A series of experiments was undertaken to determine the uptake of the photosensitizer tin ethyl etiopurpurin in the canine prostate. At increasing time intervals after intravenous injection, tissue photosensitizer levels were determined for the prostate, urinary bladder and other selected sites. Tissue effects resulting from either transurethral or transperineal delivery of light to the prostate after tin ethyl etiopurpurin-photosensitization were then assessed by light microscopy. Both resulted in hemorrhagic necrosis of the target prostatic tissue. Photodynamic therapy of the prostate using tin ethyl etiopurpurin as photosensitizer for the treatment of both benign prostatic enlargement and adenocarcinoma is worth of further investigation.

The protoporphyrin IX (PPIX) levels in rat liver, skin, and tumors in vivo in response to i.p. injection of delta-aminolevulinic acid were monitored by fluorescence spectroscopy. A pharmacokinetic model describes the PPIX dosimetry.

The results of experiments combining laser hyperthermia (LHT) wit photodynamic therapy (PDT) in the treatment of cancerous tumors have been very encouraging. Typically treatments have been sequential, employing separate hyperthermia and PDT systems. Sequential application of these therapies, however, fails to maximize the synergistic effects of combined, simultaneous treatment. This paper reports the results of an SBIR funded Phase I project intended to develop a system for precise simultaneous delivery of both LHT and PDT light, and to measure the synergistic effects os simultaneously applied LHT and PDT with both Photofrin II- and SnET2-mediated PDT. A system was developed that would allow the use to specify all treatment parameters and then deliver the combined light to the treatment site. Thermal and optical feedback in the system provide stability and precise control during the treatment. The system architecture, the computer control system, and the results obtained from bench-testing the system are presented in this paper. The system was then tested in a small animal tumor model. This paper will discuss the study protocol and the experimental results.

In this study, the effect of transdermal photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A (BPD) on the development of the immunologically mediated contact hypersensitivity (CHS) response against the hapten dinitrofluorobenzene (DNFB) and on the duration of skin allograft acceptance has been evaluated. In the CHS model it was found that the treatment of hairless strain mice with whole-body transdermal PDT using BPD (1 mg/kg) and LED light (15 J/cm²) resulted in a profound suppression of the CHS reaction if treatment was applied either 48 or 24 hours prior or up to 72 hours after sensitization of abdominal skin with DNFB. Less inhibition of the CHS response was observed if PDT was given one day before the ear challenge with DNFB which was applied 5 days following the initial DNFB sensitization. However, DNFB-exposed, PDT-treated mice retained the capacity to respond maximally to the unrelated contact sensitizer oxazolone. These results are consistent with other models of experimentally induced immune tolerance. allogeneic skin graft studies demonstrated that pretreatment of skin with BPD and light, at levels that did not cause significant tissue damage, significantly enhanced the length of engraftment. Using a separate protocol, photodynamic treatment of recipient mice at various times after transplant had no significant effect on allograft acceptance. Irradiation of skin in the presence of BPD may significantly inhibit the initiation of certain immunological responses within these tissues.

Highly phosphorescent photosensitizer Pd-tetra (o-methoxy-p-sulfo) phenyl porphyrin ( Pd-MSPP) was used to follow for interaction of its triplets with molecular oxygen in solid tumor of rats in vivo. Half an hour after intravascular injection of 2 mg of Pd-TMSPP in the rat the phosphorescence band having maximum at 710 n.m and life time of photosensitizer triplets have been measured after ns laser pulse excitation at 532 urn in tissues in vivo. The life time of triplets has varied between 100-300 js in different rats and has been independent on the time of laser pulse excitation. Then 2 mg of other photosensitizer MME cb.lorin e6 with absorption band at 660 mm have been injected and photodynamics effects have been investigated by the measurement of life time of Pd-TMSPP triplets depending on free oxygen content in tumor. Decrease of triplet life time of Pd-TMSPP have been observed during 10 mm of tumor exposition what have been interpreted as photoinduced increase of free oxygen content in the tumor. After following continuous exposition of the tumor slow continuous increase of triplet life time of Pd-TMSPP have been detected as result of developing photoinduced hypoxia. The quenching of Pd -TMSPP triplets by cells components in vivo have not been observed after blockade of tumor blood vessels and stopage of oxygen enter in tissues. The quenching of the sensitizer triplets takes place only in the presence of free oxygen in tissue. At least two exponential components of the phosphorescence decay shows that the distribution of the free oxygen is not uniform in tissues.

The effect of injection of 10 mg/g 50% glucose on photodynamic therapy of mouse transplantable S-180 sarcoma was studied. The concentration of hematoporphyrin monomethylether in plasma, skin, and tumor was measured by recording spectrofluorophotometer. tumor pathological section was made and necrosis area of tumor longitudinal section was measured by image processing after photoradiation of gold vapor laser. The results of this study suggested that the uptake of photosensitizer in tumor significantly increased while the uptake of photosensitizer in skin remained unchanged after glucose administration. Furthermore, glucose administration combined with PDT produced a greater tumor necrosis area than using PDT alone. The mechanisms and clinical significance were also discussed.

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the image contrast is derived from the fluorescence lifetime of the fluorochrome at each point in a 2D image. Lifetime images are created from a series of images obtained from two gain-modulated image intensifiers. One of them (II-1) detects the light-induced tissue fluorescence, whereas the other (II-2) detects the backscattered fluorescence excitation light. This light is modulated at the same frequency as the detectors, resulting in homodyne phase-sensitive image. These stationary phase-sensitive images are collected using two CCD cameras, digitized and manipulated with a mathematical operator in real time. A series of such images, obtained with both image intensifiers at various phase shifts between their gain modulation and the modulation of the excitation light, is used to determine phase angle and/or the modulation of the fluorescence emission at each pixel. The reference values of these phase angles and modulations are obtained with II-2, whereas II-1 enables the measurement of the phase and modulation of the fluorescence. Phase and modulation are related to the fluorescence lifetime of the fluorochrome. An advantage of the experimental method proposed here is that pixel-to-pixel scanning is not required to obtain the fluorescence lifetime image, as the information from all pixels is obtained at the same time.

AlGaInP diode lasers generate optical output in a range of the spectrum suitable for the activation of several second=generation photosensitizers including tin ethyl etiopurpurin, SnET2. The development of a system suitable for this application entails coupling the output of multiple highly divergent and astigmatic sources into a single optical fiber for most applications. Various methods of fulfilling this requirement will be discussed along with the advantages and drawbacks of each method. The incorporation into the clinical laser system of on-line dosimetry control of the light delivery parameters will be described along with the results of biological equivalency studies.

Photodynamic therapy, PDT, has mainly been done clinically using laser systems. The use of lasers to treat cutaneous lesions is not necessary. In order to provide a more efficient and cost effective light source for the PDT treatment of cutaneous lesions, a clinical based light emitting diode (LED) system has been developed. The system include a tight density packing of AlGaAs high-brightness LEDs to produce incident irradiance values up to 200 MW/cm². Initial systems have wavelengths of 665 nm and exhibit a bandwidth of 25 nm, FWHM. Other wavelengths are also possible. The system includes on-line dosimetry through an isotropic probe to permit the use to simply choose a dose, J/cm². the system has been tested against a KTP/dye laser for the photosensitizer SnET2 to determine its equivalence to a monochromatic source.

The variation in photodynamic dose achieved with protoporphyrin Ix (PPIX) and laser radiation was assessed in a cancer cell culture. The distributions of initial PPIX concentration, photobleaching light dose, and photodynamic dose were measured on 124 individual cells (rat mammary cancer cell line, MTF7) using video fluorescence microscopy.

Two significant questions arise when a drug for photodynamic therapy is introduced to the preclinical arena: (1) pharmacokinetics for the uptake and elimination and (2) relative retention by various tissues. Theses were addressed by following the pharmacokinetics in 21 rabbits with 130 tumors at different drug doses and at time intervals ranging from minutes to 24 hours. Kinetics were studied spectrofluorometrically by following extracting Protoporphyrin IX (PPIX), a metabolite of delta- aminolevulinic acid (ALA). Samples were compared to a standard, and are thus quantitative. The rise time in plasma was rapid, and reached maximum one hour post injection with drug doses of 50, 100, and 200 mg/kg. The single exponential fall time displayed a half life of fifty minutes, resulting in a complete elimination of PPIX in 24 hours. Uptake and retention in multiple tissues were measured 3 hours post injection of 100 mg/g ALA in 3 animals, with skin, tumor, and plasma measured in all 21 animals. Organs with high vascularity displayed higher values of PPIX. Results of this study indicate that ALA utilized as a systemically administered sensitizer maybe of significant value in PDT of selected tumor types.

Analysis of the results of photodynamic therapy (PDT) for treating malignant neoplasms of the skin, mammary glands, tongue, oral mucous, lower lip, larynx, lungs, urinary bladder, rectum,and other locations has been made. During 1992-1994 432 tumoral foci in 108 patients have been treated with PDT. All patients were previously treated with conventional techniques without effect or they were not treated due to contraindications either because of sever accompanying diseases or because of old age. A number of the patients had PDT because of recurrences or intradermal metastases within one to two years after surgical, radial, or combined treatment. Two homemade preparations were used as photosensitizers: Photohem (hematoporphyrin derivative) and Photosense (aluminum sulfonated phthalocyanine). Light sources were an American dye laser with argon laser pumping and homemade laser devices including a copper vapor laser-pumped dye laser, gas discharge unit, gold vapor laser for the Photohem sessions, wile for the Photosense sessions were used solid state lasers on yttrium aluminate. Up to now we have follow-up control data within 2 months and 3 years. Positive effect of PDT was seen in 90.7% of patients including complete regression of tumors in 52% and partial in 38.7%. Currently this new technique of treating malignant neoplasms is successfully being used in Russia; new photosensitizers and light sources for PDT and fluorescent tumor diagnostics are being developed as well.

From 1989 to 1994 more than 600 various malignant foci were treated by more than 400 courses of PDT in the Lithuanian Oncology Center. During the application of PDT we used the following lasers: 35 MW He-Ne laser with 630-1 nm wavelength; 100-300 MW dye lasers with a wavelength of 629-3 nm, and a 100- 300 MW gold vapor laser, with a wavelength of 629-1 nm. We noticed some differences in results between the low-power lasers and the high-energy lasers. The initial response was better in patients whose tumors were irradiated with high-energy lasers, but the was more recidivism in this group. The most interesting thin we noticed was that there were significant differences in response of tumors of various histogenesis. for example, in most cases there was full necrosis of basal cell carcinoma immediately after PDT, but there were only some necrobiotical changes at the same time in other malignant tissues. Full necrosis in squamous cell cancer was noticed ont he third to fourth day after PDT. Full necrosis of malignant melanoma was noticed on the fifth to tenth day after PDT. It is important to emphasize that full melanoma necrosis was more frequently observed in cases where the low-power He-Ne laser was used for the PDT.

The porphycenes represent a new class of photosensitizing agent with enhanced absorbance in the red. We examined the photodynamic effects of two porphycenes, a monomer and a pyrazine-linked dimer, using the P388 murine leukemia and P388/ADR, a subline that expresses the multidrug-resistance phenotype. The monomer localized at mitochondrial loci while the dimer photosensitized lysosomes and the cell membrane. the dimer was more phototoxic in terms of cells killed and photons absorbed, perhaps because of the difference in localization sites. Phototoxic effects of both porphycenes could be abolished by the addition of the oxygen scavenger trolox (a vitamin E analog) during irradiation. Both porphycenes were recognized by the multidrug transporter, an outward transport process associated with multidrug resistance. Photodynamic actio of the porphycenes in cell culture was associated with apoptosis, resulting in DNA fragmentation. Inhibition of phospholipase C activity prevented initiation of apoptosis after irradiation without protecting cells from photodamage at sites of sensitizer localization. At high concentrations, the porphycenes exhibit a 'dark toxicity' that also was associated with apoptosis, but subsequent irradiation abolished apoptosis, perhaps via destruction of endonucleases, and only necrotic cell death was observed.

Cells dissociated from tumors (carcinomas and sarcomas) growing subcutaneously in mice that have been administered Photofrin or other photosensitizers were analyzed by flow cytometry. Monoclonal antibodies were used for identification of major cellular populations contained in these tumors. The results demonstrate that a subpopulation of tumor-associated macrophages (TAMs) is unique among tumor cell populations in that it excels in the accumulation of very high levels of photosensitizers. These macrophages showed an increased expression of interleukin 2 receptor, which is indicative of their activated state. since macrophages were reported to concentrate in the periphery of human neoplasms, it is suggested that activates TAMs are the determinants of tumor-localized photosensitizer fluorescence.

Oxygen dependent phototoxicity was investigated in vivo in an orthotopic rat bladder tumor model. Two photosensitizers, benzoporphyrin derivative monoacid ring A and 5-aminolevulinic acid-induced protoporphyrin IX were studied. For a given cumulative light dose of 30 J/cm², enhanced tumor destruction was obtained for both photosensitizers by either using a low fluence rate or fractionated light delivery mode. These observations may be attributed to rapid local oxygen consumption during photochemical reactions.

We have previously reported on a microelectrode technique for mapping spatial and temporal distributions of oxygen in and around individual photosensitized multicell tumor spheroids. Steady-state and time-resolved measurements have been analyzed using diffusion theory ad have yielded estimates of the oxygen diffusion coefficient in the spheroid, the rate of metabolic oxygen consumption, and the fluence-rate-dependent rate of photochemical oxygen depletion. We have recently modified the theoretical treatment of the time-resolved measurements to include the oxygen dependence of the rate of therapy-induced oxygen consumption. The oxygen consumption term in the diffusion equation is now derived from kinetics of type II photochemistry. This expression contains the ration of two rate constants' the photosensitizer triplet decay rate (k_p) and the bimolecular rate for collisional triplet-quenching interactions with oxygen (k_ot). From fits of numerical solutions of the diffusion equation to microelectrode measurements of PDT-induced oxygen transients, k_p/k_ot, may be obtained for a photosensitizer in a multicell environment. The ration plays an important role in direct cell photosensitization by defining the concentration at which singlet oxygen formation is limited by the availability of oxygen. In a multicell environment where oxygen supply is diffusional, extremely low values of k_p/k_ot exacerbate the oxygen depletion problem. Recent experiments and analysis indicate that in some cases photosensitizer bleaching rates may also be determined from microelectrode measurements in spheroids.

Sixty-five patients with neoplastic diseases of the larynx, oral cavity, pharynx, and skin have been treated with PDT with followup to 56 months. Patients with carcinoma in situ and T1 carcinomas obtained a complete response after one PDT treatment. All but two have remained free of disease. Eight patients with T2 and T3 carcinomas treated with PDT obtained a complete response, but they all recurred locally. This is due to the inability to adequately deliver laser light to the depths of the tumor bed. five patients with massive neck recurrences of squamous cell carcinomas were treated with intraoperative adjuvant PDT following tumor resection. Only one developed recurrence with 24- month followup. PDT is highly effective for the curative treatment of early carcinomas (CIS, T1) of the head and neck. Also, intraoperative adjuvant PDT may increase cure rates of large infiltrating carcinomas of the head and neck.

The subcellular localization of endogenous protoporphyrin (endo- PP) during photosensitization in B-16 melanoma cells was analyzed by a novel spectral imaging system, the SpectraCube 1000. The melanoma cells were incubated with 5-aminolevulinic acid (ALA), and then the fluorescence of endo-PP was recorded in individual living cells by three modes: conventional fluorescence imaging, multipixel point by point fluorescence spectroscopy, and image processing, by operating a function of spectral similarity mapping and reconstructing new images derived from spectral information. The fluorescence image of ALA-treated cells revealed vesicular distribution of endo-PP all over the cytosol, with mitochondrial, lysosomal, as well as endoplasmic reticulum cisternael accumulation. Two main spectral fluorescence peaks were demonstrated at 635 and 705 nm, with intensities that differed from one subcellular site to another. Photoirradiation of the cells included point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral image reconstruction revealed the local distribution of a chosen spectrum in the photosensitized cells. On the other hand, B 16 cells treated with exogenous protoporphyrin (exo-PP) showed a dominant fluorescence peak at 670 nm and a minor peak at 630 nm. Fluorescence was localized at a perinuclear=Golgi region. Light exposure induced photobleaching and photoproduct-spectral changes followed by relocalization. The new localization at subcellular compartments showed pH dependent spectral shifts and photoproduct formation on a subcellular level.

In the problem of the use of lasers for photodynamié therapy (PDT) it will be very interesting to use compact laser without special condition for its exploitation. As previosly reported (1), a using of the gold vapour laser (GVL) is very effective for PDT. We create a GVL with 2 W average power. We use a sealed-off laser tube produced on the base of progressive metal-ceramic technology. A length of the tube is 700 mm. Active length - 400 mm. The diametre of tube is 12 mm. A pressure of neon buffer gas is 200 torn

The use of PDT for endometrial ablation has been the focus of much recent research. However, the mechanism of action, optimal treatment parameters, and long-term clinical effect are still poorly understood. This study was undertaken to further the understanding of the endometrial response to this drug/light- induced damage. Postpartum rat (Charles River) uterine horns were used as the animal model for fluorescence and treatment studies. Aminolevulinic acid was administered topically (intrauterine), and following a 0.5- to 3-hour drug incubation time, the endometrium was either removed and processed for fluorescence microscopy to assess drug localization or exposed to 150-200 J/cm² of 630-nm laser light via a 1-cm cylindrical diffusing tip. The light=treated uterine horns were removed and histologically examine 7 to 10 days following treatment. The extent and character of uterine and endometrial damage (gross and histological analysis) were recorded for the varying light doses and incubation times. With topical (intrauterine) application of photosensitizer, incubation time and penetration ability of drug were found to be crucial factors. The use of a drug penetration enhancing vehicle produced greater tissue effects (endometrial ablation). These preliminary studies also showed that tissue effect is drug and light dose related and that the most profound effects may be vascular mediated. The study provided preliminary information for the use of PDT in gynecological applications such as endometrial ablation and female sterilization through Fallopian tube occlusion.