There is a widely recognized need for new approaches to effect endometrial ablation as an alternative to hysterectomy for treatment of menorrhagia. Photodynamic therapy (PDT) offers one such approach. We have investigated the use of 5-aminolaevulinic acid (ALA)-based PDT of the endometrium in model systems and in a series of patients. In all of this work, the ALA was administrated directly into the uterine cavity to reduce any possibility of systemic photosensitization. In a series of experiments in perfused ex vivo uteri, ALA was introduced into the cavity and protoporphyrin formation was measured in the endometrium, the underlying myometrium and the perfusate. ALA transfer into the perfusate was also measured. This work demonstrated that protoporphyrin formation in the endometrium was approximately ten fold that in the underlying myometrium and that systemic photosensitization would be unlikely to result form transfer of administered ALA from the uterus into the circulation. Similar results were found in studies carried out in vivo, where ALA was administered to patients scheduled for hysterectomy. Using a specially designed light source, the first patients have now been treated by giving intrauterine ALA followed by laser light. Two series of treatments - 10 in all - have been carried out. Eight patients have one year follow up. A reduction in measured menstrual blood loss was demonstrated in all but one patient. Complete symptomatic relief was obtained in tow women who did not require further treatment.

We are reporting the results form intraoperative intracavitary PDT treatment in 56 patients with recurrent supratentorial gliomas who had failed previous surgery and radiotherapy. These patients received 2mg/kg Photofin iv. 12-36 hours prior to surgical resection of their tumor or tumor cyst drainage. The median survival times in weeks for glioblastoma (GBM), malignant astrocytoma (MA), malignant mixed astrocytoma-oligodendroglioma and ependymoma were 30, 40, >56 and >174 weeks, respectively. Eight patients with recurrent GBM who received >60 J/cm² had a median survival of 58 weeks and 24 patients who received <60 J/cm² survived 29 weeks. The survival of patients with recurrent glioblastoma who undergo surgical treatment alone is only 20 weeks. We are also reporting the results of PDT treatment in 20 patients with newly diagnosed MA or GBM treated with intracavitary Photofin-PDT at the time of their initial craniotomy. The median survival of the whole cohort was 44 weeks with a 1 and 2 year survival of 40 percent and 15 percent, respectively. The median survival of patients with GBM was 37 weeks with a 1 and 2 year actuarial survival of 35 percent and 0 percent, respectively. The median survival of patients with MA as 48 weeks with a 1 and 2 year actuarial survival of 44 percent and 33 percent, respectively. Six patients with a Karnofsky score of >70 who received a light dose of >1260J had a median survival of 92 weeks with a 1 and 2 year survival of 83 percent and 33 percent, respectively. The mortality rate in our total series of 93 PDT treatments or brain tumor is 3 percent. The combined serious mortality-morbidity rate is 8 percent.

Photodynamic therapy using Photofrin has been used as an alternative to esophagectomy for patients with dysplasia or superficial cancer associated with Barrett's esophagus. In this update we present the results in 71 patients treated and followed for 6-72 months. 54 patients had high grade dysplasia/early cancer, and 17 had low grade dysplasia. 22 Patients had early cancer and 1 had T2 cancer. Three separate PDT treatments were required in 3 patients, 2 in 20 patients and 1 in 48. All patients were maintained on omeprazole. Patients received a photofrin dose of 2 mg/kg followed two days later by 630 nm laser light from an either argon/dye laser or KTP/dye laser. The majority of patients received light from a balloon light delivery device. Dysplasia and carcinoma was eliminated or reduced in majority of the cases. 75-80 percent of Barrett's mucosa was replaced by squamous epithelium. 34 patients developed strictures. All responded well to dilation.

Lutetium texaphyrin (PCI-0123) is currently in clinical trials as a PDT agent for the treatment of cancer patients. The drug is cleared rapidly from the plasma, and photoirradiation can be performed shortly after drug administration.T He photosensitizer as yet does not appear to elicit any significant skin photosensitivity. These characteristics favor frequent multiple PDT treatments with PCI-0123. In order to support repeated PDT treatments in the clinic, the safety of multiple drug dosing was studied in rats and mice. In rats, each group received 5 consecutive daily intravenous administrations of 5, 15, 30, or 60 mg/kg/day of PCI-0123. There were no deaths in any of the groups, and no drug-related effects were detected in the 5 mg/kg/day group. In mice, there were no observable signs of toxicity after consecutive daily administration of 10 micrometers ol/kg/day of PCI-0123 for 13 days. The feasibility and efficacy of repeated PDT treatments were assessed in C3H mice bearing RIF-1 tumors. Repeated PDT proved to be superior to a single PDT treatment. Repeated PDT treatments were well tolerated. Seven PDT treatments were administered over a nine day period without significant toxicity while achieving good therapeutic responses. All six groups receiving repeated PDT treatments showed an improved response compared to groups receiving a single PDT cycle, and the improvement was statistically significant for five of these groups. Sixty-two percent of mice receiving four sequential daily treatments were cured, and daily treatments were superior to regimens with longer intervals between PDT cycles.

Lutetium texaphyrin (PCI-0123) is presently in clinical trials for the treatment of neoplasms. An argon-pumped dye laser has mostly been used to generate light for PCI-0123 photoactivation. However, lasers are expensive and produce a limited area of illumination, so the efficacy of light emitting diodes (LEDs) was investigated. An LED array was developed so that the spectral emission matched the far red absorption spectrum of PCI-0123. A preclinical PDT efficacy study comparing the laser and the LED was undertaken using EMT6-bearing animals. The LED and laser light sources were statistically comparable in eradicating the murine mammary sarcomas using PCI-0123 as the photosensitizer.

Photofrin uptake studies in brain tissues using bulk sampling techniques have demonstrated up to a 10-fold higher concentration in intracranial tumors compared to normal brain structures. This selective uptake is in part attributed to the leaky blood-brain barrier (BBB) in brain tumors while intact BBB in normal brain excludes Photofrin from the extravascular space. However, in vivo preclinical studies have shown a very high sensitivity in normal brain to photodynamic therapy (PDT) despite this selective uptake. In vivo computed tomography (CT) measurement of the blood- brain transfer constant (K) and cerebral plasma volume using an x-ray dye as a tracer was used to monitor the degradation process of the BBB in normal brain during and post PDT. In these experiments, it was observed that K increased as early as 15 minutes after the onset of PDT, peaking at approximately 3 hr post PDT treatment followed by a drop close to baseline values for 3 out of 4 animals. On the other hand, the cerebral plasma volume showed a decrease also as early as 15 min post PDT onset, but its decrease continued up to 6 hrs with no apparent vascular stasis at the end of the time period. The results suggested a continuous process of BBB breakdown and vascular shutdown shortly after start of PDT.

The applicability and limitations of the photodynamic threshold model were investigated for an intracranial tumor (VX2) and normal brain tissues in a rabbit model. Photodynamic threshold values for four different photosensitizers, i.e., Photofrin, 5(delta) -aminolaevulinic acid (5(delta) -ALA) induced Protoporphyrin IX (PPIX), Tin Ethyl Etiopurpurin (SnET₂), and chloroaluminum phthalocyanine (AlClPc), were determined based on measured light fluence distributions, macroscopic photosensitizer concentration in various brain structures, and histologically determined extent of tissue necrosis following PDT. For Photofrin, AlClPc, and SnET₂, normal brain displayed a significantly lower threshold value than VX2 tumor. For 5(delta) -ALA induced PPIX and SnET₂ no or very little white matter damage, equalling to very high or infinite threshold values, was observed. Additionally, the latter two photosensitizers showed significantly lower uptake in white matter compared to other brain structures and VX2 tumor. Normal brain structures lacking a blood- brain-barrier, such as the choroid plexus and the meninges, showed high photosensitizer uptake for all photosensitizers, and, hence, are at risk when exposed to light. Results to date suggest that the photodynamic threshold values iares valid for white matter, cortex and VX2 tumor. For clinical PDT of intracranial neoplasms 5(delta) -ALA induced PPIX and SnET₂ appear to be the most promising for selective tumor necrosis.However, the photosensitizer concentration in each normal brain structure and the fluence distribution throughout the treatment volume and adjacent tissues at risk must be monitored to maximize the selectivity of PDT for intracranial tumors.

The synthesis of protoporphyrin IX (PPIX), a photosensitizer, occurs in the mitochondria of cells. Continuous light exposure activates the PPIX while it still resides in the mitochondria. The mitochondria are especially sensitive sites for lethal damage. In tissue culture experiments, cells were treated by PDT in two ways: (1) the cells were preloaded with PPIX by overnight incubation in medium containing ALA then exposed to light for various time periods, and (2) cells were not preloaded but rather were placed in medium containing ALA at the start of light exposure. Light exposure was 100 mW/cm². After exposure the cells were plated and incubated for seven days to watch for colony formation. The results of experiment 1 showed that in preloaded cells, the large amount of PPIX that accumulates was easily photobleached in just 5 minutes which yielded a drop in cell survival to a level of 2 percent. Further light exposure up to an hour had no additional effect on cell survival. But when light exposure was continued past one hour, cell survival began to drop again. The results of experiment 2 showed that light exposure had no effect in the first hour, but thereafter continued exposure caused continued exponential drop in cell survival. In both experiments, cell survival dropped to 1/e its value for every 8 minutes of continued light exposure.

Photodynamic therapy (PDT) is rapidly becoming an accepted therapeutic modality for the treatment of some types of malignant tumors. An important feature of PDT is its absolute dependence on molecular oxygen during light irradiation.Hypoxic tumor cells, either pre-existing or photochemically depleted of their oxygen supply during light irradiation, are resistant to PDT treatment, and contribute to treatment failures. We hypothesize that tumor response to PDT can be improved by combining PDT with Hyper-oxygenation, which may simultaneously compensate for the oxygen depletion by PDT and increase oxygenation of the pre-existing hypoxic cells. The doubling time of mammary carcinoma tumors, implanted in either leg or flank of C3H mice, was evaluated after PDT treatment with/without addition of hyperoxygenation. By adding hyperoxygenation to a non- curative PDT dose, a further delay in the tumor regrowth was observed. For a sub-curative does PDT treatment, the addition of hyperoxygenation resulted in an increase in tumor cure. The results indicate that tumor response can be improved by combining PDT and hyperoxygenation.

Photodynamic therapy (PDT), which is now an approved treatment for many types of cancers, is based on the simultaneous involvement of three factors, namely: tumor tissue retention of a specific photosensitizer; local illumination of the lesion with a visible light source and the occurrence of oxygen in the triplet state. Theoretically, a change in any one of these factors may be compensated by a change in the other two factors, leading to the same therapeutic result. In practice, this is not true, since we are dealing with living tissue, but we may expect to find an ideal combination of these three factors which may give the best clinical results. In this work we present experimental results of PDT under Hyperbaric hyperoxia (HBO) in tumor masses of the dorsal subcutaneous tissue of rats. These tumors were created by previous inoculation of 'Walker 256' neoplastic cells Hematoporphyrin Ester (HpE) was administered as the photosensitizer. The rats were pressurized at up to 3 atm with a 100 percent continuous oxygen ventilation environment in a specially designed hyperbaric chamber. The skin area above the tumor was photosensitized for 45 minutes with a 7 mw HeNe laser. Twenty four hours later, the tumor was removed for study. In all the animals treated with PDT/HBO histology revealed a very important reduction in the number of tumor cells as compared with the PDT controls in normal atmospheric condition, showing numerous apoptotic as well as necrotic cells at the border of the radiated area. The observed enhancement in the PDT for this situation is, of course, related to the extra oxygen in the circulatory system.

Production of ¹O₂ during PDT may be limited as a consequence of tissue oxygen depletion by the photodynamic process. This may in turn limit cytotoxicity during PDT. One possible way of controlling oxygen consumption during treatment is through modification of fluence rate. We have studied the impact of fluence rate on tumor oxygenation and direct PDT cytotoxicity using the RIF murine tumor and the photosensitizer Photofrin. Both fluence rates caused an acute decrease in tumor pO₂ to severely hypoxic levels. With 150 mW/cm² light median pO₂ remained low during prolonged exposure, while with 30 mW/cm² light median pO₂ values recovered to above control levels. When tumors treated with 135 J/cm² at each fluence rate were tested for cell survival in a clonogenic assay, 30 mW/cm² significantly decreased both cell clonogenicity and plating efficiency compared to light-only controls. Slight but insignificant decreases were found with 150 mW/cm². During in vitro PDT the fluence rate of light delivery had no effect on cell survival. In summary, we have found that low fluence rate improves tumor oxygenation and direct cell effects during PDT.

We compared sites of photodamage with modes of PDT-induced cell death, using murine leukemia tumor cells in vitro. Photodamage to mitochondrial or mitochondrial/lysosomal sites, but not tot he outer membrane, evoked a rapid apoptotic response: DNA changes were observed within 10 min, chromatin fragmentation within 1 hr after PDT. When limited photodamage to the cell membrane also occurred, we observed a delayed apoptotic response; fragmented nuclei were not detected until 24 hr after PDT. More drastic membrane damage, resulting in impaired amino acid transport, prevented an apoptotic response to PDT. A hypothesis accounting for these observations is proposed. Results of in vivo studies suggest that the more efficacious sensitizes generally produce an apoptotic response to PDT, but this question is not yet fully resolved.

The impact of bensoporphyrin derivative monoacid ring A, and visible light was determined for mouse splenic dendritic cells (DC), potent antigen-presenting cells (APC) of the immune system. It was discovered that sub-lethal doses of BPD-MA and light significantly altered the surface receptor pattern of DC as well as diminishing the capacity of these cells to activate allogeneic T cells. Treatment of highly purified DC with BPD-MA and 690 nm wavelength light decreased DC expression of major histocompatibility (MHC) Class I and II antigens, leukocyte common antigen CD45, intercellular adhesion molecule-1 (ICAM-1, CD54), the co- stimulatory molecules CD80 and CD86, CD95 as well as integrin CD11c. In contrast, DC expression of leukocyte function-associated-1 (LFA-1, CD11a), CD11b, CD18, CD40, and the DC DEC-205 receptor increased after the treatment. Changes in receptor levels occurred rapidly. DC MHC Class I and ICAM-1 expression declined to 40 percent of control levels by 2 hours post-PDT. DC treated with BPD-MA and light were poor stimulators of allogeneic T cells in the mixed leukocyte reaction. BPD-MA, in the absence of light, had no effect on the immunostimulatory properties of these cells. The changes in DC receptor expression pattern produced by BPD-MA and light were comparable to those produced by ultraviolet B light, a treatment known to alter the immunostimulatory characteristics of DC. Photodynamic therapy with BPD-MA represents an innovative approach for the modification of immune reactivity.

We have studied the influence of preirradiated by visible light hematoporphyrin derivative (HpD) solution in PBS on the production of reactive oxygen species (ROS) and phagocytosis of latex particles by rat peritoneal polymorphonuclear neutrophils (PMN), and also on the delayed type hypersensitivity reaction (DTH) to sheep red blood cells in mice. The release of ROS and phagocytic activity were observed by means of registration of the luminol- enhanced chemiluminescence (ChL) in the absence and the in the presence of latex particles. Non-irradiated HpD did not influence neither spontaneous ChL response, nor latex- activated. HpD preirradiated by 135 J/m² did not affect spontaneous, but increased latex-activated ChL response by 20 percent. This fact indicates an increase in PMN phagocytic activity under the treatment with preirradiated HpD. Increase in preirradiation fluence up to 8.1 kJ/m² resulted in significant enhancement of spontaneous ChL and inhibition of latex-activated ChL response of PMN. Results of spectroscopic analysis showed negligible decease in HpD Soret band after preirradiation of HpD by the highest fluences used in this study. We could not detect any significant photoproduct formation by differential absorption spectroscopy. Earlier, we have propose the photoinduced aggragation as one of the possible mechanisms of photodegradation of aqueous porphyrin solutions. In all probability, the increase in ROS production by PMN, treated with preirradiated HpD can be attributed to the phagocytosis of aggregates formed. It is possible that ROS can influence directly the DTH-effector cells leading to the observed decrease in DTH reaction level.

Lasing action in strongly scattering media containing a fluorescent dye and pumped by a pulsed high peak power laser can be used to produce light sources which may be suitable for surface or intracavity light delivery in photodynamic therapy, eliminating the need for a dye laser to obtain selectable treatment wavelengths. The present study focuses on evaluating the effects, in cylindrical fiber tip sources for interstitial light delivery, of fluorophore concentration and scattering particle density on lasing peak power, emission wavelength and maximum deliverable, clinically useful fluence-rate and radiant exposure. The sources tested are comprised of Rhodamine 640 perchloride incorporated into a TiO₂ based scattering matrix in either ethylene glycol or methanol. The cylindrical fiber tips, 10 mm long and 2 mm in diameter, were pumped via a 320 micrometers diameter multimode optical fiber, achieving line narrowing to approximately 7 nm FWHM at approximately 617 nm, using pulse energies of 1.7 mJ, delivered in 10 nsec from a Q-switched, frequency doubled Nd:YAG laser. The results showed the dependence of the total gain length in the pump volume and reabsorption effects in the remaining volume of the fiber tip. Sources capable of delivering sufficient radiant exposure for clinical use were achieved. While these sources are promising, for clinical use of these fiber sources pump lasers delivering MW pulses at high repetition rates are required to achieve acceptable total irradiation times.

This study compares the photosensitizer concentration measured non-invasively in vivo by diffuse reflectance spectroscopy with the results of post mortem tissue solubilization and fluorometric assay. The reflectance spectrometer consists of a fiber optic surface probe, spectrometer, and CCD array detector. The surface probe has eight fibers separated from the light source fiber by distances ranging from .85-10 mm. The imaging spectrometer disperses the light from each detector fiber onto the 2D CCD array, while maintaining spatial separation of each individual spectrum. A single exposure of the CCD therefore captures the reflectance spectrum at eight distances and over a range of 300 nm. From the spectra, the tissue's optical scattering and absorption properties are determined using a diffusion model of light propagation. Changes in the tissue absorption are used to estimate the photosensitizer concentration. Normal NZW rabbits were injected with AlPcS₄ and probe measurements made 24 hours after injection on the dorsal skin, on muscle after surgically turning the skin back, and on liver. A comparison of the noninvasive concentration estimates to the post mortem assay results finds good agreement for liver tissue. For skin, the non- invasive estimate is proportional to the true concentration, but low by a factor of 3. Based on Monte Carlo modeling of multilayered systems, this underestimate is attributed to the layered structure of the skin and nonuniform AlPcS₄ distribution.

Three time-delayed fluorescence images were used to calculate the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative (HpD). This allowed us to observe that the drug has a significantly longer fluorescence lifetime when it is incorporated in neoplastic tissues with respect to healthy surrounding ones. The imaging technique can be applied noninvasively in vivo in real time. Its diagnostic potential was tested on two murine tumor models after sensitization with low doses of HpD. In all the experimental conditions considered, the tumor could be easily identified in the lifetime images. Images were acquired also after removal of the skin overlying the tumor mass. Removal of the skin led to higher values of the lifetime contrast and allowed an even easier tumor localization.

The PDT uses the LASER characteristics in order to treat locally some types of cancers.Its principle is to irradiate the tumoral zones after the injection of a photosensitizer. Two processes take place during the treatment: one of them is a photochemical process and the other is a photothermal one. The parameters of major interest involved in these processes are the light energy, the oxygen rate in the tumor and the temperature. the aim of the light dosimetry is to optimize the light dose delivered by the LASER in order to enhance the therapeutic yield of the treatment. A sensor dedicated to this task has been studied in our laboratory. Our aim is to perform the measurement of several light dosimetry parameters with a single sensor. These parameters are the light attenuation coefficient, the oxygen saturation and the temperature. The general principle of the instrumentation is based on the backscattering of the light emitted by the optical treatment fiber. The associated instrumentation is composed with three parts: an optical structure, an electronic system and a software for the digital treatment. Our goal is to use this sensor in order to define a general method to extract the different parameters from the single backscattered signal.

The primary focus of laser based oncologic PDT has been on the treatment of skin and hollow organ tumors. Extending PDT to other primary internal lesions and metastasis requires a different approach. Light Sciences has developed a series of semiconductor-based devices which will be completely implanted in the patient using established, minimally invasive surgical techniques. These devices are energized noninvasively utilizing inductive coupling. The light delivery system will allow the clinician to modulate the intensity, spatial distribution, and duration of light delivery in order to maximize the benefits derived from each PDT drug dose. Light Sciences' technology minimizes patient risk and discomfort, is cost competitive, and expands the treatment options available to the clinician. Avoidance of lengthy operations, bone marrow suppression, and an emphasis on organ preservation allow this next generation of PDT light delivery devices to be effectively integrated with other forms of cancer treatment, if desired. We have termed our technique 'Multi-treatment Extended Duration PDT'. In what follows, we shall describe Light Sciences' technology and development of minimally invasive oncologic PDT.

A methodology is presented which enables quantification of the fraction of photosensitizer which is photo-converted to the triplet state within homogeneously scattering media. The method relies on a correlation of absorbance with diffuse reflectance changes measured in samples of photosensitizer in scattering medium and can estimate the fraction of photosensitizer molecules present in the active monomeric form. The validity of this approach is demonstrated for the sensitizer tin etioporphyrin in a model system of lipid in water emulsion and is expected to be applicable to biological systems such as cell suspensions and tissue.

The fluorescence spectra from stomach mucosa have been measured for cancer diagnostics using hypericin as a photosensitizer. Hypericin was administered orally in amount of 0.1 mg/kg b.w. four hours before conventional endoscopic procedure. Fluorescence was induced by He-Cd laser coupled to optical fiber probe which was inserted into a biopsy channel of the endoscope; the output power at the distal fiber end was 6 mW. Fluorescent light was collected fiber optically and registered by a spectroanalyzer in the range up to 700 nm. Detection algorithm included a comparison of characteristic orange fluorescence of hypericin at 600 nm with fluorescence at 530 nm in conjunction with results of white-light endoscopy. Fluorescence procedure was performed in thirty-seven patients with various oncological and other stomach disorders. It has shown an average 90 percent specificity in detection of small size lesions. Developed technique is promising to detect early stomach cancers and indicate an advantage of laser induced fluorescence with photosensitizer hypericin in differential cancer diagnostics.

Generation of reactive photoproducts from the chromophores and destruction of biomolecules can be realized with high efficiency if the irradiation of the solutionis carried out by an intensive laser radiation, when two-step light absorption and photoreactions from highly excited electronic states of molecules are taken place. We have observed nonlinear decomposition of sensitizers in tissues upon picosecond laser radiation by fluorescence method. The subsequent change of the autofluorescence spectra of sensitized tissues has ben observed. Fluorescence spectra of tissues sensitized by tumor-localizing sensitizers hematoporphyrin derivative (HpD) chlorin e₆ was registered using fiber-optic laser spectrofluorometer. It has been shown that initial rate of photobleaching is depended quadratically on the irradiation intensity. Two- photon excited fluorescence from tissues, sensitized by chlorin e(subscript 6$., and HpD has ben registered during excitation by picosecond YAG:Nd laser. These spectra coincided with that at one photon excitation, however, the background autofluorescence was absent. Red and near infrared radiation allows to increase the light penetration depth and as a result one can enhance the sensitivity of fluorescence diagnosis and the efficiency of photochemical treatment of tumors.