The biochemical background of photodynamic therapy is only partly resolved. The biodynamic, which is shown to be different for lipophilic and hydrophilic photosensitizers influences the photodynamic efficiency. A complex cellular and subcellular localization and relocalization of the drugs is not only observed with varying incubation time but also during the light induced process. The dynamic behavior of lipophilic and hydrophilic phthalocyanines in cell cultures and non animal in vivo systems will be summarized within the presentation. As in vivo system the chorioallantoic membrane (CAM) of fertilized eggs was used. The dynamic behavior was observed with a confocal laser scanning microscope.

Benzoporphyrin derivative monoacid ring A [BPD-MA (verteporfin) or BPD], a second generation photosensitizer tested in clinical trials in combination with red light was compared for its PDT efficiency in vitro and in vivo upon activation with light in the UVA, blue and red spectral ranges. PDT efficiency, calculated based on the BPD absorption spectrum and spectral output of the different light sources, was compared with actual PDT efficiency determined in vitro and in vivo. Results obtained in an in vitro cytotoxicity assay, in which aliquots of murine P815 cells, pre-incubated for 1 h with BPD at 5 ng/mL, were exposed simultaneously to various light doses delivered within UVA, blue and red spectral ranges showed that in this test system PDT efficiency was governed by BPD absorption and light source emission spectra. Similar results were obtained in an in vitro BPD photobleaching test. Thus in vitro, values for calculated, theoretical PDT efficiency corresponded to the actual PDT efficiency. However, in vivo factors, such as depth of tissue penetration with light and localization of the target, had an important influence on PDT efficiency. In mouse models of skin photosensitivity and the cutaneous hypersensitivity immune response (CHS) assay, because of the thinness of mouse skin, PDT efficiency approximated the theoretical PDT efficiency, although blue light was somewhat more efficient in PDT than UVA, and red light was somewhat more efficient than blue or UVA. In a pig skin photosensitivity model, red light induced the highest skin response manifested by erythema and swelling, while blue light caused erythema and minimal swelling and UVA caused only erythema. These differences could be related to the thickness of pig skin and the depth of tissue penetration characteristic of each spectral range. Fluence rate was found to be an additional factor which modifies the effect of BPD and light. In conclusion, BPD can be efficiently activated with light within the UVA, blue and red spectral ranges. Moreover, light doses, deemed safe for red light, can be utilized with light of other spectral ranges, but only after a very careful evaluation of the conditions under which they were determined and the conditions under which they will be used.

The present study is based on the use of vital microscopy and a charge couple device camera for localization of a porphyrin in normal and neoplastic tissue in nude athymic Balb/c nu/nu mice. Granular tissue was allowed to grow into the space between two optical widows, separated by a 40 micrometer thick spacer in a transparent chamber. WiDr cells derived from a primary adenocarcinoma of the rectosigmoid colon, were used as a tumor model. Tumor cells were grown in vitro as spheroids and transplanted to the tissue in the chamber. The photosensitizer, meso(tetra-phenyl)porphine with two sulfonate groups on adjacent phenylrings (TPPS_2a), was injected i.p. to a concentration of 25 mg kg^-1, and the porphyrin localization was evaluated in vivo and in situ by a fluorescence microscopical technique. A significant difference between normal and neoplastic tissue was observed, with a much faster uptake of the photosensitizer in tumor tissue than in normal tissue.

The standard theories about the mechanisms involved in PDT concern themselves primarily with oxygen-centered species, above all singlet oxygen (¹O₂) and the hydroxyl radical (OH). The efficacy of the therapy depends very much not only on the photosensitizer used, but also to a great extent on the choice of irradiation parameters, the light source, and the tissue's degree of oxygenation. Both clinical and experimental studies point to the fact that the treatment of hypoxic areas generally results in a disappointing response, and the effects of the changes in perfusion and oxygenation induced by the PDT itself certainly complicate any analysis. The radical nature both of the secondary products generated from singlet oxygen reactions and of the other hypothesized primary products themselves mean that the physical method of choice for research on PDT is electron spin resonance (ESR). The aim of our investigations was the gathering of the particular information that this method could yield in order to make a comparison of the degrees to which various photosensitizers produce both singlet oxygen and hydroxide radicals in vitro. We also dedicated part of our efforts to an investigation into the effects of a simple variation in the partial pressure of the dissolved molecular oxygen.

According to our hypothesis the interactions of triplet photosensitizer molecules with doublet free radicals generated in vivo (native free radicals) play an important role in the overall photodynamic effect (MTO mechanism). In order to support this mechanism, experiments have been performed recently in vivo, in vitro and in chemical model systems. The precondition of such studies, however, is the measurement of the kinetic parameters of various native free radicals generated in the systems investigated. Experimental results and their kinetic evaluation indicate that the triplet-doublet interactions may actually take place in the overall processes under study.

The biological response to PDT depends on the photosensitizer accumulation to the malignant cells, the tumor vasculature, the inherent cell sensitivity to the photodynamic effect, etc. The in-vivo measurement of physical parameters related with these factors can serve in the understanding of the mechanisms involved in the cell death, as well as in the optimization of the treatment procedure. In this paper we demonstrate a modular diffuse reflection and fluorescence emission imaging colorimeter. We have used it for the in-vivo on-line quantitative measurement and mapping the time course fluorescence intensity after ALA application and for the tissue color changes associated with the erythema developed during irradiation. Based on the obtained quantitative data, we investigate the correlation between the photosensitizer accumulation before light irradiation and the hemodynamic changes, revealed as erythema development, occurring during light irradiation. Their relative significance as predicting factors for the photodynamic treatment effectiveness is also evaluated.

The photodynamic therapy (PDT) is an effective method to treat cancer and other nononcological lesions by means of light action on photosensitizer in tissue. It is considered that destroying effect is mainly due to the formation of singlet oxygen resulting from the interaction of light excited photosensitizer with molecular oxygen (triplet state in the ground state). So the destroying effect will be proportional to the rate of singlet oxygen formation which is in turn depends on light intensity, photosensitizer concentration and molecular oxygen concentration. The present work deals with the investigation of blood oxygen saturation in microcircular vessels (SO2) during light irradiation in the PDT process. It has been observed that SO2 behavior strongly correlates with the light power density applied for PDT. The high power density resulted in sharp SO2 decrease. The connection of SO2 decrease with enhanced oxygen consumption rate and vessel destruction due to PDT is discussed.

Antibacterial agents are increasingly being used for the prophylaxis and treatment of oral diseases. As these agents can be rendered ineffective by resistance development in the target organisms there is a need to develop alternative antimicrobial approaches. Light-activated antimicrobial agents release singlet oxygen and free radicals which can kill adjacent bacteria and a wide range of cariogenic and periodontopathogenic bacteria has been shown to be susceptible to such agents. In the oral cavity these organisms are present as biofilms (dental plaques) which are less susceptible to traditional antimicrobial agents than bacterial suspensions. The results of these studies have shown that biofilm-grown oral bacteria are also susceptible to lethal photosensitization although the light energy doses required are grater than those needed to kill the organisms when they are grown as aqueous suspensions.

The concept of the photodynamic surface is developed. Such surfaces are prepared by incorporating a suitable photosensitizer into a translucent thermoplastic polymer which can for subsequent evaluation and application be presented as a film, fiber, extrudate, molding or bead. The potential applications of such 'photosterile' materials in domestic, medical and industrial situations where it is desirable to maintain microbial populations at a low level are outlined. Polymer containing porphyrin and phthalocyanine sensitizers have been prepared by impregnation (dyeing), codissolution and casting, and copolymerization. Regenerated cellulose impregnated with 5,10,15,20-tetrakis(N- methylpyridinium)porphyrin tetra-p-tosylate has photobactericidal activity against S. aureus, E. coli., P. vulgaris and B. subtilis. The film retains both its mechanical strength and its photobactericidal properties after 50 h exposure to a xenon arc lamp. The effect is photobactericidal and not photobacteriostatic. A potential application in the development of safer domestic wiping cloths has been identified. The mechanisms of the microbicidal effect are considered to be (1) direct cell damage by singlet oxygen generated at the impregnated surface and diffusing to the microbial wall and/or (2) the controlled and slow release of the photosensitizer into solution and the microbial cell.

(delta) -aminolevulinic acid (ALA) induces the production of very high amounts of porphyrins in Gram-positive and Gram- negative bacteria. Accumulation of the porphyrins in the bacterial cell is a consequence of the high porphyrin production but most of the porphyrins are excreted from the cells into the medium. By fluorescence, measurements of the endogenic and of the exogenic content of the produced porphyrins can be determined. Bacteria loaded by their own accumulated porphyrins can undergo photoinactivation by various light sources. Killing of S. aureus cells by its endogenic porphyrins can be achieved by illumination with intense blue lights or by HeNe laser. E. coli cells loaded with endogenic porphyrins can be photoinactivated by intense blue and red light.

Photodynamic tumor therapy is able to induce apoptosis with many photosensitizers. Apoptosis is based on changes in gene expression and correlated to cell cycle activities. In this study, therefore, quantitative determination of the expression of the (proto)oncoges c-myc and bcl-2 in normal and transformed fibroblasts following PDT with ALA and low dose irradiation was connected with cell cycle analysis in order to investigate, if a risk for occurrence of secondary tumors by irreversibly increased oncogene expression can be found, if phases of the cell cycle show selective sensitivity to the therapy, and if changes in one of the two or both parameters may either precede or prepare an introduction of apoptosis. The results show (1) no mutagenic risk by timely limited overexpression of c-myc and bcl-2, (2) no selective cell cycle sensitivity to the therapy; but, in contrary, sustained increase of the proliferative activity of the transformed cells by the interaction of bcl-2 and c-myc, indicating a risk of promotion of tumor regrowth in sublethally damaged areas, (3) the introduction of apoptotic processes by low dose PDT in the cytoplasm/mitochondria and less in the nucleus. Transformed cells show higher constitutive gene expression and proliferative activities than normal cells.

Photodynamic therapy (PDT)-induced kinetics of apoptosis were studied in V79 cells using several differently localized photosensitizing dyes, mostly tetraphenylporphine derivatives. Apoptotic fractions were quantified by flow cytometry after staining the samples by the terminal deoxynucleotidyl transferase (TdT)-assay. Methylene blue derivative (MBD), a new dye for PDT, and 5-aminolevulinic acid (ALA)-induced protoporphyrin IX that are both localized in mitochondria, induced apoptosis rapidly within hours after PDT. With MBD it was shown that rapid apoptosis was induced only with dye concentration above a certain threshold. With a lower dye concentration apoptosis was delayed more than one day and was induced due to inhibition of oxidative phosphorylation. After PDT with two membrane localized dyes, tetra(3- hydroxyphenyl)porphyrin (3THPP) and Photofrin, maximal induction of apoptosis took about 12 h. With two lysosomal localized sulfonated meso-tetraphenylporphines (TPPS_2a and TPPS₄) no apoptosis was induced until more than 12 h after PDT. The results are discussed in relation to evidence in the literature on the nature of possible pathways involved.

In the present study it was found that human keratinocytes grown on collagen substrate, exhibited increased resistance to the hematoprophyrin-mediated photodynamic treatment, in comparison to keratinocytes grown on Petri dishes without collagen. Interestingly, no protection was afforded by the collagen gel to the cells in the corresponding control ('hematoprophyrin only' and 'light only') experiments. This observation was found to be independent of light dose and drug concentration, and the relative degree of resistance was the same for both normal and malignant cells. The degree of keratinocyte resistance was found to be closely related to the duration of cell attachment on the collagen substrate. These results are indicative of an active interference of collagen gel with the cellular evolution of the photodynamic phenomenon and they are also suggestive of variation in the photodynamic treatment efficacy according to the cellular environment.

Discovered during last year's phenomenon of PDT-induced apoptosis (programmed cell destruction) in cell culture immediately after light irradiation using phthalocyanine photosensitizers can be used for treatment of cancer. Experiments were carried out on mice with ascites. Ascitic liquid with the added photosensitizer was irradiated by light source with wavelength 660 - 680 nm and used according to ex vivo procedure. Actuation and development of apoptosis process in ascitic liquid were estimated by cytomorphological tests. It has been observed the phenomenon of growth of relative fraction of cells damage level expressed mainly as apoptosis after PDT procedure ex vivo. We suggest to call this phenomenon as PDT-induced post-irradiation apoptosis (PIP- apoptosis). Dependence between level of expressing of PIP- apoptosis and sulphonated phthalocyanine aluminum photosensitizer (Photosense) concentration at used photosensitizer concentrations has not been found out.

The study of single neuron response to photodynamic effect provides evaluation of the dynamics of cytotoxic events leading from initial threshold changes to the cell death and allows us to compare phototoxicity of different substances. The photosensitized isolated crayfish mechanoreceptor neurons were irradiated with the He-Ne laser (632.8 nm, 0.3 W/cm²), and their responses were recorded. The following photosensitizers were studied: methylene blue, janus green B, protoporphyrin IX, chlorins e6 and p6 and sulphonated aluminum phthalocyanine 'Photosens.' Crayfish neurons were insensitive to the He-Ne laser irradiation and photosensitization alone, but very sensitive to photodynamic effect: they changed firing rate and died at nanomolar concentrations of photosensitizers. The dynamics of neuron responses depended on the photosensitizer type and its concentration. Dependencies of firing acceleration rate and neuron lifetime on photosensitizer concentration allowed to compare efficiencies of different photosensitizers. Photosens and chlorin p6 were found to be the most potent photosensitizers.

Sodium pump (Na⁺/K⁺-ATP-ase) utilizing energy accumulated in macroergic bonds of ATP, maintains physiological sodium and potassium gradients across plasma membrane. This, in turn, preserves cell volume and enables functioning of all Na⁺-gradient driven cotransporters and exchangers. Therefore, any impairment of Na⁺/K⁺-ATP-ase activity results in severe consequences to cell viability. Cardiac cells are equipped with efficient enzymatic machinery capable of coping with oxidative stress. This was the rationale for examining their sodium pump activity upon photosensitization. Cells were incubated with hematoporphyrin dihydrochloride for 1 hour and irradiated using HeNe low-energy laser light (ED: 0.1, 0.3 or 1.0 J/cm²). It was found that Na⁺/K⁺-ATP-ase activity in 3-day-old cardiocytes is indirectly stimulated during photosensitization by rapid influx of sodium. The latter was determined by radiometric fluorescent probe SBFI/AM. Five-day-old cardiocytes turned out to be less resistant to photo-oxidative stress as the pump activity was inhibited by 50% in comparison to control cells.

Mitochondria constitute well-established target for photodynamic action provided that duration of incubation time with hydrophobic photosensitizer is long enough (18 - 24 hrs) to enable incorporation of the dye into these organelles. It was found that 1 hour incubation of rat cardiocytes with hematoporphyrin dihydrochloride (Hp) followed by low-energy HeNe laser irradiation (ED: 0.3, 1.0, or 3.0 J/cm²), affected mitochondrial membrane potential. The latter was monitored by means of the fast-response fluorescent probe DASPMI and the dynamic video imaging system equipped with frame-grabbing and analyzing software. The time-course of mitochondrial membrane potential decrease exhibited dependence on both: Hp concentration and ED used. In cells incubated with 1 (mu) g/ml Hp a decrease of DASPMI fluorescence occurred during 8 - 15 mins and was preceded by oscillations of fluorescence intensity. For cells incubated with 100 (mu) g/ml Hp disappearance of discrete pattern of mitochondrial fluorescence was observed 1 - 2 mins after irradiation.

Photodynamic therapy (PDT)-induced kinetics of apoptosis were studied in V79 cells using several differently localized photosensitizing dyes, mostly tetraphenylporphine derivatives. Apoptotic fractions were quantified by flow cytometry after staining the samples by the terminal deoxynucleotidyl transferase (TdT)-assay. Methylene blue derivative (MBD), a new dye for PDT, and 5-aminolevulinic acid (ALA)-induced protoporphyrin IX that are both localized in mitochondria, induced apoptosis rapidly within hours after PDT. With MBD it was shown that rapid apoptosis was induced only with dye concentration above a certain threshold. With a lower dye concentration apoptosis was delayed more than one day and was induced due to inhibition of oxidative phosphorylation. After PDT with two membrane localized dyes, tetra(3- hydroxyphenyl)porphyrin (3THPP) and Photofrin, maximal induction of apoptosis took about 12 h. With two lysosomal localized sulfonated meso-tetraphenylporphines (TPPS_2a and TPPS₄) no apoptosis was induced until more than 12 h after PDT. The results are discussed in relation to evidence in the literature on the nature of possible pathways involved.

The accumulation of antibodies or their fragments in tumor tissues is a basal requirement for immunotargeting of tumors in vivo. Immunotargeting is the first step for a polyphasic tumor therapy and diagnosis. Usually the high interstitial pressure and the insufficient blood supply of the tumor prevents the accumulation of tumor specific antibodies. To overcome these barriers the antibodies can be modified concerning their molecular weight. On the other hand, different injection techniques can influence the antibody distribution in-vivo. We developed a long term infusion technique for the testing of antibodies and antibody fragments in xenotransplanted tumors in the hen's egg test system. This in-vivo model allows to develop new protocols for the antibody administration resulting in a tumor localization without targeting of non-tumor tissues. Macroscopic and microscopic findings in the HET-system may reflect the binding sites of antibodies in-situ. Detection of tumor markers has not yet been realized due to problems inherent to the system.

It is well known that sulphophthalocyanines induce in process of PDT photochemical reaction of II type with generation of cytotoxic agent -- singlet oxygen. The combination of phthalocyanine and exogenic reductant -- sodium ascorbate may also induce other reactions, involving the formation of free radicals, and thus intensify the antitumor effect. The mechanism of action of the ascorbate may be connected also with the direct tumor cells damage (apoptosis) and with the stimulation of antiblastome resistance. We conducted the experimental study on 150 white mice with Erlich carcinoma. Macroscopic and microscopic data showed that ascorbate significantly increases the effect of PDT in comparison with control group due to the higher tumor damage, inhibition of its growth and stimulation of antitumor desmoplastic reaction.

The photosensitizer m-THPC (temoporfin, Foscan^R) is probably the most potent compound currently being tested for clinical use in photodynamic therapy. However, this compound is not freely water soluble and is extensively taken up and excreted by the liver following injection. This paper describes the initial characterization of a group of water- soluble poly(ethylene glycol) conjugates of m-THPC (SC102) in a mouse model system. The use of a carbonate linking group results in a labile compound which produced very high systemic photosensitivity at early times after injection. Triazine linked conjugates were stable both in vitro and in vivo and, although less potent than m-THPC on a molar basis, were capable of producing equivalent tumor necrosis accompanied by relatively low levels of muscle damage. In addition, SC102 tumor necrosis was seen over a wide range of drug-light intervals suggesting that these compounds may be less sensitive to treatment conditions than is m-THPC.

The effects of photodynamic therapy (PDT) of cancer needs objective estimation and its unification in experimental as well as in clinical studies. They must include not only macroscopical changes but also the complex of following morphological criteria: (1) the level of direct tumor damage (direct necrosis and apoptosis); (2) the level of indirect tumor damage (ischemic necrosis); (3) the signs of vascular alterations; (4) the local and systemic antiblastome resistance; (5) the proliferative activity and malignant potential of survival tumor tissue. We have performed different regimes PDT using phthalocyanine derivatives. The complex of morphological methods (Ki-67, p53, c-myc, bcl-2) was used. Obtained results showed the connection of the tilted morphological criteria with tumor regression.

The photodynamic therapy in the spectral range 690 - 750 nm is more perspective for treatment of the deeply located tumors because the absorption of tissue has a significant minimis in this spectral range. A series of phosphosubstituted phthalocyanine derivatives were studied in vitro and in experiment on white mice. It has been shown that photodynamic therapy using these photosensitizers was very effective and induced the prominent tumor damage (necrosis, apoptosis), inhibition of its growth and stimulation of antitumor desmoplastic reaction.

Certain types of cells may accumulate relatively high concentrations of protoporphyrin IX (PpIX) when exposed to 5- aminolevulinic acid or certain of its derivatives. PpIX is a photosensitizing agent which can be photoactivated in vivo by ultraviolet and/or visible light, thereby initiating a photodynamic reaction that may lead to the destruction of cells and tissues containing high concentrations of PpIX. This process is referred to as ALA-induced PpIX photodynamic therapy (ALA-PDT), a rapidly growing field of research. ALA is effective when administered topically, orally, by intravenous, subcutaneous, or intradermal injection, or by infusion into accessible body cavities. PpIX is the main photosensitizer produced under most conditions, although others may be present also especially during split dose therapy. Multiple wavelength excitation sources are required to produce and subsequently activate the far red absorbing chlorin type photoproducts that are produced under certain conditions.

Carcinoma in situ (CIS) of the bladder is a treacherous entity, that will develop into invasive cancer. Early treatment is mandatory in order to prevent progression. When conservative measures, such as Bacillus Calmette Querin (BCG) instillations have failed, radical cystectomy and urinary diversion is recommended. Whole bladder wall photodynamic therapy (PDT) with Photofrin II has been shown to be effective in eradicating carcinoma in situ, but often resulted in bladder shrinking. We wanted to evaluate the effects of PDT after aminolevulinic acid (ALA) sensitization. Six patients with refractory carcinoma in situ of the bladder were treated with whole bladder wall photodynamic therapy, after intravesical sensitization with aminolevulinic acid. The total light dose (scattered plus non scattered) was 75 J/cm². No skin sensitization occurred, nor loss of bladder capacity. One patient did not respond and was successfully treated with BCG. Another patient developed distant metastases. Carcinoma in situ was completely absent after 3 months in four patients (66%).

Twenty nine skin malignancies, 14 solar keratoses (SK) and 15 basal cell carcinomas (BCC) were treated with ALA-PDT. The overall cure response rate for SK was 85.7% and 66.6% for BCC. Specific histologic findings in sequential histology were indicative of the destructive and restoration mechanisms. A multispectral imaging system (MUSIS) was developed to perform diffuse reflection and fluorescence studies. Most of the lesions developed peak fluorescence 1.5 - 4 hours after ALA application. Hue was the most sensitive parameter to record the kinetics of the photosensitizer concentration in the atypical cells. It was worth noticing that the peak fluorescence differed among patients and this was an indication to start the irradiation more objectively. Erythema inspection and quantification during irradiation and afterwards was achieved by means of MUSIS. Erythema development was shown to include a new peak in the Hue histogram corresponding to a color range between red and violet. Time course erythema elicitation was found to differ between SK and BCC in accordance with recorded histologic difference relative to PDT-destruction mechanisms. Erythema development was shown to correlate significantly with the phototoxic effect and might be considered a reliable predictor of PDT efficacy.

This paper deals with the results of clinical trials for sulfated aluminum phthalocyanine (PHS) (Photosens, Russia) and Photogeme (PG) in Russia. The results of photodynamic therapy (PDT) of head and neck tumors (HNT), side effects and ways of their correction and prevention, as well as possibility to work out less toxic regimes of PDT with photosense, choice of laser and type of irradiation are discussed. PDT have been provided in 79 patients with different head and neck tumors. Efficacy of PDT depended on tumor size and its histological type. Undesirable changes in plasma content of antioxidants by means of high pressure liquid chromatography (HLPC) have been found in patients after PHS injection. Influence of short-term and long-term supplementation with beta-carotene and vitamin E on this parameters are discussed.

The frequency of tumor recurrences after conventional treatment is high (up to 35 - 60% depending on the stage of tumor progression) and the efficacy of conventional treatment of tumor recurrences and/or tumor 'remains' is low. In the State Research Center for Laser Medicine photodynamic therapy (PDT) is being applied to the patients with recurrent tumors of tongue, oral mucose and lower lip since 1992. In the last five years 26 patients with morphologically proven recurrent tumors of tongue, oral mucose and lower lip have been treated with PDT. The age of the patients ranged 50 to 84. Russian photosensitizers were used: photoheme (haematoperphyrine derivative in dosage 1.5 - 5.0 mg/kg body weight) and photosense (sulphonated aluminum phthalocyanine in dosage 0.7 - 1.5 mg/kg body weight) and Russian lasers. No complications of photosensitizer administration have been noted. In all cases PDT was carried out as an out-patient procedure. Therapeutic effect has been achieved in 22 patients. Complete resorption of tumors took place in 15 cases (57.7%). In 7 cases (26.9%) partial resorption of tumors took place. In four cases tumor resorption was less than 50% of elementary size. These results have been estimated as 'no response.' Mentioned results allow one to estimate PDT as a stand-alone technique for treating recurrent tumors of oropharyngeal localization. Although the results in some patients were estimated as 'no response,' there were no cases of absolute tumor resistance to PDT. The ability to perform PDT on the out-patients basis makes this technique cost-effective.

Photodynamic therapy (PDT) has the potential to be applied as an adjuvant treatment modality in advanced cancer cases and recently its role in the management of pancreatic cancer, has been evaluated. Nevertheless PDT remains an experimental therapy and one of the main reasons is its complexity, as far as it relies on the combined action of the photosensitizing drug and light source. In this study was evaluated the effect of various photosensitizer and photoirradiation doses, in the lethality of MIA PaCa cell line by using ZnPcS₄ as photosensitizer and a novel diode laser system as illumination source. No lethality was observed when only laser light or only photosensitizer was applied to cells. Cells treated with various concentrations of ZnTSPc and a light dose of 9 J/cm² indicated more than 70% lethality, 24 hrs after irradiation and with a light dose of 6 J/cm² presented almost 90% lethality, 72 hours after irradiation. The results show that low light and photosensitizer (drug) doses are enough to obtain lethality of more than 90% of MIA PaCa cells.

The results of application of photodynamic therapy (PDT) for treatment of malignant tumors of skin, breasts, tongue, oral mucose, lower lip, larynx, stomach, bladder, rectum and other localizations were assessed. In 1992 - 1997 more than 1200 tumoral foci in 288 patients have been treated with PDT. Most of the patients have been taken for PDT for tumoral recurrences or intradermal metastases after surgery, gamma- therapy or combined treatment. A certain number of patients had not been treated before due to severe accompanying diseases or old age. Russian photosensitizers Photoheme in dosage 1.0 - 5.0 mg/kg body weight, and Photosense in dosage 0.5 - 1.5 mg/kg body weight were used. Laser irradiation was performed using Coherent 'Innova-200' and Russian laser devices: copper vapor-pumped dye laser (wavelength 630 nm, output power -- 5 W), gold-vapor lasers (wavelength 628 nm, output power -- 2 W), solid-state laser (wavelength 670 nm, output power -- 2 W). In several cases non-laser light emitting devices have been employed. Up to date we possess the follow-up data in term from 2 months to 5 years. Therapeutic effect took place in 94.4% of the cases, including complete tumor resorption in 56.2% and partial resorption in 38.2% of the cases. The results of PDT application for treating malignant tumors allow one to estimate PDT as an adequate technique and in some tumor localizations PDT might become a method of choice. This new promising technique of cancer treatment is successfully applied in Russia. New photosensitizers and sources of light for PDT and fluorescent diagnostics are being developed.

Tumor suppressor gene p53 expression in a mouse fibrosarcoma following in-vivo photodynamic therapy has been studied using the immunohistochemical method. Photodynamic treatment involved injections of the well known sensitizer -- hematoporphyrin derivative at the doses 1.25 and 2.5 mg/kg of body weight and irradiations at the doses 25 and 50 J/sq cm. Glass slide preparations from PDT-treated tumors were obtained at different time points (15, 60 minutes, 2 and 24 hours) after therapy, subsequently stained for wild type/mutant p53, and assessed for positive reaction. High PDT doses (HpD -- 2.5 mg/kg; light dose -- 50 J/sq cm) correlated with decreased expression of p53 in tumor cells. The other part of the study was directed to measure the xanthine oxidase (XO) activity in the tumor cells. PDT included injections of HpD and light exposure at the same doses as for p53 study. We observed a complete inhibition of the enzyme activity. The slight increase in XO activity was found following treatment with either light or HpD alone.

The application of nonradioactive in situ hybridization with chromosome-specific probes for cytogenetic analysis has increased significantly in recent years. In the field of photodynamic therapy (PDT) the hypothesis is that after PDT the remaining viable malignant cells are potentially metastatic cells. Therefore, we performed in vitro experiments on human bladder carcinoma cells to evaluate numerical chromosomal abnormalities before and after PDT. The possible genotoxic effect of PDT with porphycene (AamTPPn) appears to be small based on criteria such as numerical chromosomal abnormalities for chromosome 1 and 18.

Seven oral antidiabetics (chlorpropamide, glibenclamide, glipizide, gliquidone, glymidine, tolazamide, and tolbutamide), and 14 diuretics (bemetizide, bendroflumethiazide, benzylhydrochlorothiazide, bumetanide, butizide, chlortalidone, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, piretanide, polythiazide, trichlormethiazide, and xipamide) were investigated for potential phototoxicity in vitro using a cell culture model and in vivo in hairless mice. After exposure to broad band UVA, the majority of the substances tested in vitro yielded phototoxic action leading to loss of culture forming ability. In vivo, all tested substances induced edema or ulceration, and lead to a significant increase in skin fold thickness of the mouse skin. In all a number of substances not described to induce clinical photosensitivity nor phototoxicity in vitro or in vivo were detected in our testing. In determining potential photosensitizers, it seems important to utilize different test methods, as not all substances will exhibit action in a given assay.

In the present study cellular uptake and intracellular localization of a liposomal formulation of Zn-PC, was studied in human cervix carcinoma cells of the line NHIK 3025. The cellular uptake of Zn-PC was found to be completed after 4 hours of incubation. Seventy to eighty percent of the dye taken up by the cells during 18 hours of incubation was retained after 1 hour of further incubation in sensitizer-free medium. The cellular uptake was attenuated by the presence of serum and at low temperature of the incubation medium. The uptake was not enhanced by decreasing the pH of the incubation medium. The present study indicated that Zn-PC is rapidly transferred from liposomes to lipoproteins. Analysis of inactivation of subcellular marker enzymes indicated that Zn- PC is located in the Golgi apparatus and mitochondria.

For the application of photosensitizers in PDT attention must be given to the stability of these compounds. For various substituted and annelated tetraazaporphyrins zinc complexes the experimentally determined first order rate constant of the photo-oxidative decomposition in DMF were compared with the calculated values of the HOMO energy level. The method described allows one to predict the photo-oxidative stability by calculating their HOMO levels which is important for the use of macrocyclic metal complexes in the photodynamic therapy of cancer. To high photo-oxidative stability leads to risks in healthy tissues and reduces photodynamic effects in tumor tissues. To low photo-oxidative stability cause problems in preparation, purification and in vivo persistency of photosensitizers for tumor treatment.

Suitable substituted phthalocyanines are promising and widely investigated photosensitizers (PS) for the photodynamic therapy of cancer (PDT). However, selective accumulation of the PSs in tumor tissues, avoiding contamination of healthy tissues, is still an unsolved central problem. We present first results on the synthesis of new biotinylated phthalocyanines as potentially selective photosensitizers when applied in a polyphasic tumor targeting (tumor cell plus first step: biotinylated/monoclonal antibody, second step: streptavidin, and third step: biotinylated PS). The binding and the photodynamic activity of biotinylated PS in this three step model is shown in tumor cell lines.

The interaction of aromatic amino acids, superoxide dismutase (SOD) and bovine serum albumin (BSA) with chlorin e₆ (Chl) and formation of complexes were studied. The decrease of the efficiency of photochemical destruction in the absence of oxygen was observed. The changes of SOD absorption spectra in UV region agree with our former results on irreversible decrease of protein's tryptophan (Trp) luminescence under irradiation. At the same time no changes in enzymatic activity of irradiated SOD in the presence of Chl were detected. The irradiation effect on K⁺ and H⁺ ions movement across the membrane of erythrocytes loaded with Chl was studied.

The pharmacokinetics of 13²-hydroxy-bacteriopheophorbid-a methyl ester (13²-OH-BPME) and octa-(alpha) -butyloxy-zinc phthalocyanine (8-(alpha) -bo-Zn-Pc) were studied in mice bearing Lewis lung carcinoma. Absorption spectroscopy was used to measure the photosensitizers concentrations. High 13²- OH-BPME and 8-(alpha) -bo-Zn-Pc uptakes were recorded in the parenchymatous organs (liver, lung), with 8-(alpha) -bo-Zn-Pc long retention. The malignant tissues accumulated 13²-OH- BPME of more than 20 folds than 8-(alpha) -bo-Zn-Pc at all incubation times. 8-(alpha) -bo-Zn-Pc concentrations in the skin and the muscle were lower than 13²-OH-BPME concentrations at any interval period, but also, the 8- (alpha) -bo-Zn-Pc retained longer until 168 h. PDT with 13²-OH-BPME will be more effective than with 8-(alpha) -bo- Zn-Pc where it has more concentrations in the malignant tissues.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)- induced photosensibilization may be used to selectively eliminate Barrett's epithelium. To find optimal treatment parameters, the kinetics, localization and mechanism of ALA- induced protoporphyrin IX (PpIX) accumulation in rat oesophagus were studied. After a single dose of ALA (200 mg/kg i.v. or oral), porphyrin accumulation in the mucosa was 3.4 fold higher than in the muscularis, with a maximum at 3 hours after ALA administration. Enzyme measurements showed a higher ratio of PBGD:ferrochelatase in mucosa. In conclusion, the rat oesophageal mucosa can be selectively photosensitized with PpIX after a single dose of ALA, with a peak concentration at 3 hours. Selectivity may be caused by the higher PBGD:ferrochelatase ratio. This makes ALA most suitable for PDT treatment of Barrett's oesophagus.

Insertion of cholesterol into phosphatidylcholin vesicles modifies the kinetics of intermembrane porphyrin movement due to as its effects on the rate of transmembrane and intermembrane pigment transfer, as the change of pigment intramembrane distribution pattern. The cholesterol-induced alteration of porphyrin intra- and intermembrane redistributions depends strongly on pigment polarity. According to flow cytometric study results, the reduction of cellular cholesterol through the incubation of cells with phosphatidylcholin liposomes enhances the cells' affinity for porphyrins and at that time efficiently promoted pigments clearance from cells. The potential role for cholesterol and its disproportionation within the cellular membranes as determinants of porphyrin binding and retention by cells is suggested.

Using fluorescence activated cell sorting we have compared the binding of a number of porphyrins with different polarity by blood cells. According to pigment level blood cells may be arranged in order granulocytes greater than or equal to monocytes greater than lymphocytes greater than erythrocytes. Cellular accumulation of selected porphyrins in blood cells was remarkably different. The equilibrium level of chlorin e6 dimethylester in blood cells was about 15 times higher compared with chlorin e6. As a result, the percentage of pigment binding by blood cells varied from 0% (of total amount) in the case of polar pigments to about 50% for moderately apolar porphyrins. The results obtained show that pigment binding to blood cells may be of certain value when the pharmacokinetic behavior of porphyrin sensitizer is analyzed.

Combined treatment of PDT and hyperthermia was examined on Ehrlich ascites tumor cell viability and A22 hepatoma tumor growth inhibition. Histological evaluations of tumors after different treatments have shown tumor necrosis, congestion of blood vessels and hemorrhage. The most drastic damages were observed after simultaneous PDT and hyperthermia action. At these experimental conditions tumor growth for 5 days was absolutely inhibited.

It is well established that the selectivity of accumulation of photosensitizer in malignant tissues determines largely the efficiency of photodynamic therapy. Existing photosensitizers have insufficient selectivity. Thus the conjugates of photosensitizers with different carriers are promising for targeting delivery into tumor. Recently the potential of the antibody-avidin-biotin system is well studied for in vivo diagnostics. In this work we suggest to use this system for targeted delivery of the polybiotinylated compounds into cancer cells. The avidin-biotin system is highly flexible from the point of view of the pharmacokinetics of uptake of photosensitizers and the pharmacodynamics of their action. This makes it possible to select optimal conditions for phototoxic action of polybiotinylated photosensitizers and some other drugs, which are derivatives of 'Photosens' and 'Teraphthal.'

The influence of drugs acting as (beta) -receptors agonists or antagonists on the uptake of Photofrin II in C6 glioma cultured cells was studied by microspectrofluorometric analysis. The pharmacological effect was evaluated on the semiconfluently grown cells, characterized by a long lasting uptake process and higher values of fluorescence intensity with respect to the solitary ones. Isoproterenol treatments resulted in a significant enhancement (by 50%) of the intracellular fluorescence signal of Photofrin II. This effect was hindered by contemporary treatments with equimolar alprenolol or propranolol, two (beta) -receptor antagonists, indicating a specific effect of isoproterenol. Both pharmacological activation of vesicular transport and changes in the membrane physical-chemico properties can explain the effects induced by drugs interacting with (beta) -receptor.

Photodynamic therapy (PDT) has become an approved treatment for different types of cancer in many countries over the last few years. As an example one might mention PDT of the early stages of bronchial or esophageal cancer which have been treated with only about 20% recurrence being observed over several years of follow-up. The low degree of invasion of PDT, as compared to most alternative treatments as well as minimal sided effects, and good repeatability, all speak for this treatment modality. Improved and cheap screening procedures, that are now being developed for the early stage disease, will lead to a more frequent application of PDT for these indications. Detailed studies of PDT showed that certain dyes, after systematic or topical application, could be taken up more in neoplastic tissue as compared to the surrounding normal tissue in the clinical context, thus leading to 'selective' or at least partially selective destruction of the tumor following light application. This selectivity of uptake of certain compounds in hyperproliferative tissue, as well as the observation that PDT can lead to blood vessel stasis, suggested that photodynamic therapy might be worth trying in non-tumor disease. Some of the diseases associated with hyperproliferation and/or neovascularization which are being considered for PDT are listed in table I.

POPC, DPPC/DMPC and POPC/OOPS liposomes containing gadolinium(III) meso-tetraphenylporphyrin and gadolinium(III) meso-tetra(4-pyridyl)porphyrin have been prepared by different techniques, their porphyrin and lipid contents have been evaluated, and the size and stability of the liposomes have been determined. The results show the influence of the method of preparation on the amount of Gd-porphyrin incorporated, and that of the lipid composition on the incorporation efficiency and stability of the Gd(III) porphyrin trapped in the liposomes, as well as the presence of strong lipid to porphyrin association at least in the case of the DPPC/DMPC liposomes.