Nsaids

Benoxaprofen, Naproxen, Piroxicam, Ketoprofen, Carprofen, Suprofen, Tiaprofenic Acid, Butibufen, Fenbufen, Tolmetin, Benorilate,

Antimicotics

Griseofulvine

Topical bacteriostatics

Bithionol, Phentichlor, Esachlorophen

Antimalarics

Quinine, Chloroquine, Hydroxychloroquine

Antiangine

Nifepidine

Immunosuppressants

Azothiaprine

Antilipemics

Fenofibrate

We are particularly interested in two largely used families of drugs, antibacterial fluoroquinolones and NSAIDs. The former is a modern class of antibiotics, continuously under development with the aim of improving their pharmacological activity and, in the meanwhile, decreasing their (photo) toxicity.6"5 The latter represents the main therapeutic agent for controlling the pain and the inflammation of rheumatic diseases.16 Many of them are known to cause both phototoxic and photoallergic reactions. A screening of the in vivo and in vitro photosensitizing activity of many NSAIDs was achieved by several techniques such as the mouse tail technique,4 photohemolysis3 and the photo-basophil-histamine-release test.17

As previously outlined, naturally incident light absorption by drugs is directly related to their chemical structure and, if sufficient concentrations are reached in a particular biological compartment, a photodegradation after radiation absorption can occur.18 This can lead to the formation of noxious transient or stable species, such as free radicals and photoproducts, and/or the promotion of energy transfer to form singlet oxygen (even if this last mechanism is co-operative with the first). Thus, it can be easily explained why ibuprofen, for example, is not phototoxic: although it can form toxic photoproducts when UVB irradiated;1 this happens for short wavelengths which are not able to penetrate skin.16 On the contrary, indomethacin absorbs above 310 nm, but it is very photostable, so it cannot start a photosensitization process related to its photodegradation. 0

A molecular mechanism of photosensitization induced by drugs can proceed through several pathways, which can be divided into two different types of mechanisms: photosensitized reactions via radicals (type I) and photosensitized reactions via singlet oxygen ('02), (type II) (Scheme 1).

Type I mechanism. This mechanism can be further distinguished whether the reaction occurs in the presence or in the absence of oxygen.

Reactions in the presence of oxygen. In this case there are four possible pathways:

1. An interaction between the excited sensitizer and oxygen occurs with formation of a charge transfer complex. This complex can dissociate in polar solvents with formation of the superoxide anion *02". This process is in general not very efficient because direct interaction between sensitizer and oxygen more likely leads to energy transfer with formation of singlet oxygen.

2. An interaction of the excited sensitizer (A*) with a substrate (B) can occur, with subsequent electron release or capture (depending on the redox potential of the A*/B system) and formation of anionic radicals which, reacting with oxygen, produce *02~.

3. An interaction between the sensitizer and the substrate can lead to radicals (for example via hydrogen abstraction), which can react with oxygen, thus generating peroxides.

4. The excited sensitizer dissociates generating radicals. These can in turn transform the substrate into a radical or be oxidized by oxygen.

In reactions 1 and 2, superoxide anion formation occurs. This species, because of its instability, is highly reactive and noxious and can decay, promoting oxidation, in four ways:21

• Hydrogen abstraction with formation of hydroperoxide HO2"

• Electron capture with formation of 022" and hence H202

• Electron release in redox reaction e.g. with Fe2+ in the Haber-Weiss cycle22

• Dismutation with formation of lC>2 and H2O2

Scheme 1 Molecular mechanism of photosensitization.

Reactions in the absence of oxygen. By using deaerated media in order to avoid the oxygen action, thus mimicking processes occurring in anaerobiosis, we can observe four typical pathways:

1. Redox interaction between sensitizer and substrate with subsequent formation of radical anions.

2. Hydrogen abstraction from the substrate with formation of radicals.

3. Electron release in an aqueous medium.

4. Photoaddition (as in the case of furocumarines with DNA).23 Type II mechanism. Once formed, singlet oxygen can decay by:

1. Emission of phosphorescence (Xem = 1.27 p.m): however, this process has a low quantum yield.

2. Non-radiative deactivation by collisions with solvent.

3. Physical quenching by quenchers (Q) which can occur by formation of charge transfer complexes or by energy transfer to Q; in the latter case, an efficient intersystem crossing process is necessary, promoting decay of 3Q* before this one is oxidized by '02.

4. Photooxidation of a substrate. This includes a) 1-2 and 1-4 addition to unsaturated compounds with formation of peroxides24'25 and b) reactions with heteroatom-containing compounds.

3 MOLECULAR TARGETS OF PHOTOSENSITIZATION 3.1 Membrane Targets

Whatever the photodamaging species (stable or transient) involved in the sensitization is, cell membrane is primarily involved as regards the chemical alterations of its principal constituents: lipid and proteins.26

3.1.1 Erythrocyte hemolysis. A first check of the photodamaging activity can be obtained through irradiation of aerated, deaerated and oxygen saturated samples of red blood cell (RBC) suspension containing the sensitizer and measure of the absorbance decrease at 650 nm, where the optical density is proportional to the number of intact RBCs27 (Figure 1). The study of the rate of delayed hemolysis as a function of the time of irradiation, of the sensitizer and oxygen concentrations and of the presence of antioxidants, free radical scavengers, singlet oxygen quenchers, and D2O (causing an increase of the lifetime of 02), provides information about the mechanism.28'29 These data, together with the elucidation of the photodegradation pathway of the drug (with the isolation and characterization of potentially toxic photoproducts), give a first picture of the photosensitization mechanism in membrane.

3.1.2 Lipid peroxidation. A simplified system used to discriminate target molecules on membrane is given by artificial bilayers (unilamellar liposomes of phosphatidylcholine from egg yolk prepared with the solvent injection technique)30 which permit the evaluation of the photosensitized damage on the lipid portion. The integrity of the liposome membrane can be measured by efflux of trapped markers, such as glucose-6-phosphate (determined spectrophotometrically at 340 nm), after addition of glucose-6-phosphate dehydrogenase and NADP. Photodynamic lipid peroxidation has been extensively studied and an excellent source of information can be found in a work of Girotti.32 The formation of peroxides in the irradiated samples can be followed through the reaction with thiobarbituric acid (TBA),33'34 also if this method is subjected to limits, particularly when cells and tissues are analysed. The iodometric approach, also if more complicated and subjected to interference with O2, allows determination of total peroxides, including those derived from cholesterol.35 Moreover, the oxidation products of cholesterol by 1C>2 and radical attack are different29 and their characterization provides evidence for the participation of these two species in the photosensitization mechanism.

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