SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—IV. THE PHOTOLYSIS OF BENOXAPROFEN, AN ANTI-INFLAMMATORY DRUG WITH PHOTOTOXIC PROPERTIES

Abstract— Benoxaprofen [2-(4-chlorophenyl)-α-methyl-5-benzoxazole acetic acid] is an anti-inflammatory drug that causes acute phototoxicity in many patients. Photolysis studies in organic solvents (ethanol, benzene, dimethylsulfoxide) showed that benoxaprofen underwent both Type I and Type II reactions. Irradiation of an anerobic solution of benoxaprofen in ethanol resulted in hydrogen abstraction from the solvent to yield hydroxyethyl and ethoxyl radicals. In the presence of oxygen, superoxide, singlet oxygen and hydroxyethyl radicals were detected. Photolysis of benoxaprofen in air-saturated benzene or dimethylsulfoxide gave superoxide. However, under anerobic conditions the drug yielded a carbon-centered radical in benzene that could not be identified. These findings suggest that both oxygen-dependent and oxygen-independent processes may be important in the phototoxic reactions of benoxaprofen.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–XVI. ISPERSE BLUE 35

The photochemistry (Type I and II) of the phototoxic textile dye Disperse Blue (DB-35) and its purified components has been studied using electron spin resonance in conjunction with spin trapping technique and the direct detection of singlet oxygen (1O2) luminescence. The main components of DB-35 (which is synthesized by the successive nitration, reduction and methylation of 1,8-dihydroxy-anthraquinone) were separated by HPLC and identified by mass spectrometry and 2-D NMR as 4,5-diamino-1,8-dihydroxyanthraquinone (4,5-DDHAQ; 62% of total dye) and 2,7-diamino-1,8-dihydroxyanthraquinone (2,7-DDHAQ; 31% of total dye). Minor components included 2,5-diamino-1,8-dihydroxyanthraquinone (2,5-DDHAQ) and a monomethylated derivative of either 4,5-DDHAQ or 2,7-DDHAQ. Irradiation (624 nm) of 4,5-DDHAQ and 2,7-DDHAQ in dimethylsulfoxide resulted in the generation of superoxide which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Visible light irradiation of the components in ethanol generated 1O2 with the yields decreasing in the following order: 4,5-DDHAQ greater than 2,5-DDHAQ greater than 2,7-DDHAQ. These findings indicate that upon irradiation by visible light DB-35 can generate active oxygen species which may be responsible for the photocontact dermatitis caused by this dye.     

AN ESR STUDY OF THE VISIBLE LIGHT PHOTOCHEMISTRY OF GILVOCARCIN V

Photolysis of gilvocarcin (GV) at 405 nm in argon saturated dimethylsulfoxide (DMSO) or 50% DMSO-water solutions in the presence of the sodium salt of 3,5-dibromo-2,6-dideutero-4-nitrosobenzene sulfonic acid (DBNBS-d2) generates the CH3-DBNBS-d2.spin adduct. It is postulated that this spin adduct is produced by photoreduction of DMSO by GV and the consequent formation and trapping of the generated methyl radicals. Gilvocarcin V also photoreduces oxygen and methyl viologen with quantum yields of 0.019 and 0.0012 respectively. The quantum yield for singlet oxygen formation by GV in DMSO, determined by measuring the rate of production of the nitroxyl radical produced by the reaction of 2,2,6,6-tetramethylpiperidinol with singlet oxygen, was found to be 0.15. Thus, GV photochemistry proceeds by both Type I and Type II pathways which could contribute to the reported GV phototoxicity in biological systems.     

ANTHRALIN-DERIVED TRANSIENTS—II. FORMATION OF THE RADICAL BY SPONTANEOUS FRAGMENTATION OF BOTH SINGLET AND TRIPLET STATES OF THE 10,10''-DEHYDRODIMER: RADICAL PAIR MULTIPLICITY EFFECTS

The singlet and triplet states of the anthralin (1,8-dihydroxy-9-anthrone) dehydrodimer have been produced selectively in benzene via pulsed laser excitation and pulse radiolysis respectively. The lifetime of S1 is less than or equal to 30 ps, that of T1 short but unspecified. Both states fragment spontaneously to yield a pair of anthralin radicals. The singlet radical pair predominantly undergoes geminate recombination within the solvent cage. In contrast, the corresponding triplet radical pair undergoes essentially exclusive cage escape to give the anthralin free radical (lambda max 370, 490 and 720 nm) which recombines under normal diffusive conditions. Both recombination processes lead, at least in part, to one or more species which have been assigned as tautomeric forms of the original dimer. The anthralin free radical in benzene is insensitive to the vitamin E model 6-hydroxy-2,2,5,7,8-pentamethylchroman and reacts only slowly with oxygen.     

SENSITIZED PHOTOOXYGENATION: CHANGE FROM TYPE I (RADICAL) TO TYPE II (SINGLET OXYGEN) MECHANISM

Abstract— As we have shown in previous papers, thionine-sensitized photooxygenation reactions follow a Type I (radical) mechanism. We now demonstrate that, by an appropriate choice of the acceptor and its concentration (solvent: pyridine) or by working in a rigid matrix (ethyl cellulose), the reaction can be switched to a Type II (singlet oxygen) mechanism. The system studied in the present investigation, thionine and 9,10-dimethylanthracene, represents to a certain extent an intermediate type. Photooxygenation at low DMA-concentration occurs according to a Type II mechanism as verified by the method of competitive photooxygenation, while in oxygen-free solutions, the sensitizer is partially photoreduced by the acceptor, which is typical for Type I systems. Whereas the photooxygenation of allylthiourea (ATU) with thionine as sensitizer takes place via radicals at high ATU concentrations, a change to the singlet oxygen mechanism could be observed at low ATU concentrations in pyridine solution.     

THE PHOTO-OXIDATION OF N, N-DIETHYLHYDROXYLAMINE BY ROSE BENGAL IN ACETONITRILE AND WATER

The Rose Bengal photosensitized oxidation of N,N-diethylhydroxylamine has been investigated in water and acetonitrile using the techniques of oxygen uptake, singlet oxygen phosphorescence and electron spin resonance. In both solvents H₂O₂ is the major oxidation product and diethylnitroxide is an intermediate. In water, superoxide dismutase decreases oxygen uptake suggesting involvement of superoxide anions in the oxidation process. Results indicate that in water the photo-oxidation proceeds mainly by a Type I(electron transfer) mechanism, while in acetonitrile a Type II(energy transfer) mechanism has been confirmed (Encinas et al., 1987, J. Chem. Soc. Perkin Trans. II,1125–1127).     

Photodynamic Crosslinking of Proteins. III. Kinetics of the FMN- and Rose Bengal-sensitized Photooxidation and Intermolecular Crosslinking of Model Tyrosine-containing N-(2-Hydroxypropyl)methacrylamide Copolymers

As part of a study on the role of Tyr residues in the photosensitized intermolecular crosslinking of proteins, we have surveyed the kinetics of the rose bengal- and flavin mononucleotide (FMN)-sensitized photooxidation and crosslinking of a water-soluble N-(2-hydroxypropyl)methacrylamide copolymer with attached 6-carbon side chains terminating in tyrosinamide groups (thus the -OH group of the Tyr is free, but both the amino and carboxyl groups are blocked, simulating the situation of a nonterminal Tyr in a protein). The intermolecular photodynamic crosslinking of the Tyr copolymer can result only from the formation of Tyr-Tyr (dityrosine) bonds, because the copolymer itself is not photooxidizable. Rose bengal, primarily a Type II (singlet oxygen) sensitizer, sensitized the rapid photooxidation of the Tyr residue in the Tyr copolymer only at high pH, where the Tyr phenolic group is ionized; crosslinking did not occur with rose bengal under any of the reaction conditions used. In contrast, FMN, which can sensitize by both Type I (free radical) and Type II processes, sensitized the photooxidation of the Tyr copolymer over the pH range 4-9.5. Also, significant photocrosslinking occurred, but only from pH 4 to 8, with a maximum rate at pH 6. Crosslinking required the presence of oxygen. Studies with inhibitors, D2O as solvent, catalase and superoxide dismutase indicated that the photooxidation and photocrosslinking of the Tyr copolymer with FMN at pH 6 were not mediated by singlet oxygen, superoxide or hydrogen peroxide. It appears that crosslinking involves the abstraction of an H atom from the Tyr phenolic group to give Tyr and FMN radicals. The Tyr radical in one Tyr copolymer can then react with a Tyr radical in another Tyr copolymer to give an intermolecular dityrosine crosslink.     

A COMPARATIVE STUDY OF THE VISIBLE LIGHT PHOTOCHEMISTRY OF GILVOCARCINS V and M

Quantum yields for the formation of superoxide ions, O₂^?1, and singlet oxygen, ¹O₂, were determined during the photolyses of gilvocarcin M (GM) in air-saturated dry dimethylsulfoxide (DMSO) and in 45:55 (vol/vol) DMSO-water mixtures. The quantum yield for the photoreduction of methyl viologen by GM in nitrogen-saturated dry DMSO was also determined. These values are not different, within experimental error, from those corresponding to gilvocarcin V (GV). Because GV is a strong photocytotoxic agent and GM is not, these results imply that Type I and Type II mechanisms are not important pathways in the cytotoxicity of GV.     

SPECTRAL AND PHOTOCHEMICAL PROPERTIES OF CURCUMIN

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione, is a natural yellow-orange dye derived from the rhizome of Curcuma longa, an East Indian plant. In order to understand the photobiology of curcumin better we have studied the spectral and photochemical properties of both curcumin and 4-(4-hydroxy-3-methoxy-phenyl)-3-buten-2-one (hC, half curcumin) in different solvents. In toluene, the absorption spectrum of curcumin contains some structure, which disappears in more polar solvents, e.g. ethanol, acetonitrile. Curcumin fluorescence is a broad band in acetonitrile (λ_max= 524 nm), ethanol (λ_max= 549 nm) or micellar solution (λ_max= 557 nm) but has some structure in toluene (λ_max= 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (φ= 0.011) but higher in acetonitrile (φ= 0.104). Curcumin produced singlet oxygen upon irradiation (φ > 400 nm) in toluene or acetonitrile (Φ= 0.11 for 50 μM curcumin); in acetonitrile curcumin also quenched ¹O₂ (k_q, = 7 × 10⁶ M^?1 s^?1). Singlet oxygen production was about 10 times lower in alcohols and was hardly detectable when curcumin was solubilized in a D₂O micellar solution of Triton X-100. In SDS micelles containing curcumin no singlet oxygen phosphorescence could be observed. Curcumin photogenerates superoxide in toluene and ethanol, which was detected using the electron paramagnetic resonance/spin-trapping technique with 5,5-dimethyl-pyrroline-.N-oxide as a trapping agent. Unidentified carbon-centered radicals were also detected. These findings indicate that the spectral and photochemical properties of curcumin are strongly influenced by solvent. In biological systems, singlet oxygen, superoxide and products of photodegradation may all participate in curcumin phototoxicity depending on the environment of the dye.     

Photochemical determination of the solubility of oxygen in various media

A photochemical method for determining the oxygen concentration in air-saturated non-aqueous solvents has been developed. Solutions containing a sensitizer (Rose Bengal or Methylene Blue) and 1,3-diphenylisobenzofuran (DPIBF) as an oxygen acceptor are irradiated at 546 or 633 nm and the absorbance at 404 nm is monitored. The dissolved oxygen content is found from the change in absorbance and the known 1:1 stoichiometry of addition of singlet oxygen to DPIBF. The solubilities found, accurate to +/- 6%, for oxygen in air-equilibrated solvents, are (mM): acetone, 2.37; acetonitrile, 2.42; dimethylsulfoxide, 0.33; ethanol, 1.94; N-methylformamide, 1.31. Measurements on mixed acetone-N-methylformamide solvents showed that the solubility of oxygen does not vary with solvent composition in a predictable manner.     

CUTANEOUS PHOTOTOXICITY OF TETRACYCLINE ANTIBIOTICS: GENERATION OF FREE RADICALS and SINGLET OXYGEN DURING PHOTOLYSIS AS MEASURED BY SPIN-TRAPPING and THE PHOSPHORESCENCE OF SINGLET MOLECULAR OXYGEN

Chlortetracycline (CTC) generated an aryl radical in aqueous buffer (pH 7.4) during near UV irradiation, as evidenced by the formation of 2-methyl-2-nitrosopropane spin adducts. The radical was produced via dechlorination, a photoprocess not previously reported for tetracyclines. Demeclocyc-line (DEM), another chlorinated tetracycline, did not produce detectable aryl radicals. Relative ¹O₂ yields obtained by direct luminescence measurements at 1268 nm for five tetracyclines in alkaline ethanol (demeclocycline · tetracycline · chlortetracycline · doxycycline · minocycline) showed that DEM produced approximately three times as much singlet oxygen as CTC. This constitutes direct evidence that tetracyclines sensitize both Type I and Type II photoreactions.     

Synthesis, photophysical and photochemical properties of poly(oxyethylene)-substituted zinc phthalocyanines

The synthesis, photophysical and photochemical properties of the tetra- and octa-poly(oxyethylene)substituted zinc (II) phthalocyanines are reported for the first time. The new compounds have been characterized by elemental analysis, IR, 1H and 13C NMR spectroscopy, electronic spectroscopy and mass spectra. General trends are described for photodegradation, singlet oxygen, triplet state and fluorescence quantum yields, and triplet and fluorescence lifetimes of these compounds in dimethylsulfoxide (DMSO). Photophysical and photochemical properties of phthalocyanine complexes are very useful for PDT applications. The effects of the substituents on the photophysical and photochemical parameters of the zinc(II) phthalocyanines (3a, 5a and 6a) are also reported. The singlet oxygen quantum yields (Phi(Delta)), which give an indication of the potential of the complexes as photosensitizers in applications where singlet oxygen is required (Type II mechanism) ranged from 0.60 to 0.72. Thus, these complexes show potential as Type II photosensitizers. The fluorescence of the complexes was quenched by benzoquinone (BQ).     

ANTHRALIN-DERIVED TRANSIENTS–I. THE TRIPLET STATE AND THE PRODUCTS OF ITS REACTION WITH OXYGEN IN BENZENE

Direct laser excitation in benzene of 1,8-dihydroxy-9-anthrone (anthralin) does not lead to transient species with lifetimes in the nanosecond time regime or longer. The triplet state has been produced in benzene by pulse radiolysis and characterised in terms of its absorption spectrum (lambda max 560 nm), natural lifetime (11 microseconds), self-quenching properties (kmicrosecond = 2.6 x 10(7) l mol-1 s-1) and triplet energy (234 kJ mol-1). There is no tendency in the non-polar medium for production of either the triplet or ground state in a tautomeric form. The observed triplet state reacts with oxygen with a typical rate constant, 2.2 x 10(9) l mol-1 s-1. The products of this reaction are singlet oxygen (approximately 64%) and the anthralin radical (approximately 14%).     

THE HEMOLYSIS OF ERYTHROCYTES BY SINGLET OXYGEN GENERATED IN THE GAS PHASE

Many sensitizers cause photodynamic hemolysis of erythrocytes. As these sensitizers usually participate in Type I as well as Type II processes, the determination of the mechanism(s) of photosensitized hemolysis is always ambiguous. Here, human erythrocytes were proved to hemolyze upon treatment with singlet oxygen (1 delta g) generated with fluoranthene in the gas phase. These conditions rigorously exclude the participation of superoxide anion. The standard diagnostic tests for singlet oxygen (enhanced effect in D2O and protection by NaN3) gave the anticipated results when the erythrocytes were treated with 1O2 generated in the gas phase. When the erythrocytes were irradiated in a buffer solution containing fluoranthene, the results of the diagnostic tests depended on the sensitizer concentration.     

Photodynamic action of a water-soluble hypocrellin derivative with enhanced absorptivity in the phototherapeutic window II. Photosensitized generation of active oxygen species

Cysteine-substituted hypocrellin B (Cys-HB) is a water-soluble perylenequinonoid derivative with significantly enhanced absorptivity in the range of wavelength longer than 600 nm. Electron paramagnetic resonance (EPR) measurements, quenching experiments and 9,10-diphenyl-anthracene bleaching studies were used to investigate the photodynamic action of Cys-HB in the presence of oxygen. Illumination of Cys-HB solution, in the presence of oxygen, generated singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. The accumulation of active oxygen species was transformed into that of the semiquinone anion radical with the depletion of oxygen, detected by the spin counteraction of TEMPO radical formed via the reaction of TEMP with singlet oxygen produced by Cys-HB photosensitization. Oxygen content, Cys-HB concentration and reaction environment affected the transformation and the competition between the Type I and Type II reactions. Compared with hypocrellin B (HB), Cys-HB primarily remained similar and slightly lower capability of active oxygen-generation, confirmed to be a favorable phototherapeutic agent.     

Antimicrobial photodynamic therapy and photodynamic inactivation, or killing bugs with dyes and light--a symposium-in-print

In antimicrobial photodynamic therapy, the photosensitizer (PS) in its ground singlet state absorbs light to give the excited singlet state that can transition to the long-lived triplet state. This PS triplet may undergo energy transfer (Type 2) or electron transfer (Type 1) to oxygen to form reactive oxygen species (singlet oxygen and/or hydroxyl radicals) that can kill both Gram-positive and Gram-negative bacteria and fungi. Infections in animal models can also be treated.     

Photoinduced Oxidation of Sterically Hindered Amines in Acetonitrile Solutions and Titania Suspensions (An EPR Study)

The reactions of sterically hindered amines (SHA) were investigated in acetonitrile solutions and TiO₂ suspensions upon exposure to monochromatic radiation, λ = 365 nm, by means of in situ EPR spectroscopy. The formation of singlet oxygen, as one of the possible oxidation agents for SHA, in these systems is affected significantly by solvent used and the experimental conditions. Experiments in homogeneous media evidenced alternative pathways for the SHA oxidation with a variety reactive oxygen species involved. In anhydrous acetonitrile solutions containing KO₂, the SHA oxidation was negligible not only in the dark but also on continuous exposure. However, the presence of water, even at low concentrations, led to the transformation of O₂^?? to singlet oxygen and hydrogen peroxide, which served as a source of hydroxyl radicals. These species participated in oxidation of SHA resulting in the generation of nitroxide radicals. To investigate the influence of different competitive reactions of SHA with other ROS formed upon TiO₂ photoexcitation, a series of experiments using different additives (e.g. KO₂, H₂O₂, NaN₃, dimethylsulfoxide, methanol as organic cosolvents) under air or argon were performed. The detailed analysis of paramagnetic intermediates formed upon the irradiation of the studied systems was accomplished using EPR spin trapping technique.     

The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.     

Electric magnetic resonance and spectrophotometry evidence on the photodynamic activity of a new perylenequinonoid pigment

Di-cysteine substituted hypocrellin B (DCHB) is a new water-soluble photosensitizer with significantly enhanced red absorption at wavelengths longer than 600 nm over the parent compound hypocrellin B (HB). The photosensitizing properties (Type I and/or Type II mechanisms) of DCHB have been investigated in dimethylsulfoxide (DMSO) and aqueous solution (pH 7.4) using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic DMSO solution, the semiquinone anion radical of DCHB (DCHB^•−) is predominantly photoproduced via self-electron transfer between excited- and ground-state DCHB species. The presence of an electron donor significantly promotes the formation of the reduced form of DCHB. When a deoxygenated aqueous solution of DCHB and an electron donor are irradiated with 532 nm light, the hydroquinone of DCHB (DCHBH₂) is formed via the disproportionation of the first-formed DCHB^•− and second electron transfer to DCHB^•− from the electron donor. When oxygen is present, singlet oxygen (¹O₂), superoxide anion radical (O₂^•−) and hydroxyl radical (^•OH) are produced. The quantum yield of ¹O₂ generation by DCHB photosensitization is estimated to be 0.54 using Rose Bengal as a reference, a little lower than that of HB (0.76). The superoxide anion radical is also significantly enhanced by the presence of electron donors. Moreover, (O₂^•−) upon disproportionation generated H₂O₂ and ultimately the highly reactive ^•OH via the Haber-Weiss reaction pathway. The efficiency of (O₂^•−) generation by DCHB is obviously enhanced over that of HB. These findings suggest that the photodynamic actions of DCHB may proceed via Type I and Type II mechanisms and that this new photosensitizer retains photosensitizing activity after photodynamic therapy-oriented chemical modification.     

A thermodynamic study of complexation process between <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>′-dipyridoxylidene(1,4-butanediamine) and Cd<Superscript>2+</Superscript> in some binary mixed solvents using conductometry

Complexation of the Cd²⁺ ion with N,N′-dipyridoxylidene(1,4-butanediamine) Schiff base was studied in pure solvents including acetonitrile (AN), ethanol (EtOH), methanol (MeOH), tetrahydrofuran (THF), dimethylformamide (DMF), water (H₂O), and various binary solvent mixtures of acetonitrile–ethanol (AN–EtOH), acetonitrile–methanol (AN–MeOH), acetonitrile–tetrahydrofuran (AN–THF), acetonitrile–dimethylformamide (AN–DMF), and acetonitrile–water (AN–H₂O) systems at different temperatures using the conductometric method. The conductance data show that the stoichiometry of complex is 1: 1 [ML] in all solvent systems. A non-linear behavior was observed for changes of log K_f of [Cd(N,N′-dipyridoxylidene(1,4-butanediamine)] complex versus the composition of the binary mixed solvents, which was explained in terms of solvent–solvent interactions. The results show that the thermodynamics of complexation reaction is affected by the nature and composition of the mixed solvents.