Entwicklung und Charakterisierung von Buparvaquon Nanosuspensionen für die pulmonale Anwendung bei der Behandlung von Pneumocystis Pneumonia

0\. Title and Index of Contents i 1\. Introduction, Aims and Objectives 1 2\. Literature review 7 3\. Materials and methods 37 4\. Results and Discussions 85 5\. Summary / Zusammenfassung 155 6\. References 163 7\. Appendices, Acknowledgements 185In this thesis, the use of nanocrystals in the pulmonary delivery of poorly soluble drugs was investigated in vivo and in vitro using the model drug buparvaquone (BPQ). BPQ is a naphthoquinone that has previously shown in vitro effectiviness against Pneumocystis pneumonia. 1) IN VITRO EXPERIMENTS WITH AQUEOUS NANOSUSPENSIONS: During the present work, suitable aqueous nanosuspension formulations were developed using emulsifying agents with an acceptable toxicological status to be administered to the lung. Stable formulations could be prepared containing up to 70 mg/mL drug content. Typically, nanocrystals with an average diameter of approximately 400 nm were obtained, with a diameter d99% of the crystals below 2 µm. This nanocrystal particle size has shown high efficiency when being nebulized by systems like the Pari LC Star nebulizer. Wet aerosols produced from the buparvaquone nanosuspensions prepared here, have shown average particle diameters (MMD) between 1.7 and 2 µm, which is a particle size ~1 µm smaller than the droplet size obtained using surfactant-free water or saline solution.Two physically and chemically stable nanosuspensions were tested using four commercially available nebulizer systems, which used three different nebulization technologies. It was determined that both, the formulation composition and the nebulization technology influence the size of the droplets produced and the aggregation of the nanocrystals contained in the aerosol produced. It was experimentally determined that using nebulization of nanosuspensions high amounts of drug (up to 220 mg) can be delivered to the lung by mean of aerosol droplets with an appropriate droplet size (<3 µm) in a relatively short time (~7min). 2) IN VIVO EXPERIMENTS WITH AQUEOUS NANOSUSPENSIONS: In an attempt to improve understanding of the mechanism of action and distribution of BPQ nanocrystals, Pneumocystis murina infected immunosupressed mice were used as a test model. Inhalation and oral administration of BPQ nanocrystals were compared. Positive and negative control therapies were included in the study using a placebo formulation (administered via inhalation) and oral atovaquone microsuspension (oral commercial formulation), respectively. These experiments demonstrated that both groups treated with BPQ nanosuspensions (inhalation and oral) had an improvement of the infection status, being this effect slightly faster in the group receiving inhalation therapy. The formulation was well tolerated by the animals (both routes). No statistically significant improvement could be demonstrated when comparing atovaquone microsuspension and BPQ nanocrystals via the oral route. A reason for that could be the relative small particle size of the microcrystals contained in the commercial product (d50% ~1 µm). 3) DEVELOPMENT OF SOLID DISPERSIONS CONTAINING NANOCRYSTALS: Solid dispersions containing BPQ nanocrystals were prepared by using techniques such as spray-drying (SD) and spray-freeze drying (SFD) (also known as spray-drying into liquid). Different kinds of spherical particles using inulin and PVP as matrix materials were prepared and showed different porosity and surface morphology. It was proven that the increased rate of dissolution of the nanocrystals remains in the solid state formulations produced with these technologies and therefore, can be considered as a suitable formulation alternative.In dieser Arbeit wurde die Anwendung von Nanokristallen zur pulmonalen Applikation sehr schlecht löslicher Wirkstoffe in vivo und in vitro anhand der Modelsubstanz Buparvaquone (BPQ) untersucht. BPQ ist ein Naphtoquinone das in vivo Wirksamkeit gegen Pneumocystis pneumonia gezeigt hat. 1) IN VITRO EXPERIMENT MIT WÄSSRIGEN NANOSUSPENSIONEN: Es wurden geeignete wässrige Nanosuspensionen für die Inhalationstherapie entwickelt, wobei nur Emulgatoren mit akzeptabler Toxizität geprüft wurden. Es gelang stabile Formulierungen mit bis zu 70 mg/mL Wirkstoffgehalt herzustellen. Der durchschnittliche Durchmesser der Nanokristalle in diesen Formulierungen beträgt etwa 400 nm und der Durchmesser d99% der Kristalle liegt unter 2 µm. Diese Teilchengrößenverteilung hat eine hohe Effizienz beim Einsatz in Verneblern wie dem Pari LC Star gezeigt. Die mit den BPQ Nanosuspensionen hergestellten feuchten Aerosole wiesen einen durchschnittlichen Durchmesser (MMD) zwischen 1.7 and 2 µm auf. Die Tröpfchen dieser Suspensionen sind nach Vernebelung ~1 µm kleiner als die Tröpfen, die man mit reinem Wasser oder Kochsalzlösung erhält. Zwei physikalisch und chemisch stabile Nanosuspensionen wurden mittels vier marktüblicher Verneblermodelle, die drei verschiedene Vernebelungstechnologien repräsentieren untersucht. Es zeigte sich hierbei, dass sowohl die Formulierungszusammensetzung, als auch die Vernebelungstechnologie einen Einfluss sowohl auf die Tröpfchengröße, als auch auf die Aggregation der Nanokristalle innerhalb des Aerosols hat. Zusammenfassend hat sich weder eine der Nanosuspensionen, noch eine Vernebelungstechnolgie als eindeutig überlegen herausgestellt. Es gelang in Experimenten zu zeigen, dass es möglich ist mittels der Vernebelung von Nanosuspensionen große Mengen (bis zu 220 mg) nanokristallinen Wirkstoffes in Tröpfchen geeigneter Größe (