Analyzing proteolytic stability and metabolic hotspots of therapeutic peptides in two rodent pulmonary fluids

Peptides and peptide drug conjugates are emerging modalities to treat pulmonary diseases. Peptides are sus-ceptible to proteolytic cleavage. Expression levels of specific proteases in the lung can be significantly increased in disease state and may lead to exaggerated peptide proteolysis. To support optimization of peptides for inhaled administration, we have recently reported a streamlined high-throughput LC-HRMS protocol to determine enzymatic protease stability of peptides. This method has now been complemented with profiling of peptide metabolic stability in two respiratory fluids, a lung supernatant (lung S9) and a bronchioalveolar lavage fluid (BALF) taken from rats. We have tested a set of 28 peptides with high structural diversity, analyzed the whole data set for formed metabolites, and identified the differences of cleavage pattern in the two test fluids. Com-parison of our experimental results and literature-derived cleavage site estimates based on e.g. MEROPS show significant differences for a number of peptides. This indicates the need for an experimental workflow using both protease panels and testing of metabolic stability in lung fluid (BALF) to guide peptide optimization and selection of peptides for inhaled in vivo PK/PD studies in our drug discovery projects.

Automated summary

Peptides and peptide drug conjugates are being used to treat pulmonary diseases, but they are susceptible to proteolytic cleavage. This study developed a high-throughput LC-HRMS protocol to determine enzymatic protease stability of peptides for inhaled administration. Testing of metabolic stability in lung fluid (BALF) was also conducted. The results showed significant differences compared to literature-derived estimates, indicating the need for experimental workflows to guide peptide optimization in drug discovery projects.