Direct lung delivery of para-aminosalicylic acid by aerosol particles.

Para-aminosalicylic acid (PAS), a tuberculostatic agent, was formulated into large porous particles for direct delivery into the lungs via inhalation. These particles possess optimized physical properties for deposition throughout the respiratory tract, a drug loading of 95% by weight and physical stability over 4 weeks at elevated temperatures. Upon insufflation in rats, PAS concentrations were measured in plasma, lung lining fluid and homogenized whole lung tissue. Systemic drug concentrations peaked at 15 min, with a maximum plasma concentration of 11+/-1 microg/ml. The concentration in the lung lining fluid was 148+/-62 microg/ml at 15 min. Tissue concentrations were 65+/-20 microg/ml at 15 min and 3.2+/-0.2 microg/ml at 3h. PAS was cleared within 3 h from the lung lining fluid and plasma but was still present at therapeutic concentrations in the lung tissue. These results suggest that inhalation delivery of PAS can potentially allow for a reduction in total dose delivered while providing for higher local and similar peak systemic drug concentrations as compared to those obtained upon oral PAS dosing. Similar particles could potentially be used for the delivery of additional anti-tuberculosis agents such as rifampicin, aminoglucosides or fluoroquinolones.

Interaction of lung surfactant proteins with anionic phospholipids.

Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.

Synchrotron X-ray study of lung surfactant-specific protein SP-B in lipid monolayers.

This work reports the first x-ray scattering measurements to determine the effects of SP-B(1-25), the N-terminus peptide of lung surfactant-specific protein SP-B, on the structure of palmitic acid (PA) monolayers. In-plane diffraction shows that the peptide fluidizes a portion of the monolayer but does not affect the packing of the residual ordered phase. This implies that the peptide resides in the disordered phase, and that the ordered phase is essentially pure lipid, in agreement with fluorescence microscopy studies. X-ray reflectivity shows that the peptide is oriented in the lipid monolayer at an angle of approximately 56 degrees relative to the interface normal, with one end protruding past the hydrophilic region into the fluid subphase and the other end embedded in the hydrophobic region of the monolayer. The quantitative insights afforded by this study lead to a better understanding of the lipid/protein interactions found in lung surfactant systems.

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability.

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.

Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy.

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.

Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers.

Fluorescence, polarized fluorescence, and Brewster angle microscopy reveal that human lung surfactant protein SP-B and its amino terminus (SP-B[1-25]) alter the phase behavior of palmitic acid monolayers by inhibiting the formation of condensed phases and creating a new fluid protein-rich phase. This fluid phase forms a network that separates condensed phase domains at coexistence and persists to high surface pressures. The network changes the monolayer collapse mechanism from heterogeneous nucleation/growth and fracturing processes to a more homogeneous process through isolating individual condensed phase domains. This results in higher surface pressures at collapse, and monolayers easier to respread on expansion, factors essential to the in vivo function of lung surfactant. The network is stabilized by a low-line tension between the coexisting phases, as confirmed by the observation of extended linear domains, or "stripe" phases, and a Gouy-Chapman analysis of protein-containing monolayers. Comparison of isotherm data and observed morphologies of monolayers containing SP-B(1-25) with those containing the full SP-B sequence show that the shortened peptide retains most of the native activity of the full-length protein, which may lead to cheaper and more effective synthetic replacement formulations.

Phase and morphology changes in lipid monolayers induced by SP-B protein and its amino-terminal peptide.

Both human lung surfactant protein, SP-B, and its amino-terminal peptide, SP-B1-25, inhibit the formation of condensed phases in monolayers of palmitic acid, resulting in a new fluid phase. This fluid phase forms a network, separating condensed-phase domains at coexistence. The network persists to high surface pressures, altering the nucleation, growth, and morphology of monolayer collapse structures, leading to lower surface tensions on compression and more reversible respreading on expansion. The network is stabilized by the low line tension between the fluid phase and the condensed phase as confirmed by the formation of "stripe" phases.