Mapping spatially resolved transcriptomes in human and mouse pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. Here we generated spatially resolved transcriptome maps of human IPF (n = 4) and bleomycin-induced mouse pulmonary fibrosis (n = 6) to address these limitations. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by transforming growth factor beta signaling alongside predicted regulators, such as TP53 and APOE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.

Spray dried lipid nanoparticle formulations enable intratracheal delivery of mRNA.

RNA therapies have recently taken a giant leap forward with the approval of Onpattro™, a siRNA therapy delivered using a lipid nanoparticle (LNP), and the LNP-enabled mRNA vaccines against COVID-19, which are the first mRNA drugs to reach the marketplace. The latter medicines have illustrated that stability is a significant challenge in the distribution of RNA drugs using non-viral delivery systems, particularly in areas without cold chain storage. Here, we describe a proof-of-concept study on the engineering of an LNP mRNA formulation suitable for spray drying. This process produced a dry powder formulation that maintained stability and preserved mRNA functionality with increased performance compared to liquid formulations stored two weeks at 4 °C. Intratracheal delivery of spray dried LNPs loaded with eGFP mRNA to rats resulted in the production of the eGFP protein in a range of cell types including bronchiolar epithelial cells, macrophages and type II pneumocytes; cell types involved in adaptive immunity and which would be valuable targets for inhaled vaccines against respiratory pathogens. Together, these data show that spray drying of LNPs enhances their stability and may enable RNA delivery to the lung for protein replacement therapy, gene editing, vaccination, and beyond.

Same lung deposited dose in dog dosing a fine and coarse aerosol indicates no difference in intranasal filtration.

A novel inhalation exposure system was developed with the aim to increase the efficiency of pharmacokinetic (PK) evaluations of inhaled drugs in a large species such as the dog. It enables collecting PK data for multiple drug candidates in a single experiment by simultaneous administration of the drugs to the same animal. This facilitates a direct PK comparison of the same lung dose of different drugs using the same blood samples, which can be considered to be a refinement measure from an animal research perspective. The system design was inspired by a clinical precision dosing dosimeter systems, which enhance dosing precision by synchronizing the aerosol delivery from the jet nebulizer with the inhalation to maximize the inhaled fraction of the nebulized dose. The performance of the novel system was validated in an in-vivo study, which included a comparison of the same nebulized dose delivered as a fine and a coarse aerosol. The drugs selected for this study were developed for local treatment of the lung via inhalation and were known to have low oral bioavailability due to being extremely poorly soluble and therefore expected to also have low nasal bioavailability. This would result in systemic exposure derived primarily from pulmonary absorption, which facilitated the PK assessment applied to determine the lung deposited dose. The jet nebulizer selected to generate a fine aerosol was designed for alveolar lung deposition and approved by U.S. Food and Drug Administration for lung ventilation imaging, and the nebulizer selected to generate a coarse aerosol was a standard nebulizer. The drugs were wet milled to a particle size considerably smaller than the nebulized droplets and the dispersed drug particles were therefore homogenously distributed in the droplet size distribution. Higher initial plasma concentrations were observed for the fine aerosol. This was expected, as the smaller droplets should deposit more efficiently in the peripheral regions of the lung, which consequently should lead to a faster absorption compared with the coarse aerosol from the standard nebulizer that should deposit more centrally. The fact that this could be observed supports that the novel system is an excellent tool in PK evaluations. Our study indicated that there was no difference in the systemic exposure between the fine and the coarse aerosol for the same nebulized dose, and thus the lung deposition was also the same. The considerable difference in the nebulized size distribution within the range relevant for available inhalation devices resulted in a negligible difference in intranasal filtration. The fraction of the nebulized dose that deposited in the lung was observed to be high in this study (mean of 21-30% and about 50% for one dog with a distinguished slow and deep breathing), which supports that the intranasal filtration was low. That a high fraction of the nebulized dose deposited in the lung indicates that an enhanced dosing precision was obtained with the novel system. The similar achieved lung doses of all three drugs, shows that the simultaneous administration of multiple drugs worked well. That the intranasal filtration was low is an important finding considering that devices for oropharyngeal delivery in dogs are applied in order to eliminate intranasal filtration. Oropharyngeal dosing is more invasive compared with oronasal dosing and avoiding utilizing that method can be considered a refinement measure from an animal research perspective.

Airway Epithelial Lining Fluid and Plasma Pharmacokinetics of Inhaled Fluticasone Propionate and Salmeterol Xinafoate in Mechanically Ventilated Pigs.

Background: The lower respiratory tract of the landrace pig has close anatomical and physiological similarities with that of the human, and hence, for inhalation studies this species is well suited for biopharmaceutical research. Methods: The objective of this study was to evaluate pharmacokinetics in pigs following one dose of Diskus™ Seretide™ forte device, labeled 500/50 fluticasone propionate (FP) and salmeterol xinafoate (SX), respectively. The PreciseInhale™ (PI) instrument was used to actuate the inhaler for in vitro testing and aerosol dosing to pigs. In vitro, the aerosol was characterized with a cascade impactor with respect to mass median aerodynamic diameter, geometric standard deviation, and fine particle dose. In vivo, dry powder inhalation exposure was delivered as a short bolus dose, to anesthetized and mechanically ventilated landrace pigs. In addition to plasma PK, PK assessment of airway epithelial lining fluid (ELF) was used in this study. ELF of the depth of three to fourth airway generation of the right lung was accessed using standard bronchoscopy and a synthetic absorptive matrix. Results and Conclusions: Dry powder inhalation exposures with good consistency and well characterized aerosols to the pig lung were achieved by the use of the PreciseInhale™ instrument. Drug concentrations of ELF for both FP and SX were demonstrated to be four to five orders of magnitude higher than its corresponding systemic plasma drug concentrations. Clinical PK following inhalation of the same dose was used as benchmark, and the clinical study did demonstrate similar plasma PK profiles and drug exposures of both FP and SX as the current pig study. Two factors explain the close similarity of PK (1) similiar physiology between species and (2) the consistency of dosing to animals. To conclude, our study demonstrated the utility and translational potential of conducting PK studies in pigs in the development of inhaled pharmaceuticals.