The effect of photodynamic therapy on rat urinary bladder with orthotopic urothelial carcinoma

OBJECTIVE: To assess the effect of whole-bladder photodynamic therapy (PDT) on a rat model with orthotopic superficial bladder cancer, as PDT is an alternative intravesical therapy for treating superficial bladder cancer, based on an interaction between a photosensitizer and light energy to induce oxygen radicals that destroy tissue by lipid peroxidation. MATERIALS AND METHODS: In all, 76 female Fischer F344 rats were inoculated intravesically with AY-27 tumour cells. After establishing superficial tumour, 24 rats were treated with PDT using aminolaevulinic acid (ALA)-induced protoporphyrin IX as a photosensitizer, and a continuous-wave argon pumped-dye laser (638 nm). At 4 h after intravenous (300 mg/kg) or intravesical (100 mg/mL) administration of ALA the bladders were intravesically exposed to a 40 J/cm(2) light dose; 12 rats received no ALA but were exposed to the same light dose. Before administering ALA, urine cytology samples were taken for analysis. At 3 or 21 days the treated rats were killed and morphological changes in the bladder walls analysed by light microscopy. Forty rats served as controls to examine the presence of tumour. RESULTS: The tumour established in 33 of 40 rats (83%) in the controls, but after PDT with intravesical ALA there was carcinoma in only in one of 12 (P < 0.001, Pearson's chi(2) test). After PDT with intravenous ALA there was carcinoma in five of 11 rats (P = 0.063, Pearson's chi2 test). In the control group of 12 rats receiving only light energy there was carcinoma in three (P = 0.001, Pearson's chi(2) test). Histologically, at 3 days after PDT there was only mild superficial damage in all six rats treated intravesically. Bladder wall destruction reached the muscular layer, with an abscess in one of six rats treated intravenously. After 3 weeks of PDT there was muscular necrosis with perforation and abscess from catheterization two of six rats treated intravesically and in three the bladder wall totally recovered. In the intravenous group the bladder walls were normal or had only mild superficial damage. Cytology of the urine sediment failed to detect half the tumours in the treatment groups. CONCLUSION: These results support the use of PDT with intravesical ALA-induced protoporphyrin X for treating superficial bladder carcinoma. Intravesical was better than intravenous ALA in eradicating bladder carcinoma with PDT.     

A phase-1 study of sequential mitomycin C and 5-aminolaevulinic acid-mediated photodynamic therapy in recurrent superficial bladder carcinoma

OBJECTIVE: To report a phase-1 study of patients with recurrent superficial bladder cancer treated with photodynamic therapy (PDT) using sequential mitomycin C and 5-aminolaevulinic acid (ALA). PATIENTS AND METHODS: Twenty-four patients were treated, the primary endpoint being the safety and tolerability of combined therapy at increasing doses of ALA and light. RESULTS: Mitomycin C instillation was followed by ALA concentrations of 6%, 8% or 10%; there was no effect on toxicity. The light dose, at a wavelength of 635 nm, was increased from zero to 25 J/cm(2), with the upper fluences producing transient symptoms. There were no episodes of skin photosensitivity or systemic toxicity. A total fluence of 25 J/cm(2) represented the upper light dose for the tolerability of this procedure by patients. There were no persistently high urinary symptom scores or reduction in functional bladder capacity up to > or =24 months of follow-up. In this group, cumulative tumour recurrences were none at 4, two at 8, six at 12, nine at 18 and 11 at 24 months after PDT, respectively. CONCLUSION: Sequential mitomycin C and ALA-PDT is a safe and well tolerated treatment, with potential for managing difficult-to-control superficial transitional cell carcinoma and carcinoma in situ of the bladder.     

Effect of photodynamic therapy on urinary bladder function: an experimental study with rats

Photodynamic therapy (PDT) produces localized necrosis with light after prior administration of a photosensitizing drug. The problems with laser light dosimetry and complications relating to bladder function appear to be important limiting factors of PDT in urology. Photodynamic therapy on urinary bladder with normal epithelium of rats was performed using an argon ion laser as an energy source, with aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photosensitizer. Four hours after ALA intravenous administration, the bladders were intravesically radiated with light doses 20, 40, or 80 J/cm². Animals in the control group did not receive ALA and were radiated with 20 J/cm² light dose. Three weeks prior to PDT, the bladder capacity and pressure changes during filling cystometry were assessed. Cystometrics were repeated 1, 3, 7, or 21 days after laser therapy. The light dose 20 J/cm² and 40 J/cm² together with the used ALA dose caused no reduction in bladder capacity, whereas 80 J/cm² light dose produced up to 50% reduction in the capacity at 3 weeks postoperatively. In control group without ALA, the animals did not regain more than 34% of the capacity of their control values at 3 weeks. The light dose of 20 J/cm² and 40 J/cm² with ALA induced functional changes that subsided after day 1. Our results indicate that with proper dosing of photosensitizing drug and light energy, the functional impairment of urinary bladder may be reduced as transient. These findings support the use of PDT as safe therapy of superficial bladder cancer. Received: 10 April 2000 / Accepted: 16 November 2000     

Photodynamic therapy for superficial bladder cancer under local anaesthetic

OBJECTIVES: To evaluate the use of local anaesthesia (LA) in 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) for superficial transitional cell carcinoma (TCC) of the bladder, and to provide further toxicity and tolerability data on this new method within the context of a phase 1 trial. PATIENTS AND METHODS: ALA PDT was administered to 19 patients with recurrent superficial TCC (stage Ta/carcinoma in situ, grades 1-3) using escalating doses of ALA (3-6%) and 633 nm laser light (25-50 J/cm2) under various LA (lignocaine) protocols. Pain was assessed using a linear analogue scale from 0 to 10. The endpoints of tolerability and toxicity were assessed for the different LA, light and ALA doses, with lignocaine levels. RESULTS: ALA PDT is painful and requires some form of anaesthesia. The discomfort was immediate, associated with bladder spasm, and was a function of the ALA concentration rather than the total light dose given. Simple passive diffusion (PD) of 2% lignocaine instilled for 40 min before PDT gave adequate anaesthesia with 3% ALA (n=8; median pain score 1, range 0-2). With 6% ALA the pain was dramatically increased using PD (n=6; median pain score 8, range 5-10) and therefore the more potent LA technique of electromotive drug administration (EMDA) of 2% lignocaine was used, with excellent results (n=3; median pain score 1, range 0-2). All patients had transient bladder irritability that typically lasted 9-12 days, with no subjective/objective change in long-term bladder function. No other toxicity was reported. Serum lignocaine levels were minimal. CONCLUSION: Bladder ALA PDT is both safe and feasible under LA. At a dose of 3% ALA, the procedure was well-tolerated using PD of lignocaine. At higher doses (6% ALA) more effective anaesthesia is required and this can be obtained satisfactorily with EMDA of lignocaine. With refinement, ALA PDT may be feasible as an outpatient treatment for superficial bladder TCC.     

Photodynamic therapy of superficial bladder tumors

Photodynamic therapy (PDT), using hematoporphyrin derivative (HPD) and the red light (wavelength 630 nm) of an argon-dye laser as the source of excitation energy was performed on 46 patients with superficial bladder tumors. Two methods of laser irradiation, (1) focal PDT using a 400 micron quartz fiber through a cystourethroscope in 22 patients with superficial bladder tumors and (2) whole bladder wall total PDT using a motor-driven laser light scattering device in 24 patients with multifocal carcinoma in situ and/or dysplasia of bladder mucosa associated with multicentric concurrent superficial tumors, were used. The patients in (2) had been referred for total cystectomy, and 19 of these 24 patients had a history of several transurethral resections, hyperthermia and/or instillation therapy. HPD 2-4 mg/kg was i.v. injected 48 to 72 hours before PDT. Judging from the results of 60 protrusions treated by focal PDT, the light power should be 200 mW/cm2 for 5-10 minutes or more and the total light energy should be 100 J/cm2 or more in tumors up to 2 cm in size. With focal PDT, 4 of the 22 patients had no recurrence with the mean tumor free time of 20.8 months. In 6 of the 24 patients treated with total PDT using 10, 20 or 30 J/cm2 of light energy, there was no recurrence with a mean tumor-free time of 7.5 months and there was no significant relationship between the recurrence rate and total light energy used.     

The morphological and functional changes in rat bladder following photodynamic therapy with phthalocyanine photosensitization

We have studied photodynamic therapy (PDT) in the rat bladder with a new photosensitizer, aluminium sulfonated phthalocyanine (AlSPc) given intravenously and intravesically. The microscopic distribution of photosensitizer fluorescence in the bladder wall was studied by laser fluorescence microscopy. Prior to PDT the bladder capacity and compliance were assessed by filling cystometry. Intravesical red light (675 nm.) from a copper vapour pumped dye laser was used to activate the photosensitizer using light doses of 20 to 200 J/cm2. Urodynamic and histologic changes were studied at intervals for up to three months. The fluorescence studies showed that AlSPc was eliminated from the deeper muscle layers more quickly than from the superficial layers of the bladder wall so that by 24 hours there was four times as much fluorescence from the mucosa and lamina propria compared to the deeper muscle. Control bladders illuminated with laser light alone showed no effects at these light doses. Animals treated 24 hours after sensitization showed a reduction in bladder capacity of up to 78% (20 J/cm2. light and 1.5 mg./kg.AlSPc). An initial reduction in compliance recovered in two weeks after low doses (0.5 mg./kg.) of AlSPc but was still abnormal at three months after higher doses (1.5 mg./kg.); though there was no long term histologic abnormality seen. Aluminium sulfonated phthalocyanine is a promising photosensitizer for bladder photodynamic therapy and using low doses of the drug it is possible to produce a superficial necrosis without muscle damage across a range of light doses. This heals by epithelial regeneration with no long term functional impairment. Direct absorption of this photosensitizer following intravesical administration seems unreliable.     

Photodynamic Therapy on Rat Urinary Bladder with Intravesical Instillation of 5-Aminolevulinic Acid: Light Diffusion and Histological Changes

Purpose

Photodynamic therapy (PDT) has the potential to treat extensive premalignant lesions and microinvasive tumors in the bladder, but its use has been hampered by the risk of detrusor muscle damage and prolonged skin photosensitivity. We have shown that the rat urothelium can be sensitized by selectively using a 10 percent solution of 5-aminolevulinic acid (ALA) at pH 5.5 administered intravesically. This paper evaluates the photodynamic effects on sensitized bladders.

Materials and Methods

The bladders of Wistar rats were instilled with ALA solutions of different concentrations at pH 5.5 and subsequently treated with laser light at 630 nm. Bladders were harvested 1 to 7 days after PDT for histological assessment.

Results

Under optimum conditions (10 percent intralipid diffusion medium, light dose 50J) uniform urothelial necrosis was seen after 1 to 2 days; it healed in 7 days without damage to the underlying muscle layer although some increase in collagen was seen in the lamina propria. Overtreatment or poor light distribution resulted in muscle necrosis and scarring.

Conclusions

Selective urothelial necrosis is possible with PDT using intravesical ALA. There is now sufficient data for pilot clinical trials to start photodynamic therapy for management of superficial bladder cancer or carcinoma in situ.     

Whole bladder photodynamic therapy for orthotopic superficial bladder cancer in rats: a study of intravenous and intravesical administration of photosensitizers

PURPOSE: Photodynamic therapy after intravenous injection of Photofrin (QLT Phototherapeutics, Vancouver, British Columbia, Canada) results in a contracted bladder and skin photosensitivity, which limits its clinical application. In an attempt to overcome these limitations photodynamic therapy after intravesical instillation of Photofrin or 5-aminolevulinic acid (ALA) in an orthotopic rat bladder tumor model was explored and compared with intravenous Photofrin for photodynamic therapy efficacy and phototoxicity. MATERIALS AND METHODS: At 2 weeks after bladder implantation of 1.5 x 10(6) AY-27 tumor cells animals were randomly grouped. Photofrin was administered (5 mg./kg. intravenously and 2 mg./ml. intravesically). The ALA concentration for intravesical instillation was 300 mM. Whole bladder photodynamic therapy with graded doses of light (lambda = 630 nm.) was performed 4 hours after drug administration. Tumor control and complications were evaluated. RESULTS: Photodynamic therapy with intravenous Photofrin plus 100 J./cm.(2) light resulted in severe bladder damage. Of 10 rats 6 died and 2 of the 10 that received 50 J./cm.(2) died. There were no photodynamic therapy related deaths in groups receiving intravesical instillation of Photofrin or ALA that also received 50 to 100 J./cm.(2) Median survival in rats treated with ALA intravesically plus 75 J./cm.(2) (77 days), Photofrin intravesically plus 50 (67) or 100 J./cm.(2) (76) and Photofrin intravenously plus 50 J./cm.(2) (60) were significantly different from that in controls (44). CONCLUSIONS: Intravesical instillation of Photofrin or ALA can achieve the same photodynamic therapy efficacy as intravenous Photofrin in this orthotopic rat bladder tumor model with less phototoxicity to normal tissues.     

Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy

BACKGROUND AND OBJECTIVE: A human glioma spheroid model is used to investigate the efficacy of different light delivery schemes in 5-aminolevulinic acid (ALA)--mediated photodynamic therapy (PDT). The results provide the rationale for the development of an indwelling balloon applicator for optimizing light delivery. STUDY DESIGN/MATERIALS AND METHODS: Human glioma spheroids were incubated in ALA (100 or 1000 microg /ml-1) for 4 hours and subjected to various light irradiation schemes. In one set of experiments, spheroid survival was monitored as a function of light fluence rate (5-200 mW cm-2). In all cases, spheroids were exposed to fluences of either 25 or 50 J cm-2. In a second study, the effects of repeated weekly PDT treatments, using sub-threshold fluences, were investigated. One group of spheroids was subjected to three treatments using fluences of 12, 12, and 25 J cm-2. Results were compared to spheroids receiving single treatments of either 12 or 25 J cm-2. A fluence rate of 25 mW cm-2 was used for all three groups of spheroids. In all cases, the effect of a given irradiation scheme was evaluated by monitoring spheroid growth. RESULTS: Low fluence rates produce greater cell kill than high fluence rates. The minimum effective fluence rate in human glioma spheroids is approximately 10 mW cm-2. Repeated weekly PDT treatments with sub-threshold fluences result in significant cell kill. In spheroids surviving the PDT treatments, growth is suppressed for the duration of the treatment period. CONCLUSION: The results of the in vitro studies support the development of an indwelling balloon applicator for the delivery of light doses in long term multi-fractionated PDT regimens.     

Hexyl-aminolevulinate-mediated photodynamic therapy: how to spare normal urothelium. An in vitro approach

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizer concentration, irradiation parameters, and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium invitro. The aim was to determine the threshold conditions for the cell survival. METHODS: Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. RESULTS: The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm(2) (irradiance 30 and 75 mW/cm(2), respectively) and for white light 0.55 J/cm(2) (irradiance 30 mW/cm(2)). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. CONCLUSIONS: We have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.     

Photodynamic therapy with PAD-S31, a new hydrophilic chlorin photosensitizer, in an orthotopic rat bladder tumor model

PURPOSE: PAD-S31 (13,17-bis (1-carboxypropion) carbamoylethyl-3-ethenyl-8-ethoxyiminoethylidene-7-hydroxy-2,7,12,18-tetramethyl-porphyrin sodium) (Photochemical Co., Ltd., Okayama, Japan), 1 of the latest second-generation photosensitizers, has hydrophilic characteristics and excitation wavelengths of around 670 nm. Using an orthotopic rat bladder tumor model we investigated the biodistribution of PAD-S31 and assessed the antitumor effects of photodynamic therapy (PDT) with PAD-S31. MATERIALS AND METHODS: An orthotopic rat bladder tumor was established by implanting AY-27 cells in the bladder wall. After intravenous PAD-S31 administration the accumulation of PAD-S31 in the tumor and normal bladder wall was investigated by a fluorometric technique. One or 3 hours after intravenous administration of PAD-S31 (5 mg/kg) bladder tumors in rats were transurethrally irradiated at 100 mW/cm with a light dose of 50 to 200 J/cm. The efficacy of PDT was evaluated 7 days later by observation with an ultrathin cystoscope and histopathological examination. RESULTS: The ratio of PAD-S31 concentration in tumor tissue to that in normal bladder wall was more than 1 at all time points and it achieved a maximum (more than 10) 150 to 240 minutes after PAD-S31 administration. All rats that were irradiated at 100 J/cm 3 hours after PAD-S31 administration showed more than 50% tumor destruction. When the light dose was more than 150 J/cm, more than half of the rats showed complete tumor eradication, of which the average size was 6 mm. CONCLUSIONS: We report that PDT using PAD-S31 is effective for destroying bladder tumors in an orthotopic rat model. These experimental results suggest that this therapy could be a clinically promising method for the treatment of patients with bladder cancer.     

Morphological effects of photodynamic therapy on rabbit bladder using Photofrin II and Photosan intravesically and intravenously.

Photodynamic therapy (PDT) has proved effective against superficial papillary bladder tumours and focal and diffuse carcinoma in situ. Effective topical administration of the sensitiser would be a welcome improvement. The morphological effects of PDT on the normal bladder were examined in 13 rabbits when 2 photosensitisers (Photofrin II and Photosan III) were applied intravesically (5 mg/kg for 1 h) and compared with intravenous administration (3 or 5 mg/kg). Four animals served as controls without a sensitiser. Intravesical red light (630 nm) from an argon dye laser was used to activate the photosensitiser, using light doses of 12 or 24 J/cm2. The animals were sacrificed either 1 or 5 to 7 days after the laser treatment. Intravenous dosage induced bladder wall oedema/haemorrhage and total necrosis of the epithelium. There was no difference between the effects of the 2 sensitisers. Intravesical application induced superficial epithelial necrosis. The control animals treated with laser light alone showed slight superficial injury to the cell layer.     

Endovascular photodynamic therapy with aminolaevulinic acid prevents balloon induced intimal hyperplasia and constrictive remodelling.

BACKGROUND AND OBJECTIVE: intimal hyperplasia (IH) and constrictive remodelling are important causes of restenosis following endovascular interventions, such as percutaneous transluminal angioplasty. Photodynamic therapy (PDT) with 5-aminolaevulinic (ALA) may prevent restenosis by cellular depletion and the elimination of cholinergic innervation. STUDY DESIGN/MATERIALS AND METHODS: rats (n=90) were subdivided into 4 main groups. In the experimental group (n=36: 3 replications x 4 doses x 3 examination time-points), ALA was administered (200mg/kg i.v.) 2-3h before balloon injury (BI) of the common iliac artery followed by endovascular illumination with 633nm at either 12.5, 25, 50 or 100J/cm diffuser length (dl BI+PDT group). As control groups served the BI+Light only (LO) group (n=36) that received no ALA, the BI only group (n=9) (BI), and a group (n=9) that received a Sham procedure (Sham group). RESULTS: planimetric analysis showed IH of 0.28+/-0.12mm(2) (BI), 0.27+/-0.12mm(2) (BI+LO at 100J/cmdl) in contrast to 0.02+/-0.02mm(2) after BI+PDT at 100J/cmdl at 16 weeks (p<0.05). In the BI+PDT groups, a light-dose increase of a factor 2 led to an IH decrease of 17% (p<0.05). In the BI and BI+LO groups constrictive remodelling was found, in contrast to BI+PDT treated groups at 16 weeks. The staining of cholinergic innervation of the tunic media of the blood vessel wall in BI+PDT showed no damage at the highest fluence. CONCLUSION: endovascular ALA-PDT prevents IH and constrictive remodelling after BI without damage of cholinergic innervation of the tunica media. The effective light fluence rate in the rat is 50-100J/cmdl.     

Long-pulse dye laser for photodynamic therapy: investigations in vitro and in vivo.

BACKGROUND AND OBJECTIVE: Continuous wave lasers or incoherent lamps are used effectively for photodynamic therapy (PDT). As the mechanism of action of pulsed lasers in PDT is not known, we investigated the efficacy of PDT with 5-aminolevulinic acid (ALA) using a long-pulse (1.5 ms) tunable flashlamp-pumped pulsed dye laser (LPDL) in vitro and in vivo. STUDY DESIGN/MATERIALS AND METHODS: HaCaT human keratinocytes were incubated with ALA (3 mmol/l) and irradiated (0-50 J/cm(2)) using the LPDL at 585 nm, 595 nm, or 600 nm vs. an incoherent light source (580-740 nm). Topical ALA-PDT was performed on 24 patients with actinic keratoses (AK) on the head (n = 200) after incubation with a 20% ALA emulsion and irradiation by either an incoherent light source (160 mW/cm(2), 60-160 J/cm(2)) or the LPDL (585 nm, 18 J/cm(2)). RESULTS: Maximal cytotoxic effects in vitro were achieved using the LPDL at 585 nm or the incoherent lamp (50 J/cm(2)). Sodium azide, a quencher of singlet oxygen, significantly reduced cell killing, suggesting that the cytotoxic effects are mainly mediated by singlet oxygen. This is supported by an increase of lipid peroxides as determined by malondialdehyde after adding D(2)O. Complete remission was achieved in 79% of 100 AK treated by ALA and the LPDL and in 84% of 100 AK treated by ALA and the incoherent lamp. Pain during light treatment was significantly reduced by using the LPDL. Control lesions (LPDL without ALA) did not clear. CONCLUSION: These results show the in vitro and in vivo efficacy of ALA-PDT using a pulsed light source mediated by singlet oxygen.     

Effects of ALA-mediated photodynamic therapy on the invasiveness of human glioma cells

BACKGROUND AND OBJECTIVE: High-grade gliomas are characterized by rapid proliferation, angiogenesis, and invasive growth. Eradication or inhibition of infiltrating glioma cells poses a significant clinical challenge that is unlikely to be solved using conventional treatment regimens consisting of ionizing radiation and chemotherapeutic agents. In this study, we evaluated the effects of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on the invasiveness of human glioma cells migrating from implanted multicell human tumor spheroids. STUDY DESIGN/MATERIALS AND METHODS: Tumor spheroids, derived from the human glioma cell line ACBT, were implanted into a gel matrix of collagen type I. Twenty-four hours following implantation there was a significant invasion into the surrounding gel by individual tumor cells to an average distance of 400 microm. The cultures were incubated in ALA for 4 hours and then exposed to 635 nm laser light in a titration of fluence level, fluence rate, and drug concentration. RESULTS: ALA-PDT at a light fluence of 6 J/cm(2) was sufficient to inhibit gloma cell migration distance by 80-90% compared to control cultures, but did not prevent spheroid growth nor was it cytotoxic to the migrating cells. The viability of the migrating cells both in control and PDT-treated cultures receiving 6 J/cm(2) was high, 85 and 65%, respectively. ALA-PDT at fluences of 25 J/cm(2) was clearly cytotoxic for both the infiltrating cells as well as the spheroids. Low fluence rates were more effective at inhibiting tumor cell infiltration than higher ones for a given total fluence. CONCLUSION: Measurement of cell survival, and results from cultures with blocked cell proliferation, indicated a direct migratory inhibition effect on the invading cells rather than cytotoxicity as the most likely mechanism for the inhibition of invasiveness observed following ALA-mediated PDT.     

Photodynamic therapy of high-grade cervical intraepithelial neoplasia with 5-aminolevulinic acid

BACKGROUND AND OBJECTIVES: To determine the safety and efficacy of 5-aminolevulinic acid (ALA) as a topically applied photosensitizer for photodynamic therapy (PDT) of cervical intraepithelial neoplasia (CIN). STUDY DESIGNS/MATERIALS AND METHODS: Forty women, who were at least 18 years old with persistent biopsy-proven CIN 2 and CIN 3 within the previous 3 months of enrollment, underwent PDT in a phase I and II design. Five escalating radiant energies (increments of 25 J/cm(2), beginning at 50-150 J/cm(2)) using a Coherent Dye Model 920 argon pumped dye laser providing light at 630 nm (maximum output 0.8 W) were used to perform PDT with a fixed dose of ALA (200 mg/ml). ALA was placed in a cervical cap fitted to the cervix. After 90 minutes, the cap was removed and the ectocervix was illuminated for 5-16 minutes, depending on the irradiance. Success was defined as the absence of CIN on Pap smear or colposcopic examination at 12-months. Patients were monitored for toxicity. RESULTS: Thirty-two women (80%) completed the study with 1 year of follow-up. Sixty percent had CIN 3 and 40% CIN 2. Success rates at 4, 8, and 12 months were 51, 46, and 31%, respectively, and were not light-dose dependent. Three patients progressed from CIN 2 to CIN 3. Toxicity was tolerable and only consisted of spotting, vaginal discharge, mild cramping, and vaginal warmth. There was no apparent dose relationship to toxicity. CONCLUSIONS: PDT at this light and ALA dose is well tolerated but has minimal activity in the treatment of CIN 2 and CIN 3. Other doses and schedules of light and ALA or novel photosensitizers may improve efficacy.     

No generalized skin phototoxicity after intravesical application of 5-aminolevulinic acid for fluorescence diagnosis of superficial bladder cancer

INTRODUCTION: A prospective monocenter open study was carried out to evaluate if generalized phototoxic skin reactions occur after intravesical application of 5-aminolevulinic acid (ALA) for fluorescence diagnosis of superficial bladder carcinomas. PATIENTS AND METHODS: On 21 patients, skin phototoxicity was determined prior to as well as 4, 8 and 28 h after intravesical instillation of a 3% ALA solution by exposing small skin areas to a progressively graded series of defined UVA-light doses (n = 9; 5-80 J/cm(2)). RESULTS: Prior to ALA instillation, erythema or pigmentation as signs of cutaneous phototoxicity occurred at an UVA-light dose of 28 +/- 1.5 J/cm(2) (mean +/- SEM). A reduction of the minimal phototoxic dose (MPD) was not detected 4 (28 +/- 1.9 J/cm(2)), 8 (28 +/- 1.6 J/cm(2)) and 28 h (28 +/- 1.5 J/cm(2)) after ALA instillation. Consequently phototoxic reactions were not observed. CONCLUSIONS: A reduction of MPD for UVA was not detected. Therefore, it is not necessary to protect the skin of patients from ambient or daylight after intravesical instillation of ALA for fluorescence diagnosis.     

Investigation of sequential mitomycin C and photodynamic therapy in a mitomycin-resistant bladder cancer cell-line model

OBJECTIVES: To investigate the hypothesis that sequential mitomycin C and 5-aminolaevulinic acid (ALA)-mediated photodynamic therapy (PDT) interact additively in both the J82 bladder cancer cell line and its mitomycin-C-resistant derivative, J82/MMC, and to assess the theoretical basis of this interaction by measuring the relative mitochondrial density of the respective cell lines, on the basis that the mitochondria are the intracellular site where ALA is metabolized to the active photosensitizer, protoporphyrin IX. MATERIALS AND METHODS: Cell survival was assayed in J82 cell line and the J82/MMC derivative after treating them with sequential ALA-mediated PDT and mitomycin C, and with the sequence of treatments reversed. Cell survival was estimated using the tetrazolium assay. The relative mitochondrial density of the two cell lines was estimated using flow cytometry to measure 123rhodamine fluorescence. RESULTS: The effect of sequential mitomycin C followed by ALA-mediated PDT enhanced the effect of PDT in both cell lines. In J82/MMC this effect was marginally supra-additive. When ALA-mediated PDT was administered before mitomycin C, the combined effect was 'sub-additive'. 123Rhodamine fluorescence was > 10 times greater in J82/MMC than J82, suggesting a significantly higher mitochondrial density in the former than the latter. CONCLUSION: Mitomycin C appears to enhance ALA-mediated PDT when administered first. This appears to be particularly so in J82/MMC. This phenomenon may have clinical significance in recurrent superficial bladder cancer.     

Photodynamic Therapy of Cutaneous Lymphoma Using 5-Aminolevulinic Acid Topical Application

BACKGROUND: Photodynamic therapy (PDT) with topical application of 5-aminolevulinic acid (ALA) is a new and effective modality for treatment of superficial basal and squamous cell carcinomas. OBJECTIVE: We present the kinetics of ALA-induced protoporphyrin IX (PP) accumulation and the results of ALA PDT treatment on two patients with different stages (stage I and stage III) of mycosis fungoides (MF)-type cutaneous T-cell lymphoma (CTCL). METHODS: ALA-Decoderm cream was applied to the lesions for 16 hours. Spectrofluorescence measurements of PP accumulation were carried out before, during, and 1 hour after photoirradiation (580-720 nm) using the VersaLight system. RESULTS: Different patterns of PP fluorescence kinetics were observed in patients with early and advanced stages of the disease. During photoirradiation the intensity of fluorescence decreased depending on the lesion thickness. One hour after the photoirradiation procedure no PP fluorescence was observed in the stage I MF lesion, while in the thick stage III MF lesions, PP fluorescence reappeared; after an additional 10-15 minutes of irradiation PP fluorescence disappeared. Complete response with excellent cosmetic results was observed in the stage I lesion after a single irradiation with a light dose of 170 J/cm2; in five stage III lesions, complete response was achieved after fractionated irradiation with a total light dose of 380 J/cm2 (follow-up at 27 and 24 months, respectively). CONCLUSION: The results showed a high response of both stage I and stage III MF lesions to ALA PDT. This modality appears to be very effective and can be used successfully for MF treatment.     

Comparative study on the ALA photodynamic effects of human glioma and meningioma cells.

BACKGROUND AND OBJECTIVE: The purpose of this study was to compare the differential susceptibility to photodynamic therapy (PDT) mediated damage in human U-105MG glioma cells and CH-157MN meningioma cells in vitro using 5-amino-levulinic acid (ALA) as photosensitizer, and to determine if growth factors would enhance PDT-mediated damage of these cells. STUDY DESIGN/MATERIALS AND METHODS: U-105MG or CH-157MN cells were irradiated with polychromatic light in the presence of ALA. A Xenon lamp (150 W) was used as the light source. For the study on the effect of growth factor on ALA-PDT, cells were cultured in serum free medium for 24 hours. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet derived growth factor BB (PDGF-BB) was added to achieve a final concentration of 50 ng/ml. 30 minutes later, cells were incubated with ALA (100 microg/ml) for 24 hours, washed, and irradiated with light (11 J/cm2). MTT tetrazolium assays were performed 24 hours after light irradiation. RESULTS: The inhibition of metabolic cellular function in U-105MG cells by ALA depended on both light energy density and ALA concentration. The susceptibility to ALA-PDT was profoundly lower for CH-157MN meningioma cells than U-105MG glioma cells. When incubated with ALA (100 microg/ml), U-105MG cells exhibited an LD50 around 8 J/cm2 of light irradiation, whereas that of CH-157MN cells was more than 25 J/cm2. EGF, bFGF, or PDGF-BB did not have any effects on the susceptibility of these two cell lines to ALA-PDT. CONCLUSION: ALA-PDT was more effective in killing U-105MG glioma cells than CH-157MN meningioma cells. The differential susceptibility was likely due to differential accumulation of PpIX in these cells. EGF, bFGF, or PDGF-BB did not have stimulatory or inhibitory effect on the efficiency of ALA-PDT.