PurposeTo characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays.MethodsThe structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF.ResultsSLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble’s solution.ConclusionThe SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.

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ObjectivesVitamin C is an important low-molecular weight antioxidant at the air-lung interface. Despite its critical role as a sacrificial antioxidant, little is known about its transport into the respiratory tract lining fluid (RTLF), or the underlying airway epithelial cells. While several vitamin C transporters have been identified, such as sodium-ascorbate cotransporters (SVCT1/2) and glucose transporters (GLUTs), the latter transporting dehydroascorbate, knowledge of their protein distribution within the human lung is limited, in the case of GLUTs or unknown for SVCTs.Setting and participantsProtein expression of vitamin C transporters (SVCT1/2 and GLUT1-4) was examined by immunohistochemistry in endobronchial biopsies, and by FACS in airway leucocytes from lavage fluid, obtained from 32 volunteers; 16 healthy and 16 mild asthmatic subjects. In addition, antioxidant concentrations were determined in RTLF. The study was performed at one Swedish centre.Primary and secondary outcome measuresThe primary outcome measure was to establish the location of vitamin C transporters in the human airways. As secondary outcome measures, RTLF vitamin C concentration was measured and related to transporter expression, as well as bronchial epithelial inflammatory and goblet cells numbers.ResultsPositive staining was identified for SVCT1 and 2 in the vascular endothelium. SVCT2 and GLUT2 were present in the apical bronchial epithelium, where SVCT2 staining was predominately localised to goblet cells and inversely related to RTLF vitamin C concentrations.ConclusionsThis experimental study is the first to demonstrate protein expression of GLUT2 and SVCT2 in the human bronchial epithelium. A negative correlation between SVCT2-positive goblet cells and bronchial RTLF vitamin C concentrations suggests a possible role for goblet cells in regulating the extracellular vitamin C pool.

Corresponding author's email: elif.bicer@kcl.ac.uk : The epithelial lining fluid (ELF) is the first physical interface with which aerosolised drugs come into contact in the airways. IntroductionThe composition and the properties attributed to ELF vary greatly along the respiratory tract and can also vary markedly in different airway pathologies. Given the complexity of this extracellular compartment, the design of a standardised lung lining fluid simulant to assess the behaviour of inhaled particles presents many challenges. Accordingly, obtaining a comprehensive understanding of their composition, including regional variation and alterations is essential.: Parallel nasal, bronchial and alveolar lavages and plasma samples were obtained from healthy (n=16, 25.0±2.6 years, 6M/10F) Methods and mild asthmatic subjects (n=16, 26.7±6.2 years, 5M/11F). Ascorbate and urate concentrations were determined using HPLC with electrochemical detection, glutathione measurements made using the glutathione disulphide recycling assay. Total protein measurements using the bincinchoninic assay, and individual protein measurements using commercially available ELISAs. Phospholipid measurements were also made using a commercially available kit, with total phosphorous determined using ICP-MS. Using the ratio of plasma to lavage urea concentrations dilution factors for nasal lavage, bronchial wash and broncholaveolar lavage ELF concentrations for the measured moieties were derived.: In mild asthmatic and healthy ELFs the contribution of ascorbate was observed to be greatest within alveolar spaces (122.1 Results (97.7-154.3) and 79.4 (62.2-144.6) µM respectively), conversely for urate, significantly higher concentrations were observed in the proximal nasal ELFs (291.0 (193.2-351.3) and 108.9 (85.3-145.6) µM respectively). In excess of 90% of total protein content of sampled ELFs was accounted for. Significant differences (p<0.05) in ELF proteome composition were observed between healthy and asthmatic samples, with lysozyme, α1-antitrypsin and transferrin being lower within bronchial asthmatic ELFs. Variation in protein composition was also observed between respiratory compartments, with albumin, lysozyme, IgA and α1-antitrypsin concentrations significantly different (p<0.001) between bronchial and alveolar ELFs. A concentration of 6.32±2.8mM choline containing phospholipids was determined, representing 69.7% of total phosphorous pool.: Samples were analysed for the major protein constituents of the ELF (albumin, transferrin, immunoglobulin A, G and M, Conclusions lysozyme, α1-antitrypsin and surfactant protein A), low molecular weight antioxidants and phospholipids. These data will be used to inform the production of realistic ELF simulants for the screening of drug behaviour at the surface of the lung. It is clear that it will be important to tailor composition to reflect both the region of the airway targeted by a drug, as well as the underlying disease profile. This abstract is funded by: GlaxoSmithKline BBSRC Am J Respir Crit Care Med ...

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.