Rationale: CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease).Objectives: Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases.Methods: Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions.Measurements and Main Results: CC16 bound to integrin a 4 b 1 ), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site.Conclusions: We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Estimation of crystalline solute solubility is well documented throughout the literature. However, the anhydrous crystal form is typically considered with these models, which is not always the most stable crystal form in water. In this study an equation which predicts the aqueous solubility of a hydrate is presented. This research attempts to extend the utility of the ideal solubility equation by incorporating desolvation energetics of the hydrated crystal. Similar to the ideal solubility equation, which accounts for the energetics of melting, this model approximates the energy of dehydration to the entropy of vaporization for water. Aqueous solubilities, dehydration and melting temperatures, and log P values were collected experimentally and from the literature. The data set includes different hydrate types and a range of log P values. Three models are evaluated, the most accurate model approximates the entropy of dehydration (ΔSd) by the entropy of vaporization (ΔSvap) for water, and utilizes onset dehydration and melting temperatures in combination with log P. With this model, the average absolute error for the prediction of solubility of 14 compounds was 0.32 log units.

Preformulation studies on tofacitinib citrate, a small molecule JAK3 specific inhibitor, have not been previously reported in literature. We therefore conducted several preformulation studies on tofacitinib citrate, and its free base, to better understand factors that affect its solubility, stability, and solid-state characteristics. Further, the results of the preformulation studies helped facilitate the development of a nebulized formulation of tofacitinib citrate for inhalational delivery to house dust mite allergen-challenged, BALB/c mice as a potential treatment for eosinophilic asthma. The preformulation results indicated tofacitinib having a basic pKa of 5.2, with its stability dependent on pH, ionic strength, and temperature. Degradation of tofacitinib follows apparent first-order kinetics. In order to maximize stability of the drug, ionic strength and temperature should be minimized, with an optimal range pH between 2.0 and 5.0. Additionally, our findings demonstrate that tofacitinib citrate can successfully be nebulized at a suitable droplet size for inhalation (1.2 ± 0.2 μm MMAD) through a nose-only chamber. Animals dosed with tofacitinib citrate demonstrated marked reductions in BAL eosinophils and total protein concentrations following HDM challenge. These data suggest that tofacitinib citrate represents the potential to be an effective therapy for eosinophilic asthma.

No abstract

Human surfactant protein-A2 (SP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria and fungi to facilitate pathogen clearance, as well as downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M.M. Pettigrew, et al., BMC Med Genet 8:15, 2007, DOI: 10.1186/1471-2350-8-15; Y. Wang, et al., Am J Hum Genet 84:52-59, 2009, DOI: 10.1016/j.ajhg.2008.11.010). Previous work performed by our group showed differences in SP-A binding to non-live mycoplasma membrane fraction, dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. Based on these differences, we have derived 20-amino acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live M. pneumoniae (Mp) in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both TNF-α mRNA and protein levels in comparison to the 20-mer containing 223K during Mp infection. While neither 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during Mp infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggests that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during Mp infection.