Characterisation of vasodilatory responses in the presence of the CGRP receptor antibody erenumab in human isolated arteries

Abstract

Background

Migraine is associated with activation of the trigeminovascular system, release of calcitonin gene-related peptide (CGRP) and dilation of dural arteries. Novel treatments target calcitonin gene-related peptide or its receptor, which are present in all vascular beds, raising cardiovascular concerns. Erenumab is a human CGRP-receptor antibody approved for the prophylactic treatment of migraine.

Methods

We characterised the relaxant responses to CGRP in the absence and presence of erenumab (1 μM) in isolated human middle meningeal, internal mammary and (proximal and distal) coronary arteries. Furthermore, in human internal mammary arteries from cardiovascularly-compromised patients, we assessed the pharmacological specificity of erenumab by investigating whether the vasodilatory responses to acetylcholine, sodium nitroprusside, pituitary adenylate cyclase activating polypeptide-38 (PACAP), vasoactive intestinal peptide and nicardipine, along with the vasoconstrictor responses to dihydroergotamine, were modified by erenumab.

Results

Calcitonin gene-related peptide induced concentration-dependent vasodilatory responses in all vessels studied that were significantly antagonised by erenumab. In human internal mammary arteries from cardiovascularly-compromised patients, the responses to acetylcholine, sodium nitroprusside, PACAP, vasoactive intestinal peptide, nicardipine and dihydroergotamine were unaffected by erenumab.

Conclusion

Erenumab inhibits calcitonin gene-related peptide-induced vasodilatory responses in human middle meningeal arteries, human internal mammary arteries and human coronary arteries. Moreover, erenumab shows functional specificity as no interaction was observed with the relaxant responses to several vasodilators, nor the dihydroergotamine-dependent vasoconstrictor responses.

Keywords

Cardiovascular safety coronary arteries mammary arteries meningeal arteries migraine

Introduction

Migraine is a highly disabling neurovascular disorder, and its pathophysiology remains elusive. However, it has been associated with an activation of the trigeminovascular system, release of calcitonin gene-related peptide (CGRP) and increase in middle meningeal artery circumference specific to the head pain side (1). Based on the involvement of CGRP in the pain signalling pathway of migraine, small molecule-CGRP receptor antagonists (gepants) were developed for the treatment of migraine. The first gepants did not reach the market, due to hepatotoxicity cases and pharmacokinetic problems (2). Although novel gepants are currently being developed, with no reported toxicity so far (3), the most recent approach for CGRP blockade consists of the antibodies against CGRP (eptinezumab, fremanezumab, galcanezumab) or its receptor (erenumab). They have all shown to be effective for the prophylactic treatment of migraine and are either approved or likely soon to be approved for commercialization (4).

While the development of antibodies directed against the CGRP pathway (i.e., antibodies against CGRP or the CGRP receptor) represents a milestone in migraine treatment, it is important to also consider the implications of peripheral CGRP receptor blockade. To begin with, CGRP fibres innervate blood vessels, and are thought to contribute in the homeostatic responses to ischemic events (5 –7). This raises some concerns, especially as migraine patients present increased cardiovascular risk (8,9). Indeed, all the antibodies have been reported to be well tolerated even in subjects exposed for longer than one year, with no cardiovascular events reported in the clinical trials that were considered to be related to CGRP pathway blockade (10). Moreover, a study explored the effect of erenumab on exercise time during a treadmill test in mainly male patients with stable angina, and no changes were observed (11), although no evidence was provided for CGRP receptor blockade to be already established at the time of the treadmill test (12). In addition, previous studies have also shown that the vasodilatory role of CGRP in the coronary arteries is more prominent in the distal portion when compared to the proximal portion (13). While males are more prone than females to present ischemic events in the proximal portion of the coronary artery, females are more prone than men to present myocardial ischemic events in the distal portion of the coronary artery (14). As the vast majority of migraine patients are female, it is important to study the effects of erenumab in the distal portion of the coronary arteries. Also, for migraine patients with established cardiovascular disease, it is important to investigate whether blockade of the CGRP pathway could worsen their disease (6). Thus, appropriate in vitro and in vivo studies are needed to assess the vascular safety of blocking the CGRP pathway.

The aim of this study was to investigate the inhibition of the vasodilatory responses to CGRP by erenumab in the human isolated meningeal artery (HMMA), one of the proposed sites of therapeutic action. Also, in view of theoretical cardiovascular safety concerns, in human isolated proximal and distal coronary arteries (HCA), and in internal mammary arteries (HIMA) from cardiovascularly compromised patients undergoing coronary artery bypass grafting surgery. Furthermore, in HIMA, we studied the functional specificity of erenumab by comparing the relaxant responses to several vasodilators in the absence and presence of erenumab, namely: (i) acetylcholine, coupled to endothelium-dependent, nitric oxide-cGMP signalling; (ii) sodium nitroprusside, coupled to endothelium-independent, nitric oxide-cGMP signalling; (iii) pituitary adenylate cyclase activating peptide-38 (PACAP) and vasoactive intestinal peptide (VIP), peptides of interest for migraine pathophysiology that are coupled to an adenylate cyclase-cAMP signalling pathway; and (iv) nicardipine, a calcium channel blocker prescribed for the treatment of hypertension that may also be used in migraine. Finally, as migraine patients under prophylactic treatment could still use acute antimigraine medication, the vasoconstrictor responses to dihydroergotamine (DHE) in the absence and presence of erenumab were also analysed to discard a possible augmentation of the contractile responses due to the inhibition of the CGRP-mediated vasodilation.

Methods

Human isolated arteries collection

Middle meningeal arteries

Segments of HMMA (internal diameter 0.5–1.5 mm) were obtained from six patients (two male and four female, 49 ± 8 years old) who underwent neurosurgical procedures requiring a trepanation of the skull. The HMMA, attached to the dura mater, was collected in a sterile organ-protecting solution and immediately transported to the laboratory to be dissected and subsequently placed in a cold, oxygenated Krebs solution of the following composition (mM): NaCl 119, KCl 4.7, CaCl₂ 1.25, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₂ 25 and glucose 11.1; pH 7.4.

Coronary arteries

Coronary arteries were obtained from six “heart beating” organ donors (two males and four females; 52 ± 5 years old), who died of non-cardiac disorders. The hearts were provided by the Heart Valve Bank Beverwijk (at that time still located in Rotterdam) from Dutch post-mortem donors, after donor mediation by The Dutch Transplantation Foundation (Leiden, The Netherlands), following removal of the aortic and pulmonary valves for homograft valve transplantation. All donors gave permission for research. Immediately after circulatory arrest, the hearts were stored at 4℃ in a sterile organ protecting solution and were brought to the laboratory within 24 hours of death. After arrival, the right proximal (internal diameter 3–5 mm) and distal (internal diameter 0.5–1 mm) portions of the HCA were dissected and placed in a cold, carbogen oxygenated (95% O₂/5% CO₂) Krebs buffer solution of the following composition (mM): NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and glucose 8.3; pH 7.4. The studies on coronary arteries were approved by the Scientific Advisory Board of the Rotterdam Heart Valve Bank.

Internal mammary arteries

Segments of HIMA (internal diameter 2–3 mm) were obtained perioperatively from 10 male patients (72 ± 2 years old) undergoing coronary artery bypass surgery. After completion of the coronary bypass procedure, the remaining segment of internal mammary artery was immediately brought to the laboratory. Connective tissue was removed from the segment and the tissue was kept in cold Krebs solution (for composition see above), aerated with carbogen.

All vessels were used on the same day or stored overnight and used the following day for functional experiments. The Medical Ethics Committee of the Erasmus Medical Centre, Rotterdam, approved the study protocols with regard to mammary arteries and middle meningeal arteries.

Isometric tension measurements

Proximal HCAs were cut into segments of 2–4 mm length, excluding macroscopically visible atherosclerotic lesions. Segments were mounted on stainless steel hooks in 15-mL organ baths filled with oxygenated Krebs buffer solution at 37℃. After 30 min of stabilization, the vessel segments were stretched to a tension of about 15 mN, as described earlier by our group (13). Changes in tension were measured with an isometric force transducer (Harvard, South Natick, MA, USA) and recorded on a flatbed recorder (Servogor 124, Goerz, Neudorf, Austria).

The HMMA, distal HCA and HIMA were cut into circular 1–2 mm long segments and mounted in Mulvany myographs (Danish Myo Technology, Aarhus, Denmark) between two parallel small stainless-steel wires (40 µm ø). Baths were filled with oxygenated Krebs buffer (37℃) and their tension was normalised to 90% of l₁₀₀ for all segments (the diameter when transmural pressure equals 100 mm Hg) as previously reported (15). Data was recorded using a LabChart data acquisition system (AD Instruments Ltd, Oxford, UK).

Experimental protocols

A paired parallel set up (i.e., experiments with and without erenumab were performed in different segments obtained from the same artery) was used. Initially, all segments were exposed to 30 mM KCl to “prime” the tissue for stable contractions. After washout, the tissue was exposed to 100 mM KCl to determine the reference contractile response.

All vessel segments were pre-contracted with 30 mM KCl after being incubated with vehicle or a supratherapeutical concentration of erenumab (1 µM) (16) for 15 min. After 15 min of precontraction (i.e., after a total incubation time of 30 min for erenumab), a concentration response curve to human α-CGRP (0.1 nM–1 µM, half logarithmic steps) was performed.

Additionally, in HIMA, after segments were pre-contracted (30 mM KCl) and incubated with vehicle or erenumab (1 μM), concentration response curves to acetylcholine (0.1 nM–3 µM, half logarithmic steps), sodium nitroprusside (1 nM–10 µM, half logarithmic steps), PACAP (0.1 nM–1 µM, whole logarithmic steps), VIP (0.1 nM–1 µM, whole logarithmic steps), or nicardipine (1 nM–30 µM, whole logarithmic steps) were performed. Also, in vessels incubated for 30 min with vehicle or erenumab (1 μM), a concentration response curve to DHE (1 nM–100 µM, whole logarithmic steps) was performed.

Finally, at the end of each experiment; that is, after construction of a concentration response curve, and washing out, the functional integrity of the endothelium was verified by observing relaxation to bradykinin (1 µM, IMA) or substance P (10 nM, HMMA and HCA) after precontraction with the thromboxane A₂ analogue U46619 (10 nM) in every individual vessel segment.

Statistical analysis

Vasodilatory responses were expressed as percentage of the precontraction induced by 30 mM KCl. For contractile responses, the values were expressed as percentage of the contraction induced by 100 mM KCl. Curves covering the full sigmoidal range were analysed by means of a computerised curve fitting technique to obtain pEC₅₀ (negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and E_max (maximal response) values. If E_max was not reached, the contraction or relaxation obtained at the highest concentration of agonist was considered as E_max, except for CGRP in the presence of erenumab, where the respective control E_max (i.e. in the absence of erenumab) was used as E_max for fitting. The blocking potency of erenumab in each tissue was estimated by calculating EC₅₀ ratios and plotting a Schild plot (17) and constraining the slope to unity to obtain the apparent pK_b values. All data are presented as mean ± standard error of the mean (SEM). Significant differences in pEC₅₀ and E_max between control and erenumab groups were examined with a paired t-test. Differences between tissues were analysed by a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc analysis. p-values of 0.05 or less were assumed to denote significant changes.

Compounds used

Erenumab and vehicle were kindly provided by Amgen (Thousand Oaks, CA, U.S.A.). Acetylcholine chloride, sodium nitroprusside, nicardipine hydrochloride, dihydroergotamine mesylate and U46619 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human α-CGRP, PACAP and VIP were obtained from PolyPeptide (Strasbourg, France).

Results

Responses to 100 mM and 30 mM KCl resulted in a mean contraction of 19 ± 4 mN and 17 ± 3.1 mN, respectively.

Effect of erenumab on the vasodilatory responses to CGRP in human isolated middle meningeal arteries

In HMMA (Figure 1), the vasodilatory responses to CGRP were significantly shifted in the presence of 1 μM erenumab (control: pEC₅₀ 8.56 ± 0.16 vs. erenumab: pEC₅₀ 6.51 ± 0.19; n = 6 each; t(5) = 16.74, p < 0.0001). The apparent pK_b value was 8.05 ± 0.12. No significant changes were observed in the maximal responses to CGRP (control: E_max 64 ± 11% vs. erenumab: E_max 56 ± 10%; n = 6 each; t(5) = 2.11, p = 0.088). Verification of endothelial function resulted in a mean dilation of 83 ± 3% of the precontraction.
Figure 1.
Relaxant responses to CGRP in the middle meningeal artery in the presence of erenumab (1 μM) or the vehicle. Data are expressed as mean ± SEM, n = 6.

Effect of erenumab on the vasodilatory responses to CGRP in human isolated coronary arteries

Relaxant responses to CGRP in the proximal HCA (pEC₅₀ < 6.54; n = 4), were inhibited in the presence of 1 μM erenumab (pEC₅₀ < 5.24; n = 4), with no significant change in the response obtained at the highest concentration (control: E_max 36 ± 6% vs. erenumab: E_max 21 ± 3%; t(3) = 1.86, p = 0.15). Due to the limited effect of CGRP on the proximal portion of the HCA, no apparent pK_b value was calculated. In the distal coronary arteries, a significant shift was observed in the vasodilatory responses to CGRP (control: pEC₅₀ 9.04 ± 0.18 vs. erenumab: pEC₅₀ 6.81 ± 0.18; n = 6 each; t(5) = 13.46, p < 0.0001), with no change in the maximal relaxation (control: E_max 85 ± 5% vs. erenumab: E_max 83 ± 6%; n = 6 each; t(5) = 0.88, p = 0.42; Figure 2) and an apparent pK_b value of 8.22 ± 0.17. A significant difference was observed between the maximal relaxation to CGRP in proximal and distal HCA (E_max proximal: 36 ± 6% vs. E_max distal: 85 ± 5%; t(8) = 4.95, p = 0.001). Endothelial function analysis resulted in a mean dilation of 12 ± 3% of the precontraction in proximal HCA and of 89 ± 4% in distal HCA.
Figure 2.
Relaxant responses to CGRP in the proximal (left) and distal (right) coronary arteries in the presence of erenumab (1 μM) or the vehicle. Data are expressed as mean ± SEM, n = 4–6.

Effect of erenumab on the vasodilatory responses to CGRP in human isolated internal mammary artery

In HIMA, the vasodilatory responses to CGRP were also significantly inhibited by erenumab (control: pEC₅₀ 7.83 ± 0.34 vs. erenumab: pEC₅₀ 5.94 ± 0.37; n = 8 each; t(7) = 4.18, p = 0.004), with an apparent pK_b value of 7.85 ± 0.46, and no significant change in the maximal response was observed (control: E_max 32 ± 12% vs. erenumab: E_max 24 ± 10%; n = 8 each; t(7) = 1.44, p = 0.19; Figure 3). Verification of endothelium function resulted in a dilation of 6 ± 3% of the precontraction. Precontraction with 30 mM KCl was not modified by erenumab (control: 154 ± 47% of the contraction to 100 mM KCl vs. erenumab 141 ± 22% of the contraction to 100 mM KCl; t(7) = 0.47, p = 0.66; data not shown).
Figure 3.
Relaxant responses to CGRP in the human isolated internal mammary artery in the presence of erenumab (1 μM) or the vehicle. Data are expressed as mean ± SEM, n = 8.

Effect of erenumab on non-CGRP induced vasodilatory responses in human isolated mammary artery

The relaxant responses to the different vasodilators studied were not modified in the presence of erenumab (Table 1, Figure 4).
Figure 4.
Vasodilatory responses to acetylcholine, sodium nitroprusside, VIP, PACAP and nicardipine in the human isolated internal mammary artery in the presence of erenumab (1 μM) or the vehicle. Data are expressed as mean ± SEM, n = 7 each.

Table 1.
Vasodilatory responses to acetylcholine, sodium nitroprusside, VIP, PACAP and nicardipine in the HIMA in the absence and presence of erenumab; n = 7 each.

Agonist pEC₅₀
E_max (%)

Control Erenumab t(6) p-value Control Erenumab t(6) p-value

Acetylcholine 7.42 ± 0.96 6.22 ± 0.45 0.47 0.66 14 ± 4 11 ± 6 0.59 0.57

Sodium nitroprusside 6.31 ± 0.29 6.44 ± 0.44 0.03 0.98 50 ± 13 50 ± 14 0.04 0.97

VIP 6.83 ± 0.11 7.05 ± 0.45 1.19 0.29 25 ± 10 21 ± 3 0.44 0.69

PACAP 6.78 ± 0.01 7.00 ± 0.21 1.25 0.28 17 ± 7 14 ± 4 0.50 0.63

Nicardipine 5.77 ± 0.24 5.49 ± 0.06 1.34 0.23 72 ± 9 68 ± 11 0.74 0.49

E_max: maximal response; PACAP: pituitary adenylate cyclase activating polypeptide-38; pEC₅₀: negative log of the molar concentration of an agonist needed to reach half of its maximal effect; VIP: vasoactive intestinal peptide.

Effect of erenumab on the contractile responses to dihydroergotamine

Similar to above, contractile responses to DHE (control: pEC₅₀ 6.78 ± 0.40 vs. erenumab: pEC₅₀ 6.65 ± 0.40, t(6) = 0.41, p = 0.72; control: E_max 11 ± 6% vs. erenumab: E_max 17 ± 12%, t(6) = 0.66, p = 0.53; n = 7 each) were unaffected by the presence of erenumab (Figure 5).
Figure 5.
Contractile responses to DHE in the human isolated internal mammary artery in the presence of erenumab (1 μM) or the vehicle. Data are expressed as mean ± SEM, n = 7 each.

Comparison of the responses to CGRP in human middle meningeal, coronary and mammary arteries

The vasodilatory responses to CGRP were more potent in the distal portion of the HCA when compared to the HIMA (pEC₅₀ 9.04 ± 0.18 vs. pEC₅₀ 7.83 ± 0.34, respectively; F(2,17) = 5.471, p_apparent = 0.01). No significant differences were observed in the potency between HMMA and HIMA (pEC₅₀ 8.56 ± 0.16 vs. pEC₅₀ 7.83 ± 0.34, respectively; F(2,17) = 5.471, p_aparent = 0.15), nor between HMMA and the distal portion of the HCA (pEC₅₀ 8.56 ± 0.16 vs. pEC₅₀ 9.04 ± 0.18, respectively; F(2,17) = 5.471, p_aparent = 0.47).

When comparing the E_max, the vasodilatory responses to CGRP were more pronounced in the distal portion of the HCA, when compared to the HIMA (E_max distal HCA: 85 ± 5% vs. E_max HIMA: 32 ± 12%; F(3,20) = 6.302, p_aparent = 0.004). No significant differences were observed between the maximal responses to CGRP in HMMA and HIMA (E_max HMMA: 64 ± 11% vs. E_max HIMA: 32 ± 12%; F (3,20) = 6.302, p_aparent = 0.112) nor HMMA and distal HCA (E_max HMMA: 64 ± 11% vs. E_max distal HCA: 85 ± 5%; F(3,20) = 6.302, p_aparent = 0.47). Also, no significant differences were observed between the maximal vasodilatory response to CGRP in proximal HCA and HIMA (E_max proximal HCA: 36 ± 6% vs. E_max HIMA: 32 ± 12%; F(3,20) = 6.302, p_aparent = 0.991) or proximal HCA and HMMA (E_max proximal HCA: 36 ± 6% vs. E_max HMMA: 64 ± 11%; F(3,20) = 6.302, p_aparent = 0.311).

Finally, no significant difference was observed in the potency of erenumab to antagonize the responses to CGRP amongst the tissues studied (p = 0.73; pK_b HMMA: 8.05 ± 0.12 vs. pK_b HCA: 8.22 ± 0.17, F (2,17) = 0.3106, p_aparent = 0.94; pK_b HMMA: 8.05 ± 0.12 vs. pK_b HIMA: 7.85 ± 0.46, F (2,17) = 0.3106, p_aparent = 0.91; pK_b HCA: 8.22 ± 0.17 vs. pK_b HIMA: 7.85 ± 0.46, F (2,17) = 0.3106, p_aparent = 0.72).

Discussion

In this study, the inhibition of the CGRP-vasodilatory responses by erenumab was examined in human isolated arteries. We used a supratherapeutic concentration of erenumab (1 μM) (16) to allow a clear analysis of its effects on CGRP as well as other vasoactive substances of interest.

Firstly, we investigated the effect of erenumab on the vasorelaxant responses to CGRP in HMMA, with our results showing a significant shift of the concentration response curve to CGRP in the presence of erenumab. As antibodies are considered to have a BBB permeability of <0.1% (18) and erenumab has been shown to be effective for the prophylactic treatment of migraine (19,20), it is considered that the mechanisms of action of erenumab are peripheral, with one of them being possibly inhibition of the CGRP-mediated vasodilation of the dural arteries (1). While erenumab has no vasoconstrictive properties per se (21), its success as prophylactic treatment may well be (partly) by effectively preventing the vasodilatory responses to CGRP in the HMMA, associated with the onset of migraine attacks. In accordance with this, human provocation studies have shown dilation of the HMMA on the headache side at migraine onset, and headache relief after vasoconstriction of the HMMA by sumatriptan (1,22). Certainly, these studies have been performed during exogenously provoked migraine-like attacks and a magnetic resonance angiography study of the intracranial and extracranial arteries in patients with spontaneous migraine attacks failed to show extracranial arterial dilatation (23). However, as previously addressed by our group (24), in the latter study authors could not exclude dilatation of dural branches of the HMMA, as those small branches could not be analysed due to technical limitations.

Due to the theoretical cardiovascular concerns of blocking the actions of CGRP (6,25), especially since migraine patients have an increased cardiovascular risk (8,9), we further studied the effect of erenumab in proximal and distal HCA (Figure 2). Although it has been shown that erenumab does not contract the HCA (21), it is important to consider the risks of the blockade of the cardioprotective vasodilation by CGRP (7). In our study, and in accordance with previous work (13,26,27), the vasodilatory responses to CGRP in the distal portion of the HCA were significantly more pronounced than in the proximal portion of the HCA and HIMA. Moreover, in the presence of erenumab, a significant shift was observed in both portions of the HCA, which seemed to be more pronounced in the distal portion. This reinforces the importance of appropriate vascular safety studies in migraine patients, with especial emphasis on female patients, who are more prone to present ischemic events in the distal portion of the coronary arterial bed where the role of CGRP in cardioprotection seems to be more significant (7,12,14).

Furthermore, we analysed the effect of erenumab on isolated arteries from cardiovascularly compromised patients. For this, we obtained HIMA peri-operatively from coronary artery bypass surgery patients, most of them suffering from atherosclerotic disease that is usually more prominent in the proximal HCA and more common in men, as previously mentioned, thus all our experiments were performed in HIMAs obtained from male subjects. However, while the responses to CGRP are more pronounced in the distal coronary artery (where women are more prone to present ischemic events), when a patient undergoes coronary bypass surgery, a portion of HIMA is grafted and thus gets incorporated in the proximal coronary arterial bed, making it a relevant tissue to study the characteristics of the CGRP-mediated vasodilatory responses and the effects of erenumab. Based on the limited relaxation to bradykinin that was analysed in every individual HIMA segment, and in accordance with the small vasodilatory responses to acetylcholine (Figure 4), functional endothelial quality was limited in these coronary artery bypass grafts, probably associated with the endothelial dysfunction associated with cardiovascular disease (28). When analysing the responses to CGRP, a concentration-dependent vasodilation was observed, which was significantly antagonised in the presence of erenumab with no change in the maximal response. The E_max of CGRP in the HIMA was significantly lower when compared to the distal portion of the HCA and not significantly different when compared to the proximal HCA (Figures 2−3), suggesting a similar role for CGRP in HIMA and proximal HCA. The vasodilatory peptides VIP and PACAP, currently considered possible therapeutic targets for migraine (29), do not seem to play an important role in HIMA vasodilation as their maximal response was rather low. Most importantly, erenumab did not modify the responses to acetylcholine and sodium nitroprusside (nitric oxide-cGMP signalling), nor the vasorelaxant responses to PACAP and VIP (adenylate cyclase-cAMP signalling). Even though the cardiovascular safety concerns are theoretical, and trials have not reported cardiovascular events that were considered to be related to inhibition of the CGRP pathway (10), the vasodilatory responses to acetylcholine, VIP and PACAP in our study were limited. Therefore, further studies should address the vasodilatory pathways involved in ischemic conditions after long-term blockade of the CGRP pathway. Nonetheless, erenumab did not modify the vasodilatory responses to nicardipine, an antihypertensive given to cardiovascularly compromised patients (Figure 4) and did not augment the vasoconstrictor responses to 30 mM KCl or DHE, an acute-acting antimigraine drug that could be taken concomitantly with erenumab (Figure 5). Similar results have previously been reported in HCA with sumatriptan (21), which is of great importance for patients under ergot (or triptan) treatment.

Finally, as the efficacy and potency of the CGRP-dependent vasodilatory responses differ amongst arteries (13), CGRP receptor blockade by erenumab could also present differential responses depending on the vessel studied; however, erenumab had a similar potency (pK_b) across the distal HCA (8.22 ± 0.17), HMMA (8.05 ± 0.12) and HIMA (7.85 ± 0.46). When comparing these results to the gepants, similar pK_b values were obtained previously by our group for telcagepant in distal HCA and HMMA (8.43 ± 0.24 and 8.03 ± 0.16, respectively) (13,26). Interestingly, olcegepant was more potent in HMMA (pK_b: 10.59 ± 0.54) than in the distal portion of the HCA, with pK_b values ranging from 8.41 ± 0.26 to 9.29 ± 0.34, depending on the concentration studied (30,31). While we do not know the reason for this discrepancy, it may well be caused by an underlying heterogeneity in CGRP receptors that could be targeted by olcegepant (30,32,33), whereas erenumab only acts at the canonical CGRP receptor (34); thus, receptors other than the CGRP receptor to which CGRP may still bind (e.g. amylin 1 receptor), may compensate for blockade of the CGRP receptor. Conversely, in the case of the antibodies directed against CGRP, peptides other than CGRP that may also bind to the CGRP receptor may exert compensatory effects. Further studies should address whether there are clinically relevant differences (i.e., in efficacy or cardiovascular safety), between the prophylactic treatment with the antibodies directed against CGRP and against the CGRP receptor.

Our results, taken together, show a differential response profile to CGRP in human isolated arteries, being more potent in the distal portion of the HCA when compared to the proximal portion and the HMMA and HIMA. Moreover, erenumab significantly inhibits CGRP-mediated vasodilation in vitro and does not interact with responses to other vasodilatory or contractile agents of interest.

Conclusion

In conclusion, erenumab is a potent inhibitor of the vasodilatory responses to CGRP in HMMA, HCA and HIMA. While the prominent role of CGRP in the distal coronary artery warrants further safety studies, particularly in women, it is important to point out that erenumab does not interact with vasodilatory responses to other vasodilators, or with the contractions to DHE.

Key findings

Erenumab inhibits the CGRP vasodilatory responses in HMMA, HCA and HIMA.

Erenumab does not interact with the vasodilatory responses to other vasodilators.

Erenumab does not augment the vasoconstrictive responses to dihydroergotamine.

Agonist	pEC₅₀	E_max (%)
Acetylcholine	7.42 ± 0.96	6.22 ± 0.45	0.47	0.66	14 ± 4	11 ± 6	0.59	0.57
Sodium nitroprusside	6.31 ± 0.29	6.44 ± 0.44	0.03	0.98	50 ± 13	50 ± 14	0.04	0.97
VIP	6.83 ± 0.11	7.05 ± 0.45	1.19	0.29	25 ± 10	21 ± 3	0.44	0.69
PACAP	6.78 ± 0.01	7.00 ± 0.21	1.25	0.28	17 ± 7	14 ± 4	0.50	0.63
Nicardipine	5.77 ± 0.24	5.49 ± 0.06	1.34	0.23	72 ± 9	68 ± 11	0.74	0.49

Footnotes

Availability of data

The dataset supporting the conclusion of this article is available upon reasonable request to the corresponding author.

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: ERB received travel support from Novartis. AMvdB received research grants and/or consultation fees from Amgen/Novartis, Lilly/CoLucid, Teva and ATI. CX is an employee of Amgen Inc. JS is an employee of Novartis Pharma AG. All other authors declare no conflicts of interest.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: AMvdB was supported by the Netherlands Organization for Scientific Research (Vidi grant 917.113.349), whereas ALR and ERB were supported by Consejo Nacional de Ciencia y Tecnología (CONACyT; fellowships No. 410778 to ALR and No. 409865 to ERB; Mexico City). KAH was supported by a fellowship from the International Headache Society. This study was financially supported by a research grant from Amgen/Novartis.

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Agonist	pEC₅₀				E_max (%)
Control	Erenumab	t(6)	p-value	Control	Erenumab	t(6)	p-value
Acetylcholine	7.42 ± 0.96	6.22 ± 0.45	0.47	0.66	14 ± 4	11 ± 6	0.59	0.57
Sodium nitroprusside	6.31 ± 0.29	6.44 ± 0.44	0.03	0.98	50 ± 13	50 ± 14	0.04	0.97
VIP	6.83 ± 0.11	7.05 ± 0.45	1.19	0.29	25 ± 10	21 ± 3	0.44	0.69
PACAP	6.78 ± 0.01	7.00 ± 0.21	1.25	0.28	17 ± 7	14 ± 4	0.50	0.63
Nicardipine	5.77 ± 0.24	5.49 ± 0.06	1.34	0.23	72 ± 9	68 ± 11	0.74	0.49