The chlorophyll-a degradation product, purpurin-18 methyl ester (4), was converted into a series of new bacteriochlorins (18)-(20). These stable bacteriochlorins have strong long- wavelength absorptions > 800 nm, and were tested for in vivo photosensitizing activity using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. In preliminary screening, among the photosensitizers tested so far, bacteriochlorins (19) and (20) have shown promising anti-tumor activity. Preliminary attempts are described, using photo- Fenton chemistry, to synthesize photosensitizers which display both Type-I and Type-II photochemical behavior.

The in vitro photodynamic inactivation of a KB cell line is studied using a large series of neutral water-soluble glycosylated porphyrins. These compounds in which hydrophilic glycosyl moieties and hydrophobic substituents are linked at the meso positions, generate singlet oxygen in roughly 70%. Results reveal that the phototoxicity is markedly dependent on the nature, the number, and the linking position of glycosyl moieties controlling their amphiphilic characters.

The preparation of several differently substituted metallo-tetrabenzoporphyrins from the appropriate 1,3-dimethylisoindoles is described. The synthesis of these and of the required 1,4-dicarbonyl precursors is also presented. Monosulphonation has been performed on nickel(II) 1,4,8,11,15,18,22,25-octamethyltetrabenzo[b,g,l,q]porphyrin, as a means to increase the amphiphilic properties of these compounds.

Photodynamic therapy (PDT) is a promising approach for the treatment of cancer, and is presently being evaluated worldwide. PDT is based on the ability of photosensitizers to accumulate selectively in tumor tissue. On irradiation fluorescence of the sensitizer allows tumor localization. Singlet oxygen formed by sensitization causes tumor destruction. Hematoporphyrin derivatives and Photofrin II^R, at the present the only practically used sensitizers in PDT, absorb light only weakly ((epsilon) approximately equals 5.000 at (lambda) equals 620 nm). It has been shown that Photofrin II^R is a multicomponent mixture of different porphyrins with only a few therapeutically active components. In contrast to porphyrins, chlorins and azachlorins have ideal photophysical properties with strong absorption ((epsilon) approximately equals 50.000) in a spectral region ((lambda) equals 650 nm) where tissue transmits much better. Molecular properties of porphinoid structures, which are responsible for tumor uptake and tumor enrichment, are connected with lipophilicity and aggregation behavior. Therefore our aim was to synthesize well defined chlorins with lipophilic properties similar to those of the active fractions of Photofrin II^R.

The reaction of singlet oxygen with vinyl porphyrins, which was first described in the case of protoporphyrin, is reinvestigated as a route to chlorin type molecules. Various dicarboxylic chlorins were thus prepared and characterized for their chemical and photosensitizing properties. All of them efficiently photosensitize the production of singlet oxygen. Some of these compounds might be formed during photodynamic therapy with hematoporphyrin derivative or Photofrin. Their biological relevance is discussed.

The parameters of chlorin e6 and trimethylester of chlorin e6 incorporation and distribution in suspensions of unilamellar liposomes of DMPC, DPPC, and DSPC, as well as efficiency of the pigment redistribution from liposomes to cellular membranes have been studied. Determination of the fraction of pigments' fluorescence which is accessible to quenching by a watersoluble quencher indicates that for both chlorins the outer monolayer of the liposomal membrane is more populated than the inner one. Gel-liquid crystalline phase transition induces a shift of a part of the pigments' molecules toward the inner monolayer. By means of ultrafiltration technique it is shown that chlorins binding to liposomal membrane occurs as partitioning between water and lipid phases. The partition coefficient is affected strongly by the type of pigment, the phase state of the lipid bilayer. Similar results were obtained when the influence of the physical state of the lipid bilayer on the rate of chlorins redistribution from liposomes to cellular membrane was studied. These findings show that diffusive mobility of the sensitizer in suspensions of cellular and model membranes is a complex process which is dependent on structural features of both the pigment and its biological carriers.

The principal advantage of photodynamic therapy is a selective eradication of neoplastic tissues due to a high dye uptake by the tumor. A considerable excess of photosensitizer (PS) concentration in a tumor in comparison to different types of normal tissue has been demonstrated in vivo and in vitro. There are different suppositions about the mechanism of this excess. We suggest one of the likely mechanisms of a penetration of hydrophobic PS through a cell plasma membrane. Accordingly, the developed mathematical model has yielded a time dependence and steady state values of PS concentration in cell membrane and cytoplasm based on a hypothetical mechanism of PS penetration into the cell and taking into account the values of extra- and intracellular pH. A distribution of different PS ionic species was obtained as a function of external pH. Computer analysis of the model has allowed us to suggest that a selective PS accumulation in a neoplasm in vivo may be determined by the greater lipid fraction inside malignant cells relative to normal ones and low extra- and intracellular pH in cancerous tissues.

We employ classical porphyrins, as well as second-generation sensitizers, such as tetrabenzo- porphyrins, naphthaloporphyrins, texaphyrins, and sapphyrins for biological photosensitization. We studied the extent of sensitizer binding to biological membranes and bacterial cells; the singlet oxygen production yield in homogeneous solutions and in membranes; and the efficiency of the photo-cytotoxic effect of the sensitizers. In carrying out these studies, absorption and fluorescence spectra were used as monitoring techniques. We demonstrate a spectroscopic probing procedure, based on the well established chemical quencher of singlet oxygen, DPIBF, which at appropriate concentration allows a simple mathematical evaluation of the absolute quantum yield of singlet oxygen production. The method does not rely on any predetermined kinetic values and provides a result with high accuracy. The relative efficiency of competing photochemical processes in cells was established, so that the likely mechanism which is responsible for the death of the cell could be pointed out. A unique and exceptional wavelength dependence of photophysical properties is described.

One of the most important problems for clinical use of photodynamic therapy is the selective accumulation of photosensitizer molecules by the tumor tissue. Possibly cyclodextrins could be used to solubilize and transport hydrophobic dyes to the target. Until now it has not been investigated to what extent the dye-cyclodextrin complexation alters the photophysical properties of the dye molecules important for their photodynamic activity. For this reason we investigated the behavior of three sensitizers belonging to different chemical classes: pheophorbide a, octa-(alpha) -butyloxy-Zn-phthalocyanine, and tetra-tert-butyl-Zn- naphthalocyanine in cyclodextrin solutions.

A novel mode to apply photosensitizing drugs specifically to tumor tissue using the principles of polyphasic tumor therapies is outlined. Key compounds are tumor specific functionalized antibodies with reduced immunogenicity. These bind to drug inclusion complexes in a multiplicative manner. Drug inclusion complexes are designed on the basis of tethered functionalized (beta) -cyclodextrin dimers with maximum affinity to porphyrinoid photosensitizers forming monomeric chlathrates. To enhance porphyrin-cyclodextrin interaction peripheral groups of the porphyrin have to be chemically modified. The development of the method is not yet completed. First results are demonstrated.

Quantum yields for formation of singlet molecular oxygen have been measured for sodium pheophorbides (Na-Phdes) a and b in aqueous and non-aqueous media. Measurements have been made for both steady-state and pulsed laser excitation with the resultant singlet molecular oxygen being detected by photo-oxygenation reactions or time-resolved luminescence spectroscopy, respectively. Singlet oxygen production sensitized by Na-Phdes a or b is insignificant in aqueous media but occurs with a good efficiency in organic solvents. Plasmid DNA is efficiently photocleaved by Na-Phdes a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na-Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na-Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures.

The photosensitizers uroporphyrin III (UP III), coproporphyrin III (CP III), and protoporphyrin IX (PP IX) were examined with respect to their composition (by different molecular species), uptake and distribution in cells and tissues, intracellular pH value, cytotoxicity and formation of photoproducts. Whereas hydrophilic UP III and CP III were characterized by low cytotoxicity and preferential accumulation in lysosomes, hydrophobic PP IX was found to be rather cytotoxic and mainly localized in cellular membranes. Rapid uptake in tissues and some preferential location in tumor cells were detected for hydrophobic photosensitizers.

In recent years a lot of new far red absorbing sensitizers for photodynamic therapy (PDT) were synthesized, like derivatives of bacteriopheophorbide. The possibility of increasing the accumulation in tumor tissue by coupling these dyes to carrier molecules is investigated. The photophysical properties of these dyes can be dramatically changed as a result of covalent binding to carrier molecules, different solvents, or biological surroundings. In heterogeneous systems ¹O₂ is strongly quenched. Therefore, its luminescence is difficult to detect. Another difficulty is the fact that especially far red absorbing sensitizers show fluorescence at the wavelength of ¹O₂-luminescence at 1270 nm, which can be several magnitudes higher than the luminescence of ¹O₂. The efficiency of singlet oxygen (¹O₂) generation is of major interest because ¹O₂ seems to play an important role in PDT. Time resolved luminescence measurements of ¹O₂ generated by dyes in micelles and liposomes in D₂O were already made in our group. In order to investigate more complex systems and to avoid unwanted photochemistry we changed the excitation wavelength from UV (337 nm) to VIS/NIR(ND:YAG/OPO). The excitation power was increased and the detection limit was decreased. First measurements in red cell ghosts are presented.

Performance characteristics of a high average power tunable laser system designed for clinical photodynamic therapy (PDT) are described. Seventeen watts of TEM₀₀ average power at 630 nm with sulforhodamine 640 dye has been achieved. The time*power pump laser product for the dye laser's output power to degrade to one half of its initial power was recorded to be thousands of watt*hours or hundreds of thousands of photons per molecule. The system had a wall plug efficiency of .33%. The system utilizes the high efficiency KTiOPO₄ (KTP) frequency doubled Nd:YAG laser to pump a tunable dye laser system designed specifically for the low maintenance and high reliability required in PDT. Performance at other wavelengths of interest in investigational and clinical PDT is described. Comparative results obtained with sulforhodamine 640 versus rhodamine 6G under identical conditions are described. Performance of various manufacturer's preparations of sulforhodamine 640 was assessed. Comparison to other high average power tunable systems as well as long term system performance including dye stability data are presented.

The primary process in the usual photosensitized tumor therapy is sensitization from the sensitizers lowest triplet state. Alternatively, the laser light can be pulsed so that excitation occurring over short interval at high peak power can induce two-photon excitation of sensitizers via nonresonant absorption and initiate photodynamic action. The theoretical model has been developed and laser pulse parameters that ensure efficient two-step process have been calculated. In addition, the threshold that separates the two-step process through singlet manifold or triplet manifold was estimated. Both two-step excitation regimes should be initiated with tailored laser pulses. In this work we focus our attention on the characterization of the parameters (the lifetime of excited singlet state, quantum yield of interconversion to the triplet state, etc.) of the most suitable sensitizers for the proposed two-step excitation scheme. On the basis of our model the parameters of the laser pulse and the parameters of the most efficient sensitizer in two-step photosensitization process could be selected.

One of the important problems of the theory and practice of spectroscopy of live biological objects is determination of mechanisms of photochemical and biological processes under the influence of light irradiation. As a part of this the revelation of relationship between chemical peculiarities of photosensitizers, their photochemical behavior, and their biological influence character is significant.

The potential advantages of fluorinated porphyrin photosensitizers are outlined. The syntheses of fluorinated analogues of sensitizers which are known to be active in vivo are described with particular reference to compounds of the tetrakis(hydroxyphenyl)porphyrin series. Three new fluorinated (hydroxyphenyl)porphyrins are prepared [5,10,15,20-tetrakis(2-fluoro-3- hydroxyphenyl)porphyrin; 5,10,15,20-tetrakis(2,4-difluoro-3-hydroxyphenyl)porphyrin; and 5,10,15,20-tetrakis(3,5-difluoro-4-hydroxyphenyl)porphyrin], and some of their relevant properties are described.

The photosensitizer benzoporphyrin derivative monoacid ring A (Verteporfin^R or BPD) has maximum absorption characteristics (690 nm) and biodistribution characteristics which permit activation of the drug in capillaries of the skin without causing skin photosensitivity (transdermal PDT). This permits targeting of cells in the circulation for selective ablation. Since BPD has been shown to accumulate preferentially in activated lymphocytes and monocytes, studies have been undertaken to determine the effect of transdermal PDT on murine models for rheumatoid arthritis (the MRL/lpr adjuvant enhanced model) and multiple sclerosis (the experimental allergic encephalomyelitis (EAE) model in PL mice). Localized transdermal PDT with BPD was found to be completely successful in preventing the development of adjuvant enhanced arthritis in the MRL/lpr mouse as well as improving the underlying arthritic condition of these animals. In the EAE model, in which an adoptive transfer system was used, it was found that transdermal PDT of recipients was effective in preventing EAE if treatments were implemented up to 24 hours after cell transfer but was not effective if given later, indicating the requirement for circulating T cells for effective treatment.

Currently transfused cellular components of blood are not available in a sterile form and carry a small risk of transmitting viral and parasite diseases. Using phthalocyanines and red light, lipid enveloped viruses, e.g., HIV-1, can be inactivated in red blood cell concentrates (RBCC). Under conditions leading to virus sterilization the blood borne parasites Trypanosoma cruzi (Chagas disease) and Plasmodium falciparum (malaria) could be eliminated to undetectable levels (> 4 log₁₀ kill). RBC damage during treatment could be avoided by increasing the light fluence rate to 80 mW/cm², and by including the free radical scavenger glutathione and the vitamin E derivative Trolox during light exposure. Similar sterilization of platelet concentrates was achieved with the psoralen derivative AMT and UVA light. Platelet damage due to PUVA treatment was avoided by including the plant flavonoid rutin during irradiation. It is concluded that elimination of the risk of transmitting pathogens during blood transfusion is feasible with photochemical treatments.

Helicobacter pylori (H. pylori) is associated with a large number of gastroduodenal disorders. Clearance of the bacteria has been shown to benefit patients with duodenal ulcers, gastric ulcers, and certain rare types of gastric tumors. Broad-spectrum antibiotics are the mainstay of current treatment strategies but side-effects, poor compliance, and drug resistance limit their usefulness. We sensitized H. pylori with toluidine blue, haematoporphyrin derivative, aluminum disulphonated phthalocyanine, methylene blue or protoporphyrin IX prior to exposure to low-power laser light from either a gallium aluminum arsenide laser or a helium neon gas laser. All 5 sensitizers caused reductions of greater than 1000-fold in the number of viable bacteria. Light alone had no effect and only HpD caused a significant decrease in bacterial numbers without laser light. Next, we sensitized H. mustelae on explanted ferret gastric mucosa (ex vivo) with the same sensitizers and exposed them to light from a copper vapor pumped dye laser tuned appropriately. MB caused significant reductions in bacterial counts. Successful lethal photosensitization of Helicobacter pylori both in vitro and ex vivo raises the possibility of a local method for eradicating the bacteria, especially as the bacteria are only found in those parts of the upper gastrointestinal tract that are accessible to the endoscope.

The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS₄) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS₄ resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS₄ is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS₄ was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS₄ in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS₄ to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS₄ is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS₄ and light.

Uptake and release/retention of the photosensitizer, benzoporphyrin derivative, monoacid ring A (BPD; 1 - 20 (mu) g/mL) was studied using cell lines (K562, L1210) and normal, non- activated and Concanavalin A-activated murine splenocytes. Concentrations of BPD in cell lysates were determined by fluorescence (440 nm excitation, 694 nm emission). The results showed that BPD was taken up and released rapidly by all types of cells within the same time frame. Maximum of BPD depended on the type of cells and was greatest in tumor cells, lowest in normal, non-activated cells and intermediate in activated cells. In addition, the maximum uptake depended on BPD concentration in the medium, length of incubation and presence of serum. All cells, regardless of type, retained a constant proportion (20 - 30%) of the amount of BPD taken up. This proportion was independent of length of incubation, BPD concentration in the medium and presence of serum. However, due to differences in maximum amounts of BPD taken up under the same conditions, tumor cells retained more BPD than normal cells and activated cells more than non-activated. The retained BPD was able to photosensitize the cells. The results were found to be relevant to the in vivo studies.

Photodynamic therapy (PDT) of cancer has been developed during the last fifteen years. Now a second generation of photosensitizers is introduced into the clinic practice whose prime advantage is higher absorption in the far-red region of the optical spectrum. Phthalocyanines (Pc) are promising representatives of this type. Sulfonated derivatives of phthalocyanine metal complexes (MeS_nPc) have been proposed for photodynamic therapy since they demonstrate a good phototoxic efficiency, are water-soluble and have a low level of dark toxicity. It was already reported that the sulfonation degree influences the photodynamic activity of MeS_nPc. Various other sensitizers are proposed for use in PDT, but still the choice of the most efficient sensitizer is mostly an empirical decision. One of the main reasons for this is the need for a clearer and deeper understanding of the relationship between the structure of the sensitizing molecule, its interaction with the cells (and tissues) and the mode and efficiency of cell death caused by subsequent photodynamic action. The present work was undertaken to investigate some aspects of this problem. We used several phthalocyanines (phthalocyanines with various degree of sulphonation, phthalocyanine adsorbed to silver sol, phthalocyanine linked to the antibody and phthalocyanine located only in the extracellular medium) to ensure different ways of internalization by the cell and studied their intracellular localization and photodynamically induced cell damage by means of confocal laser scanning microscopy, quantitative fluorescence microscopy, and flow cytometry.

This report describes the discrepancy between pheophorbide A (PH-A) localization and photodynamically induced necrosis for the digestive tract. After an IV 9 mg/Kg^-1 sensitization, the dye was caught by the whole digestive tract and its inherent vessels, as shown by fluorescence microscopy. The dye fluorescence disappeared within 24 h from the stomach and the jejunum. It remained high in the pancreas, the portal vein, the bile duct, the arteries and the duodenal mucosae. A light dose, 660 nm, 100 J/cm^-2, 24 h after Ph-A sensitization, induced a necrosis of the duodenal mucosae. The pancreas and the hepatic pedicle were relatively unaffected by photodynamic therapy (PDT). The duodenal response to PDT results logically from its selective PH-A retention. But hepatic pedicule and pancreas immunities for PDT involve either protecting singlet oxygen scavengers or photosensitizer quenchers.

The newly synthesized potential photosensitizer octa-(alpha) -butyloxy-zinc phthalocyanine (8-(alpha) -bo-ZnPc) is characterized by a high absorption coefficient at the far red wavelength 735 nm and a good singlet oxygen quantum yield. The pharmacokinetics of 8- (alpha) -bo-ZnPc were studied on the Lewis lung carcinoma in mice after i.v. administration of 7.8 micrometers ole/kg body weight solubilized in PBS with 1% Tween 80 at 2, 6, 12, 24, 48 and 168 hour incubation intervals. The accumulated dye contents were chemically extracted from selected tissues and the concentrations were measured by absorption spectroscopy. The parenchymatous organs, liver and spleen, showed maximum 8-(alpha) -bo-ZnPc concentrations after 6 h incubation (33 nmole/g liver and 5 nmole/g spleen, respectively). An extensive uptake was detected in the lung where concentrations higher than 90 nmole/g tissue were measured in the 6, 12, 24, and 48 hour extracts. The malignant tissue did not accumulate higher 8-(alpha) -bo-ZnPc concentrations than 0.4 nmole/g tumor during the entire investigation period. The dye contents extracted from muscle, representing normal tumor surrounding tissue, and skin were even lower and never exceeded 0.11 nmole/g muscle (6 h) and 0.21 nmole/g skin (2 h), respectively.

Experiments were performed on five batches of Wistar inbred rats with Walker-256 carcinosarcoma receiving as sole treatment photodynamic therapy (PDT), tumor-infiltrating lymphocytes (TIL), or associated therapy (PDT + TIL - A; PDT + TIL - B). The control batch (HBSS) consisted of animals with untreated Walker-256 tumors. The results were as follows: the sole treatment (PDT, TIL) gave survival rates between 41.4 and 52.9%, the cure rates ranging from 13.8 to 38.2%. The `combined' therapy in multiple doses increased significantly (92.8%) the survival rate of tumor bearing rats as well as the highest incidence of complete tumor regression (82.1%). Cell-mediated immunity test values in batches III and IV exposed to multiple doses of PDT + TIL showed higher values as compared to the values noticed in batches I - II and the control batch V, performed at 10 and 21 days post-treatment. Summing up, this work demonstrates that `combined' photodynamic therapy with immunotherapy with TIL stimulates cell-mediated antitumoral activity, increases survival rates, and reduces incidence of Walker-256 carcinosarcoma in the rat model.

The analysis of the results of treatment of 379 malignant neoplasms with PDT in 89 patients has been made. Photogem (hematoporphyrin derivative) and Photosense (aluminum sulfonated derivative) -- both produced in Russia -- were used as photosensitizers. An argon-pumped dye- laser called `Innova 200' (Coherent USA), a Russian dye laser with copper vapor pumping (Yakhroma 2), a gold vapor laser (630 nm and 627.8 nm, accordingly) for Photogem, and a solid aluminate ittrium laser (672 nm) for Photosense were used. Up to now we have had follow-up control of 75 patients for the period of 2 months to 2.5 years. Positive effect of PDT was seen in 90.7% (68 out of 75); including complete regression -39 (52%), partial (50 - 100%), -in 29 (38.7%).

In the present study, the biological testing of the photodegradation of protoporphyrin IX (PP IX) is proposed. The method implies preirradiation of PP IX ((lambda) < 415 nm) in buffer solution (with or without serum) with the following adding to cells and irradiation of this mixture ((lambda) < 415 nm). The increase in survival with increase in preirradiation fluence was used for estimation of rate and quantum yield of PP IX photobleaching in solutions. The quantum yield estimated by the proposed method was compared with quantum yields of photodegradation of PP IX by decay in absorbance and decay in fluorescence. The highest quantum yield of photodegradation of PP IX was obtained by the test of cells photoinactivation, the lowest by the decay monitored by absorbance. The different values of quantum yield of photodegradation are discussed in the view of photobleaching of monomeric forms which are much more photolabile than aggregates and of photoproduct formation. Biological testing of photodegradation of monomeric species of PP IX seems to be the most specific.

PH is a factor relevant for the molecular state of the sensitizer used in PDT and so may influence its photochemical activity and its interaction with cells and tissues. Thus the measurements of microenvironmental pH of the sensitizing molecule, its dynamics during irradiation with light and subsequent development of cell damage are interesting from the point of view of the PDT molecular and cellular mechanisms. Two ways of performing such measurements are suggested: the first one is to use conventional fluorescence or transmission pH-probes, and second using the sensitizer itself as such a probe. We show the complications arising in the first approach due to the interaction between the probing and sensitizing molecules on the example of measurements of the microenvironment pH of tetrasulphonated aluminum phthalocyanine (AlPcS₄) in cultured cells with the use of neutral red. These complications are expected to be common also for the other conventional pH-probes. We studied concentrational and pH dependencies of the fluorescence emission, fluorescence excitation and absorbance spectra, and fluorescence lifetimes of AlPcS₄ in aqueous solutions (10^-6 M - 10^-4 M) by means of stationary and laser picosecond fluorescence spectroscopies. With the use of numerical deconvolution of complex spectral curves (Alentsev - Fok algorithm) three individual components were found in AlPcS₄ pH-dependent fluorescence spectra. Based on the spectral data we suggest a ratioing technique for the local pH measurements with the use of AlPcS₄ as a fluorescence pH- sensitive probe. Microenvironmental pH of AlPcS₄ molecules in cultured cells were estimated by means of the suggested technique.

The hematoporphyrin derivative (HpD) is one of the most employed fluorescent markers for cancer diagnosis due to its ability to localize itself at a higher level in most tumor tissues and to emit a characteristic red fluorescence upon excitation with light at proper wavelength. This autofluorescence is responsible for a significant decrease of the fluorescence contrast between the tumor and the surrounding normal tissue. Consequently, there is a need to develop techniques to detect this fluorescence. This paper deals with the conception of a diagnosis system to detect early cancer at the surface of the hollow organs in the human body by endoscopy. The device and the principle of the image processing are detailed, and the limits of this system are discussed through the analysis of measurements carried out on phantoms and nude mice.

Preliminary biological screening has resulted in data about the considerable difference in photodynamic activity of sulfuric acids of aluminum phthalocyanine and their different salts have been obtained. The photosensitizers have been introduced intravenously in laboratory male mice 24 hours before direct laser irradiation of the liver area through the front abdominal side. Seven-day Erlich carcinomas with a diameter of 0.5 cm transplanted hypodermically to the mice have been used as an experimental tumor model. Obtained data of preliminary biological screening on photodynamic activity and tumor selectivity of different photosensitizers has been confirmed by direct experiments on the PDT experimental tumor in mice with the use of a laser system on the basis of yttrium aluminate with the wavelength 670 nm. Preparations have been introduced as a solution 2 mg/ml intravenous with a dose 10 mg/kg 48 hours before irradiation with the laser. Irradiation dose was 90 J with a spot diameter of 1 cm. Efficiency of anti-tumor PDT has been estimated by sight and with morphological study of the material.

Methods of stomach cancer photodynamic therapy with the use of Kr laser and photosensitizer-phthalocyanine (Al-Pc) are discussed. The level of preparation accumulation in the tumor and surrounding tissues has been investigated with the use of laser spectroanalyzer LESA-4 (`Biospec'). Dynamics of (Al-Pc) accumulation investigated depend on different types of tumors and different parameters of laser irradiation.

Photodynamic therapy (PDT) is a relatively new alternative modality for palliation of rectal cancer. We evaluate a new light source for PDT. In vitro PDT: CT26 murine colon carcinoma cells were incubated with aluminum phthalocyanine (AlPcS₄) for 48 hours, subjected to photoradiation using Versa-Light and viability was assessed. There was a significant decrease in viability of treated cells as compared with controls. In vivo PDT: Balb/C mice were injected either subcutaneously or intrarectally with CT26 cancer cells. IP AlPcS₄ 2.5 mg/kg was injected when tumors were visible. After 24 hours mice were subjected to photoradiation. Massive tumor necrosis in response to PDT was observed. PDT also prolonged survival of the treated mice. Patient treatment: A 70 year old woman with recurrent local rectal carcinoma received IV Photofrin II 2 mg/kg. After 48 and 96 hours she was subjected to direct photoradiation. After the first light session there was complete macroscopic disappearance of the tumor. Biopsies up to 10 weeks after the treatment showed no cancer cells in the treated area. We believe that Versa-Light is a good light source for PDT. It was effective in both in-vitro and animal studies. It can also be safely used for clinical PDT.

New chlorins and bacteriochlorins as perspective sensitizers for photodynamic therapy were synthesized. The influence on the position and the character of the substituents on the sensitizer optical properties was studied. The series of water soluble chlorin p₆ derivatives with graded amphiphility were synthesized for studies on tumor accumulation. The influence of pH on sensitizer amphiphility was discussed. The photostability of water soluble chlorins and bacteriochlorins has been examined.

Five-Aminolevulinic acid (ALA) is being used in clinical photodynamic therapy. We have investigated if ALA has any modifying effects on x rays and found that at concentrations more than 1 mM it exerts a slight radiosensitizing effect on WiDr cells in culture.

Synthesis of a series of model ether-bonded porphyrin-chlorin dimers with graded amphiphility as advantageous sensitizers for photodynamic therapy has been performed. Two macrocycles have been condensed via porphyrin-o1 trifluoroacetate. Hydroxyoctaethylporphyrin has been chosen as hydrophobic moiety and purpurin 18 derivatives have been chosen as hydrophilic components. New dimers have intense absorption maxima in the range of 665 - 700 nm, as well as weaker peaks at 622 - 624 nm that allows us to work with these sensitizers following new laser techniques which permit deeper light penetration into the tissues. The presence of hydrophobic octaethylporphyrin moiety and hydrophilic tri-carboxychlorin part in one molecule allows us to select structure with optimal amphiphility for sensitizer penetration and accumulation.

MSH receptors are one of the possible targets to specifically attack melanoma cells by photodynamically active agents bound to melanotropic hormones. To attack effectively metastatic melanoma the presence of the hormone receptor has to be proven in metastases. Radioactive labeling of melanotropic hormones allows us to check for the presence of intact receptors. We tried to develop a non-radioactive immunological method as a first step to work out therapeutic strategies to attach photodynamically active porphyrins to the surface of melanoma cells.

In vitro experiments were performed on human bladder carcinoma cells to evaluate the efficiency of photodynamic activity of protoporphyrindisodiumsalt encapsulated within liposomes. The bladder carcinoma cell line EJ 28, (Tumorba-nk Heidelberg) was grown on DMEM + 10% FCS + 2% Glutamine + 1% Penicillin/Streptomycin as a monolayer culture. In the log phase, cells were trypsinized, counted and inoculated into 35 mm-diameter multiwells at 10⁵ cells/well and allowed to grow for 24 h. The cells were incubated for 1 h with 5, 10, 20 (mu) g/ml protoporphyrindisodiumsalt in liposomes. (DPPC/Cholesterol 7:3). After incubation cells were refed with complete medium and irradiated with 3 and 6 J/cm². After irradiation, the cells were incubated for 2 days at 37 degree(s)C, then fixed, stained, counted and compared to the control group. Mean survival rates of 7.21%, 2.99%, 1.33% after irradiation with 3 J/cm² and 4.3%, 1.48% and 0.88% after irradiation with 6 J/cm² were found. By using a fluorescence microscope, we evaluated the intracellular uptake of protoporphyrindisodiumsalt in liposomes. The vesicular fluorescence pattern exhibited a concentration of photosensitizer in the nuclear membrane and adjacent parts of the cytoplasm as well as in the plasma membrane. By transmission electron microscopy marked changes were observed at the mitochondria with dissolution of the cristae and development of vacuols.

Some photophysical properties (steady-state absorption, fluorescence and phosphorescence, fluorescence decay times and singlet oxygen quantum yields) of silicon phthalocyanines with methoxypolyethylene glycol (MPEG)-substituents of various chain length at the silicon atom [SiPc(OCH₂CH₂)_n-OCH₃; n equals 1,2,3,12], silicon phthalocyanine covalently bound to the water soluble polymer methoxypolyethylene glycol [SiPc(-O-MPEG 5000)₂] and silicon naphthalocyanine [SiNc(CH₂CH₂OCH₃)₂] have been studied. The aim of these investigations was to get information about the influence of methoxypolyethylene-glycol-coupling on photophysical sensitizer parameters.

The photodynamic therapy (PDT) of cancer is based on the reaction of dyes, light and oxygen in tumorous tissue. Currently, mainly two different photosensitizers [Photofrin II and Hematoporphyrine derivatives (HPD)] are used in clinical investigations. They are 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 (maximum transmission for human tissue). These properties could potentially be improved by monofunctionalization of the photosensitizers which would allow them to be coupled with tumor specific antibodies. Also, variation of the peripheric substituents would lead to a shift in the absorption maxima to a point nearer to 760 nm. By modification of the methods found in the literature it has been possible to synthesize two new subphthalocyanines and one new subnaphthalocyanine. Starting by reacting 3,6-Dihydroxyphthalicaciddinitrile, 3,6-Dibutyloxyphthalodi-nitrile or 2,3- Dicyanonaphthalene with boron trichloride in a solvent with a high boiling point, it has been possible to form the subphthalocyanines (I) and subnaphthalocyanine (II).

The results of ORL organ tumor photodynamic therapy with the use of Phtalocyanine Al and multiple sequences of laser irradiation are presented. The possibility of decreasing the concentration of the preparation in accordance with laser irradiation tactics was investigated. The process of tissue necrotization for big and difficult tumors for laser irradiation access has been investigated. The estimation of photosensitizer distribution character for local injection has been carried out. The principal possibility of using local injection of photosensitizer for photodynamic therapy has been shown.

As it is known the tumor model Lewis lung carcinoma (LLC) is inclined to form metastases very early after its implantation. Although photodynamic therapy (PDT) of tumors is a local method, we studied its influence on the development of lung metastases in LLC bearing C57B1/6 mice after tumor implantation. The used photosensitizer was a newly synthesized Zn(II)-naphthalocyanine (tetrabenzylamidotetranaphtho (2, 3-b:2', 3'-g: 2', 3' - 1:2'', 3''-q) porphyrazinatozinc) incorporated into DPPC-liposomes and excited with monochromatic light at its absorption maximum (lambda) _exc 774 nm). The appearance of metastases in the animal lungs was evaluated according to the method of Pal, et al. Fourteen days after LLC implantation the tumor diameter for the treated mice was 6 mm (a week after PDT) towards 13.6 mm for the untreated. The percentage of surviving animals was 100 for the treated towards 0 for the untreated (a month after the implantation). Fourteen days after LLC implantation only 25% from the treated animals developed metastases to 75% from the untreated. The summary metastasis volume was 4.5 times smaller for the treated than the untreated mice. In conclusion we consider that the used Zn(II)-naphthalocyanine could be an effective photosensitizer for LLC treatment and although the PDT-method is a local one it could influence the process of metastasis development.

After experimental investigation clinical trials of photodynamic therapy (PDT) with the new photosensitizer Photosens -- (AL-sulphonated phthalocyanine) -- have been started. Four patients (with basal cell carcinoma, sarcoma Kaposi and early stage lung cancer) were treated by PDT. After an intravenous injection of drug in dose 2.0 mg/kg b.w. tumors were exposed to frequency-doubled radiation of Nd:yttrium aluminate laser (670 nm) with power densities from 80 to 200 mW/sq cm and total energies up to 2144 J. Immunological and clinical observations of patients are performed. First results indicate the high efficacy of the new drug as a tumor photosensitizer.

The first clinical trials of photodynamic therapy (PDT) in Russia were started in P. A. Hertzen Moscow Research Oncology Institute in October of 1992. Up to now, 61 patients with primary or recurrent malignant tumors of the larynx (3), trachea (1), bronchus (11), nose (1), mouth (3), esophagus (12), vagina and uterine cervix (3), bladder (2), skin (6), and cutaneous and subcutaneous metastases of breast cancer and melanomas (6) have been treated by PDT with the photosensitizer Photogem. At least partial tumor response was observed in all of the cases, but complete remission indicating no evident tumors has been reached in 51% of the cases. Among 29 patients with early and first stage cancer 14 patients had multifocal tumors. Complete remission of tumors in this group reached 86%.

The results are described of the first clinical fluorescence diagnostic observations of tumors, carried out in P. A. Hertzen Moscow Research Oncology Institute in the course of clinical trials of a new HpD-type photosensitizer Photogem, developed for cancer treatment by photodynamic therapy. The method of tumor diagnostics using fluorescence spectra of photogem has been developed and a designed spectrometric system has been tested.

We present experimental results obtained by combining photodynamic therapy (PDT) with tumor chemotherapy or radiotherapy. Dimethoxyhematoporphyrin (DMHp) and photosan (PS) were used as photosensitizers, pharanoxi and vincristine as antitumor drugs. The therapeutic effect of the combination of PDT and antitumor drugs (pharanoxi, vincristine) slightly increases as compared to the treatment of PDT or antitumor drug alone. The additive therapeutic effect is achieved under the combination of PDT and ⁶⁰Co irradiation. It seems that the sensitizers DMHp and PS regulate lipid peroxidation in blood serum of experimental animals, which becomes more active under the influence of alkylating antitumor drugs. Therefore, they could protect an organism from negative influence of tumor chemotherapy.

Cells of the line WiDr were incubated with media containing different concentrations of (delta) -aminolevulinic acid (ALA) and the amount of the produced protoporphyrin (Pp) IX was evaluated by means of steady-state fluorescence. The photomodification process of ALA- induced Pp IX was investigated by measuring spectroscopic properties of cells suspensions incubated with 0.2 mM and 1 mM of ALA. Light exposure of cells containing Pp IX resulted in a decrease of fluorescence intensities at 635 and 705 nm ((lambda) _exc equals 413 nm) due to photobleaching and in an increase of fluorescence intensity at around 670 nm due to the formation of a photoproduct. These spectral changes arise from photochemical reactions resulting in the formation of photoprotoporphyrin and destruction of porphyrin ring. The fluorescence kinetics indicate that for ALA-induced Pp IX in cells the ring opening reaction prevails the photoprotoporphyrin formation. Singlet oxygen may be involved in both processes since the photobleaching is slowed down on bubbling N₂ through the samples and photoprotoporphyrin formation is slightly faster in suspensions in phosphate buffered saline (PBS) made of D₂O compared with suspensions in PBS made of H₂O.

The investigation of spectroscopical properties of blood which contains photosensitizers irradiated by laser light is the actual problem of photodynamic therapy. The results of physiological blood characteristics and its oxygenation degree alterations for photosensitizers of tetrasulphanated aluminum phthalocyanine obtained from the experiments are presented in this paper. Spectral characteristics were measured with the use of a double ray spectrophotometer equipped by an integrating sphere. The concentration of drug and the dose of laser light for irradiation were chosen in accordance with the therapeutic ones realized during PDT.

Oxygen supply is the most important requirement of type II photodynamic reactions. Prerequisite in photodynamic tumor therapy is an intact tumor blood flow during irradiation. Most photosensitizers destroy tumor vessels due to accumulation in endothelial cells. As a prerequisite to develop novel photosensitizing drugs an in-vivo test system is required to quantitatively assess for inertness of those sensitizers to the blood supply. We adapted and further developed a system capable of measuring the relative oxygen supply to heterotransplanted tumors on the yolk sac membrane (YSM) of fertilized chicken eggs.

In the course of clinical trials of Photosens (Al-sulphonated phthalocyanine) as a new drug for PDT of cancer fluorescence observations of patients with injected photosensitizer are performed. The aim of current investigations is to develop the clinical method of cancer diagnostics with Photosens, to study the pharmaco-kinetics of the drug in the human body and its retention in tumors.

The results of photodynamic therapy (PDT) in 48 patients bearing basal cell carcinoma (BCC) and 7 patients with squamous cell carcinoma (SCC) of the skin are described. Five- aminolevulinic acid (5-ALA) was applied topically in two formulations. The first formulation contained 20% of 5-ALA in a base cream, and the second formulation (5-ALA composite cream), contained an additional 2% of dimethylsulfoxide (DMSO) and 2% of edetic acid disodium salt (EDTA). The creams were left on the skin for 2 - 5 hours. Production of protoporphyrin (PP) was measured in situ by a laser-induced fluorescence (LIF) method. The results of fluorescence measurement clearly indicate that PP accumulation in tumors induced by the 5-ALA composite cream was markedly higher than that induced by the 5-ALA cream. The tumors were light-irradiated (600 - 720 nm) after 4 - 5 hours of cream applications, using the light delivery system Versa-Light by a light dose of 100 J/cm². The clinically superficial BCC tumors were highly responsive to PDT; the overall result in BCC patients was an 85.4% complete response. Histological examination showed an initial edematous reaction, followed by necrosis and complete disappearance of the tumor. The superficial SCC tumors showed a 100% complete response after PDT. The ulcerated nodular SCC showed partial responses.

Nine selected patients with rectal tubulo-villous adenomas were treated with Photofrin- or aminolevulinic acid (ALA)-based photodynamic therapy (PDT) after the main bulk of the primary tumors had been endoscopically resected. The distribution patterns of Photofrin and ALA-induced porphyrins in the adenomas and surrounding normal tissues were studied by means of microscopic fluorescence photometry. Nine patients were treated in a total of 14 PDT sessions. Photofrin and ALA were used in 5 and 9 sessions, respectively. The tumors in all 5 Photofrin-based PDT sessions demonstrated complete regression. However, they all recurred 4 - 20 months after PDT. Four of 9 ALA-based PDT sessions achieved complete regression and so far no recurrence of these tumors has been found, although the follow-up is only 3 - 10 months. Two of the cases of partial response were given a second ALA-based PDT and both of them obtained complete response. The microscopic fluorescence photometry of the biopsies taken from the tumor and surrounding normal tissues after administration of either Photofrin or ALA showed that there was a strong fluorescence of Photofrin in the vascular stroma of the tumor and normal tissues, whereas ALA-induced porphyrins were mainly distributed in the glandular neoplastic cells. The correlation between the distribution of Photofrin and ALA-induced porphyrins in the adenomas and their photodynamic effects is discussed.