Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C. Here, we have designed single polypeptides that combine properties of SP-B and SP-C and produced them recombinantly using a novel solubility tag based on spider silk production. These Combo peptides mixed with phospholipids are as efficient as nature-derived surfactant preparations against neonatal RDS in premature rabbit fetuses.

Efficient delipidation of a recombinant lung surfactant lipopeptide analogue by liquid-gel chromatography.

Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS.

Natural Derived Surfactant Preparation As a Carrier of Polymyxin E for Treatment of Pseudomonas aeruginosa Pneumonia in a Near-Term Rabbit Model.

BACKGROUND: Pulmonary surfactant spreads rapidly over the airway epithelium, a property that could be harnessed to transport drugs into the lungs. For efficient drug delivery, an interaction between pulmonary surfactant and the drug to be administered is likely needed. On the other hand, the interaction should not compromise the activity of surfactant or the drug once delivered in vivo. The antibiotics gentamicin (an aminoglycoside) and polymyxin E represent drugs that could benefit from being delivered directly to the lung, thereby increasing local concentrations and reducing systemic side effects. Our aim was to study how the animal-derived surfactant poractant alfa (Curosurf®) affects the activities of polymyxin E and gentamicin against Pseudomonas aeruginosa. METHODS: In vitro antimicrobial assays and a neonatal near-term rabbit model were used to evaluate the combinations of antibiotics and surfactant against Pseudomonas aeruginosa. RESULTS: The bactericidal activity of polymyxin E, but not of gentamicin, against P. aeruginosa was partly reduced in vitro in the presence of poractant alfa. In contrast, in the rabbit model of P. aeruginosa pneumonia, polymyxin E administrated together with surfactant was superior in lowering the bacterial load in the lungs compared to polymyxin E alone, without affecting plethysmographically recorded lung compliance. CONCLUSIONS: The results suggest that polymyxin E interacts with poractant alfa, which reduces the antibacterial effect in vitro. However, when polymyxin E mixed with surfactant is used in the in vivo pneumonia model, increased bactericidal effect was observed. This may be due to a more efficient spreading mediated by interactions between polymyxin E and surfactant. These results warrant further studies of surfactant preparations for drug delivery against lung infections.

Disulphide Bridges in Surfactant Protein B Analogues Affect Their Activity in Synthetic Surfactant Preparations.

BACKGROUND: Limited supply and complicated manufacturing procedure of animal-derived surfactants make the development of synthetic surfactants warranted. The synthesis of surfactant protein (SP)-B and SP-C is complicated and several analogues have been developed. Mini-BLeu is an analogue that corresponds to the first and last helix of SP-B joined by a loop and linked by 2 disulphide bridges. SP-C33Leu is an SP-C analogue that can be cost-efficiently produced, but no such analogue has yet been described for SP-B. OBJECTIVE: To design short SP-B analogues which lack disulphide bridges, are easy to produce and are efficacious in a preterm rabbit fetus model of neonatal RDS. METHODS: Synthetic surfactants were prepared by adding 2 or 8% (w/w) of synthetic variants of Mini-B27, similar to Mini-BLeu but with a short loop, or different peptides covering helix 1 of SP-B to 2% (w/w) of SP-C33Leu in 80 mg/mL of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg yolk phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 50: 40: 10 (by weight). Premature newborn rabbit fetuses were treated with 200 mg/kg of the surfactant preparations and ventilated with defined pressures for 30 min without positive end-expiratory pressure. Tidal volumes were registered during the experiments and lung gas volumes were measured at the end of the ventilation period. RESULTS: Synthetic surfactant containing the Mini-B27 analogue with 2 disulphides gives similar lung gas volumes as treatment with an animal-derived surfactant preparation, but all other SP-B analogues gave lower lung gas volumes. All synthetic surfactants studied gave no significant differences in compliances except the surfactant containing the Mini-B27 analogue without cysteines that performed somewhat better at 30 min. CONCLUSION: The helix-loop-helix SP-B analogues tested in this study require the presence of 2 disulphide bridges for optimal activity in a rabbit RDS model.