Heart rate changes mediate the embryotoxic effect of antiarrhythmic drugs in the chick embryo A significant increase in cardiovascular medication use during pregnancy has occurred in recent years but only limited evidence on its safety profile is available. We hypothesized that drug-induced bradycardia is the leading mechanism of developmental toxicity. We tested metoprolol, carvedilol, or ivabradine for embryotoxicity and their acute effect on chick embryonic model. We used video microscopy and ultrasound biomicroscopy. Significant dose-dependent mortality was achieved in embryos injected with carvedilol and ivabradine. In ED4 embryos, metoprolol, carvedilol and ivabradine reduced the heart rate by 33%, 27%, and 55%, respectively, compared to controls (6%). In ED8 embryos this effect was more pronounced with a heart rate reduction by 71%, 54%, 53%, respectively (controls 36%). Cardiac output decreased in all tested groups but only proved significant in the metoprolol group in ED8 embryos. The number of -adrenergic receptors showed a downward tendency during embryonic development but a negative chronotropic effect of tested drugs was increasingly pronounced with embryonic maturity. This effect was associated with reduced cardiac output in chick embryos, probably leading to premature death....

Le cancer du rein papillaire de type 2 (PRCC2) est un cancer très agressif avec un potentiel métastatique élevé et pour lequel il n’y a pas de traitement efficace. La forme héréditaire de PRCC2 est associée au syndrome rare de la léiomyomatose cutanéo-utérine héréditaire (HLRCC). HLRCC est due à une mutation germinale hétérozygote du gène Fumarate Hydratase (FH) qui code l'enzyme du cycle de Krebs, la Fumarase. Le déficit en fumarase induit l’accumulation de fumarate et active les voies de signalisation du facteur de transcription inductible par l’hypoxie (HIF) et des espèces réactives de l’oxygène (ROS). Néanmoins, aucune mutation du gène FH n’a été rapportée dans les cas de PRCC2 sporadiques. Le projet de recherche porte sur la caractérisation moléculaire des PRCC2 héréditaires et sporadiques. Notre analyse du transcriptome a identifié des différences entre les signatures moléculaires des PRCC2 héréditaires et sporadiques. Cependant, l’étude d’immunohistochimie n'a pas révélé de biomarqueurs potentiels. Les analyses bio-informatiques de profils d’expression génique ont révélé que les tumeurs PRCC2 héréditaires et sporadiques partagent une dérégulation de la voie principale NRF2/KEAP1. Il a été montré que la surexpression de AKR1B10 (Aldo-Keto Reductase Family 1 Membre B10) est la conséquence directe de l’activation de l'élément de réponse antioxydant (ARE). Finalement, nous avons établi un nouveau modèle in vitro de lignée cellulaire, NCCFH1 (FH-/-), issue d’un patient HLRCC. NCCFH1 représente une plateforme idéale pour les études fonctionnelles, métaboliques et thérapeutiques. Bortézomib pourrait être la meilleure alternative thérapeutique pour les patients avec PRCC2. / Papillary Renal Cell Carcinoma type 2 (PRCC2) is known to be a very aggressive type of kidney cancer with a high metastatic potential, poor outcome and absence of effective therapy. Hereditary form of PRCC2 is associated with rare hereditary leiomyomatosis and renal cell carcinoma (HLRCC). HLRCC is characterized by germline heterozygous mutations in the Fumarate Hydratase (FH) gene that encodes an enzyme of the Krebs cycle, Fumarase. It has been shown that the accumulation of fumarate induces activation of Hypoxia Inducible Factor (HIF) and ROS (Reactive Oxygen Species) pathways. Nevertheless, no FH gene mutation has been reported in sporadic PRCC2 tumors. The goal of this study is to better characterize hereditary and sporadic PRCC2. Our transcriptome analysis identified the set of genes that are differentially expressed between the two types of PRCC2. Subsequent immunohistochemistry screening did not reveal any potential diagnostics biomarkers. Further, the comprehensive computational analysis of gene profiling data revealed that hereditary and sporadic PRCC2 share the similar molecular signature with NRF2-KEAP1 axis deregulation as one of the major pathway in both forms. We demonstrated that over expression of Aldo-keto reductase family 1 member B10 (AKR1B10) is the direct consequence of the antioxidant response element (ARE) activation shared in hereditary and sporadic tumors. Finally, we have established FH-deficient cell line (NCCFH1) a new preclinical model of hereditary PRCC2. It presents the perfect platform for studying the metabolic features and testing new therapies for hereditary PRCC2, while bortezomib appears to be a potential efficient therapeutic option.

Advancements in pulmonary drug delivery technologies have improved the use of dry powder inhalation therapy to treat respiratory and systemic diseases. Despite remarkable improvements in the development of dry powder inhaler devices (DPIs) and formulations in the last few years, an optimized DPI system has yet to be developed. In this work, we hypothesize that Thin Film Freezing (TFF) is a suitable technology to improve inhalation therapies to treat lung and systemic malignancies due to its ability to produce brittle powder with optimal aerodynamic properties. Also, we developed a performance verification test (PVT) for the Next Generation Cascade Impactor (NGI), which is one of the most important in vitro characterization methods to test inhalation. In the first study, we used TFF technology to produce amorphous and brittle particles of rapamycin, and compared the in vivo behavior by the pharmacokinetic profiles, to its crystalline counterpart when delivered to the lungs of rats via inhalation. It was found that TFF rapamycin presented higher in vivo systemic bioavailability than the crystalline formulation. Subsequently, we investigated the use of TFF technology to produce triple fixed dose therapy using formoterol fumarate, tiotropium bromide and budesonide as therapeutic drugs. We investigated applications of this technology to powder properties and in vitro aerosol performance with respect to single and combination therapy. As a result, the brittle TFF powders presented superior properties than the physical mixture of micronized crystalline powders, such as excellent particle distribution homogeneity after in vitro aerosolization. Lastly, we developed a PVT for the NGI that may be applicable to other cascade impactors, by investigating the use of a standardized pressurized metered dose inhaler (pMDI) with the NGI. Two standardized formulations were developed. Formulations were analyzed for repeatability and robustness, and found not to demonstrate significant differences in plate deposition using a single NGI apparatus. Variable conditions were introduced to the NGI to mimic operator and equipment failure. Introduction of the variable conditions to the NGI was found to significantly adjust the deposition patterns of the standardized formulations, suggesting that their use as a PVT could be useful and that further investigation is warranted. / text

La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-04-04T20:45:16Z No. of bitstreams: 1 DAYSE DE LOURDES MADRGA ESPÍNOLA GUEDES - DISSERTAÇÃO PPG-CEMat 2014..pdf: 2225179 bytes, checksum: ad9f795a81034cb79500cb575de58f73 (MD5) / Made available in DSpace on 2018-04-04T20:45:16Z (GMT). No. of bitstreams: 1 DAYSE DE LOURDES MADRGA ESPÍNOLA GUEDES - DISSERTAÇÃO PPG-CEMat 2014..pdf: 2225179 bytes, checksum: ad9f795a81034cb79500cb575de58f73 (MD5) Previous issue date: 2014-12-12 / A presente pesquisa tem como objetivo, caracterizar filmes de quitosana, de aplicação sublingual, para uso em sistemas de liberação controlada de fumarato de formoterol., buscando uma nova alternativa para o tratamento emergencial das crises de asma. Sabemos que a asma é uma patologia de grande incidência no Brasil e no mundo, pois existem cerca de 300 milhões de pessoas acometidas pela doença a nível mundial e 20 milhões de brasileiros .É uma doença de caráter hereditário, crônica que não tem cura e que se apresenta muitas vezes como uma emergência médica , pois nas crises da doença o socorro deve ser imediato com o intuito de evitar o óbito do paciente. Hoje utiliza-se apenas a via inalatória como preferencial para administração dos broncodilatadores, visto que essa via tem rapidez de ação. No entando, deparamo-nos com a difícil técnica de utilização dos inaladores que veiculam o medicamento o que muitas vezes impossibilita a sua utilização especialmente em crianças, idosos e pacientes muito debilidados. Vendo esta dificuldade, propusemos com o nosso trabalho buscar uma nova via de utilização dos medicamentos broncodilatadores, que tivesse a mesma eficácia da via inalatória, dispensando a difícil técnica de utilização dos fármacos .Para isso , escolhemos a via sublingual de rápida ação e facilidade de utilização, podendo ser empregada mais adequadamente em uma crise de asma . Selecionamos a quitosana, por ser um biopolímero versátil e muito utilizado em sistema de liberação controlada de fármaco para veicular o formoterol , que é um potente broncodilatador, através da produção de um filme para deposição sublingual. E os resultados obtidos através das caracterizações apresentaram membranas com variações de cristalinidade (por DRX) de acordo com o processo de reticulação, além de apresentarem uma possível relação entre reticulação e liberação. Por FTIR pode-se observar certa interação entre o fármaco e os grupos amina da quitosana, assim como possível isomerização do fármaco pela reticulação com 5% de TPP. Pelas microscopias ótica e eletrônica, pode-se observar que o acréscimo de fármaco proporcionou alguma rugosidade a membrana. Também pelas microscopias verificou-se a reticulação não homogênea da superfície da membrana. Por EDS não se verificou nenhum elemento estranho a estrutura da quitosana e do fármaco. Por medida do ângulo de molhabilidade pode-se verificar aumento do perfil hidrofílico da membrana por adição do fármaco, perfil este que não foi modificado pelo processo de reticulação. O ensaio de citotoxicidade apresentou resultados que indicam a membrana como promissora candidata a testes in vivo. / This research aims to characterize chitosan films, sublingual application, for use in controlled release of formoterol fumarate systems., Looking for a new alternative for the emergency treatment of asthma attacks. We know that asthma is a disease of high incidence in Brazil and in the world, because we have about 300milhões of people with the disease worldwide and 20 million Brazilians .It is one hereditary disease, chronic that has no cure and that often presents as a medical emergency, because the crisis of the disease the relief should be immediate in order to prevent the death of the patient. Today only is used inhalation as preferred for administration of bronchodilators, since this route has faster action .In entando, we are faced with the difficult technique for using inhalers that deliver the medicine which often makes it impossible to use especially in children, the elderly and very debilidados patients. Buy this difficulty, we proposed in our work to seek a new route for the use of bronchodilators, which had the same effectiveness of inhaled, eliminating the difficult technique of using drugs .For this, choose the sublingual route of fast action and ease of use and can be used more appropriately in an asthma attack. Chitosan selected because it is a versatile and widely used biopolymer for controlled drug delivery system for conveying formoterol, which is a potent bronchodilator, by producing a film for sublingual deposition. And the results obtained from the characterization showed membranes crystallinity variations (XRD) according to the crosslinking process, besides presenting a possible relationship between cross-linking and release. By FTIR one can observe some interaction between the drug and amino groups of chitosan, and can isomerization of drug by crosslinking with 5% TPP. Through optical and electronic microscopy, it can be seen that addition of drug has provided some roughness to the membrane. Also by microscopy verified the inhomogeneous crosslinking of the membrane surface. EDS there was no foreign object the structure of chitosan and the drug. By measuring the wetting angle can be checked increase the hydrophilic profile of the membrane by addition of the drug, this profile has not been modified by crosslinking process. The cytotoxicity assay results presented indicate that the membrane as a promising candidate for in vivo testing.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Michaela Jahodová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: In vitro and ex vivo study of drug-drug interactions of antiretrovirals on intestinal ATP-binding drug transporters The absorption of orally administered drugs takes place especially in the intestine, where it can affect by the activity of drug's ABC transporters located on the apical membrane of the intestinal epithelium. Study of drug interactions in intestinal ABC transporters is essential to ensure effective and safe pharmacotherapy. Testing of bi- directional transport on Caco-2 cells is generally the preferred method for in vitro evaluation of substrates and inhibitors of ABC transporters. Drawbacks of the Caco-2 model increase the need and necessity to introduce new models. A great potential is the involvement of ex vivo methodologies in the human or rat intestine. The aim of the work was to introduce an in vitro methodology using the Caco-2 cell monolayer and the ex vivo methodology of precision-cut rat intestinal slices. By the bi-directional transport method, we analyzed drug interactions of the model substrate P-gp and BCRP Rhodamine 123 (RHD123) and clinically-used tenofovir...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...

Identification of predictors of treatment response in patients with major depressive disorder (MDD) may facilitate improved disease management. Data were pooled from two 6-week, double-blind, placebo-controlled studies of extended-release quetiapine (quetiapine XR; 150 or 300 mg/day) as adjunct to ongoing antidepressant therapy. Effects of psychiatric history and baseline demographic and disease characteristics on efficacy outcomes (Week 6 Montgomery Åsberg Depression Rating Scale [MADRS] total score reduction) were evaluated in population subgroups (quetiapine XR both doses pooled, n = 616; placebo, n = 303). Baseline Clinical Global Impressions-Severity (CGI-S) score and previous depressive episodes on Week 6 MADRS total score change, and baseline MADRS individual item scores on Week 6 change in CGI-Improvement score, were also evaluated. No major differences between responders and non-responders to quetiapine XR were observed for patient characteristics or demographic and disease characteristics. No suggestion of a predictive association was found between baseline CGI-S score, number of depressive episodes, and baseline MADRS item scores and efficacy outcomes. These analyses showed no major differences between responders and non-responders, and no predictive association between the parameters assessed and efficacy outcomes for adjunct quetiapine XR in patients with MDD and an inadequate response to prior antidepressant therapy.

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