INTERLEUKIN-2 IN AQUEOUS AND LIPID ENVIRONMENTS: UNDERSTANDING MOLECULAR INTERACTIONS THAT IMPACT PHARMACEUTICAL FORMULATION DEVELOPMENT
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Abstract
The continued development of pharmaceutical recombinant proteins as therapeutics necessitates a full understanding of the biomolecular interactions impacting structural and functional protein stability within different environments. Two such environments are investigated here for the therapeutic protein, interleukin-2 (IL-2). Aqueous surroundings are applicable in liquid formulations for storage and parenteral injection as well as some controlled release drug delivery strategies, while the pharmacokinetic targeting possibilities of a liposome nanoparticle drug delivery system for IL-2 dictates an understanding of molecular interactions within a lipid environment.Proteins often require excipients to preserve structural and functional stability in aqueous environments, thus we investigated a novel organic salt, choline dihydrogen phosphate (CDHP), which has been shown to impart increases in thermal stability of some model proteins. Thermal stability is of particular interest for IL-2, which irreversibly aggregates upon thermal denaturation. A significant increase in the thermal midpoint transition temperature (12.5 degrees celcius) of IL-2 was seen in the presence of high concentrations of CDHP (680mM). Control formulations in sodium dihydrogen phosphate (NaDHP), choline chloride (ChCl), and sodium chloride (NaCl) revealed that the DHP anion is the primary contributor towards IL-2 thermal stability. Initial cytotoxicity studies indicated the need for lower concentrations (<40mM), suggesting the need for dilution prior to in vivo administration. Bioactivity of IL-2 in the presence of 30mM CDHP revealed that even small amounts of CDHP can affect the bioactivity of IL-2, demonstrating that increases in thermal stability do not necessarily lead to increases in bioactivity retention.The second aim of this work was to investigate the interaction between IL-2 and lipid vesicles and the clinical implications of such an interaction, including release kinetics and bioactivity of released protein. Liposomes are of particular interest for IL-2 because of their potential as a lymphatic system targeting drug delivery strategy or as a means of decreasing the in vivo clearance rate of IL-2 in the body. Three phosphatidylcholine based lipids of various acyl chain lengths (DMPC, DPPC, and DSPC) which have a neutral charge at physiological pH and one phosphatidylglycerol based lipid (DSPG) which has a charge of (-1) at physiological pH were chosen for liposome formulations. The effect of liposome composition on IL-2 binding thermodynamics, IL-2 lipid saturation ratios, IL-2 release kinetics, lipid bilayer melting thermodynamics, and IL-2 bioactivity were studied. A positive correlation between acyl chain length and IL-2 binding and saturation ratios was found. Bioactivity of released IL-2 was not significantly altered as a result of formulation into the various lipid compositions. Aggregation phenomena of liposomes, which were altered upon IL-2 incorporation into each liposome formulation, impacted release kinetics. All phosphatidylcholine based liposome formulations disaggregated upon IL-2 addition, while formulations containing phosphatidylglycerol aggregated upon IL-2 incorporation. An IL-2 liposome interaction model is proposed to explain binding thermodynamics and aggregation behavior, which may facilitate the development of future IL-2 liposome formulations.