The preliminary results of an ongoing Phase II trial of Photofrin-mediated intraperitoneal PDT (IP PDT) are presented. The clinical endpoints of this trial are to determine the response rates of patients with carcinomatosis and sarcomatosis to IP PDT and to document the toxicities of IP PDT in a defined patient population. Photofrin, 2.5 mg/kg, was administered intravenously 48 hours prior to debulking surgery and light delivery, 57 patients with ovarian cancer, gastrointestinal cancers, and sarcomas were enrolled. 44 patients received Photofrin and received light treatment. 39 patients are valuable for response. 8 of 39 patients had a complete radiographic response to IP PDT 3 months after treatment. 3 patients are alive without evidence of disease 6, 6 and 9 months after treatment. 1 patient is alive and has no evidence of intra-abdominal disease but has developed lung metastases. Toxicities include post-operative fluid shifts, hypotension, hydronephrosis, pleural effusions, enteric fistula, transient liver function test elevation, thrombocytopenia, and wound dehiscence. Toxicity is related to pre-operative tumor bulk and to the extensiveness of surgery required. IP PDT is feasible and leads to an initial clinical response rate of 25 percent in patients with incurable peritoneal carcinomatosis and sarcomatosis.

Photodynamic therapy (PDT) is a local treatment for malignment tumors. In a phase 2 trial in patients with supratentorial gliomas treated with 2 mg/kg Photofrin i.v. and intraoperative cavitary PDT, we were able to conclude that PDT was 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. The surgical mortality rate was less than 3 percent.

Limited therapeutic options exist when chest wall recurrence form breast cancer progresses despite standard salvage treatment. As photodynamic therapy offers excellent response for cutaneous lesions this may be a possible indication for PDT. A total of 102 treatment fields were illuminated on 9 women with biopsy proven chest wall recurrence of breast cancer which was progressing despite salvage surgery, radiation, and chemi-hormonal therapy. PDT consisted of outpatient IV infusion of Photofrin at 0.8 mg/kg followed 48 hours laser by illumination at 140-170 J/cm² via a KTP Yag laser coupled to a dye unit. No patient was lost to follow up. At 6 months post PDT; complete response, defined as total lesion elimination was 89 percent, partial response 8 percent, and no response 3 percent. No photosensitivity was seen and no patient developed scarring, fibrosis, or healing difficulties. Low dose Photofrin induced PDT is very active against chest wall lesions. Despite fragile and heavily pre-treated tissues, excellent clinical and cosmetic outcome was obtained. PDT is an underutilized modality for this indication.

Limited therapeutic options exist when chest wafl recurrence from breast cancer progresses despite standard salvage treatment. As photodynamic therapy offers excellent response for cutaneous lesions this may be a possible indication for PDT. A total of 1 02 treatment fields were illuminated on 9 women with biopsy proven chest wall recurrence of breast cancer which was progressing despite salvage surgery, radiation, and chemo-hormonal therapy. PDT consisted of outpatient IV infusion ofPhotofrmn at 0.8 mg/kg followed 48 hours later by illumination at 140-170 J/cm2 via a KTP Yag laser coupled to a dye unit. No patient was lost to follow up. At 6 months post PDT; complete response, defined as total lesion elimination was 89%, partial response 8%, and no response 3%. No photosensitivity was seen and no patient developed scarring, fibrosis, or healing difficulties. Low dose Photofrmn induced PDT is very active against chest wall lesions. Despite fragile and heavily pre-treated tissues, excellent clinical and cosmetic outcome was obtained. PDT is an underutilized modality for this indication.

The silicon phthalocyanine Pc 4, a photosensitizer activated with red light, has been studied for pathogen inactivation in red blood cell concentrates (RBCC). Pc 4 targets the envelope of pathogenic viruses such as HIV. To protect RBC during the process two main approaches are used: 1) Inclusion of quenches of reactive oxygen species produced during treatment. Tocopherol succinate was found to be most effective for this purpose. 2) Formulation of Pc 4, a lipophilic compound, in liposomes that reduce its binding to RBC but not to viruses. As a light source we used a light emitting diode array emitting at 660-680 nm. An efficient mixing device ensures homogeneous light exposure during treatment of intact RBCC. Treatment of RBCC with 5 (mu) M Pc 4 a d light results in the inactivation of >= 5.5 log₁₀ HIV, >= 6.6 log₁₀ VSV, and >= 5 log₁₀ of PRV and BVDV. Parasites that can be transmitted by blood transfusion are even more sensitive than viruses. Following treatment, RBCC can be stored for 28 days at 4 degrees C with hemolysis below 1 percent. Baboon RBC circulate with an acceptable 24 hour recovery and half-life. Genetic toxicological studies of Pc 4 with or without light exposure are negative. We conclude that a process using Pc 4 and red light can potentially reduce the risk of transmitting pathogens in RBCC used for transfusion.

Graft coronary artery disease (GCAD) is the chief complication following cardiac transplantation. Presently, there are limited treatment options. Insights into more expedient diagnosis and amelioration, if only partially, of GCAD are fervently sought. The selectivity of Antrin Injection (Lu-Tex) with subsequent photoactivation has been evaluated in several preclinical atherosclerosis models. The inhibitory effect of Lu-Tex induced photosensitization was demonstrated with human bypass coronary smooth muscle cells. The biodistribution of Lu-Tex was evaluated in a rat model of heterotopic cardiac allografts 60 days following transplantation. Lu-Tex was retained in the cardiac allograft, exhibiting a five-fold increase in retention between the allograft and native heart. These findings lead us to suggest that further studies are warranted to ascertain the merits of Lu-Tex for the diagnosis and possible attenuation of chronic graft vascular disease.

The extent of the apoptotic response of normal rat brain tissue to Photofrin-mediated photodynamic therapy was determined following sub-necrotic light exposures. Wistar rats were injected with 12.5 mg kg^-1 Photofrin and the brain surface was exposed to 1-17 J cm^-2 of 630 nm light 24 hours later. In order to identify apoptotic cells on tissue sections 24 hour post treatment, the TUNEL assay was used to fluorescently label DNA cleavage, with propidium iodide as a control counter stain. Confocal laser scanning microscopy was used to quantify the local spatial density of apoptotic bodies and to determine their depth distribution in the tissue from the irradiated surface. Radiant exposures of 1-3 J cm^-2 produced the highest apoptotic response, which occurred in well-circumscribed volumes, without evidence of gross tissue necrosis. Up to 40 percent of all cells in the treatment field were positively stained, with more apoptotic bodies observed at the edge of the lesion. The number of TUNEL-positive cells was lower at 5 and 17 J cm^-2. The apoptotic depth distribution was correlated with the light fluence distribution in the tissue over a limited depth range. Beyond this range there is evidence of a threshold for apoptosis, while at small depth and height radiant exposures there is a significant reduction in observed apoptosis. Hence, there is evidence that apoptosis is 'inhibited' by the presence of PDT-induced necrosis.

In the presented study, the intracellular PDT efficiency of a new poly-ethylene glycol (PEG) derivative of meso-tetra- chlorin (mTHPC) was tested in a cell culture model and the PDT dose-efficiency dependence determined using mTHPC as a reference. Squamous cell carcinoma cells incubated with mTHPC or mTHPC-PEG in 96-well microplates were irradiated with laser light. Cell viability was determined at several timepoints after irradiation by an MTS cell proliferation assay. Equal products of irradiant energy and applied doses of mTHPC-PEG, or mTHPC respectively, yielded identical PDT effects on the cells. PDT efficiency depended in a threshold-like manner on the applied light and sensitizer doses for either compound. For mTHPC-PEG, below 250 (ng/ml)*(J/cm²) no effect could be detected; with 1250 (ng/ml)*(J/cm²) and more, all cells were killed. mTHPC was 20 fold more effective than mTHPC-PEG. Since mTHPC-PEG shows higher tumor accumulation rates in several animal models than mTHPC, appropriate parameters and clinical applications should be found where the pegylation further enhances the potential of mTHPC PDT< although mTHPC-PEG is less effective than the free compound in vitro. In conclusion, the new mTHPC-PEG conjugate as a macromolecular and water-soluble variation of mTHPC shows intracellular PDT efficiency and therefore holds the potential for an improved mTHPC PDT.

Multiphoton excitation of photosensitizers in photodynamic therapy constitutes a promising approach, because of the increasing tissue penetration for longer wavelength of illumination. In this contribution the photodynamic activity of polyethylene glycol macromolecular derivatized mTHPC upon two-photon excitation is established. To test the photo- activity of the photosensitizer, human colon carcinoma cells, HCT-116, were incubated with 2 (mu) g/ml of mTHPC- CMPEG₄ in the nutrition medium. Subsequent pulsed laser irradiation at 784 nm focused down on growing cell monolayers restricts cell vitality clearly within 24 hours after irradiation. To investigate whether an anoxic or euoxic energy transfer mechanism is involved, a uric acid assay was performed to test for the generation of singlet oxygen. Upon single-photon excitation mTHPC-CMPEG₄ in TriPEG decomposed uric acid via the generation of singlet oxygen. Using femtosecond pulsed laser irradiation no decomposition of the uric acid was found, implying an anoxic energy transfer mechanism after tow-photon excitation. However, at present, we cannot exclude local hyperthermic effects in the cells containing the photosensitizer to contribute to the photodynamic activity upon two-photon excitation.

The development and implementation of PDT dosimetry for deep-seated tumors, is limited by the availability of clinically acceptable sensors capable of monitoring all PDT- efficacy determining parameters throughout the treatment volume. Here we present a multitasking optical fiber probe, with 250 micrometers OD, for the detection of the fluence-rate, photosensitizer concentration and partial oxygen pressure at one to five positions along the length of the optical fiber. The performance characteristics and preliminary evaluation of these fiber probes are described. The fluence-rate can be measured to better than 10 percent of the true value with a detection limit of < 10 (mu) W cm^-1. The detection limit for photosensitizer concentration is determined by the fluorescent quantum yield and the fluence rate at the sensor position. For typical PDT treatment conditions the detection limit for Photofrin is approximately 1 (mu) g ml^-1. The partial oxygen can be quantified for pO₂ < 2 mmHg.

Photodynamic therapy can initiate cell death by apoptosis or necrosis. Using agents with known patterns of sub-cellular localization, we examined the correlation between sites of photodamage and the mode of cell death, using murine leukemia cells in vitro. Mitochondrial or mitochondrial/lysosomal photodamage caused the rapid release of cytochrome c. This effect was not temperature sensitive, and could be demonstrated immediately after irradiation of photosensitized cells at 10 degrees C. Subsequent warming to 37 degrees C led to a rapid apoptotic response, consistent with the known ability of cytochrome c to trigger the activation of caspase-3. In contrast, lysosomal or lysosomal/membrane photodamage resulted in the release of cathepsins and other proteolytic enzymes. A subsequent incubation at 37 degrees C resulted in mitochondrial degradation, leading to loss of cytochrome c within 30 min. The apoptotic response was both delayed and incomplete, with many dead cells not exhibiting an apoptotic morphology. The latter outcome was traced to photodamage to procaspase-3, an effect not observed with sensitizers that caused mainly mitochondrial photodamage. Studies in a cell-free system demonstrated that agents with lysosomal and/or membrane targets could bring about photoinactivation of caspase-3. These result are consistent with the proposal that photodynamic therapy can both activate and inactivate components of the apoptotic process.

It is well known that photosensitizers used for PDT are liable to photobleaching. This phenomenon should be taken into account when developing the appropriate tactics of treatment. The present paper deals with detailed investigation of photobleaching of two photosensitizers: ALA induced protoporphyrin IX (PPIX) and sulphonated aluminum phthalocyanines. The fluorescence decay curves during light irradiation in vivo and in vitro have been obtained and analyzed. The mathematical model taking into account both first and second order photobleaching as well as spatial inhomogeneity of light distribution in tissue is presented. The experimental data for fluorescence decay of ALA induced PPIX during light irradiation are fitted to this mode. As opposed to PPIX the photobleaching behavior for sulphonated aluminum phthalocyanines is rather complicated to be fitted by proposed mathematical mode. The fluorescence outburning and residual fluorescence for sulphonated aluminum phthalocyanines have been observe.d It has been shown that fluorescence maximum correlates with blood oxygen saturation decrease induced by PDT effect.

We report on the development of a fluorescence imaging system for early detection of cancers in the GI tract. The system comprises a modified light source, endoscope, camera and associated image processing software. The photosensitizing agent is 5-aminolaevulinic acid, metabolized within the tumor to form protoporphyrin IX that absorbs strongly between 390-410 nm and fluoresces at 635 nm.

Photo-oxidation was studied during PDT in a model system using NADPH as the target substrate and Photofrin II as the photosensitizer. The efficiency of NADPH oxidation per photon absorbed by photosensitizer was determined as a function of substrate concentration. The kinetics of changes in absorbance was measured to assay oxidation of NADPH. The efficiency of oxidation increased with increasing NADPH concentration and approached a stable value of 0.0048. Using typical values for the quantum efficiency of the Photofrin triplet state generation and the efficiency of singlet oxygen production we obtained a value of the efficiency of interaction between singlet oxygen and NADPH. Efficiency of diffusion of singlet oxygen was modeled with molecular diffusion theory using typical values for the singlet oxygen lifetime and the diffusion coefficient in aqueous solutions.

PDT shows considerable potential as a treatment modality for superficial tumors. PDT is based on the accumulation of a photosensitizer in the target tissue. Subsequent illumination with light of an appropriate wavelength provokes a photochemical reaction that results in tumor destruction. Aminolevulinic acid (ALA) is a porphyrin precursor, and its administration result in the endogenous production of phototoxic porphyrins, which has been exploited for PDT. We assessed PDT efficacy employing both in vivo and in vitro models. We used papillomas, keratoacanthomas and in situ carcinomas chemically induced in the skin of SENCAR mice. Using ALA lotion and cream formulations, the maximal amount of porphyrin accumulation in papillomas was 5.52 (mu) g/g tissue. An energy of 150 of J/cm² was delivered by a copper-dye laser tuned at 630 nm. Microscopically, we found several signs of tissue destruction, more markedly in the upper strata of the in situ carcinomas. Papillomas, characterized by hyperkeratinization, were resistant to PDT. In our in vitro studies, we used an epithelial adenocarcinoma cell line. We tested ALA and its hexyl and methyl derivatives with the aim of increasing porphyrin synthesis. We found that hexyl-ALA was the best compound. When cultures incubated 3 hours in 0.6 mM ALA and 0.1 mM hexyl-ALA respectively were irradiated with 3 J/cm² only 5 percent of cells survived.

Chlorine₆ has been known to be attractive as photosensitizer (PS) for PDT for long. Its usefulness as a photosensitizing part of photoimmunotoxines for targeted PDT, as a synton for further chemical modification and as a promising PS has been widely recognized. There is a patented procedure at our disposal now allowing for preparation of a stable, well soluble and filtrating form of chlorine₆- 'Photodithazine'. The elaborated production technology includes 4 steps. 'Photodithazine's 1-octanol/Phosphate buffer, pH 7.4 partition coefficient has been found to be 1.4, indicating that it must be able to localize in the plasma membrane. This may be a partial explanation of its higher photodynamic efficacy in vitro, comparing to 'Photosense' and its closeness to 'Photogem'.

In vitro and in vivo biological evaluation of a novel drug substance 'photodithazine' has been performed. In vitro photocytotoxicity (EC50) was 1 (mu) M together with some cytotoxicity. In vivo acute toxicity has been found to be 170 mg/kg for KD10, 175 mg/kg for LD16, 197 mg/kg for LD50, 220 mg/kg for LD84 following 1 percent aqueous solution i.v. injection. Pharmacokinetics and biodistribution studies have been done using the same mice bearing inoculated under the skin of the flanks T36 embryocarcinomas injected i.v. with 40 mg/kg dose of the drug. Maximal tumor and most organs' uptake was attained 1 h.p.i., however, the drug's level in the organs rapidly decreasing to zero with the best tumor/muscle ratios over 10 by 5 h.p.i. 'Photodithazine' has been found to possess rapid clearance from the organism: 94 percent elimination 24 h.p.i. and 98 percent to 48 h.p.i. PDT has been performed in vivo involving 21-23 g A/Snell mice with the same type 0.9-1 g tumors injected 40 mg/kg drug i.p., with 670 nm light being delivered 5-6 h p.i. at different doses. The best irradiation dose has been found to be 170 J/cm² with a sound necrotic effect and 1-3 week remission. Thus, 'Photodithazine' represents a potent photosensitizer for PDT.

In 1998 a new second-generation photosensitizer was introduced in Russia - a chlorine derivative named Photodithazine - water-soluble preparation with absorption peak at 668 nm. This novel preparation has passed pre- clinical in vitro and in vivo studies. In 1999 a limited series of clinical testing of photodynamic therapy with photodithazine has been performed in the State Research Center for Laser Medicine of Russian Ministry of Public Health.

Photodynamic theory of tumors uses optical excitation of a sensitizing drug within tissue to produce large deposits of singlet oxygen, which are thought to ultimately cause the tumor destruction. Predicting dose deposition of singlet oxygen in vivo is challenging because measurement of this species in vivo is not easily achieved. But it is possible to follow the concentration of oxygen in vivo, and so measuring the oxygen concentration transients during PDT may provide a viable method of estimating the delivered dose of singlet oxygen. However modeling the microscopic heterogeneity of the oxygen distribution within a tumor is non-trivial, and predicting the microscopic dose deposition requires further study, but this study present the framework and initial calibration needed or modeling oxygen transport in complex geometries. Computational modeling with finite elements provides a versatile structure within which oxygen diffusion and consumption can be modeled within realistic tissue geometries. This study develops the basic tools required to simulate a tumor region, and examines the role of (i) oxygen supply and consumption rates, (ii) inter- capillary spacing, (iii) photosensitizer distribution, and (iv) differences between simulated tumors and those derived directly from histology. The result of these calculations indicate that realistic tumor tissue capillary networks can be simulated using the finite element method, without excessive computational burden for 2D regions near 1 mm², and 3D regions near 0.1mm³. These simulations can provide fundamental information about tissue and ways to implement appropriate oxygen measurements. These calculations suggest that photodynamic therapy produces the majority of singlet oxygen in and near the blood vessels, because these are the sites of highest oxygen tension. These calculations support the concept that tumor vascular regions are the major targets for PDT dose deposition.