Saquinavir é um medicamento antirretroviral desenvolvido para inibir a protease do vírus da imunodeficiência humana. Este fármaco apresenta várias limitações em função dos seus efeitos colaterais, baixa biodisponibilidade e sensorial não agradável. A nanoencapsulação de fármacos é uma técnica promissora para superar esses problemas. O objetivo deste trabalho é desenvolver suspensões aquosas de nanocápsulas poliméricas contendo saquinavir utilizando uma mistura de poli-ε-caprolactona (PCL) e poli- ε-caprolactone triol (PCL-T) visando uma formulação líquida adequada para o tratamento de crianças infectadas. As nanocápsulas foram preparadas pela técnica de deposição interfacial de polímeros pré-formados. A análise de diâmetro médio de partícula foi realizada por diferentes técnicas, difração a laser, dispersão de luz dinâmica e análise de rastreamento de nanopartículas. O potencial zeta foi determinado por mobilidade eletroforética, o pH por potenciometria e o teor de fármaco e a taxa de encapsulação por cromatografia líquida de alta eficiência com detecção no ultravioleta. O ensaio de liberação in vitro foi realizado com membrana de diálise e coleta das amostras nos intervalos de 0-120 horas, a 37°C. A estabilidade foi verificada nos tempos 0, 15, 30, 60 e 90 dias, ao abrigo da luz, em temperatura ambiente e com o monitoramento do pH, potencial zeta, diâmetro, teor de fármaco e através da análise de retroespalhamento de luz. A morfologia foi observada por microscopia eletrônica de transmissão. A citotoxicidade em linfócitos T humano foi determinada pelo teste de exclusão de azul de tripan e o mascaramento do sabor de saquinavir pela língua eletrônica. Todas as formulações apresentaram tamanho nanométrico entre 172 ± 18 e 225 ± 4 nm e uma boa distribuição de tamanho. A taxa de encapsulação de saquinavir foi maior que 99%. As nanocápsulas apresentaram potencial zeta negativo, valores de pH levemente ácido e morfologia esférica. Os parâmetros avaliados na estabilidade mantiveram-se constantes ao longo do tempo analisado. O ensaio in vitro mostrou uma liberação controlada de saquinavir. As formulações não apresentaram toxicidade in vitro e foram capazes de mascarar o sabor do saquinavir. Portanto, a associação de polímeros é eficaz para a encapsulação de saquinavir, apresentando características nanotecnológicas adequadas para a sua administração. / Saquinavir is an antiretroviral drug designed to inhibit the human immunodeficiency virus protease. This drug presents several limitations regarding side effects, very low bioavailability and sensory not pleasant. The drug nanoencapsulation is a promising technique to overcome these issues. The aim of this work is to develop aqueous suspensions of polymeric nanocapsules containing saquinavir using poly-ε-caprolactone (PCL) and poly-ε-triol caprolactone (PCL-T) towards a liquid formulation suitable for the treatment of infected children. The nanocapsules were prepared using the technique of interfacial deposition of preformed polymers. The characterization study was conducted by examining the average particle diameter by three techniques, the laser diffraction, the dynamic light scattering and the nanoparticle tracking analysis. The zeta potential was evaluated by electrophoretic mobility, the pH by potentiometry and drug content and encapsulation rate by high performance liquid chromatography with UV detection. The in vitro release assay was carried out in a dialysis membrane with analysis at 0-120 hours, at 37 °C. The stability was checked at 0, 15, 30, 60 and 90 days, protected from light, at room temperature and by monitoring the pH, zeta potential, diameter, drug content and by multiple light scattering analysis. The morphology was observed by transmission electron microscopy. The cytotoxicity in human T lymphocytes was determined by exclusion of trypan blue test and the taste masking by the electronic tongue. All formulations presented nanometric size between 172 ± 18 and 225 ± 4 nm and a good size distribution. The drug encapsulation rate was higher than 99%. The nanocapsules presented negative zeta potential, pH values slightly acidic and spherical morphologies. The parameters evaluated in stability remained constant over time analyzed. The in vitro assay showed a controlled drug release from nanocapsules formulations. The developed formulations showed no toxicity and were able to mask the taste of saquinavir. The association of polymers is effective for saquinavir nanoencapsulation presenting suitable nanotechnological features for drug administration.

Ph.D.

Saquinavir é um medicamento antirretroviral desenvolvido para inibir a protease do vírus da imunodeficiência humana. Este fármaco apresenta várias limitações em função dos seus efeitos colaterais, baixa biodisponibilidade e sensorial não agradável. A nanoencapsulação de fármacos é uma técnica promissora para superar esses problemas. O objetivo deste trabalho é desenvolver suspensões aquosas de nanocápsulas poliméricas contendo saquinavir utilizando uma mistura de poli-ε-caprolactona (PCL) e poli- ε-caprolactone triol (PCL-T) visando uma formulação líquida adequada para o tratamento de crianças infectadas. As nanocápsulas foram preparadas pela técnica de deposição interfacial de polímeros pré-formados. A análise de diâmetro médio de partícula foi realizada por diferentes técnicas, difração a laser, dispersão de luz dinâmica e análise de rastreamento de nanopartículas. O potencial zeta foi determinado por mobilidade eletroforética, o pH por potenciometria e o teor de fármaco e a taxa de encapsulação por cromatografia líquida de alta eficiência com detecção no ultravioleta. O ensaio de liberação in vitro foi realizado com membrana de diálise e coleta das amostras nos intervalos de 0-120 horas, a 37°C. A estabilidade foi verificada nos tempos 0, 15, 30, 60 e 90 dias, ao abrigo da luz, em temperatura ambiente e com o monitoramento do pH, potencial zeta, diâmetro, teor de fármaco e através da análise de retroespalhamento de luz. A morfologia foi observada por microscopia eletrônica de transmissão. A citotoxicidade em linfócitos T humano foi determinada pelo teste de exclusão de azul de tripan e o mascaramento do sabor de saquinavir pela língua eletrônica. Todas as formulações apresentaram tamanho nanométrico entre 172 ± 18 e 225 ± 4 nm e uma boa distribuição de tamanho. A taxa de encapsulação de saquinavir foi maior que 99%. As nanocápsulas apresentaram potencial zeta negativo, valores de pH levemente ácido e morfologia esférica. Os parâmetros avaliados na estabilidade mantiveram-se constantes ao longo do tempo analisado. O ensaio in vitro mostrou uma liberação controlada de saquinavir. As formulações não apresentaram toxicidade in vitro e foram capazes de mascarar o sabor do saquinavir. Portanto, a associação de polímeros é eficaz para a encapsulação de saquinavir, apresentando características nanotecnológicas adequadas para a sua administração. / Saquinavir is an antiretroviral drug designed to inhibit the human immunodeficiency virus protease. This drug presents several limitations regarding side effects, very low bioavailability and sensory not pleasant. The drug nanoencapsulation is a promising technique to overcome these issues. The aim of this work is to develop aqueous suspensions of polymeric nanocapsules containing saquinavir using poly-ε-caprolactone (PCL) and poly-ε-triol caprolactone (PCL-T) towards a liquid formulation suitable for the treatment of infected children. The nanocapsules were prepared using the technique of interfacial deposition of preformed polymers. The characterization study was conducted by examining the average particle diameter by three techniques, the laser diffraction, the dynamic light scattering and the nanoparticle tracking analysis. The zeta potential was evaluated by electrophoretic mobility, the pH by potentiometry and drug content and encapsulation rate by high performance liquid chromatography with UV detection. The in vitro release assay was carried out in a dialysis membrane with analysis at 0-120 hours, at 37 °C. The stability was checked at 0, 15, 30, 60 and 90 days, protected from light, at room temperature and by monitoring the pH, zeta potential, diameter, drug content and by multiple light scattering analysis. The morphology was observed by transmission electron microscopy. The cytotoxicity in human T lymphocytes was determined by exclusion of trypan blue test and the taste masking by the electronic tongue. All formulations presented nanometric size between 172 ± 18 and 225 ± 4 nm and a good size distribution. The drug encapsulation rate was higher than 99%. The nanocapsules presented negative zeta potential, pH values slightly acidic and spherical morphologies. The parameters evaluated in stability remained constant over time analyzed. The in vitro assay showed a controlled drug release from nanocapsules formulations. The developed formulations showed no toxicity and were able to mask the taste of saquinavir. The association of polymers is effective for saquinavir nanoencapsulation presenting suitable nanotechnological features for drug administration.

Saquinavir é um medicamento antirretroviral desenvolvido para inibir a protease do vírus da imunodeficiência humana. Este fármaco apresenta várias limitações em função dos seus efeitos colaterais, baixa biodisponibilidade e sensorial não agradável. A nanoencapsulação de fármacos é uma técnica promissora para superar esses problemas. O objetivo deste trabalho é desenvolver suspensões aquosas de nanocápsulas poliméricas contendo saquinavir utilizando uma mistura de poli-ε-caprolactona (PCL) e poli- ε-caprolactone triol (PCL-T) visando uma formulação líquida adequada para o tratamento de crianças infectadas. As nanocápsulas foram preparadas pela técnica de deposição interfacial de polímeros pré-formados. A análise de diâmetro médio de partícula foi realizada por diferentes técnicas, difração a laser, dispersão de luz dinâmica e análise de rastreamento de nanopartículas. O potencial zeta foi determinado por mobilidade eletroforética, o pH por potenciometria e o teor de fármaco e a taxa de encapsulação por cromatografia líquida de alta eficiência com detecção no ultravioleta. O ensaio de liberação in vitro foi realizado com membrana de diálise e coleta das amostras nos intervalos de 0-120 horas, a 37°C. A estabilidade foi verificada nos tempos 0, 15, 30, 60 e 90 dias, ao abrigo da luz, em temperatura ambiente e com o monitoramento do pH, potencial zeta, diâmetro, teor de fármaco e através da análise de retroespalhamento de luz. A morfologia foi observada por microscopia eletrônica de transmissão. A citotoxicidade em linfócitos T humano foi determinada pelo teste de exclusão de azul de tripan e o mascaramento do sabor de saquinavir pela língua eletrônica. Todas as formulações apresentaram tamanho nanométrico entre 172 ± 18 e 225 ± 4 nm e uma boa distribuição de tamanho. A taxa de encapsulação de saquinavir foi maior que 99%. As nanocápsulas apresentaram potencial zeta negativo, valores de pH levemente ácido e morfologia esférica. Os parâmetros avaliados na estabilidade mantiveram-se constantes ao longo do tempo analisado. O ensaio in vitro mostrou uma liberação controlada de saquinavir. As formulações não apresentaram toxicidade in vitro e foram capazes de mascarar o sabor do saquinavir. Portanto, a associação de polímeros é eficaz para a encapsulação de saquinavir, apresentando características nanotecnológicas adequadas para a sua administração. / Saquinavir is an antiretroviral drug designed to inhibit the human immunodeficiency virus protease. This drug presents several limitations regarding side effects, very low bioavailability and sensory not pleasant. The drug nanoencapsulation is a promising technique to overcome these issues. The aim of this work is to develop aqueous suspensions of polymeric nanocapsules containing saquinavir using poly-ε-caprolactone (PCL) and poly-ε-triol caprolactone (PCL-T) towards a liquid formulation suitable for the treatment of infected children. The nanocapsules were prepared using the technique of interfacial deposition of preformed polymers. The characterization study was conducted by examining the average particle diameter by three techniques, the laser diffraction, the dynamic light scattering and the nanoparticle tracking analysis. The zeta potential was evaluated by electrophoretic mobility, the pH by potentiometry and drug content and encapsulation rate by high performance liquid chromatography with UV detection. The in vitro release assay was carried out in a dialysis membrane with analysis at 0-120 hours, at 37 °C. The stability was checked at 0, 15, 30, 60 and 90 days, protected from light, at room temperature and by monitoring the pH, zeta potential, diameter, drug content and by multiple light scattering analysis. The morphology was observed by transmission electron microscopy. The cytotoxicity in human T lymphocytes was determined by exclusion of trypan blue test and the taste masking by the electronic tongue. All formulations presented nanometric size between 172 ± 18 and 225 ± 4 nm and a good size distribution. The drug encapsulation rate was higher than 99%. The nanocapsules presented negative zeta potential, pH values slightly acidic and spherical morphologies. The parameters evaluated in stability remained constant over time analyzed. The in vitro assay showed a controlled drug release from nanocapsules formulations. The developed formulations showed no toxicity and were able to mask the taste of saquinavir. The association of polymers is effective for saquinavir nanoencapsulation presenting suitable nanotechnological features for drug administration.

No description available.

Ph.D.

Les maladies inflammatoires chroniques de l’intestin (MICI) représentent un problème de santé publique majeur touchant de jeunes patients. Aux récidives inflammatoires, à la mauvaise qualité de vie et à l’espérance de vie réduite des patients viennent s’ajouter la durée, l’efficacité parfois limitée et le coût des traitements actuellement proposés. La recherche de nouvelles stratégies permettant de prévenir les récidives inflammatoires est primordiale. Ainsi, le rôle du S-nitrosoglutathion (GSNO, donneur et forme de stockage naturelle de NO) dans le maintien de l’intégrité de la barrière intestinale est étudié dans cette thèse, en décrivant : i) l’effet concentration-dépendante du GSNO sur la perméabilité intestinale (modèle de chambre de Ussing, expression des protéines de jonctions cellulaires…), ii) des formulations innovantes de GSNO (nanoparticules composites) à base de nanoparticules d’Eudragit®RL, elles-mêmes encapsulées dans une matrice polymérique à base d’alginate. Différents procédés de formulation ont été testés. Une caractérisation physicochimique des objets développés et des études de perméabilité sur des modèles cellulaires (Caco-2) ont conduit à l’obtention de trois formulations, adaptées à la voie orale et permettant de délivrer de façon prolongée le GSNO. Cependant, la prise en charge du GSNO libre ou formulé, l’identification des cibles du NO au niveau intestinal, mais aussi les doses et durées du traitement in vivo restent encore à définir avant de proposer ce donneur de NO comme candidat pour un traitement préventif des MICI / Inflammatory bowel diseases (IBD) (Crohn’s disease, ulcerative colitis…), are disabling pathologies affecting young patients and presenting the particularity that most of the current agents act by down regulating chronic inflammation in the intestine mucosa and cannot cure the disease. As the pivotal role of S-nitrosoglutathione (GSNO) in preventing intestinal inflammation and gut barrier failure has been clearly pointed out, therapies based on pharmaceutical technology to limit chronic inflammation and prevent relapses of barrier failure by an optimized dosage form of GSNO appears as an interesting challenge. The objective of this project relies on i) studying GSNO concentration/response functions in intestine by using Ussing chamber ex vivo model and following protein cell junction expression, ii) developing polymer nanocomposite particles encapsulating GSNO and adapted to the oral route, with GSNO protection, controlled delivery and local effect in intestine as major requirements. GSNO loaded polymeric (Eudragit® RL) nanoparticles were included in polymer matrix based on alginate; different processes were tested and particles were characterized: high GSNO loading, GSNO sustained release and local retention for 4 h in cellulo study (Caco-2) were obtained. Thus, therapies based on GSNO administration should represent a novel strategy to limit chronic inflammation and prevent relapses of barrier failure in IBD patients

Les bio-médicaments sont aujourd’hui confrontés à un manque d’adhésion thérapeutique due principalement à leur voie d’administration parentérale. Pour pallier à cela, un système de double encapsulation composé d’une gélule gastro-résistante renfermant un système de nanoparticules (NPs) polymériques à base de PLGA a été développé apportant protection et absorption pour in fine une administration par voie orale. L’objectif de ce travail a été de mettre au point le système de NPs renfermant de l’insuline, en jouant sur la charge de surface afin d’augmenter la biodisponibilité. Ainsi, nous avons montré un meilleur passage intestinal des nanoparticules grâce à l’apport d’une charge de surface négative confirmé par une diminution de la glycémie in vivo. Ensuite les NPs ont été recouvertes d’acide hyaluroniqueafin de réduire la perte intestinale d’insuline. Une fois validé avec l’insuline, le système nanoparticulaire a été transposé à un second bio médicament d’intérêt, l’héparine. Ainsi, nous avons obtenu des paramètres physico-chimiques semblables à ceux obtenus avec l’insuline prouvant le transfert de notre technologie. / Biologics are administrated by parenteral route due to their degradation in the gastrointestinal tract. This administration mode leads to lowadherence, showed by the low adherence for chronic diseases treatment. In order to increase adherence by reducing pain and adverse effects associated with treatment we developed a double encapsulation system to protect and increase biologic absorption for a future oral administration. Our system consists in encapsulated biologics inside PLGA nanoparticles (NPs), themselves encapsulated inside a gastroresistant vehicle. The aim of this work was to improve the system efficiency with insulin by NPs charges modification and intestinal release reduction and to transfer the technology to another molecule: the heparin. Negative charges improved the intestinal nanoparticle crossing as shown by the insulinopenic rat model with a decrease of glycaemia. To stabilize the NPs system in intestinal medium, a hyaluronic acid coating was tested and validated in vitro. When transferred to another biologics, we showed similar NPs chacacteristics with heparin than with insulin, revealing the transposition of our technic.

La voie orale est le mode d’administration le plus utilisé pour les médicaments. Ce mode d’administration présente de nombreux avantages tels que la facilité d’administration et une grande observance par les patients. Une des principales limitations de l’utilisation de la voie orale pour l’administration de molécules actives est que ces molécules doivent traverser les membranes biologiques pour rejoindre la circulation sanguine systémique et atteindre leur site d’action. C’est cette étape de passage transmembranaire qui détermine la biodisponibilité de la molécule active (ou principe actif, PA). De nombreuses molécules présentent une activité thérapeutique intéressante mais leur développement galénique n’est pas envisageable du fait de leur biodisponibilité trop faible. Dans cette thèse, le principe de la rétention magnétique a été étudié. Nous avons cherché à prouver que le fait de retenir, à l’aide d’un aimant, une formulation magnétique contenant un principe actif, peu perméable, à proximité de son site de passage dans la circulation sanguine, permettrait d’en augmenter l’absorption à travers la membrane biologique et ainsi d’en améliorer la biodisponibilité. Des formes magnétiques contenant des nanoparticules magnétiques d’oxyde de fer ont été mises au point à l’échelle du laboratoire puis transférées à l’échelle industrielle en process galénique. L’efficacité de la rétention magnétique a été testée ex vivo et in vivo sur modèle animal. Ces études pharmacocinétiques couplées à des techniques d’imagerie ont permis de montrer que la biodisponibilité du principe actif pouvait être multipliée par trois grâce à la rétention magnétique. L’accumulation de vecteurs magnétiques à l’endroit désiré du tractus gastro-intestinal provoque une surconcentration du principe actif menant à une augmentation de son passage transmembranaire et ainsi à l’augmentation de la dose absorbée dans la circulation systémique. / Oral administration still remains the route of choice for the majority of pharmaceutical dosage forms due to higher patient comfort and reduced cost of treatment. One of the major drawbacks of using the oral route to deliver drugs is that these molecules need to cross biological membranes in order to pass to the bloodstream and reach their physiological target. A large number of drugs with high clinical potential have not yet been employed because of their limited bloodstream access. Therefore, there is a real need to develop new methodologies addressing the problem of low permeable drugs used in oral administration. In this work we studied retention using an external magnetic field as a mean to maintain magnetic carriers near the absorption site of the drug. It was proven that an effective retention can cause over-concentration of the drug near the intestinal membrane thus increasing its bioavailability. For this purpose, we designed magnetic carriers, first, at the lab scale, and then using industrial processes. Magnetic retention efficiency was subsequently studied using ex vivo and in vivo experiments. Pharmacokinetics studies coupled with imaging techniques showed that magnetic retention provided a threefold increase in drug bioavailability. Finally, it was shown that retention of magnetic carriers near the absorption window of the drug can lead to overconcentration of the drug and increased transport through intestinal membrane resulting in a higher absorbed fraction reaching the bloodstream.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20219号 / 医博第4178号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小西 靖彦, 教授 小池 薫, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM