PHOTOSENSITIZATION WITH BACTERIOCHLORINS

Abstract— Biophysical and photobiological properties of a group of bacteriochlorins were compared with efficacy of these products for photodynamic therapy of murine tumors. Predictive factors for selective photosensitization in vivo include affinity binding to lipoproteins greater than albumin, extinction coefficient at the wavelength of irradiation and tumor/skin distribution. Efficacy was correlated with circulating plasma levels of the different sensitizers but not with the photodynamic therapy response in cell culture.     

In vitro PHOTOSENSITIZATION WITH A BENZOPORPHYRIN DERIVATIVE

Biophysical and photobiological properties of two benzoporphyrin derivatives were examined. These dyes exhibit substantial absorbance in the red, and are potent photosensitizers in vitro. After brief (0.5 h) incubations, phototoxicity was more closely correlated with membrane than with mitochondrial photodamage. Affinity of these dyes toward plasma lipoproteins are consistent with a mode of localization via the LDL-mediated mechanism utilized by the hematoporphyrin-derived product, HPD.     

PHOTOSENSITIZATION WITH DERIVATIVES OF CHLOROPHYLL

The properties of several chlorophyll derivatives were examined: the methyl esters of pheophorbide A, pheophorbide B and pheophytin. In spite of structural differences, all products were equally effective sensitizers in vitro and were localized equally well by murine tumors in vivo after 1 h. But only the pheophytins persisted at neoplastic loci for 24 h. There was no evidence of hydrolysis of the methyl esters, but the phytyl ester linkage was labile in vivo.     

PHOTOSENSITIZATION WITH ETIOBENZOCHLORINS AND OCTAETHYLBENZOCHLORINS

The photophysical and photobiological properties of a series of etiobenzochlorins were evaluated in cell culture using murine leukemia L1210 cells. In the series of agents tested, the chlorin-(mono)sulfonate was the most efficacious, the tin chlorin somewhat less so and the tin chlorin-sulfonate much less active. The parent chlorin was essentially inactive at the limit of solubility. Photodamage was assessed by measuring alterations in surface hydro-phobicity ( via a two-phase partitioning procedure), amino acid transport and membrane potential. Additional information was provided from fluorescence microscopy, which was used to identify sites of sensitizer binding and effects of photodamage on the binding patterns of fluorescent probes specific for mitochondria, lysosomes and plasma membranes. Effects of photodamage on fluorescence lifetime distribution of the membrane probe trimethylamino-diphenyl hexatriene were examined. The data obtained were consistent with localization of the parent etiobenzochlorin and tin derivative at lysosomal loci. the chlorin-sulfonate at plasma and mitochondrial membranes and tin-sulfonate at the cell surface.     

CATALASE INACTIVATION FOLLOWING PHOTOSENSITIZATION WITH TETRASULFONATEDMETALLOPHTHALOCYANINES

Abstract— Catalase (CAT) in solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during photosensitization with tetrasulfonated metallophthalocyantnes (MePcS₄). The effect of added scavengers and D₂0 showed that both singlet oxygen and free radical species are involved in this process. Evidence was found that direct interactions of ground or excited-stated photosensitizers with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized protein damage included oxidation of cysteine residues as well as other amino acids, as demonstrated by the formation of carbon-centered free radicals and the loss of absorbance at λ= 275 nm. In parallel, we detected degradation of the CAT heme groups, accompanied by release of Fe(II) ions in solution. These combined phenomena initiate cross-linkages between CAT subunits and subsequent degradation of the protein with formation of irreversible aggregates in solution. Phthalocyanine-mediated photoinactivation of cell-bound CAT results in loss of protection against accumulating H₂0₂, providing an additional pathway of phototoxicity.     

ACTION SPECTRA OF PHTHALOCYANINES WITH RESPECT TO PHOTOSENSITIZATION OF CELLS

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.     

PHOTOSENSITIZATION OF MELANINS: A COMPARATIVE STUDY

Abstract— Measurements of photosensitized free-radical production and oxygen consumption have been made in several melanin systems. The melanins studied were cysteinyldopa melanin (a model for pheomelanin), natural pheomelanins extracted from red hair and from red chicken feathers, and eumelanin extracted from bovine eyes. Rose Bengal was used as photosensitizer. A significant enhancement in the rates of free radical production and oxygen consumption was found in all systems when sensitizer was present. The largest effects were found with cysteinyldopa melanin and effects on pheomelanins were shown to be greater (by factors of4–6) than on eumelanins.     

NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN PRODUCED BY PHOTOSENSITIZATION WITH PROMAZINE and CHLORPROMAZINE

A sensitive near-infrared detection system has been used to study the steady-state emission of ¹O₂ at 1268 nra produced by promazine (PZ) and chlorpromazine (CPZ) during photo-illumination. Singlet molecular oxygen could be detected in a variety of ordinary and perdeuterated organic solvents, but was not detectable in water or deuterium oxide. The emission was enhanced in the perdeuterated organic solvents and could be eliminated by rigorous degassing or by addition of the singlet oxygen scavenger 2,3-dimethylfuran. Singlet oxygen could not be detected in any of the solvents during irradiation of the sulfoxides of PZ and CPZ. We conclude that in biological systems ¹O₂ production is not a major pathway to phototoxicity for the sulfoxides, while for the parent phenothiazines the formation of ¹O₂ is much more likely to be important in nonpolar environments such as cell membranes than in the aqueous parts of the cell.     

PHOTOSENSITIZATION TISSUE MODEL

Photosensitization in a light scattering matrix was investigated with a tissue model consisting of polystyrene microsphere scattering particles, in the presence of a Photofrin II as a photosensitizing agent, and subtilisin Carlsberg as an enzyme target. The photodynamic rate constant for irradiation at 435 nm, 545 nm, and red light was measured at different microsphere concentrations. The reaction rate was almost independent of the microspheres, ruling out a significant effect of light scattering on the integrated photosensitization efficiency. Modeling with the one-dimensional diffusion approximation showed that increasing scatterer concentrations led to lower transmission and higher diffuse reflection, such that the fractional absorption was almost constant. The quantum efficiency of enzyme inactivation in the light-scattering systems was 0.0014 +/- 0.0003. In terms of incident dose, red and green light had approximately the same effectiveness and blue light was two-fold more efficient.     

MOUSE SKIN PHOTOSENSITIZATION WITH BENZOPORPHYRIN DERIVATIVES AND PHOTOFRIN®: MACROSCOPIC AND MICROSCOPIC EVALUATION

A comparative study, at both the macroscopic and microscopic level, of skin photosensitivity caused by four isomeric forms of benzoporphyrin derivative (BPD) has been carried out, and compared to effects of Photofrin. Animals injected intravenously with BPD analogues and exposed to light 3 h later showed extensive photosensitivity. Animals receiving the monoacid derivatives of BPD (BPD-MA and BPD-MB) showed markedly more photosensitivity than those receiving the diacid derivatives (BPD-DA and BPD-DB). Animals receiving BPD analogues which were exposed to light 24 h or more later showed only minimal reactivity. Histological examination of biopsies taken after photosensitizer injection and light exposure showed extensive changes in epidermis and dermis, including epidermal erosion, degranulation of the stratum granulosum, spongiosis, depletion in cellularity and mast cell degranulation. These changes were noted to be similar to changes caused by Photofrin.     

PHOTOSENSITIZATION BY METHOTREXATE PHOTOPRODUCTS

Abstract— Photoproducts induced upon excitation of methotrexate by UV light have been separated by ion exchange chromatography. They include 2,4-diamino-6-pteridinecarboxylic acid, 2,4-diamino-6-pteridine-carboxaldehyde and other unidentified pteridine derivatives. The same photoproducts can be also formed upon photodynamic reaction using hematoporphyrin as photosensitizer. In oxygen saturated aqueous solutions (pH∼7), methotrexate photoproducts sensitize the oxidation of histidine and tryptophan by UV light by a process involving singlet oxygen. In aqueous solutions containing albumin or in human serum, the same photoproducts are formed from free methotrexate but not from albumin-bound methotrexate. In the latter case the results may suggest that methotrexate covalently binds to albumin upon excitation with UV light either in absence or in presence of oxygen. These results could explain the photosensitization accompanying cancer chemotherapy with high dose methotrexate and also the synergistic effects of PUVA + low dose methotrexate in psoriasis therapy.     

DETERMINANTS OF HEMATOPORPHYRIN-CATALYZED PHOTOSENSITIZATION

Abstract Using murine leukemia L1210 cells in culture, determinants of photosensitization by hemato-porphyrin were examined. Cells incubated for 10 min with a commercial preparation of hematopor-phyrin rapidly accumulated protoporphyrin and vinyl-hydroxyethyldeuteroporphyrin present as impurities. The resulting photosensitization appears to be mainly derived from the latter product.     

PHOTOSENSITIZATION BY ANTIMALARIAL DRUGS

Abstract The antimalarial drugs, chloroquine, hydroxychloroquine, quinine, quinacrine, amodiaquine and primaquine and the local anaesthetic, dibucaine, were tested for in vitro photosensitizing capability by irradiation with 365 nm UV light in aqueous solutions. The ability of these compounds to photosensitize the oxidation of 2,5-dimethylfuran, histidine, tryptophan or xanthine, and to initiate the free radical polymerization of acrylamide was examined in the pH range 2-12. Chloroquine and hydroxychloroquine show maximal photooxidative behaviour when in the monocation form at pH 9, in contrast to quinine which is extremely efficient as the dication below pH 4. This pattern appears to relate to the fluorescence yield as a function of pH. Chloroquine in the monocation or neutral form was found to undergo dechlorination upon irradiation, and this correlates directly with its ability to initiate photo-polymerization of acrylamide. Quinine also gives rise to small polymerization rates, attributed to photo-ionization in the quinoline ring, yielding a cation radical. Amodiaquine, primaquine and quinacrine do not have significant photochemical activity in aqueous solution. Dibucaine exhibits a strong photosensitizing capability at low pH, similar to quinine.     

STRUCTURE OF A THYMINE-THYMINE ADDUCT FORMED BY MENADIONE PHOTOSENSITIZATION

Abstract— The structure of one of the dithymine adducts formed by near-UV photosensitization of aqueous oxygenated thymine in the presence of 2-methyl- 1, 4-naphthoquinone is reported. The compound, 6'β-hydroxy-5'β-1 - [5-methylpyrimidinyl- 2,4-dione]-5'-6'-dihydrothymine has one thymine ring (ring I) linked through the 1-nitrogen atom to the C5' atom of a second thymine, which has been hydroxylated af C6' (ring II). Crystals are monoclinic, space group 12/c, and the structure was refined to R_obs= 0.036 for 2072 unique reflections with intensities I greater than 2.33(I). Ring I is planar, whereas ring II is not. Ring I and the OH group on ring II are cis to ring II. The planar ring I and the OH group are attached, respectively, in an equatorial and axial manner to ring II (which is in the sofa conformation). The planar rings I of close-lying pairs of molecules stack parallel to each other. The structure is held together by a hydrogen-bonding system consisting of the water molecules, the NH groups, two of the C=O groups and the OH group. The chemical formula and relative configurations at C5' and C6 are established by this analysis.     

PHOTOSENSITIZATION OF MCF-7 CELLS WITH DIAZIQUONE USING VISIBLE LIGHT: CORRELATION WITH DNA STRAND BREAKS AND FREE RADICAL PRODUCTION

The ability of visible light to enhance the activity of diaziquone (AZQ) was evaluated in MCF-7 human breast cancer cells. MCF-7 cells were sensitive to AZQ, while visible light illumination had no appreciable effect on cell survival. In the presence of AZQ, visible light potentiated AZQ's cytotoxicity. This potentiation of AZQ activity correlated with a 2-2.5-fold increase in the formation of free radicals (hydroxyl radicals and AZQ semiquinone) and with the production of DNA strand breaks as measured by electron paramagnetic resonance and gel electrophoresis respectively. These results support the hypothesis that free radical formation is part of the mechanism of action of AZQ.     

CHARACTERISTICS OF THE PRIMARY REACTION OF HYPOCRELLIN A PHOTOSENSITIZATION

The primary reaction of photosensitization of Hypocrellin A (HA) has been studied byusing techniques of ESR, spin-trapping and spin-counteraction. The experiments show thatHA is able to generate not only ~1O_2,but also O_2~-·OH and HA~- which are observed for thefirst time. The conversion of generating active oxygen into generating nonoxygen free rad-ical is confirmed as well. Based on the characteristics of the primary reaction which gene-rates these active substances (transient products), it is proposed that the photosensitized dam-age for the biological system by HA is probably related to not only ~1O_2 itself, but also amultiple effect from ~1O_2 as well as O_2~-,·OH and HA~+ free radical.     

A FLASH-PHOTOLYSIS INVESTIGATION OF FLAVIN PHOTOSENSITIZATION OF PURINE NUCLEOTIDES

Evidence for the existence of a reactive triplet excited state of lumiflavin has been obtained by the flash-photolysis technique. The triplet state is formed in high yield on the irradiation of flavin solutions in water or chloroform by visible light, and it has been demonstrated that it can transfer its energy to a second molecular species. The flavin-sensitised oxidation of two purine nucleotides, adenylic and guanylic acids, has been studied by flash-photolysis and by long-term irradiation, and the results suggest a triplet-triplet mechanism for the transfer of energy from the excited flavin to the nucleotide. Approximate absorption spectra of the triplet state and of a semiquinone of the flavin have been calculated from the complex transient absorption curves observed on flashing the flavin solution. The triplet decays by a first-order process where k₁= 1·1 × 10^-3. The chemiluminescence spectrum of skatole is identical with the fluorescence spectrum of o-formamidoacetophenone in the same environment Similar results for 2,3-dimethylindole lead to the identification of the acylamide anion as the emitter in indole chemiluminescence. A peroxide ring cleavage excitation mechanism is proposed. 10⁴ sec^-1 and the semiquinone by a second-order process where k₂= 0·75 × 10⁹ 1.m^-1 sec^-1. The rate constants and extinction coefficients obtained enable decay curves to be calculated which fit satisfactorily those measured with the kinetic-flash apparatus.