Calcitonin gene-related peptide (CGRP) is a potent vasodilator of cranial arteries (Brain et al., 1985; Falkenberg et al., 2020) and intravenous infusion of CGRP causes headache in healthy volunteers (Petersen et al., 2005b; Falkenberg et al., 2020) and migraine attacks in migraine patients (Lassen et al., 2002; Hansen et al., 2010; Ashina, 2020; Iljazi et al., 2020; Ashina et al., 2021). The molecular mechanisms by which CGRP mediates head pain are still unclear (Ashina et al., 2019). Recent studies suggested K_ATP channels as downstream effectors in the CGRP signaling pathway (Nelson et al., 1990; Kitazono et al., 1993; Quayle et al., 1994; Kleppisch and Nelson, 1995). K_ATP channels are expressed in neurons, vascular endothelium, and smooth muscle cells. These channels are involved in diverse physiological processes including insulin secretion, regulation of vascular tone, and protecting against metabolic stress (Al-Karagholi et al., 2017, 2019a, 2020b). In human provocation studies, K_ATP channel opener levcromakalim induces headache and migraine (Al-Karagholi et al., 2019b, c, 2020a, 2021a).

The widely used antidiabetic drug, glibenclamide, is a non-selective K_ATP channel inhibitor that belongs to the second-generation of sulfonylurea (Luzi and Pozza, 1997; Hibino et al., 2010). Preclinical evidence has demonstrated that glibenclamide attenuates CGRP-induced cranial artery dilation (Gozalov et al., 2005, 2008; Al-karagholi et al., 2019d) and trigeminal pain transmission (Christensen et al., 2020). In this study, we hypothesized that glibenclamide would inhibit CGRP-induced headache and vascular changes. To investigate this, we conducted a double-blind, randomized, placebo-controlled, crossover study in healthy volunteers.

Twenty healthy volunteers were recruited through the Danish website¹. Written informed consent was obtained from all participants after detailed oral and written study information. The female participants were required to have sufficient contraception [contraceptive pill or intrauterine device/system (IUD/IUS)]. Exclusion criteria were: (1) history of cardiovascular or cerebrovascular disease, diabetes mellitus, or hypercholesterolemia, (2) electrocardiogram (ECG) changes indicative of myocardial ischemia or hypertrophy, (3) hypertension at baseline on an experimental day (defined as a systolic blood pressure above 150 mmHg or a diastolic blood pressure above 100 mmHg), (4) current pregnancy or breastfeeding, (5) daily intake of medication (except oral contraceptives), (6) daily smoking within last 5 years, (7) first-degree relatives with a history of diabetes mellitus, and (8) history of any primary headache disorders (except episodic tension-type headache for <2 days per month during the last year) or first-degree family members with migraine as defined by the third International Classification of Headache Disorders (ICHD) (Vincent and Wang, 2018). A full medical examination and ECG were performed on the day of recruitment.

The study was approved by the Ethics Committee of the Capital Region of Denmark (H-19065735) and the Danish Data Protection Agency, and was conducted according to the Declaration of Helsinki of 1964, with later revisions. The study was also registered at ClinicalTrials.gov (NCT04231617).

Twenty healthy volunteers (11 women and 9 men) completed both days of the study. The mean age was 23 years (range 18–27) and mean bodyweight was 72 kg (range 51–94, body mass index range 18–25). There was no difference between the right and left-sided MCA, and therefore the average of both sides was used. There was no carry-over or period effect for values of headache, HR, MAP, V_MCA, STA, or RA.

The major outcome of the present study was that K_ATP channel inhibitor glibenclamide did not affect CGRP-induced headache and vascular changes. The CGRP-induced headache and vascular changes reported in the present study were consistent with previous studies (Petersen et al., 2005b; Hansen et al., 2010; Ashina et al., 2018; Falkenberg et al., 2020). Petersen et al. showed that pretreatment with the CGRP receptor antagonist BIBN4096BS inhibited headache and extracranial vasodilation after intravenous infusion of 1.5μg/min CGRP over 20 min.

In vivo studies showed that glibenclamide attenuated CGRP-induced vasodilation in basilar (Kitazono et al., 1993; Faraci and Sobey, 1998), pial (Hong et al., 1996), and dural arteries (Gozalov et al., 2008). The inhibitory effect of glibenclamide on CGRP-induced vasodilation was dose-dependent, and high doses were necessary to observe an effect (Gozalov et al., 2008). Intravenous glibenclamide administration at concentrations of 20–30 mg/kg attenuated CGRP-induced vasodilation (Gozalov et al., 2008), and i.p. injection of 1 and 10 mg/kg glibenclamide attenuated trigeminal pain transmission in rats with spontaneous trigeminal allodynia (Christensen et al., 2020). Since we observed the participants for 5 h, we used 10 mg per oral glibenclamide which is the maximal single dose tested in humans (Williams et al., 1998; Bayerle-Eder et al., 2000; Gori et al., 2005; Hougaard et al., 2020). This corresponds to 0.14 mg/kg, which is substantially lower than doses used in preclinical models. This might explain the insufficient glibenclamide effect to counteract the CGRP-induced vascular changes. However, using equivalent doses in humans would cause severe hypoglycemia and cannot be reasoned.

Interestingly, in vitro studies showed that glibenclamide did not affect CGRP-induced dilation of dural arteries and MCA (Kageyama et al., 1993; Gao et al., 1994; Gozalov et al., 2008). In rat coronary arteries, only the combination of glibenclamide and a calcium-activated K⁺ (BK_Ca) channel blocker charybdotoxin could attenuate the effect of CGRP on arteries in vitro (Sheykhzade and Berg Nyborg, 2001) suggesting a dual action of K_ATP and BK_Ca channels in CGRP-induced vasodilation. The role of BKCa is recently implicated in headache and migraine patophysiology (Al-Karagholi et al., 2020d, 2021b). Possible involvement of other type of potassium channels in CGRP-induced vasodilation cannot be ruled out. Altogether, these data suggest that interspecies differences are likely to contribute to the discrepancy in the findings of glibenclamide on CGRP-induced vascular effects.

Binding of CGRP to the CGRP-receptor complex on vascular smooth muscle cells (VSMCs) (Eftekhari et al., 2013; Edvinsson et al., 2018) mediates an activation of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway resulting in potassium efflux through K_ATP channels, hyperpolarization, and eventually relaxation and vasodilation (Hong et al., 1996; Williams et al., 1998; Ashina et al., 2021). K_ATP channels consist of different isoforms of sulfonylurea receptor subunits (SUR1, SUR2A, and SUR2B) (Kokoti et al., 2020). SUR1 are expressed in the pancreas (Babenko et al., 2000), the TG and the trigeminal nucleus caudalis (TNC) (Ploug et al., 2012). The SUR2B isoform is of vascular origin and highly expressed in cranial arteries (Ploug et al., 2008a, b, 2012). Glibenclamide is a non-specific SUR subunit blocker shown to have high affinity for SUR1 and low affinity for SUR2B (Inagaki et al., 1996; Stephan et al., 2006). SUR subunits are downstream molecules in CGRP signaling cascades, and whether CGRP receptors and SUR subunits have a cross-reactivity at the same cellular level is yet to be demonstrated. Thus, the results of the present study might be explained by different affinity of glibenclamide and CGRP signaling pathway to SUR isoforms.

In conclusion, we found that pretreatment with glibenclamide did not affect CGRP-induced headache and vascular responses. Our findings could suggest that: (1) the potency of used glibenclamide concentration is too low to result in any measurable effects on CGRP-induced headache and vasodilation, (2) CGRP-induced headache and vascular responses could be initiated by activation of SUR2B K_ATP channel (Al-Karagholi et al., 2017), and/or (3) interspecies differences might influence the role of K_ATP channel in CGRP signaling pathway. To clarify the molecular interaction in the CGRP signaling pathway, more studies on specific isoforms of sulfonylurea receptor subunits of K_ATP channel in humans are needed.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Capital Region of Denmark (H-19065735). The patients/participants provided their written informed consent to participate in this study.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.