Antimicrobial Excipient-Induced Reversible Association of Therapeutic Peptides in Parenteral Formulations

New classes of therapeutic peptides are being developed to prosecute biological targets which have been inaccessible to other modalities. Higher potency and longer half-life peptides have given rise to multiuse injectable formulations that enable convenient, low volume, and self-administered dosing; however, inclusion of antimicrobial preservatives to meet bactericidal requirements can impact other attributes of peptide formulations. Peptide-preservative interactions influencing solution-phase self-association of a non-insulin, linear, palmitoylated 31 amino acid peptide and two structurally similar peptides were assessed via turbidity, intrinsic fluorescence shifts and quenching, isothermal titration calorimetry, and ¹H NMR. Meta-cresol and phenol specifically interact with the peptide, result in increased hydrophobicity near the tryptophan residue, and induce conformational changes, while benzyl alcohol does not impact tryptophan fluorescence, demonstrate any interaction enthalpy, or induce conformational changes. These same trends did not hold true for the other palmitoylated peptides evaluated, reinforcing the impacts of unique peptide sequences. Importantly, the presence of benzyl alcohol does increase the physical stability and solubility of the linear, 31 amino acid peptide under salt stress. We report new insights into the physical interactions of peptides with antimicrobial excipients, demonstrating a reversible association phenomenon and highlighting practical implications for formulation design and excipient selection.