In vitro and in vivo antitubercular activity of benzothiazinone-loaded human serum albumin nanocarriers designed for inhalation.

The aim of this study was to investigate the potential of human serum albumin (HSA) as a solubilising agent/drug delivery vehicle for pulmonary administration of antimycobacterial benzothiazinone (BTZ) compounds. The solubility of four novel BTZ compounds (IR 20, IF 274, FG 2, AR 112) was enhanced 2 to 140-fold by incubation with albumin (0.38-134 µg/mL). Tryptophan 213 residue quenching studies indicated moderate binding strength to Sudlow's site I. Nanoparticle manufacture achieved 37-60% encapsulation efficiency in HSA particles (169 nm, zeta potential -31 mV). Drug release was triggered by proteases with >50% released in 4 h. The antimycobacterial activity of IR 20 and FG 2 loaded in HSA nanoparticles was enhanced compared to DMSO/phosphate buffered saline (PBS) or albumin/PBS solutions in an in vitro M. tuberculosis-infected macrophage model. Intranasal instillation was used to achieve pulmonary delivery daily over 10 days to M. tuberculosis infected mice for FG2 HSA nanoparticles (0.4 mg/kg), FG 2 DMSO/saline (0.4 and 8 mg/kg) and a reference compound, BTZ043, DMSO/saline (0.4 and 8 mg/kg). A lower lung M. tuberculosis burden was apparent for all BTZ cohorts, but only significant for BTZ043 at both doses. In conclusion, mechanisms of HSA nanoparticle loading and release of BTZ compounds were demonstrated, enhanced antimycobacterial activity of the nanoparticle formulations was demonstrated in a biorelevant in vitro bioassay and the effectiveness of BTZ by pulmonary delivery in vivo was established with pilot evidence for effectiveness when delivered by HSA nanoparticles. Finally, the feasibility of developing an inhaled nanoparticle-in-microparticle powder formulation was ascertained.

Development of new in vitro models of lung protease activity for investigating stability of inhaled biological therapies and drug delivery systems.

Proteases play a vital role in lung health and are critically important to the metabolic clearance of inhaled protein-based therapeutics after inhalation. Surprisingly little is known about lung fluid protease composition and there is a consequent lack of biorelevant experimental models, which limits research and development in the burgeoning field of inhaled biologics. The aim of this study was to quantify proteases in human lung fluid and to use this data to design novel in vitro experimental models of lung lining fluid possessing biorelevant lung protease activity for use in biopharmaceutical stability studies. As a proof of concept, these novel models were used to investigate the effect of proteolytic activity on the stability of albumin nanoparticles, a biologic nanoparticle formulation widely investigated as a pulmonary drug delivery system. Bronchoalveolar lavage fluid was collected from healthy human volunteers and proteomic analysis was used to quantify the predominant proteases. Based on these data, four new lung protease models were constructed based on: (i) trypsin as a sole protease, (ii) dipeptidyl peptidase IV, cathepsin D, cathepsin H, and angiotensin converting enzyme in ratio and concentration to mimic the protease concentration in healthy lungs. Neutrophil elastase was used to model protease activity in inflammation. Albumin nanoparticles of 100 nm diameter remained intact over 48 h in phosphate buffered saline, but were degraded more rapidly in trypsin (50% reduction in 10 min) compared to the healthy lung protease model (50% reduction in 150 min). The addition of neutrophil elastase to the healthy lung protease model resulted in a similar, but more variable degradation profile. Nanoparticle degradation was associated with concomitant appearance of small fragments and aggregates. In conclusion, we have characterised the protease concentration in the lungs of healthy humans, designed models of lung protease activity and demonstrated their utility in studying albumin nanoparticle degradation. These methods and models have wide application to study the influence of proteases in lung disease, expression of proteases in respiratory cell culture models, stability of peptide and protein-based drugs and inhaled drug delivery systems.