Identification of an IDR peptide formulation candidate that prevents peptide aggregation and retains immunomodulatory activity

Abstract

Synthetic peptides derived from naturally occurring host defence peptides (HDPs) have garnered significant attention as novel pharmaceuticals, particularly as alternatives to antibiotics and for immunomodulatory applications. One of the barriers to advancing synthetic peptides for therapeutic applications is their tendency to aggregate under specific ionic conditions similar to those they would encounter in vivo. Formulating peptides with biocompatible excipients that prevent solvent‐induced peptide aggregation represents a possible solution to this aggregation issue; however, this strategy has not been systematically explored. In the present work, we describe the screening of various polymeric substances (including hyaluronic acid, carboxymethyl cellulose and hydroxypropyl methyl cellulose) as formulation candidates for HDPs and identified derivatized hyperbranched polyglycerols (dHPGs) as a biocompatible excipient that limits peptide aggregation. Notably, the immunomodulatory activity of a synthetic innate defence regulator peptide, IDR‐1018, formulated with dHPG was retained when evaluated against both peripheral blood mononuclear cells and human bronchiolar epithelial cells in vitro. Further characterization of dHPG polymers revealed that decreasing the negative charge density on the polymer surface potentiated the cytotoxic effects of IDR‐1018, emphasizing the need to optimize this parameter for future peptide delivery formulations. Importantly, the dHPG formulated IDR‐1018 inhibited peptide aggregation in vitro in the presence of mucin as well as in vivo when injected subcutaneously into CD1 mice. This study highlights the potential use of dHPGs as formulation candidates for synthetic HDPs and identifies important considerations regarding their physico‐chemical properties and relevance to immunomodulatory applications.

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance

“…Similarly, Haney et al found that derivatized HPGs containing a hydrophobic polymer core capped with carboxylic acid-functionalized PEG groups significantly reduced the aggregation of the synthetic AMP IDR-1018 both in vitro and in vivo while maintaining the immunomodulatory activity of the peptide [132]. This is of particular interest as peptide aggregation has been shown to inhibit the activity of certain AMPs and is thought to be partially responsible for AMP toxicity [99,132]. Notably, this formulation did not require covalent conjugation or modification of the parent peptide and was more efficacious than other tested formulations utilizing hyaluronic acid, carboxymethyl cellulose, and hydroxypropyl methyl cellulose.…”

“…Though the activity of the peptide did decrease, the most promising conjugate, a low molecular weight HPG containing 7-8 conjugated peptides, still displayed moderate activity in vitro against S. aureus. Similarly, Haney et al found that derivatized HPGs containing a hydrophobic polymer core capped with carboxylic acid-functionalized PEG groups significantly reduced the aggregation of the synthetic AMP IDR-1018 both in vitro and in vivo while maintaining the immunomodulatory activity of the peptide [132]. This is of particular interest as peptide aggregation has been shown to inhibit the activity of certain AMPs and is thought to be partially responsible for AMP toxicity [99,132].…”

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application

“…Peptide 1018 aggregated (and/or formed hydrogels) in a concentration-dependent manner in buffers containing certain anions, particularly phosphate, benzoate, citrate, and nitrate, according to the Hofmeister series of anions, whereas altering the cation had little effect except at very high concentrations. It also co-precipitated serum proteins in tissue culture medium [27] and demonstrated precipitation in vivo using a mouse skin injection model [28]. This latter observation might explain the toxicity of certain peptides due to their precipitation in the blood when applied intravenously.…”

“…Inconsistencies between in vitro and in vivo efficacy might be explained by physiological conditions within the host influencing stability and toxicity of anti-biofilm peptides and thus indirectly interfering with their activity. For instance, 1018 has been shown to precipitate in vitro in the presence of mucin and ions as well as when injected subcutaneously into mice [27,28]. In another study, topical application of the rationally de-DOI: 10.1159/000491497 signed peptide DRGN-1 significantly reduced wound size in a mixed biofilm-infected murine wound model [22].…”

“…In studies with other peptides, concentration-dependent aggregation can trigger enhanced immunogenicity which might pose a major problem for clinical use of peptide treatments by inducing chemokine activity and causing allergic responses or cytotoxic effects [54]. Again appropriate formulation can influence aggregation as well as cytotoxicity [28]. Early peptide work assessed cytotoxicity in terms of its ability to cause red blood cell lysis.…”