AbstractCystic fibrosis (CF) is a lethal hereditary disorder characterised by the unregulated production of visoelastic mucus, which poses a barrier to the effective delivery of drugs to the CF lung. The barrier properties of CF sputum are thought to be due to the high DNA and actin content that together with mucins form a tangled network, held together by electrostatic charges, hydrogen bonds and van der Waals forces. CF sputum affects the deposition pattern of aerosols on the epithelial surface of the upper respiratory tract and thereby, preventing drug diffusion. To improve drug delivery in CF it is vital to reduce or remove this barrier. Recent studies have suggested that negatively charges species such as unfractionated heparin (UFH). a member of the glycosaminoglycan family, may posess mucoactive properties and shows promise as a potential therapy for treating patients with chronic obstructive pulmonmary disease (COPD) and CF. This study investigates the potential mucoactive properties of UFH, and describes the development of dry powder inhaler (DPI) formulations of UFH that can be administered to CF patients to treat airway obstruction.
In this study, a novel barrier function assay was employed to investigate the diffusion of the corticosteroid dexamethasone through CF sputum and calf-thymus DNA. In addition, atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM) were used to investigate the ultrastructure of both whole CF sputum and calf-thymus DNA. The effect of charged oligsaccharides and glycosaminoglycans, as potential mucoactive agents, on the barrier and structural properties of CF sputum were also investigated. The feasibility of producing dry powders of UFH, which are suitable for inhalation from DPIs, using spray drying, was investigated. The effect of these powders on CF sputum rheology was also studied.
Studies investigating the barrier properties of CF sputum, showed that sputum significantly (p<0.05) reduced the diffusion of dexamethasone by 90% in comparison to diffusion through buffer. Following treatment with 1.0 and 10mg/ml UFH, the diffusion of dexamethasome through CF sputum was significantly (p<0.05) increased. CLSM analysis of untreated CF sputum showed the pressence of highly polymerised fibrous bundles of DNA, which were observed to be interacting with F-actin present in the sputum. The fibrous bundles of DNA present in CF sputum were dissipated following treatment with UFH. Therefore, it was hypothesised that UFH may be exerting its mucoactive properties by targeting the interactions between DNA and other components present in CF sputum. Results suggest that the DNA component of CF sputum may also be responsible for the barrier properties of CF sputum. Indeed, it was observed that calf-thymus DNA (5mg/ml) significantly (p<0.05) reduced the diffusion of dexamethasone. Treatment of calf-thymus DNA (5 mg/ml) with 10 mg/ml UFH, significantly (p<0.05) increased the diffusion of dexamethasone. AFM analysis of calf-thymus DNA showed the presence of DNA networks that exhibited pore sizes of 0.38 ± 0.03 μm. Treatment of DNA (5 mg/ml) with 0.01 mg/ml UFH significantly (p<0.05) increased the pore size of the DNA network to 0.78 ± 0.07 μm. Hence, these data suggest that UFH may possess mucoactive properties.
Spray drying was successfully used to produce spherical micronised UFH particles, which were suitable for inhalation. The material, however, was cohesive (forming agglomerates) and hygroscopic, which compomised its physical stability. These properties may render a UFH DPI drug delivery system ineffective, as insufficient fine material would be liberated that would penetrate the deep lung. Co-spray drying UFH with L-leucine significantly reduced the cohesive properties of UFH and improved it physical stability characteristics. Rapid formulation screening studies using a conventional DPI suggested that co-spray dried UFH/leucine formulations are easily dispersed from the inhaler, with the liberation of a large proportion of fine material. In addition, both spray dried UFH and co-spray UFH/leucine significantly (p<0.05) reduced the viscoelasticity of CF sputum.
This work suggest that UFH possess mucoactive propoerties, and that the use of L-leucine may aid the development of dry powder inhlaer formulations of UFH, which can be administered to CF patients to treat airway obstruction.
|Date of Award||May 2006|
|Supervisor||Jan Shute (Supervisor) & James Smith (Supervisor)|