Inhaled nintedanib is well-tolerated and delivers key pharmacokinetic parameters required to treat bleomycin-induced pulmonary fibrosis.

Oral nintedanib is marketed for the treatment of idiopathic pulmonary fibrosis (IPF), Systemic Sclerosis-Associated Interstitial Lung Disease and Chronic Fibrosing Interstitial Lung Diseases with a Progressive Phenotype. While effective at slowing fibrosis progression, as an oral medicine nintedanib has limitations. To reduce side effects and maximize efficacy, nintedanib was reformulated as a solution for nebulization and inhaled administration. To predict effectiveness treating IPF, inhalation was used as a tool to dissect the pharmacokinetic components required for nintedanib pulmonary anti-fibrotic activity. Following oral administration, nintedanib extensively partitioned into tissue and exhibited flip-flop pharmacokinetics, whereby resulting lung Cmax and AUC were substantially higher than plasma. By comparison, inhaled nintedanib was capable of delivering an oral-equivalent lung Cmax with lower local and systemic AUC. Using a multi-challenge bleomycin rat model, this distinct inhaled pharmacokinetic profile was dose responsive (0.05, 0.25 and 0.375 mg/kg), delivering oral-superior pulmonary anti-fibrotic activity with an equivalent delivered lung Cmax (QD inhaled 0.375 mg/kg versus BID oral 60 mg/kg). Possibly assisting this improvement, the infrequent high inhaled dose also improved bleomycin-challenged animal weight gain to levels equivalent to sham. By comparison, BID oral weight gain was substantially less than controls, suggesting a negative health impact on oral administered animals combating fibrosis. Both oral and inhaled administration exhibited anti-inflammatory activity, with oral achieving significance. In summary, inhalation (short-duration nintedanib lung Cmax without high local or systemic AUC) was well-tolerated and was effective reducing bleomycin-induced pulmonary fibrosis.

Inhaled biopharmaceutical drug development: nonclinical considerations and case studies.

Biopharmaceuticals are complex molecules often manufactured from living systems and their specificity and novelty holds great promise for the treatment of chronic diseases for which there are currently no cures. The inhalation route of biopharmaceutical drug delivery is attractive because the large surface area of the lung, and close proximity of the alveolar and vascular systems, maximizes the potential for drug delivery to the lung and/or systemic circulation. In addition, costs of delivery to the patient are potentially much reduced, in comparison with parental administration, since inhalation is non-invasive and likely to promote patient compliance. However, in comparison with small molecule drug development, developing an inhaled biopharmaceutical that is effective and safe for human use is associated with many challenges. This review considers some general principles of drug delivery to lung and issues associated with the translation of proof of concept studies to toxicology safety studies (e.g. aerosol generation, species selection, exaggerated pharmacology, and immunogenicity). This review also presents a summary of nonclinical and clinical data from inhaled biopharmaceuticals which are either marketed for human use or in Phase II clinical trials (e.g. DNase, insulin, human growth hormone, vaccines, therapeutic plasmid DNA complexes).

Differential alveolar epithelial injury and protein expression in pneumococcal pneumonia.

The integrity of the alveolar epithelium is a key factor in the outcome of acute lung injury. Here, we investigate alveolar epithelial injury and the expression of epithelial-selective markers in Streptococcus pneumoniae-induced acute lung injury. S. pneumoniae was instilled into rat lungs and alveolar type I (RTI(40)/podoplanin, MMC6 antigen) and alveolar type II (MMC4 antigen, surfactant protein D, pro-surfactant protein C, RTII(70)) cell markers were quantified in lavage fluid and lung tissue at 24 and 72 hours. The alveolar epithelium was also examined using electron, confocal, and light microscopy. S. pneumoniae induced an acute inflammatory response as assessed by increased total protein, SP-D, and neutrophils in lavage fluid. Biochemical and morphological studies demonstrated morphologic injury to type II cells but not type I cells. In particular, the expression of RTI(40)/podoplanin was dramatically reduced, on the surface of type I cells, in the absence of morphologic injury. These data demonstrate that type II cell damage can occur in the absence of type I cell injury without affecting the ability of the lung to return to a normal morphology. These data also demonstrate that RTI(40)/podoplanin is not a type I cell phenotypic marker in experimental acute lung injury caused by S. pneumoniae. Given that RTI(40)/podoplanin is an endogenous ligand for the C-type lectin receptor and this receptor plays a role in platelet aggregation and neutrophil activation, we hypothesize that the reduction of RTI(40)/podoplanin on type I cells might be important for the regulation of platelet and/or neutrophil function in experimental acute lung injury.

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types.

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.

Increased virulence of a fibronectin-binding protein mutant of Staphylococcus aureus in a rat model of pneumonia.

Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia.