RESUMEN
Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.
Asunto(s)
Surfactantes Pulmonares , Síndrome de Dificultad Respiratoria del Recién Nacido , Síndrome de Dificultad Respiratoria , Recién Nacido , Animales , Femenino , Conejos , Adulto , Humanos , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Surfactantes Pulmonares/farmacología , Surfactantes Pulmonares/uso terapéutico , Surfactantes Pulmonares/química , Tensoactivos/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/metabolismo , Péptidos/farmacología , Péptidos/químicaRESUMEN
During postnatal adaptation pulmonary surfactant may be inactivated by lipopolysaccharide (LPS). We evaluated the effect of surfactant therapy in combination with antibiotic polymyxin B (PxB) in double-hit model of neonatal lung injury. Surfactant (poractant alfa, Curosurf) was exposed to smooth (S) LPS without/with PxB and tested in captive bubble surfactometer. Preterm rabbits received intratracheally saline (control) or S-LPS and were ventilated with 100% oxygen. After 30 min, LPS-treated animals received no treatment, or surfactant (200 mg/kg) without/with 3% PxB; controls received the same dose of surfactant. Animals were ventilated for further 2 h. In vitro, addition of 5% S-LPS to surfactant increased minimum surface tension (γmin) and addition of 1-3% PxB to surfactant/S-LPS mixture restored γmin to low values. Animals only given S-LPS had lower lung compliance and lung gas volume (LGV) compared to surfactant groups. Treatment with surfactant/PxB, but not with surfactant only, restored LGV. Addition of PxB to the surfactant increased the alveolar expansion. S-LPS interferes with surface activity of the pulmonary surfactant and PxB improves the resistance of surfactant to LPS-induced inactivation. In our neonatal model of respiratory distress syndrome surfactant gives positive response even in simultaneous exposure to S-LPS, when enriched with PxB.
Asunto(s)
Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/metabolismo , Polimixina B/farmacología , Surfactantes Pulmonares/metabolismo , Animales , Animales Recién Nacidos , Productos Biológicos/administración & dosificación , Productos Biológicos/antagonistas & inhibidores , Modelos Animales de Enfermedad , Femenino , Humanos , Técnicas In Vitro , Recién Nacido , Lipopolisacáridos/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Rendimiento Pulmonar/efectos de los fármacos , Lesión Pulmonar/inducido químicamente , Masculino , Fosfolípidos/administración & dosificación , Fosfolípidos/antagonistas & inhibidores , Polimixina B/administración & dosificación , Surfactantes Pulmonares/administración & dosificación , Surfactantes Pulmonares/agonistas , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismoRESUMEN
BACKGROUND: Ascending maternofetal bacterial infections often result in premature birth and neonatal respiratory distress. These neonates are treated with exogenous pulmonary surfactant (SF) and systemic antibiotics. Polymyxins are antimicrobiotic peptides that may bind to SF phospholipids. OBJECTIVES: Does topical administration of SF/polymyxin reduce bacterial growth in neonatal rabbit pneumonia and improve pulmonary function? METHODS: Neonatal rabbits were tracheotomized and treated intratracheally with mixtures of porcine SF, SF/polymyxin E (PxE), or polymyxin B (PxB). Control animals received saline. Animals were then inoculated with Escherichia coli and ventilated for 4 h. During the experiment, peak insufflation pressures, dynamic lung compliance, and ECG were recorded. Pulmonary and renal bacterial load were determined. Lung histology was performed. Lung and kidney IL-8 were measured in subgroups. RESULTS: Eighty-five animals were included in 2 experimental series, of which 78% survived 4 h of ventilation. E. coli inoculation caused severe neonatal pneumonia with median IL-8 levels of 2.2 ng/g in the lungs compared to a median of 0.2 ng/g in the lungs of the saline controls (p < 0.01). Lung compliance after 4 h was significantly increased at a mean of 0.48 ml/(kg·cm H2O) in the SF group and 0.43 in the SF + PxE group compared to 0.35 in the E. coli group (p < 0.01). In direct comparison, bacterial growth found in the E. coli group was reduced 20-fold in the SF + PxB group compared to 75-fold in the SF + PxE group. CONCLUSION: Addition of polymyxin to SF effectively promotes antimicrobial treatment and improves lung function in neonatal pneumonia of rabbits.
RESUMEN
BACKGROUND: The development of synthetic surfactants for the treatment of lung pulmonary diseases has been going on for many years. OBJECTIVES: To investigate the effects of phospholipid mixtures combined with SP-B and SP-C analogues on lung functions in an animal model of respiratory distress syndrome. METHODS: Natural and synthetic phospholipid mixtures with/without SP-B and/or SP-C analogues were instilled in ventilated premature newborn rabbits. Lung functions were evaluated. RESULTS: Treatment with Curosurf or phospholipids from Curosurf combined with SP-B and SP-C analogues gave similar results. Treatment with phospholipids from adult rabbit lungs or liver combined with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG) gave tidal volumes (VT) well above physiological levels, but alveolar stability at end-expiration was only achieved when these phospholipids were combined with analogues of SP-B and SP-C. Treatment with egg yolk-PC mixed with DPPC with and without POPG gave small VT, but after addition of both analogues VT was only somewhat lower and lung gas volumes (LGV) similar to those obtained with Curosurf. Substitution of egg yolk-PC (≥99% PC) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and combining them with DPPC, POPG and 2% each of the SP-B and SP-C analogue gave a completely synthetic surfactant with similar effects on VT and LGV as Curosurf. CONCLUSIONS: Phospholipid composition is important for VT while the SP-B and SP-C analogues increase alveolar stability at end-expiration. Synthetic surfactant consisting of unsaturated and saturated phosphatidylcholines, POPG and the analogues of SP-B and SP-C has similar activity as Curosurf regarding VT and LGV in an animal model using preterm newborn rabbits ventilated without positive end-expiratory pressure.
Asunto(s)
Fosfolípidos/análisis , Nacimiento Prematuro/tratamiento farmacológico , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares , Animales , Animales Recién Nacidos , Estabilidad de Medicamentos , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/veterinaria , Surfactantes Pulmonares/química , Surfactantes Pulmonares/farmacocinética , Surfactantes Pulmonares/uso terapéutico , Conejos , Volumen de Ventilación Pulmonar/efectos de los fármacosRESUMEN
BACKGROUND: Plasma proteins are known to interfere with pulmonary surfactant. Studies have proven the hypothesis that fibrinogen preserves exogenous surfactant subjected to long-term surface area cycling. METHODS: The exogenous surfactant Curosurf was subjected to long-term surface area cycling without or with fibrinogen (ratio 2:1 w/w) and was tested by captive bubble surfactometer and on newborn premature rabbits. RESULTS: Surface tension increased in Curosurf (80 mg/ml) samples without fibrinogen after 6-12 d of cycling. In samples with fibrinogen the cycling time had no effect on surface tension. Addition of fibrinogen to surfactant prevented lipid peroxidation. Lung gas volumes of animals with noncycled Curosurf or Curosurf cycled with fibrinogen for 6 d were comparable and higher than in rabbits with Curosurf cycled without fibrinogen. Alveolar volume density was higher in groups with noncycled Curosurf or Curosurf cycled with fibrinogen than in Curosurf cycled without fibrinogen (both P < 0.001). CONCLUSION: The effect of fibrinogen on pulmonary surfactant cycled at 37 °C depends both on surfactant concentration and cycling time. At high phospholipid concentration used in clinical practice fibrinogen has a protective effect on biophysical and physiological properties of natural modified surfactant subjected to surface area cycling. This effect is partially mediated by reduction in lipid peroxidation.
Asunto(s)
Modelos Animales de Enfermedad , Fibrinógeno/química , Surfactantes Pulmonares/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Animales , Humanos , Recién Nacido , Surfactantes Pulmonares/química , Conejos , Tensión SuperficialRESUMEN
The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20 h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Pulmón/metabolismo , Péptidos/metabolismo , Fosfatidilgliceroles/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Medición de Intercambio de Deuterio , Femenino , Feto , Humanos , Recién Nacido , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/química , Fosfatidilgliceroles/química , Embarazo , Nacimiento Prematuro , Estructura Secundaria de Proteína , Proteína C Asociada a Surfactante Pulmonar/administración & dosificación , Proteína C Asociada a Surfactante Pulmonar/química , Conejos , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Espectroscopía Infrarroja por Transformada de Fourier , Volumen de Ventilación Pulmonar/fisiologíaRESUMEN
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.