Abstract
Understanding of peptide aggregation propensity is an important aspect in pharmaceutical development of peptide drugs. In this work, methodologies based on all-atom molecular dynamics (AA-MD) simulations and 1H NMR (in neat H2O) were evaluated as tools for identification and investigation of peptide aggregation. A series of structurally similar, pharmaceutically relevant peptides with known differences in aggregation behavior (D-Phe6-GnRH, ozarelix, cetrorelix, and degarelix) were investigated. The 1H NMR methodology was used to systematically investigate variations in aggregation with peptide concentration and time. Results show that 1H NMR can be used to detect the presence of coexisting classes of aggregates and the inclusion or exclusion of counterions in peptide aggregates. Interestingly, results suggest that the acetate counterions are included in aggregates of ozarelix and cetrorelix but not in aggregates of degarelix. The peptides investigated in AA-MD simulations (D-Phe6-GnRH, ozarelix, and cetrorelix) showed the same rank order of aggregation propensity as in the NMR experiments. The AA-MD simulations also provided molecular-level insights into aggregation dynamics, aggregation pathways, and the influence of different structural elements on peptide aggregation propensity and intermolecular interactions within the aggregates. Taken together, the findings from this study illustrate that 1H NMR and AA-MD simulations can be useful, complementary tools in early evaluation of aggregation propensity and formulation development for peptide drugs.
Highlights
The aggregation behavior of therapeutic peptides influences several critical aspects of pharmaceutical development, such as the dosage forms possible to develop, ease of manufacturing, formulation stability, and patient safety and convenience.[1−5] Self-assembly is often an unwanted effect but can be utilized to alter the pharmacokinetics of peptide and protein drugs[6−8] and can improve the chemical and physical stability of a drug.[9]
The aim of this work was to evaluate the applicability of the developed 1H NMR methodology and all-atom molecular dynamics (AA-MD) simulations, individually and in combination, in developability assessments and formulation development of therapeutic peptides, and for investigation of solution behavior and aggregation propensity
This section begins with a brief presentation of how peptide aggregation is expected to influence 1H NMR spectra, followed by an overview of representative NMR data and a discussion on the significance of the results, with respect to the aggregation behavior of each peptide
Summary
The aggregation behavior of therapeutic peptides influences several critical aspects of pharmaceutical development, such as the dosage forms possible to develop, ease of manufacturing, formulation stability, and patient safety and convenience.[1−5] Self-assembly is often an unwanted effect but can be utilized to alter the pharmacokinetics of peptide and protein drugs[6−8] and can improve the chemical and physical stability of a drug.[9] When peptides self-assemble, they can, to proteins, form various types of aggregates. Transient oligomers may be relevant for small peptides, which often show high conformational flexibility and a notably amphiphilic character. To obtain a good understanding of the aggregation behavior of a peptide, methods that can distinguish aggregates of different characters and sizes are of importance
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