Encouraging results are being reported in treatment of cancer by photodynamic therapy, consisting of the administration of hematoporphyrin photosensitizers and subsequent illumination of the neoplastic lesion with visible light. Studies in our laboratory have peen directed towards defining cellular sites of action of these photosensitizers. Results obtained using a rodent mammary carcinoma in vitro and in vivo indicate that an important target of tumor photosensitization is the mitochondrion, effects that are manifested by inhibition of enzymes involved in electron transport and oxidative phosphorylation. These events in turn lead to a significant reduction in cellular ATP, which we suggest represents an early and important mechanism that contributes to the ensuing tumor cytotoxicity. Optimization of this mechanism could provide improved efficacy of treatment.

Cationic dyes such as EDKC, which preferentially accumulate in malignant cell mitochondria, permit selective photodamage and dark toxicity to these organelles. The resultant inhibition of oxidative phosphorylation and decrease in intracellular ATP should strongly potentiated thermal injury, even at temperatures which are relatively innocuous by themselves. In human squamous carcinoma (FaDu), murine melanoma (B-16) or non-malignant monkey kidney (CV-1) cells, we examined the effect of combining treatment with EDKC ± light together with mild, 42°C hyperthermia. Heat alone for up to 60 min had no effect on any of the cell lines, and EDKC + light + heat was not toxic to the non-malignant CV-1 cells. Hyperthermia synergistically enhanced photodamage in malignant cells, with survival decreasing more than 200-fold after 20 min of heat to B-16 cells or 60 min to FaDu cells. The heating also increased dark toxicity of the dye, decreasing survival of FaDu cells by more than 10-fold after treatment with 10-7 M EDKC and 60 minutes at 42°C. The sequence of application of light and heat was important, with much greater synergy for heat after irradiation.

Photodynamic therapy (PDT) of human tumors with hematoporphyrin derivative (HpD) has achieved encouraging results. However, HpD is a complex mixture whose composition varies in different preparations and with time of storage. The future promise of PDT for cancer treatment depends on the development of new chemically defined sensitizers which absorb more strongly than HpD in the 600-800 nm region. A shift to higher wavelengths is desirable since it allows increased light penetration in human tissues. In vivo, these sensitizers should be non-toxic, localize selectively in tumors and generate cytotoxic species upon illumination with a high quantum yield. These damaging species may be singlet oxygen (1O2) produced by the transfer of energy from the triplet state of the sensitizer to oxygen (Type II) or superoxide anion radicals formed by electron transfer to oxygen or substrate radicals generated by electron or hydrogen transfer directly from the sensitizer (Type I). The recent work of several groups indicating that phthalocyanines and their water soluble derivatives are promising candidates for PDT is reviewed. The photophysics, photochemistry, photosensitized killing of cultured mammalian cells and the use for in vivo photodynamic therapy of phthalocyanines is outlined. Our studies of the post-illumination photohemolysis of human red blood cells as a model system for membrane photomodification sensitized by phthalocyanine sulfonates are consistent with the predominant role of 1O2 as the damaging species.

Two new groups of photosensitizers, purpurins and metallopurpurins were tested for photodynamic activity when combined with visible light (>590nm), in the treatment of transplantable urothelial tumors (FANFT induced) in Fischer 344 rats. In preliminary studies, animals were injected with 10 mg/kg body weight (b.w.) of the free base purpurins (NT1, NT2, NT2H2, GG1, ET2, ET2H2 and JP1) and treated with 360J/cm2 of red light. Tumors were excised, fixed in formalin and stained for light microscopic examination. Purpurin based photodynamic therapy (PDT) was found to cause extensive hemorrhagic tumor necrosis. Control tumors shielded from the light showed no histological changes. These experiments were repeated using the same experimental design for the metallopurpurins (ZnET2, SnET2, ZnNT2H2, SnNT2H2, AgNT2H2 and ZnNT1). These compounds were also found to cause extensive hemorrhagic necrosis. These studies were followed by a dose response analysis in which groups of animals were treated with decreasing doses of drug while the light dose was kept constant. Response was determined by tumor dry weight, 12 days after completion of PDT. For the free base purpurins, drug doses as low as 1.0 mg/kg b.w. caused significant tumor regression while seleted metallopurpurins caused tumor regression at doses as low as 0.5 mg/kg b.w. In a series of related experiments the effect of purpurins on tumor blood flow was investigated since it has previously been shown that tumor blood flow is disrupted during porphyrin and phthalocyanine phototherapy. By using the radioactive microsphere technique, it was shown that both the free base purpurins and metallopurpurins caused a rapid decrease in tumor blood flow.

Results are presented showing how photoaffinity labeling can be used to identify ribosomal components at functional sites within the Escherichia coli ribosome. Following brief overviews of ribosomal structure and function and of the use of photoaffinity labeling to study complex biological macrostructures, results obtained in the authors' laboratory, principally with the ribosomal antibiotic puromycin and with an aryl azide derivative of puromycin, are presented to illustrate procedures used to identify photoaffinity-labeled ribosomal components, to determine whether such labeling occurs at a functionally important site, and to localize sites of labeling in a three-dimensional sense.

Fluorescent lipophilic cations such as rhodamine 123 have been used to localize mitochondria in living cells. The specific uptake is resulted from the uniquely high membrane potential across mitochondria. Although mitochondria in all cells take up rhodamine 123, the degrees of uptake and retention vary from one cell type to the other. Many carcinoma cell types/lines have a higher uptake and longer retention of rhodamine 123 than that of normal epithelial cells, probably resulted from higher mitochondrial and plasma membrane potentials. This phenotype has been exploited for selective killing of carcinoma cells in vitro as well as in vivo. Mitochondria may be used as a reservoir and a slow-releasing device for photosensitive, lipophilic, cationic drugs.

Bladder carcinoma is considered one of the most favorable targets for the application of photodynamic therapy (PDT) due to the accessibility of the bladder for light delivery. Examination of the bladder and surgical procedures are routinely performed by the insertion of an optical instrument called cystoscope through the urethra. Thus, the treatment of bladder cancer by PDT can be conducted through the cystoscope with minimal invasion. However, to achieve optimal results from this treatment, one must consider both the structure of the bladder and the nature of the carcinoma.

Reaction of ∝-chymotrypsin with various aryl azido acylimidazoles leads to covalent attachment of the aryl azide photoaffinity label. Photolysis of the polycrystalline enzyme or a frozen solution of the enzyme at 77K leads to intense EPR signals of the triplet nitrenes. The position of the EPR resonance fields and the decay kinetics can be interpreted in terms of binding of the label within or outside the binding pocket of the enzyme.

A photochemotherapy based on the synergistic action of 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet light (UVA, 320-400 nm) has been used extensively since 1974 for the treatment of psoriasis, a hyperprolifierative disease of the skin. More recently, this photochemotherapy has been used for the extracorporeal irradiation ot the blood of cutaneous T cell lymphoma (CTCL) patients in the leukemic stage of the disease. 8-MOP is ingested; two hours later when peak blood levels have been achieved the affected tissue is irradiated (skin in psoriasis and blood in CTCL). Using 8-MCP-DNA photoadduct formation as a cheillical actinometer, the effective dose of UVA radiation delivered to lymphocytes during photochemotherapy in the presence of erythrocytes was found to be ~1 J/cm2. In addition, a monoclonal antibody prepared in our laboratory was used in competitive ELISA assays to quantify the formation of 8-MOP photoadducts during the photochemotherapy.

The chalcogenapyrylium dyes can be treated as model systems whose properties can be designed by proper choice of substituents. The use of different chalcogen atoms and substituents in the dye chromophore allows control of the wavelength of absorption maxima, redox properties, fluorescence yields, and hydrolytic stability.

Photochemotherapy (PCT) is a form of cancer therapy that employs a drug called a photosensitizer, which binds to tumor and becomes cytotoxic by the absorption of radiant energy. In order for a photosensitizer to be clinically useful, it must be non-toxic to normal tissues, be selectively taken up and/or retained by malignant tissue, be activated by light that can penetrate deeply into both tumor and tumor involved normal tissues, and be photochemically efficient.

The approach used for the development of some novel benzophenoxazinium photosensitizers is presented. Considerations that have influenced the design of these dyes include structure/function relationships pertaining to such properties as selective tumor staining, fluorescent quantum yields and photosensitized singlet oxygen formation (1O2). General trends in the relative photodynamic behavior exhibited by these new dyes in cell culture are discussed. The results indicate that several of the dyes have significant photosensitizing efficacy.

The primary structure of the tumor-localizing component of Hematoporphyrin Derivative (HPD) has been shown by fast atom bombardment (FAB) mass spectrometry to consist of hematoporphyrin (HP)-based oligomers of up to five subunits with a general formula of nHP-(n-1)H2O. The nature of the covalent bond linking the porphyrin units has been probed by LiA1H4-reduction and by diazomethane-permethylation of the oligomer-containing fraction of the drug. Results suggest that both ether and ester groups are present and that the composition may be dependent on the shelf-life and the preparative method employed. Cell assay with HPD preparations enriched in ether or ester content indicates that variations of the linkage between porphyrin units have essentially no effect on the photodynamic activity.

Unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC) incorporate a variety of hydrophobic photosensitizers (e.g. hematoporphyrin dimethylester, unsubstituted phthalo-cyanines, porphycene) into the phospholipid bilayer. The physico-chemical properties of the liposome-bound photosensitizers in the ground and electronically excited states can be characterized by steady-state and time-resolved fluorescence spectroscopy. The liposome-drug system is stable under physiological conditions and, once injected into tumor-bearing animals, selectively delivers the photosensitizer to serum lipoproteins. As a consequence, the tumor uptake of the drug via receptor-mediated endocytosis of low-density lipoproteins (LDL) is favoured. This leads to a larger ratio between the photosensitizer concentration in the tumor and adjacent normal tissues, hence to an increased efficacy of the photodynamic therapy (PDT).

Gilvocarcin V (GV) is a planar, aromatic DNA-intercalating C-glycoside isolated as a natural product antibiotic. In the presence of UVA or visible radiation, it becomes a DNA damaging agent at low doses in both bacterial and mammalian cells. In mice treated without regard to light exposure, GV exhibited antitumor activity at high doses, with little accompanying toxicity. Wavelength-dependence studies showed that lambda prophage induction profiles were similar to (part of) the absorption spectrum of GV, with a peak near 400 nm. However, significant induction at a higher wavelength (546 nm), was observed at relatively high (e.g. 1 μg/m1) concentrations of GV. The DNA damaging activity of GV was dependent on both the concentration of antibiotic and the fluence of radiation in a reciprocal manner. Mutagenesis and DNA binding experiments suggest a preference for interaction with AT-rich regions of DNA, but multiple modes of interaction seem likely. The presence of different C-glycosides on the gilvocarcin V chromophore may alter the pharmacological properties of the molecule, but photoactivation appears to be independent of these groups. The therapeutic possibilities of gilvocarcins remain largely unexplored; the demonstrated potency of these compounds when activated, the reciprocity effect, possibility of structural variation, and apparent lack of toxicity in mammalian systems are properties which could be exploited in therapeutic development.

Results of dihematoporphyrin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40-60 μg DHE/g of tissue at an injected dose of 40 mg/kg. Previously concentrations on this order have only been found in organs of the reticuloendothelial system. Two intrapancreatic carcinoma models, one of acinar origin (rat) and one of ductal orgin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared to normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630 nm light from a dye laser (75 mW/cm2, 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet, the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration. These results represent the emergence of an entirely new modality, with substantial potential for the treatment of cancer of the pancreas.

Singlet oxygen is believed to be an important intermediate responsible for the cytotoxicity of HpD phototherapy. It has been recognized as a possible intermediate in photosensitization for more than 20 years. However, it has been difficult to obtain conclusive evidence of its biological characteristics in the past because most of the methods available for its generation that are compatible with biological systems also generate other reactive intermediates whose effects are difficult to distinguish from singlet oxygen. We have used a recently devised separated-surface-sensi-tizer (S-S-S) system for singlet oxygen generation' to measure the cytotoxicity and mutagenicity of singlet oxygen in bacteria. The S-S-S system employs rose bengal as a sensitizer immobilized on one surface of a glass plate. The glass plate is placed sensitizer-side down a small distance (< 1.5 mm) above a microscopically flat membrane (MilliporeTM or NucleoporeTM) that carries a monocellular layer of bacteria. The sensi-tizer-coated plate is illuminated from above to generate singlet oxygen at the surface of the sensitizer. The singlet oxygen thus generated can diffuse the short dis-tance to the surface of the membrane to react with the bacteria. Because of the short lifetime of singlet oxygen in air, increasing the distance between the sensitizer and the membrane causes a decline in the amount of singlet oxygen reaching the membrane according to a function derived from the Einstein-Smoluchowski equation for net displacement by diffusion. Plotting the log of the effect measured (e.g., cytotoxicity) vs. the square of the distance gives a straight line. The slope of this line can be used to calculate the gas phase half life of the intermediate responsible for the observed effects. We have found that bacteria are rapidly killed in the illuminated S-S-S system and that the gas phase half life of the agent responsible for cell killing is the same as that of singlet oxygen. This observation and other simple chemical tests have conclusively estab-lished that singlet oxygen is responsible for the cytotoxicity observed with bacteria. Dosimetry measurements allow us to estimate that singlet oxygen is at least 104 times more potent as a cytotoxin for Salmonella bacteria than hydrogen peroxide, on a molar basis. We have not observed mutagenicity in these bacteria exposed to sufficient singlet oxygen to kill 60-90% using a variety of bacterial strains and assays.

Several xanthene sensitizers were compared as sensitizers of membrane function in erythrocytes and some of their physico-chemical properties were examined. Eosin derivatives that localize at different membrane sites were equally effective at sensitizing both ion leaks and inactivation of membrane cholinesterase, implying that a diffusible intermediate reacts with membrane targets. Assessments of membrane loading and calculations of diffusion distances for singlet oxygen indicate that amounts of membrane-located sensitizer are quantitatively much greater than amounts in the nearby reaction medium. Potency measurements and assessment of absorption spectra and singlet oxygen production in water-dioxane mixtures lead to the conclusion that differential sorption to membranes, photon capture in low polarity environments and conversion of excited states to singlet oxygen are they key determinants of sensitizing potency.

Photodynamic Therapy is an effective treatment of malignant tumors. However the Rophyrin dyes Erggently used are associated with phototoxic effects in the skin and the eye Numerous new dyes are being developed which will hopefully combine effectiveness in treating tumors with fewer phototoxic side effects. We report here a method to assess the relative phototxicity of porphyrins in lenticular tissues that involves both in vitro, photophysical and in vivo studies. This methodology can be used to screen for potential phototoxic side effects of new photosensitizing drugs as they are being developed and to develop means by which that damage may be prevented.

The tumor-localizing product HPD (hematoporphyrin derivative) contains both monomer and dimer/oligomer components. The former are potent photosensitizers in cell-free systems and, in some cases, in cell culture. But these porphyrins are poorly retained by neoplastic cells in vivo. Tumor localization derives from the presence, in HPD, of a series of covalently-bound hematoporphyrin dimers and oligomers which are joined by ester and ether linkages. Like many non-localizing porphyrins, these products can bind to circulating low-density lipoprotein and enter cells via LDL receptors. The initial step in tumor localization results from the presence of elevated levels of LDL receptors on the surface of neoplastic cells. But retention of the HP dimer/oligomer fraction results in long-term photosensitization. The preferential dye accumulation in mitochondria cannot be attributed to the presence of specific porphyrin receptors, but may instead result from the ability of the porphyrin dimer/oligomer fraction to undergo a conformational change as a function of its environment. These changes were probed in micellar and pre-micellar systems and the results show the HP dimer/oligomers to have properties not shared by porphyrin monomers.

Simultaneous exposure to the amphipathic fluorescent dye merocyanine 540 (MC 540) and light of a suitable wavelength rapidly kills leukemia, lymphoma, and neuroblastoma cells but spares normal pluripotent hematopoietic stem cells. Tests in several preclinical models and early results of a phase I clinical trial suggest that MC 540-mediated photosensitization may be useful for the extracorporeal purging of autologous remission bone marrow grafts.

The efficiency of pulsed (4.5 kHz) and continuous-wave (CW) laser light (680 nm) in causing phototoxicity with chloroaluminum phthalocyanine tetrasulfonate (AIPCS) was studied using colony-forming ability of Chinese hamster fibroblast cells as an endpoint. CW light was nearly 5-fold more effective than pulsed light. The difference is ascribed to the 104 higher peak power density during pulsed illumination. Two explanations are advanced to explain this result: (a) the oxygen content becomes the limiting factor for the photodynamic reaction; (b) the complete conversion of AIPCS molecules to excited states during the pulse results in underutilization of the available photons. Experimental approaches to test these hypotheses are suggested.

Photodynamic therapy (PDT) and hyperthermia have been investigated as treatments for several types of tumors. Studies have been done to determine the efficacy of each modality individually and recently in combination with each other. In this study 630 nm light was delivered by an argon-dye laser and hyperthermia was induced using a Nd:YAG laser. Both lasers offer the ability of delivering the beams through a quartz fiberoptic alone or simultaneously. This present study examines (a) the efficacy of the simultaneous administration of PDT and selective hyperthermia at 44.5°C in tumor control; (b) the effect of hyperthermia and PDT + hyperthermia on tumor and normal tissue microcirculation; and (c) the toxicity in normal tissue of PDT, hyperthermia and the simultaneous administration of the two modalities. Hyperthermia alone (44.5°C, 30 min) resulted in complete destruction of tumors with no subsequent regrowth in 12% of the mice treated. PDT alone (5 mg/kg DHE; 135 J/cm2) resulted in a cure rate of approximately 30%, and the simultaneous treatment of the modalities resulted in a 65% cure rate after 6 weeks. These values are indicative of a synergistic interaction. This study also examined the toxic effects of hyperthermia and the combination therapy to normal tissues in mice. Direct organ exposures produced much greater tissue damage than whole abdomen exposures, as expected, although there was no resulting lethality. Necrosis to a small degree occurred in the spleen and pancreas with hyperthermia alone, while extensive necrosis occurred in all of the organs with the combination. The extent of damage caused, however, was no greater than that caused by PDT alone in most tissues examined. Fluorescein angiography shows a lack of response in the surrounding normal tissue microcirculation for hyperthermia only. The combination treatments, however, shut down the microcirculation within the treatment field.

The effect of free radical quenchers (ascorbate, catalase, and mannitol) on merocyanine 540 (MC540) mediated, laser light induced photolysis of human acute promyelocytic leukemia cell line (HL-60) was investigated. Results show that in the presence of human albumin (0.25%), dye-mediated (2014/m1), laser light induced photolysis of leukemic cells resulted in a 99.9999% cell kill. Seventy percent of the normal bone marrow cells survived the treatment. The addition of free radical quenchers prior to laser irradiation procedure increases the HL-60 cell survival. Increases of 5.5% and 4.4%, respectively, were observed in the presence of catalase and ascorbate or mannitol. In the presence of a mixture of catalase and mannitol or catalase and ascorbate, this increase in viability was not observed. However, the viability of normal bone marrow cells under these conditions also decreased from 70% to 63%. These findings may be useful in ex-vivo bone marrow purging.

A series of structurally related modified porphyrins have been synthesized. Each compound was tested for in vivo cytotoxicity against the FANFT-induced rat bladder tumor (designated AY-27). An attempt was then made to correlate the amount of tumor necrosis shown on examination of histologic sections of treated tumors, both four and twenty four hours after phototherapy, with the structure of each photosensitizer.

Etiopurpurin is a photosensitizer developed from etioporphyrin I, which we have shown to be useful in PDT. Incorporation of tin into the macrocycle gives a derivative which appears to be more effective both in vitro and in vivo studies on tumor cytotoxicity, than the parent purpurin. In vitro studies have shown not only that tin etiopurpurin dichloride (SnET2C12) is an efficient photosensitizer, but also that its action spectrum appears to be consistent with both the absorption and emission spectra. In vivo studies include a histological examination of tumors treated with SnET2C12 and PDT which suggests that disruption of the tumor vasculature plays an active role in tumor necrosis; a twelve day dose response study to determine effective dose; a thirty day study to determine tumor regrowth; a study of normal tissue response to PDT and a study to determine the optimum time for phototherapy after injection of the drug. For these studies, the FANFT induced urothelial tumor line was used, although we have recently initiated studies on the R3327 AT rat anaplastic adenocarcinoma, a tumor which appears to be more resistant to PDT.

In recent years, a number of new photosensitizers have been developed for the photo-dynamic treatment of cancer. These new drugs include tetrasulfonated phthalocyanines, tetra(hydroxypheny1) porphyrins, chlorins and purpurins which are water soluble, and phthalocyanines, chlorins, purpurins and verdins which are water insoluble. In vivo or in vitro data are available to assess the relative photodynamic efficiency of either hydrophobic or hydrophilic sensitizers. Hydrophobic drugs require a more complex delivery system in order to overcome their water insolubility. In this paper, we report the suitability of both oil-based emulsions and unilamellar liposomes for in vivo delivery of three different photosensitizers (a purpurin, a verdin and a chlorin).

Photooxidation of lens crystallins has been found to induce significant changes in their tertiary structures, and is likely to play an important role in the crosslinking and aggregation of these proteins observed in aged and cataractous human lenses. Irradiation of calf lens α-, β- and γ- crystallins in the presence of visible light and the photosensitizers methylene blue or riboflavin and by irradiation with 300 nm radiation has been investigated. Of the three classes of crystallins, only the monomeric γ-crystallins have been found to undergo partial insolubilization in photosensitized reactions. The insoluble material is highly crosslinked by nondisulfide linkages. Moreover, change in the molecular charge of the protein is observed. Analysis of the soluble fraction of the irradiated γ-crystallin, by circular dichroism and fluorescence spectroscopy, reveals changes in the tertiary structure of the protein, probably involving a partial unfolding of the molecule. Active species of molecular oxygen play a role in these structural modifications. The change in conformation is different for each photosensitizer used, suggesting that the sensitizer-protein complexation may be important.

Hematoporphyrin derivative also known as Photofrin-I (Pf-I) and its more active dimeric tissue localizing component dihematoporphyrin ether, also known as Photofrin-II (Pf-II), are currently used in the diagnosis and management of a variety of epithelial neoplasms, in a modality known as photodynamic therapy (PDT). One of the drawbacks of these porphyrins for PDT is their ability to evoke prolonged cutaneous photosensitization. The mechanism of tumor ablation and cutaneous photosensitization by these photosensitizers is thought to relate to the generation of one or more reactive oxygen species such as superoxide anion, singlet oxygen and hydroxyl radical. However, the role of none of these oxygen species has been established unequivocally. In this study, groups of C3H mice were injected intraperitoneally with Pf-I or with Pf-II (5 mg/kg). Four hours after treatment with the photosensitizer, animals were divided into four groups and treated with a superoxide dismutase (SOD) mimic, bis [ (3,5 diisopropyl salicylato)(0,0))copper (II) (80 mg/kg body wt. in olive oil), Ill-carotene (133 mg/kg in olive oil) and dimethyl sulfoxide (0.3 ml/mouse in olive oil) or olive oil alone. Six hours after injection of the photosensitizer all of the mice were irradiated with visible radiation from a 4 KW metal halide light source for 2.5 hrs. Immediately after the irradiation ear swelling was measured as a marker of cutaneous photosensitization. The mice treated with Pf-I or Pf-II and light demonstrated two-fold enhancement of ear swelling whereas animals treated with the SOD mimic, b-carotene and DMSO had considerably less ear swelling (p ( 0.01). The order of protective effects was SOD mimic > dimethyl sulfoxide > b-carotene. The observed protective effect was dependent on the dose of each quencher. The histopathologic evaluation of sections of ears of Pf-I and Pf-II treated photosensitized mice revealed vascular ectasia, dermal edema, neutrophilic infiltrate, increased mast cells, and mast cell degranulation. These effects were significantly alleviated by SOD mimic in both Pf-I and Pf-II treated mice. The protection was more pronounced with Pf-II as compared to Pf-I. )B-carotene and DMSO afforded lower protection than that which occurred with SOD mimic. These data provide the first in vivo evidence for the involvement of superoxide anion in cutaneous porphyrin photosensitization. Furthermore, the greater protection against ear swelling by SOD mimic in the Pf-II treated animals suggests a more important role for superoxide anion in Pf-II mediated photosensitization.

The effects of depleting glytolytic ATP on EDKC mediated cellular phototoxicity has been investigated using squamous carcinoma cells (FaDu) in vitro. Cells were pretreated with a non-metabolizable glucose analog 2-deoxy-D-glucose (5 mM) for 13-15 hr that resulted in 40-45 % decrease of cellular ATP. Subsequent exposure of the cells to EDKC resulted in twice as much dye uptake compared to control cells and upon radiation induced 4-5 fold more killing. Pretreatment with 2-deoxy-D-glucose (2-DG) alone did not have any effect on cell survival. We conclude that if cellular glycolytic ATP is lowered prior to their exposure to EDKC and radiation, cells become more vulnerable to the dye mediated photosensitization.

A method was developed for preparing highly purified pheophorbide a using the blue-green alga Anacystis nidulans as a source of chlorophyll a which was subsequently converted to the desired compound using a cation-exchange resin (Dowex 50Wx4, H+ -form) as a proton source. Pheophorbide a was found to be 20 times more photocytotoxic than was hematoporphyrin (under red light >560 nm) against EJ cells (a cell line established from a human transitional cell carcinoma of the bladder) in vitro. Pheophorbide a accumulated rapidly in EJ and IMR-90 (a non-neoplastic cell line established from human fibroblasts) cells, reaching the maximum by 30 minutes. However, the number of EJ cells fluorescing at 5, 15 and 30 minutes was 1.7, 1.5 and 1.2 times greater than IMR-90 cells respectively. Moreover, only 17 percent of IMR-90 versus 75 percent of EJ cells fluoresced at 48 hours following 4-hour pheophorbide a exposure. These results indicate that pheophorbide a is highly photocytotoxic for EJ cells in vitro and undergoes differential uptake and retention in neoplastic as compared with non-neoplastic cells.