(T1030-07-37) Development, Characterization, and In Vitro Efficacy Evaluation of an Inhalable H1B Antimicrobial Peptide Formulation for Treating Non-Small Cell Lung Cancer

Purpose: Lung cancer is the leading cause of cancer related death around the world. Currently, many treatments are administered intravenously and orally leading to systemic toxicity and low drug concentrations at the tumor sit. Therefore, there is a need for alternative delivery approaches that can achieve high local concentrations with minimal systemic toxicity. Hymenochirin-1B (H1B) is an anti-microbial peptide that has previously shown anticancer activity in non-small cell lung cancer (NSCLC). In this study, we formulated H1B into a dry powder (H1BSD) with a spray dryer to facilitate an inhalation route of delivery for the treatment of NSCLC.

Methods: H1BSD powders were spray dried with mannitol, L-Leucine, and trehalose as carriers with a constant peptide loading of 5% w/w and varied excipient ratios, using a Büchi mini-spray dryer B-290. The spray dryer conditions were as follows: inlet temperature 90 °C, feed rate 3 mL/min, and aspirator 100%. The yield was calculated based on the weight of dry powder produced as compared to the initial weight. The drug and moisture content in the dry powders were determined using micro-BCA protein assay kit and thermogravimetric analysis, respectively. The solid-state characteristics were determined using differential scanning calorimetry and powder X-Ray diffraction. The aerosolization performance of H1BSD powders was evaluated using the NGI M−170 instrument, operated at a flow rate of 60 L/min and equipped with a throat adaptor and stainless-steel gravimetric cups. Briefly, 1 capsule with 10 mg of spray-dried powder, was inserted into an RS01® dry powder inhaler, and drawn through the induction port into the NGI using a HCP5 vacuum pump. H1BSD powders were further analyzed for Mass Median Aerodynamic Diameter (MMAD) and Fine Particle Fraction (FPF). The in vitro cell viability of H1BSD powders was evaluated in A549 cells using MTT assay. Briefly, 5000 cells/well were seeded in a 96-well plate and treated with various H1BSD concentrations (0-10 μM) for 24 hours before MTT reagent was added and data collected. The scratch assay was utilized to assess the impact of H1B on cell migration and cancer cell metastasis. Briefly, 120,000 cells/well were seeded in 24-well plate and a scratch was created in the cell monolayer. Scratch assay was then observed microscopically up to 12 h after treatment with 1.25 and 2.5 μM of H1BSD4. Statistical analysis was done using two-way analysis of variance via GraphPad Prism.

Results: The spray drying process resulted in suitable yield and moisture content, ranging from 43%-63%, and 3.2-0.6% w/w. An increase in mannitol content resulted in an increase in yield and a decrease in moisture content. The drug loadings of the H1BSD formulations ranged from 48-53 µg/mg, corresponding to about 100% peptide recovery for all the spray-dried formulations, Figure 1. The DSC thermogram of pure H1B showed no thermal events between 30 to 300 °C. Mannitol exhibited a typical endothermic melting transition near 165 °C, consistent with previous findings1. Similarly, L-Leucine and trehalose dihydrate presented endothermic peaks at 285 °C and 210 °C, respectively2,3, Figure 2A. The PXRD thermograph of H1B displayed a characteristic semi-crystalline peak at roughly 20°. Similar to published reports, excipients showed crystalline peaks ranging from 2θ of 9° to 50° both before and after spray drying further reinforcing the semi-crystalline nature of the spray dried powder1–3, Figure 2B. The MMAD of all H1BSD powders were consistent, ranging from 1.7 to 2.5 µm. Additionally, the FPF for all H1BSD powders exceeded 65%, with H1BSD5 having the largest at almost 85%, indicating that most of the powders were fine particles appropriate for inhalation, Figure 2C. These findings suggest that H1BSD4 contains the optimal excipient ratio to effectively deliver H1B to the bronchial-alveolar region of the lungs via inhalation due to its FPF being greater than 70% and MMAD close to 2 µm. The quantitative colorimetric MTT assay results indicated that the inhibitory concentration (IC50) values ranged between 2.3-2.9 µM for different excipient ratios used, which is similar to the reported IC50 of H1B alone4, Figure 3A. The scratch assay revealed after 6 hours of treatment, there was no significant difference in scratch closure for all treatment groups. However, after 12 hours of treatment, untreated control resulted in a closure of 22.77 ± 4.14% whereas 1.25 and 2.5 μM of H1BSD4 resulted in 16.43 ± 4.85% (p < 0.05) and 11.72 ± 5.13% (p < 0.001), respectively, Figure 3B. This shows the ability of H1BSD4 to inhibit cell migration.

Conclusion: The H1B AMP was successfully spray dried with mannitol and trehalose at various ratios. The results indicate that a dry powder formulation with 45:30:20 of mannitol, trehalose, and L-Leucine, respectively would result in desirable aerosolization characteristics. H1BSD4 was further tested and proven to be effective in treating NSCLC in vitro.


References: 1. Yang, Y. et al. A Critical Review on Engineering of d-Mannitol Crystals: Properties, Applications, and Polymorphic Control. Crystals 12, 1080 (2022).
2. Mah, P. T. et al. The use of hydrophobic amino acids in protecting spray dried trehalose formulations against moisture-induced changes. Eur. J. Pharm. Biopharm. 144, 139–153 (2019).
3. Raula, J., Seppälä, J., Malm, J., Karppinen, M. & Kauppinen, E. I. Structure and Dissolution of l-Leucine-Coated Salbutamol Sulphate Aerosol Particles. AAPS PharmSciTech 13, 707–712 (2012).
4. Attoub, S., Arafat, H., Mechkarska, M. & Conlon, J. M. Anti-tumor activities of the host-defense peptide hymenochirin-1B. Regul. Pept. 187, 51–56 (2013).

Acknowledgements: This study was supported by funds provided to Nitesh Kunda by Catalent Pharma Solutions (Somerset, NJ)​

Excipient Ratios, drug loading and yield of H1BSD powders after spray drying

A) Differential Scanning Calorimetry of H1BSD powders and spray drying excipients. B) Powder X-Ray diffractogram of H1BSD powders and spray drying excipients (excluding Trehalose). C) Fine particle fraction and mass median aerodynamic diameter of H1BSD powders.

A) Cytotoxicity of H1B and H1BSD powders determined using MTT assay. B) Wound healing assay of H1BSD4 over a 12-hour time period.