ABSTRACT
Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.
ABSTRACT
BACKGROUND AND PURPOSE: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice. EXPERIMENTAL APPROACH: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists. KEY RESULTS: We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and ßCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity. CONCLUSION AND IMPLICATIONS: Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary).. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.
