light doses in excess of 500 J cm-3. The overall incidence of skin necrosis was 32% and was 79% in those treated with light doses of greater than 500 J cm-3. The incidence of skin necrosis with interstitial PDT is lower than that seen with superficial photodynamic therapy but higher volumetric light doses are required to produce tumour complete responses. All treatments were well tolerated and volumes of tumour up to 60 cm3 were successfully treated. The penetration depth of 630 nm light in human breast cancer tissue was determined as 4 mm. Little true tumour tissue selectivity was detected by analysis of porphyrin levels in biopsy material.     

Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors

The rate of light delivery (fluence rate) plays a critical role in photodynamic therapy (PDT) through its control of tumor oxygenation. This study tests the hypothesis that fluence rate also influences the inflammatory responses associated with PDT. PDT regimens of two different fluences (48 and 128 J/cm(2)) were designed for the Colo 26 murine tumor that either conserved or depleted tissue oxygen during PDT using two fluence rates (14 and 112 mW/cm(2)). Tumor oxygenation, extent and regional distribution of tumor damage, and vascular damage were correlated with induction of inflammation as measured by interleukin 6, macrophage inflammatory protein 1 and 2 expression, presence of inflammatory cells, and treatment outcome. Oxygen-conserving low fluence rate PDT of 14 mW/cm(2) at a fluence of 128 J/cm(2) yielded approximately 70-80% tumor cures, whereas the same fluence at the oxygen-depleting fluence rate of 112 mW/cm(2) yielded approximately 10-15% tumor cures. Low fluence rate induced higher levels of apoptosis than high fluence rate PDT as indicated by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. The latter revealed PDT-protected tumor regions distant from vessels in the high fluence rate conditions, confirming regional tumor hypoxia shown by 2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl) acetamide staining. High fluence at a low fluence rate led to ablation of CD31-stained endothelium, whereas the same fluence at a high fluence rate maintained vessel endothelium. The highest levels of inflammatory cytokines and chemokines and neutrophilic infiltrates were measured with 48 J/cm(2) delivered at 14 mW/cm(2) ( approximately 10-20% cures). The optimally curative PDT regimen (128 J/cm(2) at 14 mW/cm(2)) produced minimal inflammation. Depletion of neutrophils did not significantly change the high cure rates of that regimen but abolished curability in the maximally inflammatory regimen. The data show that a strong inflammatory response can contribute substantially to local tumor control when the PDT regimen is suboptimal. Local inflammation is not a critical factor for tumor control under optimal PDT treatment conditions.     

5-aminolevulinic acid-mediated photodynamic therapy of intraepithelial neoplasia and human papillomavirus of the uterine cervix--a new experimental approach.

The aim of this study was to treat patients for ectocervical dysplasia [cervical intraepithelial neoplasia (CIN) grades 1 and 2] and associated human papilloma virus (HPV) infections with photodynamic therapy (PDT). In 20 patients, 5-aminolevulinic acid (5-ALA, 12% w/v) was applied topically with a cervical cap 8 h prior to illumination. A thermal light source (150 W halogen lamp) emitting a broadband red light (total energy: 100 J/cm2, fluence rate: 90 mW/cm2) was used for superficial illumination of the portio. In addition, an Nd:YAG pumped dye laser (652 nm) was used to illuminate the cervical canal (total energy: 50 J/cm2, fluence rate: 300 mW/cm2). Preliminary results of follow-ups at 1, 3, 6, and 9 months posttherapy showed a cytological improvement in the grading of the PAP smears in 19 patients and the eradication of cervical HPV in 80%. These results demonstrate that ectocervical dysplasia and associated HPV infections can be treated by PDT.     

Photodynamic therapy of early squamous cell carcinoma with tetra(m-hydroxyphenyl)chlorin: optimal drug-light interval.

The optimal drug-light interval for effective photodynamic therapy (PDT) of early squamous cell carcinomas was evaluated with tetra(m-hydroxyphenyl)chlorin (mTHPC) by means of two complementary modalities: irradiation tests and ex vivo fluorescence microscopy. A Syrian hamster cheek pouch tumour model was used in these experiments. Photodynamic therapy on both tumour-bearing and contralateral healthy cheek pouch mucosae was performed at 650 nm and 514 nm. Light doses of 12 J cm(-2) were delivered at a light dose rate of 150 mW cm(-2) and light doses of 80 J cm(-2) were delivered at a light dose rate of 100 mW cm(-2) respectively, at these two wavelengths, between 6 h and 12 days after the injection of 0.5 mg kg(-1) body weight mTHPC. Two histologically different types of tissue damage were observed: first, a non-selective and non-specific ischaemic vascular necrosis for the cases in which PDT took place during the first 48 h after the injection of the dye and, second, tissue-specific PDT damage, as a coagulation necrosis, when PDT took place more than 72 h after injection of the dye. The time-dependent biodistribution of mTHPC investigated by fluorescence microscopy shows a weak and non-significant difference in relative fluorescence intensities between early SCC and healthy mucosae. Up to 2 days after the injection, the drug is mainly localized in the endothelial cells of the blood vessels. After this period, the dye accumulates in the squamous epithelia with a concentration peaking at 4 days. At all time points, a weak fluorescence intensity is observed in the underlying lamina propria and striated muscle. The information obtained from these studies could well be relevant to clinical trials as it suggests that time delays between 4 and 8 days after i.v. injection should be optimal for PDT of early malignancies in hollow organs.     

Photo-oxidative killing of human colonic cancer cells using indocyanine green and infrared light.

Despite of the approval of Photofrin in various countries, chemically defined sensitizers for photodynamic therapy (PDT) are still needed for the absorption of light in the infrared spectrum, which provides a maximal penetration of light into tissue. Therefore, both the efficacy and the mechanism of action of the clinically approved dye indocyanine green (ICG) and laser irradiation were investigated in vitro. For the investigation of phototoxic effects, HT-29 cells were incubated 24 h prior to irradiation by using different concentrations of ICG (10-500 microM). In each experiment, cells were irradiated using a continuous wave (cw)-diode laser (lambda(ex) = 805 nm, 30 J cm(-2), 40 mW cm(-2)). After laser irradiation, cell viability of dark control and of cells incubated with 500 microM ICG was 1.27+/-0.11 or 0.28+/-0.05 respectively. Using 100 microM ICG and D2O, cell viability was further decreased from 0.46+/-0.03 (H2O) to 0.11+/-0.01 (D2O). Using D2O and 100 microM ICG, the concentration of malondialdehyde, a marker of lipid peroxidation, increased from 0.89+/-0.10 nmol 10(-6) cells to 11.14+/-0.11 nmol 10(-6) cells. Using 100 microM ICG and laser irradiation sodium azide or histidine (50 mM), quenchers of singlet oxygen reduced the cell killing significantly. In contrast, when using mannitol, a quencher of superoxide anion and hydroxyl radical, cell killing was not inhibited. According to the present results, photoactivated ICG seems to kill colonic cancer cells due to the generation of singlet oxygen and the subsequent formation of lipid peroxides. Therefore, ICG might present a promising photosensitizer for PDT; first clinical results confirm these findings.     

Influence of a haematoporphyrin derivative on the protoporphyrin IX synthesis and photodynamic effect after 5-aminolaevulinic acid sensitization in human colon carcinoma cells.

Haematoporphyrin derivatives (HPDs) are potent sensitizers in photodynamic therapy (PDT), associated with prolonged skin photosensitivity. 5-Aminolaevulinic acid (5-ALA), a natural precusor of haem, is converted intracellularly into the photosensitive agent protoporphyrin IX (PPIX), causing direct cytotoxicity after laser light irradiation but limited skin photosensitivity over 1-2 days and higher tumour selectivity. Unfortunately, the use of 5-ALA in PDT has been shown to cause only superficial tissue necrosis. Therefore, a combination of HPD and 5-ALA could be of great clinical value in the treatment of tumours if a synergistic effect of both sensitizers on tumour cell necrosis with less skin photosensitivity could be demonstrated. Human colon adenocarcinoma cells (HT-29) were cultured with either HPD or 5-ALA alone, simultaneously for 24 h with 5-ALA and HPD or in succession with 5-ALA (18 h) followed by HPD (6 h at different concentrations. Intracellular PPIX concentrations were determined by high-performance thin-layer chromatography. Furthermore, PDT was performed with an incoherent light source (lambda = 580-740 nm) using a light dose of 30 J cm(-2) and an output power of 40 mW cm(-2). The intracellular PPIX concentration correlated well with 5-ALA drug dose and incubation time and was highest after single 5-ALA sensitization. In the presence of HPD, either simultaneously or sequentially, PPIX decreased significantly. The PDT effect after simultaneous incubation with both sensitizers for 24 h was not superior to incubation with HPD alone. If 5-ALA incubation (18 h) was followed by HPD (6 h) cytotoxicity after PDT was higher than with either single drug. 5-ALA (80 microg ml(-1)) led to a decrease in tumour cell viability by 40%. A similar effect could be observed when 5-ALA and HPD were sequentially combined allowing for a reduction of the 5-ALA dose from 80 microg ml(-1) in the absence of HPD to 60 microg ml(-1) and 5 microg ml(-1) together with 0.5 microg ml(-1) and 2 microg ml(-1) HPD respectively. We speculate that the enhanced PDT effect after the combined administration of 5-ALA and HPD to cultures of colon carcinoma cells should be even more impressive in the tumour in vivo, since HPD primarily targets the tumour microvasculature and secondarily tumour cells.     

The effect of hyperthermia on murine leukaemia cells in combination with photodynamic therapy

Mouse leukaemia L1210 cells were subjected to hyperthermia (43.0 +/- 0.05 degrees C, water bath) and photodynamic therapy (50 micrograms/ml haematoporphyrin derivative (HpD), 630 +/- 5 nm at 0.1 mW/cm2) for varying lengths of treatment time in different sequences. Dose-response curves showed that the two modalities interact to make the combination more cytotoxic than the sum of the separate individual treatments, and that a determining factor is the intracellular concentration of HpD of tumour cells during hyperthermia. High cytotoxic (synergistic) effects of photodynamic therapy (PDT) were obtained when PDT was performed after hyperthermia following HpD administration. However, only additive cytotoxic effects were found, when hyperthermia was performed after HpD administration and PDT. These cytotoxic data were supported by DNA damage (the increase in single-stranded DNA) as revealed by flow cytometry in cells stained with acridine orange (AO). These in vitro experiments suggest that clinical applications of PDT after hyperthermia with HpD injection might be useful.     

Photodynamic therapy upregulates expression of Mac-1 and generation of leukotriene B(4) by human polymorphonuclear leukocytes

This study investigated the expression of Mac-1 (CD11b/CD18) and generation of leukotriene B(4) (LTB(4)) by polymorphonuclear leukocytes (PMNs) in response to Mono-L-aspartyl chlorine 6 (NPe6) mediated photodynamic therapy (PDT). PDT was carried out using a laser diode at a light dose of 10 J/cm(2) and wavelength of 664 nm. Expression of Mac-1 was significantly increased about 3-fold by PDT compared with that of Npe6 exposure. The increase in the expression of Mac-1 was not inhibited by 5-lipoxygenase inhibitor (AA861). On the other hand, generation of LTB(4) by PMNs was increased by incubation with NPe6 alone and further promoted by PDT and that was significantly inhibited by AA861. These results suggest that PDT can induce PMNs to express extensively Mac-1 and generate a certain amount of LTB(4) that may play an important cytocidal role in the treatment of oral cancer.     

Photodynamic Cell Killing Effects and Acute Skin Photosensitivity of Aluminum-chloro-tetrasulfonated Phthalocyanine and Hematoporphyrin Derivative

Aluminum-chloro-tetrasulfonated phthalocyanine (PC) showing an absorption peak at 678 nm was compared to hematoporphyrin derivative (MpD), a photosensitizer commonly used in the photodynamic therapy (PDT) of cancers. In vitro studies: KK-47 cells were exposed to long-wavelength ultraviolet (UVA) or red light (>600 nm, >640 nm and >660 nm) after drug sensitization. With UVA irradiation, a higher photodynamic cell killing effect was observed in the cells treated with HpD than with PC. However, with red light irradiation (both > 640 nm and >660 nm) PC resulted in greater cell damage. PC was less toxic to KK-47 cells in the dark. In vivo studies: Using a gold vapor laser (GVL: 627.8 nm, 200 mW/cm², 200 J/cm²), the photodynamic tumor response was determined in C3H/He mice bearing transplantable squamous cell carcinoma. No significant difference was observed in the tumor volume between the PC and HpD groups, except that the PC group (10.0 mg/kg body weight) showed a significantly higher remission rate (3/6) than the control group (0/10, P<0.05). Skin Photosensitivity test: Skin photosensitivity was estimated by measuring changes in back skin thickness due to photosensitization. With UVA irradiation, a stronger skin reaction was observed in the HpD group, while with visible light irradiation there was no significant difference between the HpD and PC groups. Based on the superior cell killing effect with red light, reduced toxicity to the cells in the dark and mild skin reaction with UVA, PC may be a more promising photosensitizer for PDT.     

光动力学疗法治疗上消化道癌的临床研究

Objective： To evaluate the clinical effectiveness and adverse effects of photodynamic therapy （PDT） for the upper gastrointestinal tract cancers. Methods： 56 patients with upper gastrointestinal cancers in different clinical stages were treated with PDT. Diode laser （630 nm） was used as the light source and the parameters were as follows： power density 200 to 400 mW/cm, energy density 100 to 300 J/cm. PHOTOFRIN was used as photosensitizer, which was given in a dose of 2 mg/kg intravenously 12-24 h before irradiation. Results： Evaluation of the 56 patients＇ therapeutic effectiveness showed that 6 patients （10.7%） had a complete response （CR）, 33 patients （58.9%） partial response （PR）, 12 patients （21.4%） mild response （MR）, and 5 patients （8.9%） no response （NR）. The total response rate （CR＋PR） was 69.6%. No patients had severe adverse effects in this group. Conclusion： PDT is an effective and safe palliative modality for upper gastrointestinal tract cancers.