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[EN] Cholesterol (Ch) is an integral part of cell membrane, where it is prone to oxidation. In humans, oxidation of Ch is commonly linked to various pathologies like Alzheimer's disease, atherosclerosis, and even cancer, which proceed via mechanisms involving enzymatic and free radical pathways. The latter begin with hydrogen abstraction (HA) from Ch by a reactive free radical. It has been established that the most efficient HA from Ch occurs at C7, although HA from C4 by peroxyl radicals has recently been observed. Conversely, HA from Ch positions other than the thermodynamically preferred C7 or C4 has never been reported. We have designed a Ch derivative where a benzophenone moiety is linked to C7 by a covalent bond. This mirrors a specific orientation of Ch within a confined environment. Product analysis and time-resolved spectroscopic studies reveal an unprecedented HA from C15, which is a thermodynamically unfavorable position. This indicates that a specific topology of reactants is crucial for the reactivity of Ch. The relative orientation of the reactants can also be relevant in biological membranes, where Ch, polyunsaturated fatty acids, and numerous oxidizing species are confined in highly restricted and anisotropic environments.

[EN] In this work, the attention is focused on UVA-photosensitized reactions triggered by a DNA chromophore-containing lesion, namely 5-formyluracil. This is a major oxidatively generated lesion that exhibits an enhanced light absorption in the UVB-UVA region. The mechanistic study combining photochemical and photobiological techniques shows that irradiation of S-formyluracil leads to a triplet excited state capable of sensitizing formation of cyclobutane pyrimidine dimers in DNA via a triplet-triplet energy transfer. This demonstrates for the first time that oxidatively generated DNA damage can behave as an intrinsic sensitizer and result in an important extension of the active fraction of the solar spectrum with photocarcinogenic potential. Overall, this raises the question of an aggravated photomutagenicity of the 5-formyluracil lesion.

[EN] 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.

A new direct and noninvasive methodology based on transient absorption spectroscopy has been developed to probe the feasibility of drug-drug interactions within a common protein binding site. The simultaneous presence of (R)-cinacalcet (CIN) and (S)-propranolol (PPN) within human or bovine alpha(1)-acid glycoproteins (AAGs) is revealed by detection of (CIN)-C-3* as the only transient species after laser flash photolysis of CIN/PPN/AAG mixtures at 308 nm. This is the result of triplet-triplet energy transfer from (PPN)-P-3* to CIN, which requires close contact between the two drugs within the same biological compartment. Similar results are obtained with nabumetone and CIN as donor/acceptor partners. This new methodology can, in principle, be extended to a variety of drug/drug/biomolecule combinations.

New photoactive dyads have been synthesized by derivatization of cholic acid (CA) or cholesterol (Ch). These compounds have proven to be efficient tools to monitor incorporation of CA and Ch to mixed micelles (MM) and to probe the microenvironment experienced inside these entities. The outstanding capability of MM to solubilize Ch has been demonstrated.

Photophysical techniques, specifically time-resolved fluorescence and laser flash photolysis, have proven to be noninvasive, straightforward, and valuable tools to demonstrate how drug encapsulation into biomimetic mixed micelles (MM) influences the dynamics of excited states and their accessibility to ionic quenchers. This concept has been illustrated by choosing a set of currently administered drugs containing a common naphthalene chromophore, namely, (S)-naproxen and its methyl ester, (R)-cinacalcet and (S)-propranolol. A remarkable increase of their triplet lifetimes is noticed when experiments are performed in MM, indicating efficient entrapment of the drugs in these supramolecular entities. Furthermore, a decrease of 1 order of magnitude in the quenching rate constant of the singlet and triplet excited states (by iodide or nitrite, respectively) is observed upon encapsulation into MM. This approach can in principle be extended to other microenvironments capable of incorporating photoactive compounds. © 2013 American Chemical Society.

The two enantiomers of the nonsteroidal antiinflammatory drug naproxen and of its methyl ester have been selected as representative probes with markedly different hydrophobicity to assess the potential of cholic acid aggregates as drug carriers by means of photophysical techniques. The different distribution of the probes between bulk solution and aggregates has been assessed by quenching of their singlet and triplet excited states by iodide and nitrite anions, respectively. This straightforward photophysical methodology can, in principle, be extended to a variety of drugs containing a photoactive chromophore. © 2012 American Chemical Society.

Generation of triplet eximers of 6-fluoro-7-piperazinylquinolone-3-carboxylic acids (FQs) have been detected in aqueous media using laser flash photolysis (LFP). These transient species (SS) are generated by self-quenching reactions of FQ triplet excited states such as pefloxacin (PFX), norfloxacin (NFX), the N-acetylated form of NFX (ANFX), and its methyl ester (EANFX) with their ground states. In this context, self-quenching rate constants in the range of (1-7) X 10(8) M-1 s(-1) were determined. The triplet excimers show transient absorption spectra with lambda(max) ca. 710 nm for SSNFX, 740 nm for SSPFX, and 620 nm for SSANFX and E-ANFX, which are red-shifted with respect to their predecessors triplet excited states. These excimers can be also observed in the presence of phosphate buffer (PB). Experiments performed with NFX and ANFX at different PB concentrations showed that deprotonation processes are not involved in the generation of SS. The triplet multiplicity of the FQ excimers was confirmed by energy transfer reactions with naproxen. The correlation between fluorescence, intersystem crossing, excimer and photodegradation quantum yields of (A)NFX indicated that FQ self-quenching reactions are mainly a deactivation pathway. On the other hand, generation of FQ radical anions absorbing at lambda(max) ca. 620 nm has been observed by an efficient electron transfer reaction from Tip to NFX, PFX, and ANFX (rate constants ca. 1 X 109 M-1 s(-1)).

Cinacalcet (CIN) is a calcimimetic drug, which contains a naphthalene chromophore and binds almost quantitatively to human serum albumin (HSA). In the present work, the excited states of CIN have been characterized in order to obtain relevant information about complexation of CIN with HSA. The fluorescence spectrum in acetonitrile, at lambda(exc) = 290 nm, displayed two bands with maxima at 332 and 439 nm, assigned to the monomer and exciplex emission. Upon protonation of the amino group, the exciplex band disappeared, with a concomitant increase of the monomer emission intensity. Time-resolved fluorescence evidenced an intramolecular dynamic quenching, attributed to exciplex formation and/or photoinduced electron transfer, in agreement with the favorable thermodynamics predicted by the Rehm-Weller equations. Diffusion controlled dynamic quenching of CINH(+) fluorescence by oxygen was observed. The emission properties in PBS were similar to those obtained for CINH(+) in acetonitrile. Laser flash photolysis (LFP) of CIN and CINH(+) in aCetonitrile/N(2), at lambda(exc) = 308 nm, gave rise to the naphthalene-like triplet excited states, with maxima at 420 nm and lifetimes of 4 and 7 mu s; they were efficiently quenched by oxygen. No significant singlet excited state interaction was observed in CINH(+)/HSA complexes, as revealed by the emission spectra, which were roughly explained taking into account the relative contributions of drug and protein in the absorption spectra. Upon LFP of the complexes, triplet excited states were generated; the decays monitored at 420 nm were satisfactorily fitted using a function containing two monoexponential terms, corresponding to a Short-lived (tau(1) = 8 mu s) and a long-lived (tau(2) = 37 mu s) component. This indicates that the drug is incorporated into two different binding sites of HSA. Despite the long triplet lifetimes of the CINH(+)/HSA complexes, the rate constant of quenching by oxygen was found to be 2 orders of magnitude

[EN] Hydrogen abstraction from the C-7 position of cholesterol (Ch) by triplet excited benzophenone (BZP) exhibits remarkable solvent-dependence in product studies. Kinetic measurements on the intramolecular version of the process in dyads containing covalently linked Ch and BZP units reveal important solvent effects and significant stereodifferentiation.

[EN] Three drugs containing the naphthalene (NP) chromophore, namely, naproxen (NPX), propranolol (PPN), and cinacalcet (CIN), but with different affinities toward serum albumins (SAs) and alpha-1-acid glycoproteins (AAGs) have been employed for the assessment of drug distribution in binary SA/AAG systems. These three drugs represent an appropriate choice for checking whether a methodology based on transient absorption spectroscopy of a given reporter can be employed for discrimination between different distribution patterns in multicompartmental biological media. Thus, upon laser flash photolysis (LFP) of NPX, PPN, and CIN in the presence or absence of proteins, the NP triplet excited state ((NP)-N-3*) at similar to 420 nm was always detected, although the kinetics of the decay traces was structure- and medium-dependent. In aerated PBS, only a very short triplet lifetime (tau(T)) was found (1-2 mu s). By contrast, in the presence of SAs, two longer triplet lifetimes (5-76 mu s) were observed, ascribed to (NP)-N-3* within site land site II. Upon binding to AAGs, only a long tau(T) (15-47 mu s) was found. When the two proteins were present simultaneously in the same media, fitting of the decay traces was clearly consistent with a distribution of the drug between the different biological compartments and the bulk solution, which correlates well with the known protein affinities of every drug. Experiments were performed in both human (HSA/HAAG) and bovine protein media (BSA/BAAG). The results showed that SAs are the major carriers for NPX; by contrast, PPN binds preferentially to AAGs. An intermediate situation was found for CIN, which presents comparable affinity for both proteins. The results obtained for the two enantiomers of each drug were very similar, although a small stereodifferentiation was observed between the triplet lifetimes in the protein binding sites.