Aqueous Remote Loading of Peptides in PLGA Microspheres

Abstract

Poly (lactic-co-glycolic) acid (PLGA) microspheres are commonly used in long acting release (LAR) products due to their biodegradable and biocompatible nature. Traditional techniques for peptide drug encapsulation in PLGA microspheres such as the double-emulsion-solvent evaporation method, expose peptides and proteins to harsh conditions such as organic solvents, sheer forces, and temperature fluctuations, which can result in degradation and aggregation of the drug. Traditional encapsulation methods are also costly owing to the need for aseptic processing, the difficulty to scale-up to large-scale manufacture, suboptimal yields, and the possibility for batch failure. An alternative method for microsphere formulation is aqueous remote loading, which is performed by placing the aqueous peptide solution in the presence of sterile drug-free PLGA microspheres, thereby minimizing peptide exposure to potential degradants and simplifying manufacturing. This work investigated aqueous remote loading to encapsulate cationic peptides in acid terminated PLGA microspheres as a means to achieve high encapsulation efficiency and peptide loading with desirable release kinetics in vitro and in vivo. Free carboxylic acid terminated PLGA (PLGA-COOH) microspheres of various molecular weight (13 kDa- 38 kDa) and lactic acid to glycolic acid ratios (50/50 and 75/25) were prepared to encapsulate the model cationic peptide, leuprolide. Cationic peptides, such as leuprolide, are able to interact with PLGA-COOH via peptide absorption in the polymer to achieve elevated loading and encapsulation after incubation for only 24 hours at 37 ºC. Leuprolide was encapsulated in PLGA-COOH microspheres by this aqueous remote loading technique. Initial studies of leuprolide absorption to PLGA microspheres achieved loading (~ 9.8 %) comparable to the commercial 1-month product Lupron Depot®. However, these microspheres did not encapsulate leuprolide at high efficiency (< 40%). Using the sorption isotherms of leuprolide, a model for the prediction of leuprolide encapsulation was developed in order to select formulation conditions to achieve elevated encapsulation efficiencies and loading. Using the model predicted conditions, encapsulation efficiency was improved to > 55%. Optimized loading conditions were then applied to low molecular weight 75/25 PLGA, the polymer used in the commercial 1-month leuprolide/PLGA product, the Lupron Depot®. Low MW 75/25 PLGA microspheres loaded at 180 mg/mL and 240 mg/mL microsphere concentrations encapsulated > 80% leuprolide and 6.5%- 8.4% loading, and exhibited controlled release over 49 days in vitro. Gamma irradiation sterilization of preformed microspheres did not significantly affect peptide loading and release kinetics. These microspheres demonstrated strikingly similar efficacy to the Lupron Depot® in terms of sustained testosterone suppression of rats when dosed monthly for 3 months. The generality of remote loading was tested in 5 peptides using loading parameters optimized for leuprolide. Medium to large basic peptides, octreotide, vasopressin, and salmon calcitonin, exhibited > 65% encapsulation efficiency with elevated loading. However, low levels of encapsulation (< 30% encapsulation efficiency) were observed for acidic and small peptides, exenatide and protirelin, respectively. Hence, this thesis develops a new and simple means to encapsulate peptides in PLGA microspheres under mild, and less complex conditions, and may be generally applicable to long-acting release microspheres for basic peptides and generic product development.