title: Design, synthesis, and evaluation of serum-stable peptide antagonists of the CGRP receptor as a novel treatment for migraine
Creators: name: D'Aloisio, V
Publisher: name: Liverpool John Moores University
Year: 2024

description: Migraine, the third most prevalent disease in the world (global prevalence of 12- 14.7%), is a complex and multifaceted neurovascular disorder, characterised by severe attacks of headache and often associated with nausea and sensory hypersensitivity. Numerous contraindications and adverse events are associated with traditional medicines and, more importantly, 40% of patients receive little-to- no benefit from them. Calcitonin gene-related peptide (CGRP) and its receptor are now recognised to play a key role in migraine pathophysiology and evidence suggests that the CGRP receptor could be antagonised with synthetic peptides obtained by truncation of the native CGRP. Peptide-based therapeutics have been an active area of research for decades and have found clinical applications in a number of disease areas, e.g., metabolic disorders, cancers, cardiovascular diseases, and infection diseases, reaching the milestone of 100 approved peptide drugs in 2020. In this project, information on regulatory approved peptide therapeutics and diagnostics was collated in an open- source database, PepTherDia (http://peptherdia.herokuapp.com), and a detailed structural analysis was performed to uncover key structural trends to help with future peptide drug design. The use of peptides as medicines is still limited due to poor bioavailability after oral administration and, more generally, poor stability due to their susceptibility to proteolytic degradation. Various strategies, including fluorine-editing, peptoid- editing, or lipidation, could be explored to prepare proteolytically stable peptide or peptidomimetic analogues. Towards this goal, a library of peptide and peptidomimetic analogues of the C-terminal portion of CGRP were designed and synthesised, starting from the structure of a potent CGRP receptor antagonist (P006) previously developed in collaboration with PharmNovo AB. These compounds were tested for antagonism at the CGRP receptor, lipophilicity and toxicity. Methods to assess peptide stability in blood and plasma protein binding were also optimised, validated, and applied to study the pharmacokinetic profile of the library of compounds. This led to the discovery of potent CGRP receptor antagonists with vii extremely favourable pharmacokinetic profiles. Among them, the most promising compounds of the series were three N-terminal benzoylated analogues (i.e., LJMU027, LJMU017, and LJMU018) presenting significantly increased estimated half- lives (&gt; 3 hours) compared to P006 (estimated half-life of ~13 min), while maintaining comparable activity against the CGRP receptor. Due to first pass metabolism and poor oral absorption, peptides are often administered parenterally, thus usually resulting in low patient acceptability and compliance. Alternatively, peptides could be delivered via the intranasal route, offering the advantages of rapid direct delivery to the brain, greater acceptability and compliance, and lower adverse event profiles due to inferior systemic effects. Nano- sized structures derived from peptide self-assembly in water were obtained (diameter size between 14 and 30 nm) and peptide-loaded dry powder microparticles prepared from low molecular weight chitosan suitable for nasal delivery were manufactured (average diameter size of 9.55 ± 0.74 μm). In conclusion, this investigation showed that benzoyl modifications performed at the N-terminus of P006 did not disrupt antagonistic activity but yielded stable analogues that were up to 90% intact after 60 min incubation in pooled human serum, with a tendency to bind to plasma proteins. The ability of these peptides to self-assemble was exploited to prepare nano-sized structures, which could be directly administered parenterally or loaded into mucoadhesive carriers for nasal delivery.