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PACAP38-induced migraine attacks are independent of CGRP signaling: a randomized controlled trial

The Journal of Headache and Pain volume 26, Article number: 79 (2025) Cite this article

Abstract

Background

Calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) are key pathogenic drivers of migraine. While CGRP has become the target of several mechanism-based therapies, less is known about PACAP38 signaling in migraine pathogenesis. Previous studies suggest that PACAP38 can modulate CGRP release, but it might also induce migraine attacks via CGRP-independent mechanisms. Whether PACAP38 signaling is independent of and parallel to CGRP signaling has implications for future therapeutic strategies. Here, we aimed to ascertain whether PACAP-38 can mediate migraine attacks independently of CGRP signaling by assessing the ability of eptinezumab to prevent PACAP38-induced migraine attacks.

Methods

In a double-blind, placebo-controlled, parallel-group study, we randomly allocated adults with migraine without aura to receive either an intravenous infusion of 300-mg eptinezumab or matching placebo (isotonic saline) over 30 min. Two hours post-infusion, all participants were administered PACAP38 intravenously at 10 pmol/kg/min for 20 min. The primary endpoint was the incidence of migraine attacks during the 24-hour observational period post-infusion of eptinezumab or placebo. Key secondary endpoints included between-group differences in incidence of headache, and area under the curve (AUC) for headache intensity scores, diameter of the superficial temporal artery (STA) and facial skin blood flow.

Results

A total of 38 participants were enrolled and completed the study. No difference was observed in the incidence of PACAP38-induced migraine attacks between the eptinezumab (10 [53%] of 19) and placebo (12 [63%] of 19) groups (Fisher’s exact test: P = 0.74). Headache of any intensity was reported by 15 (79%) participants in the eptinezumab group, compared with 16 (84%) participants in the placebo group (Fisher’s exact test: P > 0.99). The AUC for headache intensity scores did not differ between the two groups during the first 12 h post-infusion of PACAP38 (Mann-Whitney U-test: P = 0.96). No differences were observed in AUC between the eptinezumab and placebo groups with respect to changes in STA diameter and facial skin blood flow (P > 0.05). No serious adverse events occurred.

Conclusions

Our results suggest that PACAP38 may mediate its signaling independently of CGRP in migraine pathogenesis. Therapies targeting PACAP signaling are thus a promising new avenue for treating migraine.

Trial registration

ClinicalTrials.gov, NCT05635604. Registered on November 15 2022.

Graphical abstract

Peer Review reports

Introduction

Migraine is a prevalent and disabling neurological disorder characterized by recurrent headache attacks, often accompanied by photophobia, phonophobia, nausea, and vomiting [1]. Despite immense progress in understanding its pathogenesis [2], the precise molecular and cellular mechanisms underlying migraine remain incompletely elucidated [3]. Two neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP), have garnered considerable attention for their roles in migraine mechanisms [4]. CGRP– a well-established molecular migraine trigger– has been the target of several therapeutic interventions, including monoclonal antibodies (mAbs) such as eptinezumab [5,6,7]. However, the role of PACAP in migraine pathogenesis is less well understood.

PACAP, particularly its predominant isoform PACAP-38, has been linked to migraine pathogenesis through various mechanisms [8]. Experimental studies have shown that intravenous (IV) infusion of PACAP-38 can induce migraine attacks in adult persons with migraine and mild, transient headache in healthy individuals [9,10,11]. Furthermore, rodent data suggest that PACAP-38 can modulate CGRP release from trigeminal neurons [12], although recent findings suggest that PACAP-38 can mediate migraine attacks via mechanisms distinct from CGRP signaling [13, 14]. This raises the possibility that PACAP signaling serves as an independent pathway in migraine pathogenesis, offering a novel target for therapeutic interventions [8]. Notably, a recent phase II trial provided proof of concept, demonstrating that a mAb directed against the PACAP ligand was effective for migraine prevention [15].

To further dissect the role of PACAP signaling in migraine, we conducted a double-blind, placebo-controlled, parallel-group study. Adults with migraine were randomly assigned to receive either an IV infusion of eptinezumab or placebo, followed by PACAP-38 administration. Our objective was to determine whether eptinezumab could prevent migraine attacks induced by PACAP-38.

Methods

The study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki, with later revisions, at a single center in Denmark (The Danish Headache Center). The study protocol was reviewed and approved by the Regional Health Research Ethics Committee of the Capital Region of Denmark (identifier: H-22038923). All participants provided written informed consent before enrollment, ensuring that they were fully informed about the study procedures and potential risks. The study was registered with ClinicalTrials.gov (identifier: NCT05635604).

Design and participants

The study was designed as a double-blind, placebo-controlled, parallel-group trial (Fig. 1). The participants were randomly assigned to receive either a single IV infusion of 300-mg eptinezumab or placebo (isotonic saline). Given the high 300 mg dose of IV eptinezumab, its rapid time to peak concentration (30–60 min) [16], and data showing that 100 mg eptinezumab provides pain relief within 1 h post-dose [17], IV PACAP-38 was administered to all participants at 2 h after infusion of eptinezumab or placebo. These factors supported the assumption of effective CGRP pathway blockade and justified the selected timing.

Fig. 1
figure 1

Timeline and study design. This double-blind, randomized, parallel-group study included 38 adult participants with migraine without aura. The participants were randomly assigned to receive a single intravenous infusion of either 300-mg eptinezumab or placebo (isotonic saline). During the in-hospital phase, measurements included headache intensity scores, associated symptoms, mean arterial pressure (MABP), heart rate (HR), facial skin blood flow, and the diameter of the frontal branch of the superficial temporal artery.

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Eligible participants were adults aged 18 to 60 years with a diagnosis of migraine without aura according to the International Classification of Headache Disorders, 3rd edition (ICHD-3) [18]. Females of childbearing potential were required to report use of contraceptives. Adults with a personal history of any other headache disorder, except for episodic tension-type headache, were excluded. Furthermore, we excluded adults who reported current use of preventive migraine medication(s). The complete list of inclusion and exclusion criteria is available at ClinicalTrials.gov (NCT05635604).

Randomization and blinding

Simple randomization was performed at a 1:1 ratio to assign participants to either the eptinezumab or placebo group. This method involved using a computer-generated random sequence of numbers to ensure a balanced allocation to either group. The randomization code was securely stored in a lightproof envelope inside a locked cabinet at the hospital. Individual sealed envelopes for each participant were also prepared and kept for emergency use.

Eptinezumab (Vyepti®) was supplied by H. Lundbeck A/S (Copenhagen, Denmark), and drug preparation (eptinezumab or placebo) was performed by personnel unrelated to the study. Both investigators and participants were blinded to drug allocation. PACAP-38 was synthesized and supplied as powder by Bachem AG (Bubendorf, Switzerland). Independent hospital pharmacy staff were responsible for preparing and packaging PACAP-38.

Screening visit

During the screening visit, site investigators assessed the participants’ eligibility based on the predefined criteria. Participants provided written informed consent prior to the commencement of study-related procedures. The participants underwent a semi-structured interview to record clinical data, a general physical examination, and a neurological examination. Vital signs were measured, and for females of childbearing potential, a urine pregnancy test was administered. The participants were informed that PACAP-38 might cause headache or migraine in some individuals; however, the expected timing and headache characteristics were not disclosed to avoid influencing their responses. Furthermore, participants were informed that eptinezumab is a humanized mAb approved for migraine prevention in adults and that it might prevent the effects of PACAP-38. No detailed information was provided regarding its potential mechanism(s) or site(s) of action.

Experimental day

On the experimental day, participants arrived at the research lab and rested in a supine position for 30 min to stabilize their physiological parameters. A venous catheter was placed in the antecubital vein for drug administration. Baseline measurements, including vital signs and headache intensity, were recorded. Facial skin blood flow and the diameter of the left superficial temporal artery (STA) were measured using different imaging techniques. The experiment was rescheduled if the participant had experienced any headache within the previous 48 h.

The participants were then randomly assigned to receive either a single IV infusion of 300-mg eptinezumab or placebo (isotonic saline) over a 30-minute period. Two hours post-infusion, all participants received an IV infusion of PACAP-38 at a dose of 10 pmol/kg/min for 20 min. All drugs were administered using a time and volume-controlled infusion pump. Following start of the PACAP-38 infusion, participants were observed for an additional 2-hour period.

Continuous monitoring of vital signs, headache characteristics, and associated symptoms was conducted throughout the 4-hour in-hospital period following the start of the infusion with eptinezumab or placebo. This included regular assessments to ensure participant safety and accurate data collection. After completing the in-hospital monitoring, participants were discharged and provided with a headache diary. The participants were permitted to use rescue medication, if needed, at any point to treat their headache or migraine.

Headache diary

A headache diary was used to ensure detailed documentation of headache features, associated symptoms, rescue medication use, and adverse events. Diary entries were made every 30 min until 120 min post-infusion start with eptinezumab or placebo. Following this, entries were made every 10 min until 120 min after infusion start with PACAP-38. Upon discharge, participants continued hourly diary entries at home until 24 h following infusion start. This window was based on prior findings showing that PACAP-38-induced headache peaks at a median of 4 h post-infusion and progress to migraine at a mean of 6 h post-infusion [9].

The headache diary included a numerical rating scale for participants to report headache intensity, where 0 indicated “no pain” and 10 represented “the worst imaginable headache ever”. In addition, the diary captured migraine features and associated symptoms, such as pain localization, pain quality, aggravation by routine physical activity, photophobia, phonophobia, nausea, and vomiting. The participants were also asked to note if their headache mimicked their usual, spontaneous migraine attacks and to record any sensations of heat, heart palpitations, facial flushing, and adverse events.

High-frequency ultrasound scanner and laser speckle contrast imager

The STA diameter was assessed using a high-resolution ultrasound scanner, specifically the Dermascan C unit (20 MHz, bandwidth 15 MHz; Cortex Technology, Hadsund, Denmark). The ultrasound probe was positioned perpendicular to the skin surface to optimize image acquisition, and the diameter of the frontal branch of the left STA was measured. To ensure accuracy and reliability, four measurements were taken at each timepoint, and the mean value of these measurements was calculated and recorded.

Facial skin blood flow was quantified using a Laser Speckle Contrast Imager (moorFLPI; Moor Instruments, Devon, UK) [19, 20]. The entire face of the participant was recorded at a distance of 30 cm. Images were automatically captured every 5 s, and participants were instructed to lie completely still with their eyes closed for at least one minute prior to and during measurements to avoid movement artifacts. The obtained images were processed manually to produce scaled, color-coded live flux images, where red indicated high perfusion and blue indicated low perfusion.

Measurements of the STA diameter and facial skin blood flow were conducted at regular intervals. Initial assessments were performed every 30 min until 120 min post-infusion with eptinezumab or placebo. Following this, measurements continued every 20 min until 120 min post-infusion with PACAP-38.

Vital signs monitoring

During the 4-hour in-hospital period, vital signs were monitored to ensure participant safety and gather accurate data. Heart rate (HR) and blood pressure were measured with an auto-inflatable cuff (ProPac Encore; Welch Allyn Protocol). Measurements were recorded at baseline and at 30-minute intervals until 120 min after the initiation of the eptinezumab or placebo infusion. Following this, measurements were taken every 10 min for 120 min after the commencement of the PACAP-38 infusion.

Experimental criteria for a migraine attack

The criteria for defining a migraine attack induced experimentally within 0–24 h after eptinezumab or placebo were established to ensure the induced attacks resembled spontaneous migraine attacks. The criteria were based on fulfilling either or both of the following conditions [21, 22]:

  1. (I)

    ICHD-3 Criteria: The headache must meet criteria C and D for migraine without aura according to the ICHD-3 [18].

  2. (II)

    Mimicking Usual Migraine Attacks: The headache features must mimic the participant’s usual, spontaneous migraine attacks and necessitate treatment with their usual acute rescue medication, such as a triptan or a non-steroidal anti-inflammatory drug (NSAID).

Statistical analysis

All analyses were performed using SPSS Statistics, version 23. Descriptive statistics were presented as frequencies and percentages for categorical variables. For continuous variables, means with standard deviations (SD) were used if the data followed a normal distribution, while medians with interquartile ranges (IQR) were reported for non-normally distributed data. The normality of continuous data was assessed using the Shapiro-Wilk test and visual inspection.

The primary endpoint was the incidence of migraine attacks during the 24-hour observational period post-infusion of eptinezumab or placebo. Sample size calculation was performed using a one-sided Fisher’s Exact test with the “exact2×2” package in R (ver 4.2.0). Based on induction rates from previous studies with PACAP-38 [9, 11, 23, 24] and an expected ∼ 20% decrease in induction rate attributable to the interventional design [25,26,27], as well as efficacy estimates of IV eptinezumab [17], we assumed that ∼ 50% of individuals in the Placebo-PACAP-38 arm would develop a migraine attack, compared with ∼ 10% of individuals in the Eptinezumab-PACAP-38 arm. Aiming for a power of 80% and a significance level of 5%, we estimated that a sample size of at least 19 participants per group would be required. Fisher’s exact test was used to analyze the primary endpoint results.

The exploratory secondary endpoints included several key measures, including the area under the curve (AUC) for headache intensity scores from 2-14 h post-infusion of eptinezumab or placebo (i.e. the first 12 h after PACAP-38 infusion). This endpoint was modified post hoc from the study protocol due to extensive missing data from 14 to 24 h post-infusion start, primarily due to sleep. The results were analyzed using the Mann-Whitney U-test. The incidence of headache of any pain intensity during the 24-hour observational period was analyzed using Fisher’s exact test. The diameter change of the frontal branch of the left STA during the 4-hour in-hospital period was analyzed using the unpaired, two-way t-test. Changes in facial skin blood flow during the 4-hour in-hospital period were analyzed using the unpaired, two-way t-test. The incidence of facial flushing during the 24-hour observational period was analyzed using Fisher’s exact test. All statistical tests were two-tailed, and a P-value of less than 0.05 was considered statistically significant.

Results

Participants

A total of 38 participants with migraine were enrolled and completed the study from March 2023 to August 2023. The participants were randomly assigned in a 1:1 ratio to receive either a single IV infusion of 300 mg eptinezumab or placebo (isotonic saline). None of the participants experienced any headache during the first two hours after eptinezumab or placebo infusion. Two hours after the initial infusion, all participants underwent an IV infusion of PACAP-38, administered at a rate of 10 pmol/kg/min over a 20-minute period. Baseline characteristics, including age, sex, body mass index, and number of monthly headache days, were well-balanced between the two groups (Table 1).

Table 1 Characteristics of the study population
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Table 2 Individual participant outcome data in the eptinezumab group
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Table 3 Individual participant outcome data in the placebo group
Full size table

Migraine induction rate

The incidence of PACAP-38 induced migraine attacks was comparable between the eptinezumab and placebo groups (Tables 2 and 3). Specifically, 10 (53%) of 19 participants in the eptinezumab group reported experiencing a migraine attack, compared with 12 (63%) of 19 participants in the placebo group (P = 0.74). The median time to attack onset was 3.8 h (IQR, 2 to 6.5) post-infusion of eptinezumab, and 4.0 h (IQR, 2.5 to 5) after placebo.

Headache incidence and intensity

Headache of any pain intensity was reported by 15 participants (79%) in the eptinezumab group and 16 participants (84%) in the placebo group, with no significant difference between the two groups (P > 0. 99). Furthermore, the AUC for headache intensity scores during the 12-hour period post-infusion of PACAP-38 did not differ between the eptinezumab and placebo groups (P = 0.96; Fig. 2).

Fig. 2
figure 2

Headache intensity scores. Median headache intensity scores (thick lines) and individual participant headache scores (thin lines) on the numerical rating scale (NRS) following placebo-PACAP-38 (blue) and eptinezumab-PACAP-38 (red) interventions are shown for comparison.

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Hemodynamic outcomes

No differences were observed between the eptinezumab and placebo groups with respect to changes in STA diameter, facial skin blood flow, and facial flushing(all P > 0.05; Fig. 3). Graphs of mean change in mean arterial pressure and HR can be found in Fig. 4.

Fig. 3
figure 3

Vascular parameters. Comparison of mean change in facial skin blood flow and mean change in superficial temporal artery diameter between placebo-PACAP-38 (blue) and eptinezumab-PACAP-38 (red) interventions.

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Fig. 4
figure 4

Blood pressure and heart rate. Mean change in mean arterial blood pressure and mean change in heart rate following placebo-PACAP-38 (blue) and eptinezumab-PACAP-38 (red) interventions.

Full size image

Adverse events and rescue medication use

The most common adverse events reported were heart palpitations and sensations of heat (Supplemental Appendix, Table S1, see Additional file 1). The use of headache rescue medication was noted in seven (37%) participants in the eptinezumab group and six (31%) participants in the placebo group.

Discussion

Our results demonstrate that pretreatment with eptinezumab, a mAb against the CGRP ligand, failed to prevent PACAP-38-induced migraine attacks. Therefore, it is reasonable to assert that PACAP may mediate it’s signaling independently of CGRP signaling in migraine pathogenesis. This finding has significant implications for drug discovery in migraine. Novel medications targeting PACAP signaling might offer a much-needed avenue for treating migraine, particularly for patients who do not respond to CGRP-targeted therapies.

PACAP signaling is independent of CGRP signaling

Over the past three decades, research into the molecular mechanisms of migraine has identified several novel drug targets [28, 29]. CGRP is widely recognized as a pathogenic driver in migraine [29, 30], with its role in meningeal vasodilation and nociception well documented [3, 30]. The advent of CGRP-targeted therapies, such as eptinezumab, has provided efficacious, mechanism-based treatment options for people with migraine. Conversely, less is known about PACAP signaling in migraine [8].

PACAP is released from both parasympathetic efferents and primary sensory afferents [31, 32]. Upon release, PACAP binds to its G protein-coupled receptors, which are expressed at multiple levels of the trigeminovascular system [8], including vascular smooth muscle cells (VSMCs), mast cells, and neurons. The canonical receptors involved are the PACAP type 1 (PAC1R), vasoactive intestinal peptide (VIP)/PACAP type 1 (VPAC1R), and VIP/PACAP type 2 (VPAC2R) receptors, with the Mas-related G protein–coupled receptor X2 (MRGPRX2) specifically expressed on mast cells [33,34,35,36,37]. Despite this knowledge, the specific cells and PACAP-responsive receptors implicated in migraine pathogenesis remain unidentified. Furthermore, preclinical data have demonstrated that PACAP-38 induces a concentration-dependent increase in CGRP release from trigeminal neurons in rodents [12]. This might suggest a positive feedforward regulation between PACAP-38 and CGRP in relation to migraine. However, our results demonstrate that neutralizing CGRP molecules does not prevent PACAP-38-induced migraine attacks. This suggests parallel and independent signaling rather than a sequential relationship. In support, recent rodent data have demonstrated that PACAP can mediate cutaneous hypersensitivity to tactile stimulation (a surrogate marker of allodynia) and light aversive behavior (a surrogate marker of photophobia) independently of CGRP [13, 14].

Vascular smooth muscle cells within the walls of the meningeal arteries

A proposed cellular site of PACAP signaling in migraine pathogenesis is the VSMCs within the walls of the meningeal arteries [8]. Upon receptor binding, PACAP causes downstream activation of cyclic adenosine monophosphate (cAMP)-dependent signaling and subsequent opening of ATP-sensitive potassium (KATP) channels and large-conductance calcium-activated potassium (BKCa) channels [38]. The resultant potassium efflux (chemical stimuli) and vasodilation (mechanical stimuli) are hypothesized to activate and sensitize perivascular meningeal nociceptors [1]. This sequence of events aligns with insights from human experimental studies, which demonstrate that upregulation of cAMP (via phosphodiesterase 3 inhibition) and opening of KATP and BKCa channels can trigger migraine attacks in people with migraine [39,40,41]. Nonetheless, further research is needed to verify this hypothesis and determine whether meningeal vasodilation and the accompanying potassium efflux have a causal role in migraine pathogenesis.

Meningeal mast cells

Another proposed site of PACAP signaling in migraine is mast cells [8], which are known to be involved in neurogenic inflammation and nociception [42]. These cells are often situated in close proximity to meningeal nociceptors and degranulate in response to nociceptor activation [43]. Of interest, PACAP is thought to mediate mast cell degranulation via binding to its MRGPRX2 receptor [37], which has distinct orthologues in rats and mice [36]. Interestingly, rodent data have shown that PACAP-38 can degranulate meningeal mast cells and mediate migraine-like pain [37, 44]. Furthermore, PACAP-38-induced dilation of meningeal arteries was inhibited in both mast cell-depleted and antihistamine-pretreated rats [45]. This is worth noting since histamine is a well-documented molecular migraine trigger and vasodilator in humans [46, 47]. Thus, it seems plausible that the MRGPRX2 receptor is a viable, novel drug target for migraine [8].

Nociceptive neurons

A third proposed site of PACAP signaling in migraine involves the nociceptive trigeminal neurons [8], which include both 1st order neurons in the trigeminal ganglion and 2nd order neurons in the trigeminocervical complex. Of note, electrophysiological data have demonstrated that PACAP-38 can activate second-order trigeminal neurons in rats [48]. However, the study did not explore whether this activation was a result of an initial activation of 1st order trigeminal neurons. Future research should investigate whether PACAP-38 administration can activate meningeal nociceptors, whose cell bodies reside in the 1st order neurons within the trigeminal ganglion.

Mapping the precise site(s) and mechanism(s) of PACAP-38’s action is critical for determining whether drugs that can penetrate the blood-brain barrier should be developed to target PACAP signaling [8]. Central nervous system (CNS)-penetrant drugs are often avoided due to the risk of serious side effects, but they might be necessary to achieve maximum efficacy if PACAP-38 acts within the CNS. However, a mAb directed against the PACAP ligand, Lu AG09222, has proven to prevent migraine attacks in a recent phase II trial [15]. Since mAbs are large molecules that do not readily cross the blood-brain barrier, this suggests that PACAP’s cellular site of action in migraine lies outside of the CNS. This information suggests that PACAP-targeted drugs do not need to penetrate the blood-brain barrier, thus reducing the risk of CNS-related side effects.

Limitations

The results of this study should be considered in light of the following possible limitations. Although an exploratory analysis of a recent clinical trial reported that the effect of eptinezumab on pain freedom, pain relief, and MBS occurred as early as 1 h following the start of drug administration [17], the 2-hour post-eptinezumab timepoint might not have been sufficient to fully block the CGRP pathway before PACAP-38 infusion. The in-hospital observational period was limited to four hours post- eptinezumab/placebo infusion for feasibility purposes. Upon discharge, participants were instructed to record outcome data every hour until 24 h post-eptinezumab/placebo infusion. Thus, the incidence of migraine attacks might have been influenced by factors such as sleep, stress, certain foods, or specific activities. Furthermore, the reliance on self-reported outcome data can introduce variability and subjectivity, potentially affecting the accuracy of the recorded information. Future studies incorporating a PACAP-targeting antibody as a positive control could provide further insight and support for the parallel nature of PACAP- and CGRP signaling.

Conclusions

Among people with migraine, pretreatment with eptinezumab, a mAb directed against the CGRP ligand, did not prevent PACAP-38-induced migraine attacks. This suggests that PACAP may operate as an independent pathogenic driver of migraine attacks. Hence, targeting PACAP signaling might offer a novel, mechanism-based approach to treating migraine. Further research is essential to ascertain the molecular and cellular mechanisms by which PACAP-38 induces migraine attacks and to explore the clinical potential of PACAP-targeted therapies.

Data availability

Deidentified study data can be made available through the corresponding author on reasonable request.

Abbreviations

AUC:

Area under the curve

BKCa :

Large-conductance calcium-activated potassium channels

cAMP:

Cyclic adenosine monophosphate

CGRP:

Calcitonin gene-related peptide

CNS:

Central nervous system

HR:

Heart rate

ICHD-3:

International Classification of Headache Disorders, 3rd edition

IQR:

Interquartile range

IV:

Intravenous

KATP :

ATP-sensitive potassium channels

mAbs:

Monoclonal antibodies

MBS:

Most bothersome symptom

MRGPRX2:

Mas-related G protein–coupled receptor X2

PAC1R:

PACAP type 1 receptor

PACAP(-38):

Pituitary adenylate cyclase-activating polypeptide(-38)

SD:

Standard deviation

STA:

Superficial temporal artery

VIP:

Vasoactive intestinal peptide

VPAC1R:

VIP/PACAP type 1 receptor

VPAC2R:

VIP/PACAP type 2 receptor

VSMCs:

Vascular smooth muscle cells

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Acknowledgements

The authors thank all participants included in this study, and Dr Faisal Mohammad Amin for his assistance during the conduct of this study. Fig 1 (Created in BioRender. Zhuang, Z. (2025) https://BioRender.com/r64t217) and Visual Abstract (Created in BioRender. Zhuang, Z. (2025) https://BioRender.com/t36x831) were created using Biorender.

Funding

This study was supported by a research grant from Lundbeck (IIT-2022-007 to MA).

Author information

Authors and Affiliations

  1. Department of Neurology, Danish Headache Center, Copenhagen University Hospital– Rigshospitalet, Copenhagen, Denmark

    Mohammad Al-Mahdi Al-Karagholi, Zixuan Alice Zhuang, Signe Beich, Håkan Ashina & Messoud Ashina

  2. Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Zixuan Alice Zhuang, Signe Beich & Messoud Ashina

  3. Translational Research Center, Copenhagen University Hospital– Rigshospitalet, Copenhagen, Denmark

    Håkan Ashina

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Contributions

M.A. conceived the project and, together with M.M.K., initiated and contributed to the study design, protocol development, participant enrollment, data acquisition, data processing, analysis, statistics, interpretation, and drafting and revision of the paper. Z.A.Z. and S.B. contributed to participant enrollment, data acquisition, data processing, analysis, statistics, interpretation, and revision of the paper. H.A. participated in the study design and contributed to data processing, analysis, statistics, interpretation, and revision of the paper.

Corresponding author

Correspondence to Messoud Ashina.

Ethics declarations

Ethics approval and consent to participate

The study protocol was reviewed and approved by the Regional Health Research Ethics Committee of the Capital Region of Denmark (identifier: H-22038923). All participants provided written informed consent before enrollment, ensuring that they were fully informed about the study procedures and potential risks.

Consent for publication

Not applicable.

Competing interests

M.A. has received personal fees from AbbVie, Amgen, AstraZeneca, Eli Lilly, GlaxoSmithKline, Lundbeck, Novartis, Pfizer, and Teva Pharmaceuticals for consulting, advisory board participation, and speaker honoraria, outside of the submitted work. M.A. also serves as an Associate Editor of Brain and The Journal of Headache and Pain. H.A. has received personal fees from AbbVie, Lundbeck, Pfizer, and Teva for consulting, advisory board participation, and speaker honoraria, outside of the submitted work. The remaining authors declare no competing interests.

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Al-Karagholi, M.AM., Zhuang, Z.A., Beich, S. et al. PACAP38-induced migraine attacks are independent of CGRP signaling: a randomized controlled trial. J Headache Pain 26, 79 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s10194-025-02022-2

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The Journal of Headache and Pain

ISSN: 1129-2377

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