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Abstract

Objective

To determine whether glibenclamide, a non-selective adenosine 5′-triphosphate-sensitive K⁺ (K_ATP) channel blocker, attenuates pituitary adenylate cyclase-activating polypeptide-38 (PACAP38)-induced headache and vascular changes in healthy volunteers.

Methods

In a double-blind, randomized, placebo controlled and crossover design, 22 healthy volunteers were assigned to receive an intravenous infusion of 10 picomole/kg/min pituitary adenylate cyclase-activating polypeptide-38 over 20 minutes followed by oral administration of 10 mg glibenclamide or placebo. The primary endpoint was the difference in incidence of headache (0–12 hours) between glibenclamide and placebo. The secondary endpoints were a difference in area under the curve for headache intensity scores, middle cerebral artery velocity (V_meanMCA), superficial temporal artery diameter, radial artery diameter, heart rate, mean arterial blood pressure and facial skin blood flow between the two study days.

Results

Twenty participants completed the study. We found no difference in the incidence of pituitary adenylate cyclase-activating polypeptide-38-induced headache after glibenclamide (19/20, 95%) compared to placebo (18/20, 90%) (P = 0.698). The area under the curve for headache intensity, middle cerebral artery velocity, superficial temporal artery diameter, radial artery diameter, facial skin blood flow, heart rate and mean arterial blood pressure did not differ between pituitary adenylate cyclase-activating polypeptide-38-glibenclamide day compared to pituitary adenylate cyclase-activating polypeptide-38-placebo day (P > 0.05).

Conclusions

Posttreatment with 5′-triphosphate-sensitive K₊ channel inhibitor glibenclamide did not attenuate pituitary adenylate cyclase-activating polypeptide-38-induced headache and hemodynamic changes in healthy volunteers. We suggest that pituitary adenylate cyclase-activating polypeptide-38-triggered signaling pathway could be mediated by specific isoforms of sulfonylurea receptor subunits of 5′-triphosphate-sensitive K₊ channels and other types of potassium channels.

Keywords

Humans migraine glyburide pituitary adenylate cyclase activating polypeptide-38 cranial arteries

Introduction

Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) is a potent vasodilator released from trigeminal sensory and parasympathetic perivascular nerve fibers (1,2). Intravenous infusion of PACAP38 causes prolonged dilation of extracerebral arteries accompanied with headache in healthy volunteers and migraine attack in migraine patients (3,4). The cellular mechanisms underlying PACAP38-induced headache and vascular changes are still not completely understood. It has been suggested that PACAP38 triggers signaling cascades involving upregulation of cyclic adenosine monophosphate (cAMP), activation of protein kinase A (PKA) and eventually phosphorylation of several downstream molecules including adenosine 5′-triphosphate-sensitive K⁺ (K_ATP) channel (5 –7) and large conductance calcium-activated potassium channels (BKca) (8). BK_Ca channels are expressed in vascular smooth muscle cells (VSMCs) and once activated, lead to arterial dilation and headache in healthy participants (9).

K_ATP channels are expressed in pancreatic β-cells, VSMCs and first and second order neurons of the trigeminal pain pathway (10,11). Activation of K_ATP channels results in potassium efflux and thus a reversible decrease in membrane potential, denoted hyperpolarization, which depending on tissue leads to a specific cellular response. In VSMCs, potassium outflow causes a decrease in cytosolic Ca²⁺ concentration and vasodilation (12 –14). In vitro studies showed that glibenclamide, a non-specific K_ATP channel blocker and a widely used anti-diabetic drug, attenuated PACAP-induced dilation of rat cerebellar and human pulmonary arteries (8,15). The involvement of K_ATP channels in PACAP38 mediated vascular changes has not been explored in vivo.

In the present study, we aimed to elucidate the molecular mechanisms underlying PACAP38-induced headache and vasodilation. We hypothesized that glibenclamide would attenuate PACAP38-induced responses. To test this hypothesis, we conducted a randomized, double blind, placebo-controlled study in healthy volunteers.

Methods

Participants

Participants were recruited between October 2020 and March 2021, via the online Danish recruitment website www.forsoegsperson.dk. All possible candidates were pre-screened over the phone and eligible participants were invited to the Danish Headache Center for detailed screening and full medical examination including electrocardiogram (ECG) recording (Cardiofax V; Nihon-Koden). Participants were informed that PACAP38 might induce headache, but its timing and characteristics were not discussed. Participants were also informed that glibenclamide is an anti-diabetic medication, and it is unknown whether it can affect PACAP38-induced headache. Inclusion criteria for the study were i) age between 18 and 60 years old, ii) weight from 50 to 100 kg, and iii) sufficient contraceptive method for female participants (contraceptive pill or intrauterine device/system (IUD/IUS)). Exclusion criteria were i) diagnosis of primary headache disorder according to ICHD-3 (16) (except tension type headache less than 5 days per month) or first-degree relative suffering from migraine, ii) first-degree relative diagnosed with diabetes mellitus, iii) anamnestic and/or clinical signs of serious somatic or psychiatric disorder, including diabetes, iv) daily intake of medication except oral contraceptives, v) substance abuse or smoking, and vi) pregnant or breastfeeding women. All participants gave written consent. The study was approved by the Regional Health Research Ethics Committee of the Capital Region (H-19065735) and the Danish Data Protection Agency. It was conducted according to the Declaration of Helsinki of 1964, with later revisions. The study was registered at ClinicalTrials.gov (NCT04960657).

Experimental design

Twenty-two healthy volunteers were enrolled. We aimed to evaluate the physiological effect of glibenclamide after PACAP38 in healthy participants as this would provide evidence for further investigating it in migraine patients. On two different study days separated by at least one week, participants received intravenous infusion of 10 picomole/kg/min PACAP38 (Bachem AG, Bubendorf, Switzerland) over 20 minutes. Immediately after PACAP38 infusion, participants were in a balanced, crossover and double-blind design randomly assigned to receive glibenclamide (10 mg oral tablet of Hexaglucon, Sandoz) or placebo (Figure 1). Study drugs and placebo were prepared by the Capital Region Central Pharmacy. Randomization of study drugs was also performed by the Capital Region Central Pharmacy, so that glibenclamide and placebo tablets looked identical. The randomization code remained in the hospital during the study and was not accessible to the investigators until the study was completed and the data were analyzed.

Figure 1.

Experimental design of the study. Healthy participants (n = 22) were randomly allocated in a double-blind crossover design, to receive glibenclamide or placebo immediately after PACAP38 infusion on two separate study days. There was a wash-out period of at least one week between the two study days. Twenty participants (n=20) completed both study days.

All participants arrived at the clinic non-fasting, between 8 am to 11 am. The participants were at least 48 hours headache-free and refrained from coffee, tea, cocoa, alcohol, and tobacco at least 12 hours prior to study onset. Upon arrival all female participants were tested for pregnancy on both study days. All study procedures were performed in a quiet room with standardized lighting and temperature. Participants were placed in supine position and a venous catheter (BD VenflonVR, Franklin Lakes, NJ) was inserted in both antecubital veins for drug (PACAP38) and 20% w/v glucose infusion. After resting for at least 30 minutes, baseline measurements of mean arterial blood pressure (MABP) and heart rate (HR) (ProPac Encore; Welch Allyn Protocol) were performed. PACAP38 infusion was administered using a time and volume-controlled infusion pump. Vital signs were continuously monitored and recorded at baseline (T0) and every 10 minutes after the start of the infusion. Changes in facial skin blood flow were measured by laser speckle contrast imager (17) (Moor Instruments speckle contrast blood assessment, moor Full Laser Perfusion Imager) at baseline and every 10 minutes after PACAP38 administration (Figure 2).

Figure 2.

Timeline for the 260 min in-hospital phase of the experiment.

One of the authors (LK) evaluated eligibility, obtained informed consent, and enrolled the participants. Experiments were carried out at the Danish Headache Center, Department of Neurology, Rigshospitalet Glostrup, from 21 October 2020 to 24 March 2021.

Headache and accompanying symptoms

We recorded headache intensity and accompanying symptoms at baseline (T0) and every 10 minutes after the start of the infusion until T260 minutes using a standardized questionnaire. Headache intensity was recorded on a numerical rating scale (NRS) from 0–10 (0; no headache, 1; very mild headache [including a pressing or throbbing feeling], 10; worst imaginable headache). Headache characteristics including localization, pain quality, aggravation by physical activity, associated symptoms (nausea, photophobia and phonophobia) were also recorded. After being discharged from the hospital, participants were asked to complete a headache diary every hour until 12 hours after the PACAP38 infusion. Apart from headache intensity and characteristics, the diary recorded uptake of analgesics and possible adverse events (AE). Participants were allowed to take medication if the headache became intolerable. The standard rescue medication provided on site was 1000 mg paracetamol and 400 mg ibuprofen.

Vascular variables

Diameter of the frontal branch of the left superficial temporal artery (STA) and left radial artery (RA) and the mean velocity of blood flow in the middle cerebral artery (V_meanMCA) were recorded at baseline, 20 minutes and 50 minutes after and then every 20 minutes until 260 minutes following the beginning of infusion. At each time point, four measurements were performed and the mean of these is reported. STA and RA diameter was measured by a high-resolution ultrasonography unit (Dermascan C; Cortex Technology: 20 MHz, bandwidth 5 MHz) as described previously (18,19). V_meanMCA was recorded bilaterally using transcranial Doppler (TCD; Doppler BoxX) with handheld 2-MHz probes as previously described (18,20). End-tidal CO₂ was recorded simultaneously with TCD recordings using an open mask that caused no respiratory resistance (ProPaq Encore; Welch Allyn Protocol).

Plasma glucose

Blood glucose levels were monitored during the experiment. Thirty minutes after posttreatment with glibenclamide or placebo (T50) and when initial fasting glycaemia had declined by 10%, blood glucose concentrations were clamped around 4–7 mmol/L by 20% glucose infusion. Glucose infusion rates (GIRs) are based on a previous study (21). Infusion rates (IRs) necessary to maintain blood glucose after drug intake were registered throughout the ensuing 260 min. The following standard formula was used to calculate the infusion rate:

infusion rate (\frac{m l}{h r}) = \frac{GIR \frac{m g}{k g * \min} * weight (k g) * 60 \frac{\min}{h r}}{concentration \frac{g}{100 ml} * 1000 \frac{m g}{g} / 100}

Twenty-three blood samples were obtained for the determination of glucose during the experimental period. Blood samples were drawn at baseline and then with 10 min intervals starting at T50. The venous blood samples were drawn from the intravenous catheter using a blood gas aspirator (Radiometer, SafePICO, self-filling blood gas syringe) and the blood glucose concentrations were determined with a blood gas analyzer (Radiometer, ABL90 FLEX). After finishing the measurements (T260) participants were provided with a standardized meal rich in carbohydrates which they had to consume before getting discharged.

Data analysis and statistics

All absolute values are presented as mean ± standard deviation (SD) except headache intensity and duration scores which are presented as median (range). The highest variation of STA diameter, V_meanMCA and HR are reported as the highest mean percentage increase and 95% confidence interval (CI).

Calculation of sample size was based on detection of a difference between two experimental days in incidence of headache at 5% significance (two-tailed) with 90% power. A 50% difference was taken to be clinically significant. We estimated that 17 participants should be included, but we increased the sample size to 22 since this is statistically more desirable.

The primary endpoint was difference in incidence of headache (0–12 h) between the two experimental days: PACAP38-glibencamide day (infusion of PACAP38 followed by oral glibenclamide) versus PACAP38-placebo day (infusion of PACAP38 followed by oral placebo). The secondary endpoints were a difference in area under the curve (AUC) for headache intensity scores (0–12 h), V_meanMCA, STA diameter, RA diameter, HR, MABP and facial skin blood flow from baseline to the last measurement (T0-T260) between the two study days. Baseline was defined as T0 before the start of infusion.

Incidence of PACAP38-induced headache on the two study days was analyzed as categorical paired data using McNemar’s test. AUC was calculated according to the trapezium rule to obtain summary measures and to analyze the differences in response between glibenclamide and placebo. AUC was compared between study drugs using the Wilcoxon signed rank test. To further evaluate the systemic hemodynamic (V_meanMCA, STA, RA, HR, MABP) and facial blood flow between PACAP38-glibenclamide day and PACAP38-placebo day we conducted an exploratory analysis by parametric testing mixed effects models. We tested for the effects of drug as an interaction on the effect of time. The outcome variables (V_meanMCA, STA diameter, RA diameter, HR, MABP, facial blood flow) were tested in models with time as fixed effect, drug (glibenclamide/placebo) as interaction terms with time and patient as random effects.

Statistical analysis and graphs were performed using GraphPad Prism 8.3.0 (San Diego, CA, USA). Level of significance at five percent (P < 0.05, two-tailed) was accepted for all tests.

Data availability

The data supporting the findings in the present study are available from the corresponding author upon reasonable request.

Results

Participant characteristics

Twenty-two healthy volunteers were enrolled in the study (Figure 3). Twenty participants (90.9%) completed both study days (10 women and 10 men) and were included in the analysis. Mean age was 23.5 (range 19-35) and mean weight 70.7 kg (range 50-94). At baseline, there were no differences for any variable. Regarding V_meanMCA measurements, the average of left and right side was used.

Figure 3.

CONSORT flow diagram of the study.

Headache and accompanying symptoms

PACAP38 induced headache in 19/20 participants (95%, median headache duration was 8 h (range 0–12)) on the active day (glibenclamide) and in 18/20 (90%, median headache duration was 8 h (range 0–12)) on the placebo day (P = 0.698). Median peak headache intensity score was 2 (range 0–7) (Figure 4A) on glibenclamide day and 2 (range 0–8) on placebo day (Table 1). We found no difference in the AUC_0–12 h for headache intensity after glibenclamide (617.3 ± 503.7) compared to placebo (539 ± 388.5) (P = 0.798) (Figure 4B).

Figure 4.

Numerical rating scale (NRS) scores for headache intensity after infusion of PACAP38 and administration of oral glibenclamide or placebo, from baseline to 12 hours. Dotted lines represent individual headache scores of each participant on each study day. Thick line represents the median headache on glibenclamide day (A) and placebo day (B) respectively. The AUC for headache intensity did not differ between the glibenclamide (617.3 ± 503.7) and the placebo day (539 ± 388.5) (P = 0.798).

Table 1.

Clinical characteristics of headache and associated symptoms in healthy volunteers after (0–12 hours observation period).

Participants	Peak headache (Duration of headache)	Headache characteristics^a	Associated symptoms^b	Migraine-like attacks^c	Treatment (time)/efficacy^d
1
PACAP38-placebo	0,33 h (10)	Bilat/2/pres/+	+/+/−	No	Antihistamine (10 h)/NR
PACAP38-glibenclamide	0,16 h (8)	Bilat/7/throb& pres/+	+/+/−	Yes	No
2
PACAP38-placebo	2,83 h (9)	Bilat/3/throb& pres/+	+/−/−	Yes	No
PACAP38-glibenclamide	0,33 h (10)	Bilat/3/throb& pres/+	+/−/−	Yes	No
3
PACAP38-placebo	5 h (2)	Bilat/1/pres/−	−/−/−	No	No
PACAP38-glibenclamide	5 h (3,7)	Bilat/2/pres/−	−/−/−	No	No
4
PACAP38-placebo	7 h (8)	Bilat/3/throb& pres/+	−/−/−	No	Paracetamol 500 mgIbuprofen (7 h)/Yes
PACAP38-glibenclamide	5 h (6)	Bilat/2/pres/+	−/−/−	No	No
5
PACAP38-placebo	0,66 h (5,11)	Bilat/2/throb& pres/+	−/−/−	No	No
PACAP38-glibenclamide	0,66 h (12)	Bilat/4/throb& pres/+	+/+/−	Yes	No
6
PACAP38-placebo	0,5 h (12)	Bilat/7/throb& pres/+	+/+/+	Yes	Paracetamol 500 mg (7 h)
PACAP38-glibenclamide	0,33 h (2,4)	Bilat/5/throb& pres/+	+/+/−	Yes	No
7
PACAP38-placebo	4,16 h (5)	Bilat/3/other/−	−/−/−	No	Paracetamol 500 mgIbuprofen (7 h)/Yes
PACAP38-glibenclamide	6 h (8)	Bilat/2/throb/−	−/−/−	No	No
8
PACAP38-placebo	3,33 h (5)	Bilat/1/other/−	−/−/−	No	No
PACAP38-glibenclamide	4 h (7)	Bilat/1/pres/−	+/−/−	No	No
9
PACAP38-placebo	None	–	+/−/−	–	No
PACAP38-glibenclamide	None	–	−/−/−	–	No
10
PACAP38-placebo	0,33 h (9)	Bilat/2/throb& pres/−	−/−/−	No	No
PACAP38-glibenclamide	10 h (12)	Bilat/1/throb& pres/−	−/−/−	No	Paracetamol 1 g (11 h)/Yes
11
PACAP38-placebo	0,5 h (5,11)	Bilat/8/throb& pres/+	+/−/−	Yes	Paracetamol 1g 400 mg ibuprofen(5 h)/Yes
PACAP38-glibenclamide	0,66 h (5,9)	Bilat/6/pres/+	−/−/−	No	Paracetamol 1 g 400 mg ibuprofen(6,5 h)/Yes
12
PACAP38-placebo	0,33 h (10)	Bilat/3/pres/−	−/−/−	No	Paracetamol 1 g, 400 mg ibuprofen (4 h)/Νo
PACAP38-glibenclamide	3 h (5,9)	Bilat/5/pres/−	+/−/−	No	Paracetamol 1 g, 400 mg ibuprofen (3 h)/Yes
13
PACAP38-placebo	3 h (4)	Bilat/1/pres/−	−/−/−	No	No
PACAP38-glibenclamide	2,66 h (5,8)	Bilat/2/throb& pres/−	−/−/−	No	No
14
PACAP38-placebo	0.16 h (8)	Until/2/pres/−	−/−/−	No	No
PACAP38-glibenclamide	6 h (10)	Bilat/2/press/−	−/−/−	No	No
15
PACAP38-placebo	0,66 h (9,25)	Bilat/2/pres/−	+/−/−	No	No
PACAP38-glibenclamide	0,33 h (5,9)	Bilat/4/throb/+	+/−/−	Yes	No
16
PACAP38-placebo	7 h (9)	Bilat/4/pres/+	+/+/−	Yes	Paracetamol 1 g, 400 mg ibuprofen (7 h)/Yes
PACAP38-glibenclamide	6 h (2,10)	Bilat/3/pres/+	+/−/−	Yes	No
17
PACAP38-placebo	None	–	+/−/−	–	No
PACAP38-glibenclamide	0,5 h (0,3)	Difus/1/pres/−	−/−/−		No
18
PACAP38-placebo	0,16 h (10)	Difus/1/pres/−	+/−/−	No	No
PACAP38-glibenclamide	0,16 h (8)	Difus/1/pres& throb/+	+/−/−	Yes	No
19
PACAP38-placebo	0.66 h (6)	Bilat/3/pres& throb/–	−/−/+	No	Paracetamol 1 g, 400 mg ibuprofen (6h)/Yes
PACAP38-glibenclamide	0,16 h (0,5)	Bilat/1/pres& throb/–	−/−/−	No	No
20
PACAP38-placebo	4,16 h (11.4)	Bilat/5/pres/+	−/−/−	No	Paracetamol 1 g, 400 mg ibuprofen (4,5 h)/Yes
PACAP38-glibenclamide	3,6 h (5)	Bilat/6/pres/+	+/−/−	Yes	Paracetamol 1 g, 400 mg ibuprofen (3,5 h)/NoParacetamol 1 g (10 h)/Yes

h: hour, NR: not reported.

a) Localization/intensity/quality (throb = throbbing; pres = pressing; diffuse)/aggravation (by cough during in-hospital phase and by movement during out-hospital phase).

b) Nausea/photophobia/phonophobia.

c) Migraine-like attacks must fulfil criteria B and C for migraine attack without aura according to ICHD-3, or mimic the patient’s usual migraine attacks and are treated with a rescue medication.

d) Pain freedom or pain relief (≥50% decrease of intensity) within 2 h.

On glibenclamide day participants reported nausea 10/20 and photophobia 3/20. On placebo day, 9/20 reported nausea, 5/20 photophobia and 1/20 phonophobia (Table 1). Five participants reported migraine-like headache (22) associated with nausea on the PACAP38-glibenclamide day and three on the PACAP38-placebo day. Moreover, three participants experienced nausea temporally unrelated to their headache (Table 1).

Cerebral hemodynamics

PACAP38 dilated the superficial temporal artery after the infusion compared to baseline (P < 0.001), and the dilation was sustained until T250 on both study days. Peak STA dilation was 45.88% (CI: 25.27–66.5%) at T70 on glibenclamide day and 42.33% (CI: 24.17–60.49) at T130 on placebo day (Figure 5A). There was no difference in the AUC_0-250min for STA diameter after glibenclamide (383.9 ± 67.77) compared to placebo (392.7 ± 66.34) (P = 0.176).

Figure 5.

Cerebral Hemodynamic after infusion of PACAP38 and administration of oral glibenclamide/placebo, from baseline to T250. (a) Mean change in diameter of superficial temporal artery (STA). PACAP38 dilated the STA on both study days (P < 0.001). Glibenclamide failed to prevent the effect of PACAP38 on the STA. (b) Mean change in velocity of middle cerebral artery (V_meanMCA). PACAP38 decreased V_meanMCA significantly (P < 0.01). Glibenclamide did not inhibit the PACAP38 induced changes.

V_meanMCA decreased significantly after PACAP38 infusion on both study days (P < 0.01). The peak decrease occurred at T20 and was 14.42% (CI: 8.74–20.09%) on the glibenclamide day and 12.86% (CI: 6.13–19.58%) on the placebo day (Figure 5B). No difference was observed in V_meanMCA between PACAP38-glibenclamide day and PACAP38-placebo day (AUC_0–250min: 16114 ± 2194, vs, 16098 ± 2232, P = 0.766).

Systemic hemodynamics

PACAP38 failed to dilate the RA on any study day (P > 0.05) (Figure 6A). There was no difference in the AUC_{0–250 min} for RA after glibenclamide (595.9 ± 77.84) compared with placebo (610 ± 91) (P = 0.294).

Figure 6.

Systemic hemodynamic after infusion of PACAP38 and administration of oral glibenclamide/placebo, from baseline to T260. (a) Mean change in diameter of radial artery (RA). PACAP38 did not dilate RA. Glibenclamide had no effect on the diameter of RA. (b) Mean arterial blood pressure (MABP) and heart rate (HR). PACAP38 decreased MABP and increased HR (P < 0.01), whereas glibenclamide did not inhibit PACAP38-induced changes.

We found no difference in MABP between glibenclamide day and placebo day over time (AUC_0-260min, 19349 ± 1795 vs. 18512 ± 4290, P = 0.647). On both study days, PACAP38 led to a decrease in MABP compared to baseline (P < 0.01) (Figure 6B). Maximum decrease was recorded at T10 on both study days. MABP decrease was almost eradicated by T20 (end of PACAP38 infusion). There was no difference in HR between glibenclamide day and placebo day over time (AUC_{0–260 min}, 20366 ± 2475 vs. 19707 ± 2629, P = 0.647). HR increased significantly from baseline after PACAP38 infusion on both study days (P < 0.01). The increase in HR was sustained throughout the in-hospital observation period (T260) on the placebo day. On glibenclamide day, HR was not significantly increased from baseline at T250 and T260 (P > 0.05). Peak increase in HR occurred at T20 on both study days and was 63.95% (CI: 45.55–82.36%) on glibenclamide day and 62.44% (CI: 45.39–79.49%) on placebo day (P < 0.01) (Figure 6B).

PACAP38 increased facial skin blood flow on both days compared to baseline (P < 0.001) (Figure 7). We found no difference in the AUC_{0–260 min} for facial skin blood flow after glibenclamide (242409 ± 30619) compared with placebo (250539.8 ± 33855) (P = 0.089).

Figure 7.

Facial skin blood flow after infusion of PACAP38 and administration of oral glibenclamide/placebo, from baseline to T260. (a) Relative changes in facial skin blood flow measured by laser speckle after infusion of PACAP38 and administration of oral glibenclamide/placebo from baseline to T260. PACAP38 increased the facial blood flow (P < 0.001) whereas glibenclamide did not inhibit PACAP38-induced changes. (b) Direct measurement of facial flushing measured by laser speckle after infusion of PACAP38 and administration of oral glibenclamide/placebo, at indicative time points. Blue areas indicate low blood flow, green moderate blood flow, and red high blood flow.

Adverse events

All participants (20/20) reported flushing, heat sensation and palpitation on both study days. Eight participants reported hunger after glibenclamide compared to four participants after placebo. The two participants that did not complete both study days, experienced nausea and were administered 10 mg metoclopramide intravenously on the PACAP38-glibenclamide day. Three patients experienced mild orthostatic hypotension by the end of the experiment. All above-mentioned symptoms resolved within 30 minutes.

Rescue Medication

Six participants used the provided rescue medication after glibenclamide compared to nine participants after placebo (P > 0.05).

Discussion

The major finding of the present study is that posttreatment with glibenclamide failed to attenuate PACAP38-induced headache and vascular changes in healthy volunteers. Moreover, PACAP38 caused headache with a prolonged STA dilation and a decrease in V_meanMCA. These results are in line with previous studies investigating the effects of PACAP38 in humans (3,23).

PACAP38-induced headache

PACAP38’s ability to trigger headache and migraine seems to implicate several possible mechanisms. Interestingly, 20-minute infusion of vasoactive intestinal peptide (VIP) induced short lasting vasodilation but no headache in healthy participants or migraine attack in migraine patients, while 2-hour infusion of VIP led to long lasting vasodilation associated with headache in healthy participants and migraine in migraine patients (24,25). PACAP38 also causes long-lasting dilation of extracerebral arteries associated with headache and migraine (3,26). Posttreatment with sumatriptan infusion reversed PACAP38 induced vasodilation and prevented migraine induction (27). However, pretreatment with sumatriptan reduced PACAP38-induced headache without altering PACAP38-induced vascular changes (28).

In rodents, mast cell degranulation caused prolonged vasodilation and activation of meningeal nociceptors as well as downstream activation of the spinal trigeminal nucleus (29). PACAP38 promotes mast cell degranulation by the orphan-receptor MrgB3 and leads to release of histamine and dural neurogenic inflammation (30 –32). Histamine can induce migraine-like attacks in 70% of migraine without aura patients (33). Of note, pre-treatment with the H₁-antihistamine, clemastine, partly prevented PACAP38-induced migraine in humans (34). Moreover, ketorolac, a non-steroidal anti-inflammatory drug that reduces mast cell degranulation (35) and blocks dural macrophage activation (36), attenuated PACAP38-induced headache in healthy volunteers. Interestingly, ketorolac exerted its effect without affecting PACAP38-induced arterial dilation (28).

Both PACAP38 and VIP activate VPAC1 and VPAC2 receptors while PAC1 is 1000-fold more sensitive for PACAP38 compared to VIP. Interestingly, VIP was able to induce headache in healthy participants and migraine in migraine patients (24,25), while a pituitary adenylate cyclase-activating polypeptide PAC1 receptor monoclonal antibody failed prevent migraine in a phase 2 trial (37). Collectively, diverse mechanisms and multiple sites of action, including vasodilation via cAMP upregulation (24) and mast cell degranulation (34) may contribute to PACAP38-induced headache. Studies focused on regulating multiple PACAP38 activated pathways are needed to clarify the role of PACAP in headache genesis.

PACAP38 and arterial dilation

In VSMCs, PACAP38 binds to G-protein-coupled receptors (GPCRs); vasoactive intestinal polypeptide receptor 1 (VPAC1), vasoactive intestinal polypeptide receptor 2 (VPAC2) and the PACAP type I receptor (PAC1) (38). Upon activation, they lead to cAMP upregulation, phosphorylation of K_ATP channels and vasorelaxation (5). Sumatriptan, which inhibits cAMP and PKA (39 –41), was able to reverse the vasodilatory effect of PACAP38 in migraine patients (27).

In vitro studies have demonstrated that glibenclamide is able to attenuate PACAP-induced dilation of cerebral, coronary and pulmonary arteries (15,42). K_ATP channel opener levcromakalim and calcitonin gene related peptide (CGRP) are strong vasodilators and both induce migraine attacks (43,44). Interestingly, in vivo studies demonstrated that glibenclamide abolished the effect of levcromakalim and CGRP-induced vasodilation in basilar (45,46), pial (47), and dural arteries (48). However, pretreatment with glibenclamide did not prevent levcromakalim and CGRP-induced vasodilation in healthy volunteers (49,50), which suggests interspecies differences.

The 10 mg dosage of glibenclamide used in the present study is the highest well-tolerated dose for humans, but represents only a fraction of the dosage that was used in animal models (20–30 mg/kg) to achieve significant blockade of vasodilation (51). Higher doses would not be safe to use in humans because of possible risk of severe hypoglycemia. It is likely that the inability to increase the dosage could be partially responsible for the lack of effect of glibenclamide on PACAP38 induced vascular changes. Of note, glibenclamide is a non-specific K_ATP channel blocker with a higher affinity for the SUR1 subunit (expressed in β-pancreatic cells, neurons, and TG) compared to the SUR2B subunit that is vastly expressed in VSMC_S (52). We could speculate that a selective SUR2B blocker would be more capable of blocking the effect of PACAP38 in cerebral arteries. To date, no selective SUR2B blockers have been available for clinical use.

Large conductance calcium-activated potassium channels (BK_Ca) are expressed in VSMCs and upon activation lead to arterial dilation (9). Preclinical studies indicated that PACAP38-mediated cAMP upregulation and activation of PKA also activates BK_Ca channels (8). Application of BK_Ca channel blocker paxilline attenuated PACAP-induced rat cerebellar artery dilation and a combined treatment with paxilline and glibenclamide abolished the dilation completely (8). Possible involvement of other types of potassium channels in PACAP38-induced vasodilation could explain the inability to attenuate it by solely blocking K_ATP channels.

PACAP38 and cerebral hemodynamics

A decrease in V_meanMCA after PACAP38 infusion has been reported in two studies using the TCD method (53), suggesting that PACAP38 infusion might affect cerebral blood flow (CBF) (54). A single-photon emission computed tomography (SPECT) study in healthy volunteers showed that regional cerebral blood flow (rCBF) decreased after PACAP38 infusion, but after correction for end-tidal partial pressure of CO₂ the reduction in rCBF was not significant (23). A possible effect of PACAP38 in CBF could not be solely attributed to the subsequent decrease of MABP as our results demonstrate that MABP reduction after PACAP38 infusion lasts 10–20 min while the decrease in V_meanMCA is sustained throughout the experiment (T250).

Direct measurement of the MCA using magnetic resonance angiography (MRA), showed no change in the diameter of the vessel after PACAP38 infusion (4,26). Thus, the observed decrease in V_meanMCA cannot not be interpreted as dilation of the MCA. Future studies should take advantage of imaging techniques such as perfusion MR, to elucidate the effect of PACAP38 on cerebral blood flow.

Conclusion

The major finding of the present study was that posttreatment with K_ATP channel blocker glibenclamide, was not able to attenuate PACAP38-induced headache and vascular changes in healthy volunteers. Moreover, glibenclamide did not have an impact on PACAP38-induced changes in mean arterial blood pressure, heart rate and facial flushing. PACAP38 possibly exerts its physiological actions through multiple diverse mechanisms. Our findings suggest that PACAP38-induced headache and vasodilation could be mediated by the SUR2B K_ATP channel and that the used glibenclamide dose is not potent enough to prevent PACAP38-induced physiological responses. More selective K_ATP channel blockers are needed to clarify the involvement of the different isoforms of sulfonylurea receptor subunits of K_ATP channel in PACAP38 signaling pathway.

Article Highlights

Posttreatment with non-selective K_ATP channel blocker glibenclamide did not prevent PACAP38-induced headache

Posttreatment with glibenclamide did not alter PACAP38-induced vascular changes

Selective K_ATP channel blockers are needed to investigate PACAP38 signaling pathway

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: FA reports personal fee for lecturing or advisory board from Novartis, Lundbeck, Eli Lilly and Teva. FA has also acted as primary investigator (PI) for phase 4 trials for Novartis and Teva. MA has received consulting fees and honoraria for lectures/presentations from AbbVie, Allergan, Amgen, Eli Lily, Lundbeck, Novartis,Teva. MA has also received personal payments for participating on data Safety Monitoring Board or Advisory Board for AbbVie, Amgen, Eli Lily, Lundbeck and Novartis.

LK, MMK, FE, HC and HG report no conflict of interest.

Funding

The research leading to these results has received funding from Lundbeck Foundation (R155?2014?171). M.A. was also supported by the Lundbeck Foundation Professor Grant (R310-2018-3711).

ORCID iDs

Lili Kokoti

MohammadAl-Mahdi Al-Karagholi

Hashmat Ghanizada

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Effect of K ATP channel blocker glibenclamide on PACAP38-induced headache and hemodynamic

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Methods

Participants

Experimental design

Headache and accompanying symptoms

Vascular variables

Plasma glucose

Data analysis and statistics

Data availability

Results

Participant characteristics

Headache and accompanying symptoms

Cerebral hemodynamics

Systemic hemodynamics

Adverse events

Rescue Medication

Discussion

PACAP38-induced headache

PACAP38 and arterial dilation

PACAP38 and cerebral hemodynamics

Conclusion

Article Highlights

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iDs

References