(M1130-07-46) Evaluating Stability and Permeation of Exenatide in the Presence of Trehalose and Mannitol, Optimizing Aerodynamic Parameters for Engineered Mannitol as Carrier for Pulmonary Delivery

Purpose: This study evaluates exenatide stability, permeation and delivery using circular dichroism (CD), franz diffusion, and engineered mannitol carriers respectively. CD revealed exenatide oligomerizes upon heating/cooling without sugars (mannitol/trehalose), but retains its initial secondary structure with them. Franz diffusion showed sugar-containing formulations enhanced permeation through artificial pulmonary mucus. Engineered mannitol carriers, designed via ammonium bicarbonate templating, were optimized for pulmonary delivery. Higher outlet temperatures (70°C) created porous structures, while increased solid content (2.5%) enlarged particle size. SEM, aerodynamic diameter, and porosity analysis confirmed these parameters yield optimal carriers. Engineered mannitol blended with spray-dried exenatide improved aerodynamic behavior, suggesting enhanced non-invasive delivery. Sugars stabilized exenatide during spray drying and facilitated mucus permeation via concentration-dependent effects.

Methods: Stability of Exenatide in the presence of mannitol and trehalose: Circular dichroism is used to estimate the secondary structure by ramping up the temperature to 90°C and cooling it down to 10°C to observe the possibility of any oligomerization.¹ Permeation Studies: The Franz cells (donor and recipient) are to be assembled using a nitrocellulose membrane (pore size: 0.45 μm). The permeation area is 1.21 cm². The receptor compartment filled with 5 ml of a 0.05 M phosphate buffer (pH 7.4). 1 mg of Exenatide equivalent dry powder is dusted uniformly on the surface of the nitrocellulose membrane. Exenatide formulated with mannitol and trehalose in different ratios individually and only spray-dried exenatide as control were performed using franz diffusion analyzed using HPLC.² Particle and Powder properties: Mannitol engineered with ammonium bicarbonate contributes to the formation of pores in mannitol particles during spray drying was analyzed for true density using a helium pycnometer and particle diameter using Mastersizer to determine its aerodynamic properties like d_aer and porosity and morphology by SEM.^3,4

Results: 1. Stability: To understand how well these sugars were protecting Exenatide, Circular Dichroism was used to estimate the secondary structure of the exenatide in comparison with that of its native state in respective solvent. To observe the behavior of the peptide, we tested two ratios of sugars (1:4 and 1:8) with mannitol and trehalose and absence of a sugar as a control. The native secondary structure involves two characteristic minima at 208 and 222nm and upon the increase in temperature exenatide losses its minima at 222nm and upon cooling it either undergoes oligomerization by increase in minimum at 222nm or retains the native structure. The results showed that in the absence of sugars oligomerization is predominant with larger minima at 222nm (Figure 1) and in presence of sugars it retained its native structure (Table in Figure 1). This suggested that sugars were indeed stabilizing Exenatide. 2. Permeation

Results: The next critical step was to assess how well Exenatide could permeate through a membrane in the solid powder form spray dried with sugars and without sugars, mimicking its absorption through the lung membrane. Permeation was evaluating with and without mucus . Interestingly, the results showed that formulations with higher sugar content exhibited better permeation in the presence of mucus, but not in its absence (Figure 2). The explanation lies in the interaction between sugars and mucus. In the absence of mucus, sugars form strong hydrogen bonds with Exenatide, hindering permeation. However, with mucus present, sugars preferentially interact with the mucus layer, freeing Exenatide to permeate more easily. The increased permeation with higher sugar concentrations in the presence of mucus emphasized the role of sugars in enhancing drug diffusion. 3. Powder Properties: Now that the exenatide stability in powder after spray drying and the spray dried powder’s diffusion has been evaluated, next challenge is to deliver the powder to the lungs for which we need a carrier. For that the mannitol carriers for pulmonary exenatide delivery were engineered using ammonium bicarbonate to form pores during spray drying, reducing particle density and enhancing dispersibility. Particle formation, governed by the Peclet number (dependent on solvent evaporation rate and solute diffusion), was tested under varying outlet temperatures and solution concentrations. Higher temperatures (70°C) accelerated evaporation, yielding smaller particles, while lower concentrations also reduced particle size. Helium pycnometry revealed porosity by measuring true density (1.873 g/ml for formulation F8) through helium’s penetration into voids. F8 (70°C outlet, 2.5% solids) exhibited 90.7% porosity, 6.0 mm aerodynamic diameter, and uniform pores via SEM, optimizing pulmonary delivery (Figure 3). This formulation outperformed commercial DPI mannitol in particle structure and aerodynamic properties.

Conclusion: After a meticulous experimentation and analysis, we successfully steered the challenges of stabilizing, permeating and delivering Exenatide for pulmonary administration. The use of sugars, particularly mannitol and trehalose, proved crucial for maintaining peptide stability during spray drying. Furthermore, engineering mannitol particles with ammonium bicarbonate allowed us to create porous carriers with optimal aerodynamic properties for efficient lung delivery. This comprehensive approach paves the way for improved therapeutic interventions using pulmonary delivery of Exenatide and potentially other peptide drugs.

References: 1. Banerjee, R., & Sheet, T. (2017). Ratio of ellipticities between 192 and 208 nm (R1): An effective electronic circular dichroism parameter for characterization of the helical components of proteins and peptides. Proteins: Structure, Function, and Bioinformatics, 85(11), 1975–1982. https://doi.org/10.1002/PROT.25351
2. Stigliani, M., Aquino, R. P., Del Gaudio, P., Mencherini, T., Sansone, F., & Russo, P. (2013). Non-steroidal anti-inflammatory drug for pulmonary administration: Design and investigation of ketoprofen lysinate fine dry powders. International Journal of Pharmaceutics, 448(1), 198–204. https://doi.org/10.1016/J.IJPHARM.2013.03.030
3. Eslamian, M., & Ashgriz, N. (2007). Effect of Atomization Method on the Morphology of Spray-Generated Particles. Journal of Engineering Materials and Technology, 129(1), 130–142. https://doi.org/10.1115/1.2400270
4. Peng, T., Zhang, X., Huang, Y., Zhao, Z., Liao, Q., Xu, J., Huang, Z., Zhang, J., Wu, C. Y., Pan, X., & Wu, C. (2017). Nanoporous mannitol carrier prepared by non-organic solvent spray drying technique to enhance the aerosolization performance for dry powder inhalation. Scientific Reports 2017 7:1, 7(1), 1–11. https://doi.org/10.1038/srep46517


Figure 1: Secondary structure evaluation using Circular Dichroism


Figure 2: Franz diffusion evaluation in the presence and absence of mucus


Figure 3: Morphology and particles properties evaluation