A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity

Abstract

Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (sc) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. Their ease and efficiency make double-click helix stabilization chemistries a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control sc absorption and clearance rates to customize plasma pharmacokinetics.

Figure 1

Design and synthesis of stabilized and non-stabilized exendin conjugates. The modified residues are highlighted in red. Fluorophores are first functionalized with one or two alkynes using amine-NHS chemistry, followed by either single or double click reactions to generate the fluorescent conjugates.

Figure 2

Peptide characterization includes binding affinity (A), trypsin digest (B), and circular dichroism measurements (C). All conjugates maintain strong binding affinity to GLP-1R. Stabilized exendin conjugates demonstrate superior protease resistance compared to single click peptides. Affinity, digest half-lives, and helicity are tabulated (D).

Figure 3

Fluorescent exendin with and without helix stabilization were administered either intravenously or subcutaneously in mice. The plasma concentration was quantified and plotted versus time to determine the effect of helix stabilization on SC bioavailability. Stabilized AF680 and Cy7 exendin displays significantly higher bioavailability than their non-stabilized counterparts.

Figure 4

Macroscopic pancreas images demonstrate islet targeting whether peptide is administered subcutaneously or intravenously for all exendin conjugates. Beta cells are located in the islets of Langerhans and appear as distinct punctate spots.

Figure 5

A three-compartment analysis of transient peptide concentration in plasma. Experimental plasma concentrations were measured after IV administration of each peptide and used to fit the rates shown with blue arrows. These values were then fixed, and the rates for the green arrows were fit to the data following SC administration to quantify absorption and degradation rates. Fitted half-lives suggest increased proteolytic resistance resulting from helix stabilization and slowed absorption due to linker lipophilicity determine the in vivo absorption profile and absolute SC bioavailability.