Synthetic lung surfactants containing SP-B and SP-C peptides plus novel phospholipase-resistant lipids or glycerophospholipids

Synthetic peptides

The homology-modeled structures and linear sequences of S-MB DATK and SP-Css ion-lock 1 peptides are shown in Figs. 1 and 2. Both peptides were synthesized on a Symphony Multiple Peptide Synthesizer (Protein Technologies, Tucson, AZ) using a FastMoc™ protocol on a H-Ser(OtBu)-HMPB NovaPEG resin (Walther et al., 2010; Walther et al., 2014; Notter, Wang & Walther, 2016). All residues were double coupled to the resin for optimal yield. Crude peptides were cleaved from the resin using the standard phenol:thioanisole:ethanedithiol:water:trifluoroacetic acid (0.75:0.25:0.5:0.5:10 v:v) cleavage-deprotection mixture (Walther et al., 2010) and purified further (>95%) by preparative HPLC using a VYDAC diphenyl or C8 (1^′′ by 12^′′ width by length) column at 20 ml/min. Purified S-MB DATK and SP-Css ion-lock 1 were eluted from the column over one hour using a 0 to 100% linear gradient (water to acetonitrile with 0.1% TFA as an ion pairing agent added to both aqueous and organic phases). Because of the enhanced peptide molecular stability imparted by the designer-turn DATK substitution in S-MB DATK and the E20-K24 ion-lock in SP-Css ion-lock 1, further treatment to promote peptide folding and oxidation was not required. Purified peptide products were freeze-dried directly and their mass was confirmed by MALDI-TOF mass spectrometry.

Phospholipase-resistant lipids (DEPN-8, PG-1)

Phospholipase-resistant lipids used in synthetic surfactant mixtures were DEPN-8 and C16:0, C16:1 phosphono-PG (PG-1) (Fig. 3). Both lipids are structurally resistant to cleavage by phospholipases A₁, A₂ and D due to the presence of ether rather than ester linkages for the fatty chains plus a phosphono rather than phosphate moiety in the molecular headgroup. DEPN-8 [(±)-trimethyl(3-phosphonopropyl)ammonium, mono(2,3-bis(hexadecyloxy)propyl) ester] was synthesized and purified using previously reported methods (Turcotte et al., 1991; Wang et al., 2003; Notter et al., 2007; Wang et al., 2007). The chemical scheme for preparing DEPN-8 initially converted (±)-1-hexadecyloxy-2,3-propanediol to (±)-2,3-bis(hexadecyloxy)-1-propanol by way of hydroxyl protection at the 3-position, followed by alkylation at the 2-hydroxyl group and deprotection. Phosphonocholine placement involved treatment of (±)-2,3-bis(hexadecyloxy)-1-propanol with 3-bromopropylphosphono-dichloridic acid prepared from 3-bromopropylphosphonic acid and PCl₅ (Wang et al., 2003), followed by reaction with Me₃N in CHCl₃:MeOH:H₂O (10:10:1). After concentration, crude DEPN-8 was purified by exposure to Amberlite^®, flash chromatography with elution by CHCl₃:MeOH:H₂O (60:35:5), and recrystallization from CHCl₃/acetone. PG-1 [mono[2-[Z-9-hexadecen-1-yloxy]-3-(hexadecyloxy)propyl] P-(3,4-dihydroxybutyl) phosphonate] was also prepared based on our prior methods (Notter et al., 2007; Schwan et al., 2011). Dimethyl 3,4-bis(benzoyloxy)butylphosphonate was initially subjected to demethylative silylation of methoxy groups using TMS-Br, and the resulting bis(silylated) phosphonate was then directly converted to the analogous dichloride using oxalyl chloride. The dichloride was reacted without purification with (±)-2-[(Z)-9-hexadecen-1-yloxy]-3-(hexadecyloxy)–propanol to prepare a dibenzoyl-protected form of the phosphonolipid target. PG-1 was then obtained from this penultimate phosphonolipid through careful methanolysis of only the carboxylic ester functionalities while leaving the phosphonate ester intact. PG-1 was further purified by silica gel flash chromatography. The structures of DEPN-8 and PG-1 were verified by high-resolution mass spectrometry and by ¹H and ¹³C NMR spectroscopy.

Synthetic glycerophospholipids (5:3:2 DPPC:POPC:POPG)

DPPC, palmitoyl-oleoyl phosphatidylcholine (POPC), and POPG were obtained from Avanti Polar Lipids (Alabaster, AL, USA) at >99% purity.

Synthetic lipid/peptide surfactant formulations

Synthetic surfactants were formulated to contain 35 mg/ml of either 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG combined with either 1.5% (by wt) S-MB DATK + 1.5% SP-Css ion-lock 1 together or 3% (by wt) of either peptide alone. Synthetic surfactant components were combined in organic solvent (e.g., chloroform for lipids and TFE for peptides), dried under nitrogen, exposed to house vacuum to remove residual solvent, resuspended by hand vortexing in 0.15 M NaCl (normal saline) adjusted to pH 7.0 with 0.1 N sodium bicarbonate, heated to 65 °C intermittently for 30 min, and refrigerated for >12 hours prior to use.

Adsorption methods

Adsorption experiments were done at 37 °C in a Teflon dish with a 35 ml subphase (0.15 M NaCl, pH 7.0) stirred to minimize diffusion resistance (Notter, 2000; Notter, Wang & Walther, 2016). A bolus of surfactant containing 2.5 mg lipid in 5 ml of 0.15 M NaCl with pH 7.0 was injected into the stirred subphase, and adsorption surface pressure (surface tension lowering below that of the pure subphase) was measured as a function of time by the force on a partially submerged, sandblasted platinum Wilhelmy slide. The final surfactant phospholipid concentration for adsorption studies was uniform at 0.0625 mg/ml (2.5 mg surfactant phospholipid/40 ml of final subphase).

Bubble surfactometer methods

The surface tension lowering ability of synthetic surfactants under dynamic compression at 37 °C was assessed by both pulsating and captive bubble surfactometers. The pulsating bubble surfactometer (General Transco Inc, Seminole, FL, USA) used was based on the design of Enhorning (Enhorning, 1977), while the captive bubble surfactometer was a fully-computerized version of that originally detailed by Schurch and co-workers (Schurch et al., 1989; Schurch et al., 1992). Measurements of overall surface activity with both instruments reflected a physiologically-relevant combination of adsorption and dynamic film compression at 20 cycles/min and body temperature (37 °C). Specific experimental procedures followed in dynamic surface activity studies were as reported previously by our group for the pulsating bubble surfactometer (Hall et al., 1993; Wang et al., 2003; Notter, Wang & Walther, 2016) and the captive bubble surfactometer (Walther et al., 2010). Pulsating bubble studies used an area compression ratio (maximum to minimum surface area) of 2:1 and were done at a uniform surfactant phospholipid concentration of 5 mg/ml (Notter, Wang & Walther, 2016). Captive bubble studies used a higher area compression ratio of 5:1, and were done at an average surfactant phospholipid concentration of 0.050 mg/ml (Walther et al., 2010).

Shear viscosity measurements

Shear viscosities of synthetic surfactants were measured with a Wells–Brookfield cone and plate micro-viscometer (Model DV-II+; Brookfield Engineering Laboratories, Inc., Stoughton, MA) as detailed previously (King et al., 2001; King et al., 2002). Shear rate was varied by using one of two different spindles in the viscometer depending on desired torque and shear (CPE-40: 1–1500 s⁻¹; CPE-51: 1–768 s⁻¹). Before each experiment, the desired spindle and sample cup (CPE-44PSY) were washed with 95% ethanol and rinsed with nanopure water, and spindle position was calibrated to a gap height of 0.0005 inches above the sample cup. Surfactants for viscosity studies were dispersed in 0.15 M NaCl adjusted to pH 7 with sodium bicarbonate. A surfactant sample (0.5 ml volume at room temperature) was placed in the center of the viscometer sample cup using a syringe, and 10 min was allowed for temperature to equilibrate with a circulating water bath (37 °C). Viscosity was measured proceeding from low to high shear rates to minimize variability from shear-induced aggregate changes (King et al., 2001; King et al., 2002). Viscosity values at fixed shear rate were considered to be at a steady state after 15 s with no significant change. Clinical surfactants Infasurf™, Survanta™, and Curosurf™ for viscosity comparisons were obtained from the pharmacy of Strong Memorial Hospital, Rochester, NY, and used as formulated.

Ventilated, lung-lavaged, surfactant-deficient rat and rabbit models

The preclinical pulmonary activity of synthetic surfactants was defined in studies in ventilated rats (phospholipase-resistant surfactants) and rabbits (glycerophospholipid surfactants) with surfactant deficiency/dysfunction induced by in vivo lung lavage. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center (Research Projects # 12958 and 13230). All procedures and anesthesia were in accordance with the American Veterinary Medical Association (AMVA) Guidelines. Detailed methods used for anesthesia, surgery, lavage, ventilation, and animal monitoring have been reported in our prior work for rats (Walther et al., 2005; Walther et al., 2010) and rabbits (Walther et al., 2014; Notter, Wang & Walther, 2016).

In brief, rat studies utilized adult male Sprague-Dawley rats weighing 200–225 g. Rats were anesthetized, intubated, mechanically ventilated with 100% oxygen using a rodent ventilator, paralyzed, and an arterial line was placed for monitoring of blood pressure and blood gases. Airway pressures and tidal volumes were recorded continuously. Rats were gently lavaged with 0.9% NaCl to achieve a stable value of <100 mmHg for the arterial partial pressure of oxygen (arterial PO₂), consistent with clinical ARDS (Bernard et al., 1994; Artigas et al., 1998). A dose of 100 mg/kg of a phospholipase-resistant synthetic surfactant or lipidonly controls was then instilled intratracheally, and arterial blood gases, tidal volume and airway pressures were determined at 15 min intervals for the remainder of study. Dynamic lung compliance was calculated by dividing tidal volume/kg body weight by changes in airway pressure (peak inspiratory pressure minus positive end-expiratory pressure) (mL/kg/cmH₂O). Rats were euthanized 90 min after surfactant instillation, and each treatment group consisted of 8–10 animals. In analogous rabbit studies, young adult New Zealand white rabbits (weight 1.0–1.3 kg) were anesthetized and a venous line was placed via a marginal ear vein. Rabbits were intubated, received a carotid arterial line, and were placed on a Harvard volume-controlled animal ventilator with 100% oxygen. After stabilization on the ventilator under paralysis, saline lavage was performed until arterial PO₂ was stable at <100 mmHg, and rabbits were then given 100 mg/kg of glycerophospholipid synthetic surfactant or lipid-only controls by intratracheal instillation. Arterial pH and blood gases and dynamic lung compliance were subsequently obtained every 15 min until the end of study. Rabbits were euthanized 120 min after surfactant administration, and each treatment group consisted of 5–9 animals.

Statistical analysis

All data are expressed as Mean ± 1 S.E.M. Statistical analyses used Student’s t-test for comparisons of discrete data points, and functional data were analyzed by one-way analysis of variance (ANOVA) with Scheffe’s post hoc analysis to adjust for multiple comparisons. Differences were considered statistically significant if the probability of the null hypothesis (P) was <0.05 or less.

Adsorption activity of synthetic lipid/peptide surfactants

All the synthetic lipid/peptide surfactants studied had substantial activity in adsorbing rapidly to the air-water interface to generate high surface pressures (low adsorption surface tensions). Synthetic surfactants containing 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG combined with 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 peptides together, or with 3% S-MB DATK alone, had the greatest adsorption activity. These surfactants adsorbed rapidly to surface pressures of 43–46 mN/m (surface tensions of 24–27 mN/m at 37 °C) within 60 s, and had final equilibrium surface pressure values of 46–48 mN/m (equilibrium surface tensions of 22–24 mN/m at 37 °C). Equilibrium adsorption surface pressures of this magnitude are very close to the interfacial spreading limit of fluid phospholipids at body temperature. This very fast adsorption was present despite the low surfactant phospholipid concentration studied (0.0625 mg/ml, see Methods). Synthetic lipid/peptide surfactants containing 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG combined with 3% SP-Css ion-lock 1 peptide alone had lower but still substantial adsorption facility, reaching surface pressures of ∼36 mN/m after 60 sec and ∼44 mN/m at final equilibrium (equivalent to adsorption surface tensions of 34 and 26 mN/m, respectively, at 37 °C).

Dynamic surface activity of synthetic lipid/peptide surfactants

All synthetic surfactants containing 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG combined with 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 peptides together, or 3% of either peptide alone, had very high activity in reducing minimum surface tension to <1 mN/m during dynamic cycling in both pulsating and captive bubble studies. Dynamic surface tension lowering data for the first ten cycles of compression/expansion on the captive bubble surfactometer are shown in Fig. 4 (glycerophospholipid surfactants) and Fig. 5 (phospholipase-resistant surfactants). All the synthetic lipid/peptide surfactants studied generated extremely low surface tension values of <1 mN/m on the first compression cycle, which was initiated following a five minute pause to allow adsorption after surfactant was first deposited in the captive bubble sample chamber. A control mixture of glycerophospholipids alone (5:3:2 DPPC:POPC:POPG) had low surface activity on the captive bubble (Fig. 4), while phospholipase-resistant lipid-only controls (DEPN-8 and 9:1 DEPN-8:PG-1) were able to reduce surface tension to <1 mN/m even in the absence of peptides (Fig. 5). This latter finding presumptively reflects the previously reported high adsorption of DEPN-8 relative to glycerophospholipids (Turcotte et al., 1991; Wang et al., 2003), which would lead to a greater surface film concentration (at fixed subphase concentration) for this resistant lipid in bubble studies.

In vivo studies in surfactant-deficient rat and rabbit models

Synthetic surfactants were administered to ventilated animals by intratracheal instillation after the arterial partial pressure of oxygenation was reduced by in vivo saline lavages to stable levels of <100 mm Hg when breathing 100% oxygen, well-below the threshold clinical criteria for ARDS (Bernard et al., 1994). All synthetic lipid/peptide surfactants had significant pulmonary activity compared to lipid-only controls when administered intratracheally to rabbits (glycerophospholipid surfactants) or rats (phospholipase-resistant lipid surfactants) with ARDS (Figs. 6 and 7, P < 0.05 by one-way ANOVA compared to control). However, although all synthetic lipid/peptide surfactants had significant pulmonary activity compared to lipid-only controls, dual-peptide surfactants containing either glycerophospholipids or phospholipase-resistant lipids combined with 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 had greater activity than single-peptide surfactants in improving both arterial oxygenation and dynamic lung compliance (P < 0.05 by one-way ANOVA). For synthetic glycerophospholipid/peptide surfactants (Fig. 6), comparative single-peptide surfactants contained either 3% S-MB DATK or 3% SP-Css ion-lock 1. For phospholipase-resistant lipid/peptide surfactants (Fig. 7), the comparative single-peptide surfactant contained 3% S-MB DATK, since it had greater activity than 3% SP-Css ion-lock 1 in studies with glycerophospholipid surfactants in Fig. 6.

Shear viscosities of synthetic surfactant preparations

Shear viscosities of saline dispersions of synthetic lipid/ peptide surfactants were measured to help assess their potential ease of deliverability via tracheal instillation in future clinical applications. Viscosity results were defined as a function of shear rate for phospholipase-resistant lipid/peptide surfactants (Table 1) and glycerophospholipid/peptide surfactants (Table 2). Comparative viscosity measurements were also made for clinical formulations of current animal-derived surfactant drugs (Table 1). All synthetic and animal-derived surfactants studied exhibited non-Newtonian rheological behavior with higher viscosities at lower shear rates. However, dual-peptide synthetic surfactants containing phospholipase-resistant lipids or glycerophospholipids combined with 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 had equal or lower viscosity values than current clinical formulations of animal-derived surfactant drugs across the range of shear rates studied (Tables 1 and 2). Synthetic surfactants containing 3% S-MB DATK had comparable or lower viscosity values compared to dual-peptide synthetic surfactants, while preparations containing 3% SP-Css ion-lock 1 exhibited somewhat larger viscosities particularly at low shear rate.