The primary goal of this study was to investigate whether the use of oral steroids would reduce the incidence of stricture formation after balloon photodynamic therapy in patients with dysplasia and early caner in Barrett's esophagus. The effect of other treatment parameters such as light dose and multiple treatments were also investigated.

We are investigating the use of Scanning Doppler Velocimetry to evaluate changes in tissue perfusion that occur during Photodynamic Therapy (PDT). In initial studies, this technique was used to assess changes in tissue perfusion in mice given PDT using Photofrin, Purlytin, or Lutrin. There was a rapid and complete loss of perfusion in both normal tissue and tumor in animals given PDT using Photofrin. Using Purlytin, there was immediate loss of perfusion at the tumor site. For PDT using Lutrin, there was a lot of perfusion in the tumor with relatively no change in the surrounding normal skin, confirming the high degree of selectivity of PDT with this photosensitizer. These reductions in persistent through at least 24 h after PDT.

The apoptotic response of normal brain and intracranial VX2 tumor following photodynamic therapy mediated by five different photodynamic drugs, Photofrin, ALA, AlClPc, SnET₂ and mTHPC, was evaluated in a preliminary retrospective analysis. Rabbit brain, with or without tumor, was treated by PDT with interstitial light delivery. Histological sections at 24 h post PDT were assessed by the TUNEL assay. Confocal fluorescence microscopy was used to determine the total apoptotic cell count and the spatial distribution of apoptotic bodies within the tissue. The data were confirmed qualitatively by light microscopy on adjacent H&E-stained sections. Light-only and drug-only controls produced background levels. The highest apoptotic count was seen with Photofrin. The counts in AlClPc-treated animals were not above the background level, while the other 3 photosensitizers gave intermediate levels. With some, but not all, drugs the spatial distribution of apoptotic bodies correlated well with the light fluence distribution. Apoptosis was seen outside the zone of frank coagulative necrosis. There was not apparent drug-dose dependency at the relatively high doses used here. The retrospective nature of this study did not allow optimization of the treatment parameters. Nevertheless, the findings have potentially significant implications, both for understanding the mechanisms of apoptosis in brain tissue and for improving the clinical use of PDT for treatment of patients with malignant brain tumors.

Since the photosensitizing agent mTHPC is insoluble in water, a formulation procedure, usually involving poly(ethylene glycol) and ethanol, is required for clinical and pre-clinical studies. Using this vehicle, we found that drug accumulation by murine leukemia L1210 cells in vitro was slow; only non-viable cells with damaged membranes showed rapid uptake of mTHPC. The drug ultimately localized in the cytosol, and subsequent irradiation led to mitochondrial > lysosomal >> membrane photodamage, and a rapid apoptotic response. The rate-limiting step in drug accumulation appears to involve dissociation of a transient albumin-bound aggregated species which exhibited a low fluorescence yield. Initial formation of this product may explain the unusual mTHPC pharmacokinetics in man recently reported. Dilution of mTHPC with human plasma led to a gradual increase in fluorescence yield as the drug became associated with lipoproteins and proteins. When a steady-state was reached, density-gradient ultracentrifugation indicated distribution of mTHPC to HDL > LDL >> albumin.

The poor prognosis for patients with malignant brain neoplasm has led to a search for better treatment modalities. Although gliomas are considered to be disseminated tumors in the brain, most recur at the site of the previous tumor resection. Improved local control would thus be of clear benefit. The utility of photodynamic therapy (PDT) in the treatment of brain neoplasms is investigated using a human glioma spheroid model. Specifically, the effects of PDT on human glioma spheroids are investigated using Photofrin^TM and 56-aminolevulinic acid (ALA). The effects of various irradiation schemes were monitored using a simple growth assay. A growth delay was observed at an optical fluence of approximately 35 J cm^-2 for spheroids incubated in Photofrin. Spheroids incubated in ALA were unaffected by the PDT treatment regimens examined in this study. This was most likely a result of inadequate photosensitizer concentration.

Photodynamic therapy (PDT) requires tissue oxygenation during light irradiation. Tumor hypoxia, either pre-existing or induced by PDT during light irradiation, can severely hamper the effectiveness of a PDT treatment. Lowering the light irradiation does rate or fractionating a light dose may improve cell kill of PDT induced hypoxic cells, but will have no effects on pre-existing hypoxic cells. In the current study, we used hyper-oxygenation during PDT to overcome cell hypoxia in PDT. C3H mice with transplanted mammary carcinoma tumor were injected with 12.5 mg/kg Photofrin and irradiated with 630 nm laser light 24 hours later. Tumor oxygenation was manipulated by subjecting the animals to 3 a.t.p. hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyper-oxygenation. With hyper-oxygenation, up to 80% of treated tumors showed no re-growth after 60 days. In comparison, only 20% of tumors treated while animals breathed normal room air, did not re-grow. To quantitatively evaluate the effects of manipulating tumor oxygenation, tumor p02 was measured with microelectrodes positioned in pre-existing hypoxic regions before and during the PDT light irradiation. The results show that hyper-oxygenation may oxygenate pre-existing hypoxic cells and compensate oxygen depletion induced by PDT light irradiation. In conclusion, hyper-oxygenation may provide effective ways to improve PDT treatment efficiency by oxygenating both pre-existing and treatment induced cell hypoxia.

Strategies for improving the clinical efficacy of photodynamic therapy (PDT) in treatment of solid cancers include applications of different types of adjuvant treatments in addition to this modality that may result in superior therapeutic outcome. Examples of such an approach investigated using mouse tumor models are presented in this report. It is shown that the cures of PDT treated subcutaneous tumors can be substantially improved by adjuvant therapy with: metoclopramide (enhancement of cancer cell apoptosis), combretastatin A-4 (selective destruction of tumor neovasculature), Roussin's Black Salt (light activated tumor localized release of nitric oxide), or dendritic cell-based adoptive immunotherapy (immune rejection of treated tumor).

Electrical impedance spectroscopy (EIS) and high-frequency ultrasound (HFU) have been evaluated in various in vivo and in vitro models as potential methods to monitor biological changes induced by photodynamic therapy (PDT). EIS was assessed in tumor-bearing rat leg in vivo, in multicell tumor spheroids in vitro, and in normal rat liver tissue in vivo. HFU measurements, both imaging and backscatter frequency scanning, were tested in normal rat brain and skin treated in vivo. Marked changes in the EIS spectra were seen in all 3 models following PDT, depending on the photosensitizer and treatment parameters. With HFU, significant increases in echogenicity of the PDT-treated tissues were observed, with evidence of dose dependency and correlation with apoptotic cell death in vivo. While the results are encouraging for both modalities, a number of technical problems remain, particularly in the case of EIS, before these methods can be used reliably in mechanistic and clinical PDT applications.

To evaluate the role of ALA induced fluorescence diagnosis in laparoscopic surgery, we induced peritoneal carcinosis in rats by multilocular intraabdominal tumorcell implantation (CC531). The animals were photosensitized by intraabdominal ALA lavage. Laparoscopy was performed with both, conventional white and then blue light (D-Light, KARL STORZ Germany) excitation. Laparoscopy with conventional white light showed peritoneal carcinoma foci from 0.1 to 2 cm in diameter. All macroscopically visible tumors (n equals 142) were fluorescence positive after laparoscopic blue light excitation. In addition, 30 laparoscopic not visible (white light) tumors showed fluorescence and were histologically confirmed as colon carcinoma metastases. We conclude that only ALA induced laparoscopic fluorescence detection after blue light excitation is the adequate method to detect the entire extent of the intraabdominal tumor spread. Fluorescence laparoscopy is essential for laparoscopic staging of colorectal cancer because of a higher rate of cancer foci detection.

Photodynamic diagnosis by fluorescence emission from PPIX induced by 5-ALA can be done with minimal damage to cells using excitation by low power blue light. This technique uses the fluorescence signal emitted by the PPIX to obtain information about its concentration and dynamics without cell damage. The intracellular peak concentration of PPIX has a distinct time dependence for each organ. We have measured the accumulation time for maximum PPIX concentration in Wister rats (liver and skin) detecting the induced fluorescence in each organ. PPIX accumulation time varies depending on the tissue type. The liver shows a higher PPIX concentration. The detection system can measure endogenous PPIX. This fact opens the possibility of in-vivo displastic tissue diagnosis since these tissues have significant endogenous PPIX concentration.

A new photosensitizer, Trithia Sapphyrin was synthesized with 3 sulfurs and 2 nitrogen inside the macrocycle. The relative effectiveness is influenced by molecular properties, which control the penetration and distribution of the sensitizer in the system prior to the photon capture. To be precise, the effectiveness of the sensitizer depends on (1) the ability to partition from the bathing medium to a region of the cell membrane where it is exposed to a low polarity environment, (2) ability to absorb light in such a low polarity environment, (3) its triplet quantum yield in this environment. In view of these factors, the partition coefficient for different concentrations, ranging from 1 to 50 (mu) g/ml of trithia sapphyrin in the Octanol/Saline system was determined. The partition coefficient for 5 (mu) g/ml is observed to be the highest, which indicated the higher partitioning of trithia sapphyrin towards the octanol phase i.e. towards the membrane. The uptake of sensitizer was analyzed in 0.05% hematocrit for different periods of incubation ranging from 5 minutes to 5 hour, in order to find (1) binding of sensitizer to the cell membrane, (2) partition of the sensitizer molecule between the aqueous phase and lipid phase and (3) diffusion of the pigment molecule into the intracellular aqueous phase. With these observations, Photohemolysis studies were carried out for different pre and post incubation time as a function of light dose and sensitizer concentration.

Intracellular photodynamic reactions by nonlinear excitation of porphyrin photosensitizers have been induced using near infrared ultrashort laser pulses at 200 fs pulse width, 80 MHz pulse repetition rate and 2 mW mean laser power. In particular, a highly focused 780 nm pulsed laser scanning beam was employed at a frame rate of 1/16 s^-1 (60 microsecond(s) pixel dwell time) to expose Photofrin-labeled and aminolevulinic acid (ALA)-labeled Chinese hamster ovary cells. Intracellular accumulation and photobleaching of the fluorescent photosensitizers protoporphyrin IX and Photofrin have been studied by non-resonant two-photon fluorescence excitation. Subsequent scanning of the sensitizer-labeled cells resulted in reduced cloning efficiency of 50% and 0% after about 13 scans (approximately equals 10 mJ) and 50 scans, respectively, in the case of Photofrin accumulation (5 (mu) g/ml) and after about 24 scans and 100 scans in the case of ALA administration (1.5 mg/ml). Live/dead assays revealed the loss of vitality of most of cells after 50 scans for Photofrin-labeled cells and 100 scans for ALA-labeled cells. Sensitizer-free control cells could be scanned more than 250 times (1.1 h) without impact on the reproduction behavior, morphology, and vitality.

This paper describes a low-cost system for the illumination and real-time imaging of skin lesions. The light source is a mercury arc lamp, filtered to preferentially excite the fluorescence of protoporphyrin IX (PpIX) formed within the skin after the topical application of 5-aminolaevulinic acid (ALA). A video camera with a reduced-frame-rate, i.e. integrating, enables low intensity fluorescence imaging. Appropriate filtering provides independent images of the illumination uniformity, PpIX fluorescence and autofluorescence. Subsequent image processing yields false color images of the tissue surface illustrating the extent of ALA application, tumor boarder, surrounding satellites and the possible demarcation of treatment zones.

Abstract not available.