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Synthetic surfactants to treat neonatal lung disease
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MOLECULAR MEDICINE TODAY, MARCH 2000 (VOL. 6)
Synthetic surfactants to treat neonatal lung disease Bengt Robertson, Jan Johansson and Tore Curstedt
Pulmonary surfactant is a complex of surface-active lipids mixed with specific proteins. Two of these, SPB and SP-C, are essential for adsorption of surfactant lipids to the air–liquid interfaces of the lungs and, hence, are also essential for alveolar stability and effective gas exchange. Surfactant substitutes must contain at least one of these proteins (or analogues of them) to be optimally effective when administered into the airways of babies with surfactant deficiency or dysfunction. This review describes how an increased understanding of the properties of surfactant proteins has led to the development of improved synthetic surfactants with the potential to treat a wide range of respiratory disorders. Neonatal respiratory distress syndrome (RDS), a major cause of morbidity and mortality in preterm babies, is caused by lung immaturity with a deficiency of surfactant in the alveolar spaces. Pulmonary surfactant is a complex mixture that is largely composed of surface-active lipids [mainly phospholipids, particularly dipalmitoylphosphatidylcholine (DPPC)] and both hydrophilic and hydrophobic proteins. It is produced by the type II cells of the alveolar epithelium, accumulates in fetal lung liquid towards the end of gestation and facilitates air expansion of the lungs at birth. Babies with surfactant deficiency fail to establish adequate functional residual capacity in the neonatal period, and repeated collapse and reexpansion of the lungs can lead to mechanical disruption of airway epithelium and profuse leakage of proteinaceous oedema fluid into the airspaces, causing additional impairment of lung mechanics and gas exchange. These welldocumented central elements in the pathophysiology of RDS have long indicated the possible treatment, or prevention, of the disease by Bengt Robertson MD, PhD* Associate Professor of Pathology Division for Experimental Perinatal Pathology Jan Johansson MD, PhD Associate Professor of Physiological Chemistry Department of Medical Biochemistry and Biophysics Tore Curstedt MD, PhD Associate Professor of Physiological Chemistry Department of Clinical Chemistry Karolinska Institutet, Karolinska Hospital, L73, SE-171 76 Stockholm, Sweden. Tel: 146 8 517 76160 Fax: 146 8 517 76165 *e-mail: [email protected]
administration of surfactant via the airways. The efficacy of this therapeutic approach, first demonstrated in animal models of RDS (reviewed in Ref. 1), has been confirmed in a large number of randomized controlled clinical trials (reviewed in Ref. 2). Surfactant therapy is now part of routine clinical management of RDS, reducing the need for mechanical ventilation and improving survival rate. The most satisfactory results have been obtained with natural – or modified natural – surfactants extracted from mammalian lungs (i.e. with preparations containing both surface-active lipids and native surfactant-associated proteins). However, the modified natural surfactants currently on the market are relatively expensive and their supply is limited, which can restrict clinical use. There is clearly a need for new, well-defined synthetic surfactants that can be produced in large amounts at a reasonable cost, and that contain only the ingredients required for optimal therapeutic effect. In this review, we outline the biophysical properties required for surfactant substitutes under clinical conditions, with particular reference to the roles of the surfactant-associated hydrophobic proteins. We also discuss recently developed synthetic analogues of these polypeptides, designed to replace the native proteins in artificial surfactants, and briefly summarize the first clinical trials of exogenous surfactants based on such protein analogues reconstituted with surfactant lipids.
Biophysical properties of surfactant An effective lung surfactant must have at least three fundamental properties. First, it should exhibit a rate of surface adsorption matching the rapid expansion of air–liquid interfaces in the lung during the first breaths, and subsequently during the inspiration phase of each ventilatory cycle throughout life. The adsorption process should generate an equilibrium surface tension of about 25 mN m21. Second, the surfactant film should reduce surface tension to nearly 0 mN m21 during surface compression, to prevent alveolar collapse and maintain the patency of terminal bronchioles at end-expiration. Third, there must be effective replenishment of surfactant molecules at the air–liquid interfaces during surface expansion, ensuring that maximum surface tension does not rise much above the equilibrium level (low maximum surface
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The first synthetic surfactants The first exogenous surfactants tested in clinical practice were made from lipids only, simply because the structure and functional significance of the surfactant proteins were not known at the time. In two early trials, aerosolized DPPC was used as a surfactant substitute to treat neonatal RDS, without impressive effects. Later on, attempts were made to produce effective artificial surfactants for clinical use by mixing DPPC with spreading agents such as serum highdensity lipoprotein, unsaturated phosphatidylglycerol (PG) (‘ALEC’, Britannia Pharmaceuticals, Redhill, Surrey, U.K.), or hexadecanol plus 120
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tension reduces the work of breathing). These properties of lung surfactant, originally defined on the basis of in vitro surface tension measurements and theoretical considerations, are indeed expressed in the terminal air spaces, as can be shown by placing droplets of test fluids with known surface tension on the alveolar surface and observing changes in the shape of the droplets during various phases of a ‘quasistatic’ ventilatory cycle (reviewed in Ref. 3). The capacity of natural surfactant films to reduce surface tension to very low values during surface compression was originally demonstrated with a Langmuir–Wilhelmy balance system, which measures surface tension as traction force on a dipping plate. Similar data were later obtained with a ‘pulsating bubble surfactometer’, in which contractile forces are recorded in an open bubble oscillating in the sample fluid. Both these systems share the problem of film leakage with possible underestimation of film stability during surface compression3. Nevertheless, recordings made with a conventional Langmuir–Wilhelmy balance clearly demonstrate the functional significance of the hydrophobic proteins of the surfactant system. For example, a mixture of DPPC and dipalmitoylphosphatidylglycerol suspended in water does not show effective adsorption and spreading (both these mechanisms are involved in film formation) when applied as a droplet onto an air–water interface, and therefore does not reduce surface tension to very low values during surface compression. Addition of small amounts of ‘low-molecular-weight surfactant apoproteins’ (currently known as SP-B and SP-C) leads to a dramatic improvement of both spreading-rate and film stability, and near-zero surface tension is recorded after a few compression cycles4 (Fig. 1). The problem of film leakage is avoided with the captive bubble system, in which changes in surface tension during cyclic surface compression are calculated from the shape of the bubble profile (the bubble flattens at low surface tension)3. Various commercially available surfactant substitutes have been evaluated with this system, and some preparations, such as bLES (Biochemicals Inc., London, Ontario, Canada) and Curosurf (Chiesi Farmaceutici, Parma, Italy) have demonstrated seemingly optimal biophysical properties. Curosurf, at a concentration of 1 mg ml21, adsorbs extremely rapidly at an air–liquid interface. The adsorption frequently seems to occur in ‘sudden jumps’, representing simultaneous insertion of about 1014 molecules of phospholipid into the surface film5. Moreover, there seems to be a selective adsorption of DPPC from the hypophase, as only a modest compression (about 20%) might be required to reduce the surface tension to nearly 0 mN m21 during the first compression cycle5. Experiments in which the original hypophase of surfactant suspension is replaced with saline after formation of the bubble, reveal that the bubble film is probably multilamellar: it contains a surface-associated surfactant reservoir from which additional material can ‘slide’ into the surface monolayer when the area is expanded6. This latter property is probably essential for optimal physiological activity under in vivo conditions.
MOLECULAR MEDICINE TODAY, MARCH 2000 (VOL. 6)
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Molecular Medicine Today
Figure 1. Langmuir–Wilhelmy balance recordings demonstrating the functional significance of the low-molecular-weight hydrophobic surfactant proteins (currently known as SP-B and SP-C). (a) A mixture of DPPC and dipalmitoylphosphatidylglycerol (80:20, by weight), suspended in saline at a concentration of 5 mg ml21, was applied onto a hypophase of normal saline at 378C. Application of 10 ml causes only minor reduction of surface tension during a 75% surface compression cycle (cycling speed 0.33 cycles min21, red arrow). After application of an additional 90 ml there is modest reduction of surface tension during subsequent compression cycles, but minimum surface tension remains .30 mN m21 and maximum surface tension about 70 mN m21. (b) A mixture of the same phospholipids plus 5% hydrophobic surfactant proteins was studied under similar conditions. Application of 10 ml of artificial surfactant onto the surface leads to a dramatic fall in surface tension reflecting effective spreading of the surfactant material. Equilibrium surface tension is about 30 mN m21, and, from the second compression cycle onwards, minimum surface tension is well below 5 mN m21. These dynamic surface properties are nearly optimal, but the rate of spreading can be further improved with pure SP-C (or analogues of SP-C) mixed with other lipids. Reproduced with permission from Ref. 4.
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Figure 2. (a) Hypothetical model of SP-B in a membrane environment. Each of the two subunits of SP-B interact with a membrane layer via amphipathic helices, depicted as cylinders. The intersubunit Cys48–Cys48 disulphide is highlighted. (b) SPC in a phospholipid bilayer. The NMR structure of SP-C is depicted in red, the phospholipid head-groups in blue and the lipid acyl-chains in green. The palmitoyl chains bound to Cys5 and Cys6 have been inserted in the same bilayer as the peptide, but this location is not experimentally verified.
tyloxapol (‘Exosurf’, Burroughs–Wellcome, Research Triangle Park, NC, USA). These preparations, devoid of surfactant proteins, have limited acute short-term therapeutic effects in animal models of surfactant deficiency as well as in babies with established RDS, but by entering the recycling machinery of surfactant-producing alveolar type II cells they might slowly improve lung function and survival, especially when given prophylactically or during the early course of RDS (reviewed in Ref. 7).
The hydrophobic surfactant proteins The biophysical activity of synthetic surfactant lipids can be greatly enhanced by adding the hydrophobic proteins SP-B and SP-C (Fig. 1). These proteins individually accelerate film formation at an air–liquid interface, irrespective of whether the surfactant material adsorbs from the hypophase or is administered directly onto the surface. A similar effect can be exerted by hydrophobic proteins present in the surface film; by unknown mechanisms, these proteins can recruit additional lipids from the hypophase and promote tight packing of the lipids at the air–liquid interface. This type of extracellular lipid transport is apparently promoted more effectively by SP-B than by SP-C (reviewed in Ref. 8). The two water-soluble surfactant proteins SP-A and SP-D, both important constituents of the pulmonary defence system, do not seem to have immediate physiological effects. SP-A might be involved in mechanisms regulating recycling of surfactant lipids by alveolar type II cells and, furthermore, it increases resistance to surfactant inactivation by plasma proteins. Congenital absence of SP-A in knockout mice does not disturb lung mechanics or surfactant homeostasis but increases susceptibility to group B streptococcal (GBS) infection in the neonatal period9. In contrast, SP-B knockout mice die soon after birth from respiratory failure, which (as in patients with congenital SP-B deficiency) is associated with abnormal accumulation of a proSP-C processing intermediate in the airspaces, suggesting that the pathways for synthesis of these two structurally very different hydrophobic surfactant proteins are, to some extent, interdependent10. SP-D null mice do not exhibit respiratory failure, although their intra-alveolar clearance of surfactant is significantly reduced11,12. Data on SP-C knockout mice are not yet available, but deficiency of this protein has recently been demonstrated in some Belgian Blue calves which, despite being born at full term, develop respiratory problems soon after birth (Danlois, F. et al., unpublished). Together, SP-B and SP-C constitute about 1–2% of the surfactant mass and differ significantly from each other in their structural properties and the ways that they interact with lipid bilayers13,14. SP-B is a 17.4 kDa molecule with two identical disulphide-linked polypeptide chains. SP-B is a member of the family of saposin-like polypeptides, which includes several proteins with apparently widely different functions. The NMR structure of NK-lysin, a water-soluble monomeric saposin that can lyse tumour cells, was determined recently15. It remains to be seen whether data relevant to SP-B can be generated based on the NK-lysin structure. SP-B contains two subunits and it is possible that each polypeptide chain can interact with a lipid bilayer; the native protein might therefore be able to crosslink two lipid bilayers (Fig. 2a). Recent in vitro data from our laboratory indicate that this particular property of 121
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the protein can be mimicked by a crosslinking peptide that lacks structural resemblance to SP-B (Zaltash, S. et al., unpublished). SP-C is a 4.2 kDa highly hydrophobic lipopeptide that is composed of an N-terminal part containing two palmitoyl chains thioester-linked to Cys5 and Cys6 and a middle/C-terminal 37 Å long all-hydrophobic a-helix composed mainly of valine residues16 (Fig. 2b). From its size, it appears that the a-helical structure of SP-C is perfectly adapted to span a bilayer of fluid DPPC; in a DPPC monolayer, the helix adopts a tilt of about 708 (Ref. 17). Keller and co-workers18 have shown that proSP-C is a type II transmembrane protein.
The development of synthetic hydrophobic surfactant peptides The SP-B molecule is too big and structurally complex to be easily synthesized by organic chemical methods, and heterologous expression of functionally active recombinant SP-B has not yet been reported. Studies to determine whether synthetic peptides that cover different regions of the polypeptide chain can exhibit similar biophysical and physiological activity to the native protein, suggest that peptides that cover the Cterminal part and contain at least 17 amino acids, accelerate surfactant spreading and improve static lung compliance in premature newborn rabbits. Furthermore, synthetic replicas of the N-terminal part of the polypeptide chain, in particular residues 1–25, mimic most of the activities performed by native, full-length, SP-B (Ref. 19). In contrast to SP-B, SP-C is monomeric and contains only 35 residues, which makes it suitable for solid phase peptide synthesis. However, analogues of SP-C might have to be developed because syn-
0.5
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thetic peptides with the native poly-Val sequence do not fold effectively into an a-helical conformation16.
The KL4 surfactant
KL4 is a 21-residue peptide (KLLLLKLLLLKLLLLKLLLLK) that was originally designed to mimic the periodic hydrophilic and hydrophobic pattern of the N-terminal part of SP-B. This peptide is a transmembrane a-helix in surfactant-like lipids20. KL4 surfactant contains DPPC, palmitoyloleoyl-PG, palmitic acid (PA) and KL4. It has been tested in lung-lavaged piglets21 and premature rhesus monkeys22 with very promising results. In a non-controlled clinical trial, newborn babies with RDS were treated with KL4 surfactant at a dose of 200 mg kg21, resulting in a dramatic improvement in arterial-to-alveolar oxygen tension ratio (a/APO2), similar to the effect seen after treatment with natural surfactant23 (Fig. 3). Of particular interest is the recent observation that airway lavage performed with diluted KL4 surfactant seems to improve lung function in experimental and clinical meconium aspiration syndrome24.
Surfactants based on synthetic analogues of SP-C
Full-length nonpalmitoylated human SP-C analogues and various truncated forms have recently been evaluated as components of artificial surfactants25. In vitro tests using a surface balance, as well as lung pressure–volume recordings, were performed in premature newborn rabbits. A core-sequence of residues 5–31 or 6–32, including the hydrophobic part that forms a helix in native SP-C, was necessary for biophysical activity. In earlier studies on several different full-length nonpalmitoylated SP-C analogues it was concluded that formation of a transmembrane a-helix is more important than retaining the exact amino acid sequence26. Replacement of the poly-valyl part of SP-C with a poly-leucyl stretch produces an analogue that forms an a-helix, even after acid-induced denaturation. This analogue promotes spreading of (32) (37) phospholipids as efficiently as native SP-C, and, when (32) (35) mixed with the DPPC/PG/PA mixture, it increases dynamic (29) (36) lung compliance in premature newborn rabbits by about 30%27. Similar results were obtained by Takei et al.28 who synthesized analogues composed of the N-terminal portion (39) of SP-C linked to poly-Leu chains of different lengths. Artificial surfactants based on the analogues showed rapid surface-spreading in vitro and improved static lung compli(32) KL4-Surfactant ance in preterm newborn rabbits to a level comparable with that obtained by treatment with Surfacten®, a modified natu(32) ral surfactant preparation obtained from bovine lungs. The present prediction is that physiologically active SP-C analogues can be obtained by replacing the poly-valyl part with a peptide sequence that forms a nonpolar transmembrane –1 0 1 2 3 4 5 6 helix in a phospholipid bilayer and reduces the risk of selfHours post dosing aggregation. Molecular Medicine Today
Recombinant SP-C-based surfactant Figure 3. Therapeutic effect of KL4 surfactant in 39 babies with RDS. The number of observations at each time point is indicated in brackets. Before surfactant treatment the arterialto-alveolar oxygen tension ratio (a/APO2) is about 0.15, indicating severe respiratory failure requiring mechanical ventilation. After treatment with KL4 surfactant (200 mg kg21), there is a striking improvement in oxygenation, reflected by nearly threefold improvement in a/APO2. This improvement is similar to that obtained with modified natural surfactant in patients with corresponding disease severity. Reproduced with permission from Ref. 23.
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Artificial surfactant that is based on recombinant SP-C is highly effective at restoring lung function in surfactantdepleted adult rats29,30 and in premature lambs and rabbits31, with clinical trials in progress. In the early studies29,30, the recombinant peptide had the same amino acid sequence as human SP-C, but lacked the palmitoyl chains. In more recent studies31–33, Cys5 and Cys6 were replaced by Phe, and Met32 was exchanged with Ile. In contrast to natural surfactant
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MOLECULAR MEDICINE TODAY, MARCH 2000 (VOL. 6)
Glossary Arterial-to-alveolar oxygen tension ratio (a/APO2) – The measure for oxygen uptake by the lung that is independent of variations in inspired oxygen.
Meconium aspiration syndrome – Neonatal lung disease by caused aspiration of meconium, usually discharged by the baby during delivery as a consequence of asphyxia.
Captive bubble system – Equipment for determination of surface tension in a closed bubble, avoiding the problem of film leakage during surface compression. Surface tension is calculated from the shape of the bubble profile.
Positive end-expiratory pressure (PEEP) – Airway pressure maintained at the end of expiration during mechanical ventilation, to avoid alveolar collapse.
Dynamic lung compliance – Mixed parameter of lung mechanics recorded under dynamic conditions (e.g. during ventilation). It is influenced by both elastic recoil forces and resistance, and usually obtained by dividing tidal volume by inspiration pressure (peak pressure minus positive end-expiratory pressure). Functional residual capacity – Volume of gas remaining in the lungs at end-expiration.
Pulmonary surfactant – Surface-active material produced mainly by the type II cells of the alveolar epithelium. Pulsating bubble surfactometer – Equipment designed for measurements of dynamic surface tension during rapid area oscillation. Surface tension is determined by recording the pressure gradient across the wall of an open bubble, pulsating in the sample fluid. Respiratory distress syndrome (RDS) – Neonatal lung disease caused by lung immaturity with deficiency of surfactant.
Hypophase – Bulk phase of liquid below the surface film. Langmuir–Wilhelmy balance – Surface balance in which surface tension is recorded as traction force on a dipping plate. Surface area can be changed with a moveable barrier.
preparations, an SP-C-based artificial surfactant substitute is only physiologically active under experimental in vivo conditions when the animal is ventilated with positive end-expiratory pressure (PEEP)31. This indicates that the airways are stabilized more effectively by the natural surfactant product. Although this difference is intriguing, it might not be very relevant under clinical conditions because ventilation with PEEP is part of the routine management of patients with severe respiratory failure.
Perspectives for the future The range of applications for surfactant therapy is widening. Originally introduced for the treatment of severely ill, ventilator-dependent babies with RDS, exogenous surfactant is now administered as rescue therapy at a much earlier phase of the disease. It seems that, in
The outstanding questions surfactant operate as a multilayer rather than a mono• Does layer at the air–liquid interface? dimeric SP-B essential for normal surfactant function? • IsIs palmitoylation of SP-C essential for normal surfactant • function? the hydrophobic proteins cooperate to enhance adsorp• Do tion of surfactant lipids and film stability; if so, what are the
•
precise molecular mechanisms involved? What is the phenotype of the SP-C knockout mouse?
Static lung compliance – Slope of the pressure-volume loop determined under static conditions (i.e. at points of zero flow). Type II cell – Cuboidal, surfactant-producing cell of the alveolar epithelium.
babies with RDS, combined treatment with nasal continuous positiveairway-pressure and exogenous surfactant instilled during a brief period of intubation, can significantly reduce the need for mechanical ventilation during the subsequent course of the disease34,35. Surfactant therapy might also be effective in babies with meconium aspiration, neonatal GBS pneumonia, lung hypoplasia caused by diaphragmatic hernia, pulmonary haemorrhage, early chronic lung disease and the adult form of acute respiratory distress syndrome (ARDS)36. We speculate that, in the future, new artificial surfactant substitutes will be tailored to resist inactivation by meconium or leaking plasma proteins, or to maximize bacteriostatic effects. We also believe that exogenous surfactant could become used as a carrier substance for antibiotics, specific antibodies, antioxidants or other drugs administered via the airways for topical treatment of various lung diseases not primarily related to the surfactant system. Acknowledgments.We are grateful to Magnus Gustafsson for the preparation of Fig. 2. The authors’ research is supported by the Swedish Medical Research Council (Projects No. 3351 and 10371) and Konung Oscar II:s Jubileumsfond.
References 01 Robertson, B. (1995). Experimental models for evaluation of exogenous surfactant. In Surfactant Therapy for Lung Disease (1st edn), (Robertson, B. and Taeusch, H.W., eds), pp. 239–267, Marcel Dekker 02 Halliday, H. and Robertson, B. (1994) Surfactant replacement. In Fetus and Neonate (1st edn) (Hanson, M.A. et al. eds), pp. 265–302, Cambridge University Press 03 Robertson, B. and Schürch, S. (1998) Assessment of surfactant function. In Methods in Pulmonary Research (1st edn), (Uhlig, S. and Taylor, A.E., eds), pp. 343–383, Birkhäuser Verlag
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04 Suzuki, Y. et al. (1986) The role of the low-molecular weight (<15 000 daltons) apoproteins of pulmonary surfactant. Eur. J. Respir. Dis. 69, 336–345 05 Schürch, S. et al. (1994) Surface activity of lipid extract surfactant in relation to film area compression and collapse. J. Appl. Physiol. 77, 974–986 06 Schürch, S. and Bachofen H. (1995) Biophysical aspects in the design of a therapeutic surfactant. In Surfactant Therapy for Lung Disease (1st edn), (Robertson, B. and Taeusch, H.W. eds), pp. 3–32, Marcel Dekker 07 Morley, C.J. (1992) Clinical experience with artificial surfactant. In Pulmonary Surfactant. From Molecular Biology to Clinical Practice (1st edn), (Robertson, B. et al. eds), pp. 605–633, Elsevier 08 Keough, K.M.W. (1992) Physical chemistry of pulmonary surfactant in the terminal air spaces. In Pulmonary Surfactant. From Molecular Biology to Clinical Practice (1st edn), (Robertson, B. et al. eds), pp. 109–164, Elsevier 09 LeVine, A.M., et al. (1997) Surfactant protein A-deficient mice are susceptible to group B streptococcal infection. J. Immunol. 158, 4336–4340 10 Clark, J.C. et al. (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in new-born mice. Proc. Natl. Acad. Sci. U. S. A. 92, 7794–7798 11 Korfhagen, T.R. et al. (1998) Surfactant protein-D regulates surfactant phospholipid homeostatis in vivo. J. Biol. Chem. 273, 28438–28443 12 Botas, C. et al. (1998) Altered surfactant homeostatis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Natl. Acad. Sci. U. S. A. 95, 11869–11874 13 Johansson, J. and Curstedt, T. (1997) Molecular structures and interactions of pulmonary surfactant components. Eur. J. Biochem. 244, 675–693 14 Pérez-Gil, J. and Keough, K.M.W. (1998) Interfacial properties of surfactant proteins. Biochim. Biophys. Acta 1408, 203–217 15 Liepinsh, E. et al. (1997) Saposin fold revealed by the NMR structure of NK-lysin. Nat. Struct. Biol. 4, 793–795 16 Johansson, J. (1998) Structure and properties of surfactant protein C. Biochim. Biophys. Acta 1408, 161–172 17 Gericke, C.R. et al. (1997) Structure and orientation of lung surfactant SP-C and La-dipalmitoylphosphatidylcholine in aqueous monolayers. Biophys. J. 73, 492–499 18 Keller, A. et al. (1991) The pulmonary surfactant protein C (SP-C) precursor is a type II transmembrane protein. Biochem. J. 277, 493–499 19 Lipp, M.M. et al. (1996) Phase and morphology changes in lipid monolayers induced by SP-B protein and its amino-terminal peptide. Science 273, 1196–1199 20 Gustafsson, M. et al. (1996) The 21-residue surfactant peptide (LysLeu4)4Lys (KL4) is a transmembrane a-helix with a mixed nonpolar/polar surface. FEBS Lett. 384, 185–188 21 Sood, S.L. et al. (1996) Exogenous surfactants in a piglet model of acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 153, 820–828
22 Revak, S.D. et al. (1996) Efficacy of synthetic peptide-containing surfactant in the treatment of respiratory distress syndrome in preterm infant rhesus monkeys. Pediatr. Res. 39, 401–410 23 Cochrane, C.G. et al. (1996) The efficacy and safety of KL4-surfactant in preterm infants with respiratory distress syndrome. Am. Respir. J. Crit. Care Med. 153, 404–410 24 Cochrane, C.G. et al. (1996) Bronchoalveolar lavage with KL4-surfactant in models of meconium aspiration syndrome. Pediatr. Res. 44, 705–715 25 Takei, T. et al. (1996) The surface properties of chemically synthesized peptides analogous to human pulmonary surfactant protein SP-C. Biol. Pharm. Bull. 19, 1247–1253 26 Johansson, J. et al. (1995) Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C. Biochem. J. 307, 535–541 27 Nilsson, G. et al. (1998) Synthetic peptide-containing surfactants. Evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution. Eur. J. Biochem. 255, 116–124 28 Takei, T. et al. (1996) Characterization of poly-leucine substituted analogues of the human surfactant protein SP-C. Biol. Pharm. Bull. 19, 1550–1555 29 Häfner, D. et al. (1994) Effects of lung surfactant factor (LSF) treatment on gas exchange and histopathological changes in an animal model of adult respiratory distress syndrome (ARDS): comparison of recombinant LSF with bovine LSF. Pulm. Pharmacol. 7, 319–332 30 Häfner, D. et al. (1995) Dose-response comparisons of five lung surfactant factor (LSF) preparations in an animal model of adult respiratory distress syndrome (ARDS). Br. J. Pharmacol. 115, 451–458 31 Davis, A.J. et al. (1998) Lung function in premature lambs and rabbits treated with a recombinant SP-C surfactant. Am. J. Respir. Crit. Care Med. 157, 553–559 32 Häfner, D. et al. (1998) Effects of rSP-C surfactant on oxygenation and histology in a rat-lung-lavage model of acute lung injury. Am. J. Respir. Crit. Care Med. 158, 270–278 33 Häfner, D. et al. (1998) Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome. Br. J. Pharmacol. 124, 1083–1090 34 Verder, H. et al. (1994) Surfactant therapy and nasal continuous positive airway pressure for newborns with respiratory distress syndrome. N. Engl. J. Med. 331, 1051–1055 35 Verder, H. et al. (1999) Nasal continuous positive airway pressure and early surfactant therapy for respiratory distress syndrome in newborns of less than 30 weeks’ gestation. Pediatrics 103, E24 36 Robertson, B. (1996) New targets for surfactant replacement therapy; experimental and clinical aspects. Arch. Dis. Child. 75, F1–F3
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Synthetic surfactants to treat neonatal lung disease Bengt Robertson, Jan Johansson and Tore Curstedt
Pulmonary surfactant is a complex of surface-active lipids mixed with specific proteins. Two of these, SPB and SP-C, are essential for adsorption of surfactant lipids to the air–liquid interfaces of the lungs and, hence, are also essential for alveolar stability and effective gas exchange. Surfactant substitutes must contain at least one of these proteins (or analogues of them) to be optimally effective when administered into the airways of babies with surfactant deficiency or dysfunction. This review describes how an increased understanding of the properties of surfactant proteins has led to the development of improved synthetic surfactants with the potential to treat a wide range of respiratory disorders. Neonatal respiratory distress syndrome (RDS), a major cause of morbidity and mortality in preterm babies, is caused by lung immaturity with a deficiency of surfactant in the alveolar spaces. Pulmonary surfactant is a complex mixture that is largely composed of surface-active lipids [mainly phospholipids, particularly dipalmitoylphosphatidylcholine (DPPC)] and both hydrophilic and hydrophobic proteins. It is produced by the type II cells of the alveolar epithelium, accumulates in fetal lung liquid towards the end of gestation and facilitates air expansion of the lungs at birth. Babies with surfactant deficiency fail to establish adequate functional residual capacity in the neonatal period, and repeated collapse and reexpansion of the lungs can lead to mechanical disruption of airway epithelium and profuse leakage of proteinaceous oedema fluid into the airspaces, causing additional impairment of lung mechanics and gas exchange. These welldocumented central elements in the pathophysiology of RDS have long indicated the possible treatment, or prevention, of the disease by Bengt Robertson MD, PhD* Associate Professor of Pathology Division for Experimental Perinatal Pathology Jan Johansson MD, PhD Associate Professor of Physiological Chemistry Department of Medical Biochemistry and Biophysics Tore Curstedt MD, PhD Associate Professor of Physiological Chemistry Department of Clinical Chemistry Karolinska Institutet, Karolinska Hospital, L73, SE-171 76 Stockholm, Sweden. Tel: 146 8 517 76160 Fax: 146 8 517 76165 *e-mail: [email protected]
administration of surfactant via the airways. The efficacy of this therapeutic approach, first demonstrated in animal models of RDS (reviewed in Ref. 1), has been confirmed in a large number of randomized controlled clinical trials (reviewed in Ref. 2). Surfactant therapy is now part of routine clinical management of RDS, reducing the need for mechanical ventilation and improving survival rate. The most satisfactory results have been obtained with natural – or modified natural – surfactants extracted from mammalian lungs (i.e. with preparations containing both surface-active lipids and native surfactant-associated proteins). However, the modified natural surfactants currently on the market are relatively expensive and their supply is limited, which can restrict clinical use. There is clearly a need for new, well-defined synthetic surfactants that can be produced in large amounts at a reasonable cost, and that contain only the ingredients required for optimal therapeutic effect. In this review, we outline the biophysical properties required for surfactant substitutes under clinical conditions, with particular reference to the roles of the surfactant-associated hydrophobic proteins. We also discuss recently developed synthetic analogues of these polypeptides, designed to replace the native proteins in artificial surfactants, and briefly summarize the first clinical trials of exogenous surfactants based on such protein analogues reconstituted with surfactant lipids.
Biophysical properties of surfactant An effective lung surfactant must have at least three fundamental properties. First, it should exhibit a rate of surface adsorption matching the rapid expansion of air–liquid interfaces in the lung during the first breaths, and subsequently during the inspiration phase of each ventilatory cycle throughout life. The adsorption process should generate an equilibrium surface tension of about 25 mN m21. Second, the surfactant film should reduce surface tension to nearly 0 mN m21 during surface compression, to prevent alveolar collapse and maintain the patency of terminal bronchioles at end-expiration. Third, there must be effective replenishment of surfactant molecules at the air–liquid interfaces during surface expansion, ensuring that maximum surface tension does not rise much above the equilibrium level (low maximum surface
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The first synthetic surfactants The first exogenous surfactants tested in clinical practice were made from lipids only, simply because the structure and functional significance of the surfactant proteins were not known at the time. In two early trials, aerosolized DPPC was used as a surfactant substitute to treat neonatal RDS, without impressive effects. Later on, attempts were made to produce effective artificial surfactants for clinical use by mixing DPPC with spreading agents such as serum highdensity lipoprotein, unsaturated phosphatidylglycerol (PG) (‘ALEC’, Britannia Pharmaceuticals, Redhill, Surrey, U.K.), or hexadecanol plus 120
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tension reduces the work of breathing). These properties of lung surfactant, originally defined on the basis of in vitro surface tension measurements and theoretical considerations, are indeed expressed in the terminal air spaces, as can be shown by placing droplets of test fluids with known surface tension on the alveolar surface and observing changes in the shape of the droplets during various phases of a ‘quasistatic’ ventilatory cycle (reviewed in Ref. 3). The capacity of natural surfactant films to reduce surface tension to very low values during surface compression was originally demonstrated with a Langmuir–Wilhelmy balance system, which measures surface tension as traction force on a dipping plate. Similar data were later obtained with a ‘pulsating bubble surfactometer’, in which contractile forces are recorded in an open bubble oscillating in the sample fluid. Both these systems share the problem of film leakage with possible underestimation of film stability during surface compression3. Nevertheless, recordings made with a conventional Langmuir–Wilhelmy balance clearly demonstrate the functional significance of the hydrophobic proteins of the surfactant system. For example, a mixture of DPPC and dipalmitoylphosphatidylglycerol suspended in water does not show effective adsorption and spreading (both these mechanisms are involved in film formation) when applied as a droplet onto an air–water interface, and therefore does not reduce surface tension to very low values during surface compression. Addition of small amounts of ‘low-molecular-weight surfactant apoproteins’ (currently known as SP-B and SP-C) leads to a dramatic improvement of both spreading-rate and film stability, and near-zero surface tension is recorded after a few compression cycles4 (Fig. 1). The problem of film leakage is avoided with the captive bubble system, in which changes in surface tension during cyclic surface compression are calculated from the shape of the bubble profile (the bubble flattens at low surface tension)3. Various commercially available surfactant substitutes have been evaluated with this system, and some preparations, such as bLES (Biochemicals Inc., London, Ontario, Canada) and Curosurf (Chiesi Farmaceutici, Parma, Italy) have demonstrated seemingly optimal biophysical properties. Curosurf, at a concentration of 1 mg ml21, adsorbs extremely rapidly at an air–liquid interface. The adsorption frequently seems to occur in ‘sudden jumps’, representing simultaneous insertion of about 1014 molecules of phospholipid into the surface film5. Moreover, there seems to be a selective adsorption of DPPC from the hypophase, as only a modest compression (about 20%) might be required to reduce the surface tension to nearly 0 mN m21 during the first compression cycle5. Experiments in which the original hypophase of surfactant suspension is replaced with saline after formation of the bubble, reveal that the bubble film is probably multilamellar: it contains a surface-associated surfactant reservoir from which additional material can ‘slide’ into the surface monolayer when the area is expanded6. This latter property is probably essential for optimal physiological activity under in vivo conditions.
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Figure 1. Langmuir–Wilhelmy balance recordings demonstrating the functional significance of the low-molecular-weight hydrophobic surfactant proteins (currently known as SP-B and SP-C). (a) A mixture of DPPC and dipalmitoylphosphatidylglycerol (80:20, by weight), suspended in saline at a concentration of 5 mg ml21, was applied onto a hypophase of normal saline at 378C. Application of 10 ml causes only minor reduction of surface tension during a 75% surface compression cycle (cycling speed 0.33 cycles min21, red arrow). After application of an additional 90 ml there is modest reduction of surface tension during subsequent compression cycles, but minimum surface tension remains .30 mN m21 and maximum surface tension about 70 mN m21. (b) A mixture of the same phospholipids plus 5% hydrophobic surfactant proteins was studied under similar conditions. Application of 10 ml of artificial surfactant onto the surface leads to a dramatic fall in surface tension reflecting effective spreading of the surfactant material. Equilibrium surface tension is about 30 mN m21, and, from the second compression cycle onwards, minimum surface tension is well below 5 mN m21. These dynamic surface properties are nearly optimal, but the rate of spreading can be further improved with pure SP-C (or analogues of SP-C) mixed with other lipids. Reproduced with permission from Ref. 4.
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Figure 2. (a) Hypothetical model of SP-B in a membrane environment. Each of the two subunits of SP-B interact with a membrane layer via amphipathic helices, depicted as cylinders. The intersubunit Cys48–Cys48 disulphide is highlighted. (b) SPC in a phospholipid bilayer. The NMR structure of SP-C is depicted in red, the phospholipid head-groups in blue and the lipid acyl-chains in green. The palmitoyl chains bound to Cys5 and Cys6 have been inserted in the same bilayer as the peptide, but this location is not experimentally verified.
tyloxapol (‘Exosurf’, Burroughs–Wellcome, Research Triangle Park, NC, USA). These preparations, devoid of surfactant proteins, have limited acute short-term therapeutic effects in animal models of surfactant deficiency as well as in babies with established RDS, but by entering the recycling machinery of surfactant-producing alveolar type II cells they might slowly improve lung function and survival, especially when given prophylactically or during the early course of RDS (reviewed in Ref. 7).
The hydrophobic surfactant proteins The biophysical activity of synthetic surfactant lipids can be greatly enhanced by adding the hydrophobic proteins SP-B and SP-C (Fig. 1). These proteins individually accelerate film formation at an air–liquid interface, irrespective of whether the surfactant material adsorbs from the hypophase or is administered directly onto the surface. A similar effect can be exerted by hydrophobic proteins present in the surface film; by unknown mechanisms, these proteins can recruit additional lipids from the hypophase and promote tight packing of the lipids at the air–liquid interface. This type of extracellular lipid transport is apparently promoted more effectively by SP-B than by SP-C (reviewed in Ref. 8). The two water-soluble surfactant proteins SP-A and SP-D, both important constituents of the pulmonary defence system, do not seem to have immediate physiological effects. SP-A might be involved in mechanisms regulating recycling of surfactant lipids by alveolar type II cells and, furthermore, it increases resistance to surfactant inactivation by plasma proteins. Congenital absence of SP-A in knockout mice does not disturb lung mechanics or surfactant homeostasis but increases susceptibility to group B streptococcal (GBS) infection in the neonatal period9. In contrast, SP-B knockout mice die soon after birth from respiratory failure, which (as in patients with congenital SP-B deficiency) is associated with abnormal accumulation of a proSP-C processing intermediate in the airspaces, suggesting that the pathways for synthesis of these two structurally very different hydrophobic surfactant proteins are, to some extent, interdependent10. SP-D null mice do not exhibit respiratory failure, although their intra-alveolar clearance of surfactant is significantly reduced11,12. Data on SP-C knockout mice are not yet available, but deficiency of this protein has recently been demonstrated in some Belgian Blue calves which, despite being born at full term, develop respiratory problems soon after birth (Danlois, F. et al., unpublished). Together, SP-B and SP-C constitute about 1–2% of the surfactant mass and differ significantly from each other in their structural properties and the ways that they interact with lipid bilayers13,14. SP-B is a 17.4 kDa molecule with two identical disulphide-linked polypeptide chains. SP-B is a member of the family of saposin-like polypeptides, which includes several proteins with apparently widely different functions. The NMR structure of NK-lysin, a water-soluble monomeric saposin that can lyse tumour cells, was determined recently15. It remains to be seen whether data relevant to SP-B can be generated based on the NK-lysin structure. SP-B contains two subunits and it is possible that each polypeptide chain can interact with a lipid bilayer; the native protein might therefore be able to crosslink two lipid bilayers (Fig. 2a). Recent in vitro data from our laboratory indicate that this particular property of 121
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the protein can be mimicked by a crosslinking peptide that lacks structural resemblance to SP-B (Zaltash, S. et al., unpublished). SP-C is a 4.2 kDa highly hydrophobic lipopeptide that is composed of an N-terminal part containing two palmitoyl chains thioester-linked to Cys5 and Cys6 and a middle/C-terminal 37 Å long all-hydrophobic a-helix composed mainly of valine residues16 (Fig. 2b). From its size, it appears that the a-helical structure of SP-C is perfectly adapted to span a bilayer of fluid DPPC; in a DPPC monolayer, the helix adopts a tilt of about 708 (Ref. 17). Keller and co-workers18 have shown that proSP-C is a type II transmembrane protein.
The development of synthetic hydrophobic surfactant peptides The SP-B molecule is too big and structurally complex to be easily synthesized by organic chemical methods, and heterologous expression of functionally active recombinant SP-B has not yet been reported. Studies to determine whether synthetic peptides that cover different regions of the polypeptide chain can exhibit similar biophysical and physiological activity to the native protein, suggest that peptides that cover the Cterminal part and contain at least 17 amino acids, accelerate surfactant spreading and improve static lung compliance in premature newborn rabbits. Furthermore, synthetic replicas of the N-terminal part of the polypeptide chain, in particular residues 1–25, mimic most of the activities performed by native, full-length, SP-B (Ref. 19). In contrast to SP-B, SP-C is monomeric and contains only 35 residues, which makes it suitable for solid phase peptide synthesis. However, analogues of SP-C might have to be developed because syn-
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The KL4 surfactant
KL4 is a 21-residue peptide (KLLLLKLLLLKLLLLKLLLLK) that was originally designed to mimic the periodic hydrophilic and hydrophobic pattern of the N-terminal part of SP-B. This peptide is a transmembrane a-helix in surfactant-like lipids20. KL4 surfactant contains DPPC, palmitoyloleoyl-PG, palmitic acid (PA) and KL4. It has been tested in lung-lavaged piglets21 and premature rhesus monkeys22 with very promising results. In a non-controlled clinical trial, newborn babies with RDS were treated with KL4 surfactant at a dose of 200 mg kg21, resulting in a dramatic improvement in arterial-to-alveolar oxygen tension ratio (a/APO2), similar to the effect seen after treatment with natural surfactant23 (Fig. 3). Of particular interest is the recent observation that airway lavage performed with diluted KL4 surfactant seems to improve lung function in experimental and clinical meconium aspiration syndrome24.
Surfactants based on synthetic analogues of SP-C
Full-length nonpalmitoylated human SP-C analogues and various truncated forms have recently been evaluated as components of artificial surfactants25. In vitro tests using a surface balance, as well as lung pressure–volume recordings, were performed in premature newborn rabbits. A core-sequence of residues 5–31 or 6–32, including the hydrophobic part that forms a helix in native SP-C, was necessary for biophysical activity. In earlier studies on several different full-length nonpalmitoylated SP-C analogues it was concluded that formation of a transmembrane a-helix is more important than retaining the exact amino acid sequence26. Replacement of the poly-valyl part of SP-C with a poly-leucyl stretch produces an analogue that forms an a-helix, even after acid-induced denaturation. This analogue promotes spreading of (32) (37) phospholipids as efficiently as native SP-C, and, when (32) (35) mixed with the DPPC/PG/PA mixture, it increases dynamic (29) (36) lung compliance in premature newborn rabbits by about 30%27. Similar results were obtained by Takei et al.28 who synthesized analogues composed of the N-terminal portion (39) of SP-C linked to poly-Leu chains of different lengths. Artificial surfactants based on the analogues showed rapid surface-spreading in vitro and improved static lung compli(32) KL4-Surfactant ance in preterm newborn rabbits to a level comparable with that obtained by treatment with Surfacten®, a modified natu(32) ral surfactant preparation obtained from bovine lungs. The present prediction is that physiologically active SP-C analogues can be obtained by replacing the poly-valyl part with a peptide sequence that forms a nonpolar transmembrane –1 0 1 2 3 4 5 6 helix in a phospholipid bilayer and reduces the risk of selfHours post dosing aggregation. Molecular Medicine Today
Recombinant SP-C-based surfactant Figure 3. Therapeutic effect of KL4 surfactant in 39 babies with RDS. The number of observations at each time point is indicated in brackets. Before surfactant treatment the arterialto-alveolar oxygen tension ratio (a/APO2) is about 0.15, indicating severe respiratory failure requiring mechanical ventilation. After treatment with KL4 surfactant (200 mg kg21), there is a striking improvement in oxygenation, reflected by nearly threefold improvement in a/APO2. This improvement is similar to that obtained with modified natural surfactant in patients with corresponding disease severity. Reproduced with permission from Ref. 23.
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Artificial surfactant that is based on recombinant SP-C is highly effective at restoring lung function in surfactantdepleted adult rats29,30 and in premature lambs and rabbits31, with clinical trials in progress. In the early studies29,30, the recombinant peptide had the same amino acid sequence as human SP-C, but lacked the palmitoyl chains. In more recent studies31–33, Cys5 and Cys6 were replaced by Phe, and Met32 was exchanged with Ile. In contrast to natural surfactant
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Glossary Arterial-to-alveolar oxygen tension ratio (a/APO2) – The measure for oxygen uptake by the lung that is independent of variations in inspired oxygen.
Meconium aspiration syndrome – Neonatal lung disease by caused aspiration of meconium, usually discharged by the baby during delivery as a consequence of asphyxia.
Captive bubble system – Equipment for determination of surface tension in a closed bubble, avoiding the problem of film leakage during surface compression. Surface tension is calculated from the shape of the bubble profile.
Positive end-expiratory pressure (PEEP) – Airway pressure maintained at the end of expiration during mechanical ventilation, to avoid alveolar collapse.
Dynamic lung compliance – Mixed parameter of lung mechanics recorded under dynamic conditions (e.g. during ventilation). It is influenced by both elastic recoil forces and resistance, and usually obtained by dividing tidal volume by inspiration pressure (peak pressure minus positive end-expiratory pressure). Functional residual capacity – Volume of gas remaining in the lungs at end-expiration.
Pulmonary surfactant – Surface-active material produced mainly by the type II cells of the alveolar epithelium. Pulsating bubble surfactometer – Equipment designed for measurements of dynamic surface tension during rapid area oscillation. Surface tension is determined by recording the pressure gradient across the wall of an open bubble, pulsating in the sample fluid. Respiratory distress syndrome (RDS) – Neonatal lung disease caused by lung immaturity with deficiency of surfactant.
Hypophase – Bulk phase of liquid below the surface film. Langmuir–Wilhelmy balance – Surface balance in which surface tension is recorded as traction force on a dipping plate. Surface area can be changed with a moveable barrier.
preparations, an SP-C-based artificial surfactant substitute is only physiologically active under experimental in vivo conditions when the animal is ventilated with positive end-expiratory pressure (PEEP)31. This indicates that the airways are stabilized more effectively by the natural surfactant product. Although this difference is intriguing, it might not be very relevant under clinical conditions because ventilation with PEEP is part of the routine management of patients with severe respiratory failure.
Perspectives for the future The range of applications for surfactant therapy is widening. Originally introduced for the treatment of severely ill, ventilator-dependent babies with RDS, exogenous surfactant is now administered as rescue therapy at a much earlier phase of the disease. It seems that, in
The outstanding questions surfactant operate as a multilayer rather than a mono• Does layer at the air–liquid interface? dimeric SP-B essential for normal surfactant function? • IsIs palmitoylation of SP-C essential for normal surfactant • function? the hydrophobic proteins cooperate to enhance adsorp• Do tion of surfactant lipids and film stability; if so, what are the
•
precise molecular mechanisms involved? What is the phenotype of the SP-C knockout mouse?
Static lung compliance – Slope of the pressure-volume loop determined under static conditions (i.e. at points of zero flow). Type II cell – Cuboidal, surfactant-producing cell of the alveolar epithelium.
babies with RDS, combined treatment with nasal continuous positiveairway-pressure and exogenous surfactant instilled during a brief period of intubation, can significantly reduce the need for mechanical ventilation during the subsequent course of the disease34,35. Surfactant therapy might also be effective in babies with meconium aspiration, neonatal GBS pneumonia, lung hypoplasia caused by diaphragmatic hernia, pulmonary haemorrhage, early chronic lung disease and the adult form of acute respiratory distress syndrome (ARDS)36. We speculate that, in the future, new artificial surfactant substitutes will be tailored to resist inactivation by meconium or leaking plasma proteins, or to maximize bacteriostatic effects. We also believe that exogenous surfactant could become used as a carrier substance for antibiotics, specific antibodies, antioxidants or other drugs administered via the airways for topical treatment of various lung diseases not primarily related to the surfactant system. Acknowledgments.We are grateful to Magnus Gustafsson for the preparation of Fig. 2. The authors’ research is supported by the Swedish Medical Research Council (Projects No. 3351 and 10371) and Konung Oscar II:s Jubileumsfond.
References 01 Robertson, B. (1995). Experimental models for evaluation of exogenous surfactant. In Surfactant Therapy for Lung Disease (1st edn), (Robertson, B. and Taeusch, H.W., eds), pp. 239–267, Marcel Dekker 02 Halliday, H. and Robertson, B. (1994) Surfactant replacement. In Fetus and Neonate (1st edn) (Hanson, M.A. et al. eds), pp. 265–302, Cambridge University Press 03 Robertson, B. and Schürch, S. (1998) Assessment of surfactant function. In Methods in Pulmonary Research (1st edn), (Uhlig, S. and Taylor, A.E., eds), pp. 343–383, Birkhäuser Verlag
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04 Suzuki, Y. et al. (1986) The role of the low-molecular weight (<15 000 daltons) apoproteins of pulmonary surfactant. Eur. J. Respir. Dis. 69, 336–345 05 Schürch, S. et al. (1994) Surface activity of lipid extract surfactant in relation to film area compression and collapse. J. Appl. Physiol. 77, 974–986 06 Schürch, S. and Bachofen H. (1995) Biophysical aspects in the design of a therapeutic surfactant. In Surfactant Therapy for Lung Disease (1st edn), (Robertson, B. and Taeusch, H.W. eds), pp. 3–32, Marcel Dekker 07 Morley, C.J. (1992) Clinical experience with artificial surfactant. In Pulmonary Surfactant. From Molecular Biology to Clinical Practice (1st edn), (Robertson, B. et al. eds), pp. 605–633, Elsevier 08 Keough, K.M.W. (1992) Physical chemistry of pulmonary surfactant in the terminal air spaces. In Pulmonary Surfactant. From Molecular Biology to Clinical Practice (1st edn), (Robertson, B. et al. eds), pp. 109–164, Elsevier 09 LeVine, A.M., et al. (1997) Surfactant protein A-deficient mice are susceptible to group B streptococcal infection. J. Immunol. 158, 4336–4340 10 Clark, J.C. et al. (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in new-born mice. Proc. Natl. Acad. Sci. U. S. A. 92, 7794–7798 11 Korfhagen, T.R. et al. (1998) Surfactant protein-D regulates surfactant phospholipid homeostatis in vivo. J. Biol. Chem. 273, 28438–28443 12 Botas, C. et al. (1998) Altered surfactant homeostatis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Natl. Acad. Sci. U. S. A. 95, 11869–11874 13 Johansson, J. and Curstedt, T. (1997) Molecular structures and interactions of pulmonary surfactant components. Eur. J. Biochem. 244, 675–693 14 Pérez-Gil, J. and Keough, K.M.W. (1998) Interfacial properties of surfactant proteins. Biochim. Biophys. Acta 1408, 203–217 15 Liepinsh, E. et al. (1997) Saposin fold revealed by the NMR structure of NK-lysin. Nat. Struct. Biol. 4, 793–795 16 Johansson, J. (1998) Structure and properties of surfactant protein C. Biochim. Biophys. Acta 1408, 161–172 17 Gericke, C.R. et al. (1997) Structure and orientation of lung surfactant SP-C and La-dipalmitoylphosphatidylcholine in aqueous monolayers. Biophys. J. 73, 492–499 18 Keller, A. et al. (1991) The pulmonary surfactant protein C (SP-C) precursor is a type II transmembrane protein. Biochem. J. 277, 493–499 19 Lipp, M.M. et al. (1996) Phase and morphology changes in lipid monolayers induced by SP-B protein and its amino-terminal peptide. Science 273, 1196–1199 20 Gustafsson, M. et al. (1996) The 21-residue surfactant peptide (LysLeu4)4Lys (KL4) is a transmembrane a-helix with a mixed nonpolar/polar surface. FEBS Lett. 384, 185–188 21 Sood, S.L. et al. (1996) Exogenous surfactants in a piglet model of acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 153, 820–828
22 Revak, S.D. et al. (1996) Efficacy of synthetic peptide-containing surfactant in the treatment of respiratory distress syndrome in preterm infant rhesus monkeys. Pediatr. Res. 39, 401–410 23 Cochrane, C.G. et al. (1996) The efficacy and safety of KL4-surfactant in preterm infants with respiratory distress syndrome. Am. Respir. J. Crit. Care Med. 153, 404–410 24 Cochrane, C.G. et al. (1996) Bronchoalveolar lavage with KL4-surfactant in models of meconium aspiration syndrome. Pediatr. Res. 44, 705–715 25 Takei, T. et al. (1996) The surface properties of chemically synthesized peptides analogous to human pulmonary surfactant protein SP-C. Biol. Pharm. Bull. 19, 1247–1253 26 Johansson, J. et al. (1995) Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C. Biochem. J. 307, 535–541 27 Nilsson, G. et al. (1998) Synthetic peptide-containing surfactants. Evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution. Eur. J. Biochem. 255, 116–124 28 Takei, T. et al. (1996) Characterization of poly-leucine substituted analogues of the human surfactant protein SP-C. Biol. Pharm. Bull. 19, 1550–1555 29 Häfner, D. et al. (1994) Effects of lung surfactant factor (LSF) treatment on gas exchange and histopathological changes in an animal model of adult respiratory distress syndrome (ARDS): comparison of recombinant LSF with bovine LSF. Pulm. Pharmacol. 7, 319–332 30 Häfner, D. et al. (1995) Dose-response comparisons of five lung surfactant factor (LSF) preparations in an animal model of adult respiratory distress syndrome (ARDS). Br. J. Pharmacol. 115, 451–458 31 Davis, A.J. et al. (1998) Lung function in premature lambs and rabbits treated with a recombinant SP-C surfactant. Am. J. Respir. Crit. Care Med. 157, 553–559 32 Häfner, D. et al. (1998) Effects of rSP-C surfactant on oxygenation and histology in a rat-lung-lavage model of acute lung injury. Am. J. Respir. Crit. Care Med. 158, 270–278 33 Häfner, D. et al. (1998) Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome. Br. J. Pharmacol. 124, 1083–1090 34 Verder, H. et al. (1994) Surfactant therapy and nasal continuous positive airway pressure for newborns with respiratory distress syndrome. N. Engl. J. Med. 331, 1051–1055 35 Verder, H. et al. (1999) Nasal continuous positive airway pressure and early surfactant therapy for respiratory distress syndrome in newborns of less than 30 weeks’ gestation. Pediatrics 103, E24 36 Robertson, B. (1996) New targets for surfactant replacement therapy; experimental and clinical aspects. Arch. Dis. Child. 75, F1–F3
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