Background
Much research effort has been devoted to studying the pathophysiology of primary headaches using human experimental models, which have led to the discovery of novel headache-eliciting signaling pathways and new drug targets [
1]. In this context, pituitary adenylate cyclase-activating polypeptide (PACAP) has over the past decade emerged as a key signaling molecule implicated in migraine [
2] and possibly also in cluster headache [
3].
PACAP belongs to the glucagon/secretin superfamily of peptides together with vasoactive intestinal polypeptide (VIP) [
4] and exists in two bioactive forms: a 38 amino acid form (PACAP38) and a truncated 27 amino acid form (PACAP27) [
5]. PACAP38 is present in first-order neurons in the trigeminal ganglion [
6], second-order neurons in the trigeminal nucleus caudalis (TNC) [
7], and dorsal horn of the human spinal cord [
8]. In addition, PACAP38 has also been identified in the otic and sphenopalatine ganglia [
9], as well as in the cerebral cortex, cerebellum, brain stem and hypothalamus [
10].
The effect of PACAP38 is mediated through three G-protein coupled receptors (PAC
1, VPAC
1–2) [
11], two of which (VPAC
1–2) hold equal affinity for PACAP38 and VIP, while the PAC
1 receptor has a much higher affinity for PACAP38 [
12]. The distribution of all three receptors has been documented in trigeminal, otic and superior cervical ganglia [
13], as well as in cerebral and meningeal arteries [
14]. Upon activation, all receptors cause downstream production of cyclic adenosine monophosphate (cAMP) through adenylate cyclase (AC) stimulation [
15]. Studies have reported that the VPAC
1–2 receptors play a role in vasodilation and mast cell degranulation [
16‐
20], whereas one study in rats implicated the PAC
1 receptor in pro-nociceptive transmission [
21].
The headache-inducing effect of PACAP38 has been extensively studied in both healthy volunteers and migraine without aura (MO) patients. This has sparked an interest in pursuing specific treatment options targeting the PACAP38 molecule [
22] or its PAC
1 receptor [
23]. Future randomized clinical trials (RCTs) will fully uncover whether PACAP38 or PAC
1 receptor blockade could be a promising new approach in treating primary headaches.
In this review, we focus on human headache models using PACAP38 as a pharmacological “trigger” of migraine-like attacks. We then consider methodological aspects and limitations. Finally, we outline future perspectives and the therapeutic potential of anti-PACAP38 treatment to address unmet patient needs.
PACAP38 migraine models
Birk et al. [
24] for the first time systematically investigated PACAP38-induced headache and cerebral hemodynamics in 12 healthy volunteers. In this and the following described studies, healthy volunteers were identified as subjects who had no prior history of migraine and no first-degree relatives suffering from migraine. Ten out of 12 participants (83%) reported mild to moderate headache following PACAP38 infusion over 20 min, while no effect was observed on regional cerebral blood flow. There was a minor dilation of the middle cerebral artery (MCA) recorded by a transcranial Doppler (TCD) after PACAP38 infusion. However, certain limitations of the TCD method should be acknowledged. The TCD method assesses MCA velocity, which is dependent on the blood flow and the cross-sectional area of the artery. To interpret reduced velocity as arterial dilation, it requires that cerebral blood flow is constant despite heart rate variability and different angles of insonation. A more detailed description of methodological considerations on arterial measurements by TCD has recently been reviewed [
25]. In healthy volunteers, the dose-response to 5, 10, 15, and 20 pmol kg
−1 min
−1 was investigated in three participants [
24]. In all three cases the infusion was aborted after 10 pmol kg
−1 min
−1 due to 40–50% increases in heart rate – probably compensatory to the vasodilating effect of PACAP38. Following these observations, which have recently been confirmed in a dose-response study [
26]; a dose of 10 pmol kg
−1 min
−1 is considered the optimal dose for experimental provocation studies.
Given that experimentally provoked attacks are not spontaneous according to the International Headache Society (IHS) criteria [
27], different criteria for experimentally induced migraine attacks have been introduced [
28]. The provoked migraine attacks should either fulfill IHS criteria C and D for MO [
25] or mimic the usual migraine attack experienced by the patient and subsequent response to treatment with acute rescue medication [
28]. To investigate the migraine-inducing effects of intravenous PACAP38 infusion, Schytz et al. [
29] performed a double-blind, placebo-controlled crossover studies in 12 healthy volunteers and 12 MO patients. The authors hypothesized that PACAP38 infusion would induce headache in controls and migraine-like attacks in MO patients. All controls reported headache after PACAP38 infusion, while two controls also experienced migraine-like attacks. In MO patients, 7 out of 12 subjects (58%) reported migraine-like attacks after PACAP38 infusion compared with zero after placebo. Interestingly, the median time to peak headache score (4 h, range 0–12 h) in MO patients following PACAP38 provocation was similar to those reported in calcitonin gene-related peptide (CGRP) (5 h, range 2-9 h) and glyceryl trinitrate (GTN) (5.5 h, range 3–10 h) provocation studies [
30,
31]. Moreover, the authors assessed the vascular effects of PACAP38 infusion on the MCA by TCD and the superficial temporal artery (STA) by dermascan ultrasonography during the hospital phase of the study (0-2 h post infusion) [
29]. In MO patients, PACAP38 infusion caused a modest MCA dilation of 9.5% compared to baseline, while a more marked dilation of 37.5% was found in the STA. This study yielded two important findings. First, PACAP38 induced migraine-like attacks in 58% of MO patients, while no attacks were reported after placebo. Secondly, prolonged cranial artery dilation suggested a possible role of vascular mechanisms in PACAP38-induced migraine.
Magnetic resonance angiography (MRA) constitutes a superior method for measuring vessel diameter compared to TCD and provides more precise measurements of circumferential arterial changes [
32]. All of the described provocation studies using TCD and MRA only assessed vascular effects in the middle meningeal artery (MMA), STA, and MCA [
24,
29,
33,
34]. Using MRA, a double-blind, placebo-controlled study investigated the effect of PACAP38 infusion on the MCA and MMA in healthy volunteers [
33]. The MMA was selected because it is the main artery supplying the dura mater and one previous study demonstrated MMA (but not MCA) involvement in CGRP-induced headache in healthy volunteers [
35]. The major finding of the MRA study [
33] was that PACAP infusion caused a long-lasting dilation (> 5 h) of the MMA co-occurring with headache, while no effect was found on the MCA circumference. In addition, subcutaneous injection of sumatriptan reversed the MMA dilation and headache, whereas the MCA circumference was unaltered. It is possible that PACAP38 does not reach its receptors on the smooth muscle cells in the MCA. In support, in vitro studies [
36] reported a vasodilatory effect of PACAP38 on the rat and human MCA when applied abluminally but
not luminally. The question is whether MMA dilation with co-occurring headache following PACAP38 infusion and the subsequent MMA constriction with co-occurring headache relief following sumatriptan reflect the importance of the MMA in migraine generation and cessation. It should be noted that sumatriptan is a 5-HT1B/1D receptor agonist that was originally developed as vasoconstrictor acting through receptor binding on cranial vessels [
37]. However, its exact mechanism of action in relation to migraine remains a highly debated topic [
38]. In healthy volunteers, subcutaneous injection of sumatriptan caused constriction of the STA, MMA, and MCA [
39]. However, the same authors found a significantly smaller intracerebral arterial constriction compared with the constriction of extracerebral arteries – suggesting a primarily peripheral site of action for triptans. In the context of human provocation studies, subcutaneous injection of sumatriptan caused co-occurring MMA constriction and amelioration of migraine-like attacks following both PACAP38 [
33] and CGRP [
40] infusion. In both provocation studies [
33,
40], no sumatriptan effect was found on the MCA circumference.
An interesting aspect to consider is that even though VIP belongs to the same family of peptides as PACAP38 [
41], it does not induce migraine attacks in MO patients [
42]. VIP infusion only induced dilation of cranial arteries and mild headache [
42]. To further examine this issue, one MRA study investigated the response to intravenous infusion of PACAP38 or VIP in MO patients [
34]. Sixteen out of 22 patients (73%) reported delayed migraine-like attacks following PACAP38 infusion, whereas only 4 out of 22 (18%) did so after VIP infusion. Moreover, this study found that both PACAP38 and VIP induced STA and MMA dilation, while the MCA remained unaffected. The PACAP38-induced vasodilation was longer-lasting (> 2 h) than the VIP-induced vasodilation which normalized after 2 h. Interestingly, there was no difference in arterial circumference between the pain and non-pain side during PACAP38-induced migraine-like attacks in 9 patients. Subcutaneous injection of sumatriptan reduced headache intensity and caused constriction of only the extracranial arteries. Another key finding from this study was that plasma levels of PACAP38 were elevated in MO patients who developed migraine-like attacks compared to those who did not 60 min after PACAP38 infusion. Since plasma PACAP38 has a half-life of 3.5 min [
24], a complete clearance of exogenous PACAP38 is expected 60 min after the start of infusion. To explain this, the authors suggested three possible mechanisms [
34]: 1) impaired elimination; 2) endogenous release; 3) de novo synthesis. However, when the data from this study [
40] was later pooled with data from a second study from the same research group [
43] to increase power and sample size, there was no difference in pre-ictal PACAP38 plasma levels between patients who developed migraine-like attacks (
n = 39) compared to those who did not (
n = 15). To our knowledge, no study has investigated the underlying mechanisms of PACAP38-induced prolonged vasodilation.
A resting-state functional magnetic resonance imaging (fMRI) study examined the involvement of specific changes in cerebral network connectivity before and during PACAP38-induced migraine-like attacks in MO patients [
44]. VIP was used as active placebo. Resting state fMRI is a method to evaluate regional interactions in cerebral connectivity when a subject is not performing an explicit task. Patients were scanned 30 min, 130 min, and 310 min after PACAP38-infusion, unless they reported migraine-like attacks. In the event of migraine-like attacks, immediate scans were performed. The study found abnormal cerebral connectivity in all the investigated cerebral networks (salience, sensorimotor, and default mode) at the onset of migraine-like attacks after PACAP38 infusion when compared to outside of the attacks [
44]. No alterations in cerebral connectivity were found following VIP infusion. These findings are interesting because those three networks have been implicated in the processing of nociceptive and emotional signals [
45‐
48]. To solidify the importance of these findings, the authors suggested that a similar experiment should be conducted before and at the early phase of spontaneous migraine attacks.
One provocation study has also examined the incidence of premonitory symptoms induced by intravenous PACAP38 administration in MO patients [
49]. Premonitory symptoms occur hours to 2 days prior to the migraine attack [
28] and most commonly present themselves as unusual fatigue, neck stiffness, and poor concentration. It has previously been reported that 36% of migraine patients experience premonitory symptoms following GTN infusion [
50]. Following PACAP38-infusion [
49], 72% and 48% of the patients experienced migraine-like attacks and premonitory symptoms, respectively. Interestingly, CGRP did not induce premonitory symptoms in the same group of patients. In addition, there was no difference of premonitory symptoms in patients who developed attacks versus those who did not. These findings are interesting because premonitory symptoms are considered a marker of CNS involvement. However, the study did not include a healthy control group or placebo-treated patients. Therefore, we cannot exclude that the observed association between PACAP38 infusion and premonitory symptoms could be due to substance-related side effects.
As we reflect on the headache-inducing capabilities of PACAP38, it is interesting that some MO patients develop migraine-like attacks while others do not. The question is whether fluctuating susceptibility could be due to genetic variations among migraine patients. Genetic studies have documented that genetic enrichment of certain risk factor genes constitute a predisposition to developing migraine [
51‐
53]. To address this issue, one study [
54] stratified patients into two groups: one group with high family load (≥ 2 first-degree relatives with MO) and one group with low family load (≤ 1 first-degree relatives with MO). In addition, genotyped patients were stratified based on risk allele status. This study revealed no association of hypersensitivity to migraine following PACAP38 administration based on family load and migraine-associated risk allele status in 32 genotyped MO patients.