G. De Guidi

MOLECULAR MECHANISM OF DRUG PHOTOSENSITIZATION–II. PHOTOHEMOLYSIS SENSITIZED BY KETOPROFEN

Red blood cell lysis photosensitized by ketoprofen (KPF) was investigated. The photohem‐olysis was inhibited by butylated hydroxyanisole, reduced glutathione, superoxide dismutase and mannitol, and was unaffected by sodium azide; the presence of oxygen markedly enhanced the lysis. Photohemolysis was also observed under anaerobic conditions. Ketoprofen, irradiated in aqueous buffer solution at pH 7.4, underwent a decarboxylation process via intermediate radicals, leading to the compounds (3‐benzoylphenyl)ethane, (3‐benzoylphenyl)ethyl hydroperoxide, (3‐benzoylphenyl)‐ethanol and (3‐benzoylphenyl)ethanone under aerobic conditions and only to the compound (3‐benzpylphenyl)ethane under anaerobic conditions.

Molecular mechanism of naproxen photosensitization in red blood cells

Red blood cell lysis photosensitized by naproxen was investigated. The photohemolysis rate was enhanced by deuterium oxide and inhibited by butylated hydroxyanisole, reduced glutathione, sodium azide and superoxide dismutase. Photohemolysis was also observed under anaerobic conditions. In the absence of red cells the irradiation of deaerated solutions underwent a decarboxylation process via intermediate radicals, while under aerobic conditions photo-oxidation leading to the photoproduct 6-methoxy-2-acetonaphthone occurred. A molecular mechanism involving free radicals and singlet oxygen as important intermediates and consistent with the overall results is proposed.

pH Effects on the Spectroscopic and Photochemical Behavior of Enoxacin: A Steady-State and Time-Resolved Study

The spectroscopic and photochemical behavior of Enoxacin (ENX), 1‐ethyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(1‐piperazinyl)‐1,8‐naphthyridine‐3‐carboxylic acid, has been investigated in aqueous solutions between pH 3.5 and pH 12. The absorption and emission properties of ENX are strongly affected by pH. The fluorescence quantum yield, 4 x 10−3 at pH 3.5, increases by a factor of two on going to neutral pH while a strong reduction is observed at alkaline pH. The photodegradation quantum yield also depends on pH, being maximum in neutral conditions (ca 0.04). Nanosecond flash photolysis experiments confirm that the yield of absorbing transients is maximum at neutral pH while it decreases to zero at acid and alkaline pH. These results indicate that both the dissociation of the carboxylic group and the protonation of the piperazinyl residues are key steps for the formation of the photochemically active form of ENX. Loss of F− by heterolytic cleavage of the C–F bond is proposed to occur from the triplet state of the zwitterion with formation of a carbocation. A path for the evolution of this intermediate to the final product is also proposed.

The Photochemistry of Flutamide and its Inclusion Complex with β-Cyclodextrin. Dramatic Effect of the Microenvironment on the Nature and on the Efficiency of the Photodegradation Pathways¶

Abstract The photochemistry of the anticancer drug flutamide (FM), 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propan-amide, in homogeneous media and in the β-cyclodextrin (β-CD) cavity has been investigated. The photoreactivity of the free molecule has been rationalized on the basis of an intramolecular nitro to nitrite rearrangement followed by cleavage of the nitrite intermediate. The twisted geometry of the nitro group with respect to the aromatic plane plays a key role in triggering such a photoprocess. Incorporation of FM in the β-CD cavity leads to dramatic effects on both the efficiency and the nature of the photochemical deactivation pathways of the guest molecule. A 20-fold increase in the FM photodecomposition quantum yield and the formation of photoproducts originated by both reduction of the nitro group and cleavage of the amide bond were observed in the presence of the macrocycle. Such a behavior cannot be attributed exclusively to the micropolarity of β-CD and/or to its role as a reactant. The induced circular dichroism spectra and the nature of the photoproducts formed in these experimental conditions provide indications that the photoreactivity in the β-CD microenvironment could likely be mediated by structural changes of FM upon complexation.

Molecular mechanism of drug photosensitization 5. Photohemolysis sensitized by Suprofen

Red blood cell lysis photosensitized by Suprofen (SPF) and the photolysis of the drug were investigated. The photohemolysis process occurs at a higher rate in anaerobic than aerobic conditions. The effect of additives demonstrates the involvement of free radicals and, to a lesser extent, singlet oxygen and hydroxyl radicals in the process. Photolysis of the drug at 310-390 nm in deaerated buffered solutions (pH 7.4) leads to a decarboxylation process with the formation of p-ethylphenyl 2-thienyl ketone (I), whereas in aerated solutions formation of photoproduct I and of the photoproducts p-acetylphenyl 2-thienyl ketone (II) and p-(1-hydroxyethyl)phenyl-2-thienyl ketone (III) occurs. The photodegradation products, which were separated and characterized, show a moderate lytic and photolytic activity. The rate of SPF photodegradation decreases in the presence of oxygen and increases in the presence of hydrogen donors. The overall results lead us to propose a mechanism of SPF photodegradation and a hemolysis scheme in which cell damage is provoked principally by the direct attack of drug radicals and secondarily by singlet oxygen and hydroxyl radicals.

Antioxidant activity and phenolic content of strawberry genotypes from Fragaria x ananassa

The aim of this study was to compare the value in phytochemicals and antioxidant activity of two genotypes of strawberry fruit (Fragaria x ananassa, Duchesne, Rosaceae), one represented by the cultivated variety (cv) “Tudla” and the other one, “Maletto”, by a type selected in the mountain region of Etna (Italy). Moreover, we have considered the influence of soil on fruit quality. Total phenolic compounds and antocyanin content were determined by spectrophotometric methods. Phenolics, recovered from pulp juice, were determined by Folin–Ciocalteu method, whereas total anthocyanins were estimated by a pH differential method. Antioxidant activity was evaluated by DPPH and ORAC assays, using quercetin and Trolox as standard respectively. Our results indicate that the different genotypes of strawberries, in particular those cultivated in volcanic soil of the Etna mountain region, that we call “Maletto” strawberry, could be considered an important resource. They are a good source of antioxidants and could be used to prevent deleterious effects induced by free radicals. The scientific importance of these results may be significant for industry concerning food quality and disease prevention.

Molecular mechanism of photosensitization. XI. Membrane damage and DNA cleavage photoinduced by enoxacin.

The photosensitizing activity of enoxacin, 1‐ethyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(1‐piperazinyl)‐1,8‐naphthyridine‐3‐carboxilic acid (ENX), toward membranes and DNA has been studied, taking into account human erythrocyte photohemolysis, unilamellar liposome alterations and plasmid pBR322 DNA photocleavage. Hydroxyl radicals and an aromatic carbene generated from ENX photode‐fluorination seem to be the active intermediates involved in the photosensitization process. The steady‐state photolysis products do not participate in the process. The mechanism of photosensitization responsible for the membrane damage depends on the oxygen concentration and follows a different path with respect to that operative for DNA cleavage. Between oxygenated radicals, the hydroxyl seems the species mainly responsible for membrane damage, whereas DNA cleavage is mainly produced by the carbene intermediate. A molecular mechanism of the photosensitization induced by ENX is proposed.

Triplet Photochemistry of Suprofen in Aqueous Environment and in the β‐Cyclodextrin Inclusion Complex

The photodecarboxylation of suprofen in the carboxylate form was studied in aqueous medium as a function of the temperature, the concentration and the presence of oxygen by steady‐state and time‐resolved photochemical techniques. The process is characterized by an activation energy of 9–10 kcal/mol, the precursor state being the lowest triplet which is of π,π* nature. The reactivity of the drug was also studied in the β‐cyclodextrin inclusion complex and an additional photoreaction involving the macrocycle as reactive species was observed. Representative NMR and circular dichroism measurements were performed. Singlet molecular oxygen formation was also investigated.

MOLECULAR MECHANISM OF DRUG PHOTOSENSITIZATION 7. PHOTOCLEAVAGE OF DNA SENSITIZED BY SUPROFEN

Abstract— Ultraviolet‐A irradiation of a suprofen (2‐[4‐(2‐thenoyl)phenyl]propionic acid) (SPF) buffered solution (pH 7.4) in the presence of supercoiled pBR322 DNA leads to single strand breaks with the formation of an open circular form and subsequent linearization of the plasmid. On the basis of agarose gel electrophoresis data of samples irradiated in an air‐saturated solution or in an oxygen‐modified atmosphere, and the effects of sodium azide, D2O, mannitol, copper(II), superoxide dismutase, 2‐H‐propanol, deferoxamine and surfactants, we suggest a photosensitization mechanism involving singlet oxygen and free radicals. The higher rate of photocleavage in nitrogen compared to that in an air‐saturated solution and the results obtained from oxygen consumption measurements support the hypothesis that both the type I and type II photosensitization mechanisms are operative and that oxygen quenches the excited state of the irradiated drug. The photosensitization model applied was in agreement with that previously applied to cell membrane SPF photoinduced damage. Interaction of the drug with DNA, studied through circular dichroism and fluorescence anisotropy, probably occurs through a surface binding mode. The experimental techniques used for assessing the photodamaging activity of this drug may be useful for screening of phototoxic compounds in the environment and for determining the active species involved.

Effect of p-Cyclodextrin Complexation on the Photochemical and Photosensitizing Properties of Tolmetin: A Steady-State and Time-Resolved Study

The effects of beta-cyclodextrin complexation on the photochemical and photosensitizing properties of tolmetin have been investigated. Absorption, emission, circular dichroism and NMR measurements were used to characterize the host-guest complex. Nanosecond laser flash photolysis and steady-state photolysis experiments were performed to clarify the photoreactivity of the drug in the macrocycle. The decarboxylation of the drug is markedly reduced upon inclusion and the rate constants of the decay of the transient intermediates involved in tolmetin photodecomposition were slowed down due to their incorporation in the hydrophobic cavity. This also influenced the distribution of the stable photoproducts. A remarkable cyclodextrin-mediated protection against the tolmetin-photoinduced damage on biological substrates was observed. A rationale for these biological effects is provided.