In vitro studies on DNA-photosensitization by different drug stereoisomers.

The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated. The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis. Formation of single strand breaks was compared for each group of compounds. Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E. coli endonuclease III (revealing oxidized pyrimidines) and E. coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage. Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases. No pyrimidine dimers were observed. Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites. The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism. Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin. In the case of carprofen, a weak nucleoside degradation was obtained. The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin. In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer. However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

Photoreactivity of tiaprofenic acid and suprofen using pig skin as an ex vivo model.

The skin is repeatedly exposed to solar ultraviolet radiation. Photoreaction of drugs in the body may result in phototoxic or photoallergic side effects. Non-steroidal anti-inflammatory drugs, such as tiaprofenic acid (TPA) and the closely related isomer suprofen (SUP) are frequently associated with photosensitive disorders; they may mediate photosensitised damage to lipids, proteins and nucleic acids. Using ex vivo pig skin as a model, we investigated the photodegradation of TPA and SUP, and photobinding of these drugs to protein by means of HPLC analysis and drug-directed antibodies. Both with keratinocytes, which were first isolated from the pig skin and thereafter exposed to UVA and with keratinocytes which were isolated from pig skin after the skin was UVA exposed, time-dependent photodegradation of TPA and SUP was found, beside photoadduct formation to protein. The results of this work show that: (a) TPA and SUP were photodecomposed with similar efficiency; major photoproducts detected were decarboxytiaprofenic acid (DTPA) and decarboxysuprofen (DSUP), respectively. (b) Both drugs form photoadducts, as concluded from recognition by drug-specific antibodies. Pig skin appears to be a good model for studying the skin photosensitising potential of drugs.

Photobinding of ketoprofen in vitro and ex vivo.

Ketoprofen (KP), a non-steroidal anti-inflammatory drug of the 2-aryl propionic class, has been shown to produce photoallergic side effects as well as cutaneous photosensitizing properties that induce other phototoxic effects. In the present study we investigated photobinding of ketoprofen to both human serum albumin (HSA), a model protein, and to ex vivo pig skin and its photodegradation. Results demonstrate that photoadduct formation and photodegradation progressively increased with irradiation time where they reach a maximum. Maximum photobinding to the viable layer of the epidermis was about 7-8% of the initial radiolabelled KP added, in the region of 15-30 min UV irradiation. These results were comparable to in vitro results that were seen with photobinding of KP to HSA; in this case, the quantity of covalently bound material was approximately 10% of the initial, after a maximum of 18 min irradiation. It was found by HPLC analysis that the KP decrease is accompanied by an increase of the corresponding photoproduct, decarboxylated ketoprofen (DKP). The yield of DKP reaches a maximum at around 15 min. DKP appears to play an important role in vitro and ex vivo, being the major photoproduct and responsible for the photobinding process. Using micro-autoradiographical techniques we investigated the penetration and distribution of ketoprofen in ex vivo pig skin in greater detail. It was apparent that percutaneous absorption was taking place and that most of the ketoprofen was predominately localised in fibroblasts in the papillary dermis. No other specific localisation within the skin architecture was identified. Although there were differences in the quantities of bound ketoprofen within the different layers of the skin, these levels did not appear to correlate with irradiation time.

An In Vitro Approach to Drug Photoallergy: Use of Drug-directed Antibodies to Assess Photobinding of Non-steroidal Anti-inflammatories to Skin Cells.

Photobinding of drugs to biomolecules constitutes the early key event in the onset of photoallergy. This process generally involves excitation of the drug to an excited triplet state, which in turn can interact with cell constituents leading, in the case of proteins, to the formation of covalent photoadducts. The resulting photoantigens may trigger an immune response. In the present communication, we report the use of drug-directed antibodies to detect photoadduct formation in skin cells. This has been exemplified with tiaprofenic acid and suprofen as model compounds (two well known photoallergens) and human fibroblasts as representative skin cells. Upon irradiation of cells in the presence of these non-steroidal anti-inflammatories, time-dependent photoadduct formation was observed. This occurred predominantly at the cell membrane level. Most interestingly, the immunogenicity of cell photoadducts could be demonstrated by injection of Balb/c mouse fibroblasts into immunologically identical syngenic animals, where they triggered an immune response, as evidenced by the formation of specific antibodies and sensitized T-cells.

Photobinding of tiaprofenic acid and suprofen to proteins and cells: a combined study using radiolabeling, antibodies and laser flash photolysis of model bichromophores.

Drug photoallergy is a matter of current concern. It involves the formation of drug-protein photoadducts (photoantigens) that may ultimately trigger an immunological response. Tyrosine residues appear to be key binding sites in proteins. The present work has investigated the photobinding of tiaprofenic and (TPA) and the closely related isomer suprofen (SUP) to proteins and cells by means of radioactive labelling and drug-directed antibodies. To ascertain whether preassociation with the protein may play a role in photoreactivity, two model bichromophoric compounds (TPA-Tyr and SUP-Tyr) have been prepared and studied by laser flash photolysis. The results of this work show that (a) TPA and SUP photobind to proteins with similar efficiencies, (b) both drugs form photoadducts that share a basic common structure, as they are recognized by the same antibody and (c) drug-protein preassociation must play a key role in photoreactivity, as indicated by the dramatic decrease in the triplet state lifetimes of the model bichromophores compared to the parent drugs.

Drug-photosensitized protein modification: identification of the reactive sites and elucidation of the reaction mechanisms with tiaprofenic acid/albumin as model system.

Certain drugs can photosensitive the formation of protein modifications, which are thought to be responsible for the occurrence of photoallergy. In the present work, the UV irradiation of serum albumin in the presence of tiaprofenic acid has been studied as a model system for drug-photosensitized protein modifications. The photolysates evidenced that His, Tyr, and Trp are the reactive sites of the protein. The experimental results strongly suggest that formal hydrogen abstraction from the OH or NH groups of Tyr or Trp by the excited drug is the key photochemical process. Competition between cage escape and in cage recombination of the resulting radical pairs governs the final outcome: protein photo-cross-linking versus drug-protein adduct formation. These findings are highly relevant to understand the process of photohapten formation, the first event in the onset of photoallergy.

Mechanisms of photosensitization by drugs: Involvement of tyrosines in the photomodification of proteins mediated by tiaprofenic acid in vitro.

The photosensitizing potential of drugs must be related to their photoreactivity towards the target biomolecules. In this context, a representative photosensitizing drug (tiaprofenic acid) was co-irradiated with a model protein, bovine serum albumin (BSA). This led to a significant degree of protein crosslinking and to the formation of trace amounts of drug-BSA photoadducts. Amino acid analysis of the hydrolysed (HC1) protein showed that His and Tyr undergo a dramatic decrease (approx. 90%) as a consequence of drug-mediated photodynamic processes. When the drug was irradiated in the presence of the pure amino acids, extensive phototransformation of the latter was observed. Other photosensitizing drugs gave rise to similar processes when irradiated in the presence of BSA or the isolated amino acids. In conclusion, histidine and tyrosine appear to be key sites for the photosensitized damage to proteins. Photodegradation of the isolated amino acids in vitro may be an indicator of the photosensitizing potential of drugs.

[Mechanism of phototoxicity induced by drugs]. / Elucidación de los mecanismos de fototoxicidad inducida por fármacos.

First of all some general concepts are given on phototoxic activity of pharmaceutical products which full fill the structural characteristics required to decompose by light and to cause biological damage, either themselves, their photoproducts or the products of their metabolism. These considerations are important due to the fact that this field of research is fairly new. Next, a review is given on recent research carried out in this laboratory on the photochemistry and phototoxicity of fibric acid and their derivatives and finally a review is made as well on the photochemistry and phototoxicity of antibacterial quinolones. Mechanisms are postulated for the photochemical decomposition of the substances investigated and possible mechanism for the in vitro activity at cellular level are also presented.

Photodegradation and in vitro phototoxicity of the antidiabetic drug chlorpropamide.

Methanolic solutions of the phototoxic antidiabetic drug chlorpropamide (CAS 94-20-2, 1) are photolabile towards UVB light under aerobic conditions. Irradiation of 1 produces the formation of the stable compounds p-chlorobenzenesulfonamide (2), N-(p-chlorophenylsulfonyl)formamide (3) and the dimer 4. A radical intermediate was evidenced by thiobarbituric acid that was used as a radical sonde, as well as by the dimerization of cysteine. The compound 1 showed moderate lytic activity upon the photohemolysis in vitro test on human erythrocytes which was increased with the addition of traces of its aggregate excipient. Inhibition of this process on addition of reduced glutathione (GSH), superoxide dismutase (SOD) or ascorbic acid suggests the involvement of radicals and superoxide ion in the photohemolysis process. The absence of inhibition with 1,4-diazabicyclo[2.2.2]octane (DABCO) and sodium azide (NaN3), and the lack of formation of singlet oxygen during the photolysis (confirmed with 2,5-dimethylfuran) rule out the possibility of participation of 1O2 in this process. Glutathione depletion was also observed. No photohemolysis was detected in the presence of the isolated photoproduct.

Photodegradation of benzydamine: phototoxicity of an isolated photoproduct on erythrocytes.

Benzydamine hydrochloride (Tantum, 1) is a photoallergic and phototoxic anti-inflammatory and analgesic agent. This drug is photolabile under aerobic and anaerobic conditions. Irradiation of a methanol solution of benzydamine under oxygen or argon at 300 nm affords 5-hydroxybenzydamine (2) and 2-beta-dimethylaminopropyl-1-benzylindalolin-3-one (3) as the main isolated and spectroscopically identified photoproducts. A radical intermediate was evidenced by thiobarbituric acid that was used as a radical sonde, as well as by the dimerization of cysteine. Erythrocyte lysis photosensitized by 1, 2, and 3 was investigated.

Photoproducts of benzydamine and azapropazone: demonstration of their phototoxicity in vitro.

Red blood cell lysis, photosensitized by the products of the aerobic photolysis of benzydamine (1) and azapropazone (4), was investigated. Irradiation of a methanol solution of 1 and 4 under oxygen produces the photoproducts 3-hydroxy-benzydamine, (2), 2-(3-dimethylaminopropyl)-1-benzylindazolin-3-one (3) and 3-dimethylamino-7-methyl-1,2,4-benzotriazine (5). The mechanism of the photodegradation of 1 was examined. Photoproducts 3 and 5 produce singlet oxygen as demonstrated by trapping with 2,5-dimethylfuran. The photohemolysis rate for the photoproducts 3 and 5 was enhanced by deuterium oxide and oxygen. No change was observed in the presence of reduced glutathione. The photohemolysis rate was low under anaerobic conditions.