Most preclinical and clinical studies involving Photodynamic Therapy (PDT) use continuous wave light generated from argon ion pumped dye lasers or appropriately filtered lamps in order to activate photosensitizers. Flashlamp pumped dye lasers and copper vapor pumped dye lasers can induce photon saturation effects which result in suboptimal photosensitization. As Photodynamic Therapy becomes more widely accepted by the medical community, the availability of a specialized, low maintenance, high efficiency laser to the clinician has become an important issue. With an appropriate system reduced treatment time may be achieved through higher laser power output and the use of fiber splitters. In a study by Ferrario et al.,1 a quazi-continuous wave laser which couples a pulsed 532nm potassium titanyl phosphate (KTP) [Laserscope, San Jose] laser to a flow dye cell laser tuned to 630nm (PDT Systems 630) [PDT Systems, Santa Barbara] was evaluated in a variety of in-vitro and in-vivo PDT experiments. It was found that comparable results to those in argon ion laser studies were obtained in all experiments. In order to determine if these findings would translate to the clinical setting, an addendum was attached to an ongoing IRB approved study utilizing Photodynamic Therapy for the treatment of nonmelanoma skin cancer at the Western Institute for Laser Treatment. The purpose of this study was to further compare KTP pumped dye laser versus argon pumped dye laser systems utilizing the photosensitizer, Photofrin' 1 mg/kg in a series of skin cancer patients. PDT was offered as a potentially curative treatment for those patients with small, local lesions while being offered as a palliative modality for those patients with metastatic disease. 48 hours postinjection of photosensitizer, and after fluorescence detection in known and suspected tumor sites utilizing a krypton laser emitting at 408nm, the treatment areas were delineated. 630 +1 3nm of laser light (CW from an argon pumped dye laser and pulsed light from a KTP pumped dye laser) was delivered utilizing a flexible 400p. quartz forward surface microlens to the tumors. A total of 1 6 treatment sites were compared in a side by side fashion. Dosimetry factors including Total Dose, Dose Rate and lesional geometry were identical in sites compared. Tumors treated included metastatic breast adenocarcinoma, squamous cell carcinoma, basal cell carcinoma and Kaposi's sarcoma. The patients were followed up at 24 hrs., one week, four weeks, eight weeks, twelve weeks, and quarterly thereafter. Treatment selectivity, lesion response, and normal skin response were graded and recorded including photodocumentation. The conclusion drawn from our work was that in all parameters evaluated the results proved equivalent. There were no clinical differences in those patients treated with argon continuous wave or KTPpulsed dye laser systems. Subsequently a series of patients were treated with the KTP pumped laser system alone. Facilitated by the reliable delivery of 35 watts of 630nm light and the use of fiber splitters, three treatment areas could be illuminated at one time, reducing treatment time to one third of that of the argon pumped dye system.

Photodynamic therapy has the potential to treat and cure early carcinomas of the head and neck while preserving normal tissue. Thirty patients with neoplasia of the head and neck have been treated with PDT with follow-up to twenty nine months. Four patients with T3 and T4 carcinomas of the upper aerodigestive tract had a partial response. Eleven patients with T1 and T2 carcinomas of the larynx obtained a complete response and are disease free. Seven patients with T1 carcinomas of the tongue, floor of mouth, and nasal cavity obtained a complete response. Three patients with mucosal melanomas of the nasopharynx obtained a complete response and have remained disease free. Two patients with Kaposi's sarcoma of the oral cavity were treated. One obtained a complete response. Three patients with recurrent juvenile laryngotracheal papillomatosis obtained a complete response, but one recurred four months post-PDT. PDT is a promising therapy for treatment of early neoplasia of the head and neck. There are, however, limitations to this treatment based on tumor size and site. Methodology, clinical response, limitations, and complications will be discussed.

Delta Aminolevulinic acid (ALA), a precursor of Protoporphyrin IX (PP IX) in hem biosynthesis has been topically applied in urinary bladders in order to study its potential as fluorescent tumor marker. Preclinical experiments have been performed on chemically induced tumors in rats, revealing a ratio of PP IX-fluorescence intensity up to 20:1 in tumors as compared to healthy urothelium. Synthesis of PP IX has been stimulated in 56 patients by intravesical instillation of a pH-neutral ALA-solution. After an incubation time of two to four hours strong red fluorescence was endoscopically observed even in tiny superficial tumors. Brightness and contrast allows visualization of early stage urothelial diseases with naked eyes and without the necessity suppressing background fluorescence or violet excitation light.

The effect of different doses of the photosensitizer dihematoporphyrin ether (DHE) on the percent decrease of obstruction by endobronchial tumors produced by photodynamic therapy (PDT) was compared on the basis of mg/kg of body weight and mg/m² of body surface. We found statistically significant differences in the effect of different mg/m² when there was no difference when compared on the basis of mg/kg of body weight. There was an increase and then a decrease in the effectiveness as the mg/m² increased. We do not know how to interpret this. However, mg/m² provides a better parameter for effectiveness and we are now using 60 mg/m² of DHE for treating endobronchial tumors on the same day as the injection.

Four lesions of early carcinoma of the esophagus found during endoscopic biopsies in three patients were treated with photodynamic therapy. Follow-up biopsies over 9 - 24 months remain negative for carcinoma. Endoscopic ultrasonography is essential for proper staging and treatment planning for these patients. Photodynamic therapy may provide an alternative to surgical resection for early esophageal carcinoma or severe dysplasia in Barrett's esophagus.

The uptake and clearance of benzoporphyrin derivative - monoacid ring A (BPD-MA) in patients with primary and metastatic skin cancer were monitored using laser induced fluorescence in-vivo. These patients received systemic infusions of BPD-MA as part of a phase I clinical photodynamic therapy trial. The instrument used for photosensitizer detection consisted of two helium-neon (HeNe) lasers (612 and 633 nm) for fluorescence excitation and a phase sensitive detection system. The system was calibrated with various concentrations of BPD-MA using phantoms with scattering coefficients similar to that of skin. Measurements were carried out on unexposed normal skin near tumors, exposed normal skin immediately adjacent to tumors, and on the tumors themselves before drug infusion and at half hour intervals for up to three hours following infusion. Tumors were then exposed to 690 nm laser light for BPD-MA activation, during which time drug 'photobleaching' was followed by the loss of fluorescence. Following tumor irradiation, measurements were conducted for an additional two hours. Although fluorescence may be a useful means of monitoring photosensitizer concentration during photodynamic therapy, problems still remain. One is the linearity and the dynamic range of the response of the fluorescence detection device. Another is the uncertainty of the tissue depth probed by the fluorescence excitation source.

This experimental clinical stuay was started in 1985. Experiment involved 1010 mice and rats with 10 different types of inoculated a nd induced tumors as well as 10 dogs and cats with 7 malignant and 9 benign spontaneous tumors. The experimental pant of this study was aimed at comparing the action upon the tumors of Hematoporphyrin derivative ( HpD ) , when agents is excita ted in loco by light or gammaradiation . In 24 72 hours ,followina the injection of HpD into tumors bearing animals ,. the tumors were irradiated by the red light or by the gammaradiatiom of radio - active cobalt ( 60 Co ) using the dose 2 3 Gy. Such treat ment was applied only once. The positive effect of both procedures was observed in the majority of cases witn inoculated tumors and in all cases of induced and spontaneous malignant tumors. PDT has suc cessfully found its way in the Head and Neck surgery department of Lithuanian 0 ncology Centre since 1989. At first ,it was used as the palliative treatment, and later on as the radical method of treat ment. The total o 42 patients has been treated : 6 previously un treated patients, the others had the recurrent desease, or tumors non responsive to other modalities . There were 72 cancer foci in 42 pa tients treated by 108 courses of PDT in all. Three new and effective PDT methods were proposed y us I Q' Applying PDT for patients with oral cancer we injected photosensibilizator into the artery branch supplying the tumor. Canu la was installed into the superficial temporal artery and Hernatoporphyrin ( 5 mg ) was injected with immediate irradiation of tumor with laser light ( each cm2 of the tumor absorbed the energy dose of 200 J.) Within the next 24 - 4-8 hours full tumor tissue necrosis was found. If the effect of such PDT course was not adecuate , this treatment has to be repeated. 2 ) The second treatment modality was applied for the patients with cytologically verified melanoma. Following the ultrasonic exarnina tion of melanoma depth, PDT was applied preoperatively ( the energy dose to be absorbed by each cm2 of melanoma was thcr.eased up to 250 J.) with wide mela noma excision 5 to 7 days later. Histological study showed no melanoma tissue. 3 .) Hematoporphyrth ( Hp ) excitation by amrnaradiation was used. Intravenous Hp was administered and 48 hours later, the tumors were 1radiated with the gammaradlatlon of radioactive coba1t, using the single dose of 3 Gy . This treatment was named by us Gamnadynarnic therapy ( ODT ) . GDT was used both : alone and in cornbthatlon with PDT. The total of 2 patients has been treated. The best effect of GDT was observed in patients with osteoenic sarcoma ( 2 patients ) and melanoma ( 8 patients ). Keywords ; PDT , gamma irradiation, G-ammadynamic therapy, melanoma, oral cancer.

Carcinoma of the penis is one of the Commonly encountered cancer in wales, being the 10th of the male malignant tumors.The traditional surgical therapy often lead to function disturbance and psychological suffering of the patients. It is, therefore, not easily to be accepted by the patients, thereby delaying the time of the treatment. From March 1984 to January 1990 we have used photodynamic therapy (PDT)for fifteen patients of this sort of cancer and good results have been obtained.

A series of experiments have been undertaken to ascertain the influence of dietary additives on the clearance of Photofrin. Post-treatment cutaneous photosensitivity continues to be a significant side effect of photodynamic therapy (PDT) in humans. Cutaneous photosensitivity in humans is evidenced by erythema and edema in exposed areas. Murine models were chosen to investigate the differences in cutaneous photosensitivity as measured by footpad thickness in the presence or absence of dietary additives. Additionally, radiation induced fibrosarcoma (RIF) cells were implanted into the subcutaneous space on the dorsal aspect of the foot. In this case, the effect of PDT on tumor growth kinetics was assumed to be proportional to Photofrin concentration. Photofrin concentrations in tumors were measured by HPLC. Serum levels for dietary additives were obtained where analytical methods were available. Ingested ethanol increased the clearance rate of Photofrin as demonstrated by measurements of Photofrin tumor concentration and by failure of RIF tumor to respond to PDT in groups treated with ethanol compared to controls.

Normal mice received hematoporphyrin derivative (HpD) i.v. prior to red light irradiation and the platelet-rich plasma was prepared and irradiated by red light. The platelets were processed for EM examination and stereological analysis. It was shown the 16 hrs after irradiation almost all platelets were necrotized; 8 hours after irradiation about one fourth of the platelets were necrotized and the remaining were considerably damaged. Immediately after irradiation a small number of platelets became necrotic and most other platelets were swollen and deformated, showing significantly increased mean area, perimeter and short axis, and mean cell volume and cell surface area. The findings indicate that platelets are highly sensitive to PDT action and can be directly and rapidly damaged by PDT even in the absence of vascular endothelial cells. The early platelet photoactivation may play an important role in the initiation of early vascular damage and microcirculatory alterations induced by PDT in vivo.

Efficiency of Ehrlich ascites cells radiosensitization by porphyrins is a function of irradiation dose. The maximal radiosensitization was reached at the dose--2Gy. Moreover, efficiency of cell radiosensitization depends on extracellular porphyrins concentration Hematoporphyrin (HP) and hemaporphyrin dimethylether (HPde) have different radiosensitizing effect on cells. One of the reasons of such phenomenon may be the different degree of hydrofobicity of those porphyrins. The studies of intracellular porphyrins concentration show that they differ in the case of HP and HPde in the same order as cell radiosensitization efficiencies.

The new hematoporphyrin derivatives--HpD-1 and dimethoxyhematoporphyrin--DMHp were studied. Their physico-chemical properties, activity in tumor PDT and influence on therapeutic effect of tumor radiation therapy and on toxicity of chemotherapeutic agents were investigated. Hematoporphyrin--Hp was used as control. Data, obtained by absorption spectroscopy, indicated that HpD-1 and DMHp strongly aggregates in aqueous solution and HpD-1 has many covalently bound structures. HpD-1 and DMHp effects in tumor PDT and radiation therapy were studied using 8 species of transplantable tumors. The sensitizers were injected to the animals intraperitoneally in a dosage of 5 - 20 mg/kg. In 48-76 hours after sensitizers were injected, the tumors were irradiated by the pulsed Raman laser pumped by the SHG of YAG:Nd³⁺ laser. The parameters of this laser system are as follows: the wavelength 630 nm, the pulse duration (tau) equals 1 ns, energy E equals 10 mJ, the repetition rate f equals 10 Hz, average power P equals 100 mW. The goal of our investigation was to attempt to optimize tumor radiation therapy and chemotherapy.

In preliminary studies, the efficacy of a new photosensitizer, pyropheophorbide-(alpha) - hexyl ether (HPPH #23), for use in photodynamic therapy (PDT) was assessed using the rat 9L gliosarcoma tumor model in subcutaneous flank tumors, intracranial tumors, and in vivo. Flank and intracranial tumors were irradiated with 75 - 203 J/cm² 24 hours after 0.3 - 0.6 mg/kg IV injection of HPPH #23. At 24 hours post-PDT, and flank tumors showed a range of necrosis at the highest laser dose from 50 - 100%. The overlying skin and underlying muscle were spared. Intracranial tumors exhibited moderate to severe hemorrhagic necrosis. Areas of brain adjacent to tumor within the irradiated field also showed some damage. In vitro phototoxicity of HPPH #23 was compared to that of Photofrin II (PhII). Cells growing in culture dishes were exposed to HPPH #23 or PhII for 20 hours, washed free of unbound drug, then irradiated at 2.5 J/cm², 17 mW/cm² at 665 nm (HPPH #23) or 630 nm (PhII). Irradiated cultures were maintained in dark incubators for an additional 4 - 5 days, and phototoxic inhibition of cell proliferation was quantified by the sulphorhodamine B spectrophotometric assay. Under identical irradiation conditions, the IC₅₀ for HPPH #23 (0.25 (mu) g/ml) was 10-fold lower than that of PhII (2.5 (mu) g/ml). Complete cell kill was achieved at sensitizer doses of 0.5 (mu) g/ml (HPPH #23) and 5.0 (mu) g/ml (PhII).

The kinetics of protoporphyrin IX (PPIX) synthesis, bioconversion to other metabolic products, and photobleaching were measured in cell cultures after incubation in media containing the metabolic precursor for heme synthesis, (delta) -aminolevulinic acid (5 ALA). A compartmental model described the kinetics in terms of rate constants for the three processes. The maximum amount of PPIX that can be attained in the cells and the concentration of 5 ALA in the medium that obtains this maximum were determined. Using this information, two dosimetry protocols are outlined which both involve complete photobleaching of the PPIX: (1) the classical acute protocol using maximum 5 ALA to produce maximum PPIX and a light treatment of about 0.5 - 1 hr, and (2) a novel prolonged protocol using continuous low-level 5 ALA delivery to produce only slightly elevated PPIX and an extended light exposure time of over 24 hrs.

Thus far, in PDT the photosensitizer is administered systemically in its active form. A new concept is the use of endogenous photosensitizers. ALA is a precursor of protoporphyrin IX (PPIX), a potent photosensitizer. The administration of exogenous ALA may induce biosynthesis of an excess of PP IX, that leads to photodestruction upon light exposure. The goal of our study was to examine dark toxicity of ALA, and its phototoxic potential in vitro. Bladder carcinoma cells (Waf) were incubated with ALA for 3.5 h at a concentration of up to 20 mM for examination of dark toxicity. Irradiation was performed with an argon-dye laser emitting light at a wavelength of 630 nm. After irradiation, cells were incubated for two days, fixed, then stained and counted. Dark toxicity of Waf-cells was appreciated after incubation with ALA beginning at a concentration of 15 mM. Therefore, photodynamic treatment was performed at ALA-concentrations of 1, 5 and 10 mM. After incubation with an ALA- concentration of 1 mM and irradiation, there was only a slight decrease in cell survival. After incubation with 5 and 10 mM ALA and irradiation at 48 J/cm² cell survival was decreased to 11 and 1.4%, respectively.

Quantitative data are presented on uptake and phototoxicity of the photosensitizing drug, 13² hydroxy bacteriopheophorbide a methyl ester, in three cell lines of melanotic (SkMel 25) and amelanotic (A375, Melur SP 18) melanoma. A375 is the most sensitive cell easily killed by irradiation with a laser diode at 775 nm by 2 J/cm². The Melur SP 18 cell line needs 5 J/cm² for the same effect. In both cases the decay of overliving cells obeys apparently the simple exponential law. In the melanotic SkMel 25 an overshoting plateau at low energy fluence was observed signalizing a metastatic potency induced by PDT. This is documented by a series of photographs. In principle, photosensitizing drugs with absorption maxima in the far red region are suitable to treat malignant melanoma with high efficiency. A method has to be developed to overcome PDT stimulation at low energy densities in melanotic melanoma.

This study was made in order to determine whether liposomes can bind and deliver the photosensitizer to human bladder carcinoma cells and how effective the photodynamic activity of this photosensitizer is. TMHP (synthesized by Prof. Muller v.d. Haegen) was incorporated into small unilamellar vesicles of DPPC, following the procedure described by Jori et al TMHP was used in a dosage of 2.5, 5, 10, and 20 (mu) g/ml on two different cell lines. Cellular uptake of TMHP in liposomes was observed by fluorescence microscopy. Dark toxicity became evident, when doses of 10 and 20 (mu) g/ml TMHP encapsulated in liposomes were compared to control liposomes without photosensitizer. PDT was performed after sensitization of cells for one hour using an argon-pumped dye laser at 630 nm and a power density of 30 mW/cm². Irradiation with 3,6 and 7,2 Joule/cm² resulted in a decreasing survival rate. This study demonstrates PDT-efficiency being dependent on the dose of liposome-encapsulated TMHP as well as the fluence rate. There is also a difference in cell survival according to the cell line.

Different aspects of photodynamic therapy in normal rat brain tissue have been studied, in an effort to understand and improve the dosimetry of this new modality in treatment of brain tumors. dosimetry parameters, including light energy dose, fluence rate and beam size, and drug dosage were studied. PDT induced lesion depth in brain was measured as a biological endpoint. Effective attenuation depth and absolute light fluence rate distribution under superficial irradiation were measured using invasive optical probes. Photosensitizer uptake was quantified using HPLC analysis. The results indicate that normal brain have a high intrinsic sensitivity to PDT treatment, based on the estimated photodynamic threshold.

Immunophototherapy (IPT) is an experimental method of medical diagnosis and treatment the seeks to provide for the initial detection and subsequent selective destruction of diseased cancer cells such as the squamous carcinoma cells found in malignant tumors resident in cancer of the larynx. Monoclonal antibodies that are specific to squamous cells will be used to detect and treat this neck cancer. These antibodies are tagged with photosensitive compounds and metal colloids and then intravenously injected into the patient. The tagged antibodies quickly and selectively bind to the squamous cells in the larynx and other affected organs. The cancer cells are then selectively destroyed by irradiation of these complexes with light of the proper wavelength. This light activates the photosensitive material which then creates singlet oxygen that destroys the cells. Toxic products of lysis are quickly discharged from the body by activation of the reticuloendothelial system. IPT has been demonstrated to be very effective in the in vitro selective destruction of specified cell types.

A fluorescence imaging device exploiting differences in tissue autofluorescence between pre- malignant, malignant and normal tissues was developed to detect and clearly delineate the exact location and extent of these lesions. The device consists of a Helium-Cadmium laser (442 nm) for illumination and two image-intensifies CCD cameras with a red and green filter to capture a red and green fluorescence image simultaneously onto an imaging board of a computer. A pseudo-image is then computed and displayed in real time. In patients with known or suspected lung cancer, dysplastic lesions and carcinoma in-situ were localized that would otherwise escape detection by conventional white-light bronchoscopy. The extent of endobronchial spread of tumor can be accurately determined, which is important for successful application of photodynamic therapy for early lung cancer. The same device can also be used to study the uptake and distribution of fluorescent antineoplastic agents such as Taxol and Doxorubicin. Preliminary studies in mice bearing SCCVII squamous sell carcinoma suggest preferential retention of Taxol in tumor tissues 4 to 44 hours after intravenous injection.

The observation of photobleaching is affected by the tissue optics. The apparent time constant for photobleaching at a particular irradiance of light is related to the true photobleaching constant, D, in J/cm² by a factor which depends on the tissue optical properties, especially at the wavelength used to excite fluorescence. We report in vivo experimental measurements of protoporphyrin IX (PPIX) photobleaching in subcutaneous rat tumors which suggest tentative values for D of about 105 +/- 23 J/cm² during 630 nm irradiation. The effect of such a photobleaching constant on the photodynamic dose, P, in photons/cm³ absorbed by PPIX, was calculated. We estimate that an initial PPIX concentration of about 0.2 (mu) g/ml is required such that P reaches twice the toxic threshold photodynamic dose, P_th, which is assumed to be similar to the 8.6 X 10¹⁷ value cited for PhotofrinII. The above estimates are subject to further investigation. This paper presents the mathematics underlying the process of photobleaching and its observation based on fluorescence monitoring.

Lasers have traditionally been the preferred light source for activation of the photosensitizing agents used in photodynamic therapy (PDT). Their monochromaticity, high power, and the ability to couple that high power into optical fibers have dictated their use. There are however, many potential applications for PDT which do not require fiberoptic light delivery, and thus, need not incur the high cost associated with the use of laser systems. Treatment of skin cancer, cervical cancer, and cancers in the oral cavity could be effectively treated with alternative light sources, which would greatly reduce the cost of treatment. This paper will describe the features of a preclinical light emitting diode (LED) based source for photodynamic therapy, designed and built by PDT Systems. The results of an animal study, using the photosensitizer SnET2 activated at 660 nm, which compared the efficacy of PDT performed with a dye laser system with that of the LED system will be presented. Future plans for a clinical version of the LED system will also be discussed.

As Photodynamic Therapy becomes more widely accepted by the medical community the need for specialized, low maintenance high efficiency light sources increases. We report on the design and performance of a medical dye laser which can be configured for activation of photosensitizers from 630 nm to 720 nm. The dye laser is pumped by the second harmonic of a surgical Nd:YAG laser, which is fiberoptically coupled to the dye laser. Design and performance of the system will be presented along with biological comparison of its performance to that of an Argon/Dye system as an activation source for Photofrin^R.

The goal of this study was to detect the presence of benzoporphyrin derivative-monoacid (BPD-MA) in tissues of a tumor bearing animal model. Eighty one Lobund-Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used. This animal model exhibits unique predictable, unilateral, metastatic spread. The animals were injected intravenously with 0.75 mg/kg of BPD-MA. A Helium-Cadmium (He-Cd) laser (442 nm, 17 mW) was used as an excitation source and coupled to a 400 micrometers core diameter fiber. Following laparotomy, exploration of the abdominal and inguinal area was performed with laser induced fluorescence. Fluorescence spectra of the primary tumor, bilateral lymph nodes, and various organs were recorded. Fluorescence measurements were conducted four hours post injection. The spectra obtained were characterized by a broadband autofluorescence (approximately 540 nm) and a characteristic peak of BPD-MA (approximately 690 nm). Overall, the BPD-MA concentration was higher in lymph nodes than in the skin, kidney, large bowel, muscle or spleen. Skin exhibited the lowest fluorescence intensity ratio, indicative of a lower drug concentration in this tissue. In summary, our results suggest that laser induced fluorescence spectroscopy may provide an alternative way of assessing the biodistribution of BPD-MA or other photosensitizers.

Images of superficially growing tumors marked with prophyrin-based photosensitizers (Photofrin II, P4P-mD) were recorded in vivo by delayed observation of fluorescence following pulsed laser excitation. A pulsed, solid state, widely tunable laser system was used together with an intensified CCD camera. The ratio of the fluorescence radiant exitances of tumor and normal tissue taken as contrast between tumor and surrounding tissue was studied systematically by varying excitation wavelength and delay time between the optical pulse and the opening of the intensifier of the CCD camera. The dependence of this ratio on excitation wavelength was explained qualitatively using a simple one-dimentional mathematical model, taking the penetration depth of the exciting laser radiation and the thickness of the tumor and the layer of normal tissue above it into account. Besides fluorescence images, delayed fluorescence emission spectra of tumor and normal tissue were recorded in vivo.

An isotropic probe for direct measurement of fluence rate in tissue was made by melting 110 micrometers -core fiber to less than 0.4 mm sphere and painting by scattering material. Anisotropy response for the best probe of 0.35 mm diameter was +/- 10% in angular range to +/- 150 degree(s). The general position error of the probe in the tissue provided by experimental setup was less than 0.1 mm. The fluence rate was measured in the human leg in vivo at a depth of up to 5 mm for incident irradiance of 100 mW/cm² and wavelength 625 nm (dye laser) for beam diameters of 1 mm and 5 mm. Effective attenuation coefficients of about 0.6 mm^-1 (for depth of up to 1 mm) and 0.7 mm^-1 (for muscle layers) were obtained. Differences in light distribution in the skin comparative to the deeper layers were not found.

The present study explores the possibility of utilizing photodynamic therapy (PDT) in treating localized prostate carcinoma. Optical properties of ex vivo human prostatectomy specimens, and in vivo and ex vivo dog prostate glands were studied. The size of the PDT induced lesion in dog prostate was pathologically evaluated as a biological endpoint. The data indicate that the human normal and carcinoma prostate tissues have similar optical properties. The average effective attenuation depth is less in vivo than that of ex vivo. The PDT treatment generated a lesion size of up to 16 mm in diameter. The data suggest that PDT is a promising modality in prostate cancer treatment. Multiple fiber system may be required for clinical treatment.

Superficial tumors of the bladder or in situ carcinoma could be interesting indications of Photodynamic Therapy (PDT), since a total mutilating cystectomy could be avoided. The plurifocality of the lesions requires a treatment of the whole mucose; the quantity of light energy must be homogenous and sufficient to induce a therapeutic effect, still non toxic for normal tissues. We therefore developed a system of treatment and intravesical dosimetry control so that the operator can have precise information on the light repartition in the bladder in real time to enable him to optimize the positioning of the irradiation source. This intravesical device consists of twelve light sensors with optical fiber distributed symmetrically against the walls of the bladder; the emitting source is constituted of a scattering isotropic sphere. The signals emitted by the sensors are converted into tension. The acquisition part of the system values consists of two parts : an analogic part, the values are multiplexed on a same oscilloscope track to see in real time their evolution according to the position of the emitting source. The other part is constituted by the numeric acquisition of values for further analysis. We developed, from a mathematical modelisation of the bladder, a centering program of the diffusor that indicates its position in the bladder, as well as a cartography program where the bladder is re-built by interpolation with the different lighting levels.

We have investigated the feasibility of using optical spectroscopy as a method for monitoring the efficiency of Photodynamic therapy in real time.

Mechanisms of cell death have been explored in cells and tumors treated with photodynamic therapy (PDT). Photosensitizers used for these studies were Photofrin, tetrasulfonated and nonsulfonated aluminum phthalocyanine, and a new silicon phthalocyanine [SiPc(OH)OSi(CH₃)₂(CH₂)₃N(CH₃)₂], referred to as PcIV. In mouse lymphoma L5178Y cells, a dose of PDT sensitized by PcIV which causes a 90% loss of cell survival induces apoptosis (programmed cell death) over a several-hour time course, beginning within 10 minutes of irradiation. Apoptosis is a metabolic process initiated by PDT-induced damage to membranes and triggered by the activation of phospholipases A₂ and C and the release of Ca⁺⁺ from intracellular stores. An endogenous endonuclease is activated and cleaves nuclear DNA in the internucleosomal region of chromatin. Subsequent metabolic events now appear to cause the loss of cellular NAD and ATP, the former a result of the activation of a second nuclear enzyme, poly(ADP-ribose) polymerase, by the endonucleolytically generated DNA strand breaks. Loss of ATP follows upon the loss of NAD needed for energy metabolism. Although the induction of apoptosis is efficiently produced by direct PDT damage to L5178Y cells, we now find that apoptosis is also produced by treatment of certain other lymphoid-derived cells and cells of epithelial origin. Under the limited set of conditions tested, there was no evidence for PDT-induced apoptosis in a fibroblast cell line, in mouse fibrosarcoma RIF-1 and L929 cells, in human adenocarcinoma A549 cells, or in human squamous cell carcinoma cells in culture. The evidence suggests that apoptosis, a form of metabolic cell death, is an important mechanism of tumor ablation in PDT-treated tumors, and that the induction of apoptosis may involve the interaction of direct PDT damage to malignant cells with factors produced by PDT action on vascular and other host cells.

Intracellular events associated with photosensitizers and their subsequent activation with light are as yet poorly understood. Using a model photosensitizer, benzoporphyrin derivative, monoacid ring A (BPD), we have studied cellular uptake and release using both cell lines and normal murine splenocytes. These studies showed that maximum uptake was effected rapidly, peaking between 15 and 30 minutes following exposure to BPD. Absolute quantities taken up varies substantially between cell sources, malignant cell lines and activated normal cells taking up significantly higher levels. Release of BPD from cells occurred equally rapidly when they were removed from BPD-containing medium. Phototoxicity studies indicated that more sensitive cell structures were exposed to the photosensitizer with increased time of incubation. Further experiments were conducted to study protein expression in a murine tumor cell line following BPD treatment. We observed that the oxidative stress associated with photodynamic therapy resulted in the induction of a set of heat shock or stress proteins, and that the pattern of expression was similar when tumor cells were treated in vitro and in vivo.

A variety of porphyrins and related structures can sensitize cells to light; many of these agents can also promote ultrasound-induced cytotoxicity. Subcellular sites of localization sensitizers with a sufficient fluorescence yield can be assessed by fluorescence microscopy, but this becomes difficult when (Phi) _F is low. We have explored several indirect procedures for assessing examining loci of photodamage and sonodamage. Damage to lysosomal structures was probed with acridine orange, mitochondria with Rhodamine 123 and the plasma membrane with several diphenylhexatriene (DPH) derivatives. Additional information on alterations in heterogeneity of binding of diphenylhexatriene derivatives to photodamaged cells was provided by a distributed fluorescent lifetime study. Using a sulfonated benzochlorin, which photosensitizes cell-surface loci, we evaluated four DPH derivatives for their sensitivity to membrane damage. Anionic or cationic DPH derivatives were the most sensitive in this regard. Enhanced cytotoxicity associated with ultrasound + porphyrins yielded no detectable effects on mitochondrial or lysosomal structures, and barely detectable changes in membrane interactions with DPH derivatives, suggesting an 'all or none' effect.

The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD₅₀). In contrast, PC3 showed only marginal apoptosis at the LD₅₀ but had a marked response at the LD₈₅. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (< 1 hr). H322a cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

Fluorescence spectroscopic studies have been carried out on tissue sensitization by Aluminium Sulphonated Phthalocyanine (AlSPc) and endogenous Protoporphyrin IX induced by administration of exogenous 5-aminolaevulinic acid (ALA). A charge-coupled device (CCD) imaging system has been used to obtain quantitative fluorescence distributions of sensitization in frozen sections taken from rat tumors together with normal adjacent tissues. Using ALA, specific porphyrin sensitization of malignant epithelium is observed with much less sensitization present in connective tissue. Photodegradation of AlSPc and PPIX was studied by monitoring of fluorescence bleaching: in normal rat colon there is a significant reduction in AlSPc fluorescence at the edge of the photonecrosed zone which suggests that photodegradation may provide a means of diagnosing the extent of tissue damage. ALA- induced PPIX fluorescence is also observed to bleach in colon simultaneously with an increase in fluorescence emission near 675 nm which we attribute to a photoprotoporphyrin degradation product.

Sections of SCCVII tumor (squamous cell carcinoma) grown in C3H mice treated with Photofrin based photodynamic therapy (PDT) were examined by electron microscopy. The tumors were collected at different times after light treatment, and divided according to light penetration into superficial (treatment light oriented) and deep areas. The results show evidence of the progressive destruction of tumor cells occurring in both areas, with the presence of apparently intact tumor cells in the deep region 6 hours after PDT. These healthy looking tumor cells were often found adjacent to heavily damaged tumor cells, which is indicative of selectivity in their killing within the same microregion of tumor tissue. Many histiocytes and mast cells that appeared engaged in tumoricidal activity were also detected. It is suggested that in addition to lethality coming from progressive hemorrhagic necrosis, host immune cells infiltrating the tumor are elicited by PDT and contribute to the indirect killing effect.

Photohemolysis of human erythrocytes sensitized by sulfonated aluminum phthalocyanine was used as an endpoint to study possible chemical modifications of photodynamic therapy. Ascorbate, in concentrations up to 0.1 mM, had a small protective effect. In larger amounts it stimulated the rate of photohemolysis in a concentration dependent manner up to 1mM, by a factor of 2. Azide and D20 tests indicated some participation of singlet oxygen, although to a lesser extent than in the absence of ascorbate. Kinetic considerations augur for a reaction path initiated by an interaction of excited sensitizer-ascorbate, parallel to the singlet oxygen-mediated process. Because of the ubiquitous presence of ascorbate in human tissues in concentrations comparable to those of dissolved oxygen, it is a reasonable estimation that in photodynamic therapy, a fraction of the photodynamic damage proceeds via a Type I, ascorbate-assisted, mechanism. Tocopherol had an effect opposite to that of ascorbate, namely it inhibited the photohemolysis. Likewise, quercetin, a plant flavonoid, was protective against phthalocyanine-induced photohemolysis.

Improved control of tumor growth has been accomplished by modification of the irradiation scheme applied to hematoporphyrin-induced sensitization. Significantly enhanced efficacy of PDT was seen with either reduction of fluence rate or with an intermittent light-dark protocol, together suggesting that consideration must be given to photochemical oxygen depletion as a rate-limiting component in the production of singlet oxygen, the mediator of cytotoxicity resulting from PDT. A model to explain the basis for the improved efficacy was developed and is being tested by study of homograft and xenograft tumors in vivo and in multicellular tumor spheroids in vitro.

A study was conducted in the normal canine esophagus to compare continuous wave (argon- pumped dye-laser) and pulsed (KTP/532-pumped dye-laser) laser light for photodynamic therapy with PHOTOFRIN^R. 48 hours post injection, 630 nm laser light was delivered using a 24 mm diameter cylindrical esophageal PDT balloon positioned at either distal or proximal esophagus. A 1.0 cm cylindrical diffuser placed in the center of the balloon delivered 300 J/cm of light at an intensity of 400 mW/cm. Three dogs received continuous wave (CW) light proximally and pulsed light distally. Four dogs received CW light distally and pulsed light proximally. The light dose delivered to the esophageal mucosa was measured using three isotropic probes placed on the balloon wall. Similar mucosal light doses were verified for sites receiving pulsed or CW laser light. Two days after PDT, the severity of the esophageal lesions were evaluated endoscopically, grossly and histologically. While some response variability was observed among different animals, endoscopic examination of the lesions revealed comparable injury from CW and pulsed light in each subject. Based on the gross and histological examination of the lesions, the CW and pulsed laser-induced injuries could not be distinguished.

Photodynamic therapy (PDT) has emerged as a promising modality for the treatment of cancer. We are using newly synthesized and chemically defined and characterized porphyrin photosensitizers that are specifically labeled with deuterium to perform in vivo NMR studies in a murine tumor model. In vivo magnetic resonance offers the potential for repetitive, safe, noninvasive evaluation of photosensitizers, tumor metabolism, and the effect of PDT on the tumor metabolism. In an effort to monitor noninvasively the photosensitizers in an in vivo tumor model, we are synthesizing several deuterium labeled photosensitizers which absorb red light at or above 630 nm. Development of methods to test these photosensitizers directly in humans is not feasible at this time, since these photosensitizers are new and we do not yet understand the side effects. In addition, we do not understand the potential benefits compared with Photofrin II, the widely used photosensitizer. To perform our in vivo deuterium NMR studies on mouse foot tumors, we have constructed a solenoid coil which operates at 30.7 MHz for the deuterium nucleus. We have been able to detect the deuterium labeled photosensitizer in the tumor after a direct intra-tumor injection. The use of ³¹P NMR to predict the possible outcome of PDT in these tumors is also discussed.