KL4 Surfactant Research Articles

Aerosol, chemical and physical properties of dry powder synthetic lung surfactant for noninvasive treatment of neonatal respiratory distress syndrome

Inhalation of dry powder synthetic lung surfactant may assist spontaneous breathing by providing noninvasive surfactant therapy for premature infants supported with nasal continuous positive airway pressure. Surfactant was formulated using spray-drying with different phospholipid compositions (70 or 80 total weight% and 7:3 or 4:1 DPPC:POPG ratios), a surfactant protein B peptide analog (KL4, Super Mini-B, or B-YL), and Lactose or Trehalose as excipient. KL4 surfactant underperformed on initial adsorption and surface activity at captive bubble surfactometry. Spray-drying had no effect on the chemical composition of Super Mini-B and B-YL peptides and surfactant with these peptides had excellent surface activity with particle sizes and fine particle fractions that were well within the margins for respiratory particles and similar solid-state properties. Prolonged exposure of the dry powder surfactants with lactose as excipient to 40 °C and 75% humidity negatively affected hysteresis during dynamic cycling in the captive bubble surfactometer. Dry powder synthetic lung surfactants with 70% phospholipids (DPPC and POPG at a 7:3 ratio), 25% trehalose and 3% of SMB or B-YL showed excellent surface activity and good short-term stability, thereby qualifying them for potential clinical use in premature infants.

Radiation Mitigating Properties of Intranasally Administered KL4Surfactant in a Murine Model of Radiation-Induced Lung Damage

The threat of exposure to ionizing radiation from a nuclear reactor accident or deliberate terrorist actions is a significant public health concern. The lung is particularly susceptible to radiation-induced injury from external sources or inhalation of radioactive particles from radioactive fallout. Radiation-induced lung disease can manifest with an acute radiation pneumonitis and/or delayed effects leading to pulmonary fibrosis. As prior warning of radiation exposure is unlikely, medical countermeasures (MCMs) to mitigate radiation-induced lung disease that can be given in mass-casualty situations many hours or days postirradiation are needed to prevent both early and late lung damage. In this study, KL4 surfactant (lucinactant) was evaluated as a radiation mitigator in a well-characterized mouse model of targeted thoracic radiation exposure, for its effect on both early (several weeks) and late (18 weeks) lung damage. Here, 120 mg/kg total phospholipid of KL4 surfactant was administered twice daily intranasally, (enabling intrapulmonary inhalation of drug) to C57BL/6 mice 24 h after a single 13.5 Gy dose of thoracic irradiation (LD50 dose). Both early and chronic phase (2 and 4 weeks and 18 weeks postirradiation, respectively) assessments were performed. Mice were evaluated for evidence of reduced arterial blood oxygenation and early and chronic lung and systemic inflammation, lung fibrosis and oxidative stress. Analysis was done by performing lung function/respiration dynamics and measuring cellular protein content of bronchoalveolar lavage fluid (BALF), and levels of cytokines, 8-iso-prostaglandin F2α, hydroxyproline in lung and plasma, along with evaluating lung histology. The results of this study showed that intranasal delivery of KL4 surfactant was able to preserve lung function as evidenced by adequate arterial oxygen saturation and reduced lung inflammation and oxidative stress; total white count and absolute neutrophil count was decreased in BALF, as were plasma pro-inflammatory cytokine levels and biomarker of oxidative stress. KL4 surfactant is a promising MCM for mitigation of lung tissue damage after targeted, thoracic irradiation and has the potential to be developed as a broad-spectrum, multi-use MCM against chemical, biological, radiological or nuclear threat agents with potential to cause lung injury.

Aerosolized KL4surfactant improves short-term survival and gas exchange in spontaneously breathing newborn pigs with hydrochloric acid-induced acute lung injury

Surfactant therapy may be beneficial in acute lung injury (ALI). In spontaneously breathing newborn pigs with ALI supported with continuous positive airway pressure (CPAP), we evaluated the hypothesis that aerosolized KL4 surfactant (AERO KL4 S) would provide a similar therapeutic effect as intratracheal KL4 surfactant (ETT KL4 S) when compared to controls. We randomized pigs with HCl-induced ALI to: (1) 175 mg/kg KL4 surfactant via endotracheal tube (ETT); (2) AERO KL4 S (22.5 mg/min phospholipid) for 60 min via continuous positive airway pressure (CPAP); or (3) sham procedure on CPAP. We obtained physiologic data and arterial blood gases throughout the 3-hr study. At study end, lungs were excised for analysis of interleukin-8 (IL-8), myeloperoxidase (MPO) levels and histomorphometric data. Pigs treated with ETT KL4 S and AERO KL4 S had improved survival and sustained pO2 compared to controls. The AERO KL4 S group had higher pH compared to controls. Lung IL-8 levels were lower in the AERO KL4 S group compared to controls. Histomorphometric analysis showed less hemorrhage in the ETT and AERO KL4 S groups compared to controls. The AERO KL4 S group had more open lung units per fixed-field than the ETT KL4 S or controls. AERO KL4 S produced similar improvements in survival, physiology, inflammatory markers, and morphology as ETT KL4 S in an ALI model.

Aerosolized KL4 Surfactant Improves Gas Exchange and Survival in Spontaneously Breathing Piglets with HCL-Induced Acute Lung Injury

Aerosolized KL 4 Surfactant Improves Gas Exchange and Survival in Spontaneously Breathing Piglets with HCL-Induced Acute Lung Injury

Dose-Response to Aerosolized KL4 Surfactant in the Spontaneously Breathing CPAP-Supported Preterm Lamb

Dose-Response to Aerosolized KL 4 Surfactant in the Spontaneously Breathing CPAP-Supported Preterm Lamb

Beneficial effects of synthetic KL4surfactant in experimental lung transplantation

The aim of this study was to investigate whether intratracheal administration of a new synthetic surfactant that includes the cationic, hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL₄), might be effective in reducing ischaemia-reperfusion injury after lung transplantation. Single left lung transplantation was performed in Landrace pigs 22 h post-harvest. KL₄ surfactant at a dose of 25 mg total phospholipid·kg body weight⁻¹ (2.5 mL·kg body weight⁻¹) was instilled at 37°C to the donor left lung (n = 8) prior to explantation. Saline (2.5 mL·kg body weight⁻¹; 37°C) was instilled into the donor left lung of the untreated group (n = 6). Lung function in recipients was measured during 2 h of reperfusion. Recipient left lung bronchoalveolar lavage (BAL) provided native cytometric, inflammatory marker and surfactant data. KL(4) surfactant treatment recovered oxygen levels in the recipient blood (mean ± sd arterial oxygen tension/inspiratory oxygen fraction 424 ± 60 versus 263 ± 101 mmHg in untreated group; p=0.01) and normalised alveolar-arterial oxygen tension difference. Surfactant biophysical function was also recovered in KL₄ surfactant-treated lungs. This was associated with decreased C-reactive protein levels in BAL, and recovery of surfactant protein A content, normalised protein/phospholipid ratios, and lower levels of both lipid peroxides and protein carbonyls in large surfactant aggregates. These findings suggest an important protective role for KL₄ surfactant treatment in lung transplantation.

Improved gas exchange and survival after KL‐4 surfactant in newborn pigs with severe acute lung injury

To determine the effectiveness of artificial surfactant therapy using KL-4 surfactant in newborn pigs with hydrochloric acid (HCl)-induced acute lung injury (ALI). After induction of ALI via intratracheal HCl instillation, pigs were randomized to receive 5.8 ml/kg KL-4 surfactant or no surfactant prior to extubation to bubble CPAP. Clinical laboratory. Spontaneously breathing newborn pigs (<1 week of age). Treatment with KL-4 surfactant on bubble CPAP with PEEP of 6 cmH(2)O for 3.5 hr after extubation compared with controls. Physiologic parameters and arterial blood gases were measured every 15 min. At the conclusion of the study, the lungs were excised for the analysis of histopathology and morphometric data. Pigs treated with KL-4 surfactant had arterial blood gases with less acidosis (P < 0.001), higher P(a)O(2) levels (P < 0.001), and lower P(a)CO(2) levels (P < 0.001). Pigs treated with KL-4 surfactant had improved survival compared with controls (6/12 KL-4, 2/12 control, P < 0.05). Postmortem morphometric data demonstrated that pigs treated with KL-4 surfactant had larger (P < 0.05) exchange units in the caudal-dorsal lung as compared to relatively atelectatic region in the control animals. In newborn pigs with severe HCl-induced ALI, treatment with KL-4 surfactant resulted in improved respiratory parameters, less dependent atelectasis, and improved short-term survival.

Effect of Surfactant Protein A on the Physical Properties and Surface Activity of KL 4-Surfactant

SP-A, the major protein component of pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. However, it is thought that therapeutic properties of natural surfactants improve after enrichment with SP-A. The objective of this study was to determine SP-A effects on physical properties and surface activity of a new synthetic lung surfactant based on a cationic and hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK, KL 4. We have analyzed the interaction of SP-A with liposomes consisting of DPPC/POPG/PA (28:9:5.6, w/w/w) with and without 0.57 mol % KL 4 peptide. We found that SP-A had a concentration-dependent effect on the surface activity of KL 4-DPPC/POPG/PA membranes but not on that of an animal-derived LES. The surface activity of KL 4-surfactant significantly improved after enrichment with 2.5–5 wt % SP-A. However, it worsened at SP-A concentrations ≥10 wt %. This was due to the fluidizing effect of supraphysiological SP-A concentrations on KL 4-DPPC/POPG/PA membranes as determined by fluorescence anisotropy measurements, calorimetric studies, and confocal fluorescence microscopy of GUVs. High SP-A concentrations caused disappearance of the solid/fluid phase coexistence of KL 4-surfactant, suggesting that phase coexistence might be important for the surface adsorption process.

Oxidative inactivation of surfactants.

Reactive oxygen species (ROS) may play an important role in the chronic pulmonary morbidity of preterm infants. We therefore studied the magnitude and mechanisms of oxidative inactivation of a natural lung surfactant (NLS) and of two surfactants used for treatment of respiratory distress syndrome, beractant and KL4 surfactant (KL4). Incubation with Fenton reagents, 2-4 mM peroxynitrite (ONOO-) or 0.5 mM hypochlorous acid (OCl-), resulted in an increased minimum surface tension (MST) of all surfactants; the order of effect on MST was beractant > KL4 > NLS. After incubation with Fenton reagents, NLS contained a higher concentration of conjugated dienes (p < 0.01) but lower concentration of malondialdehyde (p < 0.001) than beractant. Protein carbonyl concentrations after treatment with Fenton reagents were higher in NLS and KL4 than in beractant (p < 0. 05). Surface area cycling for 24 h with 2 mM ONOO- or 0.5 mM OCl- caused both beractant and KL4 to increase the proportion of light subtypes from 8-10% to 26-29%; with Fenton reagents, there was disappearance of the light subtype and formation of ultraheavy subtype 74-91% with poor MST. Natural and therapeutic surfactants differ markedly in their sensitivity to ROS, which may be important for surfactants in therapeutic use because oxidative inactivation may limit their effect. Oxidation of natural surfactant may result in reduced function and contribute to chronic lung disease.

Protein composition of synthetic surfactant affects gas exchange in surfactant-deficient rats.

Synthetic surfactant peptides offer an opportunity to standardize the protein composition of surfactant. We tested the effect of phospholipids (PL) with synthetic full-length SP-B1-78 (B), mutant B (Bser), KL4 peptide (UCLA-KL4), and palmitoylated SP-C1-35 (C) on oxygenation and lung function in a surfactant-deficient rat model. Sixty-four adult rats were ventilated with 100% oxygen, a tidal volume of 7.5 mL/kg, and a rate of 60/min. Their lungs were lavaged with saline until the arterial PO2 dropped below 80 torr, when 100 mg/kg surfactant was instilled. Surfactant preparations included: PL (PL surfactant), PL + 3% B (B surfactant), PL + 3% B and 1% C (BC surfactant), PL + 3% UCLA-KL4 (KL4 surfactant), PL + 3% Bser (Bser surfactant), and PL + 3% B and 1% UCLA-KL4 (BKL4 surfactant). Sixty minutes after surfactant instillation, positive end-expiratory pressure was applied for 5 min, and pressure-volume curves were determined in situ. The six surfactant preparations had a minimum surface tensions <10 mN/m on a Langmuir/Wilhelmy balance. Instillation of PL, Bser, and BKL4 surfactant increased mean arterial/alveolar PO2 (aADO2) ratios by 50-100% over postlavage values, whereas KL4 surfactant increased aADO2 ratios by 118%, B surfactant by 191%, and BC surfactant by 225%. Lung volumes at 30 cm H2O pressure were highest after treatment with BC surfactant, intermediate after B and KL4 surfactants, and lowest after BKL4, Bser, and PL surfactants. These data suggest that a surfactant preparation with a combination of synthetic B and C peptides surpasses synthetic B and KL4 surfactants in improving oxygenation and lung compliance in surfactant-deficient rats.

Alterations in Lung Surfactant Disaturated Phosphatidylcholine, SP-A, and SP-B in Meconium Injured Rats Lavaged with Perfluorochemical (PFC) or KL4 Surfactant † 1600

Meconium aspiration remains a common cause of respiratory distress from lung injury and surfactant inhibition. Meconium debris in the lung can be removed by lavage with KL4 surfactant, a mixture of phospholipids (PL) and synthetic peptide (Revak, Pediatr Res, 41;264A,1997), or with PFC which are used to maintain gas exchange. In these models, however the status of lung surfactant composition remains unclear. In a rat model of meconium injury, we have evaluated if lung lavage alters the surfactant disaturated phosphatidylcholine (DSPC) and proteins SP-A and SP-B in spontaneously breathing animals. Adult rats were intubated and given 0.3 ml meconium (13.5 mg dry wt) or saline (NS, n=3) endotracheally. Ten minutes later, meconium instilled lungs (Mec) were lavaged (x 3) with 6 ml/kg aliquots of saline (NSL, n=5) or PFC (PFCL, n=5). One group of Mec rats was lavaged twice with saline suspension of KL4 at 2.5 mg/ml, and once at 10 mg/ml (KL4L, n=5); another group was lavaged twice with saline, followed by once with KL4 at 10mg/ml, in order to replenish surfactant washed out with saline lavages (SKL4L, n=5). Animals were extubated after recovering from anesthesia (1-2 hours) to spontaneously breathe room air for 18 hours, a period previously shown to correlate with maximal lung injury in this model. Thereafter, bronchoalveolar lavage fluid (BAL) was obtained on excised lungs and seperated into large(Lag) and small surfactant aggregates. BAL fractions and lung tissue were analyzed for DSPC content and Lag for SP-A and SP-B. DSPC levels (μg lipid phosphorus/lung) were similar in Lag of Mec (12.5±2.1, mean±SE, n=4) and NS animals (9.5±0.2, p=0.06). They were slightly elevated in PFCL group (14.0±0.5, p>0.05, vs Mec) and significantly elevated in KL4L (87±19, p<0.001, vs Mec) and NSKL4L groups (48±8, p<0.001, vs Mec). DSPC levels in the lung tissue were similar in Mec(138±14) and NS animals 189±17, p=0.06). The lungs of KL4L(330±85, p<0.02) and NSKL4L animals (256±42, p<0.05) also showed higher levels of DSPC compared to Mec animals (138±20). In Western blots of Lag, both SP-A and SP-B were decreased in NSL group compared with NS or Mec animals, suggesting that saline lavage depleted SP. Both proteins were elevated in KL4L compared to NSL animals. We speculate that KL4 lavage may lead to metabolic changes that result in increased retention of PL in lung tissue, and may affect SP metabolism to prevent their depletion in lung surfactant. KL4 was a gift of Acute Therapeutics, Inc, Doylestown, Pa.

Lung Lavage with KL4 Surfactant in a Neonatal Model of Meconium Aspiration; The Importance of Peep. † 960

Lung Lavage with KL4 Surfactant in a Neonatal Model of Meconium Aspiration; The Importance of Peep. † 960

Lung Mechanics and Inflammatory Response in Meconium Injured Rats following Lung Lavage with Perfluorochemical or KL4 Surfactant † 1757

Lung Mechanics and Inflammatory Response in Meconium Injured Rats following Lung Lavage with Perfluorochemical or KL4 Surfactant † 1757

Characterization of Surfactant Subtypes of Beractant and a Synthetic Peptide Containing Surfactant KL4 following Surface Area Cycling and Addition of Fibrinogen

Surfactant is not a homogeneous material and can be separated into subtypes. Subtype conversion is clinically important because it is thought to occur naturally and because surface activity varies depending on the subtype. Fibrinogen, a naturally occurring serum protein, is known to affect this conversion. In this study we studied two surfactants, beractant and KL4, to examine their subtype characteristics. Surface area cycling, an in vitro method, was used in conjunction with sucrose gradient ultracentrifugation to separate subtypes in both surfactants. Activity, expressed as minimum surface tension of these subtypes, was measured using a pulsating bubble surfactometer. The effect of fibrinogen on subtype conversion and subsequent change in activity was elucidated. Our results indicate that following surface area cycling, beractant and KL4 have different subtypes and different responses to fibrinogen. Cycling of beractant resulted in two bands, representing a heavy and a light subtype. In the presence of fibrinogen, cycling resulted in two separate heavy subtypes. Cycling of KL4 surfactant also yielded light and heavy subtypes. However, in the presence of fibrinogen, cycling of KL4 resulted in ultraheavy subtypes. These ultraheavy subtypes retained minimum surface tension comparable to that of native KL4 surfactant. We conclude that these two surfactant preparations have different subtype conversions when subjected to surface area cycling and in the presence of fibrinogen. These conversions result in different activities toward lowering surface tension. We speculate that endogenous fibrinogen will also affect these two surfactants differently in vivo and thus affect their clinical effectiveness.

Comparative Effects of Some Serum Components and Proteolytic Products of Fibrinogen on Surface Tension-Lowering Abilities of Beractant and a Synthetic Peptide Containing Surfactant KL41

The serum components of C-reactive protein, lysophosphati-dylcholine, fibrinogen, and fibrinogen proteolytic products have been shown to reduce surface tension-lowering abilities of lung surfactant. The inhibitory effects of these serum components were compared among four different surfactants: natural lung surfactant, a phospholipid mixture that had no surfactant proteins, KL4 surfactant which has a synthetic surfactant protein B (SP-B)-like peptide, and beractant (BER) which has both SP-B and SP-C. The pulsating bubble surfactometer was used to measure the surface tension of these surfactants after the addition of inhibitors. Inhibition of BER and KL4 surfactant was observed with some serum components within 1 min of pulsation, but was reversed after 3 min of pulsation for KL4 surfactant and to a lesser extent with BER. The surface tension of phospholipid mixture alone was significantly increased and did not improve with further pulsations. Natural lung surfactant was least inhibited and was affected only at very high fibrinogen concentrations (5 mg/mL). At identical concentrations of these inhibitors, KL4 surfactant was inhibited less compared with BER. We conclude that the response of a lung surfactant to inhibitory agents may depend on the presence or absence of surfactant-related protein(s) in the surfactant and the concentration of exogenous surfactant used. KL4 surfactant, which has a synthetic peptide in lieu of SP-B, resists inhibition to these serum components more than BER at similar phospholipid concentrations.

Positive end-expiratory pressure during KL4 surfactant instillation enhances intrapulmonary distribution in a simian model of respiratory distress syndrome.

Intrapulmonary distribution of a peptide-phospholipid (KL4) surfactant administered through an adapter permitting maintenance of positive end-expiratory pressure was compared with distribution by instillation with disconnection from mechanical ventilation in 10 surfactant-deficient Macaca mullata preterm infants. Animals received KL4 surfactant (200 mg/kg) when the arterial to alveolar (oxygen ratio) (a/Ao2) was < or = 0.22 (approximately 50 min after birth) on mechanical ventilation. Six rhesus infants received bolus instillation of two half doses of KL4 surfactant through an endotracheal tube adapter over 10-15 s while maintaining positive end-expiratory pressure (0.4 kPa) accompanied by turning to the right and left lateral positions for 60 s. In four rhesus premature infants KL4 surfactant was injected as two half-dose boluses through the endotracheal tube with disconnection from mechanical ventilation while positioning the infant rhesus monkey in the right and left lateral positions for 30 s of mechanical ventilation between instillation. Acute effects on oxygen saturation were monitored, and physiologic measures of a/Ao2, mean airway pressure, and the ventilatory efficiency index were monitored over the 12-h study. Intrapulmonary distribution of KL4 surfactant was determined using dye-labeled microspheres or [3H]dipalmitoylphosphatidylcholine-labeled surfactant, measured by colorimetry or by scintillation counting. Lungs of each monkey were processed into 50 +/- 5 pieces to determine distribution of radiolabel or microspheres and for scanning electron microscopy. The drop in oxygen saturation was greater among monkey infants disconnected from the ventilator for surfactant instillation.(ABSTRACT TRUNCATED AT 250 WORDS)