We are updating the results of PDT treatment in 84 patients with supratentorial gliomas. These patients received 2 mg/kg Photofin iv. 12-36 hours prior to surgical resection oftheir tumor or tumor cyst drainage. There were 64 recurrent patients who had failed previous surgery and radiotherapy. The energy density range was 8 150 J/cm2 and the delivered light energy range was 440 -7200 J (median=1700J). The median survival times in weeks for recurrent glioblastoma (37 patients), malignant astrocytoma (16 patients), malignant mixed astrocytomaoligodentroglioma (7 patients) and ependymoma (4 patients) were 3 1, 50, <64 and <261 weeks, respectively. The thirteen patients with recurrent GBM who received 50J/cm2 had a median survival of 51 weeks. The eight patients with recurrent GBM who received <60 J/cm2 had a median survival of 58weeks. The mortality rate in our total series of 96 PDT treatments for brain tumor (metastatic and non glial primary tumors included) is 3%. The combined serious mortality-morbidity rate is 8%. Patients with malignant astrocytic tumors (GBM and MA) have a very poor prognosis. Nevertheless, PDT is safe in patients with either newly diagnosed or recurrent supratentorial malignant gliomas. There appears to be prolongation of survival in selected patients when an adequate light dose is used. Further improvement in survival may be expected with higher light doses. Key Words: Photodynamic therapy ofbrain tumors, Intracavitary photo-illuminatio

Photodynamic therapy (PDT) using Photofrin and 630 nm laser light was used to treat 100 Barrett's esophagus patients with dysplasia and early cancer. Twelve patients had superficial esophageal cancers (T1-T2, NO, MO). Laser light was delivered to the esophageal mucosa by cylindrical diffuser inserted through the endoscope or via a 3, 5 or 7 cm windowed esophageal centering balloons. Nd:YAG laser ablation was used on small residual islands of Barrett's mucosa during long term follow-up after PDT. Patients were maintained on omeprazole and were followed for 6 - 84 months following photodynamic therapy. Photodynamic therapy produced destruction of normal, dysplastic and malignant mucosa in treated areas. Approximately 75 - 80% of treated Barrett's mucosa healed as normal squamous epithelium in all patients. Complete elimination of Barrett's epithelium was found in 43 patients. Nd:YAG laser was required to ablate small residual areas of Barrett's mucosa in 35 of these patients. Dysplasia was eliminated in 77 patients. Ten of the 12 malignancies were ablated with no recurrence being found during follow-up. Healing was associated with esophageal strictures in 34% but after using the 5 and 7 cm balloons, the incidence of strictures decreased to 18%. All strictures were treated successfully by dilation. In summary, PDT alone or combined with Nd:YAG laser ablation, in conjunction with long-term acid inhibition provides an effective endoscopic treatment to eliminate dysplasia and superficial cancer in Barrett's patients, and reduce the amount of or eliminate Barrett's mucosa completely.

Background and objective: To compare the effectiveness in decreasing the amount of obstruction caused by endobronchial tumors when they are retreated with photodynamic therapy (PDT) several weeks after injection of Photofrin^R (porfimer sodium). Study design, materials and methods: The percentage of endobronchial obstruction from tumors before PDT and at the end of toilet bronchoscopy of 91 sites with PDT performed within 4 days after injection of porfimer sodium was compared to that obtained when PDT was repeated without re-injection of porfimer sodium in the time frames 2 - 4 weeks after injection to 11 sites and the period 4 - 8 weeks after injection to 17 sites. All patients were injected intravenously with 60 mg of Photofrin^R per square meter of body surface and all treatments were done with a power density of 500 mW/CF and a light dose of 400 J/CF delivered from cylinder diffusing fibers. Results: Paired Student's t tests and Wilcoxon signed ranks tests showed significant decreases in the percentage of endobronchial obstruction regardless of whether the PDT was first performed or repeated. Unpaired Student's t tests and Mann-Whitney U statistical comparisons showed a significant difference between the decrease of obstruction when treatment was performed within the first 4 days after injection (mean 41%) as compared to the repeated group 2 to 4 weeks after injection (mean 16%) and the group treated 4 to 8 weeks after injection (mean 19%). However there was no significant difference in the amount of decrease of obstruction between the 2 - 4 week group and the 4 - 8 week group. Conclusions: Photodynamic therapy to relieve endobronchial obstruction can be repeated without reinjection of Photofrin^R up to 8 weeks after injection with a significant decrease in the amount of obstruction. However, it will only be about 1/3 as effective as the initial treatment performed within the first four days of injection.

Photodynamic therapy (PDT) of locally recurrent breast cancer has been limited to treatment of small lesions because of non- selective necrosis of adjacent normal tissues in the treatment field. Lutetium Texaphyrin (PCI-0123, Lu-Tex) is a photosensitizer with improved tumor localization that is activated by 732 nm light, which can penetrate through larger tumors. We have evaluated Lu-Tex in a Phase I trial and in an ongoing Phase II trial in women with locally recurrent breast cancer with large tumors who have failed radiation therapy. Patients received Lu-Tex intravenously by rapid infusion 3 hours before illumination of cutaneous or subcutaneous lesions. In Phase I, Lu-Tex doses were escalated from 0.6 to 7.2 mg/kg in 7 cohorts. Sixteen patients with locally recurrent breast cancer lesions were treated. Dose limiting toxicities above 5.5 mg/kg were pain in the treatment field during therapy, and dysesthesias in light exposed areas. No necrosis of normal tissues in the treated field was noticed. Responses were observed in 60% of evaluable patients [n equals 15, 27% complete remission (CR), 33% partial remission (PR)], with 63% of lesions responding (n equals 73: 45% CR, 18% PR). In Phase II, 25 patients have been studied to date, receiving two treatments ranging from 1.0 to 3.0 mg/kg at a 21 day interval. Treatment fields up to 480 cm² in size were treated successfully and activity has been observed. Patients have experienced pain at the treatment site but no tissue necrosis. These studies demonstrate the feasibility of Lu-Tex PDT to large chest wall areas in women who have failed radiation therapy for the treatment of locally recurrent breast cancer. Treatment conditions are currently being optimized in the ongoing Phase II trials.

Laser induced fluorescence technique was used for detection of Lutetium Texaphyrin (Lutex) is vivo. Lutex is a new generation photosensitizer with rapid tissue clearance and deep red absorption characteristic. The detection system consisted of a nitrogen/dye laser (410 nm), an optical multichannel analyzer equipped with an intensified diode array, a specially designed fiber bundle, and a computer. Tissue fluorescence was first measured before injection of the Lutex to establish a baseline spectrum for each tissue. The fluorescence was then measured at 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, 72 hours, 1, 2, 3, and 4 weeks after the injection. All spectra were normalized with respect to the total photon counts to determine the lineshape. The baseline was subtracted from all other fluorescence spectra. The Differential Spectrum showed a negative peak around 478 nm where the fluorescence of tissue was maximally absorbed by Lutex. Fluorescence was measured from mucus membrane of the lip, skin, and tumor in one case. Data for lip and tumor are presented here. As Lutex cleared the tissue, the negative peak at 478 nm approached zero. The clearance of the negative peak is presented for both lip (normal tissue) and nodular cutaneous metastatic breast is presented. In summary, absorption of tissue auto-fluorescence by Lutetium Texaphyrin may be used to detect the in vivo drug non-invasively. This technique may be used to determine the clearance of the Lutex in a variety of tissues.

Lutetium texaphyrin (Lu-Tex) photodynamic therapy (PDT) relies on the presence of the water-soluble Lu-Tex, oxygen, and light (activation around 730 nm). Cytotoxic oxygen species are produced that cause irreversible damage to biological substrates. Damage may be inflicted via direct cell kill mechanisms or through vasculature effects that cause hypoxia. The addition of hypoxia enhanced drugs, such as Mitomycin C (MMC), can potentially increase the anti-tumor response. RIF-1 bearing C3H mice received 10 micrometers ol Lu-Tex/kg and were illuminated with 100 J/cm² 3 hours postinjection. Mice received MMC (2.5 or 5 mg/kg, before and after light) in conjunction with PDT and were compared to subsets of drug alone controls. A significant improvement in PDT response was observed when MMC was added to the dosing regimen; the effect was more pronounced at the highest MMC dose of 5 mg/kg: MMC prior to PDT gave a median tumor regrowth time (10X original volume) of 28 days compared to MMC and PDT alone, 16.3 and 14.9 days, respectively. The anti-tumor activity of lutetium texaphyrin induced PDT was improved by the addition of the bioreductive alkylating agent mitomycin C.

The dynamic behavior of lipophilic and hydrophilic sensitizers in cell cultures and non animal in vivo systems with varying incubation but also during the photodynamic therapy will be summarized within the presentation. As an appropriate in vivo system we used the chorioallantoic membrane (CAM) of fertilized eggs, which served as a substrate for tumor cells. Because the CAM is a transparent membrane it is possible to view individual blood vessels and to examine tumor cells as well as structural changes of the supplying vasculature. To adapt this system to high magnification microscopy, we established a new technique for in vivo observation of the CAM tissue. This technique enables online investigations of alterations at cellular level induced by drugs with confocal laser scanning microscopy. The localization of the drugs with clinical importance was observed after different application times in the lumen of the vessels, the endothelial cells and the tumor cells. In addition light induced subcellular Ca²⁺-changes were observed and correlated with the photodynamic process.

Photodynamic treatment with the phthalocyanine Pc 4, a mitochondrially localizing photosensitizer, is an efficient inducer of cell death by apoptosis, a cell suicide pathway that can be triggered by physiological stimuli as well as by various types of cellular damage. Upon exposure of the dye- loaded cells to red light, several stress signalling pathways are rapidly activated. In murine L5178Y-R lymphoblasts, caspase activation and other hallmarks of the final phase of apoptosis are observed within a few minutes post-PDT. In Chinese hamster CHO-K1 cells, the first signs of apoptosis are not observed for 1 - 2 hours. The possible involvement of three parallel mitogen-activated protein kinase (MAPK) signalling pathways has been investigated. The extracellular- regulated kinases (ERK-1 and ERK-2), that are thought to promote cell growth, are not appreciably altered by PDT. However, PDT causes marked activation of the stress-activated protein kinase (SAPK) cascade in both cell types and of the p38/HOG-type kinase in CHO cells. Both of these latter pathways have been demonstrated to be associated with apoptosis. A specific inhibitor of the ERK pathway did not alter PDT-induced apoptosis; however, an inhibitor of the p38 pathway partially blocked PDT-induced apoptosis. Blockage of the SAPK pathway is being pursued by a genetic approach. It appears that the SAPK and p38 pathways may participate in signaling apoptosis in response to PDT with Pc 4.

Treatment of mice with certain photosensitizers combined with exposure to visible light limits the development of the immunologically-mediated contact hypersensitivity (CHS) response against topically-applied chemical haptens. Understanding of the inhibitory action of photosensitizers upon the CHS response is incomplete. Benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin), a photosensitizer with immunomodulatory activity, strongly depressed CHS responses to the hapten dinitrofluorobenzene (DNFB). However, if mice were administered 1 (mu) g of a recombinant preparation of the pro- inflammatory cytokine interleukin-12 (rIL-12), full-fledged CHS responses to DNFB ensued in animals treated with BPD-MA and light. In contrast, when rIL-12 was given in combination with an anti-IL-12 antibody the restorative effect of rIL-12 on the CHS response of PDT-treated mice was blocked. Evaluation of the cytokine status of spleen and draining lymph node cells showed for DNFB painted animals, that the release of the immunosuppressive cytokine IL-10 was increased by PDT and rIL-12 counter-acted the increase in IL-10 liberation associated with PDT. These studies indicate that IL-10 formation is upregulated and the availability of IL-12 may be limited in mice treated with PDT. These features may contribute to deficient CHS responses observed with PDT.

Oxidative stress is the term used for a sudden and intense exposure of living tissue to reactive oxygen radicals. Tumor tissue response to oxidative stress, invoked in the action of photodynamic therapy (PDT) and some other modalities for cancer treatment, at the level of vascular endothelium has important therapeutic implications. Nitric oxide (NO), a transient radical species which is an important bioregulatory molecule involved in a diverse array of physiological events, has important functions in the regulation of progression of cancerous growth. Response to cancer therapies associated with the induction of oxidative stress was suggested to be amenable to NO mediation. Events involved in antitumor effects of PDT that can be markedly affected by changes in NO availability are listed. The correlation between endogenous NO production in tumors and the response of these lesions to PDT is discussed. Results of treatments aimed at modulating NO levels in PDT treated tumors are reviewed and evaluated.

We report on photodynamically induced inactivation of the skin bacterium Propionibacterium acnes (P. acnes) using endogenous as well as exogenous photosensitizers and red light sources. P. acnes is involved in the pathogenesis of the skin disease acne vulgaris. The skin bacterium is able to synthesize the metal-free fluorescent porphyrins protoporphyrin IX (PP) and coproporphyrin (CP) as shown by in situ spectrally-resolved detection of natural autofluorescence of human skin and bacteria colonies. These naturally occurring intracellular porphyrins act as efficient endogenous photosensitizers. Inactivation of P. acnes suspensions was achieved by irradiation with He-Ne laser light in the red spectral region (632.8 nm). We monitored the photodynamically-induced death of single bacteria using a fluorescent viability kit in combination with confocal laser scanning microscopy. In addition, the photo-induced inactivation was calculated by CFU (colony forming units) determination. We found 633 nm-induced inactivation (60 mW, 0.12 cm² exposure area, 1 hour irradiation) of 72% in the case of non-incubated bacteria based on the destructive effect of singlet oxygen produced by red light excited endogenous porphyrins and subsequent energy transfer to molecular oxygen. In order to achieve a nearly complete inactivation within one exposure procedure, the exogenous photosensitizer Methylene Blue (Mb) was added. Far red exposure of Mb-labeled bacteria using a krypton ion laser at 647 nm and 676 nm resulted in 99% inactivation.

PDT with sensitizers that target mitochondria for photodamage induces a rapid apoptotic response in murine leukemia cells. Immediately after irradiation, we observed loss of the mitochondrial membrane potential ((Delta) (Psi) _m), and transfer of cytochrome c from mitochondria to the cytosol. Within 3 min, there was a marked increase in caspase-3 activity, but not of caspases 1,4,5,6 or 8. While phosphorylation of a tyrosine residue on an 85 kDa protein was a prominent consequence of PDT, this was inhibited by the protein kinase C inhibitor staurosporin, a drug which enhanced the apoptotic response to PDT. Tyrosine phosphorylation may therefore not be a requisite for PDT-induced apoptosis. These results are consistent with a mechanism whereby mitochondrial photodamage, leading to the release of cytochrome c (and perhaps other factors), can directly trigger caspase activation and apoptosis. We have previously reported that the apoptotic response to PDT was delayed when both mitochondria and the plasma membrane were targeted for photodamage. We found that the membrane photodamage resulted in inhibition of neutral sphingomyelinase activity. This enzyme is one source of ceramide biosynthesis, suggesting that ceramide formation via sphingomyelinase activity enhances the apoptotic response to PDT.

Preliminary clinical studies of 5-aminolaevulinic acid (ALA) induced photodynamic therapy (PDT) with the maximum tolerated oral dose (60 mg/kg), currently appear to only produce limited amounts of necrosis. We have studied ways of increasing this effect without increasing the drug dose. In normal, female, Wistar rats we have found it possible to increase the area of necrosis produced in the colon substantially by simply interrupting the light dose (25 J, 635 nm, 100 mW) for a short period of time, while all other variables are kept constant. It is possible to cause up to four times more necrosis with a dose of 200 mg/kg ALA i.v. by introducing a single 150 second interval which splits the light dose into two fractions after 5 J has been delivered. We have found these parameters to be optimal for this dose. Likewise, in the same model, the effect of the iron chelating agent, CP94, was also investigated and we have found it possible to produce three times the area of necrosis with the simultaneous administration of 100 mg/kg CP94 i.v. and 50 mg/kg ALA i.v. We have therefore shown, that it is possible to significantly increase the effects of ALA induced PDT without increasing the administered dose of ALA by utilizing these techniques.

Photodynamic therapy (PDT) is an effective method to treat cancer and other nononcological lesions by means of light action on photosensitizer in tissue. It is assumed that the destroying effect is mainly due to the formation of singlet oxygen resulting from the interaction of light excited photosensitizer with molecular oxygen (triplet ground state). So the destroying effect will be proportional to the rate of singlet oxygen formation which in turn depends on light intensity, photosensitizer concentration and molecular oxygen concentration. The present work deals with the investigation of blood oxygen saturation in vitro and in vivo during light irradiation in the PDT process. It has been observed that SO2 behavior strongly correlates with the light power density applied for PDT and photosensitizer concentration.

Photodynamic therapy (PDT) may provide a new approach for treatment of patients with superficial transitional carcinoma and carcinoma in situ of the bladder. The light applicator for the bladder wall (Rusch) is constructed as a balloon catheter with two concentric balloons. A new PDT applicator (Rusch) was assessed for the homogeneity and accuracy of irradiation during PDT. In an in-vitro experiment with 17 freshly harvested porcine bladders the fluence rate was measured locally with isotropic detectors. The results were compared to the light fluence detected by the PDT applicator. The increase of the fluence rate (beta) inside the bladders due to back scattering ranged between 5.3 and 7.0 with an average of 6.2. Local variations of the fluence rate in the spherical bladders were also smaller than 15%. Therefore it is concluded, that a homogeneous and accurate irradiation during PDT is possible. Blood between the outer balloon and the bladder wall reduces the local fluence rate strongly and should to be avoided. Also larger air bubbles in the applicator can lead to an inhomogeneous light distribution. In regular application the presented new catheter system provides accurate and easy light dosimetry during PDT of the bladder. Attention had to be paid to a continuous flushing of the space between balloon and bladder wall in order to prevent the accumulation of urine and blood. To avoid a malfunction of the system and large errors in light dosimetry and application, it is advisable to monitor the measured light dosage and the shape of the balloon using ultrasonography during PDT.

Lipid peroxidation (LP) is involved in cell damage induced by photodynamic treatment (PDT) sensitized by some lipophylic porphyrins. We investigated an effect of lipophylic antioxidant (alpha) -tocopherol and its water-soluble analog, trolox, on meta-tetra(hydroxyphenyl)chlorin (mTHPC) sensitized PDT (413 nm) of cultured human colon adenocarcinoma cells (HT29). Cell survival was measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion to farmazan (MTT assay). Both antioxidants in concentrations lower than 0.1 mM did not affect photokilling of HT29 cells. These data might suggest that LP is not of crucial importance in cell damage photosensitized by mTHPC. One mM (alpha) -tocopherol or trolox decreased cell survival by ca. 15 and 13% respectively. Both antioxidants increased PDT- induced damage of HT29. Potentiation was evident as the decrease in the initial shoulder part of fluence dependence curve. We propose that antioxidants at height, pro-oxidant concentrations can potentiate PDT induced killing of tumor cells.

One hundred thirty patients with neoplastic diseases of the larynx, oral cavity, pharynx and skin have been treated with photodynamic therapy (PDT) with follow-up to 79 months. Those patients with primary or recurrent leukoplakia, carcinoma-in- situ (CIS) and T1 carcinomas obtained a complete response after one PDT treatment and 87% remain free of disease. Sixteen patients with deeply invasive T2 and T3 carcinomas were treated with PDT. Of those sixteen, ten obtained a complete response, but six have recurred locally. Although a response can be achieved with PDT in the larger solid tumors, it is not a consistent complete response because of the depth of invasion of the tumor. This is due to the inability to adequately deliver laser light to the depths of the tumor bed. Fourteen patients with massive recurrences of squamous cell carcinomas were treated with intraoperative adjuvant PDT following tumor resection. Two patients developed a local recurrence within the field of treatment. PDT is highly effective for the curative treatment of early carcinomas (CIS, T1) of the head and neck. T2 and T3 superficial carcinomas, with invasion less than 0.5 cm, are also curatively treated with PDT with significantly reduced morbidity compared to conventional modes of treatment. Also, intraoperative adjuvant PDT may increase cure rates of large infiltrating carcinomas of the head and neck.

M-THPC, a second generation photosensitizer, has greater potential of phototoxicity than the first generation PS hematoporphyrinderivative because of greater light penetration depth into tissue and higher therapeutic index. The uptake, selectivity and kinetics of C-14 labeled mTHPC was investigated in a C6 glioma induced rat model. The highest amount was detected at 48 to 96 hours after intraperitoneal injection with a ratio of 150:1 of tumor to normal brain concentration (0.53 vs. 0.003 (mu) g/g tissue). A high selectivity was also confirmed by confocal laserscanning microscope in frozen sections of the human glioblastoma. Up to now 15 patients underwent mTHPC-mediated PDT presenting with primary (n equals 2), recurrent (n equals 8) glioblastoma multiform or recurrent metastatic disease of the brain (n equals 3) and of the scull base (n equals 2). After sensitization with 0.15 Foscan^R mg/kg i.v. a gross tumor removal was performed on day 4 followed by intraoperative PDT by a KTP pumped dye laser or a diode laser emitting at 652 nm (light dose of 20 J/cm²). Patients with primary glioblastomas underwent additional radiation therapy with one progressing after 5 months, the other is surviving for 6 months, patients with recurrent glioblastomas demonstrated a median time to progression of 4 months and a median survival of 6 months, patients with metastasis faired better with only one progressing after 6 months the remaining 4 patients are alive demonstrating a complete response with a median survival time of 7 months. Our first clinical results of mTHPC mediated PDT in brain tumors demonstrate that the survival time of our patients are not superior as compared to the first generation sensitizer. Due to its superior photophysical properties however, mTHPC should be intensely investigated for its use in neurosurgery.

Electrical impedance spectroscopy (EIS) has been evaluated as a non- or minimally-invasive technique to monitor the acute tissue response to photodynamic therapy (PDT). In this study the EIS spectra of normal muscle tissue in the rat hind leg were monitored immediately before and at time intervals up to 96 hours post-PDT treatment with different photosensitizers (Photofrin, ALA-induced PpIX, BenzoPorphyrin Derivative), at varying photosensitizer and light doses. EIS measurements were made using a pair of solid matrix Ag-AgCl electrodes placed parallel to one another on either side of the muscle mass and interfaced to a precision LCR impedance meter scanning the frequency range 1 - 1000 KHz. Independent histological grading of tissue injury was performed on tissue sections from treated and untreated legs at the 96 hour end point. Significant and PDT dose-dependent changes in the EIS spectra following treatment were observed, including increases in conductivity which correlated with the immediate post-PDT edematous response with Photofrin and ALA and which resolved or partially-resolved over the measurement time course. Photofrin treatments exhibited a clear drug dose response at 96 hours that was evident in both the EIS spectra and the histological sections. These changes included significant tissue necrosis as well as edema, inflammation and early fibroplasia. The BPD data were less clear, but potentially quite interesting. Most striking were below unity ratios of treated-to-untreated muscle spectra components at 24 hours which reversed to above unity by 96 hours in the through skin measurements. This phenomenon is indicative of a tissue response distinctly different than that observed with Photofrin or ALA. These data also suggest that EIS measured changes are sensitive enough to detect differences in PDT-initiated tissue damage that may be photosensitize-specific. While the data are derived from a small number of animals, the findings are quite encouraging in terms of the potential for EIS to track PDT-induced changes in tissue.