No description available.

The therapeutic effect of pulmonary drug delivery systems is limited by its rapid clearance from the lungs by robust clearance mechanisms. By controlling the release of drugs, the therapeutic effect of pulmonary drug delivery systems, as well as patient convenience and compliance could be improved by reducing the number of times drugs need to be administered. In this study, two controlled pulmonary drug delivery systems for drugs of different solubilities were investigated and they were characterised for their viability as effective controlled release pulmonary drug delivery systems, particularly in areas of aerosol performance and dissolution profile. A hybrid protein-polymer controlled release pulmonary drug delivery system was developed to sustain the release of a water-soluble anti-asthma drug, cromolyn sodium (CS). Two excipients with complementary characteristics – a protein, bovine serum albumin, and a polymer, polyvinyl alcohol – were formulated together with CS via co-spray drying, with varying protein-polymer ratios and drug loadings. The hybrid particles showed promise in combining the positive attributes of each excipient, with respirable particles shown to sustain the release of CS with a fine particle fraction of 30%. Combining the two excipients was complex, with further optimisation of the hybrid formulations possible. A commercially available polymer, Soluplus® was spray-dried with a poorly-water soluble corticosteroid, beclomethasone dipropionate (BDP). The resultant respirable powders were shown to have potential for use as a controlled release pulmonary drug delivery system with up to 7-fold improvement in the amount of BDP released compared to spray-dried BDP. The spray-dried BDP-Soluplus® powders were found to be amorphous, and physically stable against re-crystallisation for up to 9 months at accelerated stress test conditions with drug loadings of up to 15 % (w/w). Although it provided a platform to compare between formulations, the USP 4 flow-through cell dissolution apparatus was found to be inadequate to accurately study the dissolution profiles of the pulmonary drug delivery systems due to the formation of a gel in the apparatus. Preliminary work on the use of a novel technique to predict the crystallisation of amorphous formulations with terahertz time-domain spectroscopy was also conducted. The system confirmed the re-crystallisation tendencies of several hybrid CS/BSA/PVA formulations. Modification to the experimental setup to probe the formulations at different relative humidities instead of temperatures could yield improved results.

Attention has begun to focus on the pulmonary delivery of antifungal agents for invasive fungal infections as inhalation of the fungal spores is often the initial step in the pathogenesis of many of these infections. Invasive fungal infection in the lungs in immunocompromised patients has high mortality rates despite current systemic (oral or intravenous) therapies. However, drug delivery of antifungal agents directly to the lungs could potentially result in high concentrations of drug in the lungs, a quicker onset of action, and reduction of systemic side effects. Voriconazole (VRC) is a second, generation triazole antifungal agent with increased potency, a broad spectrum of antifungal activity, and a fairly poor aqueous solubility. It is the recommended therapeutic agent for the treatment of Invasive Pulmonary Aspergillosis (IPA), and its use has improved therapeutic outcomes in immunocompromised patients with IPA. Still, systemic administration by oral or intravenous delivery is limited by high inter- and intra-patient pharmacokinetic variability, many potential drug interactions, and a narrow therapeutic index with many adverse effects, leading to clinical failures. Therefore, development of novel particulate formulations containing VRC for targeted drug delivery to the lungs is critical to improving therapeutic outcomes in patients with invasive fungal infections in the lungs. Within the framework of this dissertation, two particle engineering processes, thin film freezing (TFF) and advanced evaporative precipitation into aqueous solution (AEPAS), were investigated. The goal was to investigate microcrystalline VRC, nanocrystalline VRC, and nanostructured amorphous VRC formulations suitable for pulmonary delivery and to determine the effect of morphology on the in vivo deposition and distribution of inhaled particulate VRC formulations. TFF process parameters significantly affected the solid state properties and aerodynamic performance of the dry powder formulations containing VRC. Following dry powder insufflation into the lungs of mice, microstructured crystalline TFF-VRC achieved higher and more prolonged concentrations of VRC in the lungs with slightly lower systemic bioavailability than nanostructured amorphous TFF-VRC-PVP K25. AEPAS and TFF of template nanoemulsions did not lead to production of crystalline nanoparticles, as predicted. In particular, VRC proved to be a difficult molecule to stabilize in the nanocrystalline and nanostructured amorphous states. Ultimately, this body of work demonstrated that the particle engineering process, TFF, could be used to develop voriconazole formulations suitable for dry powder inhalation with more favorable pharmacokinetic parameters compared to inhaled voriconazole solution. / text

Since the discovery of cyclosporine in 1971, calcineurin inhibitors have played a critical role in the therapeutic suppression of the immune response. Patients receiving solid organ transplants rely heavily on these medications to prevent the acute and chronic rejection of allografted tissue. Introduction of tacrolimus, the most frequently prescribed calcineurin inhibitor, has lead to improved clinical outcomes for organ transplant recipients; however, little improvement has been noted in lung transplantation. Difficulties with current oral dosing regimens for lung transplant patients stem primarily from drug systemic toxicity, heightened risk of invasive infection, and erratic oral bioavailability. We have proposed that pulmonary delivery of a tacrolimus formulation with improved solubility can provide high lung concentrations, while limiting corresponding systemic levels associated with toxicity. Chapter 2 investigates the pulmonary administration of tacrolimus dispersion for nebulization to lung transplanted rats. Resulting lung and blood levels were determined by appropriate bioanalytical methods. Limited systemic absorption was seen after pulmonary delivery, resulting in a 50 to 1 lung to blood concentration ratio. A 28 day safety and stability evaluation of tacrolimus dispersion for nebulization was conducted in Chapter 3. Results showed no signs of toxicity in Sprague Dawley rats and proved the stability of tacrolimus powder for dispersion for 3 months. For cases of severe asthma, immunosuppression is also necessary to restore normal lungs function and is typically treated with corticosteroids. Corticosteroids, however, are well known for their untoward side effects and can prove ineffective in severe asthmatics that have developed corticosteroid resistance. Chapter 4 investigates the use of tacrolimus dispersion for nebulization for prophylactic treatment of asthma. Efficacy was determined in an asthma-induced animal model by quantification of inflammatory cells and signaling chemicals. In Chapter 5, tacrolimus powder for inhalation is investigated in a novel dry powder inhalation platform. Respirable particles are produced when bulk particles (500 [micrometer]) comprising a matrix of drug/excipient are sheared apart by a marketed inhalation device to produce particles of the appropriate geometric diameter (50 [micrometer]). Biocompatible material with brittle properties were found to produce fine particle fractions (FPF) up to 70.3% and total emitted doses (TED) higher than 95%. / text

Pneumonia is one of the main causes of death worldwide, specially of the elderly and the children under 5 years old. The traditional antibiotic-based therapy faces a crisis due to the increase in resistance and a lack of new molecules approved. Recently, our research group demonstrated the photodynamic inactivation of streptococcal pneumonia in vivo, a technique to which the development of resistance is described to be unlikely. This study proposed to investigate the applicability of nebulization as a delivery method for photosensitizers, in the hope to advance the research of the photodynamic inactivation of bacterial pneumonia. First, the critical attributes for nebulization (droplet size and delivery rate), the extent of nebulization, and the stability of three photosensitizers were stablished, and they were all found to be compatible with the technique. Then, the delivery was validated in an animal model using the most promising compound. It was possible to activate it using extracorporeal infrared light without causing acute lung or liver damage. In conclusion, nebulization presented itself as a promising tool for the delivery of photosensitizers to the respiratory tract. / A pneumonia é uma das principais causas de morte no mundo, sobretudo de idosos e crianças menores de cinco anos. A terapia tradicional, baseada em antibióticos, enfrenta uma crise diante do aumento da resistência e do número reduzido de novas moléculas que são aprovadas. Recentemente, este grupo de pesquisa demonstrou a inativação fotodinâmica da pneumonia pneumocócica in vivo, uma técnica para a qual o surgimento de resistência é descrito como pouco provável. Este estudo se propôs a investigar a aplicabilidade da nebulização como método de entrega de fotossensibilizadores, na esperança de avançar a pesquisa da inativação fotodinâmica da pneumonia bacteriana. Inicialmente, os atributos críticos da nebulização (tamanho de gotícula e taxa de entrega), a extensão da dose nebulizada, e a estabilidade de três fotossensibilizadores foram estabelecidas. Todos eles se mostraram compatíveis com a técnica. Então, a entrega foi validada em um modelo animal, utilizando o composto mais promissor. Foi possível ativá-lo usando luz infravermelha extracorpórea sem que houvesse dano agudo pulmonar ou hepático. Em conclusão, a nebulização se mostrou uma ferramenta promissora na entrega de fotossensibilizadores ao trato respiratório.

Targeted delivery of drugs directly to the lung epithelium is a promising, though challenging, strategy for the treatment of diseases that affect the lung tissues, such as infections caused by cell-penetrating pathogens, cystic fibrosis, and cancer. With appropriate surface functionality, such as through the attachment of ligands that recognize receptors on cellular surfaces, particulate carriers show improved efficiency in penetrating cells in vitro. A useful class of ligands is produced by many natural human pathogens that infect the respiratory tract. A variety of phylogenetically distinct respiratory bacterial pathogens, such as Haemophilus influenzae, invade host cells in the upper airways by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms. By expressing host carbohydrate structures, including phosphorylcholine (ChoP), as a terminal structure on the LOS, the bacteria exhibit molecular mimicry of the host and are able to evade the host immune system. The effectiveness of LOS to induce cellular uptake of the bacteria is dependent on the specific glycoform, with higher ChoP content inducing more bacterial adherance into the lung epithelial. These ligands naturally expressed on bacterial cell surfaces can be isolated and utilized as targeting ligands for delivery vehicles. The studies described in this thesis focus on the development of particulate drug carriers coated with LOS bacterial ligands to enhance the targeting and binding of the carriers to the lung epithelium. Three NTHi clinical isolates were screened to select the strain with the highest ChoP level, and NTHi 3198, an isolate from a patient with chronic obstructive pulmonary disease (COPD), was selected due to its high ChoP activity. LOS from NTHi 3198 was isolated from the bacterial cell membrane, and its activity verified using dot immunoblot and ELISA techniques. Particles (0.2 and 1 µm) composed of polystyrene or poly(lactic-co-glycolic acid) were passively coated with 0.005-50 µg/mL of the isolated LOS 3198 with or without gelatin, coated with gelatin alone, or left uncoated. The LOS coating on the particles was verified using either XPS or ELISA. The association of particles with human bronchial epithelial cells was investigated using two cell culture models, 16HBE14o- and Calu-3, as a function of particle concentration and incubation time. The expression of PAFR on both cells types was confirmed, though the expression of PAFR on 16HBE14o- cells was significantly greater than on Calu-3 cells. Enhancement of 0.2 µm particle-cell association was achieved through coating of the particles with LOS. However, no significant difference in particle-cell association was observed for the 1 µm particles based on particle coating. Control particles of 0.2 µm size, those coated with gelatin (with or without LOS) or uncoated, exhibited low cell binding with a maximum of about 10-18% of cells associated with particles. The ability of the LOS ligand to enhance particle-cell association was coating concentration dependent, with a low coating concentration of LOS having little effect on association, but a concentration 1000-fold higher causing a doubling of the percentage of cells associated with particles at 24 hours. This enhancement was attributed to increased cellular binding of the 0.2 µm particles to the cell surface by confocal microscopy, and was further increased by activating the PAFR prior to incubation with particles. These results suggest the potential application of LOS as a targeting ligand for lung epithelial cells, especially under conditions where PAFR has been activated, such as occurs in lungs infected with Haemophilus influenzae. A significant reduction in particle-cell association was observed when particles were incubated with Calu-3 cells due to the presence of mucus on the cellular surface. This suggests that further optimization of the drug carrier system is needed to efficiently overcome the mucosal fluids.

Invasive Pulmonary Apsergillosis (IPA) is caused by inhalation of fungal conidia to the deep lung followed by germination and invasive hyphal growth in heavily immunosuppressed patients (e.g. those with hematologic malignancies, hematopoietic stem cell transplant recipients, and those undergoing solid organ transplantation). Hyphal growth into pulmonary capillaries often leads to dissemination of the infection and high mortality rates despite current treatment and prophylactic modalities. In addition, systemic antifungal therapy is often limited by drug toxicities, low and variable bioavailability, erratic pharmacokinetics, and drug interactions. Although targeted drug delivery to the lungs has been investigated to reduce adverse events and promote drug efficacy, inconsistent pharmacokinetic properties following inhalation of poorly water soluble antifungals has prompted variable drug efficacy. In this dissertation, inhaled voriconazole was investigated through in vitro and in vivo testing to evaluate pharmacokinetic properties, characterize drug safety and, determine drug efficacy as prophylaxis against IPA. In Chapter 2, the in vitro evaluation of solution properties and aerosol characterization of aqueous voriconazole was evaluated. Subsequent in vivo single and multiple dose pharmacokinetic studies demonstrated high drug concentrations were achieved in lung tissue and plasma following inhalation in contrast to previous reports of inhaled antifungals. Inhaled voriconazole was then administered twice daily (BID, at 08:00 and 16:00) in a murine model of IPA as described in Chapter 3 with significant improvements in animal survival over 12 days compared to both positive and negative control groups. As described in Chapter 4, voriconazole was then chronically administered BID at a high and low dose to rats over 21 days with a 7 day recovery period to assess dose tolerability through laboratory tests and histopathological changes to lung, liver, kidney, and spleen tissues. Inhaled voriconazole was well tolerated through all assessments but with signs of mild acute histiocytosis in lung tissue without other signs of inflammation. Chapter 5 expanded the single inhaled dose pharmacokinetic profile in lung tissue and plasma with determination of additional pharmacokinetic parameters through compartmental modeling. Peak and trough voriconazole concentrations were also evaluated in mice as well as rats following multiple doses administered over 12 hours (Q12H) as opposed to BID. / text

The use of lung transplantation, a life saving intervention, has been increasing over the last thirty years with a disappointing median survival of only 4.8 years. Despite the progress made in immunosuppressive therapies, allograft rejection following transplantation is the leading cause of death. As part of the immunosuppressive therapy, mycophenolate mofetil (MMF), the ester prodrug of mycophenolic acid (MPA) has proven its efficacy among heart, liver, kidney as well as lung transplanted patients. However, due to its rapid excretion, high daily doses are necessary and lead to serious side effects, forcing the patient to stop and change their course of treatment. Administration of drugs to the lungs is known to minimize local and systemic side effects by employing a lower amount of drug, to increase patient compliance and to improve the efficacy of the treatment. Therefore, developing novel MMF formulations for targeted delivery to the lungs will broaden the therapeutic options against lung transplant rejection. Within the framework of this dissertation, the development of an inhaled formulation of MMF was investigated. MMF must be metabolized by carboxylesterases to become active and its metabolism suffers from high inter- and intra-patient variability. The first objective of this dissertation was to investigate the occurrence of MMF hydrolysis in the lung. The second objective was to study the in vivo deposition,metabolism and distribution in rats, of an inhaled micron-size MMF suspension in comparison to inhaled IV Cellcept® and oral Cellcept®, the currently marketed products. According to the in vitro results, MMF is metabolized in human lung cells by carboxylesterases. The in vivo results showed an incomplete metabolism of MMF when delivered as a suspension due to the limited dissolution of the drug in the lungs. Following inhalation, the MMF suspension achieved higher and more prolonged concentration of the total drug in the lungs and lymphoid tissues as compared to the inhaled IV Cellcept®. The pulmonary delivery of the MMF suspension was able to achieve similar levels of drug in the lungs, higher levels in the lymphoid tissues and significantly lower levels in the systemic circulation when compared to the levels obtained from the oral gavage of oral Cellcept®. Ultimately, this dissertation demonstrated that the administration of micron-size MMF suspension offers a great potential for pulmonary administration. / text

Pneumonia is one of the main causes of death worldwide, specially of the elderly and the children under 5 years old. The traditional antibiotic-based therapy faces a crisis due to the increase in resistance and a lack of new molecules approved. Recently, our research group demonstrated the photodynamic inactivation of streptococcal pneumonia in vivo, a technique to which the development of resistance is described to be unlikely. This study proposed to investigate the applicability of nebulization as a delivery method for photosensitizers, in the hope to advance the research of the photodynamic inactivation of bacterial pneumonia. First, the critical attributes for nebulization (droplet size and delivery rate), the extent of nebulization, and the stability of three photosensitizers were stablished, and they were all found to be compatible with the technique. Then, the delivery was validated in an animal model using the most promising compound. It was possible to activate it using extracorporeal infrared light without causing acute lung or liver damage. In conclusion, nebulization presented itself as a promising tool for the delivery of photosensitizers to the respiratory tract. / A pneumonia é uma das principais causas de morte no mundo, sobretudo de idosos e crianças menores de cinco anos. A terapia tradicional, baseada em antibióticos, enfrenta uma crise diante do aumento da resistência e do número reduzido de novas moléculas que são aprovadas. Recentemente, este grupo de pesquisa demonstrou a inativação fotodinâmica da pneumonia pneumocócica in vivo, uma técnica para a qual o surgimento de resistência é descrito como pouco provável. Este estudo se propôs a investigar a aplicabilidade da nebulização como método de entrega de fotossensibilizadores, na esperança de avançar a pesquisa da inativação fotodinâmica da pneumonia bacteriana. Inicialmente, os atributos críticos da nebulização (tamanho de gotícula e taxa de entrega), a extensão da dose nebulizada, e a estabilidade de três fotossensibilizadores foram estabelecidas. Todos eles se mostraram compatíveis com a técnica. Então, a entrega foi validada em um modelo animal, utilizando o composto mais promissor. Foi possível ativá-lo usando luz infravermelha extracorpórea sem que houvesse dano agudo pulmonar ou hepático. Em conclusão, a nebulização se mostrou uma ferramenta promissora na entrega de fotossensibilizadores ao trato respiratório.

Philosophiae Doctor - PhD / Introduction: Tuberculosis (TB) is a serious bacterial infections caused by the Mycobacterium Tuberculosis (MTB) organism affecting mainly the lungs. Occasionally, MTB bacilli may be transported out of the pulmonary region and infect peripheral organs causing extra-pulmonary tuberculosis. Many therapeutic agents were developed over the years to combat TB, however the rapid emergence of resistant strains hampered their use. Furthermore, most of the current anti-TB drugs experience many challenges, which can be summarized in treatment regimen factors, drug-drug interactions, and physicochemical characteristics factors (such as hydrophobicity and low permeability into alveolar macrophages). These challenges have a significant role in treatment failure and the emergence of resistant TB. Due to the lack of newly discovered anti-TB drugs, and the absence of effective vaccines, many scientists have suggested the use of novel modalities for the current anti-TB drugs to enhance their efficacy and overcome some of the drawbacks. One of these modalities is nanotechnology-based drug delivery systems. Most of the anti-TB drugs experience low drug distribution to the lung and particularly alveolar macrophages within which the MTB resides, leading to treatment failure. Employing nanoparticles as drug delivery systems can have a significant impact on improving the pharmacokinetic profile of anti-TB drugs, the feasibility of different routes of administration, enhancing drug permeability, controlled/sustained drug release, and targeting specific disease sites. Collectively, these impacts will aid in enhancing drug concentration at the site of infection and reduce dosing and regimen duration. Dendrimers, such as polyamidoamine (PAMAM) dendrimers, are synthetic polymeric nanoparticles that have unique features that afford a dendrimer-conjugate complex the possibility to overcome the most common hurdles associated with drug delivery and treatment of diseases. Obstacles associated with solubility, permeability, inadequate biodistribution associated side effects may be enhanced. Manipulating the outermost surface functional groups with various ligands and polymers, will enhance the dendrimer properties and targeting potential. Aim: This study aims to develop a novel pulmonary delivery system for the anti-TB drug rifampicin using surface-modified G4 PAMAM dendrimer nanoparticles (polyethylene glycol (PEG) or mannose moieties), to improve drug solubility, prolong-release, enhance permeability into the macrophages, and decrease the toxicity of the drug-dendrimer conjugates. Methods: PAMAM dendrimers having increasing concentrations of poly(ethylene glycol) (PEG) 2 kDa or mannose residues were synthesized. The 4-nitrophenyl chloroformate was used as an activator in the case of PEG functionalization, while for the mannose conjugation the 4-isothiocyanatophenyl alpha-D-mannopyranoside (4-ICPMP) directly interacted with the primary amines of the dendrimer. The conjugated PEG polymers and mannose moieties on the dendrimer periphery were confirmed using FTIR and 1H NMR analytical techniques. Thereafter, rifampicin was loaded into the native and surface-modified dendrimers via a simple dissolution solvent evaporation method. Rifampicin-loaded dendrimers were then characterized using several analytical techniques namely; FTIR, DSC, NMR, SEM, and DLS. The polymer encapsulation efficiency (EE%) and percentage of drug loading (DL%) were determined directly using a validated HPLC method. In vitro drug release was studied at pH 7.4 and pH 4.5. The MTT technique was used to assess the cytotoxicity of the dendrimer formulations against raw 264.7 cell lines. Finally, the uptake of dendrimer nanoparticles by raw macrophages was studied using a flow cytometer and fluorescence microscopy techniques. Results: The percentage coverage of 4.0 G PAMAM dendrimer peripheral with PEG was achieved in a range of 38% - 100%, while for mannose moieties was from 44% - 100%. The EE% of unmodified dendrimer was 7.5% (w/w). The EE% of PEGylated dendrimers ranged from 65.0% - 78.75% (w/w), whereas for mannosylated dendrimers was from 43.43% - 57.91% (w/w). The size of the unloaded dendrimer nanoparticles was less than 25 nm, a gradual increase in the size after drug conjugation followed. The zeta potential of dendrimers was positive with values greater than 12 mV, the nanoparticle's zeta potential decreased upon increasing the density of PEG/mannose and after drug loading. FTIR and NMR data showed that rifampicin molecules were conjugated to the dendrimer at three sites; at the surface amines via electrostatic linkages, within the PEG/mannose, and into the dendrimer interior. SEM images of dendrimer nanoparticles confirmed the spherical shape of particles, and DSC data verified drug entrapment. Drug release was found to be affected by the pH of the medium and the extent of dendrimer functionalization. At the physiologic pH, surface-modified dendrimers showed a slower release rate compared to the unmodified dendrimer and free drug. Among surface-modified dendrimers, the release rate was inversely associated with the density of PEG/mannose molecules. At pH 4.5, a relatively higher drug release from all formulations was observed which suggests a burst release inside the alveolar macrophages. Toxicity studies showed that the unmodified dendrimer experienced time-dependent and concentration-dependent cytotoxicity against raw 264.7 cells. The toxicity gradually decreased upon increasing the density of PEG/mannose, and negligible toxicity was detected for formulations with 100% functionalization. Dendrimer nanoparticles were successfully internalized into raw cells after 24 hrs of incubation. The order of nanoparticles permeability was PEG 100% < PEG 85% < PEG 70% < PEG 49% < PEG 38% < unmodified dendrimer < mannose 44% < mannose 69% < mannose 93% < mannose 100%. The significant increase in the uptake of mannosylated dendrimers was due to the interaction with lectin receptors at the surface of raw macrophages, whereas the lower internalization of PEGylated dendrimers was due to the shielding of the surface positive charges. Conclusion: The in-vitro and ex-vivo data studies suggested that the developed novel surface-modified G4 PAMAM dendrimers are suitable drug carriers in terms of biocompatibility, release behaviour, and site-specific delivery of the anti-TB drug rifampicin.