Novel Exenatide Analogs with Peptidic Albumin Binding Domains: Potent Anti-Diabetic Agents with Extended Duration of Action

Abstract

The design, synthesis and pharmacology of novel long-acting exenatide analogs for the treatment of metabolic diseases are described. These molecules display enhanced pharmacokinetic profile and potent glucoregulatory and weight lowering actions compared to native exenatide. [Leu14]exenatide-ABD is an 88 residue peptide amide incorporating an Albumin Binding Domain (ABD) scaffold. [Leu14]exenatide-ABP is a 53 residue peptide incorporating a short Albumin Binding Peptide (ABP). [Leu14]exenatide-ABD and [Leu14]exenatide-ABP exhibited nanomolar functional GLP-1 receptor potency and were metabolically stable in vitro in human plasma and in a pancreatic digestive enzyme mixture. Both molecules displayed picomolar and nanomolar binding association with albumin across multiple species and circulating half lives of 16 and 11 hours, respectively, post a single IV dose in rats. Unlike exenatide, both molecules elicited robust glucose lowering when injected 1 day prior to an oral glucose tolerance test, indicative of their extended duration of action. [Leu14]exenatide-ABD was compared to exenatide in a Lep ob/ob mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu14]exenatide-ABD displayed superior glucose lowering and weight loss in diabetic mice when compared to continuously infused exenatide at the same total weekly dose. A single oral administration of each molecule via an enteric coated capsule to cynomolgus monkeys showed superior pharmacokinetics for [Leu14]exenatide-ABD as compared to [Leu14]exenatide-ABP with detectable exposure longer than 14 days. These studies support the potential use of these novel long acting exenatide analogs with different routes of administration for the treatment of type 2 diabetes.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

“…One significant difference was the higher liver uptake of the Cy7 monomer. An alternative approach to slow clearance is to add an albumin binding peptide sequence through solid-phase synthesis [60], but increased liver uptake due to longer plasma exposure and/or scavenger uptake[75–76] and down-regulation of GLP-1R may not provide any targeting benefit over the more rapidly cleared monomer. In the pancreas, total uptake of 800CW and Cy7 monomers was not statistically different (p = 0.26).…”

Intramolecular Thioether Crosslinking of Therapeutic Proteins to Increase Proteolytic Stability

“…[29][30][31] The very long in vivo circulation half-life is probably a result of high-affinity binding to albumin, thereby resulting in the formation of a tight complex in vivo. [30,32] The long circulation half-life, comparable to albumin itself, is a strong indication of the very high serum stability of ABD. However, as demonstrated here, the protein is sensitive to degradation by gastrointestinal proteases.…”

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

“…Azidohomoalanine is easily incorporated during solid phase peptide synthesis (SPPS) and has been previously used to generate fluorescent imaging agents via alkyne fluorophore conjugation as well as stabilized fluorescent and non-fluorescent peptides such as exendin and inhibitors of Mdm2 31, 32, 37, 38 . Based on past results focused on the glucagon-like peptide-1 receptor (GLP-1R) ligand exendin and FDA approval of multiple subcutaneously administered exendin analogues for treating type 2 diabetes including exenatide, liraglutide, and dulaglutide, exendin was chosen as the model system for investigating the effect of lipophilicity and stability on SC bioavailability 39, 40 . Helix stabilization and lipophilicity impact the absorption and bioavailability in complex ways.…”