Sildenafil and calcitonin gene-related peptide dilate intradural arteries: A 3T MR angiography study in healthy volunteers

Abstract

Background

Sildenafil and calcitonin gene-related peptide are vasoactive substances that induce migraine attacks in patients. The intradural arteries are thought to be involved, but these have never been examined in vivo. Sildenafil is the only migraine-inducing compound for which cephalic, extracranial artery dilation is not reported. Here, we investigate the effects of sildenafil and calcitonin gene-related peptide on the extracranial and intradural parts of the middle meningeal artery.

Methods

In a double-blind, randomized, three-way crossover, placebo-controlled head-to-head comparison study, MR-angiography was recorded in healthy volunteers at baseline and twice after study drug (sildenafil/ calcitonin gene-related peptide/saline) administration. Circumferences of extracranial and intradural middle meningeal artery segments were measured using semi-automated analysis software. The area under the curve for circumference change was compared using paired t-tests between study days.

Results

Twelve healthy volunteers completed the study. The area under the curve_{Baseline-120min} was significantly larger on both the sildenafil and the calcitonin gene-related peptide day in the intradural middle meningeal artery (calcitonin gene-related peptide, p = 0.013; sildenafil, p = 0.027) and the extracranial middle meningeal artery (calcitonin gene-related peptide, p = 0.0003; sildenafil, p = 0.021), compared to placebo.

Peak intradural middle meningeal artery dilation was 9.9% (95% CI [2.9–16.9]) after sildenafil (T_30min) and 12.5% (95% CI [8.1–16.8]) after calcitonin gene-related peptide (T_30min). Peak dilation of the extracranial middle meningeal artery after calcitonin gene-related peptide (T_30min) was 15.7% (95% CI [11.2–20.1]) and 18.9% (95% CI [12.8–24.9]) after sildenafil (T_120min).

Conclusion

An important novel finding is that both sildenafil and calcitonin gene-related peptide dilate intradural arteries, supporting the notion that all known pharmacological migraine triggers dilate cephalic vessels. We suggest that intradural artery dilation is associated with headache induced by calcitonin gene-related peptide and sildenafil.

Keywords

Neuroimaging middle meningeal artery human migraine models

Introduction

Sildenafil and calcitonin gene-related peptide (CGRP) are vasoactive substances known to provoke headache in healthy volunteers (1,2) and migraine attacks in migraine patients (3,4). The vasoactive actions of sildenafil and CGRP are mediated predominately by activation of intracellular second messengers; cyclic guanosine monophosphate (cGMP) (5) and cyclic adenosine monophosphate (cAMP) (6), respectively. To what extent vasoactive properties of both substances contribute to headache or migraine development remains unknown. The dura mater is one of the important intracranial pain-sensitive structures that have been extensively studied in relation to headache in humans (7). Trigeminal nociceptive afferents are densely localized in the perivascular space in the dura mater (8), which is supplied mainly by the middle meningeal artery (MMA). The intracranial part of the MMA is embedded in layers of dura mater and expansion is partly limited by the confines of the cranial vault. Release of chemicals including neuropeptides in the perivascular space may dilate intradural arteries and activate dural trigeminovascular nociceptors (9). Ultrasound studies reported that CGRP (10), but not sildenafil (3), dilated extracerebral arteries and questioned the role of vasoactive properties in headache development. However, to our knowledge, no studies in humans have evaluated the effect of sildenafil or CGRP on the intradural arteries in vivo. Ultrasonographic doppler studies and magnetic resonance angiography (MRA) studies have found no dilation of the middle cerebral artery (MCA) after administration of sildenafil or CGRP respectively, in healthy volunteers (1,2) which suggests that the dural vasculature might be of greater importance in developing headache.

In the present study, we tested the hypothesis that both sildenafil and CGRP dilate the intradural MMA and induce headache in healthy volunteers. Furthermore, we hypothesized that neither intervention would dilate the MCA. We used advanced high-resolution MRA and investigated the effect of sildenafil and CGRP on intradural MMA as well as MCA in a randomized, double-blind, double-dummy, placebo-controlled, three-way crossover study.

Methods

Participants

We recruited healthy volunteers through a Danish recruitment website (www.forsoegsperson.dk). Inclusion criteria: Healthy men and women between ages 18 and 50; weight from 50 to 100 kg. Exclusion criteria were: A history of somatic and/or psychiatric disease; any primary headache disorder, other than infrequent episodes of tension type headache (less than 2 days/month); first-degree relatives with migraine; pregnant or breastfeeding women; daily medication intake, other than oral contraceptives; absence of safe contraceptive methods; contraindications for MRI scans (i.e. ferromagnetic implants, recent surgical procedures, claustrophobia, etc.); any condition perceived by the investigator to be incompatible with participation in the study. All participants gave informed consent in the essence of the Helsinki Declaration, and the study was approved by the Ethical Committee of the Capital Region of Denmark (H-15019063). The study was registered at ClinicalTrials.gov (NCT03143465). This study is part of a larger study, consisting of other imaging parameters, which will be published separately elsewhere.

Design

We randomly allocated participants to oral sildenafil 100 mg (STADA, Bad Vilbel, Germany), intravenous infusion of CGRP 1.5 µg/min for 20 min (PolyPeptide, Strasbourg, France), or placebo (isotonic saline infusion/calcium capsules) on three separate study days. On each experimental day, participants were scanned at baseline (T_Baseline) before receiving both an infusion (CGRP/placebo) and two capsules (2 × 50 mg sildenafil/2 × placebo) to allow for a drug combination of either CGRP/placebo, sildenafil/placebo or placebo/placebo at T₀. Participants were subsequently scanned 30 min after administration (T_30min) and two hours after administration (T_120min).

A study flow chart is shown in Figure 1.

Figure 1.

Study day flow chart. MRA was timed at T_Baseline, T₃₀ and T₁₂₀.

Experimental procedures

Participants reported headache free to the clinic on each study day. Abstinence from coffee, tea, cocoa, medication (other than contraceptives) and tobacco 12 h before the baseline scan and total fasting 4 h before the baseline scan was required. All subjects were scanned within the same hour of the day on each of the three study days. Upon arrival, participants were placed in supine position and a peripheral venous catheter (18G Vasofix® Safety, B. Braun, Melsungen, Germany) was inserted in a cubital vein. Participants were monitored every 10 min with end-tidal carbon dioxide, blood oxygen saturation, blood pressure and heart rate (Veris® monitor, Medrad, Warrendale, PA, USA). MRA recordings were chosen to identify the MMA and the MCA and subsequently measure the circumference of each vessel segment at all time points. Subjects were instructed to lie still and stay awake while the recordings were obtained.

Headache characteristics (intensity, throbbing/constant pain, aggravation by activity, location, photo/phonophobia, nausea/vomiting) were recorded via a purpose-developed questionnaire between scans throughout the study day. Headache intensity was recorded on a numerical rating scale (NRS 0–10) rating pain from non-existing (NRS 0) to maximum imaginable (NRS 10).

Data acquisition

All MRI scans were performed on the same 3.0 Tesla Philips Achieva Scanner (Philips Medical Systems, Best, Netherlands) using a 32-channel phased-array head coil. A three-dimensional time-of-flight MRA was used for vessel imaging. First, a scout MRA was performed with field of view (FOV) 200 × 200 ×150 mm³, acquired matrix size 200 × 134, acquired voxel resolution 1.00 × 1.50 × 2.00 mm³, reconstructed resolution 0.39 × 0.39 × 1.00 mm³, repetition time (TR) 23 milliseconds, echo time (TE) 3.5 milliseconds, flip angle 18°, SENSE p reduction 2, 2 chunks, duration 2 min 46 sec. The subsequent MMA and MCA scans were planned on the scout MRA. MMA scan used FOV 200 × 200 × 36.75 mm³, acquired matrix size 800 × 570, acquired voxel resolution 0.25 × 0.35 × 0.70 mm³, reconstructed voxel resolution 0.20 × 0.20 × 0.35 mm³, TR 23 milliseconds, TE 3.5 milliseconds, flip angle 18°, SENSE p reduction 2.5, 3 chunks, duration 13 min 06 sec. MCA scan used FOV 200 × 200 × 12.25 mm³, acquired matrix size 784 × 571, acquired voxel resolution 0.26 × 0.35 ×0.70 mm³, reconstructed voxel resolution 0.20 × 0.20 ×0.35 mm³, TR 23 milliseconds, TE 3.5 milliseconds, flip angle 18°, SENSE p reduction 2.5, 1 chunk, duration 2 min 59 sec.

Image analysis

After acquisition, data was transferred to a separate workstation in DICOM format and analyzed by LKEB-MRA software (11,12). The software operates by allowing the user to input start- and endpoint in a desired vessel, after which the software automatically detects the center line and vessel contours perpendicular to this line. A circumference measurement is calculated for every 0.2 mm along the vessel segment. The segment was reviewed by the user and for each vessel a subsegment was chosen to ensure the most reliable measurement. If the chosen segment contained noisy or otherwise immeasurable slices, these slices were excluded from the segment. If the entire segment was immeasurable due to noise or artifacts, the segment was discarded altogether. The same subsegment was chosen within each subject between scans and days. For MMA, three segments of interest were chosen: Extracranial MMA between the main trunk of the maxillary artery and the base of the skull; cranial floor MMA from the point where the vessel enters the cranial cavity after traversing the foramen spinosum; and the intradural convexity MMA, the innermost part of the vessel measurable within the chosen FOV. To ensure that the same subsegments were chosen across repeated scans, the FOV was placed and measurements were performed relative to the major anatomical hallmarks. Terminal branches of the external carotid arteries were used to guide FOV placement. The main trunk, as well as the curvature from the intraforaminous to the intracranial part, of MMA were used to place contour analysis start- and endpoints. For MCA, we chose the first part of the M1 segment (Figure 2).

Figure 2.

MR angiography depicting the four arterial segments in red.

Statistics

All absolute values are presented as mean with 95% confidence interval (CI).

Calculation of sample size was based on previous findings of ∼10% dilation of MMA after CGRP (2). We chose to analyze at 5% significance level with 90% power and calculated a required sample size of 8 participants. To increase confidence in our findings we included 12 participants in total.

The primary endpoints of the study were a difference in the area under the curve (AUC) for the arterial (MCA and three MMA segments) circumference changes from baseline through 30 min to 120 min scans on the active (i.e. CGRP and sildenafil) days compared with placebo. Area under the curve was calculated using the trapezium rule (13). The secondary endpoints were mean difference in arterial circumference between baseline and each of the subsequent scans on each day, as well as headache incidence on placebo and both active days. Changes in heart rate and mean arterial blood pressure were analyzed using differences in AUC between days.

We tested for differences in mean arterial circumference and vital signs using Student’s paired samples t test, which was computed in IBM SPSS Statistics (Version 22) for all analyses. Level of significance was accepted at 0.05 and no corrections were made for multiple comparisons.

Results

Twelve volunteers completed all three study days (six females, six males), mean age 23 years (range 19–29 years). Median time between study days 1 and 3 was 23.5 days (range 7–40 days) (Figure 3). Seven participants reported headache on the sildenafil day, eight on the CGRP day and two on the placebo day (headache incidence is shown in Figure 4). We found no changes in AUC_{Baseline-120min} of mean arterial pressure or heart rate between sildenafil or CGRP and placebo (p > 0.05) (Figure 5).

Figure 3.

Recruitment flow chart. Number of vessel pairs depicted is number eligible for AUC analysis, where vessel segments were measurable at all three scans on both active day and placebo.

Figure 4.

Proportion of subjects reporting headache at given time points. Missing values for sildenafil and placebo headache induction at 180 min represent subjects finishing their scan days without reporting at the last time point before discharge. Of the three missing values after sildenafil, only one had reported headache up until 180 min. Of the missing values on placebo, none had reported headache before 180 min.

Figure 5.

Mean arterial pressure (MAP) and heart rate (HR) depicted as means over time from drug administration.

Effect of sildenafil on intradural and extracranial MMA and MCA

AUC_{Baseline-120min} of sildenafil dilation in intradural MMA was significantly larger than placebo (p = 0.027). AUC_{Baseline-120min} of dilation in cranial floor MMA (p = 0.01) and extracranial MMA (p = 0.021) was significantly larger after sildenafil than after placebo. We did not detect dilation of MCA by sildenafil compared to placebo (AUC_{Baseline-120 min}, p = 0.627).

Exploratory analyses showed that the cranial floor MMA was not dilated at T_30min (p = 0.189) compared to baseline, but only at T_120min by 0.46 mm (95% CI [0.22–0.71], p = 0.001). Absolute and relative circumference changes are shown in Table 1 and Figure 6.

Table 1.

Overview of secondary end points with absolute and relative differences in circumference between baseline and 30 and 120 mins. Difference in mm in each of the four arterial segments; extracranial MMA, cranial floor MMA, dural MMA and middle cerebral artery (MCA).

			Paired differences				Mean of % difference from baseline
		Number of pairs (n)	Mean difference from baseline (mm)	95% CI			Mean of % difference from baseline
Lower	Upper	p
Extracranial MMA	CGRP – T30	21	0.78*	0.57	0.99	<10⁻⁴	15.7%
	CGRP – T120	21	0.63*	0.40	0.85	<10⁻⁴	12.1%
	Sildenafil – T30	19	0.45	0.19	0.71	.002	9.5%
	Sildenafil – T120	21	0.94*	0.63	1.24	<10⁻⁴	18.9%
	Placebo – T30	21	0.18	0.00	0.36	.047	4.3%
	Placebo – 120	21	0.36	0.19	0.54	<10⁻⁴	7.6%
Cranial floor MMA	CGRP – T30	21	0.47*	0.27	0.67	<10⁻⁴	10.0%
	CGRP – T120	21	0.32*	0.08	0.56	.013	6.7%
	Sildenafil – T30	21	0.16	−0.09	0.41	.189	4.2%
	Sildenafil – T120	21	0.46*	0.22	0.71	.001	10.1%
	Placebo – T30	21	−0.07	−0.25	0.11	.445	−1.0%
	Placebo – 120	21	0.00	−0.13	0.12	.955	0.4%
Intradural MMA	CGRP – T30	21	0.48*	0.32	0.65	<10⁻⁴	12.5%
	CGRP – T120	21	0.33	0.16	0.50	.001	8.1%
	Sildenafil – T30	21	0.40*	0.10	0.71	.013	9.9%
	Sildenafil – T120	21	0.36	0.11	0.62	.008	9.4%
	Placebo – T30	21	−0.08	−0.40	0.24	.613	−0.1%
	Placebo – 120	21	0.03	−0.28	0.35	.820	2.7%
MCA	CGRP – T30	24	0.09	−0.14	0.31	.429	1.1%
	CGRP – T120	24	0.37	0.12	0.62	.006	4.0%
	Sildenafil – T30	24	0.06	−0.16	0.28	.587	0.9%
	Sildenafil – T120	24	−0.10	−0.47	0.27	.595	−0.6%
	Placebo – T30	24	0.04	−0.10	0.18	.527	0.5%
	Placebo – 120	24	0.14	−0.04	0.31	.117	1.5%

Denotes changes significantly different from placebo response (p < 0.05).

Figure 6.

Relative change in arterial circumference from baseline across time in four arterial segments (mean ± SE).

Effect of CGRP on intradural and extracranial MMA and MCA

We found that AUC_{Baseline-120min} for dilation of intradural MMA was significantly larger for CGRP than placebo (p = 0.013). In addition, AUC_{baseline-120min} for dilation of cranial floor MMA (p = 0.003) and for extracranial MMA (p = 0.0003) was significantly larger after CGRP compared to placebo. We did not detect changes in MCA after CGRP compared to placebo (AUC_{Baseline-120 min}, p = 0.353).

CGRP increased the circumference of intradural MMA from baseline to T_30min by 0.48 mm (95% CI [0.32–0.65]) and from baseline to T_120min by 0.33 mm (95% CI [0.16–0.50]). Exploratory analyses revealed that circumference increase in intradural MMA from baseline to T_30min was significantly larger than placebo (p = 0.005), but difference between baseline and T_120min was not (p = 0.144). Absolute and relative changes for all time points are shown in Table 1 and Figure 6.

Discussion

The major findings of the present study were that both sildenafil and CGRP dilated intradural and extradural MMA, while we found no dilation of the MCA. These data suggest that sildenafil- and CGRP-induced headache may be associated with dilation of intradural arteries. We suggest that dilation of the intradural arteries may reflect activation of perivascular afferents.

Sildenafil is a highly selective inhibitor of the cGMP hydrolyzing enzyme phosphodiesterase 5 (PDE5) and causes accumulation of cGMP in vascular smooth muscle cells (VSMC), leading to relaxation (Figure 7) (1). In vitro myography studies in precontracted human cerebral arteries showed responses to sildenafil (maximum dilation 25.1 ± 8.1%) and CGRP (maximum dilation 88 ± 3%), when the drugs were administered abluminally (14,15). The difference in MCA dilation between abluminal drug administration (14) and our findings suggest that, in the present study, CGRP and sildenafil either do not reach the target VSMC in MCA or that counter regulatory mechanisms to maintain the VSMC tone in MCA could be in effect in the in vivo setting. Sildenafil is lipophilic, and should thus readily cross over the endothelial membranes to VSMC in MCA, but it could be that alternate isoforms of PDE5 or other PDEs, less susceptible to sildenafil, are more important in regulating cGMP and vascular tone in MCA (3).

Figure 7.

Vascular actions of sildenafil and CGRP. Sildenafil inhibits intracellular phosphodiesterase 5 (PDE5) and prevents the breakdown of cyclic guanosine monophopshate (cGMP), which lowers intracellular calcium concentration [Ca²⁺] and opens potassium (K⁺) channels, causing dilation. Luminal CGRP binds to endothelial CGRP receptors and activates adenylate cyclase, causing a rise in cyclic adenosine monophosphate (cAMP), which promotes a transfer of nitric oxide to the vascular smooth muscle cells (VSMC). Here, nitric oxide activates guanylate cyclase, raising [cGMP], which leads to dilation. Abluminal CGRP binds to receptors on VSMC and activates adenylate cyclase causing a rise in cAMP leading to dilation.

Intravenously administered CGRP binds to the endothelial CGRP receptor complex and activates adenylate cyclase, causing accumulation of cAMP (6). This leads to production and release of nitric oxide from endothelial cells to VSMC. The following activation of guanylate cyclase and accumulation of cGMP in VSMC lowers intracellular Ca²⁺ and opens K⁺ channels, causing relaxation (6). CGRP can also act directly on the VSMC, where binding to the CGRP receptor complex activates adenylate cyclase. The subsequent rise in cAMP activates protein kinase A, which opens potassium channels, raises intracellular Ca²⁺ and leads to relaxation of VSMC (Figure 7) (6). A recent animal study suggests that endothelial CGRP receptors are not present in MCA since abluminal, but not luminal, application of CGRP induced dilation of rat MCA, which is in accordance with our findings in humans (16).

Previous headache provocation studies revealed differences in cephalic artery responses between sildenafil and CGRP (1 –3,17). While sildenafil induced headache (1,3) without dilation of cephalic arteries (MCA and superficial temporal artery), CGRP-induced headache was associated with dilation of extracranial MMA (2,17). In rats, both sildenafil (intravenous infusion) and CGRP (intravenous bolus) dilated the dural part of MMA in vivo (18 –23), and CGRP increased the diameter of dural MMA both from resting tone and after preconstriction with either potassium or prostaglandin F2alfa in vitro (18,19,24). Moreover, in vitro studies in rat and human arteries reported that both CGRP and sildenafil relaxed preconstricted MMA (24 –26). In the present study we found that sildenafil dilated the intradural MMA by 8.7% and CGRP by 8.2% 120 min after drug administration.

We observed an increase in extracranial MMA circumference by 7.5% after placebo, which may reflect body temperature increase and movement artifacts during the long scan sessions. Dilation after sildenafil and CGRP was, however, still significantly larger than after placebo (Table 1). Particularly, dilation of the extracranial MMA after placebo has been reported previously (2,27).

Six in 12 and five in 12 of the participants reported headache after sildenafil and CGRP, respectively, at 120 min. Correlation analyses between arterial circumference change and headache require larger sample sizes and our study was not powered for such calculations. Moreover, a lack of direct correlation between immediate headache and changes in arterial diameter (superficial temporal artery) or blood flow velocity (MCA) after infusion of pharmacological substances has been reported (28). Arterial circumference changes may reflect activation of perivascular dural afferents, which are likely to generate head pain.

Differences in timing of dilation response (Figure 6) can, in part, be attributed to the different administration forms of sildenafil (oral) and CGRP (intravenous). Peak plasma concentration of 100 mg oral sildenafil is reached after ∼1 h (29), whereas CGRP is infused intravenously and has a plasma half-life of ∼10 min (6). In rats, intravenous administration of sildenafil caused peak dural artery dilation after 1–2 min (23).

Variability in dilation patterns along the MMA indicates that dilation potential differs along the course of the artery. This could be due to anatomical confines being less adaptive to dilation in the intradural segment than in the immediate intracranial or extracranial segments of the artery. Alternatively, the variability might stem from methodological limitations, as the intradural convexity MMA is small (∼4 mm in circumference) and has a more varying course than any other arterial segment examined, which can affect acquired image quality.

Strengths and limitations

Strengths in the present study constitute the placebo-controlled, three-way, double blind, crossover design with direct measurement of the cranial arteries. As we did not study migraine patients, data cannot readily be extrapolated to this group. A similar study in migraine patients is needed to better understand pathophysiological mechanisms in the sildenafil and CGRP models of migraine.

We were limited by the poor temporal resolution of MRA compared to ultrasonography or in vitro myography. However, the high spatial resolution enabled us to measure the intradural MMA directly in vivo in humans, which can also be applied to migraine patients for a better understanding of dural vascular changes in migraine. Our sample size was based on detecting dilation of the MMA, which means that we are not able to conclude whether there is no dilation of MCA or we are simply underpowered for detecting these changes. However, our findings regarding MCA are consistent with previous studies (1,2).

It is challenging to detect small changes in the intradural arteries, as minimal movements by the participants can cause artifact in terms of, for instance, partial volume. A post hoc power analysis revealed that that change in extracranial MMA from baseline to 120 min was detected after sildenafil and CGRP with 99% and 98% power respectively. It has been discussed whether dilation relating to the extracranial MMA is a transferable reflection of vascular changes occurring in the intradural segment. While we found similar patterns in the extracranial and intracranial dilation, suggesting that the extracranial MMA could be a marker of dural MMA changes, further studies are needed to verify this.

Conclusion

Sildenafil and CGRP dilated intradural and extradural segments of MMA and induced immediate headache in healthy volunteers. The dura mater is densely innervated by trigeminal nociceptors, and intradural MMA dilation might be associated with their activation. Future examinations in migraine patients may delineate whether intradural MMA dilation is directly related to ipsilateral head pain.

Clinical implications

Sildenafil- and CGRP-induced headache is accompanied by dilation of intradural middle meningeal artery.

Intradural artery dilation may be a marker of perivascular trigeminal activation in pharmacological models of migraine.

Footnotes

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: MA reports personal fees from Alder BioPharmaceuticals, Allergan, Amgen, Eli Lilly, Novartis and Teva. MA acted as principal investigator in the following clinical trials: Alder ALD403-CLIN-011 (Phase 3b), Amgen 20120178 (Phase 2), 20120295 (Phase 2), 20130255 (OLE), 20120297 (Phase 3), 20150308 (Phase 2a), Electrocore GM-11 gamma-Core-R, Novartis CAMG334a2301 (Phase 3b), Teva TV48125-CNS-30068 (Phase 3). MA has no ownership interest and does not own stocks of any pharmaceutical company. MA serves as associated editor of Cephalalgia and co-editor of the Journal of Headache and Pain. MA is President-elect of the International Headache Society and General Secretary of the European Headache Federation. The remaining authors report no conflicts of interest.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Lundbeck Foundation (R155-2014-171 and R249-2017-1608). Funding sources played no role in study design, data collection, analysis, interpretation, manuscript preparation, or submission.

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