Photosensitisation by voriconazole-N-oxide results from a sequence of solvent and pH-dependent photochemical and thermal reactions.

The phototoxicity of voriconazole (VN) prescribed in the treatment of severe fungal infections is frequently reported. Its major metabolite, a N-oxide derivative (VNO), was suspected to be the photosensitizer because it shows a maximum absorbance at ~310 nm in aqueous solutions. It was reported that the VNO photoproduct (VNOP) was phototoxic to human keratinocytes. Steady state and laser flash photolyses were performed to shed light on the phototoxic properties of VNO and VNOP. The quantum yield of the VNOP production by UVB-UVA light in buffered or alcoholic solutions is 0.6. VNOP has been identified as (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-7-oxa-1,3-diazabicyclo[4.1.0]hepta-2,4-dien-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol. VNOP undergoes a marked thermal degradation and an efficient UVA photolysis with well differentiated kinetics and end-products. The temperature-dependent VNOP dark degradation produces a single product VNOPD identified as 6-[(2S,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-5-fluoropyrimidin-4-ol with absorbance maximum at 308 nm and ε = 2700 M-1 cm-1. Under UVB-UVA irradiation, VNOPD, the stable end-product, is a remarkable photodynamic photosensitizer towards Trp and His. The Trp photo-oxidation (Φox(Trp) = 0.13) mainly involves type I radical reactions whereas His is oxidized by 1O2 (Φox(His) = 0.012). These results force us to question the validity of the in vitro photosensitization of human keratinocytes by VNO and VNOP previously reported.

A molecular insight into the phototoxic reactions observed with vemurafenib, a first-line drug against metastatic melanoma.

The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 µs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 µs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ âˆ¼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).

Experimental functional response and inter-individual variation in foraging rate of teal (Anas crecca).

The functional response, i.e. the change in per capita food intake rate per time unit with changed food availability, is a widely used tool for understanding the ecology and behaviour of animals. However, waterfowl remain poorly explored in this context. In an aviary experiment we derived a functional response curve for teal (Anas crecca) foraging on rice (Oryza sativa) seeds. We found a linear relationship between intake rate and seed density, as expected for a filter-feeder. At high seed densities we found a threshold, above which intake rate still increased linearly but with a lower slope, possibly reflecting a switch from filter-feeding to a scooping foraging mode. The present study shows that food intake rate in teal is linearly related to food availability within the range of naturally occurring seed densities, a finding with major implications for management and conservation of wetland habitats.

Photoreactivity of the nonsteroidal anti-inflammatory 2-arylpropionic acids with photosensitizing side effects.

The photoreactivity of the nonsteroidal anti-inflammatory 2-arylpropionic acids benoxaprofen, carprofen, naproxen, ketoprofen, tiaprofenic acid, and suprofen is reviewed with special emphasis on fundamental photophysical and photochemical properties. The absorption and emission properties of the excited states of these drugs as well as their main photodegradation routes are summarized. The photochemical mechanisms are discussed on the basis of product studies and detection of short-lived intermediates by means of laser flash photolysis. After dealing with the unimolecular processes, attention is focused on the photosensitized reactions of key biomolecules, such as lipids, proteins or nucleic acids. Finally, a short section on the photobiological effects on simple biological models is also included. Although some earlier citations are included, the literature coverage is in general limited to the last decade.

Photochemistry of 2-hydroxy-4-trifluoromethylbenzoic acid, major metabolite of the photosensitizing platelet antiaggregant drug triflusal.

Triflusal is a platelet antiaggregant drug with photoallergic side effects. However, it is considered a prodrug since it is metabolized to 2-hydroxy-4-trifluoromethylbenzoic acid (HTB)--the pharmacologically active form. HTB was found to be photolabile under various conditions. Its major photodegradation pathway appears to be the nucleophilic attack at the trifluoromethyl moiety. The involvement of the triplet state in the photodegradation has been unequivocally proved by direct detection of this transient in laser flash photolysis and by quenching experiments with oxygen, cyclohexadiene and naphthalene. Finally, the photobinding of HTB to proteins such as bovine serum albumin has been demonstrated using ultraviolet-visible (UV-Vis) and fluorescence spectroscopy. Nucleophilic groups present in the protein appear to be responsible for the formation of covalent drug photoadducts, which is the first step involved in the photoallergy shown by triflusal.

A laser flash photolysis and pulse radiolysis study of primary photochemical processes of flumequine.

The 355 nm laser flash photolysis of argon-saturated pH 8 phosphate buffer solutions of the fluoroquinolone antibiotic flumequine produces a transient triplet state with a maximum absorbance at 575 nm where the molar absorptivity is 14,000 M(-1) cm(-1). The quantum yield of triplet formation is 0.9. The transient triplet state is quenched by various Type-1 photodynamic substrates such as tryptophan (TrpH), tyrosine, N-acetylcysteine and 2-deoxyguanosine leading to the formation of the semireduced flumequine species. This semireduced form has been readily identified by pulse radiolysis of argon-saturated pH 8 buffered aqueous solutions by reaction of the hydrated electrons and the CO2*- radicals with flumequine. The absorption maximum of the transient semireduced species is found at 570 nm with a molar absorptivity of 2,500 M(-1) cm(-1). In argon-saturated buffered solutions, the semireduced flumequine species formed by the reaction of the flumequine triplet with TrpH stoichiometrically reduces ferricytochrome C (Cyt Fe3+) under steady state irradiation with ultraviolet-A light. In the presence of oxygen, O2*- is formed but the photoreduction of Cyt Fe3+ by O2*- competes with an oxidizing pathway which involves photo-oxidation products of TrpH.

Substituent effects on electrophilicity of flavins: an experimental and semi-empirical molecular orbital study.

The reactivities of the triplet excited states of several flavins with different N(10)-phenyl substituents have been found to be very similar. There is a good correlation between semi-empirical molecular orbital calculations at the MINDO/AM1 level and observed redox potentials. However, the most interesting observation is that the substituent effects are very small; this suggests that the N(10)-phenyl substituents are nearly perpendicular to the plane of the main ring due to their steric interactions with the main isoalloxazine system. Computed calculations as well as the fact that similar absorption maxima are obtained for 3-methyl-10-phenylisoalloxazine and 3-methyl-10-methylisoalloxazine are in agreement with this prediction.

Tiaprofenic acid-photosensitized damage to nucleic acids: a mechanistic study using complementary in vitro approaches.

In order to determine whether or not tiaprofenic acid (TPA) could cause cellular DNA damage, human fibroblasts were irradiated in the presence of the drug and subsequently examined by means of the comet assay. This led to the observation that TPA actually sensitizes cellular DNA to the subsequent irradiation. When TPA was irradiated in the presence of supercoiled plasmid DNA, it produced large amounts of single-strand breaks (SSB); this is consistent with the effects observed on cellular genomic DNA by the comet assay. More importantly, low concentrations of TPA, unable to produce direct SSB, caused photo-oxidative damage to DNA as revealed by the use of excision-repair enzymes. The fact that TPA-irradiated DNA was a substrate of formamidopyrimidine glycosylase as well as endonuclease III revealed that both purine and pyrimidine bases were oxidized. This was further supported by the TPA-photosensitized oxidation of 2'-deoxyguanosine which led to a product mixture characteristic of mixed type-I/II mechanisms. Thymidine was less reactive under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucleosides with clearly different rate constants (10(8) vs 10(7) M-1 s-1, respectively). As cellular RNA also contains oxidizable bases, it could be the target of similar processes, thus interfering with the biosynthesis of proteins by the cells. Extraction of total RNA from TPA-irradiated human fibroblasts, followed by gel electrophoresis and PCR analysis, confirmed this hypothesis. Finally, photosensitization experiments with Saccharomyces cerevisiae showed that, in spite of an efficient drug-yeast interaction leading to cytotoxicity, neither intergenic recombination nor gene conversion took place. Thus, while TPA-photosensitized damage to nucleic acids can result in genotoxicity, the risk of mutagenicity does not appear to be significant.

A photophysical and photochemical study of 6-methoxy-2-naphthylacetic acid, the major metabolite of the phototoxic nonsteroidal antiinflammatory drug nabumetone.

Nabumetone is a phototoxic nonsteroidal antiinflammatory drug used for the treatment of osteoarthritis. However, nabumetone is considered a prodrug with its metabolite 6-methoxy-2-naphthylacetic acid the active form. Photophysical and photochemical studies on this metabolite have been undertaken. It undergoes photodecarboxylation in aerated aqueous and organic solvents. In addition to the accepted photodegradation pathway for related molecules, a new mechanism that implies generation of the naphthalene radical cation from the excited singlet and addition of O2 prior to the decarboxylation process has been demonstrated. Evidence for the involvement of the excited singlet state in this mechanism have been obtained by steady-state and time-resolved fluorescence experiments. The fluorescence quenching by O2 and the shorter singlet lifetime in aerated solvents support this assignment. Laser flash photolysis also supports this mechanism by showing the noninvolvement of the triplet in the formation of the naphthalene radical cation. Finally, the well-known electron acceptor CCl4 acts as an efficient singlet quencher, enhancing the route leading to the radical cation, preventing intersystem crossing to the triplet and thus resulting in a dramatic increase in the yield of 6-methoxy-2-naphthaldehyde, the major oxidative decarboxylation product; this constitutes unambiguous proof in favor of the new mechanistic proposals.

Involvement of type I and type II mechanisms in the linoleic acid peroxidation photosensitized by tiaprofenic acid.

Analysis of the photomixtures resulting from irradiation of aqueous solutions of linoleic acid sensitized by tiaprofenic acid (TPA) or its major photoproduct (DTPA) by HPLC has shown the formation of all the four possible conjugated dienic hydroperoxides. According to laser flash photolysis experiments the rate constants for hydrogen abstraction from linoleic acid by the excited triplet states of TPA and DTPA are 2 x 10(5) and 3.2x 10(5) M(-1) s(-1), respectively. These data, together with the known rate constants for oxygen quenching of triplet (D)TPA and for the reaction of singlet oxygen with linoleic acid, show that the mechanism is mixed type I/type II. Finally, typical radical scavengers such as BHA and singlet oxygen quenchers such as DABCO and sodium azide are efficient quenchers of the triplet excited state of DTPA. This shows the risk of assigning mechanisms based on indirect 'evidences' using 'specific' additives.

Photobinding of carprofen to protein.

Carprofen is a non-steroidal antiinflammatory drug with marked photosensitising properties. In order to elucidate the mechanisms underlying the phenomenon of drug-protein photobinding, mixtures of the drug and human serum albumin were irradiated under different experimental conditions. After irradiation and subsequent gel-filtration chromatography of the photomixture, the eluting protein fraction was analysed by means of fluorescence spectroscopy. The formation of drug-protein photoadducts could be evidenced by the characteristic emission properties of the carbazole chromophore. The photobinding of the drug to human serum albumin appears to involve the formation of aryl radicals resulting from carbon-halogen photocleavage. This mechanistic interpretation is supported by the observed variations in the intensity of the fluorescence spectra, which can be correlated with the lower quantum yield emission of chlorocarbazoles as compared to non-halogenated analogues. The results from laser flash photolysis studies are also in agreement with this proposal.

Phototoxicity associated with diclofenac: a photophysical, photochemical, and photobiological study on the drug and its photoproducts.

Diclofenac (1) is a photosensitizing nonsteroidal antiinflammatory drug. Its photodecomposition gives rise to chlorocarbazole 2a. This product undergoes photodehalogenation to 3a in a subsequent step. When the photobiological activities of 1, 2a, and 3a are compared by means of the photohemolysis test, it is clearly observed that chlorocarbazole 2a causes cell lysis with a markedly higher efficiency than the parent drug or the secondary photoproduct 3a. Laser flash photolysis studies suggest that photodehalogenation of 2a occurs from its excited triplet state via quenching by ground-state 2a and formation of an excimer. As a consequence, an aryl radical plus an N-centered carbazolyl radical are formed. These radical intermediates appear to be responsible for the observed photobiological effects of diclofenac, via hydrogen abstraction from the target biomolecules, which initiates a type-I photodynamic effect. The efficient peroxidation of model lipids, such as linoleic acid, photosensitized by 2a are in favor of this proposal. Thus, the photosensitizing properties of diclofenac appear to be associated with the photochemical and photobiological activity of its major photoproduct.

Photosensitizing drugs containing the benzophenone chromophore.

The nonsteroidal anti-inflammatory agents ketoprofen, tiaprofenic acid, suprofen and tolmetin, together with the anti-hyperlipoproteinemic drug fenofibrate and the anti-arrhythmic amiodarone can be included in the group of benzophenone-derived photosensitizing drugs. They contain a diaryl ketone chromophore and mediate the development of phototoxic reactions. In some cases, photoallergic responses have been reported. These properties have been substantiated in clinical reports, as well as by means of in vivo and in vitro assays. Tolmetin is phototoxic in vitro, however there are no reports on photosensitization by this drug in humans. In general, photochemical and photobiological studies strongly suggest that photosensitization involves formal hydrogen abstraction (either in a single step or via electron transfer followed by proton transfer) by the benzophenone-like chromophore from the excited triplet state. In the case of amiodarone, the radicals generated by photodehalogenation from the triplet are responsible for the photosensitivity side-effects.

Drug-photosensitized protein modification: identification of the reactive sites and elucidation of the reaction mechanisms with tiaprofenic acid/albumin as model system.

Certain drugs can photosensitive the formation of protein modifications, which are thought to be responsible for the occurrence of photoallergy. In the present work, the UV irradiation of serum albumin in the presence of tiaprofenic acid has been studied as a model system for drug-photosensitized protein modifications. The photolysates evidenced that His, Tyr, and Trp are the reactive sites of the protein. The experimental results strongly suggest that formal hydrogen abstraction from the OH or NH groups of Tyr or Trp by the excited drug is the key photochemical process. Competition between cage escape and in cage recombination of the resulting radical pairs governs the final outcome: protein photo-cross-linking versus drug-protein adduct formation. These findings are highly relevant to understand the process of photohapten formation, the first event in the onset of photoallergy.

Photophysical and photochemical characterization of a photosensitizing drug: a combined steady state photolysis and laser flash photolysis study on carprofen.

Carprofen (1a) is a photosensitizing nonsteroidal anti-inflammatory drug. It undergoes photodehalogenation from its triplet excited state. The resulting aryl radical (II) is able to abstract hydrogen atoms from model lipids, mediating their peroxidation by a type I mechanism. This aryl radical intermediate appears to be responsible for the observed photobiological effects of carprofen. The active involvement of the triplet state has been confirmed by direct detection of this species in laser flash photolysis and by quenching experiments with cyclohexadiene and naphthalene. Carprofen also photosensitizes singlet oxygen production with a quantum yield of 0.32. A minor reaction pathway is photodecarboxylation, which occurs from the excited singlet state and leads to an acetyl derivative (1b). In the case of the dehalogenated photoproduct (2a), photodecarboxylation to the ethyl (2d) and acetyl (2b) derivatives, together with singlet oxygen production (quantum yield = 0.18), is also possible. However, the biological activity of 2a in the linoleic acid photoperoxidation and photohemolysis tests is markedly lower than that of 1a, which constitutes further evidence in favor of the important role of photodehalogenation in the adverse photobiological effects of carprofen.

Photochemical and photobiological properties of ketoprofen associated with the benzophenone chromophore.

Irradiation of ketoprofen in neutral aqueous medium gave rise to 3-ethylbenzophenone as the major photoproduct. Its formation is justified via protonation of a benzylic carbanion or hydrogen abstraction by a benzylic radical. Minor amounts of eight additional compounds were isolated. Four of them are derived from the benzylic radical: 3-(1-hydroperoxyethyl)benzophenone, 3-(1-hydroxyethyl)benzophenone, 3-acetylbenzophenone and 2,3-bis-(3-benzoylphenyl)butane. The other four products involve initial hydrogen abstraction by the excited benzophenone chromophore of ketoprofen: 1,2-bis-(3-ethylphenyl)-1,2-diphenyl-1,2-ethanediol, 2-(3-benzoylphenyl)-1-(3-ethylphenyl)-1-phenylpropan-1-ol, alpha-(3-ethylphenyl)phenylmethanol, 1,2-bis-[3-(2-hydroxycarbonylethyl) phenyl]-1,2-diphenyl-1,2-ethanediol. The latter process was found to mediate the photoperoxidation of linoleic acid through a type I mechanism, as evidenced by the inhibition produced by the radical scavengers butylated hydroxyanisole and reduced glutathione. The major photoproduct, which contains the benzophenone moiety but lacks the propionic acid side chain, also photosensitized linoleic acid peroxidation. Because lipid peroxidation is indicative of cell membrane lysis, the above findings are highly relevant to explain the photobiological properties of ketoprofen.

Photosensitization by fenofibrate. II. In vitro phototoxicity of the major metabolites.

Fenofibric acid, the major metabolite of fenofibrate, was found to be photolabile. Its irradiation in aqueous solution gave rise to two photoproducts, whose formation involves photodecarboxylation of the dissociated acid to an aryloxy-substituted carbanion, which is directly protonated or, alternatively, undergoes a Wittig rearrangement. A comparative in vitro phototoxicity study has been carried out on the anti-hyperlipoproteinemic drug fenofibrate, its metabolites and the photoproducts of fenofibric acid. Fenofibrate, fenofibric acid and its two photoproducts were found to be active when examined by the photohemolysis test and were able to photosensitize peroxidation of linoleic acid, as evidenced by the UV monitoring of dienic hydroperoxides. In summary, the major metabolite of fenofibrate (fenofibric acid), as well as its photoproducts, are phototoxic in vitro. This behavior can be attributed to the fact that the four compounds retain the benzophenone chromophore present in fenofibrate and is indicative of free radical-mediated photosensitization. In agreement with this rationalization, the metabolites with a reduced ketone functionality exhibit no detectable in vitro phototoxicity.

In vitro phototoxicity of clofibrate. Photochemical and photohemolytic studies on its metabolite clofibric acid.

Aqueous or methanolic solutions of clofibrate and clofibric acid are photolabile towards UVB light under aerobic as well as anaerobic conditions. Nine photoproducts have been identified; their formation involves primary cleavage of the carbon-halogen bond or of the aryloxy-carbon bond, followed by hydrogen abstraction and/or radical recombination. Clofibric acid is phototoxic in vitro as indicated by the photohemolysis test, under both oxygen and argon atmospheres, although the photohemolysis rate is markedly higher under aerobic conditions. Partial inhibition of this process on addition of butylated hydroxyanisole (BHA), reduced glutathione (GSH), sodium azide (NaN3) or 1,4-diazabicyclo[2.2.2]octane (DABCO) suggests the involvement of type I as well as type II mechanisms.

Photodegradation and in vitro phototoxicity of fenofibrate, a photosensitizing anti-hyperlipoproteinemic drug.

The phototoxic anti-hyperlipoproteinemic drug fenofibrate was found to be photolabile under aerobic and anaerobic conditions. Irradiation under argon of a methanol solution of this drug produced the photoproducts isopropyl 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate, 1,2-bis(4-chlorophenyl)-1,2bis (4-[isopropoxycarbonylisopropoxy]phenyl)ethane-1,2-diol and 4-(4-chlorobenzoyl)phenol, while under oxygen the photoproducts were 4-chloroperbenzoic acid, methyl 4-chlorobenzoate, 4-chlorobenzoic acid and singlet oxygen, as evidenced by trapping with 2,5-dimethylfuran. These results can be rationalized through hydrogen abstraction by excited fenofibrate, to afford a free radical as key intermediate. Biologically active antioxidants such as glutathione and cysteine efficiently reduced 4-chloroperbenzoic acid to 4-chlorobenzoic acid. The involvement of an electron transfer mechanism is suggested by detection (UV-vis spectrophotometry) of the radical cation TMP+. during the oxidation of tetramethylphenylenediamine (TMP) with 4-chloroperbenzoic acid. Fenofibrate was phototoxic in vitro when examined by the photohemolysis test, both under oxygen and argon atmosphere, although the photohemolysis rate was markedly lower under anaerobic conditions. The photoproducts 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate and 4-chloroperbenzoic acid induced hemolysis in the dark; however, this effect was quantitatively less important than photohemolysis by fenofibrate. On the other hand, fenofibrate photosensitized peroxidation of linoleic acid, monitored by the UV detection of dienic hydroperoxides. Based on the inhibition of this process upon addition of butylated hydroxyanisole, a radical chain (type I) mechanism appears to operate. In summary, fenofibrate is phototoxic in vitro. This behavior can be explained through the involvement of free radicals, singlet oxygen and stable photoproducts.