La stéatose hépatique et la détérioration du profil lipidique plasmatique sont des pathologies métaboliques favorisées par la carence œstrogénique post-ménopausique. Cependant les mécanismes à la base de ces pathologies n’ont été que très peu étudiés. Le but de cette thèse a été d’investiguer les mécanismes moléculaires possibles à l’origine de l’hypercholestérolémie et de l’accumulation des lipides (triglycérides : TG et cholestérol) dans le foie en utilisant un modèle animal de la ménopause, la rate Sprague Dawley ovariectomisée (Ovx). Nous avons également examiné si le changement des habitudes de vie comme la pratique de l’exercice physique pouvait prévenir ou corriger les modifications induites par l’Ovx. Enfin, rosuvastatine (statine) a été utilisée comme thérapie pharmacologique de l’hypercholestérolémie dans le but de comprendre son effet au niveau moléculaire chez la rate Ovx. L’objectif de la première étude était de déterminer comment l’Ovx peut affecter les niveaux de TG et de cholestérol dans le foie des rates nourries avec une diète riche en lipides (HF : 42% gras). Les rates ont été soumises à la diète HF ou normale pendant 6 semaines avant d’être Ovx ou Sham (ovariectomie simulée), puis maintenues aux mêmes conditions diététiques pour 6 autres semaines. L’Ovx a provoqué une accumulation de TG dans le foie, mais pas la diète HF seule. Cependant, lorsque l’Ovx était combinée à la diète HF, l’accumulation des TG était beaucoup plus importante comparé à ce qui était observé chez les rates Ovx soumises à la diète normale. L’expression génique (ARNm) de CPT1 (Carnitine palmitoyltransferase 1), PGC1α (Peroxisome proliferator-activated receptor gamma, coactivator 1) et PPARα (Peroxysome proliferetor activated receptor alpha) intervenant dans l’oxydation des acides gras dans le foie était augmentée par la diète HF (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectivement) ; mais atténuée (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectivement) lorsque les rates ont été Ovx, favorisant ainsi l’accumulation des TG dans le foie. La combinaison de la diète HF à l’Ovx a également provoqué une hypercholestérolémie et une accumulation de cholestérol dans le foie malgré la diminution de l’expression de la HMGCoA-r (3-hydroxy-3-methylglutaryl-CoA reductase), enzyme clé de la synthèse du cholestérol. Ceci était associé à l’inhibition de l’expression génique de CYP7a1 (Cytochrome P450, family 7, subfamily a, polypeptide 1), suggérant une diminution de la synthèse des acides biliaires. Ayant constaté dans la première étude que l’Ovx élevait les niveaux de cholestérol hépatique et plasmatique, nous nous sommes fixés comme objectif dans la deuxième étude d’évaluer les effets de l’Ovx sur l’expression génique des transporteurs et enzymes responsables du métabolisme du cholestérol et des acides biliaires dans le foie et l’intestin, et de vérifier si l’exercice sur tapis roulant pouvait prévenir ou corriger les changements causés par l’Ovx. L’hypercholestérolémie constatée chez les rates Ovx comparativement aux Sham était accompagnée de la diminution de l’expression génique des récepteurs des LDL (R-LDL), des résidus de lipoprotéines (LRP1), de SREBP-2 (Sterol regulatory element binding protein 2) et de PCSK9 (Proprotein convertase subtilisin/kexin type 9) dans le foie, suggérant une défaillance dans la clairance des lipoprotéines plasmatiques. L’Ovx a aussi inhibé l’expression génique de la MTP (Microsomal triglyceride transfer protein) et stimulé celle de SR-B1 (Scavenger receptor class B, member 1); mais aucun changement n’a été observé avec CYP7a1. Ces changements moléculaires pourraient par conséquent favoriser l’accumulation de cholestérol dans le foie. L’exercice physique n’a pas corrigé les modifications causées par l’Ovx sur l’expression génique de ces molécules au niveau hépatique à l’exception de SREBP-2. Par contre, au niveau intestinal (iléum), l’exercice sur tapis roulant a inhibé l’expression génique des marqueurs moléculaires intervenant dans l’absorption des acides biliaires (OSTα/β, FXR, RXRα, Fgf15) et du cholestérol (LXRα, NCP1L1) au niveau de l’iléum chez les rates Sham entraînées. Ces adaptations pourraient prévenir le développement de l’hypercholestérolémie protégeant en partie contre la survenue de l’athérosclérose. Au vue des effets délétères (hypercholestérolémie et diminution de l’expression du R-LDL, PCSK9, LRP1, SREBP-2 et HMGCOA-r dans le foie) causés par l’Ovx sur le métabolisme du cholestérol constatés dans l’étude 2, la 3ième étude a été conçue pour évaluer l’efficacité de rosuvastatine (Ros) sur l’expression génique de ces marqueurs moléculaires chez les rates Ovx sédentaires ou soumises à l’entraînement volontaire. Ros a été administrée aux rates Ovx pendant 21 jours par voie sous-cutanée à la dose de 5mg/kg/j à partir de la 9ième semaine après l’Ovx. Ros n’a pas diminué la concentration plasmatique de LDL-C et de TC chez les rates Ovx. Par contre, Ros a stimulé (P ˂ 0.05) l’expression génique de PCSK9, SREBP-2, LRP1, HMGCoA-r et ACAT2 (Acyl-CoA cholesterol acyltransferase) mais pas significativement (P = 0.3) celle du R-LDL dans le foie des rates Ovx sédentaires et entraînées. Ros n’a pas réduit la concentration plasmatique de LDL-C probablement à cause de l’induction plus importante de PCSK9 par rapport au R-LDL. Cependant, la stimulation de LRP1 par Ros protège partiellement contre la survenue des maladies cardiovasculaires. En conclusion, les études de cette thèse indiquent que la baisse du niveau des œstrogènes entraîne des changements radicaux du métabolisme hépatique des TG et du cholestérol provoqués par des altérations de l’expression des gènes clés des voies métaboliques associées. / Hepatic steatosis and plasma lipid profile deterioration are metabolic diseases favored by post-menopausal estrogen deficiency. However, mechanisms underlying these diseases have not been systematically adressed. The aim of this thesis was to investigate molecular mechanisms causing hypercholesterolemia and lipids (triglycerides: TG and cholesterol) accumulation in the liver using animal model of menopause, the ovariectomized (Ovx) Sprague Dawley rat. We also examined whether lifestyle modifications such as physical activity can prevent or correct changes induced by Ovx. Finally, rosuvastatin (statine) was used as a pharmacological therapy of hypercholesterolemia in order to understand its effect at the molecular level in Ovx rats. The first study was designed to determine how the Ovx may affect levels of TG and cholesterol in the liver of rats fed a high-fat diet (HF: 42% fat). Rats were submitted to a HF or a normal diet for 6 weeks prior to Ovx or being sham operated, and then kept on the same diets for another 6 weeks. The Ovx increased liver TG content, but not the HF diet alone. However, the combination of Ovx and HF diet resulted in a greater liver TG accumulation than that observed in Ovx submitted to normal diet. The mRNA levels of CPT-1, PGC1 and PPARα involved in liver lipid oxidation significantly increased in rats fed the HF diet (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectively); but this increase was substantially less if HF fed rats were Ovx (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectively), thus favouring TG accumulation in the liver. The combination of HF diet and Ovx also induced hypercholesterolemia and an increase in liver total cholesterol content, in spite of the reduction of liver HMGCoA-r gene expression, the key enzyme for cholesterol synthesis. This was also associated with a decrease of liver CYP7a1 gene expression, suggesting a reduction in bile acids synthesis. Having found in the first study that the Ovx increases liver and plasma cholesterol levels, we aimed in the second study at determining the effects of Ovx on gene expression of hepatic and intestinal transporters and enzymes involved in cholesterol and bile acids metabolism; and to verify whether treadmill exercise could prevent or correct changes induced by Ovx. The Ovx resulted in hypercholesterolemia associated with a reduction in gene expression of hepatic low-density lipoprotein receptor (LDL-R), lipoprotein remnants receptor (LRP1), SREBP-2 and PCSK9, suggesting a failure in the clearance of plasma lipoproteins particles. The Ovx also inhibited the expression of MTP and stimulated that of SR-B1 in the liver, but no change was observed with CYP7a1. These molecular changes might, therefore, favor cholesterol accumulation in the liver. Exercise training did not correct the deleterious effects caused by Ovx on gene expression of these molecular markers in the liver with the exception of SREBP-2. However, in the intestine (ileum) treadmill exercise reduced gene expression of molecular markers involved in the absorption of bile acids (OSTα/β, FXR, RXRα, Fgf15) and cholesterol (LXRα, NCP1L1) in Sham trained rats compared to sedentary rats. This could prevent the development of cholestasis and hypercholesterolemia protecting partially against the onset of atherosclerosis. In view of the deleterious effects (hypercholesterolemia and decreased in gene expression of LDL-R, PCSK9, LRP1, SREBP-2 and HMGCoA-r in the liver) caused by Ovx on cholesterol metabolism observed in the second study, the 3rd study was designed to test the effect of rosuvastatin (Ros) on gene expression of these molecular markers in Ovx sedentary rats or in Ovx rats submitted to voluntary training. Ros was injected to Ovx rats subcutaneously at dose of 5mg/kg/day during 21 days from the ninth week after ovariectomy. Ros failed to decrease plasma LDL-C and TC in Ovx rats. In contrast, Ros increased (P ˂ 0.05) PCSK9, SREBP-2, LRP1, HMGCoA-r and ACAT2 but not significantly (P ˂ 0.3) LDL-R mRNA in the Ovx sedentary and trained rat liver. Ros failed to decrease plasma LDL-C in Ovx rats probably because of a stronger induction of PCSK9 than LDL-R gene expression. However by increasing LRP1 expression, Ros could decrease circulating lipoprotein remnants and, therefore, protects partially against the onset of cardiovascular diseases. In conclusion, the studies of this thesis indicate that the decrease of ovarian estrogen levels causes radical changes in hepatic TG and cholesterol metabolism caused by alterations in the expression of key genes associated with metabolic pathways.

Les transporteurs membranaires sont des éléments importants dans le devenir, l’efficacité, et la toxicité du médicament. Ils influencent la pharmacocinétique et la pharmacodynamie de ces derniers. Plusieurs interactions médicamenteuses observées cliniquement sont attribuables à la fois aux enzymes responsables du métabolisme des médicaments et aux transporteurs membranaires. Il est connu qu’une variabilité existe entre différents individus dans la réponse à un médicament et les polymorphismes génétiques retrouvés dans les gènes codant pour les transporteurs membranaires peuvent partiellement expliquer cette variabilité. OATP1A2 est un transporteur membranaire exprimé sur des organes importants, comme le cerveau et le rein. Plusieurs médicaments utilisés en clinique sont des substrats d’OATP1A2 et l’expression localisée de ce transporteur suggère un rôle important dans le devenir du médicament. Donc, mon projet de doctorat consistait à caractériser l’activité d’OATP1A2 en relation avec ses substrats et inhibiteurs, et de plus, à évaluer l’impact de différents variants génétiques d’OATP1A2 sur leur transport. Dans le premier article, la rosuvastatine a été utilisée comme substrat-type pour étudier le transport d’OATP1A2. Les expériences ont été menées en introduisant la rosuvastatine en compétition avec différent β-bloqueurs, une classe de médicaments rapportée dans la littérature comme substrats d’OATP1A2. Parmi les β-bloqueurs évalués, le carvédilol était l’inhibiteur le plus puissant. Dans la deuxième partie de l’étude, des médicaments ayant une structure similaire au carvédilol, tels que les antidépresseurs tricycliques, ont été évalués quant à leur potentiel d’inhibition sur OATP1A2. Une relation structure-activité a été définie à l’aide de ces données. Nous avons démontré que des composés tricycliques avec une courte chaîne aliphatique pouvaient inhiber OATP1A2. Dans le deuxième article, OATP1A2 a été étudié en considérant son expression et son rôle au sein de la barrière hémato-encéphalique (BHE). Des études précédentes ont démontré qu’OATP1A2 est exprimé sur la membrane luminale des cellules endothéliales formant la BHE. Nos données démontrent que les triptans, une classe de médicaments couramment utilisées pour traiter la crise migraineuse, sont des substrats d’OATP1A2 et que les composés tricycliques identifiés comme inhibiteurs d’OATP1A2 dans nos études précédentes peuvent inhiber le transport des triptans par OATP1A2. Ces résultats sont importants puisque: 1) il a été suggéré que les triptans peuvent agir au niveau du système nerveux central en se liant aux récepteurs trouvés sur les neurones centraux; 2) comme les triptans sont des molécules hydrophiles, un mécanisme de transport facilité est nécessaire pour qu’ils pénètrent la BHE et OATP1A2 pourrait être l’élément clé; 3) l’inhibition d’OATP1A2 par les composés tricycliques pourrait limiter l’accès des triptans à leur site d’action. Le troisième article caractérise l’activité associée à deux variants génétiques d’OATP1A2 (OATP1A2*2 et *3). Leur capacité à transporter les triptans et leur potentiel d’inhibition par les médicaments tricycliques ont été évalués. Des résultats supplémentaires caractérisant OATP1A2, mais sans liens directs avec les trois articles, seront présentés en annexe. Dans l’ensemble, les résultats présentés dans cette thèse servent à caractériser le transporteur membranaire OATP1A2 en relation avec ses substrats et inhibiteurs, et en fonction de ses variants génétiques. / Drug transporters are important determinants in drug disposition, efficacy, and toxicity. They influence the pharmacokinetics and pharmacodynamics of drugs. Several clinically-observed drug-drug interactions are mediated through drug metabolizing enzymes and drug transporters. It is well known that there is an interindividual variability in the response to medications and polymorphisms found in genes encoding for drug transporters partially account for it. OATP1A2 is a membrane drug transporter expressed on important organs, such as the brain and the kidney. A wide spectrum of drugs used in the clinic are substrates of OATP1A2. Its localisation suggests an essential role in drug disposition. Thus, my PhD project consisted of characterizing the activity of OATP1A2 in regards to its substrates, inhibitors, and different protein variants due to genetic polymorphisms. In the first article, rosuvastatin was used as the probe substrate to study OATP1A2 transport activity. Experiments were conducted by putting rosuvastatin in competition with different β-blockers, a class of drugs known in the literature to be transported by OATP1A2. One of the drugs evaluated, carvedilol, inhibited OATP1A2 with much more potency than the others. In the second part of the study, drugs with a structure similar to carvedilol, such as tricyclic antidepressants, were tested for their potential to inhibit OATP1A2. A structure-activity relationship was defined using the data. It was demonstrated that drugs composed of a tricyclic ring with a short aliphatic amine chain were potent OATP1A2 inhibitors. In the second article presented, OATP1A2 was studied in the context of its localization at the blood-brain barrier (BBB). OATP1A2 expression at the luminal membrane of the endothelial cells making up the BBB was demonstrated in the literature. Our article showed that triptans, a class of commonly used anti-migraine drugs, were OATP1A2 substrates. The tricyclic drugs previously evaluated were shown to potently inhibit triptan transport through OATP1A2. These findings are important for three reasons: 1) it has been postulated that triptans may act at the central nervous system by binding to receptors found on central neurons; 2) as triptans are hydrophilic molecules, a facilitated transport mechanism is required for them to penetrate the BBB and OATP1A2 may be the key player; and 3) the inhibition of OATP1A2 by the tricyclic drugs may limit the entrance of triptans to their site of action. The third article characterized the transport activity of two OATP1A2 protein variants (OATP1A2*2 and *3). Their capacities to transport triptans and their potential of being inhibited by tricyclic drugs were evaluated. Additional data characterizing OATP1A2 but considered out of the scope of the three articles will be presented in appendices. In overall, the central theme of this thesis looks into the characterization of the OATP1A2 membrane drug transporter in regards to its substrates, inhibitors, and proteins variants.

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 no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535 / TESIS