Unveiling the important roles of coexisting contaminants on photochemical transformations of pharmaceuticals: Fibrate drugs as a case study.

Pharmaceuticals are a group of ubiquitous emerging pollutants, many of which have been shown to undergo efficient photolysis in the environment. Photochemically produced reactive intermediates (PPRIs) sensitized by the pharmaceuticals in sunlit natural waters may induce photodegradation of coexisting compounds. In this study, the roles of coexisting contaminants on the phototransformation of pharmaceuticals were unveiled with the fibrate drugs gemfibrozil (GMF), fenofibrate (FNF), and fenofibric acid (FNFA) as model compounds. GMF undergoes initial concentration dependent photodegradation due to the involvement of singlet oxygen (1O2) initiated self-sensitized photolysis, and undergoes pH dependent photodegradation due to dissociation and hydroxyl radical (OH) generation. The decarboxylated intermediates of GMF and coexisting FNFA significantly accelerated the photodegradation of GMF. The promotional effects of the decarboxylated intermediates are attributed to generation of PPRIs, e.g. 1O2, superoxide (O2-), that subsequently react with GMF. Besides, FNFA can also promote the photodegradation of GMF through the electron transfer reaction from ground state GMF to excited state FNFA, leading to the formation of decarboxylated intermediates. The formed intermediates can subsequently also facilitate GMF photodegradation. The results presented here provided valuable novel insights into the effects of coexisting contaminants on the photodegradation of pharmaceuticals in polluted waters.

Enzymatic reactive oxygen species assay to evaluate phototoxic risk of metabolites.

The present study aimed to verify the feasibility of an enzymatic reactive oxygen species (eROS) assay to evaluate the phototoxic risk of compounds after their metabolization. The eROS assay was designed based on the combined use of an in vitro drug metabolism system and a ROS assay. The incubation time of compounds with human hepatic S9 fractions was optimized with the use of fenofibrate (FF), a typical phototoxicant with metabolite-related phototoxicity, and the reproducibility and robustness of the eROS assay were examined using FF. The eROS assay was applied to 12 phototoxic compounds, including 7 phototoxicants with metabolite-related phototoxicity, to clarify the assay performance. According to the eROS data on singlet oxygen generation from FF and metabolic conversion profiles of FF and fenofibric acid, the incubation time of chemicals with human hepatic S9-mix was determined to be 4min. The singlet oxygen-based evaluation system in the eROS assay was found to be acceptable as a high-throughput assay because of its favorable intra-/inter-day reproducibility (coefficient of variation: ca. 8%) and robustness (Z'-factor: 0.23). Singlet oxygen data on phototoxicants with phototoxic metabolites tended to exceed 120% of control, suggesting the feasibility of the eROS assay to evaluate metabolite-related phototoxic potentials. However, further data accumulation is still needed to improve the assay performance because the eROS assay provided false predictions for some compounds. The present eROS assay may be applicable in part for evaluating the phototoxic risk of drug candidates after their metabolization in the early stage of drug discovery.

Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid: Photoreactivity with Serum Albumins.

The photoreactivity of fenofibric acid (FA) in the presence of human and bovine serum albumins (HSA and BSA, respectively) has been investigated by steady-state irradiation, fluorescence, and laser flash photolysis (LFP). Spectroscopic measurements allowed for the determination of a 1:1 stoichiometry for the FA/SA complexes and pointed to a moderate binding of FA to the proteins; by contrast, the FA photoproducts were complexed more efficiently with SAs. Covalent photobinding to the protein, which is directly related to the photoallergic properties of the drug, was detected after long irradiation times and was found to be significantly higher in the case of BSA. Intermolecular FA-amino acid and FA-albumin irradiations resulted in the formation of photoproducts arising from coupling between both moieties, as indicated by mass spectrometric analysis. Mechanistic studies using model drug-amino acid linked systems indicated that the key photochemical step involved in photoallergy is formal hydrogen atom transfer from an amino acid residue to the excited benzophenone chromophore of FA or (more likely) its photoproducts. This results in the formation of caged radical pairs followed by C-C coupling to give covalent photoaducts.

Toxic and genotoxic impact of fibrates and their photoproducts on non-target organisms.

Lipid regulators have been detected in effluents from sewage treatment plants and surface waters from humans via excretion. This study was designed to assess the ecotoxicity of fibrates, lipid regulating agents. The following compounds were investigated: Bezafibrate, Fenofibrate and Gemfibrozil and their derivatives obtained by solar simulator irradiation. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while SOS Chromotest and Ames test were utilized to detect the genotoxic potential of the investigated compounds. The photoproducts were identified by their physical features and for the first risk evaluation, the environmental impact of parental compounds was calculated by Measured Environmental Concentrations (MEC) using the available data from the literature regarding drug occurrence in the aquatic environment and the Predicted No Effect Concentrations (PNEC) based on our toxicity data. The results showed that acute toxicity was in the order of dozens of mg/L for all the trophic levels utilized in bioassays (bacteria, rotifers, crustaceans). Chronic exposure to these compounds caused inhibition of growth population on rotifers and crustaceans while the algae seemed to be slightly affected by this class of pharmaceuticals. Genotoxic and mutagenic effects were especially found for the Gemfibrozil photoproduct suggesting that also byproducts have to be considered in the environmental risk of drugs.

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.

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.

Formation of a perbenzoic acid derivative in the photodegradation of fenofibrate: phototoxicity studies on erythrocytes.

The phototoxic antihyperlipoproteinemic drug fenofibrate (1) is photolabile under aerobic conditions. Irradiation of a methanol solution of 1 produces, under oxygen, photoproducts 2, 3, and 4. A peroxidic photoproduct 2 was isolated and identified. The biologically active antioxidants glutathione and cysteine efficiently reduce 2 to its acid. This photoproduct was also capable of efficiently oxidizing tetramethyl phenylendiamine (TMP) through an electron transfer mechanism, detecting a TMP+ species by UV-visible spectrometry. Fenofibrate was screened in vitro at different concentrations for UV-visible-induced phototoxic effects in a photohemolysis test, under oxygen as well as argon. The photohemolysis rate was low under anaerobic conditions. No hemolysis occurred without irradiation. The isolated photoproduct 2 induced hemolysis without irradiation.