Synthetic surfactant based on analogues of SP-B and SP-C is superior to single-peptide surfactants in ventilated premature rabbits.

BACKGROUND: Respiratory distress syndrome (RDS) is currently treated with surfactant preparations obtained from natural sources and attempts to develop equally active synthetic surfactants have been unsuccessful. One difference in composition is that naturally derived surfactants contain the two hydrophobic proteins SP-B and SP-C while synthetic preparations contain analogues of either SP-B or SP-C. It was recently shown that both SP-B and SP-C (or SP-C33, an SP-C analogue) are necessary to establish alveolar stability at end-expiration in a rabbit RDS model, as reflected by high lung gas volumes without application of positive end-expiratory pressure. OBJECTIVES: To study the efficacy of fully synthetic surfactants containing analogues of both SP-B and SP-C compared to surfactants with only one protein analogue. METHODS: Premature newborn rabbits, treated with synthetic surfactants, were ventilated for 30 min without positive end-expiratory pressure. Tidal volumes as well as lung gas volumes at end-expiration were determined. RESULTS: Treatment with 2% Mini-B (a short-cut version of SP-B) and 2% SP-C33, or its C-terminally truncated form SP-C30, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 68:31 (w/w) resulted in median lung gas volumes of 8-9 ml/kg body weight, while animals treated with 2% Mini-B surfactant or 2% SP-C33/SP-C30 surfactant had lung gas volumes of 3-4 ml/kg, and those treated with Curosurf, a porcine surfactant, 15-17 ml/kg. In contrast, mixing SP-C33 with peptides with different distributions of positively charged and hydrophobic residues did not improve lung gas volumes. CONCLUSIONS: The data indicate that synthetic surfactants containing analogues of both SP-B and SP-C might be superior to single-peptide surfactants in the treatment of RDS.

Inactivation of pulmonary surfactant by silicone oil in vitro and in ventilated immature rabbits.

OBJECTIVE: Surface activity of pulmonary surfactant is impaired by exposure to syringes lubricated with silicone oil (SO). These syringes are used daily in clinical practice. DESIGN: In vitro experiments were used for detection of SO, determination of surface activity, and semiquantitative measurement of surfactant protein (SP)-B and -C in SO/surfactant mixtures. Randomized, controlled animal studies were applied for determination of in vivo activity. SETTING: University research laboratory. INTERVENTIONS: Mass spectrometry of SO originating from syringes with and without surfactant was performed. The surface activity of SO plus surfactant phospholipids (PLs) or modified natural surfactant (Curosurf) was measured. SO/Curosurf preparations were further analyzed for changes in the content of SP-B and SP-C using immunoblotting. Neonatal rabbits received mixtures of SO/Curosurf (ratio 0-1.3 mg/mg PL) intratracheally and were then ventilated with a standardized sequence of peak insufflation pressures. Tidal volume curves were recorded, gas volumes of excised lungs were measured, and histologic analysis was performed. MEASUREMENTS AND MAIN RESULTS: Dissolved SO was found after rinsing syringes with organic solvents or Curosurf. Surface activity of Curosurf was significantly reduced after addition of 0.13-1.3 mg SO/mg PL. Immunoblotting revealed interference of SO with SP-B, but not with SP-C. With increasing SO/Curosurf ratios, patchy alveolar air expansion was observed, lung gas volumes were reduced, and time to inflate the lungs was increased, whereas compliance and tidal volumes remained unimpaired. CONCLUSIONS: In vitro SO impairs surface activity of Curosurf and leads to interference with SP-B. SO contamination of exogenous surfactant impairs lung function in animal studies and should be avoided.

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome.

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.

Concentration dependence of a poly-leucine surfactant protein C analogue on in vitro and in vivo surfactant activity.

BACKGROUND: Modified natural surfactants currently used for treatment of respiratory distress syndrome contain about 0.5-1% (w/w phospholipids) of each of the surfactant proteins SP-B and SP-C. The supply of these preparations is limited and synthetic surfactant preparations containing lipids and peptides are under development. OBJECTIVES: To investigate the potential of different concentrations of the SP-C analogue SP-C33 in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (68:31, w/w). METHODS: Surface activity was evaluated in pulsating and captive bubble surfactometers and in immature newborn rabbits. RESULTS: Preparations containing >or=1% SP-C33 achieve minimum surface tension <5 mN/m indicating good biophysical activity, and increase tidal volumes in premature rabbit fetuses to the same level as a modified natural surfactant preparation does. Alveolar patency at end expiration, as evaluated by measurement of lung gas volumes, histological assessment of alveolar expansion and determination of alveolar volume density, was lower in the animals treated with synthetic surfactant than in those receiving modified natural surfactant. CONCLUSIONS: These data suggest that SP-C33 is similarly efficient as the native peptide in improving surface properties of phospholipids mixtures and in increasing lung compliance in surfactant-deficient states, but that other components are needed to maintain alveolar stability at low airway pressures.

Polymyxin B/pulmonary surfactant mixtures have increased resistance to inactivation by meconium and reduce growth of gram-negative bacteria in vitro.

Pulmonary surfactant is inactivated in meconium aspiration syndrome and neonatal pneumonia. Development of an exogenous surfactant less sensitive to inactivation might be useful for treating these diseases. We investigated in vitro whether addition of the cationic cyclic membrane cross-linking peptide polymyxin B (PxB) and/or calcium chloride (CaCl2) to modified porcine surfactant Curosurf increases resistance to meconium-induced inactivation of surface activity while antimicrobial activity of PxB is maintained. To study bacterial proliferation, Escherichia coli, group B streptococci (GBS), or Staphylococcus aureus were incubated 0-5 h in saline or in meconium in the presence or absence of Curosurf with or without PxB. PxB and CaCl2 improved spreading and adsorption of Curosurf. Curosurf plus CaCl2/PxB needed a 4-fold increase of meconium concentration to increase dynamic surface tension significantly compared with Curosurf plus CaCl2 alone, indicating that PxB further increases the resistance of Curosurf to meconium-induced inactivation. Meconium alone like meconium/Curosurf promoted growth of E. coli and GBS, but addition of Curosurf/PxB or PxB alone significantly reduced the growth of E. coli. Biophysical and antibacterial properties of Curosurf and PxB may be combined into a useful adjunct in the treatment of neonatal Gram-negative pneumonia and/or meconium aspiration syndrome.

Dextran or polyethylene glycol added to curosurf for treatment of meconium lung injury in rats.

BACKGROUND: In experimental lung injuries, improvement of lung function after treatment with surfactant/polymer mixtures may depend on both type of polymer and the specific surfactant. In vitro studies suggest that dextran is more effective when mixed with Curosurf, and polyethylene glycol (PEG) is more effective when mixed with Survanta. We therefore wanted to find out whether these results held true in an animal model of acute lung injury. OBJECTIVE: To compare the response to therapy of PEG vs. dextran when added to Curosurf after meconium lung injury. METHODS: Lung injury was produced by intratracheal instillation of meconium (30 and 4 ml/kg). One hour after injury, Curosurf (35 mg/ml) with or without 5% dextran (68 kDa) or 5% PEG (10 kDa) was given. Arterial blood gases and peak inspiratory pressures were measured for 3 h after treatment while animals were supported by volume-regulated ventilation. Then animals were sacrificed and pressure volume relationships, lung wet/dry weights, and histology were assessed. RESULTS: Initially, improved PaO2 and inspiratory pressure occurred for both Curosurf/PEG and Curosurf/dextran groups compared with Curosurf, but at three hours, peak inspiratory pressure and PaO2 remained significantly improved for the Curosurf/dextran but not for Curosurf/PEG groups when compared with Curosurf alone. Total lung capacity at the end of the experiment was also significantly increased in the Curosurf/dextran group, but not the Curosurf/PEG group when compared with Curosurf. CONCLUSION: Under these experimental conditions, Curosurf/dextran mixtures provided a better therapeutic response than Curosurf/PEG or Curosurf.

Spontaneous breathing or mechanical ventilation alters lung compliance and tissue association of exogenous surfactant in preterm newborn rabbits.

In preterm infants with respiratory distress syndrome, surfactant administration followed by immediate extubation to spontaneous breathing with nasal continuous positive airway pressure reduces the need for mechanical ventilation. With this treatment approach, repeated doses of surfactant are rarely indicated. We used a rabbit model to test the hypothesis that exogenous surfactant therapy followed by spontaneous breathing results in a more sustained initial treatment response compared with treatment followed by mechanical ventilation. Preterm rabbits (gestational age 28.5 d) were treated with pharyngeal deposition of 200 mg/kg radiolabeled surfactant (14C-Curosurf) and randomized to 4 h of spontaneous breathing or mechanical ventilation or to a control group, killed immediately after surfactant administration. With pharyngeal deposition, 46 +/- 10% (mean +/- SEM) of the administered surfactant reached the lungs. The dynamic lung-thorax compliance was higher in spontaneously breathing compared with mechanically ventilated animals (median, 9.9 and 0.75 ml x cm H2O(-1) x kg(-1), respectively; p < 0.05). The relative distribution of 14C-Curosurf in bronchoalveolar lavage fluid and homogenized lung tissue showed a higher degree of tissue association in the spontaneously breathing animals [53 +/- 4 versus 26 +/- 3% (mean +/- SEM)] than in mechanically ventilated animals (p < 0.01), the latter figure being very similar to that of the control group (25 +/- 5%). There was a higher degree of lipid peroxidation and fewer microbubbles in bronchoalveolar lavage fluid from mechanically ventilated animals. We conclude that the initial lung tissue association of exogenous surfactant is impaired by mechanical ventilation. This is associated with a reduction of dynamic compliance and evidence of increased surfactant inactivation.

Incomplete protection by prophylactic surfactant against the adverse effects of large lung inflations at birth in immature lambs.

OBJECTIVE: To investigate whether preceding surfactant instillation prevents the harmful effect of large lung inflations at birth in immature lambs, and, if not, to find out for how long the immature lung remains sensitive to large inflations. DESIGN: In an exploratory study, 12 preterm lambs given surfactant at birth were randomized to receive five large lung inflations at four different times: at birth just before or immediately after surfactant treatment; at 10 min; or at 60 min of age. In a confirmatory study, 10 pairs of preterm lamb twins were all given surfactant before the first breath. One lamb in each pair was randomised to receive large lung inflations immediately after surfactant while the other twin underwent similar inflations at 10-15 min of age. SETTING: Animal laboratory. EXPERIMENTAL ANIMALS: Anaesthetized lambs delivered by cesarean section at a gestational age of 127 days. INTERVENTIONS: Surfactant supplementation at birth. Five sustained lung inflations of 16 ml/kg at different times in relation to surfactant instillation. Pressure-limited mechanical ventilation for 4 h. MEASUREMENTS AND RESULTS: The response to surfactant was assessed by ventilator settings, lung mechanics and lung histology. Preceding surfactant supplementation did not prevent the adverse effect of large lung inflations at birth on ventilatory efficiency and lung mechanics, but seemed to protect against severe lung injury. No adverse effect was seen from large lung inflations given at 10 min of age or later. CONCLUSION: Prophylactic surfactant supplementation does not fully protect against the harmful effect of large lung inflations during a short sensitive period immediately after birth.

Intratracheal albumin reduces interleukin-8 in tracheobronchial aspirates in piglets after meconium aspiration.

Meconium aspiration induces pulmonary inflammation and reduces surfactant function. We hypothesized that albumin mixed with meconium attenuates pulmonary inflammation and improves surfactant function after meconium aspiration. We measured the concentration of free fatty acids (FFA) in the meconium (110 mg dry weight/mL) and added albumin to provide a molar FFA:albumin ratio of 1:1. Newborn piglets, 0-2 day of age, artificially ventilated and exposed to hypoxemia by ventilation with 8% O2, were randomized to group A receiving meconium (n = 12), or group B receiving meconium + albumin (n = 12), 3 ml/kg intratracheally. The animals were reoxygenated for 8 h. Reoxygenation was started when mean arterial blood pressure was < 20 mm Hg or base excess was < -20 mmol/L. During 8 h of reoxygenation the interleukin-8 concentrations in tracheobronchial aspirates increased 5-fold more in the meconium vs. the meconium + albumin groups (93 +/- 56 vs. 18 +/- 4 pg/mL, p < 0.005). There were no differences between the groups for tumor necrosis factor alpha in tracheobronchial aspirates, recruitment of inflammatory cells in the airspaces or surfactant function in bronchoalveolar lavage fluid. In conclusion, albumin significantly decreased interleukin-8 concentrations in tracheobronchial aspirates after meconium aspiration.

Influence of modified natural and synthetic surfactant preparations on bacterial killing by polymorphonuclear leucocytes.

In addition to its biophysical functions, surfactant plays an important role in pulmonary host defense. In this investigation we studied the influence of various commercially available surfactants on the phagocytosis of bacteria that are common pathogens in the neonatal period. Group B streptococci (GBS), Escherichia coli and Staphylococcus aureus were cultured with isolated human polymorphonuclear leucocytes (PMN) and non-specific serum in the presence or absence of different modified natural (Curosurf, Alveofact, Survanta) or totally synthetic, protein-free surfactant preparations (Exosurf, Pumactant). Prior to and after 30 and 60 min of incubation with PMN at different surfactant concentrations (1, 10 or 20 mg/ml), the number of viable bacteria was determined by colony counting. Killing of S. aureus by PMN was not influenced by any of the surfactants. Alveofact and Curosurf had no significant negative impact on phagocytosis. At 20 mg/ml, Curosurf even reduced the number of viable E. coli. Survanta at 10 and 20 mg/ml and Exosurf at all concentrations impaired the killing of non-encapsulated GBS and E. coli. Pumactant at 1-20 mg/ml interfered with the phagocytosis of E. coli. In further experiments we demonstrated that Curosurf did not interfere with the phagocytosis of an encapsulated GBS-strain opsonised by a specific antiserum either. In additional experiments we analysed the influence of the different surfactants on the release of reactive oxygen metabolite by PMN and found that the changes in nitroblue tetrazolium reduction did not necessarily correlate with the findings of the studies on killing. In conclusion, we found that killing by PMN was influenced by the bacterial species and the composition and concentration of the different surfactant preparations. The strongest impairment in phagocytic function of PMN was observed with the protein-free synthetic surfactant Exosurf, a phospholipid preparation that contains the alcohols hexadecanol and tyloxapol as spreading agents.

Influence of partial liquid ventilation on bacterial growth and alveolar expansion in newborn rabbits with group B-streptococcal pneumonia.

Partial liquid ventilation (PLV) with perfluorocarbons has been considered as an alternative therapy for severe inflammatory lung disease. The present study was performed to test whether PLV influences bacterial growth and lung histology in a rabbit model of congenital pneumonia caused by group B streptococci. Near-term newborn rabbits were tracheotomized, inoculated via the airways with group B streptococci, and subsequently ventilated for 5 h with either PLV or conventional ventilation. At 30 min after group B streptococci administration, animals in the PLV group (n = 16) received 30 mL/kg body weight of perfluorocarbon (PF 5080) via the tracheal tube. Evaporative losses were substituted with 20 mL/kg perfluorocarbon at hourly intervals. Identical volumes of air were injected in control animals at the same times (n = 15). The number of colony-forming units in left lung homogenate, evaluated at the end of the experiments, tended to be lower in PLV-treated animals than in controls (6.8 x 109 versus 6.4 x 1010 colony-forming units/g body weight; p = 0.06). Comparison of these numbers with the colony-forming units injected at the beginning of the experiments revealed a reduction in bacterial number in the PLV group and proliferation in the controls (-2.2 x 108 versus +5.6 x 1010 colony-forming units/g body weight; p < 0.05). Histologic examination demonstrated less inflammation and more homogeneous lung expansion in PLV-treated animals. Two animals in the PLV group had focal interstitial emphysema. Our results suggest that PLV with PF 5080 reduces bacterial proliferation in experimental group B streptococcal pneumonia.

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.

Modified protocols for surfactant therapy in experimental meconium aspiration syndrome.

In adult rats with experimental meconium aspiration syndrome, we investigated whether the therapeutic effect of exogenous surfactant was increased by addition of dextran or preceding airway lavage with diluted surfactant. Animals (n = 72) ventilated with pure oxygen were given human meconium suspension (50-75 mg kg(-1)) through the airways. When the PaO(2) had decreased to <20 kPa (mean +/- SD 12 +/- 3.9 kPa), the rats were randomly allocated to ten groups (G). G 6-10 underwent lung lavage with diluted Curosurf (5 mg ml(-1), 20 ml kg(-1)), whereas G 1-5 did not. G 1 and 6 received no additional material through the airways. G 2 and 7 received Curosurf (100 mg kg(-1)), and G 3 and 8 received Curosurf (100 mg kg(-1)) plus dextran (75 mg kg(-1)); G 4 and 9 received Curosurf (200 mg kg(-1)), and G 5 and G 10 received Curosurf (200 mg kg(-1)) plus dextran (75 mg kg(-1)). All rats in G 1 died before 180 min after randomization. In G 2, 3, 6, 7, and 8, the PaO(2) transiently increased to 30-40 kPa. In G 4, 5, 9, and 10, the PaO(2) remained >30 kPa for 180 min. Both airway lavage and supplementation with dextran improved the therapeutic effects of surfactant; however, a large dose (200 mg kg(-1)) was nevertheless required to optimize gas exchange.

Syringes with rubber-coated plungers and inactivation of surfactant.

Injectable drugs are generally administered to newborns with 1 mL syringes. During in-vitro measurements of surface tension with a pulsating bubble surfactometer, we noticed that surfactant was inactivated after repeated aspiration into a 1 mL syringe with a rubber-coated plunger. Inactivation did not take place, however, when we used rubber-free two-part syringes with polyethylene pistons. Results of further studies showed that the silicone fluid used to lubricate the rubber surface of the syringe was inactivating the surfactant. Our findings suggest that contact with rubber surfaces should be avoided in the handling of surfactant material for biophysical assessment or clinical use.