Beeinflussung der Ex-vivo-Chemoresponse von Plattenepithelkarzinomen der Kopf-Hals-Region auf Cisplatin und Docetaxel durch 5-Fluorurazil

Continuous monitoring of physiological parameters such as pH and oxygen (O2) are of great importance in determining the health status of a patient. Arterial blood gas analysis is a current clinical method used to measure pH, PCO2, PO2, and the concentration of variety of ions, typically with blood withdrawn from an artery. The need for robust, and a rapidly responding technology to enable bed-side monitoring has driven considerable efforts to produce better sensor devices. Optical sensing systems have experienced rapid growth, with drivers including low-cost optical fibres, and the availability of miniature optical set-ups (light sources, detectors, etc.). Herein, polymer-based optical fibre sensors for pH and O2 sensing were developed. The pH and/or oxygen reporters were immobilised at the end of an optical fibre by photo-polymerisation, and their performance in measuring pH and O2 concentration investigated. pH sensing was based on fluorescence detection using single excitation/single emission (Chapter 2), and single excitation/dual emission (Chapter 3). O2 sensing was based on the luminescence quenching of metalloporphyrins by oxygen (Chapter 4). In the last chapter, the in vivo applicability of an O2 sensor was investigated by measuring O2 level changes inside an ex vivo lung.

Aus pharmakokinetischer Sicht sind neben Parametern wie der oralen Bioverfügbarkeit und der systemischen Clearance, für die Effektivität und Sicherheit eines inhalativ angewendeten Wirkstoffes unter anderem das Ausmaß der pulmonalen Deposition und seine pulmonale Umverteilungskinetik entscheidend. Wird eine topische Wirkung des Arzneistoffes angestrebt, so trägt eine lange Verweilzeit des Arzneistoffes im Zielgewebe, verbunden mit einer langsamen Umverteilung in den systemischen Kreislauf zu einer Wirkungsoptimierung mit gleichzeitiger Minimierung systemischer Nebenwirkungen bei. In-vitro- und ex-vivo-Modelle eignen sich hervorragend zur isolierten Untersuchung solcher pharmakokinetischer Vorgänge ohne den Einfluss verschiedener in-vivo-Faktoren, wie der Verteilung in andere Gewebe, Metabolisierungs- oder Eliminationsprozessen. Das Ziel der vorliegenden Arbeit war es daher, Modelle der humanen Lunge zu etablieren bzw. weiterzuentwickeln, die möglichst realitätsnah die Untersuchung der Pharmakokinetik pulmonal applizierter Wirkstoffe ermöglichen. / From a pharmacokinetic point of view, the extent of pulmonary deposition and the pulmonary redistribution are crucial for an inhaled drug’s effectiveness and safety besides parameters such as oral bioavailability and systemic clearance. Aiming at a local effect, a long residence time in the target tissue combined with a slow redistribution into systemic circulation contribute to a drug’s optimal potency while simultaneously minimizing systemic adverse effects. In-vitro and ex-vivo models are particularly suitable for examining single pharmacokinetic aspects without the influences occurring in-vivo such as distribution into other tissues and processes of metabolism or elimination. Therefore, the aim of the present thesis was to establish, respectively enhance models of the human lung, which were able to describe the pharmacokinetics of pulmonary applied drugs close to reality.

Zugl.: Dresden, Techn. Univ., Diss., 2007

Zugl.: Berlin, Charité, Univ.-Med., Diss., 2008

Zugl.: Berlin, Freie Univ., Diss., 2009

Ulm, Univ., Diss., 2004.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tatiana Sloukgi Supervisor: PharmDr. Jana Pourová, Ph.D. Title of Diploma Thesis: The effect of Silymarin Flavonolignans and their sulfated conjugates on blood vessels ex vivo. Silymarin flavonolignans have recently shown some positive effects on the cardiovascular system. In this work, we studied the vasodilatory effect on rat aorta ex vivo of three silymarin conjugates, silybin A-20-sulfate, silybin B-20-sulfate and 2,3- dehydrosilychristin-19-O-sulfate, and one parent flavonolignan 2,3- dehydrosilychristin. For each substance, a concentration response curve was created and the concentration that produces 50% of maximum relaxation was determined (EC50). All substances exerted very low or no vasodilatory activity. Finally, we focused on the mechanism of action of silybin A. We tested whether its vasorelaxant activity depends on the presence of intact endothelium. The vasorelaxant effect of silybin A on isolated rat aorta ex vivo was clearly endothelium-dependent.

O aumento da incidência do câncer de pele está associado à maior exposição à luz solar e a adoção de ações de proteção ao Sol é uma estratégia para minimizar os níveis cumulativos de danos à pele. Os raios ultravioletas (UV), ao alcançarem o tecido cutâneo, podem causar eritema, inflamação, fotoenvelhecimento, formação de rugas e imunossupressão, entre outros, devido à formação de espécies reativas de oxigênio (ERO´s). A formação de ERO´s, como o oxigênio singleto, radical ânion superóxido, peróxido de hidrogênio e radical hidroxil, elevam o risco dos danos foto-oxidativos. O desequilíbrio entre a formação de ERO´s e os mecanismos antioxidantes do organismo desencadeia o estresse oxidativo. Na pele, as ERO´s são as responsáveis pelo dano oxidativo no DNA, proteínas e lipídeos. Identificar e quantificar biomarcadores do estresse oxidativo cutâneo é essencial para a correlação entre os raios UV e seus efeitos. Deve-se isto, em parte, à limitação de métodos para quantificar os parâmetros que são diretamente afetados pela exposição aos raios UV, tais como a peroxidação lipídica. São necessários métodos complementares para avaliação da eficácia de fotoprotetores perante os danos causados por este tipo de estresse. Esta pesquisa projeto compreendeu a avaliação ex vivo da eficácia de filtros solares UVB por meio da quantificação da peroxidação lipídica proveniente do estrato córneo removido por tape stripping. Foram preparados sistemas emulsionados do tipo O/A com os filtros octocrileno, metoxicinamato de octila e salicilato de octila. A caracterização funcional da eficácia fotoprotetora in vitro demonstrou que o filtro octocrileno manteve-se estável, mesmo após exposição solar artificial. Os filtros octocrileno (10% p/p), metoxicinamato de octila (10% p/p) e salicilato de octila (5% p/p) alcançaram, após irradiância, respectivamente, os valores de FPS 5,7 ± 2,1; 4,7 ± 1,5 e 1,0± 0,0. As formulações foram utilizadas na avaliação da eficácia fotoprotetora ex vivo. O método por CLAE, para quantificação da peroxidação lipídica no estrato córneo, possuiu linearidade e demonstrou exatidão e precisão satisfatórias. O estresse pela radiação UV desencadeou a peroxidação lipídica no estrato córneo. Em função do protocolo aplicado, não houve diferenças entre as amostras. A eficácia, com relação à inibição da peroxidação lipídica, foi similar em todas as amostras. / The increasing of incidence of skin cancer is associated with greater exposure to sunlight and the adoption of sun protection actions is a strategy to minimize cumulative levels of skin damage. Ultraviolet (UV) rays, when they reach the cutaneous tissue, can cause erythema, inflammation, photoaging, wrinkling and immunosuppression, among other things, due to the formation of reactive oxygen species (ROS). The formation of ROS, such as singlet oxygen, superoxide anion radical, hydrogen peroxide and hydroxyl radical, raise the risk of photooxidative damage. The variation between the formation of ROS and the antioxidant mechanisms of the organism triggers oxidative stress. In the skin, ROS are responsible for oxidative damage in DNA, proteins and lipids. Identifying and quantifying biomarkers of cutaneous oxidative stress is essential for the correlation between UV rays and their effects. This is partially due to the limitation of methods for quantifying parameters that are directly affected by exposure to UV rays, such as lipid peroxidation. Complementary methods are needed to evaluate the effectiveness of photoprotectors because of the damage caused by this type of stress. This research project had the ex vivo evaluation of the efficacy of UVB sunscreens by quantifying the lipid peroxidation from the stratum corneum removed by tape stripping. Emulsified O/A type systems were prepared with the octocrylene, octyl methoxycinnamate and octyl salicylate filters. The functional characterization of photoprotective efficacy in vitro revealed that the octocrylene filter remained stable even after artificial sun exposure. Octocrylene (10% w / w), octyl methoxycinnamate (10% w / w) and octyl salicylate (5% w / w) respectively reached the values of FPS 5.7 ± 2.1; 4.7 ± 1.5 and 1.0 ± 0.0. The formulations were used in the evaluation of ex vivo photoprotective efficacy. The method by HPLC, for quantification of the lipid peroxidation in the stratum corneum, had linearity and demonstrated satisfactory accuracy and precision. UV radiation stress triggered lipid peroxidation in the stratum corneum. Due to the protocol applied, there were no differences between the samples. The efficacy, compared to the inhibition of lipid peroxidation, was similar in all samples.

L’ischémie reperfusion pulmonaire et sa traduction clinique la dysfonction primaire du greffon sont responsables d’une morbi-mortalité importante en transplantation pulmonaire aussi bien à court terme qu’à long terme. Nous avons voulu faire la démonstration de la pertinence des microparticules comme marqueur de l’ischémie reperfusion pulmonaire. Nous avons reproduit et validé la stabilité d’un modèle de perfusion ventilation ex vivo de poumon de rat aussi bien en conditions normales (pas d’ischémie pulmonaire avant reperfusion) qu’en conditions extrêmes (1 h d’ischémie chaude avant reperfusion pulmonaire). Nous avons étudié la génération de microparticules par des poumons soumis à des conditions variables d’ischémie froide et d’ischémie chaude. Les poumons soumis à de fortes conditions d’ischémie froide (20h) produisent un pic précoce de microparticules d’origine épithéliale alvéolaire, leucocytaire et endothéliale. Nous en concluons que le modèle de perfusion ventilation ex vivo de poumons de rats est un modèle pertinent pour l’étude des réactions d’ischémie reperfusion. Les microparticules apparaissent comme un marqueur précoce des lésions d’ischémie reperfusion pulmonaires dans ce modèle. / Lung ischemia reperfusion and its clinical expression as primary graft dysfunction are provider of immediate and long term morbidity and mortality for patients. We aimed at demonstrating the usefulness and relevance of microparticles as biomarkers for lung ischemia reperfusion injury. We first reproduced an ex vivo rat lung perfusion and ventilation experimental model. Stability of the model was validated for normal conditions (no ischemia before reperfusion) as well as for extreme conditions (1 hour warm ischemia before reperfusion). The generation of microparticles was studied in that model for variable conditions of cold ischemia and for warm ischemia. Lung submitted to strong ischemic injury (20hours cold ischemia) generate an early pike of microparticles originated from leukocyes, endothelial cells, and epithelial alveolar cells. We may conclude the ex vivo model of rat lung perfusion and ventilation is relevant for the study of lung ischemia reperfusion injury. Microparticles are relevant markers of rat lung ischemia reperfusion injury in our model.