En raison de leur petite taille, les nanoparticules (NP) (< 100 nm) peuvent coaguler très rapidement ce qui favorise leur pénétration dans l’organisme sous forme d’agglomérats. L’objectif de cette recherche est d’étudier l’influence de l’état d’agglomération de NP de dioxyde de titane (TiO2) de trois tailles de départ différentes, 5, 10-30 ou 50 nm sur la toxicité pulmonaire chez le rat mâle (F344) exposé à des aérosols de 2, 7 ou 20 mg/m3 pendant 6 heures. Dans une chambre d’inhalation, six groupes de rats (n = 6 par groupe) ont été exposés par inhalation aiguë nez-seulement à des aérosols ayant une taille primaire de 5 nm, mais produits sous forme faiblement (< 100 nm) ou fortement (> 100 nm) agglomérée à 2, 7 et 20 mg/m3. De façon similaire, quatre autres groupes de rats ont été exposés à 20 mg/m3 à des aérosols ayant une taille primaire de 10-30 et 50 nm. Les différents aérosols ont été générés par nébulisation à partir de suspensions ou par dispersion à sec. Pour chaque concentration massique, un groupe de rats témoins (n = 6 par groupe) a été exposé à de l’air comprimé dans les mêmes conditions. Les animaux ont été sacrifiés 16 heures après la fin de l’exposition et les lavages broncho-alvéolaires ont permis de doser des marqueurs d’effets inflammatoires, cytotoxiques et de stress oxydant. Des coupes histologiques de poumons ont également été analysées. L’influence de l’état d’agglomération des NP de TiO2 n’a pu être discriminée à 2 mg/m3. Aux concentrations massiques de 7 et 20 mg/m3, nos résultats montrent qu’une réponse inflammatoire aiguë est induite suite à l'exposition aux aérosols fortement agglomérés. En plus de cette réponse, l’exposition aux aérosols faiblement agglomérés à 20 mg/m3 s’est traduite par une augmentation significative de la 8-isoprostane et de la lactate déshydrogénase. À 20 mg/m3, les effets cytotoxiques étaient plus importants suite à l’exposition aux NP de 5 nm faiblement agglomérées. Ces travaux ont montré dans l'ensemble que différents mécanismes de toxicité pulmonaire peuvent être empruntés par les NP de TiO2 en fonction de la taille de départ et de l’état d’agglomération. / Given their small size, nanoparticles (NP) (< 100 nm) can coagulate quickly, which promotes their entry into the body in the form of agglomerates. The objective of this study is to evaluate the influence of the agglomeration state of three different primary particle sizes (5, 10-30 and 50 nm) of titanium dioxide (TiO2) NP on the pulmonary toxicity of male rats (F344) exposed to aerosols at 2, 7 or 20 mg/m3 for 6 hours. In an inhalation chamber, six groups of rats (n = 6 per group) were acutely exposed by nose-only inhalation to aerosols with a 5-nm primary particle size, produced in the form of small agglomerates (< 100 nm) (SA) or large agglomerates (> 100 nm) (LA) at 2, 7 and 20 mg/m3. Similarly, four other groups of rats were exposed to aerosols at 20 mg/m3 with a primary particle size of 10-30 and 50 nm. The different aerosols were generated by nebulization of suspensions or by dry dispersion. For each mass concentration, one group of control rats (n = 6 per group) was exposed to compressed air under the same conditions. The animals were sacrificed 16 hours after the end of exposure, and analysis of the bronchoalveolar lavage fluids was used to measure markers of inflammatory, cytotoxicity and oxidative stress effects. Lung sections were also analyzed for histopathology. The influence of the agglomeration state of TiO2 NP (5 nm) could not be determined at 2 mg/m3. For mass concentrations of 7 and 20 mg/m3, our results showed that an acute inflammatory response was induced following exposure to LA aerosols. In addition to this response, exposure to SA aerosols resulted in a significant increase in 8-isoprostane and lactate dehydrogenase. At 20 mg/m3, the cytotoxic effects were greater after exposure to the 5-nm NP in the SA aerosol. This study showed that TiO2 NP use different mechanisms to induce their pulmonary toxicity as a function of their primary particle size and their agglomeration state.

La circulation extracorporelle (CEC) est une technique utilisée en chirurgie cardiaque effectuée des milliers de fois chaque jour à travers le monde. L’instabilité hémodynamique associée au sevrage de la CEC difficile constitue la principale cause de mortalité en chirurgie cardiaque et l’hypertension pulmonaire (HP) a été identifiée comme un des facteurs de risque les plus importants. Récemment, une hypothèse a été émise suggérant que l'administration prophylactique (avant la CEC) de la milrinone par inhalation puisse avoir un effet préventif et faciliter le sevrage de la CEC chez les patients atteints d’HP. Toutefois, cette indication et voie d'administration pour la milrinone n'ont pas encore été approuvées par les organismes réglementaires. Jusqu'à présent, la recherche clinique sur la milrinone inhalée s’est principalement concentrée sur l’efficacité hémodynamique et l'innocuité chez les patients cardiaques, bien qu’aucun biomarqueur n’ait encore été établi. La dose la plus appropriée pour l’administration par nébulisation n'a pas été déterminée, de même que la caractérisation des profils pharmacocinétiques (PK) et pharmacodynamiques (PD) suite à l'inhalation. L'objectif de notre recherche consistait à caractériser la relation exposition-réponse de la milrinone inhalée administrée chez les patients subissant une chirurgie cardiaque sous CEC. Une méthode analytique par chromatographie liquide à haute performance couplée à un détecteur ultraviolet (HPLC-UV) a été optimisée et validée pour le dosage de la milrinone plasmatique suite à l’inhalation et s’est avérée sensible et précise. La limite de quantification (LLOQ) était de 1.25 ng/ml avec des valeurs de précision intra- et inter-dosage moyennes (CV%) <8%. Des patients souffrant d’HP pour lesquels une chirurgie cardiaque sous CEC était prévue ont d’abord été recrutés pour une étude pilote (n=12) et, par la suite, pour une étude à plus grande échelle (n=28) où la milrinone (5 mg) était administrée par inhalation pré-CEC. Dans l'étude pilote, nous avons comparé l'exposition systémique de la milrinone peu après son administration avec un nébuliseur pneumatique ou un nébuliseur à tamis vibrant. L’efficacité des nébuliseurs en termes de dose émise et dose inhalée a également été déterminée in vitro. Dans l'étude à plus grande échelle conduite en utilisant exclusivement le nébuliseur à tamis vibrant, la dose inhalée in vivo a été estimée et le profil pharmacocinétique de la milrinone inhalée a été pleinement caractérisé aux niveaux plasmatique et urinaire. Le ratio de la pression artérielle moyenne sur la pression artérielle pulmonaire moyenne (PAm/PAPm) a été choisi comme biomarqueur PD. La relation exposition-réponse de la milrinone a été caractérisée pendant la période d'inhalation en étudiant la relation entre l'aire sous la courbe de l’effet (ASCE) et l’aire sous la courbe des concentrations plasmatiques (ASC) de chacun des patients. Enfin, le ratio PAm/PAPm a été exploré comme un prédicteur potentiel de sortie de CEC difficile dans un modèle de régression logistique. Les expériences in vitro ont démontré que les doses émises étaient similaires pour les nébuliseurs pneumatique (64%) et à tamis vibrant (68%). Cependant, la dose inhalée était 2-3 fois supérieure (46% vs 17%) avec le nébuliseur à tamis vibrant, et ce, en accord avec les concentrations plasmatiques. Chez les patients, en raison des variations au niveau des facteurs liés au circuit et au ventilateur causant une plus grande dose expirée, la dose inhalée a été estimée inférieure (30%) et cela a été confirmé après récupération de la dose de milrinone dans l'urine 24 h (26%). Les concentrations plasmatiques maximales (Cmax: 41-189 ng/ml) et l'ampleur de la réponse maximale ΔRmax-R0 (0-65%) ont été observées à la fin de l'inhalation (10-30 min). Les données obtenues suite aux analyses PK sont en accord avec les données publiées pour la milrinone intraveineuse. Après la période d'inhalation, les ASCE individuelles étaient directement reliées aux ASC (P=0.045). Enfin, notre biomarqueur PD ainsi que la durée de CEC ont été identifiés comme des prédicteurs significatifs de la sortie de CEC difficile. La comparaison des ASC et ASCE correspondantes a fourni des données préliminaires supportant une preuve de concept pour l'utilisation du ratio PAm/PAPm comme biomarqueur PD prometteur et justifie de futures études PK/PD. Nous avons pu démontrer que la variation du ratio PAm/PAPm en réponse à la milrinone inhalée contribue à la prévention de la sortie de CEC difficile. / Cardiopulmonary bypass (CPB) is a technique used during cardiac surgery performed thousands of times each day worldwide. Hemodynamic complications associated with difficult separation from CPB represent a leading cause of mortality in cardiac surgery and pulmonary hypertension (PH) was identified as one of the most important predictor and risk factor. Recently, inhaled milrinone administration prior to CPB was hypothesized to have a preventive effect and facilitate separation from CPB in patients with PH. However, this indication and route of administration have not yet been approved by regulatory agencies for milrinone. So far, research efforts on inhaled milrinone have mainly focused on evidence supporting hemodynamic efficacy and safety in cardiac patients although no biomarker has been established. The most appropriate dose for nebulization has never been determined, nor have pharmacokinetic (PK) and pharmacodynamic (PD) profiles been characterized after inhalation. The objective of the current research consisted of characterizing the exposure-response relationship for milrinone administered by inhalation in patients undergoing cardiac surgery. An improved high-performance liquid chromatography (HPLC) analytical assay using UV detection was validated for the quantification of milrinone in plasma after inhalation and proved to be sensitive and accurate. The lower limit of quantification (LLOQ) was 1.25 ng/ml with mean intra-assay and inter-assay precisions (CV%) <8%. Pulmonary hypertensive patients scheduled for cardiac surgery with CPB were first recruited for a pilot (n=12) and, subsequently, a full-scale (n=28) study where milrinone (5mg) was administered by inhalation pre-CPB. In the pilot study, milrinone early systemic exposure was investigated using a jet nebulizer or a mesh nebulizer. Nebulizers performance in terms of emitted and inhaled doses were also determined in vitro. In the full-scale study, using a mesh nebulizer exclusively, in vivo inhaled dose was estimated and milrinone definite pharmacokinetics fully characterized based on blood sampling and urine collection. Mean arterial pressure to mean pulmonary arterial pressure ratio (mAP/mPAP) was selected as the PD biomarker. Milrinone exposure-response relationship was characterized during the inhalation period by studying the relationship between individual area under the effect-time curve (AUEC) and corresponding area under the plasma concentration-time curve (AUC). Finally, the mAP/mPAP ratio, among other variables, was explored as a potential predictor of difficult separation from bypass in a multiple logistic regression model. In vitro experiments demonstrated that emitted doses were similar for the jet (64%) and the mesh (68.0%) nebulizers. However, the inhaled dose was 2-3 fold higher (46% vs 17%) after mesh nebulization, which was in agreement with plasma concentrations. In patients, due to variations in circuit-related and ventilator-related factors, the inhaled dose was estimated to be lower (30%) and this was confirmed by 24-h recovery in urine (26%). Milrinone peak plasma concentrations (Cmax: 41-189 ng/ml) and magnitude of peak response ΔRmax-R0 (0-65%) were observed at the end of inhalation (10-30 min). Data obtained from PK analysis agreed with published data for intravenous milrinone. After the inhalation period, individual AUEC were directly related to AUC (P=0.045). Finally, our PD biomarker, expressed as ΔRmax-R0, as well as CPB duration, were both identified as significant predictors of DSB. The comparison of corresponding AUC and AUEC provided preliminary evidence of a proof of concept for the use of the mAP/mPAP ratio as a promising PD biomarker and warrants future PK/PD studies. Indeed, mAP/mPAP ratio variation in response to inhaled milrinone was found to contribute in the prevention of DSB.

En raison de leur petite taille, les nanoparticules (NP) (< 100 nm) peuvent coaguler très rapidement ce qui favorise leur pénétration dans l’organisme sous forme d’agglomérats. L’objectif de cette recherche est d’étudier l’influence de l’état d’agglomération de NP de dioxyde de titane (TiO2) de trois tailles de départ différentes, 5, 10-30 ou 50 nm sur la toxicité pulmonaire chez le rat mâle (F344) exposé à des aérosols de 2, 7 ou 20 mg/m3 pendant 6 heures. Dans une chambre d’inhalation, six groupes de rats (n = 6 par groupe) ont été exposés par inhalation aiguë nez-seulement à des aérosols ayant une taille primaire de 5 nm, mais produits sous forme faiblement (< 100 nm) ou fortement (> 100 nm) agglomérée à 2, 7 et 20 mg/m3. De façon similaire, quatre autres groupes de rats ont été exposés à 20 mg/m3 à des aérosols ayant une taille primaire de 10-30 et 50 nm. Les différents aérosols ont été générés par nébulisation à partir de suspensions ou par dispersion à sec. Pour chaque concentration massique, un groupe de rats témoins (n = 6 par groupe) a été exposé à de l’air comprimé dans les mêmes conditions. Les animaux ont été sacrifiés 16 heures après la fin de l’exposition et les lavages broncho-alvéolaires ont permis de doser des marqueurs d’effets inflammatoires, cytotoxiques et de stress oxydant. Des coupes histologiques de poumons ont également été analysées. L’influence de l’état d’agglomération des NP de TiO2 n’a pu être discriminée à 2 mg/m3. Aux concentrations massiques de 7 et 20 mg/m3, nos résultats montrent qu’une réponse inflammatoire aiguë est induite suite à l'exposition aux aérosols fortement agglomérés. En plus de cette réponse, l’exposition aux aérosols faiblement agglomérés à 20 mg/m3 s’est traduite par une augmentation significative de la 8-isoprostane et de la lactate déshydrogénase. À 20 mg/m3, les effets cytotoxiques étaient plus importants suite à l’exposition aux NP de 5 nm faiblement agglomérées. Ces travaux ont montré dans l'ensemble que différents mécanismes de toxicité pulmonaire peuvent être empruntés par les NP de TiO2 en fonction de la taille de départ et de l’état d’agglomération. / Given their small size, nanoparticles (NP) (< 100 nm) can coagulate quickly, which promotes their entry into the body in the form of agglomerates. The objective of this study is to evaluate the influence of the agglomeration state of three different primary particle sizes (5, 10-30 and 50 nm) of titanium dioxide (TiO2) NP on the pulmonary toxicity of male rats (F344) exposed to aerosols at 2, 7 or 20 mg/m3 for 6 hours. In an inhalation chamber, six groups of rats (n = 6 per group) were acutely exposed by nose-only inhalation to aerosols with a 5-nm primary particle size, produced in the form of small agglomerates (< 100 nm) (SA) or large agglomerates (> 100 nm) (LA) at 2, 7 and 20 mg/m3. Similarly, four other groups of rats were exposed to aerosols at 20 mg/m3 with a primary particle size of 10-30 and 50 nm. The different aerosols were generated by nebulization of suspensions or by dry dispersion. For each mass concentration, one group of control rats (n = 6 per group) was exposed to compressed air under the same conditions. The animals were sacrificed 16 hours after the end of exposure, and analysis of the bronchoalveolar lavage fluids was used to measure markers of inflammatory, cytotoxicity and oxidative stress effects. Lung sections were also analyzed for histopathology. The influence of the agglomeration state of TiO2 NP (5 nm) could not be determined at 2 mg/m3. For mass concentrations of 7 and 20 mg/m3, our results showed that an acute inflammatory response was induced following exposure to LA aerosols. In addition to this response, exposure to SA aerosols resulted in a significant increase in 8-isoprostane and lactate dehydrogenase. At 20 mg/m3, the cytotoxic effects were greater after exposure to the 5-nm NP in the SA aerosol. This study showed that TiO2 NP use different mechanisms to induce their pulmonary toxicity as a function of their primary particle size and their agglomeration state.

Thesis (M.A.)--University of South Florida, 2006. / Title from PDF of title page. Document formatted into pages; contains 52 pages. Includes bibliographical references.

Les tumeurs pulmonaires, qu’elles soient primaires (principalement représentées par le cancer du poumon non-à-petites cellules) ou secondaires (métastases), causent la mort de plusieurs centaines de milliers de personnes par an à travers le monde. Malgré les modalités de traitements existantes, un plateau thérapeutique a été atteint avec un taux de survie à 5 ans de maximum 15%. Actuellement, il est connu que le cancer du poumon non-à-petites cellules ainsi que les métastases sont intrinsèquement résistants à l’apoptose.<p>Pour apporter des réponses aux principales problématiques rencontrées avec l’administration systémique de la chimiothérapie conventionnelle qui est principalement constituée d’agents pro-apoptotiques, nous avons développé des formulations à base d’agents antinéoplasiques aux propriétés anticancéreuses non pro-apoptotiques qui sont destinées à être administrées de manière localisée par la voie inhalée. Il faut savoir que l’inhalation est la voie d’administration préférentielle des principales affections respiratoires telles que l’asthme, la bronchopneumonie chronique obstructive et la mucoviscidose. <p>La première partie de ce travail a consisté à produire et à évaluer des formulations à base de témozolomide destinées à être administrées chez la souris porteuse de pseudo-métastases pulmonaires (issues d’un mélanome expérimental, le modèle B16F10), soit via la voie intraveineuse (iv) conventionnelle soit via la voie inhalée à l’aide d’un dispositif endotrachéal approprié. La suspension pour inhalation a été produite à l’aide de technique de réduction de taille et a été stabilisée à l’aide de phospholipides compatibles avec la voie pulmonaire. L’activité anticancéreuse in vitro a été vérifiée pour le témozolomide formulé sous forme de suspension pour inhalation et de solution intraveineuse par rapport à du témozolomide non formulé sur des lignées de cellules cancéreuses de cancer humain NSCLC A549, de glioblastome humain T98G et de mélanome murin B16F10. Cette dernière lignée a été utilisée pour générer les pseudo-métastases pulmonaires chez la souris en injectant les cellules de mélanomes dans la voie systémique via la veine caudale. La reproductibilité de la dose et de l’aérosol générés à partir de la suspension pour inhalation à l’aide du dispositif d’administration endotrachéal et la déposition des gouttelettes dans les poumons de la souris ont pu être respectivement évaluées avec précision par une méthode de quantification du témozolomide qui a été validée par nos soins, par des techniques de diffraction laser et par des techniques de microscopie à fluorescence et d’analyse d’images histologiques. Enfin, l’activité antitumorale in vivo et la tolérance des traitements conventionnels ou localisés ont été vérifiées chez la souris porteuse de ces pseudo-métastases pulmonaires B16F10. Les résultats ont montré que le dispositif endotrachéal utilisé permettait de produire des doses et des aérosols reproductibles et de déposer les gouttelettes d’aérosol profondément dans les poumons des souris. De plus, lors de l’étude in vivo, les traitements administrés étaient bien tolérés et la dose de témozolomide administré sous forme de suspension pour inhalation à l’aide du dispositif endotrachéal avait permis d’obtenir une efficacité antitumorale similaire à une dose similaire de témozolomide administrée par la voie iv conventionnelle. De plus, 11% (3/27) de souris « long-survivantes » avaient été observées avec le groupe traité par inhalation trois fois par semaine pendant trois semaines consécutives et les poumons de ces long-survivants avaient présenté une éradication quasi complète des tumeurs pulmonaires. Ce phénomène n’avait pas été observé dans les groupes de souris traitées de manière conventionnelle.<p>Ensuite, la seconde partie de notre travail a consisté en l’élaboration du témozolomide sous forme de poudres sèches pour inhalation destinées à être délivrées à l’aide d’un dispositif à poudre sèche à usage humain. Pour ce faire, nous avons développé les poudres sèches pour inhalation à l’aide de techniques de réduction de taille pour microniser la poudre de départ et d’atomisation pour évaporer le solvant et élaborer un enrobage autour des particules micronisées. La nature de l’enrobage était soit hydrophile soit lipophile. Ensuite les caractéristiques physicochimiques telles que les propriétés thermiques, les propriétés cristallines, la distribution de taille particulaire et la morphologie des formulations de poudre sèche pour inhalation ont été évalués à l’aide de techniques appropriées telles que la calorimétrie différentielle à balayage, la diffraction des rayons X sur poudre, la diffraction de la lumière laser et la microscopie électronique à balayage. Les profils de déposition pulmonaire et de dissolution ont été respectivement déterminés in vitro à l’aide de l’essai de la pharmacopée européenne utilisant l’impacteur à cascade multi-étages et d’un test de dissolution adapté aux formes pulmonaires. Les quatre formulations élaborées présentaient des caractéristiques physicochimiques intéressantes pour la stabilité à long-terme de la substance active et des formulations. De plus, deux formulations de poudre sèche pour inhalation (les formulations F1 et F2) présentaient des propriétés aérodynamiques tout à fait attrayantes avec une fraction minimale de poudre déposée au niveau du tractus respiratoire supérieure et une fraction maximale de poudre déposée au niveau du tractus respiratoire inférieur où se localisent les tumeurs pulmonaires. De plus, l’ensemble des formulations ont montré que la fraction sélectionnée des particules fines des poudres sèches pour inhalation libérait 75% du témozolomide dans le liquide simulant le fluide pulmonaire endéans les dix premières minutes du test de dissolution in vitro adapté aux formes pulmonaires. <p>Enfin, nous avons comparé l’efficacité et la tolérance in vivo d’une de nos formulations de poudre sèche de témozolomide pour inhalation administrée soit sous forme de suspension, soit sous forme de poudre sèche, à l’aide du dispositif endotrachéal approprié chez la souris porteuse de pseudo-métastases pulmonaires. L’uniformité de la dose délivrée par les différents dispositifs a été évaluée à l’aide d’une technique quantitative validée. Les résultats de cette étude ont montré qu’en administrant une formulation de poudre sèche sous forme d’un mélange de poudres plutôt que sous forme d’une suspension liquide, les doses en témozolomide à administrer pour obtenir une efficacité comparable était au moins deux fois moins élevées. Cependant, le dispositif endotrachéal pour les formulations de poudre présentait plus de variabilité au niveau de la dose délivrée que le dispositif endotrachéal pour les formulations liquides ce qui induit une variabilité dans les doses délivrées. Pour clôturer ce travail, nous avons appliqué certaines techniques que nous avons développées pour le témozolomide à une nouvelle molécule de synthèse, le trivanillate polyphénolique 13c, qui montre des propriétés anticancéreuses intéressantes dans le cadre des tumeurs pulmonaires. En effet, une méthode quantitative a été développée et a été validée. Une étude de pré-formulation et des essais de formulation pour produire une suspension, des complexes d’inclusion et des microparticules lipidiques ont été entrepris avec de relativement bons résultats pour les complexes d’inclusion élaborés avec des gamma cyclodextrines qui permettaient d’augmenter la solubilité dans l’eau de la molécule de 13c d’un facteur d’au moins 1,5×106. De plus, les particules de 13c montraient la particularité de se solubiliser dans un mélange dichlorométhane/éthanol (1 :1 v/v) ce qui nous a permis d’élaborer des microparticules lipidiques pour lesquelles les propriétés de mouillage devront être améliorées dans l’avenir./<p>Primary lung tumors, mainly represented by non-small-cell lung cancers (cancers NSCLC), or secondary lung tumors (metastasis) cause the death of hundred thousand human beings worldwide. Despite the therapeutic modalities used, the five-year survival rate reaches only 15%. Nowadays, it is known that cancers NSCLC and metastasis are intrinsically resistant to apoptosis.<p>To overcome the main problems occurring with the systemic delivery of conventional chemotherapy which are mainly constituted of non-specific and non selective pro-apoptotic agents, we have developed some formulations based on non pro-apoptotic antineoplasic drugs which are designed to be delivered by a localized administration, the inhalation. Indeed, inhalation is the preferential route to treat the main pulmonary affections such as asthma, chronic obstructive pulmonary disease or cystic fibrosis.<p>The first part of this work consisted to produce and evaluate temozolomide-based formulations designed to be delivered to mice bearing pulmonary pseudo-metastases (using a experimental melanoma, the B16F10 model), either by the conventional intravenous (iv) route or by inhalation using an endotracheal device appropriate to mice. The suspension for inhalation was produced by means of a high pressure homogenizing technique using phospholipids compatible with the lungs to stabilize the suspension. The in vitro anticancer activity was evaluated for both temozolomide-based formulations in comparison with non-formulated temzolomide on three cancer cell lines, a human NSCLC cancer cells (A549), a human glioblastoma cancer cells (T98G) as positive control and a murine melanoma cancer cells (B16F10). The latter was used to generate lung tumors in mice by injecting the melanoma cells by iv. Reproducibility of delivered dose and generated aerosol by the endotracheal device from the suspension for inhalation and the deposition of droplets in the mouse lungs were precisely evaluated by means of a validated HPLC determination method, a laser diffraction technique and fluorescent microscopy and histological image analysis, respectively. Then, the tolerance and the antitumor efficacy of iv or inhaled temozolomide-based treatments were evaluated on mice bearing pulmonary pseudo-metastases B16F10. The results showed that endotracheal device produced reproducible doses and aerosols and that the aerosol droplets were deposited deeply in the mouse lungs. Moreover, the temozolomide-based treatments were well tolerated in terms of weight evolution and the inhaled based-temozolomide treatments were able to get the same antitumor efficacy in terms of median survival rate as the conventional iv based-temozolomide treatments delivered at a same frequency. Moreover with the group treated by inhalation three times a week during three consecutive weeks, 11% (3/27) mice survived with an almost complete eradication of lung tumors which was not observed with the groups treated by conventional route.<p>Then, the second part of our work consisted to produce temozolomide-based dry powders for inhalation able to be delivered with a dry powder inhaler for human use. We developed the dry powders for inhalation using a high-pressure homogenizing technique to micronize temozolomide particles and then spray-drying technique to coat temozolomide microparticles. The coating was either hydrophilic or lipophilic. Then, the physicochemical characteristics such as thermal or crystalline properties, the particle size distribution and the particle morphology were evaluated for the four dry powders for inhalation by means of differential scanning calorimetry, x-ray powder diffraction, laser light scattering and scanning electron microscopy, respectively. The in vitro pulmonary deposition and dissolution were respectively determined by European pharmacopeia assay for the aerodynamic assessment of fine particles using a multi-stage liquid impinger and by dissolution test optimized for inhaler products. The four formulations produced presented physicochemical properties promoting long-term stability of temozolomide and formulations.Moreover, two of them (dry powder without coating or with a thin lipid coating) showed attractive aerodynamic properties with a minimal fraction of powder deposited in the oropharyngeal and tracheal zones and maximal fraction deposited in the lungs (almost 50% of the nominal dose) where the lung tumors are localized. Moreover, fine particle fraction of all formulations showed a fast release and dissolution of temozolomide with more than 75% of temozolmide dissolved within 10 minutes in the simulated lung fluid during the in vitro dissolution test optimized for dry powders for inhalation.<p>Then, we compared the in vivo antitumor efficacy and tolerance of one of dry powders for inhalation on mice bearing pulmonary pseudo-metastases B16F10. The dry powder for inhalation was administered either by dispersing it as a extemporaneous suspension able to be delivered by the endotracheal device for liquid forms or by mixing it with a spray-dried diluent able to be delivered by the endotracheal device for dry powders. The uniformity of delivered dose by the different endotracheal device was evaluated by a validated quantitative method. The results showed that the delivery of the powder mixture presented the same antitumor efficacy as the extemporaneous suspension but for a half dose of temozolomide. However, the endotracheal device for dry powders presented a higher variability of delivered dose than the endotracheal device for liquid forms.<p>Finally, we apply the pulmonary application on a polyphenol developed in the Faculty of Pharmacy, the molecule 13c, that showed very interesting in vitro anticancer properties against lung tumors. So, a quantitative method was developed and was validated. A preformulation studie was performed and formulation developements are on-going.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

While realistic in vitro testing of dry powder inhalers (DPIs) can be used to establish in vitro–in vivo correlations (IVIVCs) and predict in vivo lung doses, the aerodynamic particle size distributions (APSDs) of those doses and their regional lung deposition remains unclear. Four studies were designed to improve testing centered on the behavior of Novolizer®. Different oropharyngeal geometries were assessed by testing different mouth-throat (MT) models across a realistic range of inhalation profiles (IPs) with Salbulin® Novolizer®. Small and large Virginia Commonwealth University (VCU) and Oropharyngeal Consortium (OPC) models produced similar ranges for total lung dose in vitro (TLDin vitro), while results for medium models differed significantly. While either group may be selected to represent variations in oropharyngeal geometry, OPC models were more difficult to use, indicating that VCU models were preferable. To facilitate simulation of human IPs through DPIs, inhalation profile data from a VCU clinical trial were analyzed. Equations were developed to represent the range of flow rate vs. time curves for use with DPIs of known airflow resistance. A new method was developed to couple testing using VCU MT models and simulated IPs with cascade impaction to assess the APSDs of TLDin vitro for Budelin® Novolizer®. This method produced IVIVCs for Budelin’s total lung dose, TLD, and was sufficiently precise to distinguish between values of TLDin vitro and their APSDs, resulting from tests using appropriately selected MT models and IPs. For example, for slow inhalation, TLD values were comparable in vivo and in vitro; TLDin vitro ranged from 12.2±2.9 to 66.8±1.7 mcg aerosolized budesonide while APSDs in vitro had mass median aerodynamic diameters of 3.26±0.27 and 2.17±0.03 µm, respectively. To explore the clinical importance of these variations, a published computational fluid dynamic (CFD) model was modified and coupled to accept the output of realistic in vitro tests as initial conditions at the tracheal inlet. While simplified aerosol size metrics and flow conditions used to shorten CFD simulations produced small differences in theoretical predictions of regional lung deposition, the results broadly agreed with the literature and were generally consistent with the median values reported clinically for Budelin.

Human health risk assessments are used by environmental regulatory agencies to determine risk from Hazardous Air Pollutants (HAPs). In this study, the Human Exposure Model (HEM-3) was used to compare the cancer and non-cancer inhalation health effects of a single organic chemical manufacturing facility in Geismar, Louisiana prior to and after Maximum Achievable Control Technologies (MACT) were implemented. The results indicate significant reductions in both cancer risk and non-cancer hazards. The analysis also indicated that the equivalent cancer risk reduction could have been achieved by addressing MACT in only one production process and one single pollutant (ethylene dichloride) within that process. This demonstrates the value that these risk assessments have at evaluating emissions at the facility level, and how they could be used in the control strategy decision making process.

Este estudo objetivou avaliar a condição clínica pré-operatória dos cães idosos submetidos à anestesia inalatória e relacionar as alterações encontradas na avaliação pré-operatória com a ocorrência de complicações e óbito perioperatório. O estudo foi prospectivo, observacional e realizado no período de abril de 2007 a abril de 2008. Os cães idosos foram avaliados por meio de anamnese, exame físico, mensuração da pressão arterial sistólica (Doppler), eletrocardiograma (ECG) e análises laboratoriais previamente ao procedimento cirúrgico. O procedimento anestésico, cirúrgico e a ocorrência de complicações transoperatórias foram analisados pelo registro nas fichas de anestesia. Incluíram-se no estudo os animais submetidos à anestesia inalatória por no mínimo 30 minutos. As complicações pós-operatórias foram avaliadas por meio da consulta pós-operatória, do registro nos prontuários e relato dos proprietários. Os grupos foram comparados por meio do teste de Mann-Whitney. Nas variáveis categóricas foi empregado o teste do qui-quadrado ou o teste exato de Fisher. O grau de significância foi de 5%. Foram incluídos no estudo 169 cães, 97 machos e 72 fêmeas, com idade média de 10,4&plusmn;2,2 anos (125,0&plusmn;26,6 meses) e com peso médio de 19,8&plusmn;12,3 kg. A idade e peso não apresentaram diferença entre os sexos (p&gt;0,05). O hematócrito abaixo dos valores normais ocorreu em 16,0% (27/169) dos animais e houve associação com a ocorrência de complicações (21/169=12,43%) e óbito (4/169=2,37%) perioperatório (p&lt;0,01). A concentração de hemoglobina abaixo dos valores normais (4/169=2,37%) e a necessidade de transfusão de hemocomponentes foram associadas com o desfecho de óbito no pós-operatório (p&lt;0,01). Os animais classificados com categoria de risco anestésico (ASA) III (34/169=20,12%) e IV (2/169=1,18%) apresentaram maior ocorrência de complicações (p&lt;0,05) e óbito (p&lt;0,01) perioperatório quando comparado a ASA II (133/169=78,70%). Nos cães categorizados com risco cirúrgico médio (96/169=56,80%) e alto (9/169=5,33%) ocorreu maior morbidade e mortalidade perioperatório quando comparado ao baixo (64/169=37,87%) (p&lt;0,01). Houve menor mortalidade com o emprego da acepromazina (89/169=52,66%) na medicação pré-anestésica (p&lt;0,05). A hipotensão arterial foi a complicação mais frequente (78/169=46,15%) no transoperatório, mas não foi associada ao uso de acepromazina e desfecho de óbito (p&gt;0,05). A administração de fentanil em bolus (54/169=31,95%) foi associada com a hipotensão arterial transoperatória (p&lt;0,05). A duração do procedimento anestésico acima de 75 minutos apresentou maior ocorrência de complicações perioperatórias (p&lt;0,01). A morbidade e mortalidade perioperatória observada nos cães idosos foi de 56,21% (IC95%: 48,38-63,82%) e 2,96% (IC95%: 0,97-6,77%), respectivamente. A partir dos resultados pode-se concluir que: o hematócrito abaixo do valor de referência indica maior ocorrência de complicações e óbito perioperatório; a concentração de hemoglobina abaixo do valor de referência e a necessidade de transfusão de hemocomponentes determinam maior mortalidade perioperatória; a classificação de risco anestésico e cirúrgico são métodos válidos na determinação dos pacientes mais propensos a ocorrência de complicações e o desfecho de óbito; a administração de acepromazina reduz a mortalidade; o procedimento anestésico superior a 75 minutos aumenta a ocorrência de complicações; e a morbidade e mortalidade perioperatória são elevadas nos cães idosos. / This study aimed to evaluate the preoperative clinical condition in elderly dogs undergoing inhalation anesthesia and relate the changes found in the preoperative evaluation with the occurrence of perioperative complications and death. This study was prospective, observational and accomplished from April 2007 to April 2008. The elderly dogs were evaluated by clinical history, physical examination, measurement of systolic blood pressure (Doppler), electrocardiogram (ECG) and laboratory tests before surgery. The anesthetic and surgical procedure and the occurrence of intraoperative complications were analyzed according to the anesthesia records. This study included the animals submitted to inhalation anesthesia for at least 30 minutes. The postoperative complications were evaluated by postoperative visit, enrollment in medical records and owners&#39; reporting. The groups were compared using the Mann-Whitney test. For the categorical variables it was used the chi-square or Fisher&#39;s exact test. The level of significance was 5%. The study included 169 dogs, 97 males and 72 females, mean age 10.4&plusmn;2.2 years old (125.0&plusmn;26.6 months old) and mean weight was 19.8&plusmn;12.3 kg. The age and weight did not differ between the genders (p&gt;0.05). The hematocrit below normal values occurred in 16.0% (27/169) of the animals and was associated with the occurrence of perioperative (p&lt;0.01) complications (21/169=12.43%) and death (4/169=2.37%). The hemoglobin concentration below normal values (4/169=2.37%) and the need for blood components transfusion were associated with the outcome of postoperative death (p&lt;0.01). The animals classified as anesthetic risk category (ASA) III (34/169=20.12%) and IV (2/169=1.18%) had a higher perioperative complication (p&lt;0.05) and death (p&lt;0.01) compared to ASA II (133/169=78.70%). The dogs categorized as moderate (96/169=56.80%) and high (9/169=5.33%) surgical risk had higher perioperative morbidity and mortality when compared to low (64/169=37.87%) (p&lt;0.01). There was a lower mortality with the use of acepromazine (89/169=52.66%) in premedication (p&lt;0.05). Hypotension was the most frequent complication (78/169=46.15%) during surgery, but was not associated with the use of acepromazine and outcome of death (p&gt;0.05). The administration of fentanyl (54/169=31.95%) was associated with intraoperative hypotension (p&lt;0.05). The duration of anesthesia for over 75 minutes had a higher incidence of perioperative complications (P&lt;0.01). The perioperative morbidity and mortality observed in older dogs was 56.21% (95% CI: 48.38-63.82%) and 2.96% (95% CI: 0.97-6.77%), respectively. According to the results it can be concluded that: the hematocrit below the reference value indicates a higher incidence of perioperative complications and death; the hemoglobin concentration below the reference value and the need for blood components transfusion determine higher perioperative mortality; the classification of anesthetic and surgical risk are valid methods in determining the most likely patients to complications and outcome of death; acepromazine administration reduces mortality; anesthesia for over 75 minutes increases the complications; and perioperative morbidity and mortality are high in elderly dogs.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-11-01T14:43:59Z No. of bitstreams: 1 TESE REVISADA 11.pdf: 1670065 bytes, checksum: b89c59f7fd857c80bace24d161f11331 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-11-06T16:27:20Z (GMT) No. of bitstreams: 1 TESE REVISADA 11.pdf: 1670065 bytes, checksum: b89c59f7fd857c80bace24d161f11331 (MD5) / Made available in DSpace on 2018-11-06T16:39:53Z (GMT). No. of bitstreams: 1 TESE REVISADA 11.pdf: 1670065 bytes, checksum: b89c59f7fd857c80bace24d161f11331 (MD5) Previous issue date: 2018-09-28 / Objective: To evaluate the level of knowledge in asthma and health literacy of parents of children and adolescents diagnosed with the disease. Methods: A cross-sectional study that evaluated 120 children and adolescents aged between one and 17 years and their legal guardians in the city of Vit?ria / ES. The following questionnaires were applied: Brazil's Economic Classification Criteria (CCEB), Clinical Questionnaire, Global Initiative for Asthma (GINA), Knowledge in Pediatric Asthma (QCAP), Short Assessment of Health Literacy for Portuguese Adults (SAHLPA-18). To evaluate the inhalation techniques, a questionnaire on theoretical inhalotherapy and practical analysis was applied. For the purpose of analyzing the understanding of the medical prescription, the type of prescription (rescue or preventive) and the understanding about the medical prescription were evaluated with the parents. Results: Most asthmatic patients (75%) do not have disease control. In the last 12 months the results showed that the controlled asthma group had the highest percentages of continuous treatment (p <0.01), with lower scores for use of oral corticosteroids (p = 0.01) and bronchodilator use (p = 0.01). In the evaluation of the level of reading of the medical prescription and understanding about the prescription and the type of treatment, the results demonstrate that the greater the control of the disease, the better the percentage of understanding on the part of those responsible. Conclusion: The difficulty of control is a relevant problem in the management of children with asthma. Parents of children with controlled asthma demonstrate a higher level of knowledge about health and asthma, as well as having a better understanding about medical prescription, treatment techniques for the disease. / Objetivo: Avaliar o n?vel de conhecimento em asma e alfabetismo em sa?de de pais de crian?as e adolescentes com diagn?stico da doen?a. Met?dos: Estudo transversal que avaliou 120 crian?as e adolescentes com idade entre um a 17 anos e seus respons?veis legais no munic?pio de Vit?ria/ES. Foram aplicados os seguintes question?rios: Crit?rio de Classifica??o Econ?mica Brasil (CCEB), Question?rio Clinico, Global Initiative for Asthma (GINA), Conhecimento em Asma Pedi?trica (QCAP), Short Assessment of Health Literacy for Portuguese Adults (SAHLPA-18). Para avalia??o das t?cnicas inalat?rias foi aplicado um question?rio sobre inaloterapia te?rico e an?lise pr?tica. Para fins de an?lise do entendimento do receitu?rio m?dico, foi avaliado junto aos pais o tipo de receitu?rio (resgate ou preventivo) e o entendimento sobre a prescri??o m?dica. Resultados: A maior parte dos pacientes asm?ticos (75%) n?o possui controle da doen?a. Nos ?ltimos 12 meses os resultados demonstram que o grupo de asma controlada possui os maiores percentuais de tratamento cont?nuo (p<0,01), com menores escores para uso de corticoides orais (p=0,01) e uso de broncodilatadores (p=0,01). Na avalia??o do n?vel de leitura do receitu?rio m?dico e entendimento sobre a prescri??o e o tipo de tratamento os resultados demonstram que quanto maior ? o controle da doen?a, melhores s?o os percentuais de entendimento por parte dos respons?veis. Conclus?o: A dificuldade de controle ? um problema relevante no manejo de crian?as com asma. Pais de crian?as com asma controlada demonstram possuir maior n?vel de conhecimento sobre sa?de e sobre asma, al?m de possu?rem melhor entendimento sobre prescri??o m?dica, t?cnicas de tratamento para a doen?a.

Three main projects are included in this dissertation. Though seemingly broad in scope, this research afforded a unique opportunity for comprehensive study of urban and rural environmental inhalation exposures to endotoxin (lipopolysaccharide LPS) and the associated immune response. In the LA PM10 Endotoxin Study, ambient concentration of LPS in PM (particulate matter) was quantified through analysis of air samples collected in Southern California. Endotoxin concentrations measured were lower than recognized thresholds for adverse health effects in occupational exposures, but in the same range as for indoor effects. This study provides the first extensive characterization of endotoxin concentration across a large metropolitan area in relation to PM10 and other pollutant monitoring, and supports the need for studies of the role of endotoxin in childhood asthma in urban settings. The Mouse Whole Blood Assay (WBA) Study replicated LPS-induced airway inflammation in a laboratory model. Presently, there is a need for less invasive options for evaluating pulmonary responses to occupational exposures. The whole blood assay (WBA), which measures cytokine production of leukocytes after ex vivo stimulation with LPS, may be one such option. This study used an endotoxin-tolerance model to demonstrate the efficacy of the WBA as a biomarker of inhalation exposure to swine concentrated animal feeding operation (CAFO) dust and showed the utility of the WBA for assessing susceptibility to organic dust-induced lung inflammation. Finally, The Human WBA Study applied the WBA outside the controlled environment of the laboratory. This study utilized pulmonary function testing (PFT), symptom questionnaires and the WBA to evaluate inflammatory responses following an inhalation exposure to purified LPS in CAFO workers and controls. Subjects were stratified into response groups for analysis of WBA results based on PFT response. All subjects demonstrated significant WBA LPS-stimulant dose-responses for all 3 cytokines measured. This study demonstrated that LPS-induced pulmonary and WBA responses are variable among individuals and offered insight into the use of the WBA in future studies. Information gained from these studies provides much insight into urban endotoxin concentrations, the use of the WBA as a biomarker of pulmonary exposure in the rural environment, and offers possibilities for further research.