DP synthetic surfactant powders were designed and formulated at Acorda Therapeutics, utilizing its ARCUS ^® Pulmonary Dry Powder Technology. The challenge that was overcome via the utilization of the ARCUS ^® technology was to produce phospholipid-based DP lung surfactant formulations, with and without SP-B peptide mimetic, that both replicated the fluidizing surface activity of endogenous lung surfactant yet were both physically and chemically stable as well as dispersible and aerosolizable in a solid-state DP form, in contrast to the liquid-based forms currently utilized for surfactant replacement therapy. An extensive screening process ¹⁰ was conducted utilizing various combinations of phospholipids, fatty acids and stabilizing excipients that resulted in a lead set of optimized peptide-free candidate DP synthetic surfactant powders. SP-B peptide mimic-containing formulations were later produced utilizing this lead set of powders. Lead DP synthetic surfactant powders were formulated by adding the following components to the organic solvent used for spray drying: 49 wt% DPPC and 21 wt% POPG-Na (Avanti Polar Lipids, Alabaster, AL); an optional third phospholipid, e.g., 7 wt% POPC (Avanti Polar Lipids, Alabaster, AL 35007); an excipient, e.g. polyglycitol Stabilite™ SD-30 (Grain Processing Corp., Muscatine, IA 52761); an optional fatty acid, e.g., 5 wt% palmitic acid (PA) (Sigma-Aldrich Co, Saint Louis, MO 63103); 2 wt% NaCl (Sigma-Aldrich Corporation, Saint Louis, MO 63103); and optionally 3 wt% of a SP-B peptide mimic (SMB); with all components amounting to 100 wt%.

SMB was synthesized at the Los Angeles Biomedical Research Institute using a standard Fmoc protocol with a Symphony Multiple Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ 87514) or a CEM Liberty microwave synthesizer (CEM Corporation, Mathews, NC 28104), cleaved-deprotected, purified using reverse phase HPLC, freeze-dried, and had its mass confirmed by MALDI TOF mass spectrometry and concentration quantitated using UV absorbance ^{4, 5} .

Respirable dry synthetic surfactant particles were produced at Acorda Therapeutics using a GEA Niro PSD-1 spray dryer (Niro Inc., Copenhagen, Denmark) or Buchi B-290 mini (Buchi Corpration, New Castle, DE 19720) spray dryer. After spray drying, the synthetic surfactant powders were filled into size 00 capsules. Testing of aerosol properties and solid-state properties of the DP surfactant formulations included particle sizing, X-ray diffraction, thermogravimetric analysis and differential scanning calorimetry. Respirable dry particles with a mass median aerodynamic diameter of between 1 and 4 microns were preferred. A set of lead optimized DP synthetic surfactant formulations were then provided to Los Angeles Biomedical Research Institute in a blinded manner for further in vitro and in vivo characterization.

Next, three DP synthetic surfactants containing SMB were chosen for more extensive in vitro and in vivo testing and compared to a lipid control powder without SMB (DPPC:POPG-Na:SD-30:NaCl 49:21:28:2 wt:wt) as a negative control (DP-C) and the clinical surfactant Curosurf ^® , a porcine surfactant containing both SP-B and SP-C, as a positive control, where appropriate. The three surfactant formulations with SMB were: DPPC:POPG-Na:SD-30:SP-B:NaCl 49:21:25:3:2 wt:wt (DP-2L); DPPC:POPG-Na:POPC:SD-30:SP-B:NaCl 49:21:7:18:3:2 wt:wt (DP-3L); and DPPC:POPG-Na:SD-30:PA:SP-B:NaCl 49:21:20:5:3:2 (DP-PA).

Because there is currently no commercially available system designed specifically for inhalation therapy during nCPAP, a proprietary DP delivery device was designed and produced by Acorda Therapeutics (Chelsea, MA 02150). This low flow aerosolization chamber (LFAC) consists of a cylindrical chamber with one or more holes at one end that can accommodate a perforated capsule containing powder and disperse it at low flow rates (4–10 l/min) and can also be incorporated into a ventilatory system like nCPAP in lieu of being utilized as a stand-alone device. Depending on the flow rate, DP surfactant is delivered as a fine mist over 1–3 minutes per capsule filled with about 30 mg of surfactant. Dosing was based on the lipid content of the DP surfactant and amounted 100 mg/kg bodyweight. In consideration of the potential applications of this work, in addition to its efficacy in aerosolizing and delivering DP synthetic surfactants, additional design criteria for the device included the following (1) simplicity of design and use, (2) minimum number of parts, and (3) low cost of goods/manufacture.

ATR-FTIR spectra were recorded at 37°C using a Bruker Vector 22 FTIR spectrometer (Pike Technologies, Fitchburg, WI 53719) with a deuterium triglyceride sulfate (DTGS) detector. The spectra were averaged over 256 scans at a gain of 4 and a resolution of 2 cm ^{-1 5} . For FTIR spectra of SMB in DP synthetic lung surfactant, the powder was solvated with deuterated water (D ₂O) and transferred onto a germanium ATR crystal. The aqueous solvent was then removed by flowing nitrogen gas over the sample to produce a thick lipid-peptide (lipid:peptide ratios of 10:0.3, mole:mole) ⁵ . The multilayer film was then hydrated to ≥35% with deuterated water vapor in nitrogen for 1 h before acquiring the spectra ¹¹ . The spectra for the SMB peptide in the lipid film were obtained by subtracting the spectrum of a peptide-free control sample from that of the peptide-bound sample. The relative amounts of α-helix, β-turn, β-sheet, or random (disordered) structures in lipid-peptide films were estimated using Fourier deconvolution (GRAMS AI 8, version 8.0, Thermo Fisher Scientific, Waltham, MA 02451). The respective areas of component peaks were calculated using curve-fitting software (Igor Pro, version 1.6, Wavemetrics, Lake Oswego, OR 97035) ¹² . FTIR frequency limits were: α-helix (1662-1650 cm ^-1), β-sheet (1637-1613 cm ^-1), turn/bend (1682-1662 cm ^-1), and disordered or random (1650-1637 cm ^-1) ¹³ .

Surface activity of the DP surfactant formulations was measured with a captive bubble surfactometer built by Schürch and coworkers ^{14, 15} . DP surfactant formulations were dissolved in distilled water at a concentration of 35 mg/mL and quasi-static and dynamic compression cycling of the air bubble was performed at an average surfactant lipid concentration of 70 µg/mL in the bubble chamber (~1.0 mL volume) ^{5, 16} . All measurements were performed in quadruplicate.

Animal experiments were performed according to protocols that were reviewed and approved by the Institutional Animal Care and Use Committee of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center (LA BioMed protocol # 020645) ¹⁶ . All procedures and anesthesia were in accordance with the American Veterinary Medical Association (AMVA) guidelines. Treatment allocation was done by dynamic randomization and minimum sample size per treatment group was based on effect size ¹⁶ .

Lung lavaged rabbits are surfactant-deficient for at least 6–8 hours, but need respiratory support and surfactant treatment for survival. A total of 30 young adult, male New Zealand white rabbits with a weight of 1.0–1.4 kg were purchased from IFPS Inc. (Norco, CA 92860). Veterinary care; anesthesia, sedation, and muscle paralysis; and provision of maintenance fluid have been reported previously ^{5, 16} . After inducing anesthesia, a venous line was placed via a marginal ear vein, an orotracheal tube was inserted, mechanical ventilation was started, and a carotid arterial line was inserted via a small incision in the skin of the anterior neck. Heart rate, arterial blood pressures and rectal temperature were monitored continuously (Labchart ^® Pro, ADInstruments Inc., Colorado Springs, CO 80906). Respiratory support was provided with volume-controlled ventilation (Harvard Apparatus, Holliston, MA 01746) using a tidal volume 7.5 mL/kg, a positive end-expiratory pressure of 3 cm H ₂O, an inspiratory/expiratory ratio of 1:2, 100% oxygen, and a respiratory rate sufficient to maintain the partial pressure of carbon dioxide (PaCO ₂) at ∼40 mmHg ^{5, 16} . Airway flow and pressures and tidal volume were monitored with a pneumotachograph (Hans Rudolph Inc., Shawnee, KS 66227) connected to the orotracheal tube during mechanical ventilation. After a partial pressure of oxygen in arterial blood (PaO ₂) >500 mmHg was reached at a peak inspiratory pressure <15 cm H ₂O in 100% oxygen, surfactant-deficiency was obtained with repeated lung lavages with 30 mL/kg of warmed normal saline. When the PaO ₂ was stable at <100 mmHg (average four lavages), one of two surfactant treatment approaches was chosen in a random fashion: (1) insufflation of surfactant via the orotracheal tube using the LFAC device with continuation of mechanical ventilation (n=13), or (2) extubation after establishment of adequate spontaneous breathing, followed by placement on nasal continuous positive airway pressure (nCPAP) using a F&P bubble CPAP system (Fisher & Paykel Healthcare Inc., Irvine, CA 92618) with short home-made nasal prongs and insufflation of surfactant with the LFAC device at its lowest possible flow setting via the nCPAP system ⁹ (n=17). DP surfactant was administered at a dose of 100 mg lipids/kg body weight. Oxygenation was followed by measuring arterial pH and blood gases and, if the rabbit was intubated and mechanically ventilated, dynamic lung compliance at 15 min intervals over a 2 h period. At 2 h after surfactant administration, rabbits were euthanized with 100 mg/kg of pentobarbital intravenously. Animals supported on nCPAP were re-intubated via a tracheotomy and mechanically ventilated to measure dynamic lung compliance. Oxygenation and dynamic lung compliance were used as end-points.

Preterm lambs at 135 days of gestation (term is 150 d) are surfactant-deficient, but able to breathe spontaneously while supported with CPAP delivered with nasal prongs (nCPAP) ^{17, 18} . A total of 12 date-mated pregnant ewes with twin pregnancies were treated with 12 mg of betamethasone by intramuscular injection 48 and 24 h prior to delivery by cesarean section. Anesthesia of the ewes was induced with 10 mg/kg of ketamine IM and maintained with oxygen and isoflurane after placement of a cuffed endotracheal tube and starting mechanical ventilation. The uterus was then exposed through a ventral midline incision, the fetus(es) were located and their legs and abdomen were exposed through a hysterotomy, followed by insertion of an arterial and a venous umbilical catheter, exteriorization, insertion of an orotracheal tube (ID 4.0 mm) for mechanical ventilation, and cutting of the umbilical cord. Prior to the cesarean section, heparinized blood was collected from the jugular vein of the ewe to be administered to the lambs in case of hypotension or blood loss. After the cesarean section, ewes were euthanized by infusing 100 mg/kg of pentobarbital IV.

The lambs were dried, weighed, placed in a warmer on a heating pad and under heating lamps, and started on mechanical ventilation after a sustained inflation at 40 cm H ₂O pressure for 20 sec. Initial ventilator settings were: peak inspiratory pressure (PIP) of 30 cm H ₂O, positive end-expiratory pressure (PEEP) of 8 cm H ₂O, inspiratory time of 0.4 sec, and respiratory rate of 60/min. PIP and respiratory rate were adjusted to achieve tidal volumes of 6 (5–10) mL/kg, a pH >7.20 and PaCO ₂ <60 mmHg. Both nostrils of the lambs were sprayed with 10% lidocaine spray and custom-made binasal prongs (shortened Portex tracheal tubes, ID 3.0 mm) were inserted 6–7 cm for CPAP delivery ¹⁷ . Spontaneous breathing was stimulated with 20 mg/kg of caffeine and 5 mg/kg of doxapram IV, followed by continuous infusion of 2.5 mg/kg/h of doxapram. Heart rate, arterial blood pressure, oxygen saturation (pulse oximetry) and rectal temperature were recorded continuously. Anesthesia was provided by administering 30 mg/kg/h of propofol IV as needed. Maintenance fluid consisted of Lactated Ringer’s solution at a rate of 10 mL/kg/h. Airway flow and pressures and tidal volume were monitored during mechanical ventilation with a pneumotachograph (Hans Rudolph Inc., Shawnee, KS 66227).

Respiratory failure was monitored by observing oxygen saturation and confirmed by PaO ₂ levels <150 mmHg in 100% oxygen, at which time lambs were treated with synthetic DP lung surfactant. Surfactant was given either (1) with the LFAC device attached to the orotracheal tube, followed by extubation to nasal intermittent positive pressure ventilation (NIPPV) and then to nCPAP (heated and humidified bubble CPAP with PEEP 8-9 cm H ₂O and 100% oxygen), or (2) during nCPAP (after extubation and transition via NIPPV) with the LFAC device attached to one of the nasal prongs of the CPAP system and a short period of mononasal CPAP support, or (3) same as (2) with a second dose of surfactant 1 h after the initial dose. So, the location of aerosol delivery of synthetic DP lung surfactant (IT or via nCPAP) and the number of surfactant doses during nCPAP (1 or 2) were the only differences between the three treatment groups. Aerosol delivery of surfactant powder usually took about 15 min from start to finish. After surfactant treatment, arterial pH and blood gas measurements were done every 15–30 minutes. At 3 h after surfactant treatment, the lambs were sacrificed with 100 mg/kg of pentobarbital IV. After euthanasia, the thorax was opened by a midline incision, an endotracheal tube (ID 4.0 mm) was re-inserted, and a pressure volume curve was recorded. End-points of the experiment included: gas exchange (arterial pH and blood gases) and pulmonary mechanics (dynamic compliance during mechanical ventilation, postmortem pressure-volume curve).

All data are expressed as mean ± SEM. Student's t-tests were used for comparisons of discrete data points and functional data were analyzed with one-way analysis of variance (ANOVA) with Scheffe’s post-hoc test. Differences with a P value <0.05 were considered statistically significant.

Aerosol properties and solid-state properties of the DP surfactant formulations are summarized in Table 1. Geometric particle size (gPSD) and fine particle fraction (FPF) were well within the margins for respiratory particles. The FPF of DP-2L remained relatively constant across all stability time points whereas a drop in FPF was observed for DP-PA and DP-3L. The solid-state properties are similar among the various formulations. The DP formulations are semi-crystalline with a characteristic diffraction peak at 21° 2theta, which is attributed to the presence of phospholipids in a bilayer structure. Residual solvent (ethanol and water) content is less than 1.5 wt%. The DP formulations contain phase transitions at ~45–53°C, calculated as the intercept of a step transition, and 61–67°C, calculated as a peak extremum of an endotherm. The stability data of these formulations indicate that the powders would be viable for long-term storage.

The secondary structures for SMB in DP-2L, DP-3L, and DP-PA were studied with conventional ¹²C-FTIR spectroscopy of samples solvated in D ₂O. Representative FTIR spectra of the amide I band for SMB in these environments were all similar ( Figure 2), each showing a primary component centered at ~1654-1656 cm ^-1, with a small low-field shoulder at ~1622-1626 cm ^-1. Because prior FTIR studies of proteins and peptides ^{13, 19} have assigned bands in the range of ~1650-1659 cm ^-1 as α-helical, while those at ~1613-1637 cm ^-1 denote β-sheet, SMB probably assumes α-helical and β-sheet structures and possibly other conformations in these environments. Self-deconvolutions of the Figure 2 spectra confirmed that SMB is polymorphic, primarily adopting α-helix but with significant contributions from β-sheet, loop-turn and disordered components ( Table 2). Interestingly, the relative proportions of secondary conformations determined from FTIR spectra of SMB (i.e., α-helix > loop-turn ~ disordered ~ β-sheet) in both lipid mimetics and surfactant lipids of varying polarity are all comparable ( Table 2) ^{5, 8} , suggesting overall stability of the SMB structure that is remarkably conserved. It should also be noted that the proportions of these secondary conformations are entirely compatible with SMB principally assuming an α-helix hairpin ⁸ , and which has been shown to be surface active in in vivo experiments involving intratracheal instillation of liquid surfactants ⁵ .

All three DP surfactant formulations with 3 wt% SMB, i.e. DP-2L, DP-3L and DP-PA, exhibited a minimum surface tension below 1 mN/m during quasi-static cycling and were comparable with the positive control, i.e. Curosurf ^®, indicating that there was no loss in in vitro surface activity during the surfactant production process ( Figure 3). Minimum surface tension values of DP surfactant without SMB (DP-C), our negative control, far exceeded those of the surfactant formulations with SMB and amounted to ~18 mN/m (p<0.001), comparable to equivalent non-spray dried surfactant lipid formulations.

First, second, fifth and tenth surface tension-area relations produced by dynamic cycles (20 cycles/min) are shown in Figure 4. The dry powders DP-2L, DP-3L, and DP-PA ( Figure 4) and the clinical surfactant Curosurf ^® ( Figure 4) all needed an area compression of approximately 30% to reach a minimum surface tension of 1 mN/m in the first cycle. Dynamic cycling showed a wide difference between the compression and expansion curves for both DP-2L and Curosurf ^®, with a relatively smaller hysteresis area for DP-PA and an even smaller hysteresis area for DP-3L. The lipid control (DP-C) ( Figure 4) failed to reach low surface tension values as seen during quasi-static cycling in Figure 3.

A total of 30 surfactant-deficient rabbits (mean weight 1.3 ± 0.02 kg) received 100 mg lipids/kg bodyweight of one of the three DP synthetic lung surfactant containing SMB via intratracheal (IT) or nCPAP aerosol delivery. Of the 30, 13 rabbits underwent IT aerosol delivery while supported with mechanical ventilation: 6 received DP-2L, 5 DP-PA and 2 DP-3L. IT delivery was followed by continuation of mechanical ventilation and we observed a quick and large improvement in oxygenation and lung compliance after aerosolization of the three DP synthetic surfactants containing SMB (DP-2L, DP-A and DP-3L) ( Figure 5). Oxygenation in the IT groups increased from 10–16% to 75–85% and lung compliance from 46–52% to 69–71% of baseline values measured prior to lung lavage. The surfactant response was comparable to our recent experience with liquid synthetic surfactant preparations containing 3 wt% of SMB or its sulfur-free derivative B-YL or the clinical surfactant Curosurf ^{® 4, 5, 16} . The remaining 17 rabbits received DP synthetic surfactant by aerosol delivery via the nCPAP system: 8 received DP-2L, 6 DP-PA and 3 DP-3L. Nasal CPAP led to a consistent, but smaller increase in oxygenation and dynamic compliance ( Figure 5). Oxygenation in the nCPAP groups increased from 12–13% to 41–43% and lung compliance from 44–50% to 63–67% of baseline values measured prior to lung lavage. Improvement in lung function in the rabbits receiving mechanical ventilation was independent of the active DP synthetic surfactant administered and the differences among the rabbits treated with surfactant while on nCPAP support were not statistically different either. The differences in oxygenation and lung compliance between IT and nCPAP aerosol delivery of DP-2L, DP-PA and DP-3L were statistically significant for each surfactant (p<0.05).

A total of 12 pregnant ewes delivered 22 preterm lambs; one ewe carried a single fetus and one ewe had a stillbirth. Of the lambs, three died quickly after birth despite mechanical ventilation and resuscitation efforts, so 19 preterm lambs (weight 3.1 ± 0.2 kg, gestational age 135.5 ± 0.3 days, 11 males and 8 females) received DP synthetic surfactant. In total, 6 lambs were dosed IT and then weaned to nCPAP, 7 lambs received one dose of surfactant while supported with nCPAP and 6 lambs received two doses of surfactant 1 h apart while supported with nCPAP. Based on the available in vitro data and experience with surfactant-deficient rabbits, all preterm lambs received DP-2L surfactant at 100 mg lipids/kg bodyweight/dose. All lambs were first weaned to NIPPV before initiating nCPAP support, and, if necessary, were returned for short periods to NIPPV in case of insufficient breathing effort or rising PaCO ₂ values. The response to surfactant treatment was monitored with pulse oximetry and arterial pH and blood gases every 15 min during the 1 ^st and 2 ^nd hour and every 30 min during the 3 ^rd hour after initial surfactant treatment. Oxygenation quickly improved, reaching PaO ₂ values of 385 ± 20 mmHg in 100% oxygen at 3 h after IT aerosol delivery of DP-2L versus 204 ± 19 mm Hg after 1 dose and 310 ± 16 mm Hg after 2 doses of DP-2L administered via the nCPAP system (p<0.02) ( Figure 6). PaCO ₂ values decreased more slowly and were 58.0 ± 2.2, 56.8 ± 2.8 and 54.8 ± 2.1 mm Hg (NS), respectively, at 3 h after surfactant treatment ( Figure 6) and this reduction was accompanied by an equivocal increase of pH values (data not shown). Data from postmortem pressure-volume curves are shown in Figure 7. Only the differences in lung volumes at pressures of 40 and 20 cm H ₂O between IT or two doses of nCPAP surfactant treatment and one dose of nCPAP surfactant were statistically significant (p=0.003 and 0.035, respectively).