La photosynthèse artificielle est considérée comme étant un atout capable de fournir des carburants alternatifs et renouvelables par conversion et stockage de l'énergie solaire. Une approche prometteuse consiste en un développement de photo-catalyseurs moléculaires inspirés par des enzymes photosynthétiques naturelles. La première partie de cette thèse concerne les modèles artificiels du photosystème II (qui catalyse l'oxydation d'eau), composé d'un chromophore et d’un relais d’électrons comme équivalent synthétique correspondant à l'ensemble P680-TyrZ/His190 du photosystème II. Trois complexes ruthénium polypyridyl - imidazole - phénol avec un groupe méthylique à différentes positions sur l'anneau phénolique (Ru-xMe) ont été synthétisés et caractérisés par des méthodes électrochimiques et photophysiques. L’augmentation, comparée aux complexes précédents, du potentiel redox des groupes phénols (0.20 V->0.9 V par rapport à l’électrode de ferrocène) rend leur fonction de relais d’électron dans un système photocatalytique pour l'oxydation d'eau thermodynamiquement possible. Des études d’absorption transitoire ont révélé que le transfert d’électron intramoléculaire est rapide (5-10 µs dans solvant aprotique et < 100 ns dans l'eau) malgré la faible force motrice, mettant en evidence l'importance de la liaison hydrogène entre le phénol et le groupe imidazole. Les légères différences entre les trois complexes Ru-xMe ainsi que l’étude de l'effet de bases externes nous ont permis d’établir un mécanisme dans laquelle l'imidazole est impliqué dans une réaction de transfert de proton en cascade. L'acceptation du proton phénolique durant l'oxydation du ligand rend son deuxième site azote plus acide et seulement la déprotonation de ce dernier bascule l’équilibre réactionnel complétement vers l'oxydation du ligand. La deuxième partie de cette thèse consiste en la synthèse d’un complexe chromophore-tryptophane en utilisant une approche de chimie dite « click ». On a montré que l'oxydation, induite par la lumière, du Trp au sein du complexe Ru-tryptophane suit un mécanisme ETPT. Selon le pH, les radicaux du tryptophane (Trp• ou TrpH•⁺) ont été détectés et les mesures spectrales à différents temps ont montrés la transition entre les deux formes radicalaires. La déprotonation du radical dépend de la concentration d'eau assurant la fonction d’accepteur de proton. La dernière partie de la thèse concerne nos efforts à lier, par une liaison covalente, une unité catalytique au module de chromophore- relais électronique caractérisé précédemment. L'approche de chimie « click » n’était pas efficace pour l’obtention de l’assemblage photocatalytique final. Donc, l'activation biomoléculaire d'un catalyseur Mn salen a été effectuée et la formation de l’espèce Mn(IV) a été observée. Etant une étape vers l'utilisation de ces types de photocatalyseurs dans une cellule photoélectrochimique, un chromophore [Ru(bpy)₃]²⁺ avec des groupes d’ancrage phosphonate a été synthétisé (Ru-phosphonate) et greffé sur la surface méso-poreuses d'un semi-conducteur de TiO₂ pour effectuer des mesures du photocourant. / Artificial photosynthesis is often considered to have great potential to provide alternative, renewable fuels by harvesting, conversion and storage of solar energy. One promising approach is the development of modular molecular photocatalysts inspired by natural photosynthetic enzymes. The first part of this thesis deals with artificial mimics of the water oxidizing photosystem II composed of a chromophore and an electron relay as synthetic counterpart of the P680-TyrZ/His190 ensemble of photosystem II. Three ruthenium polypyridyl – imidazole - phenol complexes with varying position of a methyl group on the phenol ring (Ru-xMe) were synthesized and characterized by electrochemical and photophysical methods. As an improvement compared to earlier complexes the increased redox potential (~0.9 V vs. Ferrocene) of the phenol groups makes their function as an electron relay in a photocatalytic system for water oxidation thermodynamically possible. Time-resolved absorption studies revealed fast intramolecular electron transfer (<5-10 µs in aprotic solvent and <100 ns in water) despite the low driving force and the importance of the hydrogen bond between the phenol and the imidazole group was put in evidence. Slight differences between the three Ru-xMe complexes and investigation of the effect of external bases allowed to derive a mechanistic picture in which the imidazole is involved in a “proton domino” reaction. Accepting the phenolic proton upon ligand oxidation (within the H-bond) renders its second nitrogen site more acidic and only deprotonation of this site pulls the overall equilibrium completely towards oxidation of the ligand. Another part of this thesis comprises a chromophore-tryptophan construct synthesized using a click chemistry approach. Light-induced oxidation of Trp in this Ru-tryptophan complex was shown to follow ETPT mechanism. Depending on the pH conditions tryptophan radicals, either Trp• or TrpH•⁺ were detected and spectral measurement at different time showed the transition between the two forms. Deprotonation of the radical was dependent on the concentration of water as proton acceptor. Later part of the thesis deals with efforts to covalently bind a catalytic unit to the previously characterized chromophore-electron relay module. The click chemistry approach was not successful to obtain the final photocatalytic assembly. Therefore bimolecular activation of a Mn salen catalyst was performed and formation of Mn(IV) species was observed. As a step towards utilization of these types of photocatalysts in a photoelectrochemical cell a [Ru(bpy)₃]²⁺ chromophore with phosphonate anchoring groups (Ru-Phosphonate) was synthesized and grafted on the surface of a TiO₂ mesoporous semiconductor surface anode to perform photocurrent measurements.

Ao ser excitado do estado fundamental para o estado singlete excitado, o Pka do equilíbrio ácido-base das antocianinas apresenta um deslocamento de ca. 4 para ca. -1. Esta característica de superfotoácido, aliado à técnica de fotólise por pulso de laser, permite a perturbação da posição do equilíbrio ácido-base no estado fundamental. O acompanhamento da cinética de reestabelecimento do equilíbrio inicial, existente antes do pulso do laser, permite a determinação das constantes de desprotonação kd e protonação kp no estado fundamental. Este método foi aplicado para a determinação das constantes de protonação e desprotonação das seguintes antocianinas sintéticas e naturais: 4-metil-7-hidroxiflavílio (HMF), 4\', 7-dihidroxiflavílio (DHF), malvidina-3-glucosídeo (Oenina), malvidina-3,5-diglucosídeo (Malvina), pelargonidina-3,5-diglucosídeo (Pelargonina) e cianidina-3 ,5-diglucosídeo (Cianina). Assim, as constantes de desprotonação do estado fundamental são: kd = 1,4 x 106 s-1 (HMF), 3,1 x 106 s-1 (DHF), 5, 1 x 106 s-1 (Oenina), 3,8 x 106 s-1 (Malvina), 1,3 x 106 s-1 (Pelargonina) e 1,8 x 106 s-1 (Cianina). As constantes de protonação do estado fundamental são:kp = 3,56 x 1010 L mol-1 s-1 (HMF), 3,06 x 1010 L mol-1s-1 (DHF), 2,51 x 1010 L mol-1s-1 (Oenina), 2,9 x 1010 L mol-1s-1 (Malvina), 3,6 x 1010 L mol-1s-1 (Pelargonina) e 2,2 x 1010 L mol-1s-1 (Cianina). Nosso método é a única técnica que permite a determinação direta destas constantes. Este método também permite a determinação de kd e kp em meios micro-heterogêneos. Através de medidas de kd , foi possível provar que ocorre a estabilização do cátion flavílio de HMF em micelas aniônicas de SOS (dodecilsulfato de sódio). Em micelas não iônicas e catiônicas, por outro lado, ocorre a desestabilização do cátion flavílio, que se manifesta através do aparecimento de reatividade ausente em solução aquosa. Os resultados da aplicação deste método revelam ainda que o principal processo de dissipação da energia da luz, absorvida pelas antocianinas, é a transferência de próton no estado excitado e não a fluorescência. / Upon excitation from the ground state to the first excited singlet state, the pKa of the acid-base equilibrium of anthocyanins exhibits a large shift, from ca. 4 to ca. -1. This characteristic of super-photoacidity, coupled with the technique of laser flash photolysis, can be employed to perturb the position of the acid-base equilibrium in the ground state. Monitoring the kinetics of relaxation back to the initial equilibrium position that existed prior to the laser pulse permits determination of the rate constants for protonation (kp) and deprotonation (kd) in the ground state. This method was applied to the following natural and synthetic anthocyanins: 4-methyl-7-hydroxyflavylium (HMF); 4\', 7-dihydroxyflavylium (DHF); malvidine-3-glucoside (Oenin); malvidine-3,5-diglucoside (Malvin); Pelargonidine-3,5-diglucoside (Pelargonin); and. cyanidine-3,5-diglucoside (Cyanin). Thus, the ground state deprotonation rate constants (kd) for the acid form were found to be: 1.4 x 106 s-1 (HMF); 3.1 x 106 s-1 (DHF); 5.1 x 106 s-1 (Oenin); 3.8 x 106 s-1 (Malvin); 1.3 x 106 s-1 (Pelargonin); 1.8 x 106 s-1 (Cyanin). The corresponding rate constants for protonation of the ground state of the base (kp) were: 3.6 x 1010 M-1s-1 (HMF); 3.1 x 1010 M-1s-1 (DHF); 2.5 x 1010 M-1s-1 (Oenin); 2.9 x 1010M-1s-1 (Malvin); 3.6 x 1010 M-1s-1 (Pelargonin); 2.2 x 1010 s-1 (Cyanin). Our method is currently the only one that permits direct determination of these rate constants. This method also allows the determination of kpand kd in microheterogeneous media. Thus, employing measurements of kd, it was possible to demonstrate that anionic SDS (sodium dodecyl sulfate) micelles stabilize the flavylium cation form of HMF. In cationic and nonionic micelles, however, the flavylium cation is destabilized, which manifests itself via the appearance of reactivity that is absent in aqueous solution in the absence of these surfactants. Finally, the results of the application of this method show that the primary energy wasting process for radiant energy absorbed by anthocyanins is excited state proton transfer rather than fluorescence.

Photosensitizing effects of xenobiotics are of increasing concern in public health since modern lifestyle often associates sunlight exposure with the presence of chemical substances in the skin. An important number of chemicals like perfumes, sunscreen components, or therapeutic agents have been reported as photosensitizers. In this context, a considerable effort has been made to design a model system for photosafety assessment. Indeed, screening for phototoxicity is necessary at the early phase of drug discovery process, even before introducing drugs and chemicals into clinical therapy, to prevent undesired photoreactions in humans. In the case of new pharmaceuticals, their phototoxic potential has to be tested when they absorb in the regions corresponding to the solar spectrum, that is, for wavelengths >290 nm. So, there is an obvious need for a screening strategy based on in vitro experiments. The goal of the present thesis was the photochemical study of different photoactive drugs to investigate the key molecular aspects responsible for their photosensitivity side effects. In a first stage, rosuvastatin was considered in chapter 3 as representative compound of the statin family. This lipid-lowering drug, also known as “superstatin”, contains a 2-vinylbiphenyl-like moiety and has been previously described to decompose under solar irradiation, yielding stable dihydrophenanthrene analogues. During photophysical characterization of rosuvastatin, only a long-lived transient at ca. 550 nm was observed and assigned to the primary photocyclization intermediate. Thus, the absence of detectable triplet-triplet absorption and the low yield of fluorescence ruled out the role of the parent drug as an efficient sensitizer. In this context, the attention was placed on the rosuvastatin main photoproduct (ppRSV). Indeed, the photobehavior of this dihydrophenanthrene-like compound presented the essential components needed for an efficient biomolecule photosensitizer i.e. (i) a high intersystem crossing quantum yield (ΦISC =0.8), (ii) a triplet excited state energy of ca. 67 kcal mol−1 , and (iii) a quantum yield of singlet oxygen formation (Φ∆) of 0.3. Furthermore, laser flash photolysis studies revealed a triplet-triplet energy transfer from the triplet excited state of ppRSV to thymidine, leading to the formation of cyclobutane thymidine dimers, an important type of DNA lesion. Finally, tryptophan was used as a probe to investigate the Type I and/or Type II character of ppRSV-mediated oxidation. In this way, both an electron transfer process giving rise to the tryptophanyl radical and a singlet oxygen mediated oxidation were observed. On the basis of the obtained results, rosuvastatin, through its major photoproduct ppRSV, should be considered as a potential sensitizer. Then, itraconazole (ITZ), a broad-spectrum antifungal agent, was chosen as main character of chapter 4. Its photochemical properties were investigated in connection with its reported skin photosensitivity disorders. Steady state photolysis, fluorescence and phosphorescence experiments were performed to understand ITZ photoreactivity in biological media. The drug is unstable under UVB irradiation, suffering a primary dehalogenation of the 2,4-dichlorophenyl moiety that occurs mainly at the ortho-position. In poorly H-donating solvents, as acetonitrile, the major photoproduct arises from intramolecular attack of the initially generated aryl radical to the triazole ring. In addition, reduced compounds resulting from homolytic cleavage of the C-Cl bond in ortho or para positions and subsequent Habstraction from the medium are obtained to a lesser extent. In good H-donating solvents, such as ethanol, the main photoproducts are formed by reductive dehalogenation. Furthermore, irradiation of a model dyad containing a tryptophan unit and the reactive 2,4-dichlorophenyl moiety of itraconazole leads to formation of a new covalent link between these two substructures revealing that homolysis of the C-Cl bond of ITZ can result in alkylation of reactive amino acid residues of proteins, leading to formation of covalent photoadducts. Therefore, it has been established that the key process in the photosensitization by itraconazole is cleavage of the carbon-halogen bond, which leads to aryl radicals and chlorine atoms. These highly reactive species might be responsible for extensive free radical-mediated biological damage, including lipid peroxidation or photobinding to proteins. In chapter 5, photobehavior of imatinib (IMT) was addressed. This is a promising tyrosine kinase inhibitor used in the treatment of some types of human cancer, which constitutes a successful example of rational drug design based on the optimization of the chemical structure to reach an improved pharmacological activity. Cutaneous reactions, such as increased photosensitivity or pseudoporphyria, are among the most common nonhematological IMT side effects; however, the molecular bases of these clinical observations have not been unveiled yet. Thus, to gain insight into the IMT photosensitizing properties, its photobehavior was studied together with that of its potentially photoactive anilino-pyrimidine and pyridyl-pyrimidine fragments. In this context, steady-state and time resolved fluorescence, as well as laser flash photolysis experiments were run, and the DNA photosensitization potential was investigated by means of single strand breaks detection using agarose gel electrophoresis. The obtained results revealed that the drug itself and its anilino-pyrimidine fragment are not DNA-photosensitizers. By contrast, the pyridyl-pyrimidine substructure displayed a marked photogenotoxic potential, which was associated with the generation of a long-lived triplet excited state. Interestingly, this reactive species was efficiently quenched by benzanilide, another molecular fragment of IMT. Clearly, integration of the photoactive pyridyl-pyrimidine moiety in a more complex structure strongly modifies its photobehavior, which in this case is fortunate as it leads to an improved toxicological profile. Thus, on the bases of the experimental results, direct in vivo photosensitization by IMT seems unlikely. Instead, the reported photosensitivity disorders could be related to indirect processes, such as the previously suggested impairment of melanogenesis or the accumulation of endogenous porphyrins. Finally, a possible source of errors in the TEMPO/EPR method for singlet oxygen detection was analyzed. For many biological and biomedical studies, it is essential to detect the production of 1O2 and to quantify its production yield. Among the available methods, detection of the characteristic 1270 nm phosphorescence of singlet oxygen by time-resolved near infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with trapping. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6- tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been largely employed, it can produce misleading data. This was demonstrated by the present study, where the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method were compared for a set of well-known photosensitizers. The results revealed that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizers were efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP+• radical cation, followed by deprotonation and reaction with molecular oxygen gives rise to a EPR detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological and medical studies. / Nardi, G. (2014). MECHANISTIC STUDIES ON THE PHOTOTOXICITY OF ROSUVASTATIN, ITRACONAZOLE AND IMATINIB [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535

[ES] En la presente tesis doctoral se ha desarrollado una metodología multidisciplinar para la evaluación del potencial fotoquímico y fotobiológico in vitro de los fármacos inhibidores de la poli (ADP-ribosa) polimerasa (PARP), combinando tanto estudios fotofísicos y de caracterización como estudios fotobiológicos en biomoléculas modelo y en cultivos celulares, concretamente en queratinocitos humanos inmortalizados (HaCaT). Así, el objetivo general es investigar si estos fármacos, en combinación con la radiación solar, son capaces de inducir reacciones de fotosensibilidad y, por tanto, poder alertar a los oncólogos de estos hallazgos para que puedan indicar pautas de fotoprotección adecuadas a sus pacientes, y así prevenir estos efectos indeseados. Esta tesis se divide en un total de 7 capítulos. En el primero (introducción), se recogen los hitos más importantes relacionados con los inhibidores de la PARP, así como un resumen acerca de las reacciones de fotosensibilización y de los mecanismos químicos y biológicos involucrados en las mismas. Tras una exhaustiva búsqueda bibliográfica acerca de estos fármacos, se hizo un primer cribado de los cinco de la tercera y última generación (rucaparib, talazoparib, niraparib, olaparib y veliparib), identificando así tres de ellos como posibles agentes fotosensibilizantes: rucaparib, talazoparib y niraparib, cuyo estudio se describe en profundidad en los capítulos 4, 5 y 6 de la tesis. En el capítulo 4 se evalúa la fototoxicidad del rucaparib (RCP), un fármaco que, a pesar de tener un cromóforo indol en su estructura química, posee un desplazamiento batocrómico hacia la región UVA de la luz solar, siendo así capaz de inducir reacciones de fotosensibilidad. Se observó mediante experimentos de fluorescencia y fotólisis de destello láser que, tras absorción de luz en esta región del espectro, tiene lugar la generación de especies reactivas de oxígeno (ROS), que podrían ser las responsables de generar un daño fotooxidativo hacia el ADN celular y a las proteínas transmembranales, originando como resultado una foto(geno)toxicidad. Además, se estableció que el mecanismo de la muerte celular fotoinducida por RCP es por apoptosis. El capítulo 5 se centra en la evaluación del perfil de fotoseguridad de talazoparib (TLZ), el cual, tras su irradiación con luz UVA da lugar a un fotoproducto que, tras su aislamiento y caracterización, resultó no presentar fotorreactividad. Este fotoproducto resultó además ser el responsable de reducir significativamente el potencial fototóxico del fármaco original, ya que no se detectaron especies transitorias tras su irradiación con luz UVA. Además, para el TLZ se reveló la producción in vitro de ROS, especies que podrían ser las responsables de generar un daño fotooxidativo tanto hacia el ADN celular como a las proteínas de membrana, originando como resultado una foto(geno)toxicidad. En el capítulo 6 se evalúa la fototoxicidad del niraparib (NRP) y su principal metabolito (N-M1). A pesar de que ambos compuestos resultaron ser fototóxicos, dicha fototoxicidad no afectó igual a las principales dianas celulares, ya que el daño fotooxidativo de NRP se observó en lípidos, en proteínas transmembranales y en el ADN celular, mientras que en N-M1 se aprecia fundamentalmente en las proteínas de membrana. Además, la toxicidad observada del NRP en células de cáncer de ovario (A2780 y A2780cis) indicó que este fármaco podría contemplarse como candidato para su uso en un futuro en la terapia fotodinámica para el tratamiento del cáncer de ovario. Finalmente, con todo ello se concluye que estos fármacos de la familia de los inhibidores de la PARP (RCP, TLZ y NRP) son capaces de inducir reacciones de fotosensibilidad, con valores de factor de fotoirritación (PIF) de 41, 7 y 46, respectivamente. / [CAT] En la present tesi doctoral s'ha desenvolupat una metodologia multidisciplinària per a l'avaluació del potencial fotoquímic i fotobiològic in vitro dels fàrmacs inhibidors de la poli(ADP-ribosa) polimerasa (PARP), combinant tant estudis fotofísics i de caracterització com estudis fotobiològics en biomolècules model i en cultius cel·lulars, concretament en queratinòcits humans immortalitzats (HaCaT). Així, l'objectiu general és investigar si aquests fàrmacs, en combinació amb la radiació solar, són capaços d'induir reaccions de fotosensibilitat i, per tant, poder alertar als oncòlegs d'aquestes fites perquè puguen indicar pautes de fotoprotecció adequades als seus pacients, i així previndre aquests efectes no desitjats. Aquesta tesi es divideix en un total de 7 capítols. En el primer (introducció), es recullen les fites més importants relacionades amb els inhibidors de la PARP, així com un resum sobre les reaccions de fotosensibilització i dels mecanismes químics i biològics involucrats en aquestes. Després d'una exhaustiva cerca bibliogràfica sobre aquests fàrmacs, es va fer un primer estudi dels cinc de la tercera i última generació (rucaparib, talazoparib, niraparib, olaparib i veliparib), identificant així tres d'ells com a possibles agents fotosensibilizants: rucaparib, talazoparib i niraparib, l'estudi del qual es descriu en profunditat en els capítols 4, 5 i 6 de la tesi. En el capítol 4 s'avalua la fototoxicitat del rucaparib (RCP), un fàrmac que, malgrat tindre un cromòfor indole en la seua estructura química, posseeix un desplaçament batocròmic cap a la regió UVA de la llum solar, sent així capaç d'induir reaccions de fotosensibilitat. Mitjançant experiments de fluorescència i fotòlisi de flaix làser es va observar que, després de l¿absorció de llum en aquesta regió de l'espectre, té lloc la generació d'espècies reactives d'oxigen (ROS), que podrien ser les responsables de generar un dany fotooxidatiu cap a l'ADN cel·lular i a les proteïnes transmembranals, originant com a resultat una foto(geno)toxicitat. A més, es va establir que el mecanisme de la mort cel·lular fotoinduïda per RCP és per apoptosi. El capítol 5 se centra en l'avaluació del perfil de fotoseguretat de talazoparib (TLZ), el qual, després de la seua irradiació amb llum UVA dona lloc a un fotoproducte que, després del seu aïllament i caracterització, va resultar no presentar fotorreactivitat ninguna. Aquest fotoproducte va resultar a més ser el responsable de reduir significativament el potencial fototòxic del fàrmac original, ja que no es van detectar espècies transitòries després de la seua irradiació amb llum UVA. A més, per al TLZ es va revelar la producció in vitro de ROS, espècies que podrien ser les responsables de generar un dany fotooxidatiu tant cap a l'ADN cel·lular com a les proteïnes de membrana, originant com a resultat una foto(geno)toxicitat. En el capítol 6 s'avalua la fototoxicitat del niraparib (*NRP) i el seu principal metabòlit (N-M1). A pesar que tots dos compostos van resultar ser fototòxics, aquesta fototoxicitat no va afectar per igual a les principals dianes cel·lulars, ja que el dany fotooxidatiu de NRP es va observar en lípids, en proteïnes i en l'ADN cel·lular, mentre que en N-M1 s'aprecia fonamentalment en les proteïnes de membrana. A més, la toxicitat observada del NRP en cèl·lules de càncer d'ovari (A2780 i A2780cis) va indicar que aquest fàrmac podria contemplar-se com a candidat per al seu ús en un futur en la teràpia fotodinàmica per al tractament del càncer d'ovari. Finalment, amb tot això es conclou que aquests fàrmacs de la família dels inhibidors de la PARP (RCP, TLZ i NRP) són capaços d'induir reaccions de fotosensibilitat, amb valors de factor de fotoirritació (PIF) de 41, 7 i 46, respectivament. / [EN] In this doctoral thesis, a multidisciplinary methodology has been performed for the evaluation of the in vitro photochemical and photobiological potential of the poly (ADP-ribose) polymerase (PARP) inhibitors, combining photophysical and photobiological studies in biomolecules and cell cultures, specifically in human immortalized keratinocytes (HaCaT). Thus, the general objective is to investigate if these drugs, in combination with solar radiation, can trigger photosensitivity reactions; this will allow oncologists to indicate appropriate photoprotective guidelines to their patients in order to prevent these undesirable effects. This thesis is divided into 7 chapters. The first one (introduction) contains the essential issues concerning PARP inhibitors, a background of the photosensitization reactions and a description of the involved chemical and biological mechanisms. After in-depth bibliographical research about these drugs, a screening of the five PARP inhibitors (rucaparib, talazoparib, niraparib, olaparib and veliparib) of the third and last generation was carried out, identifying three of them as possible photosensitizing drugs: rucaparib, talazoparib and niraparib. Their behaviour is thoroughly described in chapters 4, 5 and 6. Chapter 4 evaluates the phototoxicity of rucaparib (RCP), a drug that possess an indole chromophore in its chemical structure but displays a bathochromic shift towards the UVA region of sunlight, which makes it able to induce photosensitivity reactions. Generation of reactive oxygen species (ROS) after UVA light absorption was detected by fluorescence and laser flash photolysis experiments. These species could generate photooxidative damage to cellular DNA and transmembrane proteins, resulting in photo(geno)toxicity. In addition, it was established that the mechanism of RCP photoinduced cell death is by apoptosis. Chapter 5 focuses on the photosafety profile of talazoparib (TLZ), a compound that, after UVA irradiation, gives rise to a photooxidized product; after isolation and characterization, the photoproduct did not display any photoreactivity, and no transient species were detected after UVA light irradiation. Therefore, it was responsible for the significantly reduced phototoxic potential of the parent drug. Additionally, for TLZ, in vitro ROS production was detected. These species could lead to photooxidative damage to both cellular DNA and membrane proteins, resulting in photo(geno)toxicity. Chapter 6 deals with the phototoxicity of niraparib (NRP) and its main metabolite (N-M1). Although both compounds are phototoxic, the phototoxicity was found to be different for the main cellular targets: thus, the photooxidative damage of NRP was noticed in lipids, transmembrane proteins and cellular DNA, whereas in N-M1 it was mainly observed in membrane proteins. In addition, NRP was cytotoxic in ovarian cancer cells (A2780 and A2780cis), indicating that this drug could be considered as a future candidate for its use in photodynamic therapy to treat ovarian cancer. Finally, it can be concluded that these PARP inhibitors (RCP, TLZ and NRP) are able to induce photosensitivity disorders, with PIF values of 41, 7 and 46, respectively. / We thank the Agencia Estatal de Investigación (PID2020-115010RB-I00/AEI/10.13039/501100011033 and the Generalitat Valenciana (CIAICO/2021/061 and ACIF/2018/153 fellowship for A. M.-P. / Mateos Pujante, A. (2023). Evaluación del potencial fotoquímico y fotobiológico de los inhibidores de la poli (ADP-ribosa) polimerasa [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196092

[ES] En las últimas décadas, la aprobación de los inhibidores de la tirosina quinasa (del inglés TKI) como una nueva clase de terapia dirigida ha mejorado la calidad de vida y las tasas de supervivencia de los pacientes con cáncer. Sin embargo, los efectos adversos asociados a éstos, como son las reacciones cutáneas, siguen siendo un desafío para la terapia controlada. De acuerdo con anteriores estudios fotofísicos y fotobiológicos de TKI realizados por el grupo de investigación, esta tesis sigue un enfoque multidisciplinar para investigar nuevos fármacos fotoactivos dentro de esta familia. En la etapa inicial, se seleccionaron cuatro TKI, gefitinib, axitinib, dasatinib y avapritinib, por su capacidad para absorber luz UVA y por su potencial fototóxico. Los estudios, tanto fotofísicos como fotobiológicos, se llevaron a cabo en estos fármacos. Gefitinib (GFT), un TKI con un cromóforo quinazolina, reveló cambios significativos en la fototoxicidad debido a modificaciones metabólicas en su estructura. Así, la desalquilación de la cadena lateral propoxi-morfolina (DMOR-GFT) presentó el valor más alto de factor de fotoirritación (PIF), aprox. 48, mientras que el metabolito desmetilado (DMT-GFT) mostró un valor de PIF mucho menor (~7), casi la mitad del valor de PIF del fármaco inalterado (~13). Por el contrario, el metabolito que presenta un grupo hidroxilo en lugar de flúor (DF-GFT) resultó no ser fototóxico. Notablemente, solo se confirmó que DMOR-GFT induce fotoperoxidación lipídica mediante un mecanismo oxidativo de Tipo I, basado en la escasa producción de oxígeno singlete y la eficiente desactivación del estado excitado triplete por un modelo lipídico. La fotooxidación de proteínas se evidenció en el caso de GFT y, en menor medida, en DMOR-GFT, pero resultó insignificante para DMT-GFT. Sin embargo, a diferencia de GFT, el daño al ADN inducido por el metabolito desmetilado no se reparó incluso después de varias horas. Axitinib (AXT), comercialmente disponible como el isómero (E)-AXT, tiende a fotoisomerizar a (Z)-AXT, especialmente en presencia de proteínas. Así, se revelaron dos mecanismos de fototoxicidad. En primer lugar, la conversión del (E)-AXT (no citotóxico) en el (Z)-AXT (citotóxico) tras irradiación. En segundo lugar, la fototoxicidad intrínseca exhibida por (Z)-AXT. Además, la fotooxidación de proteínas se atribuyó al isómero Z debido a la similitud en el contenido de carbonilo entre ambos isómeros y la alta afinidad del isómero Z por las proteínas. Finalmente, la fotogenotoxicidad solo se reveló mediante la detección de histonas ¿-H2AX. Dasatinib (DAS) es un TKI propuesto para el uso tópico en enfermedades cutáneas. Tras establecer un PIF inicial de 5, se confirmó la fototoxicidad de DAS en una emulsión oleo-acuosa en epidermis humana reconstruida (RhE), la cual se redujo sustancialmente al incorporar un filtro solar de amplio espectro. DAS presenta capacidad de generar tanto oxígeno singlete como radicales, desencadenando fotooxidación tanto en lípidos como en proteínas. Asimismo, se evidenció daño fotoinducido al ADN tanto mediante el ensayo cometa como la detección de ¿-H2AX. Avapritinib (AVP), un TKI de nueva aprobación, demostró ser un fármaco fototóxico con un valor de PIF de aproximadamente 11. Además, fue capaz de inducir tanto fotooxidación a las proteínas como daño en el ADN. En definitiva, el estudio de la toxicidad cutánea de los TKI en combinación con la luz solar se llevó a cabo mediante una exhaustiva evaluación de su fotocomportamiento tanto en disolución como en células de piel. El objetivo es proporcionar a los profesionales de la salud información actualizada sobre la foto(geno)toxicidad y alentarlos a evaluar e implementar estrategias de fotoprotección para los pacientes sometidos a la terapia basada en TKI. / [CA] En les últimes dècades, l'aparició d'inhibidors de la tirosina cinasa (de l'anglès TKI) com una nova classe de teràpia dirigida ha millorat la qualitat de vida i les taxes de supervivència dels pacients amb càncer. No obstant això, els efectes adversos associats a aquests, com les reaccions cutànies, continuen sent un desafiament per a la teràpia controlada. D'acord amb estudis fotofísics i fotobiològics prèvis de TKI realitzats pel grup de recerca, esta tesi segueix un enfocament multidisciplinari per a investigar nous fàrmacs fotoactius dins d¿aquesta familia. En l'etapa inicial, es van seleccionar quatre TKI, gefitinib, axitinib, dasatinib i avapritinib, per la seua capacitat per absorbir llum en la regió UVA i el seu potencial fototòxic. Gefitinib (GFT), un TKI amb un cromòfor quinazolina, va experimentar canvis significatius en la fototoxicitat a causa de modificacions metabòliques en la seua estructura. La desalquilació de la cadena lateral propoxi-morfolina (DMOR-GFT) va presentar el valor més alt de factor de fotoirritació (PIF), aprox. 48, mentre que el metabòlit desmetilat (DMT-GFT) va mostrar un valor de PIF molt menor (~7), quasi la meitat del valor de PIF del fàrmac inalterat (aprox. 13). Al contrari, el metabòlit que presenta un grup hidroxil en lloc de fluor (DF-GFT) va resultar no ser fototòxic. Notablement, només es va confirmar que DMOR-GFT induïx fotoperoxidació lipídica mitjançant un mecanisme oxidatiu de Tipus I, basat en l'escassa producció d'oxigen singlet i l'eficient desactivació de l'estat excitat triplet per un model lipídic. La fotooxidació de proteïnes va ser evident per a GFT i, en menor mesura, per a DMOR-GFT, però va resultar insignificant per a DMT-GFT. No obstant això, a diferència de GFT, el dany a l'ADN induït pel metabòlit desmetilat no es va reparar fins i tot després de diverses hores. Axitinib (AXT), comercialment disponible com a (E)-AXT, tendeix a fotoisomeritzar a (Z)-AXT, especialment en presència de proteïnes. Així, es van revelar dos mecanismes de fototoxicitat. En primer lloc, la conversió de l'(E)-AXT (no citotòxic) en el (Z)-AXT (citotòxic) després d'irradiació. En segon lloc, la fototoxicitat intrínseca exhibida per (Z)-AXT. A més, la fotooxidació de proteïnes es va atribuir a l'isòmer Z a causa de la similitud en el contingut de carbonil entre ambdós isòmers i l'alta afinitat de l'isòmer Z per les proteïnes. Finalment, la fotogenotoxicitat només es va revelar mitjançant la detecció de histones ¿-H2AX. Dasatinib (DAS) és un TKI proposat per a l'ús tòpic en malalties cutànies. Després d'establir un PIF inicial de 5, es va confirmar la fototoxicitat de DAS en una emulsió oli-aquosa en epidermis humana reconstituïda (RhE), la qual es va reduir substancialment en incorporar un filtre solar d'ample espectre. DAS presenta capacitat de generar tant oxigen singlet com radicals, desencadenant la fotooxidació tant en lípids com en proteïnes. Així mateix, es va evidenciar mitjançant l'assaig cometa i la detecció d'H2AX dany fotoinduït a l'ADN. Avapritinib (AVP), un TKI de segona generació, va demostrar ser un fàrmac fototòxic amb un valor PIF d'aproximadament 11. A més, va ser capaç d'induir tant la fotooxidació a les proteïnes com induir dany en l'ADN. En definitiva, l'estudi de la toxicitat cutània dels TKI en combinació amb la radiació solar es va dur a terme mitjançant una exhaustiva avaluació del seu fotocomportament tant en dissolució como en cèl·lules de pell. L'objectiu és proporcionar als professionals de la salut informació actualitzada sobre foto(geno)toxicitat i fomentar l'avaluació e implementació d'estratègies de fotoprotecció per als pacients sotmesos a la teràpia basada en TKI. / [EN] In recent decades, the emerge of tyrosine kinase inhibitors (TKIs) as a new class of targeted therapy has substantially enhanced the quality of life and survival rates for cancer patients. However, associated adverse effects, such as dermatological reactions, remain a challenge to sustained therapy. In light of our research group established insights into the photophysical and photobiological aspects of some TKIs, this thesis follows a similar multidisciplinary approach to investigate other photoactive drugs within the TKI family. In the initial stage, four TKIs, gefitinib, axitinib, dasatinib, and avapritinib, were selected based on their ability to absorb in the UVA region of the solar spectrum and their phototoxic potential. Consequently, photophysical and photobiological studies were conducted on these TKIs. Gefitinib (GFT) is a TKI with a quinazoline moiety, in which modifications resulting from metabolism significantly alter the phototoxicity potential. Dealkylation of the propoxy-morpholine side chain (DMOR-GFT) exhibited the highest photoirritant value (PIF), reaching approximately 48, while the demethylated metabolite (DMT-GFT) displayed much lower phototoxicity (PIF ~7), nearly half the PIF value of the parent drug (ca. 13). In contrast, replacing the fluorine substituent with OH (DF-GFT) resulted in the absence of phototoxic activity. Surprisingly, only DMOR-GFT was confirmed to induce lipid photoperoxidation which occurred through a Type I oxidative mechanism, based on the weak singlet oxygen production and the efficient quenching of the triplet excited state by a lipid model. Furthermore, protein photooxidation was evident for GFT and, to a lesser extent, for DMOR-GFT, but negligible for DMT-GFT. However, unlike the parent drug, DNA photodamage induced by the demethylated metabolite exhibited limited repair even after several hours. Axitinib (AXT), commercially available as (E)-AXT, showed a tendency for photoisomerization to (Z)-AXT, particularly within proteins. Thus, two phototoxicity mechanisms were unveiled. Firstly, the transformation of the initially non-cytotoxic (E)-AXT into the cytotoxic (Z)-AXT upon radiation. Secondly, the intrinsic phototoxicity exhibited by (Z)-AXT. Moreover, protein photooxidation was unequivocally attributed to the (Z)-isomer due to the similarity in carbonyl content between E/Z-isomers and the high protein affinity of the (Z)-isomer. Finally, the photogenotoxicity was only revealed through the detection of ¿-H2AX histone foci. Dasatinib (DAS) is a TKI suggested for topical treatment of dermatological diseases. Given this context and having determined a PIF value ca. 5, an evaluation of DAS phototoxicity in reconstructed human epidermis (RhE) was conducted. DAS formulated in an oil-in-water emulsion exhibited high phototoxicity, which was substantially reduced upon incorporating a broad-spectrum sunscreen. DAS, capable to generate both singlet oxygen and radicals, triggered photooxidation in both lipids and proteins. Similarly, DNA photodamage was evidenced through comet assay and H2AX foci detection. Avapritinib (AVP), a newly approved TKI, was proven to be a phototoxic drug with a PIF value ca. 11, which was highly photooxidative toward proteins and capable to induce DNA photodamage. All in all, the study of skin toxicity of TKIs in combination with sunlight was achieved through a comprehensive evaluation of their photobehavior both in solution and within skin cells. The aim is to provide healthcare professionals with updated information on photo(geno)toxicity and encourage them to assess and implement photoprotection strategies for patients undergoing TKI-based therapy. / Agradezco a la Universitat Politècnica de València por la ayuda para la formación de doctores dentro del subprograma 1 (PAID-1- 2019) y al Ministerio de Ciencia, Innovación y Universidades por la ayuda para la formación del profesorado universitario (FPU19/00048). Ambas subvenciones resultaron fundamentales para la elaboración de mi tesis doctoral. / Ouardi El Hamidy, ME. (2024). In Vitro Photobehavior of Tyrosine Kinase Inhibitors in Solution and within Skin Cells [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/206167