A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.

A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.

Depuis une quinzaine d’années, les polymères chiraux sont utilisés dans de nombreuses applications, comme phases stationnaires pour la séparation d’énantiomères ou encore en tant que catalyseurs en synthèse asymétrique. Aux vues de ces intérêts grandissants, de nombreuses méthodes ont émergé afin de les synthétiser. Nous nous sommes concentrés sur des méthodes organocatalytiques originales de synthèse de polymères chiraux par réaction de polyaldolisation et par désymétrisation de bis-anhydrides. Nous avons ainsi développé des processus de désymétrisation itératifs et ainsi généré une chiralité C-centrée sur la chaine polymérique. Des polyesters chiraux ont ainsi été obtenus avec de très bonnes séléctivités. / Chiral polymers are used in many applications such as stationary phases for chiral HPLC and catalysts in asymetric synthesis. The synthesis of chiral polymers traditionally deals with metal catalysts-based methodologies and often involved sensitive substrates. On the other hand, only a limited number of publications has been reported through environmentally-friendly organocatalytic pathways.The goal of this Ph.D. studies was devoted to the design of new routes toward chiral polymers under organocatalysis. We chose polyaldolisations and anhydride desymmetrizations with alcohols as key reactions to obtain original polymers with a C-centered chirality in the main polymer chain.

Malaria is the most important parasitic disease in man and it kills approximately 2,000 people each day. Pregnant women are especially vulnerable to malaria with increased incidence and mortality rates. There are indications that pregnancy alters the pharmacokinetic properties of many antimalarial drugs. This is worrisome as lower drug exposures might result in lowered efficacy and lower drug exposures can also accelerate the development and spread of resistant parasites. The aim of this research was to study the pharmacokinetics and pharmacodynamics of the most commonly used drugs for the treatment of uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy using a pharmacometric approach. This thesis presents a number of important findings that increase the current knowledge of antimalarial drug pharmacology and that may have an impact in terms of drug efficacy and resistance. (1) Lower lumefantrine plasma concentrations at day 7 were evident in pregnant women compared to that in non-pregnant patients. Subsequent in-silico simulations with the final pharmacokinetic-pharmacodynamic lumefantrine/desbutyl-lumefantrine model showed a decreased treatment failure rate after a proposed extended artemether-lumefantrine treatment. (2) Dihydroartemisinin exposure (after intravenous and oral administration of artesunate) was lower during pregnancy compared to that in women 3 months post-partum (same women without malaria). Consecutive in-silico simulations with the final model showed that the underexposure of dihydroartemisinin during pregnancy could be compensated by a 25% dose increase. (3) Artemether/dihydroartemisinin exposure in pregnant women was also lower compared to literature values in non-pregnant patients. This further supports the urgent need for a study in pregnant women with a non-pregnant control group. (4) Quinine pharmacokinetics was not affected by pregnancy trimester within the study population and a study with a non-pregnant control group is needed to evaluate the absolute effects of pregnancy. (5) Finally, a data-dependent power calculation methodology using the log likelihood ratio test was successfully used for sample size calculations of mixed pharmacokinetic study designs (i.e. sparsely and densely sampled patients). Such sample size calculations can contribute to a better design of future pharmacokinetic studies. In conclusion, this thesis showed lower exposures for drugs used to treat uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy. More pharmacokinetic studies in pregnant women with a non-pregnant control group are urgently needed to confirm the current findings and to enable an evidence-based dose optimisation. The data-dependent power calculation methodology using the log likelihood ratio test can contribute to an effective design of these future pharmacokinetic studies.

L'amertume fait partie intégrante de notre alimentation. Elle est par exemple fortement représentée dans certaines boissons (ex: café) ou dans certains légumes tels les crucifères. Néanmoins, la perception de l'amertume varie entre les individus et certains aliments considérés comme bénéfiques pour la santé peuvent être rejetés en raison de leur goût amer. Des facteurs génétiques (ex : polymorphisme génétique des récepteurs du goût amer) ou environnementaux (ex : âge, prise de médicaments) expliquent en partie les variations interindividuelles dans la perception de l'amertume. Cependant, d'autres facteurs péri-récepteurs pourraient intervenir, notamment la composition salivaire. Afin d'investiguer dans un premier temps le lien existant entre le protéome salivaire propre à un individu et sa sensibilité à l'amertume, le seuil de détection du goût amer de la caféine a été mesuré sur 29 hommes sains. Leur salive au repos a été étudiée par électrophorèse mono- et bidimensionnelle. L'analyse par électrophorèse bidimensionnelle de la salive au repos des 6 sujets les plus sensibles et 6 les sujets les moins sensibles à la caféine a permis la détection de 255 spots, dont 26 étaient significativement différents entre hyper- et hyposensibles. L'identification de ces 26 spots a révélé la surexpression de fragments d'alpha amylase, de fragments d'albumine sérique, et de sous-unités alpha de l'immunoglobuline A ainsi que la sous-expression de cystatine SN chez les hypersensibles. Ce dernier résultat a été confirmé par Western Blot. Ceci a permis de formuler une hypothèse sur le rôle de la protéolyse en bouche sur la sensibilité à l'amertume. Dans un deuxième temps et afin d'étudier l'effet des molécules amères sur la composition salivaire, une étude in vitro a été menée sur la lignée cellulaire de glandes salivaires humaines HSG différenciées en acini ou non. Après une mise au point des conditions de différenciation (culture dite en 3D), la cystatine SN a été détectée dans les cellules HSG par Western blot après traitement des cellules à la caféine, à la quinine, et à l'urée. Après traitement à la caféine à 5, 50 ou 100µM, une quantification par ELISA a mis en évidence que la cystatine SN était toujours plus abondante dans les cellules HSG différenciées que dans les cellules non-différenciées. Spécifiquement dans les cellules différenciées, l'exposition à la caféine induisait une sur-expression de cystatine SN, la teneur maximale en cystatine SN étant observée avec la caféine à 50 µM. La présence de cystatine SN a également été détectée dans les milieux de culture

This diploma thesis focuses on quinine solutions and quality of these solutions after applying plasma discharge. These electric discharges can be used to destroy some substances from the water. The theoretical part is focused on description of electric discharges in liquids and on the properties of quinine. In the experimental part, the properties of quinine solutions containing different electrolytes were analysed by UV-VIS spectrophotometer and fluorescence spectroscopy. Two different types of configurations of plasma discharges in liquids were used. Solutions were compared on the base of different concentration of quinine, different electrolytes, different configurations of plasma discharges and pH values of the solutions. Furthermore, the experimental work focuses on quinine solutions with the sodium nitrate. The time instability of the solutions was analysed. Also, the influence of the solution age and different types of the plasma discharge on the excitation and emission spectra of quinine were investigated.

No description available.