(M1330-02-11) Formulation and Characterization of Non-invasive Nebulization Based Antiviral Drug, NSF207 Loaded Pegylated Nanoliposomal Suspension Product for Treatment of COVID-19

Purpose: SARS-CoV-2 has had a significant impact on the world. Based on the WHO data up to April 18, 2023, there have been nearly seven million COVID-19 deaths worldwide. Although geriatric patients and people with underlying health condition have been most affected, healthy adults and the pediatric population have also been significantly impacted. NSF207 is a dual-target inhibitor which has affinity and selectivity towards two SARS-CoV-2 co-receptors, and treatment has been demonstrated to have a major effect on viral cell entry¹. Owing to the high selectivity of NSF207 to Cathespin L, which reduces the potential toxicity towards the host cell and higher antiviral potency, we intended to use NSF207 in a nebulization-based non-invasive formulation product for localized delivery as antiviral therapy. The aim is to develop and characterize the NSF207 loaded pegylated extended release nanoliposomes (NLs) consisting of a cationic lipid, using ethanol injection method intended for non-invasive nebulization based localized delivery antiviral therapy for treatment of COVID-19. We hypothesize that the cationic lipid will cleave the cell membrane and aid in the internalization of the drug to induce better efficacy with minimal adverse effects. While the drug acts on host cell Cathespin L, which has critical role in viral entry.

Methods: NSF207-cSS-peg-NLs preparation and analysis: We have prepared the NSF207 loaded nanoliposomes using the coatsome-SS based lipid (NSF207-cSS-peg-NLs) with the composition we developed using the Quality by Design approach using a model drug in our previous study for inhalation delivery. For the quality by design, we used the concentrations of DPPC, cholesterol and DOPE-PEG2000 as our factors and varied them on 3 levels. Using a Box-Behnken Design, we were able to develop a suspension product having a particle size of less than 200nm, polydispersity index of less than 0.4, zeta potential between 0-5mV, entrapment efficiency greater than 90% and drug loading of greater than 80%. Along with DPPC which is an endogenous lipid found in the lungs, we also added equal concentration of a coatsome-SS cationic lipid. This was introduced to the formulation for ease of internalization of the lipidic globules into the cells. The lipid materials DPPC (4.322 mg), Coatsome-SS (5.322), Cholesterol (3.43 mg), DOPE-PEG2000 (2mg) and NSF207 were dissolved in 1.3 ml ethanol. The aqueous phase was made by dissolving 0.9%w/v NaCl in the 10.0ml of milli Q water. Both organic and aqueous solutions were maintained at 55^oC. The ethanolic solution was injected (0.15 ml/min) in the aqueous solution using a syringe pump at 600 rpm mixing speed at 55^oC. The formed liposome suspension mixed at 600 rpm for 30min while maintaining the temperature at 55^oC. The liposome suspension was subjected to extrusion through a Genizer mini extruder using 0.1µm Whatman filter to yield smaller particle size and narrower polydispersity index (PDI). The NLs were analysed for their particle size, polydispersity index (PDI) and zeta potential (ZP) using Malvern Zetasizer. The unentrapped NSF207 was analyzed using LC-MS method we developed. Sample preparation for entrapment efficiency and drug loading: Centrifugation of Nano-liposomes (0.5ml) was performed by using Amicon ultra filters (10kDa) at 13000 rpm for 15min filtrate was analysed by LC-MS. Sample preparation was assay: 0.5mL of formulation was added to 0.25mL of Methanol and 50% w/v TritonX-100, which was sonicated for an hour and centrifuged at 13000 rpm for 15min, supernatant was analysed using LC-MS. We performed the in-vitro release studies and aerodynamic characterization studies for the developed formulation using the 100kD float-a-lyzer membranes and next generation impactor respectively.

Results: NSF207-cSS-peg-NLs suspension was found to have the size of 299.9±6.98nm, zeta potential of 0.840±0.04mV, and PDI of 0.167±0.03. After subjecting the liposome suspension through the extrusion process using a mini extruder, NLs of 183.5±7.85nm and PDI of 0.112±0.02 were produced. The entrapment efficiency of the formulation was found to be 63.860±0.952%w/w before extrusion and 96.258±0.382%w/w after extrusion. The drug loading which plays a role in determination of dose of the formulation was found to be 57.143±0.641%w/w before extrusion. After extrusion, the drug loading increased to 92.555±0.367%w/w. We performed release study using the Simulated Lung Fluid with a pH of 7.4. We observed an extended release of the formulation compared to immediate release of the control drug solution. The formulation was observed to follow a Korsmeyer-Peppas model. The formulation showed a MMAD of < 5µm implying that the deep lung delivery target will be achieved.

Conclusion: The NSF207-cSS-peg-NLs were successfully developed with target characteristics with particle size range of less than 250nm, and PDI less than 0.4 implying that the formulation is monodisperse with high entrapment efficiency and high drug loading. The product was observed to have am extended release over 12 hours implying a twice a day dosage regimen which is pediatric and geriatric patient compliant. The formulations seems to deposit in the deeper lung region for a better efficacy of the developed product.

References: Li Z, Zhang Y, Wurtz W, et al. Characterization of Nebulized Liposomal Amikacin (ArikaceTM) as a Function of Droplet Size. J Aerosol Med Pulm Drug Deliv. 2008;21(3). doi:10.1089/jamp.2008.0686

Acknowledgements: This work was partially supported by National Heart, Lung, and Blood Institute of the National Institutes of Health (R15HL138718) and start up funds. This content is solely responsibility of the authors and does not necessarily represent the official views of the National institute of Health.

Preparation of MPI8-cSS-pegNL using ethanol injection method. Lipids and drugs were added to ethanol while the aqueous phase is 0.9%w/v NaCl. Both phases were maintained to 55°C. Ethanol is injected into the aqueous phase at 0.15mL/min rate while stirring continuously.

This figure represents the observed sustained release of the formulation. The control solution shows >90% release in 2 hours and follows a first order release kinetic. The formulation, on the other hand, follows a Korsmeyer-Peppas release kinetic showing a sustained release by achieving >95% release in 12 hours.