New synthetic surfactants--basic science.

The hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of surface-active lipids to the air-liquid interface of the alveoli and are essential for alveolar stability and gas exchange. Synthetic surfactant preparations must contain at least one of these hydrophobic proteins, or analogs thereof, to have optimal effects when administered into the airways of patients with lung diseases. However, development of clinically active artificial surfactants has turned out to be more complicated than initially anticipated since the native hydrophobic proteins are structurally complex or unstable in pure form. The proteins have been replaced by different analogs which have the right conformation without forming oligomers. Increased understanding of the surfactant proteins will hopefully lead to development of effective synthetic surfactants which can be produced in large quantities for treatment of a wide range of respiratory disorders. Furthermore, the lipid composition seems to be important, as well as a high lipid concentration in the suspension. For successful treatment of many respiratory diseases, it is also desirable that the synthetic surfactant resists inactivation by plasma components leaking into the alveoli.

New synthetic surfactants: the next generation?

Surfactant preparations have been proven to improve clinical outcome of infants at risk for or having respiratory distress syndrome (RDS). In clinical trials, ani mal-derived surfactant preparations reduce the risk of pneumothorax and mortality when compared to non-protein-containing synthetic surfactant preparations. In part, this is thought to be due to the presence of surfactant proteins in animal-derived surfactant preparations. Four native surfactant proteins have been identified. The hydrophobic surfactant proteins B (SP-B) and C (SP-C) are tightly bound to phospholipids. These proteins have important roles in maintaining the surface tension-lowering properties of pulmonary surfactant. Surfactant protein A (SP-A) and D (SP-D) are extremely hydrophilic and are not retained in the preparation of any commercial animal-derived surfactant products. These proteins are thought to have a role in recycling surfactant and improving host defense. There is concern that animal-derived products may have some batch-to-batch variation regarding the levels of native pulmonary surfactant proteins. In addition, there is concern regarding the hypothetical risk of transmission of viral or unconventional infectious agents from an animal source. New surfactant preparations, composed of synthetic phospholipids and essential hydrophobic surfactant protein analogs, have been developed. These surfactant protein analogs have been produced by peptide synthesis and recombinant technology to provide a new class of synthetic surfactants that may be a suitable alternative to animal-derived surfactants. Preliminary clinical studies have shown that treatment with these novel surfactant preparations can ameliorate RDS and improve clinical outcome. Clinicians will need to further understand any differences in clinical effects between available products.

Surfactant protein C: basics to bedside.

Development of clinically active synthetic surfactants has turned out to be more complicated than initially anticipated. Surfactant protein analogues must have the right conformation without forming oligomers. Furthermore, the lipid composition, as well as a high lipid concentration in the suspension seem to be important. For successful treatment of many respiratory diseases, it is desirable that the synthetic surfactant may stabilize the alveoli at end-expiration and may resist inactivation by components leaking into the alveoli.

Mutation of SFTPC in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease.

Pulmonary surfactant protein C (SP-C) is a highly hydrophobic peptide produced by type-II alveolar cells through the processing of a high-molecular weight precursor (pro-SP-C), that enhances surface tension and facilitates the recycling of pulmonary surfactant in vitro. Recently, two seemingly dominant-negative mutations of the pro-SP-C-encoding gene (SFTPC, MIM 178620), were reported in families with vertically-inherited interstitial lung disease (Nogee et al. [2001: N Engl J Med 344:573-579]; Thomas et al. [2002: Am J Respir Crit Care Med 165:1322-1328]). We have examined the SP-C protein and its precursor as well as the encoding gene, in a cohort of 34 sporadic or familial cases with unexplained respiratory distress (URD) in which surfactant protein B (SP-B) deficiency related to SFTPB mutation had been ruled out. One patient with complete SP-C deficiency had no detectable mutation of SFTPC. Of the 10 patients with abnormal pro-SP-C processing, as suggested from analysis of broncho-alveolar lavage (BAL) fluid, two distinct heterozygous SFTPC missense mutations were identified. The first, g.1286T > C (p.I73T), was de novo and resulted in progressive respiratory failure with intra-alveolar storage of a granular, protein- and lipid-rich, periodic acid Schiff (PAS)-positive material (pulmonary alveolar proteinosis (PAP)), and interstitial lung disease. The second, g.2125G > A (p.R167Q), was found in two PAP patients from the endogamous white settler population of Réunion Island in which URD has an unexpectedly high prevalence. Since this mutation was diagnosed in subjects from this subpopulation who did not have evidence for lung disease, we propose environmental exposures or modifier genes to play a role in the phenotype, as suggested from murine models lacking the SP-C protein, although we cannot rule out a rare polymorphism, hitherto restricted to that subpopulation. Most remarkably, these observations extend the phenotypic spectrum related to SFTPC mutation from interstitial lung disease to PAP. Notably, the reported mutations do not appear to be dominant negatives. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/index.html.

A synthetic surfactant based on a poly-Leu SP-C analog and phospholipids: effects on tidal volumes and lung gas volumes in ventilated immature newborn rabbits.

Available surfactants for treatment of respiratory distress syndrome in newborn infants are derived from animal lungs, which limits supply and poses a danger of propagating infectious material. Poly-Val-->poly-Leu analogs of surfactant protein (SP)-C can be synthesized in large quantities and exhibit surface activity similar to SP-C. Here, activity of synthetic surfactants containing a poly-Leu SP-C analog (SP-C33) was evaluated in ventilated premature newborn rabbits. Treatment with 2.5 ml/kg body wt of 2% (wt/wt) SP-C33 in 1,2-dipalmitoyl-sn-3-glycero phosphoryl choline (DPPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl choline (POPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl glycerol (POPG), 68:0:31, 68:11:20, or 68:16:15 (wt/wt/wt) suspended at 80 mg/ml gave tidal volumes (Vt) of 20-25 ml/kg body wt, with an insufflation pressure of 25 cmH2O and no positive end-expiratory pressure (PEEP), comparable to the Vt for animals treated with the porcine surfactant Curosurf. Nontreated littermates had a Vt of approximately 2 ml/kg body wt. The Vt for SP-C33 in DPPC-egg phosphatidylglycerol-palmitic acid [68:22:9 (wt/wt/wt)], DPPC-POPG-palmitic acid [68:22:9 (wt/wt/wt)], and DPPC-POPC-POPG [6:2:2 (wt/wt/wt)] was 15-20 ml/kg body wt. Histological examination of lungs from animals treated with SP-C33-based surfactants showed incomplete, usually patchy air expansion of alveolar spaces associated with only mild airway epithelial damage. Lung gas volume after 30 min of mechanical ventilation were more than threefold larger in animals treated with Curosurf than in those receiving SP-C33 in DPPC-POPC-POPG, 68:11:20. This difference could be largely counterbalanced by ventilation with PEEP (3-4 cmH2O). An artificial surfactant based on SP-C33 improves Vt in immature newborn animals ventilated with standardized peak pressure but requires PEEP to build up adequate lung gas volumes.

Surfactant with SP-B and SP-C analogues improves lung function in surfactant-deficient rats.

The use of mammalian lung surfactant extracts has sharply reduced mortality and morbidity from respiratory distress syndrome in premature infants. Synthesis of surfactant protein B and C (SP-B and SP-C) analogues may lead the way to a synthetic surfactant preparation. Dimeric SP-B(1-25) (dSP-B(1-25)) is based on the N-terminal domain of human SP-B and SP-Cfc is a modified human SP-C in which a single phenylalanine is substituted for a palmitoylated cysteine residue in the N-terminal segment (Phe-4 > Cys-4 variant). We tested the effects of synthetic surfactants with 1 or 2% dSP-B(1-25) and 1% SP-Cfc on lung function in surfactant-deficient rats. Four experimental surfactant preparations were prepared by mixing 1% dSP-B(1-25), 2% dSP-B(1-25), 1% dSP-B(1-25) +1% SP-Cfc, and 2% dSP-B(1-25) +1% SP-Cfc with phospholipids (PL). PL and Survanta, a bovine lung extract, were controls. Groups of 8 rats were ventilated, lavaged until surfactant deficiency, and treated with 100 mg/kg surfactant. Arterial blood gas values and dynamic compliance were measured every 15 min and after 2 h of ventilation, the rats were killed and pressure-volume curves performed. Oxygenation improved quickly after instillation of surfactant with synthetic peptides and Survanta. Oxygenation and lung volumes were consistently higher in the 2% than in the 1% dSP-B(1-25) groups. Addition of 1% SP-Cfc to the synthetic surfactants further improved oxygenation and lung volume, but to a lesser extent than increasing the dSP-B(1-25) content from 1 to 2%. These data indicate that improvements in oxygenation and lung volume in lavaged rats are dependent on the concentration of dSP-B(1-25) in the surfactant preparation and that the presence of SP-Cfc has a relative minor effect on these parameters.

Effects of the human pulmonary surfactant protein-C (SP-C), SP-CL16(6-28) on surface activities of surfactants with various phospholipids.

We previously reported that a human analogue of pulmonary surfactant protein-C (SP-C), SP-CL16(6-28), with 23 residues was the most active analogue in a reconstituted lipid mixture and had the shortest chain among the poly-leucine-analogues examined. There has been little research on the chemical components of synthetic lung surfactants (SLSs). In the present study, we attempted to compare SLS with various phospholipids in surface activity. That is, SP-CL16(6-28) plus various phosphatidylglycerols (PG) were tested for surface activity in a Langmuir-Wilhelmy surface balance (WSB) apparatus and pulsating bubble surfactmeter (PBS). Further, SLSs were examined for biological properties using an animal model of surfactant deficiency, infant respiratory distress syndrome (IRDS), in vivo. Palmitoyl-oleoyl-phosphatidylglycerol (POPG)-SLS exhibited minimum and maximum surface tensions of 1.7 mN/m and 28.6 mN/m in WSB and 8.5 mN/m and 36.2 mN/m in PBS, respectively. Moreover, in the IRDS model, POPG-SLS remarkably improved the lung volume (LV) of a premature lagomorph fetus at LV30 cmH2O and LV5 cmH2O. That is, a significant improvement equal to the LV of a full-term fetus was observed. The level of LV exhibited respiratory improvement equivalent to surfactant-TA. SLS seemed comparable in surface activity with Surfacten (Surfactant-TA), a modified surfactant preparation which has been used for the treatment of RDS.

cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal).

In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of Mr = 5,000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. We have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNA encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated an Mr = 22,000 precursor protein, the active hydrophobic peptide being produced by proteolytic processing to Mr = 5,000-6,000. Two classes of cDNAs encoding SPL(pVal) were identified. mRNA of approximately 900 bases was identified on Northern analysis of fetal and adult RNA. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.

Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung.

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.