(W1030-01-04) In Vitro Characterization and In Vivo Efficacy Study of a 2nd Generation Synthetic Lung Surfactant (SLS) Excipient Enhanced Growth (EEG) Dry Powder Formulation and Device

Purpose: Instilled surfactant replacement therapy (SRT) is the first-line treatment for neonatal respiratory distress syndrome (NRDS), the most common lung disease in preterm neonates [1,2]. However, non-uniform distribution of the surfactant in the lung airways, respiratory related side effects arising from intratracheal instillation of a high liquid volume through the endotracheal tube and the requirement for multiple doses of surfactant are the most common problems in liquid bolus instillation of SRT [3]. These problems could be eliminated by aerosol delivery of a surfactant formulation. This study describes our 2^nd generation synthetic lung surfactant excipient enhanced growth (EEG) powder formulation produced with a nozzle-based spray dryer and a dry powder inhaler capable of uniform high efficiency powder aerosol delivery over a series of low air volume actuations.

Methods: Three batches of the synthetic lung surfactant EEG dry powder aerosol formulation were prepared by spray drying (Buchi Mini Spray Dryer S-300) of a feed dispersion of 0.125 %w/v solids containing phospholipids (DPPC and POPG) and surfactant protein mimic (B-YL) as active ingredients, L-leucine as a dispersion enhancer and D-mannitol and sodium chloride as the hygroscopic excipients. Briefly, following a previously reported procedure, the feed dispersion was prepared in 5% ethanol in water [4]. The spray-dried powders were collected and, following 30 minutes equilibration at 20±5%RH / 24±2ºC, stored at 0%RH / −20ºC. The particle size of the formulation was measured using the Sympatec HELOS laser diffraction apparatus using 0.5 and 4.0 bar dispersion pressure. The DPPC and B-YL content of the synthetic lung surfactant EEG powders were determined using a HPLC method and solid-state properties were evaluated by X-ray powder diffraction (XRPD) conducted using the Rigaku Miniflex 6G. In preparation for in-vivo testing, the aerosolization performance and lung delivery efficiency of the powders were evaluated by delivering 30 mg of formulation from the VCU D3 air-jet DPI to the 3D-printed rabbit upper tracheobronchial (TB) airway model at 3 LPM flow rate using a 10 mL air actuation volume. Briefly, the DPI was connected to the airway model by an endotracheal tube (ETT), and aerosol was delivered through the ETT and tracheobronchial region (TB) to a lung chamber and outlet filter (Figure 1). The aerosolization performance was determined based on the DPPC emitted dose, and in vitro lung delivery efficiency was measured from the DPPC deposited on the chamber plus outlet filter. The formulation / device combination was also characterized for aerosol aerodynamic properties using a next generation impactor (NGI). The in-vivo efficacy of the surfactant EEG powder was evaluated by measuring changes in PaO₂ as primary outcome after delivering the EEG powder to rabbit surfactant depletion model (n=7) and compared to the current standard of care using 200 mg / kg liquid instillation of Curosurf (n=5).

Results: Spray drying of the feed dispersions to produce three batches of powder formulation resulted in a mean spray rate of ~2.1 mL/min with powder bulk density of ~0.10 g/mL. The primary particle size (mean Dv50 ± SD) of the formulations measured at 4 bar dispersion pressure was 1.16±0.03 µm with %CV of 2.6 suggesting the reproducibility of the spray drying batches. The mean ± SD DPPC and B-YL content of the formulations were 100.2±3.3% and 99.7±2.3%, respectively, of the theoretical content with no significant differences among the three batches. X-ray powder diffraction showed evidence of crystalline structure in the synthetic lung surfactant powder formulations. The in vitro rabbit model study showed 70.0±2.7% dose emitted from the rabbit device with 56.8<±2.8% total lung delivery efficiency, based on loaded dose (Figure 2a). The NGI study showed 2.0 µm mass median aerodynamic diameter (MMAD) of EEG powder delivered from the VCU D3 air-jet DPI with a fine particle fraction < 5 µm of 87.9% (Figure 2b). The in-vivo rabbit study revealed that surfactant depletion was reversed with a rapid (30 mins) near-full (99%) recovery of blood gas oxygenation (PaO₂) and two-fold greater pulmonary compliance with the aerosol formulation at doses ~10-fold lower than the standard of care liquid instillation (Figure 3).

Conclusion: We have successfully produced a synthetic lung surfactant EEG formulation with high lung delivery efficiency and potent in-vivo efficacy when aerosolized using a novel low actuation air volume dry powder inhaler. This formulation / device combination may be suitable for treating premature neonates and could overcome the challenges of liquid bolus instillation of surfactant replacement therapy.

References: 1. Sardesai S et al., Evolution of surfactant therapy for respiratory distress syndrome: past, present, and future. Pediatr Res 2017, 81: 240-8.
2. Dyer J: Neonatal respiratory distress syndrome: tackling a worldwide problem. Pharm Ther 2019, 44: 12.
3. El-Gendy N et al., Delivery and performance of surfactant replacement therapies to treat pulmonary disorders. Ther Deliv 2013, 4: 951-980.
4. Boc S et al., Development and Characterization of Excipient Enhanced Growth (EEG) Surfactant Powder Formulations for Treating Neonatal Respiratory Distress Syndrome. AAPS PharmSciTech 2021, 22: 1-12.

Acknowledgements: This study was funded by the Bill and Melinda Gates Foundation. The funding body was not involved in the collection, analysis, and interpretation of data and in writing the abstract. All experimental animal procedures were conducted according to the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Seattle Children’s Institutional Animal Care and Use Committee. Virginia Commonwealth University is currently pursuing patent protection of formulations, devices and methods described in this study, which if licensed and commercialized, may provide a future financial interest to the authors.


Experimental setup for the in-vitro rabbit delivery study.


In-vitro rabbit delivery (a) and NGI-measured aerodynamic properties (b) of spray-dried synthetic lung surfactant EEG powder after delivering 30 mg of formulation using VCU air-jet DPI (D3) (bars represent the mean values; error bars represent standard deviations, n=3).


In-vivo efficacy of the spray-dried synthetic lung surfactant EEG powder delivered at 3 LPM flow rate using the D3 air-jet DPI.