Photochemically Generated Stable Cation Radical of Phenothiazine Aggregates in Mildly Acid Buffered Solutions

Abstract

This work characterizes, for the first time, the photochemical behavior of the antipsychotic drugs thioridazine (TR), trifluoperazine (TFP), and fluphenazine (FP) influenced by the aggregation state of the molecules. Samples of monomeric and aggregated forms of phenothiazines were submitted to 20 min of irradiation at 254 nm for intervals of 1, 5, 10, 15, 20, or 25 days. In high phenothiazine concentrations, the irradiation led to the appearance of absorbance bands in the visible region peaking at 633 nm for TR and 509 nm for FP and TFP. In the dark, at room temperature and at 4 degrees C, these bands disappeared, after approximately 15 and approximately 60 min, respectively, but reappeared after a new irradiation session. These visible bands were assigned to stable cation radicals that were characterized by direct EPR measurements and by flash photolysis. Photogenerated stable cation radicals in the phenothiazine aggregates at room temperature are formed probably due to the stacking of the thiazine phenyl moieties. For the monomeric forms of phenothiazines, the spectral changes observed during the irradiation suggested the formation of sulfoxide and hydroxylated derivates. Oxidized derivates were detected by mass spectrometry of the aggregated forms of phenothiazines (>100 microM) only in the samples irradiated for more than 20 days. In contrast, monomeric phenothiazines were totally converted to the oxidized forms after 20 min of irradiation. Surface tension measurements of phenothiazines revealed that, in concentrations above 100 microM, the drugs formed aggregates. In the case of TR, small-angle X-ray scattering measurements indicated that this compound forms large lamellar-like aggregates in aqueous solutions.

UV-Light Effects on Cytochrome C Modulated by the Aggregation State of Phenothiazines

“…Upon irradiation, the spectral changes of cytochrome c at pH 4.0 were characterized by a blue shift and bleaching. The spectral changes of cytochrome c could be observed concomitantly with the spectral changes of TR previously assigned to the formation of sulfoxide and the derivative with the hydroxylated ring [11](Figure 2B). The aforementioned spectral changes of TR and cytochrome c were not detected when the sample was maintained in the dark conditions (not shown).…”

“…The aforementioned spectral changes of TR and cytochrome c were not detected when the sample was maintained in the dark conditions (not shown). Considering that TR and FP form aggregates that stabilize the respective cation radical derivatives [11], cytochrome c underwent UV irradiation in the presence of different concentrations of the phenothiazine. Figure 2C illustrates the differentiated effect of high concentrations of TR.…”

“…Figure 2 C also shows the TR 615 nm absorbance band in the spectrum of a mixture containing 5 µM cytochrome c and 2.5 mmol/LTR that was subjected to UV irradiation. TR cation radicals stabilized by the aggregation of the drug are related to the appearance of a band at 620 nm region while FP and the parent compound TFP cation radicals are associated to bands peaking at around 520 nm [11]. Figure 2D shows the rate of a Soret band blue shift plotted as a function of TR concentration.…”

“…Conversely, the photo-induced damage was mitigated by the presence of 2.5 mmol/L FP (Figure 3B). In the presence of 25 µmol/L FP, the spectral changes indicative of the formation of FP derivative sulfoxide [11] were observed concomitantly with a Soret band blue shift. These results are consistent with the low stability of photo-generated free radicals of the drug.…”

“…In a biological system, these reactive species can cause damage in biomolecules such as lipids, proteins, and DNA [6-10]. Previously, it was demonstrated that the photo-excitation of the aggregate state of three phenothiazine derivatives (thioridazine (TR), trifluoperazine (TFP), and fluphenazine (FP) generates the corresponding cation radicals, that remained stable during hours [11]. However, TR, TFP, and FP cation radicals are not stabilized when the drugs are associated to lipid bilayers and promote oxidative damage in membranes [12].…”