Le mitotane, ou o,p’DDD, médicament historique dérivé d’un insecticide, reste le traitement de référence du corticosurrénalome (CS), tumeur rare et de mauvais pronostic. Le mitotane exerce un effet anti-sécrétoire associé à un effet anti-tumoral dont les mécanismes d’action sous-jacents sont mal compris et ses cibles moléculaires restent inconnues. Néanmoins, de nombreux arguments semblent désigner la mitochondrie comme organite cible du mitotane. Dans ce travail de thèse, nous nous sommes intéressés à l’impact mitochondrial du mitotane par des approches complémentaires sur plusieurs modèles expérimentaux. Nous avons ainsi pu mettre en évidence une inhibition sélective des complexes I et IV de la chaîne respiratoire, une fission du réseau mitochondrial ou encore une activation de la biogenèse mitochondriale. Ces données nous ont permis d’identifier les mitochondrial-associated membranes (MAM) et le transducéosome comme organites cibles du mitotane. Parallèlement, en réévaluant le rôle potentiel du métabolite acide o,p’DDA, nous avons pu exclure son implication dans l’action cytotoxique du mitotane. Nous avons également démontré que le mitotane libre, non lié aux lipoprotéines, était le plus actif in vitro et pouvait ainsi être celui responsable de l’action pharmacologique in vivo. L’ensemble de ces résultats apportent une meilleure compréhension des mécanismes d’action du mitotane et devrait permettre d’identifier des facteurs prédictifs de réponse à ce traitement pour une meilleure prise en charge des patients atteints de CS. / Mitotane is o,p’DDD, an historic drug derived from an insecticide and represents the treatment of choice for adrenocortical carcinoma (ACC), a rare adrenal tumor associated with bad prognosis. Mitotane is responsible for an inhibition of steroidogenesis associated with an antitumoral effect but its exact molecular mechanisms of action remain unclear and its molecular target remains unknown. However, mitochondria could be an organite targeted by mitotane. In this study we evaluated mitochondrial impact of mitotane using complementary approaches on several experimental models. We showed a mitotane-induced selective inhibition of respiratory chain complexes I and IV, an increased fission of the mitochondrial network and an activation of the mitochondrial biogenesis. These data helped us to identify mitochondrial-associated membranes (MAM) and so-called transduceosome as targeted organites of mitotane. We also reevaluated the potential role of the acid metabolite o,p’DDA and excluded its implication in the cytotoxic action of mitotane. Finally, we demonstrated that free mitotane which is not linked to lipoproteins is more efficient in vitro and could be therefore responsible for pharmacological action in vivo. Altogether, these results allow to better understand mitotane mechanisms of action and should help to identify predictive markers of response to mitotane for a better care of patients with ACC.

Le mitotane ou o,p'-DDD est un dérivé organochloré très peu soluble dans l'eau. Il est utilisé dans le traitement du cancer corticosurrénalien métastasé ou inopérable (Lysodren®). En thérapeutique, il est nécessaire d'utiliser de fortes doses pour atteindre généralement au bout de trois mois, la mitotanémie efficace. Cela entraine le plus souvent, des effets indésirables gastroduodénaux et neuromusculaires, qui rendent les patients très peu compliants.L'objectif principal de cette thèse est de mettre au point, d'évaluer et de comparer les différentes formulations de mitotane afin d'améliorer la biodisponibilité du mitotane par rapport à la forme conventionnelle Lysodren®. Dans cette optique, le but recherché en clinique humaine serait de concevoir une forme galénique pouvant permettre d'utiliser de faibles doses journalières afin d'éviter les effets indésirables liés à la toxicité cumulative du mitotane. Pour cela, dans le but d'augmenter sa solubilité et de le rendre plus biodisponible, nous avons encapsulé le mitotane sous formes de particules polymériques et de microémulsions.Nous avons préparé des nanocapsules à partir de polymères biodégradables (la poly-epsilon-caprolactone) (PCL) et d'une association de PCL et de polymères non biodégradables (L'Eudragit® RL). Nous avons également préparé des microparticules de PCL et des systèmes autoémulsionnants ou SMEDDS.L'évaluation des caractéristiques physico-chimiques des particules montre des diamètres de 300 nm pour les nanocapsules et pour les microparticules, des diamètres variant de 40 à 76 µm. Le potentiel zêta est négatif pour les particules de PCL et positif pour celles associant les polymères PCL et RL. Pour les microémulsions, les diagrammes pseudoternaires ont permis le choix d'une association comportant du Capryol®, Tween® 20 et Crémophor® EL (33, 33, 33%). Les microémulsions ont un diamètre d'environ 40 nm. Les profils de libération in vitro du mitotane montrent une cinétique rapide et une quantité de mitotane libérée plus importante pour les microémulsions et les formes particulaires par rapport à la forme conventionnelle Lysodren®.De même, la réalisation de la pharmacocinétique à dose unique de 100 mg/kg chez des lapins montre des biodisponibilités relatives de 339% plus importantes pour les microémulsions, 195% pour les nanocapsules et 187% pour les microparticules. La quantification du mitotane absorbé dans des modèles Caco-2 montre une absorption complète du mitotane au bout de 4h lorsque le mitotane est formulé sous forme de microémulsions. Pour les microparticules et les nanocapsules, 50 et 45% de la dose initiale ont été respectivement absorbées par les cellules Caco-2. Cette évaluation sur le modèle Caco-2 a également confirmé le faible taux de passage de la poudre de mitotane (10%). Enfin, la réalisation des études de passage sur des coupes de jéjunum de rat en chambre de Ussing confirme que la quantité de mitotane qui a diffusé à travers la membrane jéjunale à partir des microémulsions est 5 fois supérieure à celle obtenue à partir de la poudre de mitotane.En conclusion, les microémulsions présentent un intérêt comme forme orale pour améliorer la biodisponibilité du mitotane. Elles ont pour avantage de multiplier la biodisponibilité par un facteur 3 chez le lapin et sont de fabrication peu coûteuse. Elles constituent une réelle alternative à la forme conventionnelle Lysodren® disponible actuellement sur le marché européen / Mitotane or o, p'-DDD is a organochlorine drug, very slightly soluble in water. It is used in the treatment of non resectable and metastasized adrenocortical carcinoma (Lysodren®). In therapy, to achieve therapeutic plasma level, high cumulative doses of mitotane were usually used during 3-5 months. This regimen causes gastrointestinal and neuromuscular side effects and make patients to be less compliants.The main objective of this work is to developp differents formulations of mitotane in order to improve the relative bioavailability when compared with conventional form Lysodren®. To shorten this equilibration time and reduce side effects, it's necessary to develop a new formulation. In order to increase mitotane solubility and make it more bioavailable, we encapsulated mitotane in polymeric particles and microemulsions.We prepared nanocapsules with biodegradable polymers (poly-epsilon-caprolactone) (PCL) and an association of PCL and non-biodegradable polymers (Eudragit®RL). We have also prepared PCL microparticles and a Self Microemulsifying Drug Delivery System or SMEDDS.Nanocapsules and microparticles diameters were respectively 300 nm and 40 to 76 µm. The zeta potential is negative for PCL particles wheras particles combining PCL and Eudragit®RL polymers exhibited positive zêta potential. For microemulsions, we investigated by constructing ternary phase diagrams and choosing the optimal formulation consisted of a mixture of Capryol®, Tween® 20 and Cremophor® EL (33, 33, 33%) with an emulsion diameter of 40 nm. The release of mitotane from SMEDDS and particles was higher and faster than from the conventional form Lysodren®.Pharmacokinetics after single-dose of oral mitotane formulations (100 mg/kg) in rabbits showed a 339% increase of relative bioavailability with microemulsions, 195% with the nanocapsules and 187% with the microparticles. Caco-2 cell culture showed a complete absorption of mitotane after 4h with microemulsions. For microparticles and nanocapsules, 50 and 45% of the initial dose, were respectively absorbed by Caco-2 cells. Caco-2 cells evaluation confirmed the low absorption of the mitotane powder (10%). Finally, Ussing chamber showed that microemulsions pass through the intestinal barrier 5 times higher than a solution of mitotane. In conclusion, microemulsions showed improvement of bioavailability of mitotane by a factor 3 in rabbits and could allow cost effective production. Microemulsions are a real alternative to Lysodren® which is currently available on the European market

O carcinoma adrenocortical (ACC) é um tumor maligno raro. O objetivo foi testar a ação de drogas antitumorais na linhagem H295R, em cultura 2D e 3D, analisadas por MTS, ensaios multiparamétricos, e microscopia confocal. Em monocamada (2D), as drogas tiveram maior efeito quando utilizadas com o mitotano 10 mM, sendo: everolimus 10 mM (50±0,02% p &#8804 0.001), imatinib (10 mM 52±0.01% p &#8804 0.001), sunitinib 5 mM (47±0.02% p &#8804 0.001), e nilotinib 5 mM (59±0,03% p &#8804 0.001), com evidência de apoptose. Nos esferoides (3D) foi necessário mitotano 30 M com everolimus 10 mM para se obter diminuição de 32±0.02% p &#8804 0.001 na viabilidade das células, e com nilotinib 10 mM para redução de 57±0.03% p &#8804 0.001, com evidência de necrose e apoptose. Em resumo, os dados sugerem que os esferoides são mais resistentes aos tratamentos, como ocorre com tumores in vivo, e podem representar uma importante abordagem para estudos de ACC. O nilotinib foi o que induziu as melhores respostas, tanto no modelo em 2D quanto 3D, resultados que se apresentam promissores para o tratamento dos carcinomas adrenocorticais. / Adrenocortical carcinoma (ACC) is a rare malignant tumor. The objective was to test the antitumor drugs action in the H295R cell line, in 2D and 3D culture system, by using MTS, multiparameter assay (High Content Screening) and confocal microscopy. In monolayer, all drugs were more effective in combination with mitotane 10 mM: everolimus 10 mM (50±0.02%), 10 mM imatinib (52±0.01%), 5 mM sunitinib (47±0.02%), and 5 mM nilotinib (59±0.03%). The spheroids required mitotano 30 mM with everolimus 10 mM to decrease cell viability (32±0.02% p &#8804 0.001); and with 10 mM nilotinib to inhibit cell viability in 57±0.03% (p &#8804 0.001), with induction of apoptosis and necrosis. In summary, the spheroids were more resistant to treatment and may represent an important approach for studies of ACC. Nilotinib was the tyrosine kinase inhibitor that, either alone or in combination with mitotane, induced higher cytotoxicity, in both 2D and 3D cell cultures, showing promising results for the treatment of adrenocortical carcinoma, as well as for the studies of its mechanism of action.

abstract: In wild birds, the stress response can inhibit the activity of the innate immune system, which serves as the first line of defense against pathogens. By elucidating the mechanisms which regulate the interaction between stress and innate immunity, researchers may be able to predict when birds experience increased susceptibility to infections and can target specific mediators to mitigate stress-induced suppression of innate immune activity. Such elucidation is especially important for urban birds, such as the House Sparrow (Passer domesticus), because these birds experience higher pathogen prevalence and transmission when compared to birds in rural regions. I investigated the role of corticosterone (CORT) in stress-induced suppression of two measures of innate immune activity (complement- and natural antibody-mediated activity) in male House Sparrows. Corticosterone, the primary avian glucocorticoid, is elevated during the stress response and high levels of this hormone induce effects through the activation of cytosolic and membrane-bound glucocorticoid receptors (GR). My results demonstrate that CORT is necessary and sufficient for stress-induced suppression of complement-mediated activity, and that this relationship is consistent between years. Corticosterone, however, does not inhibit complement-mediated activity through cytosolic GR, and additional research is needed to confirm the involvement of membrane-bound GR. The role of CORT in stress-induced inhibition of natural antibody-mediated activity, however, remains puzzling. Stress-induced elevation of CORT can suppress natural antibody-mediated activity through the activation of cytosolic GR, but the necessity of this mechanism varies inter-annually. In other words, both CORT-dependent and CORT-independent mechanisms may inhibit natural antibody-mediated activity during stress in certain years, but the causes of this inter-annual variation are not known. Previous studies have indicated that changes in the pathogen environment or food availability can alter regulation of innate immunity, but further research is needed to test these hypotheses. Overall, my dissertation demonstrates that stress inhibits innate immunity through several mechanisms, but environmental pressures may influence this inhibitory relationship. / Dissertation/Thesis / Doctoral Dissertation Biology 2017

Orientador: Cesar Costapinto Santana / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-08T16:18:50Z (GMT). No. of bitstreams: 1 Dias_RaquelMacedo_M.pdf: 1158217 bytes, checksum: 17fc0268f6869b95e17b301e65d69e9c (MD5) Previous issue date: 2007 / Resumo: O mitotano (o,p?-diclorodifenildicloroetano) é um fármaco utilizado no tratamento de carcinoma adrenocortical. Ele é comercializado na forma racêmica, ou seja, na proporção 1:1 dos seus enantiômeros R e S. A influência da quiralidade da molécula sobre seu efeito farmacológico ainda não foi estudada. Portanto, a separação dos enantiômeros é importante para testes biológicos comparativos de efeitos colaterais. Este trabalho foi desenvolvido com o intuito de estudar a separação deste fármaco pela técnica de cromatografia líquida de alta eficiência utilizando coluna recheada com a fase estacionária quiral O,O?-bis[4-tercbutilbenzoil]-N,N?-dialil-L-tartardiamida. Diferentes combinações de fase móvel foram testadas e os melhores resultados foram obtidos com hexano/acetato de etila na proporção 95/5 (v/v). Experimentos de pulsos com soluções diluídas do traçador e dos enantiômeros do mitotano foram realizados variando a vazão de fase móvel e a temperatura do sistema. Foram determinados as porosidades do sistema, os parâmetros cromatográficos, os dados de equilíbrio, coeficientes de dispersão axial e parâmetros de transferência de massa. Os resultados mostraram separação satisfatória, com número de pratos superando 9000 e fatores de separação na ordem de 1,13. Os valores dos coeficientes de Henry foram maiores que a unidade para ambos os enantiômeros, sendo que o enantiômero mais retido R-(+)-mitotano, apresentou maior afinidade pela coluna quiral. Valores de km superiores a 300 min-1 revelaram baixo efeito dos fenômenos de transferência de massa e consequentemente predomínio dos efeitos termodinâmicos (energia entálpica superior -10 kJ/mol para os enantiômeros). Experimentos a altas concentrações foram realizados com a finalidade de se determinar às isotermas pelo método da análise frontal e também os cromatogramas sob estas condições. Para a concentração da mistura até 16 g/L as isotermas mostraram um bom ajuste ao modelo de Langmuir. A partir da separação em batelada determinaram-se as regiões de separação dos enantiômeros para um sistema cromatográfico contínuo do tipo leito móvel simulado para diferentes concentrações de alimentação da mistura racêmica. Avaliaram-se as variáveis desempenho (consumo de solvente e produtividade) no sistema contínuo e compararam-se com as obtidas em separações em batelada. Melhores resultados foram obtidos para um sistema contínuo do tipo leito móvel simulado / Abstract: Mitotane (o, p'-dichlorodiphenyldichloroethane) is a drug used in the treatment of adrenocortical carcinoma. It is marketed in the racemic form, proportion 1:1 of their R and S enantiomers. The influence of the molecule quirality on its pharmacology effect was not studied yet. For this reason, this separation is important for comparative biological tests of collateral effects. This work was developed with intention to study the separation of this drug via liquid chromatography using columns packed with the chiral stationary phase, O,O?-bis[4-terc-butylbenzoyl] - N, N' - diallyl-L-tartardiamide. Different combinations of mobile phase was tested and better results were achieved with hexane/ethyl acetate in ratio 95/5 (v/v). Pulses experiments with diluted solutions of the inert and the enantiomers were accomplished at different flow rates and temperature. The system porosities, chromatographic parameters, equilibrium constants, axial dispersion and mass transference parameters were obtained. The results showed satisfactory separation, with number of plates overcoming 9000 and separation factors in the order of 1,13. The values of Henry coefficients were greater than one for both enantiomers, the most retained was R (+) -mitotane, and it presented greater affinity for the chiral column. The overall mass transfer coefficient achieved values higher than 300 min-1, demonstrating low mass transfer effect rates and consequently a prevalence of the thermodynamic effects (enthalpy energy greater than -10 kJ/mol for the enantiomers). Experiments in overload conditions were realized in order to determining the isotherms using the method of the frontal analysis as well the chromatograms under these conditions. For the concentration of the mixture smaller than 16 g/L the isotherms showed a good adjusted to the Langmuir model. The separation regions for a chromatographic continuous system like simulated moving bed were determined with different feed concentrations. This permits the comparison of the performance parameters using the continuous system and batch ones / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE) is bioactivated by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of mice and forms irreversibly bound protein adducts, reduces glucocorticoid secretion, and induces cell death selectively in cortisol-producing adrenocortical cells. 3-MeSO2-DDE has therefore been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) therapy. The aims of this thesis were to (1) develop in vitro test systems based on murine and human adrenocortical cell lines and to (2) investigate the mechanisms behind 3-MeSO2-DDE toxicity in adrenocortical cells. The cytotoxic and endocrine-modulating effects of 3-MeSO2-DDE were compared to those of o,p′-DDD (mitotane), the current ACC therapy, and to those of several structurally analogous compounds in both murine and human cell lines. 3-MeSO2-DDE bioactivation and cytotoxicity proceeded in a similar manner in the murine adrenocortical Y-1 cell line as in mice in vivo. The effects were highly structure-specific. Moreover, 3-MeSO2-DDE formed irreversibly bound protein adducts and caused cell death also in the human H295R cell line, and was slightly more cytotoxic than o,p′-DDD. However, 3-MeSO2-DDE toxicity in human cells was not affected by the CYP11B1 inhibitor etomidate, suggesting that bioactivation in human cells is performed by additional/other enzyme(s) than CYP11B1. 3-MeSO2-DDE generated biphasic responses in cortisol and aldosterone secretion and in expression levels of the steroidogenic genes CYP11B1, CYP11B2, and StAR. Such hormesis-like responses were not seen for o,p′-DDD or the precursor DDT metabolite p,p′-DDE. In addition, the two o,p′-DDD enantiomers (R)-(+)-o,p′-DDD and (S)-(-)-o,p′-DDD exhibited slight differences in cytotoxic and endocrine-modulating activity in H295R cells. In conclusion, this thesis  provides  extended  knowledge  on  the  mechanisms  of  action  of 3-MeSO2-DDE and points out important differences in effects between murine and human cells. Lead optimisation studies of 3-MeSO2-DDE using the herein presented in vitro test systems are ongoing.

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenasewildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.