| Pulmonary arterial hypertension (PAH) is a rare disorder characterized by progressive obliteration of small pulmonary arteries that leads to elevated pulmonary arterial pressure and right heart failure. Current therapeutic intervention for PAH involves use of various categories of drugs. Unfortunately, none of these medications is optimal with regard to ease of administration, pulmonary selectivity, drug stability, safety and efficacy. As a result, the present thesis hypothesizes that mPEG-PLGA micelles via the pulmonary route are most promising nanocarriers to minimize the systemic side effects but also to achieve effective pulmonary vasodilation. In this study, curcumin acetate (CA), an ester prodrug of curcumin, is used as a model drug. Curcumin is the major active ingredient of Curcuma longa rhizome (popularly known as turmeric). As an NF-κB inhibitor, curcumin has exhibited protective effects in chronic hypoxic hypercapnic and monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) in rats and it is thus considered as a potential therapeutic agent for PAH. The main objective of the present thesis was to test the hypothesis that inhaled CA-loaded mPEG-PLGA micelles were effective carriers for providing sustained levels of curcumin in the lung and thus increasing the local accumulation of the drug in the pulmonary arteries with also a view to elucidatethe absorption and transport mechanisms for the polymeric micelles after delivery to the lungs.Firstly, effective methods were developed to determine curcumin acetate, crucumin, Nile red and coumarin-6, respectively. An HPLC method was used for simultaneous determination curcumin and CA in rat plasma and tissues. The assay was linear over the concentration ranges in plasma and tissues with the accuracy being between93.5-111.9%and the precision1.56-16.60%, respectively. The absolute recovery of curcumin and CA in plasma and tissues were over70%. In addition, two fluorescent methods were satisfied to determine Nile red and coumarin-6in biosamples in terms of linearity, accuracy, precision and limit of quantification.Secondly, following the encapsulation of CA or the mixture of CA and Nile red into PEG-PLGA micelles by a solvent evaporation method, the micellar formulation increased the stability of CA in water and physiologically relevant fluids and led to a sustained drug release in vitro. The stabilizing effects conferred by micelles appeared to be affected by the presence of amino acids or surfactants with leucine being the optimal additive. The micelles with or without leucine exhibited hydrodynamic particle size of -28nm, excellent encapsulation efficiency and process robustness. However, the presence of leucine in the micelles provided not only better stabilization of CA but also more sustained release than the formulation in the absence of leucine in vivo. Thirdly, the pharmacokinetic data indicated that CA loaded micelles led to sustained plasma levels of CA for up to24h after intratracheal (IT) administration whereas micelles provided plasma levels only12h after intravenous (Ⅳ) administration. In addition, following IT or Ⅳ administration of CA loaded micelles, converted curcumin could be determined up to6-10h. Tissue distribution data suggested that the IT administration of CA loaded micelles achieved prolonged pulmonary retention with AUC values almost400-fold higher than that by IV route and were able to achieve curcumin tissue concentrations that might be sufficient to treat PAH. Moreover, this result also suggested that IT micelles could achieve high targeting efficiency in the lung but did not markedly increase the accumulation in non-targeted tissues, potentially leading to an extended therapeutic window.Fourthly, the laser scanning confocal microscope images showed that both CA and Nile red with high co-localization degree were distributed in pulmonary arterial endothelial tissue, suggesting the uptake of micelles into vascular endothelial cells. In addition, IT micelles showed higher and more sustained local concentrations of CA in pulmonary arterial endothelia in terms of fluorescent intensity, relative to IV micelles, suggesting that IT micelles conferred to the higher accummulation of CA to the pulmonary vascular endothelium relative to IV given micelles. Furthermore, the high co-localization degree of CA and Nile red in the confocal micrographs of brain slices indicated that the drug can penetrated through the BBB as encapsulated in the intact micelles.Fifthly, the cytotoxicity of PEG-PLGA and blank micelles in Calu-3and NCI-H441cell lines were evaluated by MTT and LDH assays. The results showed that the cell toxicity of PEG-PLGA and blank micelles were low (cell viability>75%) up to5mg/ml. In addition, following IT administration to rats, blank or CA loaded micelles exhibited comparable biocompatibility to saline in terms of the LDH activity in bronchialalveolar lavage fluids.Sixthly, the uptake and transport mechanisms of micelles in both Calu-3and NCI-H441cell lines were investigated using different types of inhibitors. The results showed that the clathrin-mediated pathway, energy and the cholesterol might play the great role in the cellular uptake mechanisms of the micelles in the two cell lines. Interestingly, some inhibitors (such as chlorpromazine) were found to inhibit transcytosis of micelles significantly but not the endocytosis. The results suggested that the mechanisms of uptake and transport may be different.Finally, the CA loaded micelles were prepared as a dry powder inhaler (DPI) or a formulation for nebulization after either spray-drying or freeze-drying. Both spray-drying and freeze-drying process in the presence of leucine did not significantly affect the physicochemical property of CA loaded micelles in terms of the particle size, encapsulation efficiency and in vitro release profiles. The present data demonstrated that the use of spray-dried micellar particles in carrier free dry powder inhaler resulted in a fine particle fraction of55%whereas the reconstituted solutions of lyophilized micellar powders were ready to be nebulized with an average median droplet diameter of3.2μm. The results suggested that CA loaded micelles were inhalable either in a DPI or a nebulized formulation.In conclusion, the use of polymeric micelles as a carrier for preparing inhalable formulations enabled to achieve passive targeting to pulmonary vascular endothelium, where sustained release effect was brought about. In addition, the inhaled micelles might elicit sustained and efficient translocation of intact micelles vesicles across the air-blood barrier into the bloodstream, with subsequent distribution to extra-pulmonary organs including the brain, and CA-loaded micelles might, therefore, be interesting candidates for new treatment strategies of PAH. |
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