Background
Migraine is a neurovascular disorder characterized by recurrent attacks of incapacitating unilateral headaches, recently interconnected with an overall increased risk of stroke and cardiovascular disease [
1,
2]. Although its exact pathophysiology has not been elucidated completely, migraine headache has been associated with activation of the trigeminovascular system and increased release of calcitonin gene-related peptide (CGRP), resulting in dysfunctional nociceptive transmission and neurogenic dural vasodilatation [
3].
The triptans, serotonergic agonists with selective affinity for 5-HT
1B/1D/(1F) receptors, are specific drugs for the acute treatment. Their mechanism of action has been attributed to a dural perivascular inhibition of CGRP release, an inhibition of central nociception and/or a postjunctional constriction of (cranial) blood vessels [
4‐
6]. Because of the latter, the triptans are contraindicated in patients with cardiovascular risk factors or a history of cardiovascular disease.
Isometheptene is a sympathomimetic racemic drug available by prescription or over the counter in several countries, that has long been used for the acute treatment of primary headaches [
7,
8]. Nevertheless, a few case reports of acute intracranial vasoconstriction after its use [
9,
10] highlight its presumed vasoactive properties [
11]. Given that the development of new antimigraine agents with a beneficial cardiovascular safety profile is crucial, Tonix Pharmaceuticals™ separated isometheptene racemate into its enantiomers, (
S)-isometheptene and (
R)-isometheptene, a mixed-acting (tyramine-like/minor direct α
1-adrenoceptor) and an indirect-acting (tyramine-like) adrenergic receptor agonist, respectively. Additionally, (
R)-isometheptene is an imidazoline I
1 receptor agonist [
12], and previous studies have shown that: (i) imidazoline I
1 receptor knockout mice have a potentiated nociceptive perception, suggesting that this receptor could be associated with an endogenous analgesia system [
13]; (ii) (
R)-isometheptene decreased trigeminal sensitivity in two rat models of chronic migraine [
14]; and (iii) imidazoline I
1 receptor agonists, like moxonidine and agmatine induced a prejunctional inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats [
15]. Together, these findings suggest that a potential antimigraine action of (
R)-isometheptene could be mediated by inhibition of the trigeminal system. Hence, we hypothesized that the use of only (
R)-isometheptene will maintain its antimigraine therapeutic effect, while the major side effects associated with the racemate or (
S)-isometheptene (i.e. cranial vasoconstriction) will be diminished [
16].
On this basis, the present study set out to analyse the effects of the isometheptene enantiomers and the racemate on human isolated blood vessels (i.e. middle meningeal artery, proximal and distal coronary arteries, as well as saphenous vein) and trigeminal CGRP-induced neurogenic dural vasodilation in anaesthetized rats (through a closed cranial window).
Materials and methods
Human isolated blood vessels
Middle meningeal arteries [internal diameter (ID) 0.5–1.5 mm] were obtained from 11 patients (3 males, 8 females; mean age 53 ± 5 years) who underwent neurosurgical interventions requiring a trepanation of the skull. During surgery, the dura mater together with a small piece of meningeal artery was collected in a sterile organ protecting solution and was immediately transported to the laboratory. The meningeal arteries were dissected and placed in a cold (4 °C) oxygenated Krebs bicarbonate solution with the following composition (mmol/L): NaCl 119, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO2 25 and glucose 11.1; pH 7.4.
Saphenous veins (ID 0.5–3 mm) were obtained from 11 patients (10 males, 1 female; mean age 71 ± 2 years) who underwent coronary artery bypass surgery. Immediately after surgery, veins were placed in cold (4 °C) oxygenated Krebs buffer solution with the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO2 25 and glucose 8.3; pH 7.4.
Proximal (ID 2–3 mm) and distal (ID 0.5–1.0 mm) coronary arteries were obtained from 10 heart valve donors (6 males, 4 females; mean age 40 ± 5 years) who died of non-cardiac disorders: four traumatic brain injury, one benzodiazepine overdose, three anoxic encephalopathy and two cerebrovascular accident. The hearts were provided by the Heart Valve Bank Beverwijk (at that time still located in Rotterdam) from Dutch post-mortem donors, after donor mediation by The Dutch Transplantation Foundation (Leiden, The Netherlands), following removal of the aortic and pulmonary valves for homograft valve transplantation. All donors gave permission for research. Immediately after circulatory arrest, the hearts were stored at 4 °C in a sterile organ protecting solution and were brought to the laboratory within the first 24 h of death. The coronary arteries were dissected and placed in Krebs buffer with the same composition as the one used for the saphenous veins (see above). All blood vessels were used on the same day or stored overnight and used the following day for functional experiments.
The middle meningeal arteries and the distal coronary arteries were cut into ring segments of 1–2 mm length and suspended in Mulvany myographs on two parallel steel wires. The tension was normalized to 90% of the estimated diameter at 100 mmHg [
17]. The proximal coronary arteries and saphenous veins were cut into ring segments of about 3–4 mm length and suspended on stainless steel hooks in 15-mL organ baths. The vascular rings were stretched to a stable pretension of 10–15 mN, the optimal pretension as determined earlier [
17], and changes in tissue force were measured with an isometric force transducer (Harvard, South Natick, MA, U.S.A.) and recorded on a flatbed recorder (Servogor 124, Goerz, Neudorf, Austria). The buffer was aerated with 95% O
2 and 5% CO
2 and was maintained at 37 °C. The segments were allowed to equilibrate for at least 30 min and were washed every 15 min.
In vitro experimental protocols
Initially, segments were exposed to 30 mM KCl, followed by 100 mM KCl to determine the reference contractile response in each segment. Cumulative concentration response curves were constructed to (
S)-isometheptene, (
R)-isometheptene, isometheptene racemate, sumatriptan and noradrenaline, using whole logarithmic steps (1 nM to 100 μM). Sumatriptan and noradrenaline were used as positive controls, as previously reported [
17]. Finally, the functional integrity of the endothelium was assessed by observing the relaxation to substance P (10 nM) in arteries or bradykinin (10 μM) in saphenous veins after precontraction with the thromboxane A
2 analogue U46619 (10–100 nM).
Animals
Twelve male Sprague-Dawley rats (300–350 g; 8–10 weeks of age), purchased from Harlan Netherlands (Horst, the Netherlands), were maintained at a 12/12-h light-dark cycle in a special room at constant temperature (22 ± 2 °C) and humidity (50%), with food and water ad libitum. Only male rats were used to avoid crosstalk between CGRP and hormonal fluctuations of the oestrus cycle previously described in this model [
18,
19]. Experimental protocols were approved by the Erasmus Medical Center’s institutional ethics committee (EMC permission protocol number 3393), in accordance with the European directive 2010/63/EU and the ARRIVE guidelines for reporting experiments in animals [
20].
After anaesthesia with sodium pentobarbital (60 mg/kg i.p. followed by 18 mg/kg i.v. per hour), the trachea was cannulated and artificially ventilated (58 strokes/min.; small animal ventilator SAR 830 series, CWE Inc., Ardmore, PA, U.S.A). The adequacy of anaesthesia was judged by the absence of ocular reflexes and a negative tail flick test.
End-tidal pCO2 was monitored with a capnograph (Capstar 100 CWE Inc., PA, U.S.A.) and kept between 35 and 45 mmHg. The left femoral vein and artery were cannulated for i.v. administration of drugs and monitoring of mean arterial pressure (MAP), respectively. The animals’ body temperature was maintained at 37 °C by a homeothermic blanket (Harvard Instruments, Edenbridge, Kent, U.K.). The head of each rat was fixed in a stereotaxic frame and the parietal bone overlying a segment of the dural middle meningeal artery was drilled thin, applying cold saline until the artery was clearly visible. As skull drilling induces vasodilation, animals were allowed to rest at least for 1 h before the experimental protocol started. The artery diameter was recorded with an intravital microscopy setup (MZ16, Leica microsystem Ltd., Heerbrugg, Switzerland) using a cyan blue filter on a cold light source. A zoom lens (80-450x magnification) and a camera were used to display images on a standard PC monitor. The artery diameter (30–40 μm at baseline) was continuously monitored and measured with an intravital dimension analyser (IDA 1.2.1.10; U.K.). For periarterial electrical stimulation (ES), a bipolar stimulating electrode (NE 200X, Clark Electromedical, Edenbridge, Kent, U.K.) was placed on the surface of the bone approximately within 200 μm from the artery. The surface of the closed cranial window was stimulated at 5 Hz, 1 ms for 10 s (Stimulator model S88, Grass Instruments, West Warwick, RI, U.S.A.) with increasing voltage until maximum dilation was observed.
In vivo experimental protocols
After a stable hemodynamic condition for at least 60 min, baseline values of dural artery diameter and MAP were determined. Subsequently, the 12 rats were randomly divided into three sets (n = 4 each). In each set, a control vasodilator response of the middle meningeal artery was produced by either endogenous [released by ES (150–300 μA) or capsaicin (10 μg/kg, i.v.)] or exogenous CGRP (1 μg/kg, i.v.). A 30-min interval between control and each subsequent vasodilation was allowed for the recovery of baseline values, and 5 min before the next vasodilation, (R)-isometheptene, (S)-isometheptene or the racemate (3 mg/kg, i.v., each) were injected. The administration of the isometheptene enantiomers was alternated, and followed by racemate. In each case, there was a time interval of 5 min to allow the dural artery diameter and MAP to return to baseline, before the next vasodilator was administered. We have previously shown that repeated (up to 5 times) ES and treatment with capsaicin or CGRP produced reproducible increases in dural artery diameter (data not shown).
Statistical evaluation
All data are presented as mean ± SEM. The concentration response curves obtained in the vessels were analysed using GraphPad software (GraphPad software Inc., San Diego, CA, U.S.A.) to calculate the maximal effect (Emax) and pEC50 values. In case a concentration response curve did not reach a plateau, the contraction to the highest concentration was considered as Emax. Emax and pEC50 values were compared by unpaired t-test.
The peak increases in dural artery diameter (measured in arbitrary units) in anaesthetised rats are expressed as percent change from baseline. Changes in MAP are expressed in absolute values (mm Hg). A repeated measures one-way analysis of variance (ANOVA) followed by Tukey’s test was performed to examine the different effects per se between isometheptene enantiomers and the racemate. The dural vasodilator differences between the variables within one group were compared using an ANOVA followed by Dunnett’s test. Statistical significance was accepted at P < 0.05 (two-tailed).
Compounds
Apart from the anaesthetic (sodium pentobarbital), the drugs used in the present study were: isometheptene racemate, (R)-isometheptene and (S)-isometheptene (Tonix Pharmaceuticals Inc., New York, N.Y., U.S.A.); sumatriptan, bradykinin, noradrenaline, capsaicin, U46619 and substance P (Sigma Chemical Co., St. Louis, MO, U.S.A); and rat/human α-CGRP (NeoMPS S.A., Strasbourg, France). Capsaicin was dissolved in a mixture of tween 80, ethanol 70% and water (1:1:8), while the rest of the compounds were dissolved in either distilled water (in vitro) or physiological saline (in vivo). The doses mentioned in the text refer to the free base of substances in all cases.
Acknowledgements
Dr. Antoinette MaassenVanDenBrink was supported by the Netherlands Organisation for Scientific Research (Vidi grant 917.113.349), whereas Prof. Carlos M. Villalón, Eloísa Rubio-Beltrán and Alejandro Labastida-Ramírez were supported by Consejo Nacional de Ciencia y Tecnología (CONACyT; Grant No. 219707 to CMV and fellowship No. 409865 to ERB and 410778 to ALR; Mexico City). Dr. Kristian A. Haanes was supported by a postdoctoral fellowship from the International Headache Society.