Numerous photosensitizing pigments that absorb visible light and are selectively retained in neoplastic tissue are being investigated as potential photochemotherapeutic agents. While much emphasis is being placed on the synthesis of new, far-red absorbing photosensitizers, an alternative approach has been to stimulate the human body to produce its own natural photosensitizer, namely protoporphyrin IX (PpIX). Exogenous 5-aminolevulinic acid (ALA) is rapidly bioconverted into PP by mitochondria, the process being particularly efficient in tumor cells. Since PpIX has a natural and rapid clearing mechanism (via the capture of iron in the process of being converted into heme), ALA-PDT does not suffer from lingering skin phototoxicity. ALA may be introduced orally, intravenously, or topically, and ALA-PDT has been shown to be effective in the treatment of both malignant and non-malignant lesions.

A family of benzoporphyrins formed by differently substituted metallo tetrabenzoporphyrins and one opp-dibenzoporphyrin has been prepared. The former benzoporphyrins, and meso- tetra(m-hydroxy-phenyl)chlorin (m-THPC) to act as reference, have been encapsulated into liposomes and subjected to preliminary in vitro and in vivo assays to test their efficacy as photosensitizers in the photodynamic therapy of cancer. The results of the photocytotoxicity test shows that, with the exception of the nickel complexes 5, and 7/8, the other porphyrins are photobiologically active, the Mg-tetrabenzoporphyrin 1 and the opp-dibenzoporphyrin 10 being the most active. The dark toxicity of the photoactive porphyrins is in the range described for many photosensitizers, including HPD. The in vivo assays show no toxicity in the liver for any of the compounds tested, and also that 2 is the most promising photosensitizer among them, because of an efficient localization in an experimental mouse tumor.

A series of novel sensitizers, which absorb in the range of 660 - 820 nm, derived from natural occurring chlorophyll and bacteriochlorophyll was synthesized. Biomass of blue-green algae Spirulina platensis was used to prepare chlorophyll a derivatives, and biomass of purple bacteria Rhodobacter capsulatus was applied for preparation of bacteriochlorophyll a. The influence of different substituents on spectral characteristics and the amphipility of the sensitizer was investigated. The route for the synthesis of porphyrin macrocycle with the spacer that bears the isothiocyanate group capable for binding with proteins was proposed. Photophysical properties of chlorin p₆, purpurin 18 and their esters in different solvents are investigated. Accumulation of two chlorins in the model Erlich tumor was studied.

The chick chorioallantoic membrane (CAM) is a convenient model for the study of photodynamic therapy (PDT). This membrane has a rich vasculature, which mimics the tumor neovasculature, and can also serve as a host for implanted tumors. The transparency of the CAM enables in-vivo monitoring of vascular changes during and post PDT, without the need to sacrifice test animals at each time point. Video documentation and analysis of events occurring during and after irradiation permit the quantification of changes in vessel morphology, blood perfusion and tumor development. The compounds tested in this study belong to a family of potential sensitizers -- the porphycenes. These are phorphyrin isomers based on a 16-membered macrocycle, in which the four methine moieties linking the pyrrole rings have been replaced by two direct bonds and two ethine bridges. Experiments were performed on blood vessels of the intact CAM and on recurrent human melanoma cells implanted on the CAM. Tumor selectivity was demonstrated by measuring drug uptake using fluorescence methods. A sensitizer injected systemically into the embryo yolk sac could be detected in the blood vessels 30 min after injection; 1 h later the sensitizer had preferentially accumulated in the tumor. Tumors were irradiated at the optimal uptake time (after 1 h) for 16 min with a 20 mW HeNe laser. Video image analysis showed that 96 h after irradiation tumors had decreased to 5% of their original size. In contrast, non-irradiated control tumors on the same CAM, continued to proliferate and grew to more than twice their original size. In addition, we observed a difference in the damage mechanism after systemic compared to topical administration. Topical application followed by irradiation caused fast necrosis of tumors, which might suggest direct damage to tumor cells, whereas after systemic administration, PDT damage was manifested by slower necrosis, presumably caused by vascular destruction.

Photodynamic therapy (PDT) with topically applied 5-aminolevulinic acid (ALA) is of growing interest, in particular in dermatology. Due to the fact that PDT with intravenously administered Photofrin is the only clinically approved sensitizer so far and is performed at a wavelength of 630 nm, this wavelength is also used in most experimental and clinical trials with ALA. In this study influence of irradiation with coherent light from a tunable dye laser at different wavelengths ranging from 625 to 649 nm was investigated. In in vitro experiments HaCaT immortalized human keratinocytes were sensitized with 30 (mu) g/ml ALA for 24 hrs. By determination of cell viability with the MTT test, best cell-killing effects were observed following irradiation at 635 nm. In an in vivo setting using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, these results were confirmed: tumor growth determined by measuring tumor volume increase after 28 days was less pronounced in animals treated with 100 mg/kg ALA i.v. and irradiated 2.5 hrs. later at 635 nm, as compared to animals receiving an equal dose and irradiated at 630 nm. This observation in vitro is probably due to large amounts of photosensitizing protoporphyrin IX (PP) localized in cell membranes which is visualized by confocal laser scanning microscopy (CLSM) and determined by HPLC analysis. These results suggest that in ALA-PDT when a coherent light source is used probably better results are achieved irradiating at 635 nm.

In this first clinical study performed according to GCP- (good clinical practice) guidelines, efficacy, and tolerability of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) were tested in the treatment of actinic keratoses. Ten patients (6 f, 4 m) with 36 lesions (19 located on hands and arms, 17 on the head) received ALA-PDT once. Five to six hours after occlusive application of ALA (water-in-oil-emulsion containing 10% ALA) irradiation was performed with an incoherent light source. Up to 3 months after treatment patients were monitored. A score evaluating infiltration and keratosis of treated actinic keratoses allowed us to estimate therapeutic efficacy. Compared to the initial score (100%) significantly lower score-sums were observed at the 28 day follow-up at both localizations (head: 15%; hand: 67%). Complete remission (score sum 0) resulted in 71% of actinic keratoses localized on the head. Except for slight pain and burning sensations during and after irradiation there were no notable side effects. This study proved good efficacy and tolerability of topical PDT in the treatment of actinic keratoses. Whether PDT is able to compete with established treatment modalities remains to be shown in further studies.

Topically applied ALA-PDT for human nodular basal cell carcinoma has encountered some problems, one of which is that the concentration of ALA-induced PpIX is in some cases too low to sensitize complete tumor destruction. Our present study, by means of an optical-fiber based point monitoring system in situ, has shown that the fluorescence intensity of PpIX was significantly higher in the normal skin of nude mice after topical application of ALA and desferrioxamine (DF) or in combination with DMSO than that of ALA alone or ALA plus DMSO. These data indicate that DF has an effect on enhancing the production of ALA- induced PpIX in the mouse skin. Thus, there may clinically be advantages of using ALA and DF over ALA alone in PDT of superficial skin lesions.

The purpose of this study was to determine the optical dose needed for both lasting endometrial destruction and prevention of implantation by photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) as a photosensitizer. Three hours after topical drug administration 74 female Sprague-Dawley received varying optical doses of 630 nm light delivered by an intrauterine cylindrical light diffusing fiber. Histologic evaluation of the endometrium 1 and 21 days after PDT as well as the number of implantation sacs after mating were assessed. Irreversible endometrial destruction was determined measuring the thickness of the endometrial layer 3 weeks after treatment. An in situ dose of 64 J/cm² was required to eradicate endometrial glands and prevent regeneration. In contrast, a 43 J/cm² in situ dose visibly damaged the endometrial stroma and myometrium but the endometrial glands survived and the endometrium regenerated to its full thickness within 21 days. However, implantation potential was significantly reduced at these low light levels. Due to differential cell photosensitivity, the optical threshold for lasting endometrial destruction is higher than for functional damage. For lasting endometrial destruction the endometrial glands must be destroyed, whereas for reproductive impairment, damage to the endometrial stroma seems to be sufficient.

The agent 5-aminolevulinic acid (5-ALA) can be an alternative drug in whole bladder wall (WBW) photodynamic therapy (PDT), as its good tumor selectivity and the short time skin photosensitivity after systemic administration are advantageous for clinical use. To determine the maximum drug and light doses for reversible normal tissue damage, a pre-clinical study was performed using an in vivo normal piglet bladder model. First, the kinetics of PpIX production in 2 pigs was determined in vitro after oral administration of 75 and 150 mg/kg ALA respectively. The concentration of PpIX in plasma, and erythrocytes was determined by reversed phase high-performance liquid chromatography (HPLC) and the maximum was reached at approximately equals 5 hours after the administration of ALA. This provided a guideline for the optimum interval between ALA administration and light application. Next, various ALA doses were either administered orally or instilled in the bladder and different light doses were applied. Bladder biopsies were taken at regular intervals and normal tissue damage was investigated histologically. Reversible tissue damage was obtained using 60 mg/kg of 5-ALA in combination with a light dose of 100 J cm^-2 (non-scattered plus scattered 630 nm wavelength light) in the case of oral administration. In the case of intravesical instillation, a drug dose of 2.5 gram and a light dose of 100 J cm^-2 are still too high to obtain reversible tissue damage.

In vitro experiments were performed on human bladder carcinoma cells (EJ, 28, Tumorbank Heidelberg, Germany) to evaluate the efficiency of photodynamic activity of AamTPPn encapsulated within liposomes. After irradiation the cells were incubated for 2 days at 37 degrees Celsius, then fixed, stained, counted and compared with the control group. By using a fluorescence microscope, we evaluated the intracellular uptake of AamTPPn in liposomes. After incubation of cells with liposomes-administered AamTPPn at a concentration of 40 (mu) g/ml dark toxicity occurred. In all experiments a drug and light dose dependent decrease in cell survival was found. By transmission electron microscopy marked changes were observed at the mitochondria with dissolution of the cristae and development of vacuols.

Photodynamic therapy (PDT) in dermatology is of growing interest and there are efforts to develop new sensitizers for topical application. In vitro parameters for photodynamic therapy with 9-acetoxy-2,7,12,17-tetrakis-((beta) -methoxyethyl)-porphycene (ATMPn) using two human skin derived cell lines (HaCaT keratinocytes and dermal fibroblasts) were established. Short time flow cytometry analysis (0-2,000 s) for both cell lines revealed an immediate increase of intracellular ATMPn-fluorescence after start of incubation (100 (mu) g/ml). Long time flow cytometry analysis (0-24h) in both cell lines showed a constant increase of fluorescence up to 12h, with a steady-state up to 24h. Confocal laser scan microscopy showed spotty, granular fluorescence inside the cytoplasm after different incubation times, similar to the pattern of rhodamine dye which stains mitochondria. However, uptake of ATMPn in fibroblasts was slower than in keratinocytes. These results confirm the rapid intracellular uptake of ATMPn in vitro and indicate there is a sufficient photodynamic effect when ATMPn-sensitized cells are exposed to light.

PDT in dermatology is of growing interest and there are efforts to develop new sensitizers for topical application. Human keratinocytes and dermal fibroblasts were incubated with different concentrations of Acetoxy-tetrakis-(methoxyethyl)-porphycene (ATMPn) for different times and subsequently irradiated with red light (580-740 nm) of different light doses and intensities to determine photodynamic efficacy using a MTT-assay. First, concentrations ranging from 0 ng/ml to 1,000 ng/ml ATMPn (solved in DMEM) were tested (incubation time 24 h, irradiance 80 mW/cm², total light dose 60 J/cm²). Second, three different light intensities and doses were used for irradiation (47 mW/cm²/35 J/cm²; 80 mW/cm²/60 J/cm²; 107 mW/cm²/80 J/cm²; incubation time 24 h, 100 ng/ml ATMPn). Third, different incubation times of ATMPn (100 ng/ml, 60 J/cm²) were tested (1, 2, 4, 6, 8, 16, 24 h). Using 1 ng, 10 ng and 25 ng/ml, a PDT-effect could not be seen as compared to the dark control group (100%). A significantly high PDT cell mortality rate (94%) was achieved with 100 ng/ml ATMPn yielding a low dark toxicity rate (11%), results with concentrations between 50 and 150 ng/ml were comparable. However, compared to photofrin which served as control (10 (mu) g/ml), dark toxicity rates were significantly lower (Photofrin: 67%). Using different light intensities and doses, best cell killing results were achieved with 60 J/cm². Comparing different incubation times, no significant differences in cell killing were found. Thus, optimal in vitro parameters for PDT with ATMPn were established using 100 ng/ml ATMPn, about 1- 8 h incubation time and irradiation with 60 J/cm². As compared to photofrin in previous studies, this novel sensitizer exhibits reduced dark toxicity and similar phototoxicity at a 100-fold lower concentration.

Syntheses, singlet oxygen ¹O₂ quantum yields ((Phi) _(Delta )) and photostability of new monofunctional, long wavelength absorbing (680 - 750 nm) porphyrazines are reported. For the coupling to biomolecules without crosslinking reactions the sensitizers contain one reactive polar sulfo, carboxy, amino, hydroxy, or pyridyloxy group. Besides, the sensitizers are substituted by lipophilic tert-butyl, tert-butylphenoxy or alkoxy groups in order to introduce an intramolecular polarity axis for better cellular uptake and to increase solubility in organic solvents.

Two possibilities to accumulate photosensitizing drugs at the surface of cultured tumor cells were exploited: biotinylation of the cell surface followed by avidin and dibiotin, and reaction with a biotinylated antibody followed by avidin and biotinylated sulfophthalocyanin. Both reactions were performed successfully. The latter leads to enhanced phototoxicity of the accumulated drug.

Pheophorbide a ethyl ester, pyropheophorbide a ethyl ester, and bacteriopheophorbide ethyl ester were substituted in 3¹-position with tert.butyl phenoxy or tert.butyl benzoic acid ester groups resp. in order to enhance affinity to (beta) -cyclodextrin dimers which form inclusion complexes with these photosensitizing drugs. This is a first step to construct inert transport complexes in order to photosensitize specifically cancer cells.

Various tetrapyrrolic compounds are well-known photosensitizers in PDT. One of the important unsolved problems in PDT research is the selective accumulation of such compounds in tumor tissues. In this context different carrier systems for efficacious and directed transport of the dyes to the tumor tissue are still under investigation. From these investigations new problems have arisen like change of the sensitizer's photoactivity and photostability. As a result the relation between energy and electron transfer processes (that means the relative efficacy of sensitization via type I and II) during illumination can change. The photophysical properties of Pheoporbide a in different sensitizer-carrier systems (antibodies and liposomes), are investigated.

The changes of the fluorescence emission spectra detected after the irradiation of buffered Hp solutions (C equals 10^-4 M) in a suspension of resealed erythrocyte cell ghosts (ECG) showed that the emission decay of Hp incorporated in ECG is faster than that of Hp in aqueous medium. The disappearance of emission bands at 634 nm and 700 nm and the development of a new emission band at 642 nm precede a period when the photobleaching of aqueous Hp becomes dominant. Besides the possible photodestruction of Hp, these spectroscopic changes might be related to the formation of a photoproduct and the light- induced relocalization of Hp in ECG. A fraction of aqueous Hp is transformed to the photoproduct with emission band at around 640 nm. It is likely that the HP fraction incorporated in ECG mostly undergo a relocalization, which may lead to a strong interaction of Hp with proteins and lipids of ECG resulting in a disappearance of characteristic porphyrin emission bands and a decrease of the emission capacity. However, a possible formation of a small amount of the new photoproduct (similar to that formed in aqueous medium) and photodestruction of Hp could not be excluded.

An analysis of the spectral changes of porphyrins in aqueous solutions detected upon irradiation with visible light shows that a few photoprocesses take place. The decrease in absorption and emission intensity throughout the whole spectra reflects: (1) a photoinduced destruction of the porphyrin macrocycle and (2) a photoinduced chemical modification leaving the porphyrin macrocycle intact. The photodestruction of the porphyrin macrocycle is related to: (1) the formation of di- and mono-pyrrolic photoproducts absorbing in the UV and (2) the formation of negligible amounts of bilirubin-like photoproducts with the emission maximum at 545 nm. Another photoprocess is related to the formation of chlorin and bacteriochlorin type photoproducts with absorption maxima at about 640 - 660 nm, maintaining the porphyrin macrocycle intact. A scheme of the photomodifications of porphyrins is presented. The phototransformations of sensitizers should be taken into account in clinical photochemotherapy.

In order to compare the efficiency of the three different mechanisms suggested for photosensitization in biology and medicine: the sensitizer radical, (Type I), the singlet oxygen (Type II), and the native free radical-triplet sensitizer interaction (MTO mechanism) mediated effects, a possible mechanism is suggested including the primary steps of all three mechanisms. Simulation and sensitivity tests based on this mechanism have revealed: that (i) the main contributing elementary steps to the overall photodynamic effect are the decay of the triplet sensitizer; the interaction of sensitizer radicals with biomolecules and the interactions of native free radicals with biomolecules. (ii) The extent of the contribution of the latter is determined by the concentration of the native free radicals in the tissue preceding illumination. (iii) By changing the assumed rate constants of the triplet-doublet interactions it has been established, that with the decrease of their rate constants the overall photodynamic effect decreases. And (iv) its contribution might become negligible. Kinetic consideration has shown, that while continuous illumination results in steady states, illumination by pulsing light leads to non-steady states of the transient species and the latter enables the distinction between the mechanisms provided one of them is predominant under conditions of the measurements.

Photodynamic therapy (PDT) has shown promising results in treatment of malignant tumors. However, the mechanisms leading to tumor destruction during PDT are still not completely understood. In addition to effects on the microcirculation, damage to cellular structures has been observed following exposure of cells to PDT. A phenomenon preceding these events might possibly be cell swelling. We therefore studied the influence of treatment with Photofrin (PF) and laser light on volume changes and cell viability of endothelial cells. Endothelial cells were obtained from human umbilical cord veins (HUVEC) by an adaption of the method of Maruyama (1963). After subcultivation the cells were harvested and transferred as a cell suspension into a specially designed incubation chamber. Cells received either PF in concentrations of 1.5 or 3.0 (mu) g/ml and laser illumination (630 nm; 40 mW/cm², 4 Joule), PF alone, or laser treatment only. Following start of PF incubation and after phototreatment cell samples were taken for volume measurements using flow cytometry and for studies of cellular morphology using scanning electron microscopy. Simultaneously, cell viability was monitored by the trypan blue exclusion test and colorimetric MTT assay. (abstract truncated)

All photosensitizers applied in experimental and clinical photochemotherapy (PCT) of cancer are degraded during light exposure. Under certain conditions this may be a disadvantage since larger light fluences are needed to destroy the malignant tissue. However, photodegradation may also offer an advantage: if the applied dose of sensitizer is so low that most of it is photodegraded before normal tissue is destroyed, but still large enough to sensitize the tumor to destruction, one may achieve a larger tumor to normal tissue therapeutic ratio than when using a higher dose of sensitizer. Tumors usually contain two to ten times higher concentrations of sensitizers than do the surrounding normal tissues. We have studied the photodegradation of a number of sensitizers, including Photofrin (PII), benzoporphyrin derivative mono acid ring A (BPD), chlorin e₆ (Chle₆) 5-aminolevulinic acid (ALA)- induced protoporphyrin IX (PpIX), meso-tetrahydroxyphenyl-chlorin (m-THPC), meso- tetrahydroxyphenyl-porphyrin (m-THPP) tetraphenylporphine tetrasulfonated (TPPS₄), aluminum phthalocyanine disulfonated (AlPcS₂), tetrasulfonated (AlPcS₄) and zinc phthalocyanine (ZnPc) in liposomes. The sensitizers were injected in Balb/c nude mice and exposed to light from an argon pumped dye laser, tuned to the appropriate therapeutic wavelength at a fluence rate of 100 mW/cm². The sensitizer fluorescence in the laser- exposed skin was monitored by a fiberoptic probe coupled to a fluorescence spectrometer. The kinetics of the fluorescence decay during PCT were, in all cases, nonexponential but differed from dye to dye. Chle₆ and m-THPC were found to be the most photolabile sensitizers. AlPcS₄ and AlPcS₂ and, to a minor degree, TPPS₄ showed a peculiar fluorescence increase during PCT, similar to what we have found earlier for these sensitizers in cells in vitro. The fluorescence increase is indicative of lysosomal localization and perforation of the lysosomes during PCT.

The photodynamic therapy (PDT) of cancer is based on the reaction of dyes, light and oxygen in tumorous tissues. Currently mainly two types of photosensitizers [Photofrin II and Hematoporphirine derivatives (HPD)] are used in clinical investigations. But they are still far from being ideal for this purpose as they do not have the required specificity and the absorption maxima do not lie in the ideal region of 760 nm (maximal transmission for human tissue). Because of their absorption maxima, the high extinction coefficient and the good rate of singlet oxygen generation phthalocyanines (PCs) seem to be good photosensitizers for the photodynamic therapy of cancer. At this time the main problem for the use of PCs is the leak of solubility in physiological media. By partial sulfonation it is in fact possible to increase the hydrophilic attributes, but not seriously the selectivity to tumorous tissue. For these reasons it is absolutely necessary to synthesize monofunctionalized PCs, so that covalent coupling to carrier systems (e.g. monoclonal antibodies) are possible. By the synthesis of PCs via coupling to modified polystyrene as a carrier, it is possible, after linking a phthalodinitrile with a spacer (alkylchain C₄ and longer) to the polymer to prepare exact defined monofunctionalized PCs.

The two photosensitizers Octa-(alpha) -Butyloxy-Zincphthalocyanine and 13²-Hydroxy- Bacteriopheophorbide-a-methylester with the maxima of absorption at wavelengths 735 nm and 750 nm, respectively, are promising candidates for photodynamic therapy (PDT). Photobleaching of these dyes was investigated with a pulsed Titan:Sapphire laser (duration of the pulse: 5 microseconds, repetition rate: 4 Hz) at 750 nm. Different concentrations of the dyes, in each case dissolved in ethanol, were irradiated with this laser. The variation of the bleaching rate (eta) with the total number of pulses at constant irradiation energy per pulse (alteration of the irradiation time at constant pulse duration and repetition rate) as well as with irradiation energy at constant total number of pulses was investigated. The degree of laser- induced bleaching was determined by the decrease of absorption after irradiation. A nearly linear increase of the bleaching rate with the energy per pulse was found for Octa-(alpha) - Butyloxy-Zincphthalocyanine. For 13²-Hydroxy-Bacteriopheophorbide-a-methylester at higher concentrations the bleaching rate was smaller (in comparison to Octa-(alpha) - Butyloxy-Zincphthalocyanine) with increasing sensitizer concentration at the same energies. The increase of bleaching rate with energy was approximately linear, again. No saturation effects occurred. The influence of the total number of pulses on the bleaching rate was investigated for Octa-(alpha) -Butyloxy-Zincphthalocyanine at the highest concentration. With longer irradiation times a saturation was observed. No new maxima in the absorption spectrum (monitored region 200 nm less than or equal to lambda less than or equal to 1100 nm) were found after irradiation; so the fragments of the dyes have probably no photoactivity in this range.

Meso-tetrahydroxyphenylchlorin (mTHPC) was evaluated in vitro in HT29 human colon adenocarcinoma cell line and compared with hematoporphyrin derivative (HpD). The cellular distribution of mTHPC was analyzed by fluorescence microscopy and revealed that mTHPC distributed diffusively in the cytoplasm. A lower proportion of the photosensitizer (50%) was found in the nuclear area. In both areas, mTHPC fluorescence decay was monoexponential. The incorporation kinetics, evaluated by flow cytometry showed that mTHPC cellular uptake is related to the incubation time until 12 hours, then a plateau appeared. For 12 hr-incubation period, the cellular uptake of mTHPC was found to be linearly related to the extracellular concentration, suggesting passive diffusion mechanism. Cytotoxicity assays were performed using MTT assay after photoirradiation at 650 nm for mTHPC and 630 nm for HpD. The photodynamic activity of mTHPC was influenced by serum protein of the culture medium and time-delayed with a maximal activity 48 hrs after the photoirradiation. In optimized experimental conditions i.e. 2% serum protein-containing culture medium, 12 hr-incubation period, cytotoxicity measured 48 hrs after exposure to 10 J/cm² light fluence, mTHPC appeared approximately 50-fold more active than HpD with IC₅₀ values of 0.06 (mu) g/ml and 3.30 (mu) g/ml, respectively.

The fluorescence emission of hydrophilic tetrasulphonated aluminum phthalocyanine (AlPcS₄) and hydrophobic zinc phthalocyanine (ZnPc), bound to the membrane of liposomes were investigated in vivo in an appropriate tumor model of the rat bladder and in addition in RR 1022 epithelial cells of the rat. The sensitizers were administered systemically to the rats and PDT was performed 24 h later. During PDT treatment the fluorescence was measured every 15 s. Fluorescence was excited with the 633 nm light of a HeNe laser and the fluorescence spectra were recorded with an optical multichannel analyzer system. PDT was performed in the case of both sensitizers with 672 nm light from an Ar⁺-Dye laser. Fluorescence changes during PDT were significantly different for the two phthalocyanines. In the case of AlPcS₄ an initial fluorescence intensity increase followed by subsequent photobleaching could be observed. In contrast, ZnPc fluorescence showed an exponential decrease and no increase in the beginning of the treatment. Tumor necrosis 24 h after PDT was significant only for ZnPc. RR 1022 cells incubated 24 h with AlPcS₄ revealed a granular fluorescence pattern, whereas ZnPc was localized diffusely in the cytoplasm of the cells. In correlation to the measurements in vivo subcellular relocalization and fluorescence intensity increase could be detected exclusively in the case of AlPcS₄.

The influence of spacers with different length (6, 10, or 13 C-atoms) on complex formation between (beta) -cyclodextrin dimers and t-butylphenoxy substituted Zn-phthalocyanine was studied by computer simulations. In complexes with C10-spacer the length of the sensitizer with end groups allowed an insertion in all cases. The dye fitted well into dimers with bent geometry. In the other cases (C6, C13) the length ratio of dimer and sensitizer was inappropriate.

The success reported for the treatment of superficial skin carcinomas by photodynamic therapy (PDT), following topical application of 5-aminolaevulinic acid (ALA), has therapeutic implications for the treatment of other skin disorders. This presentation describes the accumulation of the photosensitizing agent protoporphyrin IX (PpIX) in areas of psoriatic plaque, by monitoring the fluorescence emission induced by low-intensity laser excitation at 488 nm. We present the results from 15 patients, with a total of 42 plaques. These results show that PpIX fluorescence increases in intensity within the 6 hour period following application of ALA, which implies there is a potential for PDT. The emission is localized to the area of ALA application and the effect of occlusion appears insignificant. Also, the rate of increase, and maximum intensity of fluorescence emission, is not directly related to the applied quantity of ALA. The variability of the fluorescence intensity is as great between plaques at different sites on the same patient as between different patients. We also present measurements of the depletion in intensity of fluorescence emission during PDT treatment, using white light, at an irradiance of 25 mW cm^-2, that is a consequence of the molecular photo-oxidation of PpIX. The use of fluorescence measurements in predicting the therapeutic effect of treating plaque psoriasis by ALA-PDT is discussed.

The process of photo-degradation, during photodynamic therapy, has important implications for both the diagnostic and therapeutic potential of specific photosensitizers. Monitoring photo-degradation may provide a useful indicator of the local concentration of singlet oxygen, and therefore a direct measure of photodynamic effectiveness. The spectroscopic properties of protoporphyrin IX (PpIX), the photosensitizer generated by the precursor 5-aminolevulinic acid, have been well documented. By recording the fluorescence emission spectrum of PpIX during illumination (at 630 nm) the photo-degradation of the sensitizer can be recorded. The rate of PpIX photo-degradation is dependent on the concentrations of both the sensitizer and molecular oxygen, but the decay cannot be described by a simple function that is valid under a variety of experimental conditions. By numerically solving differential equations describing the instantaneous concentrations of species in the photo-oxidation pathway of PpIX, we have been able to model the dynamics of sensitizer fluorescence under varying conditions of sensitizer concentration, oxygen concentration and illumination irradiance. Results are consistent with those measured in aqueous solution.

Ultrasounds were described by a few authors as possibly inducing sonodynamic reaction, with singlet oxygen production, as photodynamic therapy. The aim of this project was to evidence this effect and to try to explain its different mechanisms. A specific device was developed with a strict control of temperature to avoid hyperthermia and of acoustical intensity: the characteristics of the US beam and the reproducibility of treatment conditions were strictly evaluated. We studied the frequency of 2.21 MHz using an antiresonance frequency of a transducer. US treatment was applied continuously or in pulsed mode. Human colorectal adenocarcinoma cells (HT-29) were used to test the cytotoxicity using trypan blue exclusion test. Analyses were performed using cell suspensions. Different intensities were studied ranging from 0 to 3.7 W/cm². Moreover, fluorescence emission spectra of hematoporphyrine derivative (HpD) were recorded before and after US treatment. Results of viability showed a higher cytotoxicity with US alone or with HpD in cell suspensions from 3.7 W/cm² (20% survival). These results show that cavitation alone can account for the cytotoxic effects of sonotherapy. In fact, cavitation is higher with continuous than with pulsed US treatment. No significant difference was found with or without HpD. HpD fluorescence spectra did not differ before and after US treatment suggesting that no modification of HpD structure was induced by US. Fluorescence spectra showed a very slow and small decrease in fluorescence intensity with time probably caused by the low interfering light used for the experiment. In conclusion, in our experiments, ultrasounds do not seem to induce any chemical reaction with photosensitizers, conversely to what was already reported. However, other photosensitizers, molecules and different cell lines (less resistant) must be studied in order to conclude about the absence of cytotoxicity of this technique.

Benzoporphyrin derivative monoacid ring A (BPD) is currently in Phase II clinical trials for the treatment of cutaneous malignancies (basal cell carcinoma and cutaneous metastases) and psoriasis. Results to date suggest that this photosensitizer has potential in both of these areas. Recently, a clinical trial with BPD was initiated for the treatment of age related macular degeneration, a neovascular condition in the eye which leads to blindness. BPD is a lipophilic photosensitizer which is rapidly taken up by activated cells and the vascular endothelium of neovasculature. The PDT effects seen with BPD appear to be a combination of vascular occlusion and direct killing of target cells. Since many diseases involve either activated cells and/or neovasculature, PDT with photosensitizer with characteristics like those of BPD, has applications far wider than oncology. A new area of interest involving photosensitizers is that of immune modulation. A number of photosensitizers have been shown to effect immune modulation in animal models of immune dysfunction including autoimmunity (rheumatoid arthritis, lupus), cutaneous hypersensitivity and allografts. BPD and PHOTOFRIN^R have both been shown to be effective in ameliorating arthritic symptoms in a number of animal models. The mechanisms by which immune modulation is affected in these studies still remains to be resolved.

Cytotoxicity, uptake, intracellular location and phototoxicity were compared for the naturally occurring porphyrins uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), as well as for tetra-, tri-, di- and monosulphonated meso-tetraphenyl porphyrins (TPPS_n with n equals 4, 3, 2a, 1) in cultivated endothelial cells from calf aorta (BKEz-7). In addition, the uptake of CP and PP in the chick chorioallantoic membrane (CAM) was studied after intravenous application. Hydrophilic porphyrins (UP, CP, TPPS₄ and TPPS₃) exhibit low cellular uptake as well as low cyto- and phototoxicity as compared with the more hydrophobic compounds. UP, CP, TPPS₄ and TPPS₃ were mainly located in lysosomes, whereas PP, TPPS_2a and TPPS₁ were preferentially observed in the cytoplasm -- supposed to be located in cellular membranes of organelles -- and exhibited pronounced light-induced reactions. A rapid uptake by the CAM vasculature (5 min. - 30 min. after application) and some delayed uptake by the CAM matrix (30 min. - 6 h with a maximum at 60 min.) were measured for CP and PP.

Photodynamic therapy of neoplastic tissues is a new treatment modality that combines the in- vivo administration of a photosensitizer followed by its excitation with visible light, which leads to a photochemical reaction and tissue destruction. Naphthalocyanine derivatives are a class of second-generation photosensitizers that have excellent prospects as photodynamic therapeutic agents. Relevant to these types of applications are their photochemical properties, their tumor-localizing abilities, and their ability to elicit photodynamic responses. Bis(di- isobutyloctadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) illustrates some of the above promising photoproperties: absorption in the red at 776 nm with an extinction coefficient greater than 10⁵ M^-1 cm^-1, a triplet state lifetime of 331 microseconds and singlet oxygen yields of approximately 0.20. Due to their high degree of hydrophobicity, metallonaphthalocyanines require a variety of approaches before they can be administered to cells in vitro or injected in vivo. One approach is the selection of solubilizing agents or vehicles such as a solution of Tween 80 in saline or emulsions of Cremophor EL in saline. This paper describes studies in (a) drug uptake by tumors and other tissues as a function of isoBOSINC's dose; (b) drug levels in normal versus tumor-bearing rats; (c) in-vitro photostability of isoBOSINC; (d) effects of delivery systems on photosensitizer tissue levels and pharmacokinetics, and PDT outcome.

Having successfully completed an extensive three year study of the pharmacokinetics and efficacy of m-THPC as a photosensitizer in three different animal models (rabbit, dog and nude rats) we began a phase one human trial in two centers. At the Orebro Medical Center Hospital, Sweden ten patients were selected for the treatment of bronchial, prostate, skin, laryngeal and nasopharyngeal tumors while at Long Island Jewish Medical Center Hospital four patients were treated for laryngeal cancers. These studies were designed to study the optimal parameters for human treatment and as such relied on data from the animal studies mentioned above. De-escalating drug doses of 0.3, 0.15, 0.075 and 0.0375 mg/kg were chosen and the pharmacokinetics of the patients plasma, tumor and adjacent healthy tissues were measured spectrofluorometrically following chemical extraction of the drug. The half life of the drug in our Cotton tail rabbit model was measured as 24.7 hours as opposed to the human half life of 44.5 hours within the studied dosing range. This illustrates the extreme care that must be exercised before translating animal pharmacokinetics data to human dosing decision.

Photodynamic therapy (PDT) is a new treatment modality for bladder carcinoma, especially carcinoma in situ. The goal of our study was to compare two different phthalocyanines for PDT of bladder carcinoma cells and to evaluate phototoxic potential. Material and methods: For PDT hydrophilic chlor-aluminum sulfonated phthalocyanine (CASP) and lipophilic zinc- phthalocyanine (ZnP, CPG 55 847) were compared. For ZnP liposomes as drug carrier were used. The photosensitizer concentrations for both substances used for PDT experiments were 5, 10 and 20 (mu) g/ml. Irradiation was performed with a Penta lamp emitting at wavelengths between 590 and 900 nm at a fluence of up to 12 J/cm². After irradiation cells were incubated for two days, counted and compared with a control group. Results: The cell survival rate was decreased depending on the light and drug dose. After incubation with the highest drug dose and irradiation with 12 J/cm² cell survival was 0.03 and 5.5% after incubation with CASP or ZnP, respectively. Light, CASP or ZnP alone had no effect on cells. Studies of the PDT effect by electron microscopy showed intracellular vacuolization caused by mitochondrial damage after incubation with either photosensitizer. Conclusion: The hydrophilic and lipophilic phthalocyanine tested here showed very similar effects on our cell line. ZnP is a pure compound and hence has some advantage over CASP, which is a mixture of a tri- and tetrasulfonated phthalocyanine. In addition, ZnP is administered in liposomes which should enhance tumor selectivity by binding to low density lipoprotein receptors on tumor cells.

Phthalocyanines and m-TPPs were substituted with two or three t-butyl phenyl anchor groups to build up inclusion complexes with dimeric (beta) -cyclodextrins. These complexes proved to be stable enough to hinder complexation of the porphyrinoids with plasma lipoproteins and, so, offer an independent pathway to transfer porphyrinoids to photosensitize tumor tissue.

The importance of pH as a factor relating to porphyrin binding and distribution in different models was examined using for sensitizers, derivatives of chlorin e6 differing in their lateral substitutors. On the basis of experimental data obtained with the use of several different methods (spectral fluorescence, fluorescence quenching, ultrafiltration) we have investigated the influence of pH on pigments affinity to serum proteins (including serum albumin, high- and low-density lipoproteins), porphyrin intramembrane distribution pattern and its mobility across membrane and when membrane-membrane exchange of porphyrin molecules occurs. The affinity of chlorin e6 to serum proteins is very sensitive to the protonation of side carboxylic groups. A fraction of the dye that is bound to serum albumin decreases with decreasing pH. In contrast to serum proteins, there is a significant increase of chlorin e6- binding capacity to the model membrane when pH shifts from 7.0 to 5.5. In acid medium, deeper penetration of chlorin molecules into lipid is observed. As compared with neutral medium, more pigment molecules are localized in inner monolayer bulk. The pH dependence of the rate of chlorin e6 molecules exchange from outer lipid layer of donor vesicles to acceptor vesicles is markedly different from that of transmembrane movement. The rate of the latter, slower process increases greatly in acid medium, whereas the rate of intervesicle exchange decreases.

Porphyrins and porphine analogs have been shown to induce cytotoxic effects on cells and tissues after exposure to light, an effect which is currently being studied as a new modality for treatment of cancer, termed photodynamic therapy (PDT). One of the important factors in PDT is the preferential uptake of sensitizers by rapidly proliferating tissues. Previous studies showed that cytoskeletal structures are affected by porphyrin-induced PDT. In the present study we investigate the inhibitory efficiency of porphines on tubulin assembly in vitro. We analyze the efficiency of several sulfonated porphine isomers: tetraphenylporphine n-sulfonate (TPPS_n) where n equals 4, 2a and 2o (a and o refer to adjacent and opposite substitution, respectively) and the structural isomers of tetra(o-,m-, and p-hydroxyphenyl)porphine (o-,m- ,p-THPP), in order to find a possible structure-activity relationship. The efficiency of the sensitizers was assayed by their capacity to inhibit microtubule assembly. Binding to monomeric tubulin is essential for effective inhibition of assembly, with or without exposure to light. Without exposure to light, TPPS_2o was found to be the most potent inhibitor, followed by TPPS_2a and to a much smaller extent by TPPS₄. All THPP isomers have negligible inhibitory effect. Upon exposure to white light, microtubule assembly was inhibited in the same order:TPPS_2o greater than TPPS_2a greater than TPPS₄ greater than THPP. All porphines were found to have high affinity to the same site on tubulin even those who had almost no dark effect on tubulin assembly (THPP). Addition of the porphines to assembled microtubules did not lead to their depolymerization even after prolonged irradiation. Since it was previously suggested that porphines may share the same binding site on tubulin as bis-ANS, a known tubulin assembly inhibitor, we performed competition studies with this inhibitor and the porphines. It was shown that bis-ANS does not share the same site on tubulin as the porphines and therefore their effects are additive.

Tumors are characterized by an insufficient neoangiogenesis. Therefore targeting of the fragile tumor microcirculation by photodynamic therapy (PDT) may induce easily tumor ischemia leading to tumor necrosis. Nine-acetoxy-2,7,12,17-tetrakis-((beta) -methoxyethyl)- prophycene (ATMPn) is a chemically pure, lipophilic substance and revealed superior photodynamic characteristics in vitro as compared to Photofrin^R. In this study pharmacokinetics, photodynamic effects and localization of ATMPn incorporated in small unilamellar liposomes in tumor and surrounding normal tissue were evaluated. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters (70 - 80 g b.w.). Fluorescence kinetics of ATMPn administered intravenously (1.4 micrometers ol/kg b.w.; n equals 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 micrometers ol/kg was injected 24 h (n equals 3), 3 h (n equals 3), 1 min (n equals 6) and 2.8 micrometers ol/kg 1 min (n equals 6) before PDT (630 nm, 100 mW/cm², 100 J/cm²). Tumor growth was measured over 28 days. Solid tumors (n equals 3) were excised 1 min after injection of ATMPn (1.4 micrometers ol/kg) and cryostat sections (10 micrometers) were analyzed by confocal laser scanning microscopy (CSLM) to determine tissue localization of dye. Maximal fluorescence (mean plus or minus S.E.) arose in tumor (94 plus or minus 7%) and surrounding host tissue (67 plus or minus 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable after 12 h. Only PDT 1 min after injection of ATMPn was effective yielding 1/6 complete remission (1.4 micrometers ol/kg) and 3/6 complete remissions (2.8 mmol/kg), respectively. At that time dye is primarily localized in vessels and vessel walls as shown by CSLM. ATMPn in liposomes reveals very rapid kinetics thus suitable for intraoperative PDT. Moreover, PDT (2.8 micrometers ol/kg) at time, when dye is localized in tumor microcirculation, exhibits best tumor killing effects.

Combination effects of photodynamic therapy (PDT) with meso-tetra (di-adjacent- sulfonatophenyl) porphine (TPPS_2a) and the microtubule (MT) inhibitor, vincristine (VCR), were studied in the CaD2 mouse tumor model in mice. A synergistic effect was found when VCR, at an almost nontoxic dose (1 mg/kg), was injected i.p. into the mice 6 hr before PDT. The data on mitotic index show a 4 - 5 fold accumulation of the cells in mitosis 6 hr after injection of VCR into the mice. Cell cycle and ploidy distributions in tumor tissues were determined by means of image analysis with measurement of integrated optical density after Feulgen reaction on monolayers. Ploidy distribution of the tumors was not significantly changed 6 and 12 hr after administration of VCR only, while an increasing aneuploidy was observed 24 and 48 hr after VCR treatment. No prominent changes of the cell cycle and ploidy distributions were found in the tumor tissues after PDT or PDT combined with VCR.

A main factor to consider before photochemotherapy (PCT) is chosen to treat a tumor is whether sufficient fluences of light can reach the base of the tumor. The choice of optimal wavelengths for PCT should also be based on a knowledge of the optical penetration spectrum of the tumor. By means of a fiberoptic probe, adapted to a commercial fluorescence spectrometer, it is possible to measure the penetration depths of visible light into living, human tissues. We have performed such experiments, using the light from a 1000 W Xenon lamp. The light was guided from the lamp to the tissue by a perspex rod and the fluence rate penetrating different thickness of tissue was monitored by the fiberoptic probe. From penetration curves obtained at a number of wavelengths, it was possible to construct an accurate optical penetration spectrum of the tissue.

Skin photosensitivity is the most common side effect of photodynamic therapy (PDT) and in clinical situations needs to be avoided or at least minimized. However, because of the accessibility of skin tissue, skin photosensitivity represents a useful test system in vivo for evaluation of the pharmacokinetics of photosensitizers and light sources. Pig skin resembles in many aspects human skin and, therefore, is most suitable for these tests. Using pig skin photosensitivity as an end point, we evaluate the effect of cell loading with a photosensitizer, benzoporphyrin derivative (BPD verteporfin) following its intravenous administration either as a rapid bolus or slow infusion. Skin response to light activation indicated a very similar cell content of BPD. These results were in agrement with those obtained in an in vitro model. In addition, in the same pig skin photosensitivity model we compared the efficiency of activation of BPD with either laser (690 plus or minus 3 nm) or light-emitting diode (LED; 690 plus or minus 12 nm) light. Results indicated the equivalency of the two light sources in this test system, with LED light being slightly more efficient, due possibly to a fluence rate lower than laser light.

Experiments were performed on five batches of Wistar inbred rats with Walker-256 carcinosarcoma receiving photodynamic therapy (PDT), rMuIFN-gamma activated macrophages (AM(Phi) ) or associated therapy (PDT-AM(Phi) -A; PDT-AM(Phi) -B); the control batch (HBSS) consisted of animals with untreated Walker-256 tumors. The results were as follows: the sole treatment (PDT, AM(Phi) ) gave survival rates between 57.2 and 57.7% and cure rates ranging from 23.1 to 34.3%. The 'combined' therapy in multiple doses increased significantly (87.9%) the survival rate of tumor bearing rats as well as the rate of complete tumor regression (72.7%). Cell-mediated immunity test values in batches III and IV exposed to multiple doses of PDT-AM(Phi) showed higher values as compared to the values noticed in batches I - II and the control batch V, performed at 12 and 21 days post-treatment. Summing up, these results demonstrate that 'combined' photodynamic treatment and biotherapy with interferon activated macrophages stimulate cell-mediated antitumoral activity, increase survival rates and reduce incidence of Walker-256 carcinosarcoma in rat model.

We report a preliminary study on the introduction of a new, blue-green fluorescent lamp with high phototherapeutic efficiency in the treatment of neonatal hyperbilirubinemia. The lamp (New Lamp) has an emission spectrum, peaked at 490 nm and about 40 nm wide, that was not previously investigated in clinical trials. Our study demonstrates the significantly greater efficacy of the New Lamp in decreasing the bilirubin serum level, in comparison with the most commonly used blue fluorescent lamp. The rate of decline of bilirubin concentration with the New Lamp was twice that with Philips/BB light. The success of the blue-green PT is mainly due to the combined effects of the (1) increase from blue to green of the quantum yield for lumirubin, that is the bilirubin photoproduct rapidly excreted from the organism; (2) corresponding decrease of the configurational photoisomer, formed with high concentration but not excreted from the organism; (3) filtering effect of the skin, which attenuates more blue than green light. Our results represent the first significant improvement of phototherapy efficiency following the development and introduction of the special-blue lamp by Sisson in 1970. The phototherapy exposure time has now been reduced to less than 1-day in preterm infants, ensuring less stress to the infant and less interference with nursing care.

Photodynamic therapy may induce in in-vivo conditions the cytokine Interleukin-1-alpha in Balb/c mice. The sensitizer, i.e. chlorin e₆, in the doses 2.5 and 5.0 mg/kg of body weight followed by light treatment with doses 50 and 100 J/sq.cm resulted in the increase in serum levels of the cytokine. The levels of Interleukin-1-alpha have been determined at different time points using enzyme-linked immunosorbent assay (ELISA). These levels in control animals did not exceed the mean value of 15 pg/ml, whereas in photodynamically treated mice the levels were almost 3 - 4 times higher. The entire experiment has been carried out on healthy animals.

Irradiation of pheophorbide a-sensitized melanotic M2Rcells inside threshold dose leads to development of a minimum of three of the properties of metastasing tumor cells: cell proliferation, synthesis of tPA, and loss of alpha 4 integrins. Collagenase synthesis can be assumed from loss of contact to the growth substrate. The suspected metastatic potential has to be proven now by animal experiments.

The 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 1992-1995 543 tumoral foci in 146 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 severe accompanying diseases or because of old age. A part of the patients had PDT because of recurrences or intradermal metastases in 1-2 years after surgical, radial or combined treatment. Two home-made preparations were used as photosensitizers: Photohem (hematoporphyrine derivative) and Photosense (aluminum sulfonated phthalocyanine). Light sources were: the argon pumped dye laser ('Innova-200,' 'Coherent') and home-made laser devices: copper-vapor laser-pumped dye laser ('Yakhroma-2,' Frjazino), gas-discharge unit 'Xenon' (wavelength 630 nm), gold-vapor laser (wavelength 627.8 nm) for Photohem; while for Photosense sessions we used solid-state laser on ittrium aluminate 'Poljus-1' (wavelength 670 mn). Up to now we have follow-up control data within 2 months and 3 years. Positive effect of PDT was seen in 92.4% of patients including complete regression of tumors in 62.3% and partial -- in 30.1%. Currently, this new perspective technique of treating malignant neoplasms is successfully being used in Russia; new photosensitizers and light sources for PDT and fluorescent tumour diagnostics are being developed as well.

Photodynamic therapy (PDT) with the use of laser endoscopic spectrum analyzer (LESA-5), the spectral-analyzing video-imaging system, Kr laser and various types of catheters for different tumor localizations, and Phthalocyanine aluminum photosensitizers in patients with gastric cancer was discussed. PDT was carried out in fifteen patients with gastric cancer. There were the following indications for PDT: early gastric cancer (3 patients), malignant stenosis of the cardia or pyloric portion of the stomach (4 patients), cancer of gastric stump with stenosis of gastrojejunal anastomosis (1 patient), preoperative treatment of patients with large but probably resectable gastric tumor size (7 patients). Usually we used 3 - 4 seances of laser treatment 10 - 30 minutes long. Concentration of photosensitizer in normal and malignant tissue was controlled by LESA-5. Treatment was monitored by spectral-analyzing video- imaging system in fluorescent light. The results show high efficiency of PDT especially in patients with early gastric cancer (necrosis of all tumor mass, i.e. complete regression of tumor). For all other patients we obtained partial regression of gastric cancer.

Photodynamic therapy (PDT) with the use of laser endoscopic spectrum analyzer (LESA-5), spectral-analyzing video-imaging system, Kr laser and various types of catheters for different tumor localizations, and Phthalocyanine aluminum photosensitizers in patients with lung cancer was discussed. PDT was carried out in twenty patients with lung cancer. Usually we used 1 - 3 seances of laser treatment 10 - 30 minutes long. Concentration of photosensitizer in normal and malignant tissue was controlled by LESA-5. Treatment was monitored by spectral- analyzing video-imaging system in fluorescent light. The results show high efficiency of PDT especially in patients with early lung cancer (necrosis of all tumor mass, i.e. complete regression of tumor). For all other patients we obtained partial regression of lung cancer.

One of the most promising directions in modern experimental and clinical oncology is a photodynamic therapy of cancer. The affect of this method rests on the capability of malignant cells to accumulate and to store any photosensitizers more than cells of normal tissues can do. Photobiological and photomedical progress far extends the capabilities of traditional methods for treatment of new malignant formations. However problems with creation and optimization of photosensitizers that meet modern medical technology's requirements are not solved up to now. That is the reason to continue searching for new more active and less toxic photosensitizers.

Free radicals are the main basis of anticancer effect of photodynamic therapy (PDT). At the same time, they cause different complications. The goal of this study is to investigate the changes in homeostasis of cancer patients under the influence of PDT. It was shown, as a result of study of antioxidizing and immune status of these patients, that there are significant deviations in their indices even before PDT. The treatment leads to further development of disbalance in these systems which demands correction. Several remedies have been offered for correction therapy. The application of these remedies causes the reduction of overstrain in antioxidizing defence and leads to decrease in cases of complications.

The paper presents the results of photodynamic therapy (PDT) of early-stage cancer of lung (17 patients), esophagus (8 patients) and stomach (10 patients). Fifteen patients had second primary tumors. New drugs photoheme and photosens were used as photosensitizers. Complete remission was obtained in 87%. The patients are followed up without relapses to 2.5 years.

In the course of the clinical trials of Photosens (Al-sulphonated phthalocyanine) as a drug for PDT of cancer fluorescence examinations of 45 patients with injected photosensitizer (0.5 - 2.0 mg/kg b.w.) were performed. The aim of these investigations was to study the drug accumulation in normal, inflammatory tissues and tumors of different localizations. The data concerning kinetics of photosensitizer accumulation and its removal from normal skin and lip mucosa are also presented.

Photodynamic therapy with the use of laser endoscopic spectrum analyzer (LESA-5), spectral- analyzing video-imaging system, Kr laser and various types of catheters for different localizations and different geometry of tumor, and phthalocyanine aluminum photosensitizers in patients with malignant strictures of esophagus is discussed. Photodynamic therapy was carried out to four patients: with esophageal cancer (3 patients) and gastric cancer with infiltration of lower esophagus (1 patient). All patients suffered from severe dysphagia. Photosensitizer was used in a dose 1-1.5 mg/kg of weight. Usually we used 3-4 seances of laser treatment 10-30 minutes long. The accumulation of photosensitizer was controlled by LESA-5. Laser induced fluorescent image was monitored by the video-imaging system in order to control laser treatment. There were no side-effects. The results show high efficiency of photodynamic therapy. There was marked reduction of dysphagia symptoms in all cases. It seems that photodynamic therapy is a good alternative to palliative surgical treatment of patients with malignant strictures of esophagus.

Photodynamic therapy (PDT) using quantoscope (scanning electron-beam pumped semiconductor laser, (lambda) equals 670 plus or minus 2 nm, P equals 10 W) and Phtalocyanine Al as photosensitizer (PS) have been provided in nine patients (27 tumor sites) with spread skin malignancies (basal cell, squamous cell cancer, melanoma, metastases of breast cancer) and cancer of the lip T2 N0 M0. During PDT power density has been from 200 to 450 mW/cm², light doses ranging from 150 to 700 J/cm². We have fulfilled diagnostic of tumor after injection of PS and have controlled treatment using spectral- fluorescent video complex. In five patients (23 sites) we had complete clinical response and in four patients (4 sites) partial response after PDT. Results of our study show that use of scanning electron-beam-pumped semiconductor laser is promising in the treatment of spread skin malignancies.

Photodynamic therapy (PDT) is proved to have potential for managing various malignancies. We investigated tissue biodistribution and photodynamic effects on a canine model in vivo using second generation photosensitizers, meso-tetra(m-hydroxyphenyl)chlorin (mTHPC) and 5-aminolaevulinic acid (ALA) to evaluate the feasibility and possible future application of PDT on the prostate. Using fluorescence microscopy, the optimal sensitization time of the prostate was between 24 - 72 hours with mTHPC and, 3 hours with ALA. After optimum time of sensitization, prostates of mature beagle were treated with laser at various sites by placing fiber interstitially under the guidance of transrectal ultrasound. The light dose for each treatment site was 100 J (100 mW for 1,000 seconds at the wavelength of 650 and 630 nm, respectively). With mTHPC, single laser fiber was able to induce organ confined PDT lesion as large as 20 by 18 by 18 mm in size. However, the PDT lesion with ALA was negligible 3 days after treatment. Physical distress manifested as urinary retention, poor appetite and body weigh loss, was more prominent with increasing number of treatment sites as a result of extensive prostatic swelling and urethral damages. However, these problems usually alleviated spontaneously 7 to 10 days after PDT. The characteristic histological changes were hemorrhagic necrosis and glandular destruction with preservation of interlobular collagen fibers. Urethral damage seen at the early stage healed by regeneration of urothelium in 4 weeks. We conclude that interstitial PDT with mTHPC is technically possible to produce extensive glandular necrosis in the normal prostate which heals safely and does not change the prostatic architecture. ALA, although it seems promising for bladder tumors, is much less effective than mTHPC on the prostate. With mTHPC, it might have the potential for treating prostate cancers localized in the periphery of the gland.

The phototoxicity, darktoxicity and uptake kinetics of three natural porphyrins (Uropoprhyrin III; UP III, Coproporphyrin III; CP III and Protoporphyrin IX; PP IX) were investigated in vitro using the BKEz-7 aorta endothelial cells of the calf. The cells were incubated with the porphyrins in different concentrations (0.5 (mu) M PP IX; 50 (mu) M UP III and CP III). After 24 h incubation they were irradiated in the case of PP IX with an Ar⁺-dye- laser at 635 nm and in the case of UP III and CP III with a Kr⁺-laser at 407 nm: While PP IX was phototoxic at low concentrations (0.5 (mu) M) and low energies (10 J/cm²), irradiation of UP III and CP III hardly induced phototoxicity even at higher concentrations. The same could be observed for the darktoxicity. PP IX was darktoxic at relatively low concentrations (1 (mu) M). In addition PP IX was taken up much faster and in greater amounts into the endothelial cells than UP III and CP III. These results could be due to the different structures of the sensitizers and/or to different uptake mechanisms. PP IX is a hydrophobic sensitizer while UP III and CP III are both hydrophilic molecules. A different uptake mechanism and accumulation in endothelial cells is quite probable. This hypothesis was confirmed with video-microscopy. In addition to the experiments in vitro, the cellular uptake and distribution of the sensitizers were observed in an appropriate in vivo model of the Chorioallantoismembrane (CAM).

The phototransformation of photodrugs during irradiation can influence the photosensitization reactions: (1) the photobleaching has been proposed to be taken advantage of to enhance the therapeutic ratio and to protect a normal tissue during photosensitized tumor therapy (PTT), (2) the formation of a photoproduct with absorption in the red spectral region can introduce new sensitization pathways. Therefore, the spectroscopic characteristics of the photodrugs in tumors and photostability under the irradiation by visible light were investigated. A few photoprocesses were studied under the irradiation of tumors ex vivo. Efficient photobleaching of porphyrin emission bands at 630 and 690 was detected in all cases. This process was accompanied by the formation of a broad emission band in the spectral region 660 - 680 nm. Careful examination of the emission spectra of tumors ex vivo (excitation at 488 nm) shows that the emission around 660 nm was related with photoproduct formation. The increase of emission intensity at around 680 nm and the observed redistribution of emission intensity after prolonged irradiation can be caused by photoinduced relocalization of the sensitizers in the tumor.

For successful application of photodynamic therapy (PDT) and photosensitized fluorescent diagnostics (PFD) of tumors a great number of various parameters affecting the efficiency of therapy and diagnostics should be controlled. This paper presents what parameters are important for photodynamic therapy protocol and how these parameters could be measured during PDT. For storing and analysis of these data it is desirable to use database program. This program allows one to save and represent information in a convenient way, make statistical analysis, and compare results obtained in various clinics.

An isolated crayfish stretch receptor neuron was used as a new test-object for cytophysiological study of various photosensitizers. This large cell is very suitable for complex electrophysiological and cytological investigation. It generates spikes with a nearly constant frequency, and dynamics of impulse activity shifts under the laser irradiation may be precisely studied at this stable background. The experimental procedure was as follows: 30 min control spike frequency registration, 30 min neuron staining, He-Ne-laser irradiation with continuous registration of cell response dynamics. The typical response of photosensitized neuron to laser irradiation was impulse activity acceleration after some latency and then irreversible block of spike generation. Dependencies of spike frequency acceleration and neuron lifetime on photosensitizer concentration allowed us to compare different photosensitizer efficiencies. As the first set of photosensitizers methylene blue, janus green, bengal rose, and chlorine e6, were studied. Chlorine e6 was the most potent photosensitizer among them. Such an approach provides evaluation of both: initial threshold alteration in cell membrane and cytotoxic events leading to the cell death.

Photodynamic therapy of skin tumors requires a technique which enables us to measure photosensitizer concentration in the tissue. This paper presents such a technique. The possibility of using two wavelengths of Kr⁺-laser (647 and 675 nm) to calculate the concentration of Phthalocyanine aluminum photosensitizer at different depth in the tissue is discussed. Scattering laser lines and photosensitizer fluorescence intensity with respect to the distance between illumination point and signal output point were measured. A special system has been developed for irradiation of flat surfaces and measuring light intensity. The results are useful in the basic experimental research and in the photodynamic therapy in clinics.

Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that ¹O₂ has a short radius of action. Since practically all PCT sensitizers act via the ¹O₂ pathway, only targets with significant sensitizer concentrations can be damaged. A given number of ¹O₂ entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar ¹O₂ yield.

Efficient photodynamic therapy (PDT) of malignant melanoma may be possible with photosensitizers having absorption maxima in the far-red region e.g., above 700 nm. Bacteriochlorin a (BCA), a non toxic derivative of bacteriochlorphyllin a, has a high molecular absorption coefficient (32.000 M^-1.cm^-1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. In several series of experiments BCA was proven to be a very effective photosensitizer, in vitro and in vivo. It is preferentially retained in experimental hamster Greene melanoma, rhabdomyosarcoma, RIF- and mamma tumors. Its fluorescence can be detected in vivo, thus enabling early tumor detection and it is rapidly cleared from the tissues which promises no, or minor skin photosensitivity. The effects of BCA-PDT were studied in vitro and in vivo using the heavily pigmented Hamster Greene Melanoma (HGM) cell line as a model. In vitro it was found that the uptake of BCA was time, concentration and temperature dependant. Upon illumination (10 Mw/cm², 756 nm) after incubation with 2.5 (mu) g/ml BCA for 1 h, almost complete cell kill was obtained within seconds. Hamster Greene Melanoma implanted in the anterior eye chamber of rabbits is an accepted in vivo model for ocular melanoma. The effects of BCA-PDT using this model were studied by light- and electron microscopy. Immediately after PDT intracellular spaces were enlarged and blood vessels were clotted with swollen erythrocytes. Electron microscopy showed fused inner and outer membranes and affected cristae mitchondriales of some mitochondria. With time, the severity of tissue and cell damage increased. One day after irradiation tumor growth had stopped; fluorescein angiography showed non perfusion of the tumor. Histopathology showed almost complete tumor necrosis with occasionally viable cells at the tumor periphery. It is concluded that the direct mitochondrial damage and the vascular damage both contribute to BCA-PDT induced tumor necrosis.

Numerous photoactivatable dyes have been proposed as photodynamic therapy (PDT) candidates during the past few years. Very few will ever graduate to approved drug status. Critical issues exist post-research which can be factored into the selection of suitable photosensitizers for development and substantially enhance prospects of success. Examples are provided by the novel porphycene family of synthetic dyes. Non-research factors such as patents, regulatory agencies and finance require early consideration. Porphycene photosensitizers have been designed chemically to ensure purity, avoid isomers, offer rapid metabolism, minimize phototoxicity and enhance target tissue selectivity while addressing key issues of safety, efficacy and economics in the development of a topically applied photosensitizer for dermatological disease.

The use of photosensitizers to generate cytotoxic species in situ following exposure to light is finding a wide range of applications. The photophysics of these sensitizers have been well characterized in homogeneous solution and in some microheterogeneous phases, however there have only been limited studies of the photophysics of the sensitizers inside living cells. Such studies are crucial to the unambiguous elucidation of the photodynamic mechanism within the cell and to the development of improved sensitizers and treatment modalities. We have constructed a laser flash photolysis system designed specifically to probe the fate of the triplet state of excited sensitizer molecules in living cell suspensions. Steady state and time resolved fluorescence techniques have also been applied to characterize the photophysics of aluminum disulphonated phthalocyanine (AlPcS₂) in aqueous suspensions of bacteria, yeast and murine cell lines. We have observed considerable aggregation of AlPcS₂ in all cell lines. We report triplet lifetimes in the absence of oxygen which suggest that sensitizer triplet state deactivation is primarily oxygen dependent. Fluorescence lifetime measurements suggest that there is no quenching of the singlet state. We have also made progress towards the reduction of early time spikes which render singlet oxygen luminescence detection in aqueous solution difficult.

PDT in dermatology is of growing interest and there are efforts to develop new sensitizers for topical application. Penetration potency of 9-acetoxy-2,7,12,17-tetrakis-((beta) - methoxyethyl)-porphycene (ATMPn), a novel sensitizer for topical photodynamic therapy, was studied in perilesional normal human skin from 58 melanoma patients and basal cell carcinomas of 12 patients. Single specimens of freshly excised perilesional skin (n equals 368) and basal cell carcinomas (n equals 28) were evaluated after topical application of an ATMPn solution for different time intervals (2, 6, 16 h) and subsequent preparation of cryostate sections (10 micrometer). Fluorescence distribution in tissue sections was estimated qualitatively using a score system respecting the morphological structure of human skin. In all examined sections of perilesional skin red fluorescence indicative of ATMPn was seen down to the basal layer of the epidermis using epifluorescence microscopy and optical multichannel analysis. Longer incubation times (16 - 20 h) revealed significant higher score sums as compared to incubation times of 2 or 6 h. However, basal cell carcinomas did not show time dependent differences in penetration. After 6 h penetration of ATMPn into the tumor, cell nests in the deep dermis were detected. These results indicate that there might be a faster penetration of this sensitizer in tumorous tissue as in normal tissue after topical application.

Photosensitizer Photosens is a mixture of sulphonated Al-phthalocyanines with a different number of substituents per phthalocyanine molecule. In the beginning of 1994, this photosensitizer was approved for clinical trials. Since that time till May 1995, 45 patients with 120 tumors were treated by PDT-Photosens. The main tumor localizations were lung (5/6), head and neck (4/4), esophagus (8/8), stomach (2/2), vulva (2/2), bladder (1/1), breast cancer (3/3), skin (basalioma, melanoma, sarcoma Kaposi, mts breast cancer) (20 patients/94 tumors). The lesions were photoirradiated 48-72 h after intravenous injection of Photosens in doses from 0.5 to 2.0 mg/kg b.w. (1.0 mg/kg b.w., on average). PDT was performed by laser power density from 20 to 1400 mW/sq cm (300 mW/sq.cm, on average), energy density varying from 15 to 200 J/sq cm (100 J/sq.cm, on average). The therapeutical effect of PDT was evaluated histologically, endoscopically, roentgenologically and sonographically 3 - 4 weeks after the treatment. Complete regression of tumors was reached in 56%, significant remission was reached in 34%, and partial remission was observed in 10% of cases. The follow-up of patients with complete tumor regression was to 15 months.

Recent studies have shown that cariogenic bacteria can be killed when exposed to low power laser light in the presence of a photosensitizing agent. The purpose of this study was to determine the mechanism by which the cariogenic bacterium Streptococcus mutans can be killed by toluidine blue O and helium neon laser light. To determine whether membrane damage occurred, suspensions of sensitized S. mutans were exposed to a 7.3 mW HeNe laser for 30 mins and samples removed every 5 mins. Survivors were enumerated by viable counting on tryptone soya agar plates and cell free filtrates were assayed for phosphate and (beta) -galactosidase. Lipid peroxidation was assessed by assaying for malondialdehyde, a by- product of lipid peroxidation. The role of oxygen and reactive oxygen species was studied by exposing sensitized bacteria to laser light (1) under different atmospheric conditions, (2) in the presence of deuterium oxide, and (3) in the presence of inhibitors of reactive oxygen species. Following exposure of sensitizede S. mutans to 13.2 J of HeNe laser light, 2.6 nmoles of phosphate and 228 nmoles of (beta) -galactosidase were detected in the cell free filtrates. Ten micrometers oles of malondialdehyde were also detected. When the sensitized bacteria were exposed to laser light under anaerobic conditions there was no significant decrease in the viable count compared to a 60% kill in the presence of oxygen. In the presence of D₂O there was a 15-fold increase in the numbers of bacteria killed. O.1 M methionine and 0.5 M sodium azide each afforded 98% protection from lethal photosensitization. These results imply that lethal photosensitization results from membrane damage due to lipid peroxidation and that reactive oxygen species are mediators of this process.

Photodynamic therapy (PDT) following surgical tumor resection is leading to improved local tumor control and might be useful for selected intrathoracic malignancies. However, optimal tumor selectivity of PDT is mandatory to avoid injury of adjacent normal tissues. (1) PDT was applied on human tumor xenografts (malignant mesothelioma, squamous cell carcinoma of the neck, adenocarcinoma of the colon). M-tetrahydroxyphenylchlorin (mTHPC) and polyethylene glycol-derived mTHPC (MD-mTHPC) were administered i.p. The tumor and normal tissue of the hind leg were irradiated with 652 nm laser-light. Drug and light doses and drug-light intervals were varied. The extent of necrosis was assessed histologically. (2) Intrathoracic PDT was performed in minipigs with drug-light doses optimized in nude mice. After administration of the sensitizers i.v., intrathoracic structures were irradiated and analyzed histologically. The tumor selectivity of PDT increased in the xenograft model by: (1) choosing an appropriate drug light interval; (2) decreasing the drug dose while increasing the light dose; and (3) applying MD-mTHPC instead of mTHPC. In the minipig model, the extent of injury of intrathoracic structures was equally related to modulation of treatment conditions. The modification of chlorins and the modulation of the drug-light conditions improved the tissue selectivity of PDT. Nevertheless, further methodological optimizations are prerequisites for clinical use of PDT, especially for intraoperative application in thoracic surgery.

Photodynamic therapy (PDT) consists in the administration of a photosensitizer and subsequent irradiation of the tumor with visible light. Routinely, the photosensitizer is given intravenously (i.v.), but the major drawback of this procedure is the resulting skin photosensitivity. The goal of our study was to examine whether intravesical (i.b.) instillation of the photosensitizer for PDT of bladder cancer might be feasible in order to target the tumors and to avoid the photosensitization phenomenon. After studying the normal bladder histology of pig and rat, not much described so far, we studied the diffusion and localization of hematoporphyrin derivative (HpD) in vitro on the pig bladder and the biodistribution of HpD in vivo in the rat bladder, two and four hours after intravesical administration, by spectrofluorimetry and fluorescence microscopy. We have the following results: (1) no diffusion through the pig bladder wall was detected; (2) the penetration depth of HpD into the pig bladder wall was 450 plus or minus 44 micrometers (n equals 8), including urothelium and chorion in totality and a small part of the muscles; (3) the penetration depth of HpD into the rat bladder wall was 55 plus or minus 9 micrometer (n equals 9) after two hours and 960 plus or minus 118 micrometer (n equals 9) after four hours, corresponding respectively to the totality of the urothelium and a small part of the chorion or almost completely in the bladder wall, a small part of the adventicia being excluded. In conclusion, intravesical instillation is feasible and, as superficial bladder cancer, especially carcinoma in situ particularly occur in the urothelium or in the chorion, a bladder instillation of two hours should be advantageous.

The effects of a classic two-stage carcinogenesis protocol on the formation of skin tumors in hairless female SKH-1 mice were studied in order to carry out photochemotherapy on the mice bearing tumors later. Mice were initiated with a single application of 100 nmol of 7,12- dimethylbenz[a]anthracene in 0.1 ml acetone and promoted one week later, twice weekly with topical applications of 1.8 nmol (first protocol) or 5 nmol (second protocol) 12-o- tetradecanoylphorbol-13-acetate in 0.1 ml acetone. The first tumors occurred between 4 and 6 weeks after the beginning of the promotion process depending on the protocol and the percentage of mice bearing tumors increased and reached 41% and 100% at the end of the treatment respectively for the first and the second protocol. Depending on the protocol, the tumor yield was 0.8 for the first one and approximately 10 for the second one whereas we expected 3 tumors per mouse. Histology of some skin tumors revealed that all were papillomas, hence benign tumors. These papillomatous lesions seem characteristic of a viral attack as seen in other strains of mammals including humans.

All photosensitizers applied in experimental- and clinical-photochemotherapy (PCT) have broad absorption spectra stretching from the ultraviolet up to 6 - 700 nm. Light of wavelengths in the red part of the spectrum is chosen for PCT even though the extinction coefficients of the sensitizers are usually smaller in this wavelength region than at shorter wavelengths. Thus, if one wants to treat superficial tumors or skin disorders, this may be a wrong choice. Two pieces of information are needed in order to make a proper choice of wavelength to treat a lesion of a given depth: the wavelength dependence of the optical penetration depth into tissue, and the action spectrum for tumor destruction. Additionally, the skin photosensitivity induced by the drug should be considered. We have non-invasively measured the optical penetration spectra of human tissues in vivo and the fluorescence excitation spectra for several sensitizers, including protoporphyrin (PpIX), in cells. Assuming that the action spectrum for cell inactivation can be approximated by the fluorescence excitation spectrum of the sensitizer -- which is indeed the case for a number of sensitizers in cells in vitro -- we have considered the situation for 5-aminolevulinic acid-induced PpIX in human tissue. All the way down to about 2 mm below the surface light in the Soret band (-410 nm) would give the largest cell inactivation, while at depth exceeding 2 mm, the conventional 635 nm light would be optimal. Light at the argon laser wavelength 514.5 nm is more efficient than light at 635 nm down to 1 mm. From the surface and down to 6 mm, the 635 nm peak of the excitation spectrum of PpIX, as evaluated per photon incident on the skin surface, is redshifted by less than 2 nm. In some cases photosensitizing photoproducts are formed during PCT, such as photoprotoporphyrin during PCT with PpIX. In such cases it may be advantageous to apply a broad-band light source with a spectrum that covers also part of the action spectrum of this photoproduct.

Antibody targeted chemotherapy is a relatively new technique which involves the specifically carrier mediated delivery ofchemotherapeutic agents to tumors or other pathogens for the treatment of such deseases. Conjugation of antibodies with photosensitizers (PSs) can also lead to potential therapeutic agents which combine photodynaniic cytotoxicity with specific antibody binding. The antibody mediated delivery of a photosensitizer molecule and the target destruction upon irradiation by light followed by production of singlet oxigen or other radicals, results in a higher therapeutic ratio compared to the conventional photodynarnic therapy (PDT). In this study the naturally occurring PS Alphà-Terthienile (ATT) was chemically derivatized with an amino group specific reactive side arm and in order to exploit its toxicity as an effector function suitable for targeted photolysis, covalently conjugated to the 225.28S monoclonal antibody (inAb) specific for the high molecular weight melanoma associated antigen (HMW-MAA). The 225-28S-ATT conjugate prepared was then tested against the melanoma cell line Co1o38 in comparison with HT29 tumor cells, not recognized by 225-28S mAb, as negative control. The selective uptake of labelled niAb 22528S-ATF and the melanoma cells death following irradiation can be observed. In conclusion the 225-28S-ATT conjugate, as far as one can judge from the effect on cells grown in vitro, seems a good candidate as a model to test the antibody targeted photolysis of melanoma cells for developing specific antimelanoma therapeutic agents.