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

Abstract

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 1 H 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.

Molecular Mechanism of Antimicrobial Excipient-Induced Aggregation in Parenteral Formulations of Peptide Therapeutics

“…Likely, the hydrophobic interaction with m -cresol is extended to the acyl chain of the peptide. As reported in our previous study, peptide A without an acyl chain is less sensitive to the addition of m -cresol, forming detectable insoluble aggregates only at a higher PER . Further higher-order acyl-peptide A oligomers appear to form with exposure to m-cresol, which eventually develop into insoluble aggregates.…”

“…Although this study has focused on elucidating the molecular mechanism of m -cresol-induced peptide association, it is interesting to explore the different observations from titrating BA preliminarily. Our previous study has suggested the increase in turbidity and blue shift of the Trp fluorescence emission upon addition of m -cresol into the acyl-peptide A formulation, while no change in solutions containing BA was observed . In the current study, NMR results have identified site-specific interaction between m-cresol and acyl-peptide A (Figure ).…”

“…In contrast, BA does not exhibit significant chemical shift perturbations in the 2D 1 H– 1 H NOESY spectrum of acyl-peptide A (Figure ). Moreover, when the palmitoyl side chain is removed, both the propensity of this peptide to aggregate and fluorescence emission maximum shifts are reduced, as observed in our previous study . These findings suggest a hypothesis that hydrophobic interactions between m-cresol and lipid sidechains likely play a role in the peptide association.…”

“…This observation is in agreement with the lack of observed spectral changes for the peptide in the presence of BA (Figure 3). 28 3.3. Peptide−Excipient Complex from 2D DOSY Spectroscopy.…”

“…We have previously evaluated the impact of APs on the stability of 31-residue acyl-peptide A formulations through assessments of turbidity, intrinsic fluorescence shifts and quenching, isothermal titration calorimetry, and proton nuclear magnetic resonance ( 1 H NMR) . Interestingly, BA does not induce measurable peptide aggregation or conformational change.…”