16th European Headache Congress

Chair

Neurokinins and PACAP/VIP in relation to the trigeminovascular system

Professor

Invited speaker

Invited lecture

Amylin and amylin receptors in migraine

Prof.

The role of non-CGRP neuropeptides in the pathophysiology of migraine

Development and pharmacological characterisation of bifunctional CGRP-PACAP receptor antagonists in transfected cells and spinal cord cultures

Abstract text (incl. figure legends and references) Question: The neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) are both implicated in migraine. Blocking the activity of these peptides simultaneously may provide a clinical advantage over individual blockade. One strategy is to develop a bifunctional ligand, capable of antagonizing both systems at once. As a starting point we utilized the known antagonism imparted by CGRP and PACAP peptide fragments, exploring different lengths of PACAP. From this, we selected CGRP8-37 and PACAP6-38 to attach together and assessed these molecules as bifunctional antagonists. Methods: Peptides were synthesized in-house and CGRP8-37 was linked to PACAP6-38 using 1,3-dipolar cycloaddition at amino acid positions 21, 34 and 38. The potency of these peptides as bifunctional antagonists was then tested, and compared to the parent fragments. We tested antagonism against CGRP at the human CGRP and AMY1 receptors and against PACAP-27, PACAP-38 and VIP at the human PAC1, VPAC1 and VPAC2 receptors in Cos7 cells (cAMP production). Translational relevance was assessed by measuring antagonism of agonist-stimulated cAMP production in primary rat spinal cord cultures. Results: The bifunctional antagonists generally displayed similar antagonist activity to CGRP8-37 and PACAP6-38 in receptor transfected Cos7 cells and spinal cord cultures. Interestingly, linking CGRP8-37 to position 38 of PACAP6-38 generated a peptide with greater antagonist potency than CGRP8-37 at CGRP and AMY1 receptors in Cos7 cells. Conclusions: This study provides proof-of-concept that bifunctional antagonists capable of blocking both CGRP and PACAP activity can be generated.

Basic science, animal models in headache research

Neuropeptides, channels in headache

Abstract lecture

Prof. Dr.

Crosstalk between cannabinoid and vanilloid systems: role of CB receptors in the capsaicin‑induced relaxation responses in human coronary arteries

Abstract text (incl. figure legends and references) Background: The use of cannabis and its derivatives has increased during the last years due to their therapeutic potential. However, the exact mechanisms of action of cannabinoids are still limited. It has been suggested that cannabinoids can exert their effects via the activation of cannabinoid receptors (i.e. CB1 or CB2 receptors) and/or transient receptor potential vanilloid 1 (TRPV1) channels, suggesting an interaction between both systems. We investigated the role of CB receptors in the vasodilatory effects induced by capsaicin in human isolated coronary arteries (HCAs). Methods: In HCAs (female, n=5; 56±5 years and male, n=4; 57±4 years), the vasodilatory responses to capsaicin (TRPV1 channel agonist) were evaluated in the absence or presence of the antagonists capsazepine (TRPV1, 5 µM); AM6545 (CB1 receptor, 1 µM); AM630 (CB2 receptor, 1 µM); O-1918 (putative endothelial CB receptor, 10 µM) or cannabidiol (GPR55 receptor, 1 µM) to obtain the maximum contractile response (Emax). Results: Capsaicin induced concentration-dependent relaxation responses (Emax 109±8%), which were significantly reduced by AM6545 (Emax 87±4%) or cannabidiol (Emax 86±3%), but not by capsazepine (Emax 103±6%), AM630 (Emax 100±3%) or O-1918 (Emax 93±5%). Moreover, pilot experiments (n=2) showed that the maximal response induced by N-arachidonoylethanolamine, (ACEA, a CB1 receptor agonist; Emax 43±7%) is inhibited by AM6545 or capsazepine: Emax 16±3% and 21±4%, respectively. Conclusions: (i) Capsaicin-induced relaxation responses are mediated by CB1 and GRP55 receptors; and (ii) TRPV1 channels may be involved in the modulation of the ACEA-induced relaxation responses. Thus, we can suggest that the CB and vanilloid systems may share a signaling pathway to modulate the vascular tone, which may provide novel therapeutic targets for vascular disorders (e.g. migraine and its related cardiovascular events). Further studies should elucidate additional mechanisms involved in these responses.

Second messenger signaling bypasses blockade of the calcitonin gene-related peptide (CGRP) receptor to provoke migraine attacks in humans

Abstract text (incl. figure legends and references) Numerous endogenous molecules trigger migraine attacks when administered to humans. Of therapeutic importance, this has led to the concept of a &quot;migraine attack signaling cascade&quot; with the calcitonin gene-related peptide (CGRP) acting via a downstream second messenger cyclic adenosine monophosphate (cAMP) in intracranial vascular smooth muscle cells and other cells. However, whether intracellular cAMP signaling acts strictly downstream or is dependent on CGRP receptor activation during a migraine attack has never been tested directly in humans. Here, using a human provocation model (CGRP and phosphodiesterase 3 inhibitor, cilostazol, an agent known to accumulate intracellular cAMP by inhibiting its degradation) in a randomized, double-blind, placebo-controlled, parallel trial design, we demonstrate that migraine attacks can be provoked by intracellular cAMP-mediated mechanisms using cilostazol in the presence of CGRP receptor blockade (erenumab). Consistent with these findings, cilostazol-induced dilation of cranial arteries was unaffected by a CGRP receptor blockade. Our work provides clinical evidence that cAMP-evoked migraine attacks act downstream of the CGRP receptor, and that these cAMP-evoked migraine attacks appear independent of CGRP-receptor activation. These findings open new avenues for mechanism-based drug development for migraine.

SciSe 2
The role of non-CGRP neuropeptides in the pathophysiology of migraine

Appointment

Chair

Programme

SciSe 2The role of non-CGRP neuropeptides in the pathophysiology of migraine

Appointment

Chair

Programme

SciSe 2
The role of non-CGRP neuropeptides in the pathophysiology of migraine