(M1430-09-49) Remote Encapsulation of Exenatide in PLGA Microspheres Using Cationic Excipients

Purpose: Glucagon-like-peptide-1 (GLP-1) and related peptides have shown great promise in the treatment of diabetes and, more recently, obesity. The growing need for medications in this area supports further development of long-acting formats of GLP-1 peptides. Our lab has previously made significant progress in the high efficiency aqueous remote encapsulation of net cationic peptides in acid-terminated PLGA microspheres for long-acting release. Microencapsulation by remote loading offers a multitude of advantages, such as simple unit operations, limited organic solvent exposure, and minimal drug losses during encapsulation. However, most GLP-1 peptides are acidic, limiting their capacity for remote loading in blank PLGA microspheres. One such GLP-1 peptide approved in a PLGA formulation is exenatide (Bydureon®), indicated for the treatment of type 2 diabetes. In this study, we used exenatide as a model net anionic peptide to evaluate the co-incorporation of cationic excipients—the polymer polyethyleneimine (PEI) and the lipid 18:1 TAP (DOTAP)—in PLGA for remote loading. We evaluated particle characteristics and the encapsulation advantages relative to PLGA microspheres for the acidic peptide to extend the remote loading paradigm to a previously incompatible peptide class.

Methods: Single and double emulsion solvent evaporation techniques were used to prepare PLGA microspheres containing DOTAP and PEI, with varying excipient concentration and microsphere porosity. Low molecular weight (2.5 kD) linear PEI was used to mitigate cytotoxicity concerns from the cationic polymer. Control microspheres were prepared using acid- and amine-terminated PLGA. Exenatide was remote-loaded by incubating microspheres in peptide solutions at varying pH and buffer concentration. Loading and encapsulation efficiency (EE) were determined by ultra-performance liquid chromatography (UPLC) after extracting the drug from microspheres with acetonitrile. Scanning electron microscopy (SEM) was used to determine particle morphology, and particle size was evaluated by laser diffraction. In vitro release was assessed under physiological conditions using a high-volume filter bag method developed in our lab.

Results: Microspheres in the size range of 20-90 µm were collected, with a mean diameter close to 40 µm. Consistent with previous reports, exenatide does not remote load well into acid-terminated PLGA microspheres due to electrostatic repulsion (EE < 2%). Microspheres made from amine-capped PLGA marginally improved the EE but still with insufficient quantities for a useful controlled release dosage form. PLGA with co-incorporated DOTAP or PEI were both capable of loading the drug, with increased loading at higher excipient concentration, loading solution pH, and lower buffer concentration. 5% DOTAP and 10% PEI relative to PLGA, and 0.1M MES buffer at pH 6 in the loading solution provided optimal loading of exenatide. DOTAP and PEI improve EE roughly 60- and 100-fold, respectively, relative to PLGA microspheres. PLGA/DOTAP released approximately 80% of the encapsulated drug in 24-h, and PLGA/PEI was able to release the drug continuously over one month with a modest initial burst ( < 15%). A low level of exenatide acylation was observed at the time of loading and release, which was likely related to the presence of the basic excipients. This degradation pathway is well known from previous studies with the commercial formulation and could potentially be limited with the use of peptides without reactive amino groups or modifying microsphere material selection and remote loading protocols.

Conclusion: The incorporation of low molecular PEI in PLGA microspheres significantly improves the encapsulation of the acidic peptide, exenatide, relative to PLGA and PLGA amine-capped microspheres. To our knowledge, this is the first report of remote encapsulation in PLGA for a large, net anionic peptide. The remote loading paradigm demonstrated here, with novel materials co-incorporated in remote-loading PLGA microspheres, may be extended to other net anionic peptides for new long-acting release formulations.

References: 1. Giles, Morgan B., et al. "Efficient aqueous remote loading of peptides in poly (lactic-co-glycolic acid)." Nature Communications 13.1 (2022): 3282.
2. Kunath, Klaus, et al. "Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine." Journal of Controlled Release 89.1 (2003): 113-125.
3. Chandrashekar, Aishwarya, et al. "In vitro performance of composition-equivalent PLGA microspheres encapsulating exenatide acetate by solvent evaporation." International Journal of Pharmaceutics 643 (2023): 123213.
4. Hong, Justin KY, et al. "Mapping in vivo microclimate pH distribution in exenatide-encapsulated PLGA microspheres." Journal of Controlled Release 352 (2022): 438-449.

Exenatide can be remote loaded in PLGA microspheres with added DOTAP or PLGA with high encapsulation efficiency relative to blank acid- and amine-capped PLGA microspheres.