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Biochemical Determinants of the Function of Pulmonary Surfactant

C.G. Cochrane, S.D. Revak

Pulmonary surfactant is a monolayer of phospholipids and proteins, unique in biological systems, that forms at the air-water interface of the pulmonary alveolus. Its major function is to prevent collapse of the alveoli. Rigid acyl side chains of the phospholipids, a large percentage of which are saturated palmitoyl groups of phosphatidylcholine, resist compression of the alveolus. However, incorporation of hydrophobic proteins in the phospholipid monolayer is essential for sustained surfactant activity. Several laboratories, including ours, found that of the 4 proteins of surfactant, surfactant protein B (SP-B), when recombined with appropriate phospholipids, has the greatest activity in this regard.

SP-B is soluble only in organic solvents and consists of approximately 80 residues (Fig. 1). Experiments showed that 15-residue synthetic peptides taken from along the entire sequence had activity when combined with the phospholipids. Thus, the pattern of SP-B provides the essential function. In this pattern, intermittent basic residues, both lysine (K) and arginine (R), occur between stretches of hydrophobic residues. Simple peptides consisting of stretches of hydrophobic leucine (L) with intermittent positively charged lysines or arginine that mimic SP-B were synthesized. The peptides had activity similar to that of SP-B or synthetic SP-B peptides. An example of a simple peptide that has been extensively studied is KLLLLKLLLLKLLLLKLLLLK (KL₄).

When tryptophan residues were incorporated into the center of the leucine stretches in the synthetic peptides, fluorescence analysis indicated that the peptides lay in the hydrophobic part of the phospholipid layer, presumably in the acyl side chains, but close to the polar-head groups. Deletion of the basic residues, or replacement of basic residues with negatively charged aspartic acid residues, greatly diminished the surfactant activity. This finding suggested the existence of electrostatic interactions between the basic residues and presumably the phosphates of the polar-head groups, a hypothesis that was supported by nuclear magnetic resonance studies. Raman vibrational spectroscopy indicated that SP-B and the synthetic peptides induce order in the phospholipid molecules and increase lateral stability. These data support the hypothesis of the role of SP-B in surfactant function as diagramed in Figure 2.

In preterm rhesus monkeys with pulmonary collapse due to the prematurity of the animals, instillation of KL₄-Surfactant increased pulmonary function into the normal range within 12 hours, and direct examination of the lungs at 20--24 hours confirmed excellent expansion of the lungs. In addition, in phase 1 and phase 2 clinical trials in 47 preterm infants in 6 centers in the United States, KL₄-Surfactant induced a rapid and sustained increase in pulmonary function. Normal levels were reached within 12 hours after instillation of the surfactant. Chest radiographs revealed increasing expansion of the lungs during the 12-hour period. Phase 3 trials in human preterm infants are in preparation.

Meconium aspiration syndrome in newborn infants is perhaps the most severe disease in newborns to treat, and no approved therapeutic agent is available. Meconium degrades intrinsic surfactant and induces a marked inflammatory response in the lungs. We have attempted to treat this disease in 2.5-kg rabbits and newborn rhesus monkeys by lavaging the meconium-injured lungs with diluted KL₄-Surfactant. Lavage of the lungs with KL₄-Surfactant partially removed meconium and provided expansion of otherwise collapsed lungs in both species. Lavage with saline removed meconium but did not result in expansion of the lungs. The inflammatory response in rabbits was diminished in the lungs lavaged with KL₄-Surfactant but not in the lungs lavaged with saline. A phase 1/phase 2 clinical trial in human newborn infants with meconium aspiration syndrome is under way.

In the acute respiratory distress syndrome and acute lung injury in humans, the inflammation that develops in the lungs inactivates intrinsic surfactant. The lungs undergo atelectatic changes and fill with inflammatory exudate. Approximately 40% of patients die, a total of 50,000 per year, close to the mortality rate of AIDS and cancer. We have produced a model of acute lung injury in 2.5-kg rabbits by partially removing intrinsic surfactant and then instilling bacterial lipopolysaccharide intratracheally. The lungs become atelectatic, and proteinaceous exudate develops during a period of 6 hours. Treatment with a bolus of KL₄-Surfactant produced partial restoration of pulmonary function but did not diminish the inflammatory response. However, bronchoalveolar lavage with KL₄-Surfactant removed part of the inflammatory exudate and led to expansion of alveoli. Pulmonary function greatly improved. A protocol similar to that used in the rabbit studies was used in a phase 1 clinical trial in adult humans with acute respiratory distress syndrome, and a phase 2/phase 3 trial in approximately 50 centers in the United States is under way.

PUBLICATIONS

Cochrane, C.G. Surfactant protein B and mimic peptides in the function of pulmonary surfactant. FEBS Lett. 430:424, 1998.

Cochrane, C.G., Revak, S.D. Surfactant lavage treatment in a model of respiratory distress syndrome. Chest, in press.

Cochrane, C.G., Revak, S.D., Merritt, T., Schraufstatter, I.U., Hoch, R.C., Henderson, C., Andersson, S., Takamori, H., Oades, Z.G. Bronchoalveolar lavage with KL₄-Surfactant in models of meconium aspiration syndrome. Pediatr. Res. 44:1, 1998.