La pyrazinamide est le seul anti-tuberculeux de première intention actif sur la formedormante de Mycobacterium tuberculosis. Sa prescription par voie orale permet de réduire la durée du traitement de 9 à 6 mois. Nous avons développé des formes galéniques de pyrazinamide administrables directement au niveau des poumons afin d'augmenter localement la concentration de pyrazinamide au site pathologique afin de réduire la durée du traitement. Deux formes galéniques de pyrazinamide ont été optimisées: une poudre sèche pour inhalation et des nanoparticules polymères administrables par nébulisation liquide ou sous forme de poudre sèche.La poudre sèche pour inhalation est composée de particules obtenues par atomisation-séchage. La pyrazinamide a été solubilisée dans un mélange 70/30 v/véthanol/eau. Après atomisation-séchage de cette solution, nous avons obtenu des particules cristallines instables et non adaptées à l'administration pulmonaire du fait de leur grande taille. Afin d'obtenir des poudres adaptées à une administration pulmonaire dans le poumon profond, et stables en termes de taille et de caractéristiques physico-chimiques, nous avons passé en revue toute une série d'excipients: phospholipides, bicarbonate d'ammonium, leucine, acide hyaluronique.Nous avons montré qu'en associant tous ces excipients au principe actif, on pouvait obtenir des particules d'environ 6 microns, de faible densité tassée et stables pendant 4 semaines dans des conditions de stockage classiques.L'évaluation aérodynamique in vitro de la poudre optimisée a révélé l'existence de deux populations de particules: de grosses particules pauvres en pyrazinamide et de petites particules riches en pyrazinamide. Ces deux populations proviennent d'une ségrégation des différents composants lors du processus de séchage. Pour remédier à ce phénomène et obtenir des particules de composition homogène, la vitesse de séchage a été diminuée. En conséquence, nous avons obtenu des poudres homogènes avec de bonnes propriétés aérodynamiques pour délivrance dans les poumons: fraction de particules fines de 40,1 ± 1,0% et fraction alvéolaire de 29,6 ±3,1%. Cette poudre a alors été évaluée in vivo chez le rat sain et nous avons mesuré les concentrations de pyrazinamide dans le plasma et le liquide de lavage bronchoalvéolaire après insufflation intratrachéale de la poudre, par comparaison avec une administration intraveineuse d'une solution de pyrazinamide. L'insufflation intratrachéale de poudre et l'administration intraveineuse conduisent à des paramètres pharmacocinétiques similaires prouvant que les particules se dissolvent rapidement lors du dépôt et que la molécule traverse efficacement la barrière pulmonaire pour atteindre la circulation systémique. De manière surprenante, la pyrazinamide est éliminée plus rapidement du liquide pulmonaire lorsqu'elle est administrée par insufflation intratrachéale que par voie intraveineuse. La délivrance pulmonaire de pyrazinamide apparaît comme une alternative intéressante à l'administration orale de la molécule et doit maintenant être testée dans un modèle d'animal infecté pour évaluer son efficacité contre Mycobacterium tuberculosis.En parallèle, nous avons optimisé l'encapsulation de pyrazinamide dans des nanoparticules polymères de poly(lactide-co-glycolide) PLGA monodisperses de taille inférieure à 200nm, grâce un plan d'expériences. Les nanoparticules de PLGA chargées en pyrazinamide ont été préparées par la méthode d'émulsion double. La méthode de Taguchi a été utilisée pour optimiser les paramètres de formulation. Le type de solvant, le rapport en poids pyrazinamide/ PLGA et le rapport des volumes des phases aqueuse et organique étaient les paramètres pertinents. La méthode de Taguchi s'est avérée efficace pour optimiser les nanoparticules d'environ 170nm avec un indice de polydispersité ˂ 0,1, un potentiel zêta d'environ -1mV et une efficacité d'encapsulation de 7-8% soit 3% de taux de charge de la pyrazinamide. / Pyrazinamide is the only first intention anti-TB drug active on the dormant form ofMycobacterium tuberculosis. Its oral prescription reduces treatment duration from 9to 6 months. We have developed dosage forms of pyrazinamide to administer directlyto the lungs to locally increase the concentration of pyrazinamide at the diseased siteand further reduce the duration of treatment. Two dosage forms of pyrazinamidewere optimized: a dry powder for inhalation and polymer nanoparticles administrableeither by liquid nebulization or as a dry powder.The dry powder for inhalation is composed of particles obtained by spray-drying.Pyrazinamide was dissolved in a mixture 70/30 v/v ethanol/water. After spray-dryingthe solution, we obtained large crystalline particles that were unstable and notsuitable for pulmonary administration because of their large sizes. To obtain powderssuitable for pulmonary delivery to the deep lungs, and stable in terms of size andphysico-chemical characteristics, we reviewed a variety of excipients: phospholipids,ammonium bicarbonate, leucine, hyaluronic acid. We have shown that by combiningall these excipients with the drug, one could obtain particles of about 6 microns, witha low tapped density and stable for 4 weeks under conditions of conventionalstorage.The in vitro aerodynamic evaluation of the optimized powder showed the existence oftwo populations of particles: large particles with a low content of pyrazinamide andsmall particles with high pyrazinamide content. These two populations derived fromthe segregation of different components during the drying process. To obtainparticles of uniform composition, the drying rate was decreased. As a result, weobtained homogeneous powders with good aerodynamic properties for delivery intothe lungs: fine particle fraction of 40.1 ± 1.0% and alveolar fraction of 29.6 ± 3.1%.This powder was then evaluated in vivo in healthy rats and we measured theconcentrations of pyrazinamide in plasma and bronchoalveolar lavage fluid afterintratracheal insufflation of the powder in comparison with intravenous administrationof a solution of pyrazinamide. The intratracheal insufflation of the powder and theintravenous injection lead to similar pharmacokinetic parameters proving that theparticles dissolve rapidly after deposition and pyrazinamide crosses efficiently thelung barrier to reach the systemic circulation. Surprisingly, pyrazinamide disappears4faster form lung lining fluid when administered by pulmonary insufflation than afterintravenous administration. Pulmonary delivery of pyrazinamide appears as anattractive alternative to oral administration of the drug and must now be tested in ananimal model of infection to assess its efficacy against Mycobacterium tuberculosis.In parallel, we have optimized the encapsulation of pyrazinamide in polymericnanoparticles of poly (lactide-co-glycolide) PLGA lower than 200 nm andmonodisperse, using experimental design. The pyrazinamide-loaded PLGAnanoparticles were prepared by the double emulsion method. The Taguchi methodwas used to optimize the formulation parameters. The type of solvent, thepyrazinamide / PLGA weight ratio and aqueous to organic phases volume ratio wererelevant parameters. The Taguchi method has proven effective to optimizenanoparticles of about 170nm with a polydispersity index < 0.1, a zeta potential ofapproximately -1mV and an encapsulation efficiency of 7-8% or 3% pyrazinamide drugloading.

Local delivery of medication to the lung is highly desirable as the principal advantages include reduced systemic side effects and higher dose levels of the applicable medication at the site of drug action. This administration could be particularly useful for patients with specifically chronic pulmonary infections or pulmonary diseases, such as cystic fibrosis, asthma or lung cancer. In order to deliver a high dose range of medication for highly-dosed drugs such as antibiotics, “carrier-free” DPI formulations of tobramycin were developed with the aim of minimizing the use of excipients. Briefly, dry powders were prepared by spray drying various suspensions of tobramycin in isopropanol. First, as particle size is a key parameter in defining drug deposition in the lungs, the new Spraytec® laser diffraction method specifically modified for measuring the PSD of aerosolized drug was evaluated. The dispersion properties of various dry powder formulations were investigated using different laser diffraction and impaction apparatuses at different flow rates and using different inhalator devices. Different correlations between geometric and aerodynamic size data were demonstrated in this study. As a potential application, for the flow rate, the different inhalation devices and the drug formulations examined, the tobramycin fine particle fraction could be predicted from measurements obtained from the Spraytec® using linear relationships. Correlations (R² > 0.9) between the MMAD and the percentage of particles with a diameter below 5 µm could be demonstrated between the results obtained from the laser diffraction technique and the impaction method. Consequently, the Spraytec® laser diffraction technique was proved to be an important tool for initial formulation and process screening during formulation development of DPIs. In order to modify the surface properties of the raw tobramycin powder, different powder compositions were formulated with the aim of studying the influence of the concentration of tobramycin in drug suspensions used for spray-drying, the lipid film composition (cholesterol:Phospholipon ratio) and the coating level (in percentage) on the physicochemical and aerodynamic characteristics of the antibiotic. The results indicated that the application of a lipid coating around the active particles allowed an improvement in particle dispersion from the inhalator, decreasing raw powder agglomeration and thus enhancing drug deposition deep in the lungs. Moreover, these results seemed to be influenced by the amount and composition of the lipids in the formulations. The evaluation of the influence of the coating level showed that the deposition of only 5% w/w lipids (on a dry basis) was sufficient to improve particle dispersion properties during inhalation. The FPF, which is around 36% for the uncoated micronized tobramycin, was increased to up to about 68% for the most effective lipid-coated formulation. Of particular importance, these results revealed the need to add sufficient amounts of covering material in order to significantly modify the particle surface properties and reduce their tendency to agglomeration, while limiting the lipid level in the formulations in order to avoid any undesirable sticking and to allow the delivery of more of the active drug to the deep lung. Another approach used to modify the surface properties of raw tobramycin was to coat the micronized particles with nanoparticles of the drug, produced by high pressure homogenization. The evaluation of the influence of the level of nanoparticle coating of the micronized particles showed that the presence of nanoparticles in the formulations improved the particle dispersion properties during inhalation. One microparticle was completely covered with a single layer or several layers of nanoparticles, in function of the percentage of nanoparticles in the mixture. Coating the fine drug particles with particles in the nanometer range was believed to reduce Van Der Waals forces and powder agglomeration. These various layers of nanoparticles also allowed a decrease in the cohesion of the powder by improving the slip between the particles. On the other hand, suspensions containing solely nanoparticles were spray dried with various concentrations of surfactant in order to produce easily dispersible and reproducible micron-size agglomerates of nanoparticles during inhalation. The evaluation of the influence of the concentration of surfactant showed that deposition of only 2% w/w (on a dry basis) of Na glycocholate is sufficient to improve particle dispersion properties during inhalation. Consequently, the use of nanoparticles in dry powder formulations increased the FPF from 36% for the uncoated micronized tobramycin to about 61% for this latter formulation. To modify the balance between the different forces of interactions without the need for any excipient, the influence of formulation components on the aerosolization characteristics of spray-dried tobramycin through the use of various proportions of water in the solvent used to prepare initial suspensions was investigated. These results showed that it is possible to modify the surface properties of the particles by coating the particles of drug with a homogeneously distributed film of the active compound dissolved in a solvent system containing a mixture of different solvents such as isopropanol and water. During nebulization of the suspension, droplets are composed of one or more particles in solid state surrounded with solvent containing the dissolved drug. It is hypothesized that during the drying step, dissolved tobramycin forms a coating of the amorphous drug around particles in suspension. The coating of drug particles can thus be used as an alternative approach that permits the modification of the surface properties of the particles, increasing the flowability, the desagglomeration tendency and the fine particle fraction deposited in the deep lung. So, the evaluation of the influence of the water content of the suspensions and the effect of the inlet temperature during spray-drying showed that the addition of 2% water v/v is sufficient to improve particle dispersion during inhalation. Of particular interest, as tobramycin is a very hygroscopic drug, the addition of water turned out to be a critical step. It was thus important to add a small amount of water to the solvent system and to process the drying step at a high temperature to produce formulations containing solely the active drug and showing a FPF of up to 50%. Moreover, stability studies demonstrated that these optimized formulations (lipid-coated formulation, nanoparticle formulation and amorphous drug-coated formulation) were stable over a long time period at various ICH temperature and relative humidity storage conditions (25°C/60% RH, 30°C/65% RH and 40°C/75% RH). The formulations were shown to keep their crystalline state, initial PSD, redispersion characteristics and deposition results for more than twelve months. In order to confirm these encouraging results, two optimized formulations (one with a lipid coating and another with amorphous drug coating) were selected and compared to the only commercially available tobramycin formulation for inhalation, Tobi® (nebulizer solution), by performing a combined in vivo scintigraphic and pharmacokinetic evaluation of tobramycin DPIs in nine CF patients. In comparison with Tobi®, it was estimated that lung deposition, expressed as a percentage of the nominal dose, was 7.0 and 4.5 times higher for the lipid-coated and amorphous tobramycin-coated formulations, respectively. Moreover, the pharmacokinetic data, adjusted to the same drug dose as that of the Tobi® deposited in the lungs, showed that the AUC values were found to be 1.6 times higher for Tobi® than for DPI formulations. So this evaluation confirmed the superiority of dry powder formulations in terms of drug deposition and reduced systemic exposure in comparison with the conventional comparator product, Tobi®. Thus, these new and orginal tobramycin DPI formulations based on the use of very low excipient levels and presenting very high lung deposition properties, were shown to offer very good prospects for improving the delivery of drugs to the pulmonary tract and to the widest possible patient population.

Local delivery of medication to the lung is highly desirable as the principal advantages include reduced systemic side effects and higher dose levels of the applicable medication at the site of drug action. This administration could be particularly useful for patients with specifically chronic pulmonary infections or pulmonary diseases, such as cystic fibrosis, asthma or lung cancer.<p>In order to deliver a high dose range of medication for highly-dosed drugs such as antibiotics, “carrier-free” DPI formulations of tobramycin were developed with the aim of minimizing the use of excipients. Briefly, dry powders were prepared by spray drying various suspensions of tobramycin in isopropanol.<p><p>First, as particle size is a key parameter in defining drug deposition in the lungs, the new Spraytec® laser diffraction method specifically modified for measuring the PSD of aerosolized drug was evaluated. The dispersion properties of various dry powder formulations were investigated using different laser diffraction and impaction apparatuses at different flow rates and using different inhalator devices. Different correlations between geometric and aerodynamic size data were demonstrated in this study. As a potential application, for the flow rate, the different inhalation devices and the drug formulations examined, the tobramycin fine particle fraction could be predicted from measurements obtained from the Spraytec® using linear relationships. Correlations (R² > 0.9) between the MMAD and the percentage of particles with a diameter below 5 µm could be demonstrated between the results obtained from the laser diffraction technique and the impaction method. Consequently, the Spraytec® laser diffraction technique was proved to be an important tool for initial formulation and process screening during formulation development of DPIs.<p><p>In order to modify the surface properties of the raw tobramycin powder, different powder compositions were formulated with the aim of studying the influence of the concentration of tobramycin in drug suspensions used for spray-drying, the lipid film composition (cholesterol:Phospholipon ratio) and the coating level (in percentage) on the physicochemical and aerodynamic characteristics of the antibiotic.<p>The results indicated that the application of a lipid coating around the active particles allowed an improvement in particle dispersion from the inhalator, decreasing raw powder agglomeration and thus enhancing drug deposition deep in the lungs. Moreover, these results seemed to be influenced by the amount and composition of the lipids in the formulations. The evaluation of the influence of the coating level showed that the deposition of only 5% w/w lipids (on a dry basis) was sufficient to improve particle dispersion properties during inhalation. The FPF, which is around 36% for the uncoated micronized tobramycin, was increased to up to about 68% for the most effective lipid-coated formulation. Of particular importance, these results revealed the need to add sufficient amounts of covering material in order to significantly modify the particle surface properties and reduce their tendency to agglomeration, while limiting the lipid level in the formulations in order to avoid any undesirable sticking and to allow the delivery of more of the active drug to the deep lung. <p><p>Another approach used to modify the surface properties of raw tobramycin was to coat the micronized particles with nanoparticles of the drug, produced by high pressure homogenization. The evaluation of the influence of the level of nanoparticle coating of the micronized particles showed that the presence of nanoparticles in the formulations improved the particle dispersion properties during inhalation. One microparticle was completely covered with a single layer or several layers of nanoparticles, in function of the percentage of nanoparticles in the mixture. Coating the fine drug particles with particles in the nanometer range was believed to reduce Van Der Waals forces and powder agglomeration. These various layers of nanoparticles also allowed a decrease in the cohesion of the powder by improving the slip between the particles.<p>On the other hand, suspensions containing solely nanoparticles were spray dried with various concentrations of surfactant in order to produce easily dispersible and reproducible micron-size agglomerates of nanoparticles during inhalation. The evaluation of the influence of the concentration of surfactant showed that deposition of only 2% w/w (on a dry basis) of Na glycocholate is sufficient to improve particle dispersion properties during inhalation. Consequently, the use of nanoparticles in dry powder formulations increased the FPF from 36% for the uncoated micronized tobramycin to about 61% for this latter formulation.<p>To modify the balance between the different forces of interactions without the need for any excipient, the influence of formulation components on the aerosolization characteristics of spray-dried tobramycin through the use of various proportions of water in the solvent used to prepare initial suspensions was investigated. These results showed that it is possible to modify the surface properties of the particles by coating the particles of drug with a homogeneously distributed film of the active compound dissolved in a solvent system containing a mixture of different solvents such as isopropanol and water. During nebulization of the suspension, droplets are composed of one or more particles in solid state surrounded with solvent containing the dissolved drug. It is hypothesized that during the drying step, dissolved tobramycin forms a coating of the amorphous drug around particles in suspension. The coating of drug particles can thus be used as an alternative approach that permits the modification of the surface properties of the particles, increasing the flowability, the desagglomeration tendency and the fine particle fraction deposited in the deep lung. So, the evaluation of the influence of the water content of the suspensions and the effect of the inlet temperature during spray-drying showed that the addition of 2% water v/v is sufficient to improve particle dispersion during inhalation. Of particular interest, as tobramycin is a very hygroscopic drug, the addition of water turned out to be a critical step. It was thus important to add a small amount of water to the solvent system and to process the drying step at a high temperature to produce formulations containing solely the active drug and showing a FPF of up to 50%.<p><p>Moreover, stability studies demonstrated that these optimized formulations (lipid-coated formulation, nanoparticle formulation and amorphous drug-coated formulation) were stable over a long time period at various ICH temperature and relative humidity storage conditions (25°C/60% RH, 30°C/65% RH and 40°C/75% RH). The formulations were shown to keep their crystalline state, initial PSD, redispersion characteristics and deposition results for more than twelve months.<p><p>In order to confirm these encouraging results, two optimized formulations (one with a lipid coating and another with amorphous drug coating) were selected and compared to the only commercially available tobramycin formulation for inhalation, Tobi® (nebulizer solution), by performing a combined in vivo scintigraphic and pharmacokinetic evaluation of tobramycin DPIs in nine CF patients.<p>In comparison with Tobi®, it was estimated that lung deposition, expressed as a percentage of the nominal dose, was 7.0 and 4.5 times higher for the lipid-coated and amorphous tobramycin-coated formulations, respectively. Moreover, the pharmacokinetic data, adjusted to the same drug dose as that of the Tobi® deposited in the lungs, showed that the AUC values were found to be 1.6 times higher for Tobi® than for DPI formulations. So this evaluation confirmed the superiority of dry powder formulations in terms of drug deposition and reduced systemic exposure in comparison with the conventional comparator product, Tobi®.<p><p>Thus, these new and orginal tobramycin DPI formulations based on the use of very low excipient levels and presenting very high lung deposition properties, were shown to offer very good prospects for improving the delivery of drugs to the pulmonary tract and to the widest possible patient population. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished