Effect of the calcitonin gene-related peptide (CGRP) receptor antagonist telcagepant in human cranial arteries

Abstract

Introduction: Calcitonin gene-related peptide (CGRP) is a neuronal messenger in intracranial sensory nerves and is considered to play a significant role in migraine pathophysiology.

Materials and methods: We investigated the effect of the CGRP receptor antagonist, telcagepant, on CGRP-induced cranial vasodilatation in human isolated cerebral and middle meningeal arteries. We also studied the expression of the CGRP receptor components in cranial arteries with immunocytochemistry. Concentration response curves to αCGRP were performed in human isolated cerebral and middle meningeal arteries in the absence or presence of telcagepant. Arterial slices were stained for RAMP1, CLR and actin in a double immunofluorescence staining.

Results: In both arteries, we found that: (i) telcagepant was devoid of any contractile or relaxant effects per se; (ii) pretreatment with telcagepant antagonised the αCGRP-induced relaxation in a competitive manner; and (iii) immunohistochemistry revealed expression and co-localisation of CLR and RAMP1 in the smooth muscle cells in the media layer of both arteries.

Conclusions: Our findings provide morphological and functional data on the presence of CGRP receptors in cerebral and meningeal arteries, which illustrates a possible site of action of telcagepant in the treatment of migraine.

Keywords

CGRP receptors cerebral arteries meningeal arteries telcagepant MK-0974

Introduction

The intracranial circulation is supplied by calcitonin gene-related peptide (CGRP)-containing nerve fibres that originate in the trigeminal ganglion (1,2). Activation of the intracranial trigeminovascular system results in release of CGRP (3); this occurs in primary headaches and following subarachnoid hemorrhage (4–7). CGRP is a potent vasodilator in several species (8–10), increases heart rate (11) and has positive inotropic effects on isolated human trabeculae (12). CGRP initiates vascular responses through interaction with G-protein coupled receptors of the B-type that are primarily coupled to the activation of adenylyl cyclase and is independent of endothelium in human cerebral, meningeal (1,13,14) and coronary (10) vessels. CGRP receptor characterisations have relied on the use of the antagonist CGRP_8–37; more recently, however, several small dipeptide CGRP receptor antagonists have been reported such as olcegepant (BIBN4096BS) (15). Although this dipeptide derivative is effective in migraine (16), due to its molecular structure it has until now only be used parenterally. Recently, an orally bio available CGRP receptor antagonist, telcagepant (MK-0974), was described (17) and shown to be effective as an antimigraine drug (18,19).

The aims of the present study were to: (i) compare the functional responses to αCGRP and the antagonistic effects of telcagepant in human cerebral and meningeal arteries; and (ii) examine the expression of the receptor elements calcitonin-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) with immunohistochemistry in human cerebral and meningeal arteries.

Materials and methods

Human isolated arteries

Human cerebral (cortex) arteries (5 male, 4 female; age 45–76 years; internal diameter 300–500 µm) were removed at neurosurgical tumour operations in Lund, Sweden. Human meningeal arteries (2 male, 2 female; aged 42–62 years; internal diameter 500–750 µm) were obtained peri-operatively from patients undergoing neurosurgical procedures at Erasmus MC, Rotterdam, The Netherlands. All vessels were placed in buffer solution, for cerebral arteries, composition in mM: NaCl, 119; KCl, 4.7; CaCl₂, 1.5; MgSO₄, 1.17; NaHCO₃, 25; KH₂PO₄, 1.18; EDTA, 0.027; glucose, 5.5, pH 7.4; for meningeal arteries, composition in mM: NaCl 119, KCl 4.7, CaCl₂ 1.25, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and glucose 11.1; pH 7.4 aerated with 5% CO₂ in O₂ (carbogen) and transported to the laboratory for investigation. The Swedish part of the study was approved by Lund University Ethics Committee (LU99) and had the individual patients’ approval, while the ethics committee dealing with human experimentations at Erasmus Medical Centre, Rotterdam, approved the Dutch part of the study.

Functional experiments

The arteries were cut into cylindrical segments of 1 or 2 mm in length for in vitro pharmacological experiments. Each segment was mounted on two metal prongs, one of which was connected to a force displacement transducer and attached to a computer, and the other to a displacement device. The position of the holder could be changed by means of a movable unit allowing fine adjustments of vascular tension by varying the distance between the metal prongs (20,21).

The mounted specimens were immersed in temperature-controlled tissue baths (37°C) containing the buffer solution continuously gassed with carbogen, and the artery segments were allowed to equilibrate for approximately 30 min. The vessel tension was continuously recorded and the distance between the pins or wires was adjusted to maintain a resting tone of 4 mN for cerebral arteries or stretched to a tension normalised to 90% of l100 (the diameter when transmural pressure equals 100 mmHg (21)) for the meningeal segments.

Following the 30-min equilibration period, the contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution which had the same composition as the standard solution except that the NaCl was exchanged for an equimolar concentration of KCl for the experiments performed in the cerebral artery. In the experiments performed in the meningeal artery, vessel segments were exposed to 30 mM KCl once. Subsequently, the tissue was exposed to 100 mM KCl to determine the maximum contractile response to KCl.

The relaxant effect of human αCGRP was examined by cumulative application of increasing concentrations of the peptide in the absence or presence of various concentrations of the antagonist telcagepant. Segments were precontracted with 1 µM U46619 (cerebral arteries) or 30 mM KCl (meningeal arteries) before αCGRP was added. In the meningeal artery, each segment was exposed to a single cumulative concentration-effect curve and a matched pair’s protocol was used where one segment acted as control (no antagonist present) while in another segment from the same artery, the agonist response was assessed following equilibration (20–30 min) with 1 µM of the antagonist. After wash out, the functional integrity of the endothelium was verified by observing relaxation to substance P (1–10 nM) after precontraction with the thromboxane A₂ analogue U46619 (10 nM). In the cerebral artery, due to the scarcity of the tissue, cumulative concentration-effect curves in the absence or presence of the antagonist were performed in the same segments. The first curve acted as control (no antagonist present). After washout, the next curve was then performed in the presence of the antagonist (10 nM, 100 nM or 1 µM).

Compounds

The following materials were used in the in vitro experiments: human αCGRP (NeoMPS S.A., Strasbourg, France, and Sigma, St Louis, MO, USA, for the Dutch and Swedish experiments, respectively) and U46619 (Sigma). Telcagepant (MK-0974) was synthesized by the Medicinal Chemistry Department, Merck Sharp and Dohme Research Laboratories, USA. The αCGRP and U46619 were dissolved in water and stored in aliquots at −20°C. Telcagepant was dissolved in dimethylsulphoxide (DMSO) and stored in aliquots at −20°C. When the compounds were to be used, they were diluted in saline.

Analysis of data

The vasodilator response to CGRP was expressed relative to the contraction evoked by U46619 or KCl, respectively (=100%). For each segment, the maximum vasodilator effect (E_max) was calculated. The concentration-response curves for all agonists were analysed using non-linear regression analysis and the potency of agonists was expressed as pEC₅₀ (i.e. negative logarithm of the molar concentration of agonist inducing half maximum response) using Graph Pad Prism v.4.0 (Graph Pad Software Inc., San Diego, CA, USA). The blocking potency of the antagonists was estimated by calculating EC₅₀ ratios and plotting a Schild-plot (22) using linear regression to get the slope value. The pA₂ represents the negative logarithm of the concentration of antagonist that induces a 2-fold shift of the concentration response curve to the right. This parameter can be calculated in the case of competitive antagonism, i.e. when the slope of the Schild-plot is equal to unity. In meningeal arteries, only one concentration of telcagepant was studied; in these cases, ‘apparent pK_B’ (a parameter similar to the pA₂, which is used in cases where antagonism has not been demonstrated to be competitive in nature) values were calculated, constraining the Schild slope to unity. Since it was not feasible to use agonist concentrations higher than 3 µM, concentration response curves in the presence of higher concentrations of antagonist did not always reach a plateau. In these cases, the concentration response curves were extrapolated, considering the maximal response in the absence of antagonist as E_max.

Data are expressed as mean values ± SEM and ‘n’ refers to the number of patients from whom the vessels were collected. Statistically significant differences in pEC₅₀ values were examined by Mann–Whitney U-test.

Immunohistochemistry

For immunofluorescence, the cerebral and meningeal artery segments obtained peri-operatively from patients were directly snap-frozen immediately after arrival in the laboratories and were then embedded in Tissue TEK (Gibco, Invitrogen A/S, Taastrup, Denmark), frozen at −80°C and subsequently sectioned into 10-µm thick slices. Cryostat sections were fixed for 10 min in ice-cold acetone (−20°C) and thereafter rehydrated in phosphate-buffered saline (PBS, pH 7.2) containing 0.25% Triton X-100 (PBST), for 3 × 5 min. The sections were then permeabilised with PBST and blocked for 1 h in blocking solution containing PBS and 5% normal donkey serum and then incubated overnight at 4°C with either of the following primary antibodies: rabbit anti RAMP1 (Santa Cruz Biotechnology, CA, USA; sc-11379) diluted at 1:50, or rabbit anti CLR (Alpha Diagnostic International, SA, USA; CRLR-11A) diluted at 1:100. The dilutions of the primary antibodies were done in PBST, 1% bovine serum albumin (BSA) and 3% normal donkey serum. On the second day, sections were raised to room temperature and rinsed in PBST (3 × 15 min). Sections were subsequently incubated with the secondary antibody (1 h, room-temperature). The secondary antibody used was Cy™² conjugated donkey anti rabbit (Jackson ImmunoResearch, West Grove, PA, USA; 711-165-152) diluted 1:200 in PBST and 1% BSA. The sections were washed subsequently with PBST and mounted with Crystal mounting medium (Sigma). To determine the cellular localisation of RAMP1 and CLR, double immunofluorescence was performed by addition of a mouse anti-smooth muscle actin antibody (Santa Cruz; sc-53015) diluted 1:200 in PBST, 1% BSA and 3% normal donkey serum. As secondary antibody, Texas Red-conjugated donkey anti-mouse was used (Jackson ImmunoResearch; 715-076-150), diluted 1:200 in PBST and 1% BSA. In order to co-localise RAMP1 and CLR, a new CLR antibody was used (anti-goat, 1:50; Santa Cruz; sc-18007). Vectashield medium containing 4′,6-diamidino-2-phenylindole (DAPI) staining nucleuses was used on some sections (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA).

Immunoreactivity was visualised and photographed with an Olympus microscope (BX 60, Japan) at the appropriate wavelength (FITC Filterblock N B-2EC, EX465–495, EM515–555; Filterblock N G-2EC, EX540/25, EM605/55; DAPI Filterblock N UV-2EC, EX340–380, EM435–485). Adobe Photoshop CS3 was used to visualise co-labelling by superimposing the digital images. Negative controls for all antibodies were made by omitting primary antibodies. In all cases, no specific staining was found; only autofluorescence in lamina elastica interna was seen (not shown). To evaluate the autofluorescence in lamina elastica interna, controls were made with only primary antibodies.

Results

Functional studies to αCGRP in human isolated arteries

The cumulative administration of αCGRP caused a concentration-dependent relaxation of cerebral arteries precontracted with U46619, yielding a pEC₅₀ value of 9.0 ± 0.2 and an E_max of 56 ± 6% (Figure 1). Also, in the meningeal artery, αCGRP induced a concentration-dependent relaxation, the E_max amounting to 50 ± 11% of precontraction with 30 mM KCl and pEC₅₀ value of 8.7 ± 0.1 (Figure 2).

Figure 1.

Relaxant effect of αCGRP on human cerebral arteries that were precontracted with U46619. Concentration response curves to αCGRP in the absence or presence of increasing concentrations of telcagepant (left). There was a clear shift to the right in the concentration effect curve. The average Schild plot of the concentration response curves in the cerebral arteries (right). Values given represent mean ± SEM; number of subjects, n = 9.

Figure 2.

Relaxant effect of αCGRP on human meningeal arteries that were precontracted with 30 mM KCl. Concentration response curves to αCGRP in the absence or presence of 1 µM telcagepant (left). There was a clear shift to the right in the concentration effect curve. The apparent pK_B value was calculated with a slope (dotted line) constrained to the unity (right). Values given represent mean ± SEM; number of subjects, n = 4.

Effects of telcagepant in human isolated arteries

The CGRP receptor antagonist telcagepant, tested in concentrations up to 10 µM, did not show any marked vasomotor responses in any of the isolated vessel segments at basal tone (contraction response with E_max of 2.0 ± 2.9%; n = 9). Pretreatment with telcagepant at increasing concentrations (10 nM to 1 µM) shifted the concentration response curves to αCGRP to the right without changing the maximal effect (Figure 1). In the meningeal arteries, CGRP induced an E_max of 55 ± 7% in the presence of 1 µM telcagepant (Figure 2). The pA₂ value amounted to 9.37 ± 0.12 in cerebral artery; the slope of the Schild plot did not differ from unity (0.97 ± 0.17). In the meningeal arteries, the apparent pK_B value was 8.03 ± 0.16 (pA₂ could not be calculated since only one concentration of telcagepant was studied, the slope of the plot was constrained to unity to calculate the apparent pK_B).

Immunohistochemistry of human arteries

The distributions of RAMP1 and CLR in human cerebral (Figure 3A) and meningeal (Figure 3B) arteries were studied by immunohistochemistry. We observed positive immunoreactions for RAMP1 and CLR in the smooth muscle cell layer (media layer) of cerebral and meningeal artery segments. Localisation of the CGRP receptor components in the smooth muscle layer was confirmed by double staining with an antibody specific for actin. In separate experiments using another CLR antibody, we verified that the two receptor components RAMP1 and CLR co-localised in the smooth muscle cells (Figure 4). There were no obvious positive immunoreactions in the endothelium or in the lamina elastica interna; the latter is strongly autofluorescent, especially in the green filter.

Figure 3.

Immunohistochemistry of human cerebral (A) and meningeal (B) artery segments. Antibodies specific for RAMP1 and CLR showed positive staining in the walls of the artery segments (insert, a higher magnification). Co-staining with actin-specific antibody revealed the localisation of the immunoreactions in the smooth muscle cells. We observed no staining in endothelium or in the adventitial layers. Marker, 100 µm.

Figure 4.

Immunohistochemistry of human cerebral (A) and meningeal (B) artery segments. Antibodies specific for RAMP1 and CLR showed positive staining in the cytoplasm of the smooth muscle cells in walls of the artery. The receptor components co-localised (merged, arrows). DAPI (blue), staining of the nuclei, Marker, 100 µm.

Discussion

CGRP receptors have long been regarded as a useful target for the development of novel antimigraine therapies (23). In this study, we have shown that CGRP induced vasodilatation and that telcagepant had no direct vasoconstrictor or vasodilator effect per se on isolated human cerebral and meningeal arteries. However, telcagepant was able to block, in a competitive manner, the vasodilator effect of αCGRP on these blood vessels. We also showed that the CGRP receptor complex, consisting of CLR and RAMP1, is co-expressed in the smooth muscle cells of human cerebral and meningeal arteries and not present in the endothelium or in the adventitia, which is in agreement with a previous study of human intracranial arteries (24).

As stated above, telcagepant did not show any evidence of direct vasoconstrictor or vasodilator effect when given alone. If the same will apply to observations made in vivo, these findings suggest that CGRP receptors normally do not have a tonus role (25). This profile of telcagepant agrees well with previous studies of CGRP receptor antagonists such as CGRP_8–37, Compound 1 (26), and olcegepant (10,13,27).

The relaxant responses to αCGRP were not different in cerebral and meningeal arteries and were in accordance with previous studies (13,26,28). Telcagepant antagonised relaxations induced by αCGRP with a potency that seemed somewhat lower in the middle meningeal than in the cerebral artery. Because of the small methodological differences between the protocols used for the both tissues, no statistical comparison was made. However, since the potency of telcagepant was determined using the control curves in each respectively tissue, the results obtained in the two laboratories should be comparable. Further, in pilot experiments we demonstrated that the different precontractions used in the two laboratories do not affect the response to CGRP (data not shown). When comparing the effects of telcagepant with those of the CGRP receptor antagonist olcegepant, telcagepant shows activity in higher concentrations (about one to two log units) (13,26), thus being less potent than olcegepant which agrees with the K_i of respective compounds (telcagepant, 0.77 nM; olcegepant, 0.014 nM) (29,30). However, telcagepant has the advantage that it can be given as an oral medication (19), whereas olcegepant is not orally available (16).

In comparing the clinical effects, it is interesting that both compounds required substantially higher plasma concentrations relative to their in vitro pA₂ to achieve clinical efficacy for the acute treatment of migraine (16,19). For example, plasma concentrations of telcagepant associated with clinical efficacy are in the micromolar range, which is substantially higher than the pA₂ that we have seen for the cranial vascular effect (in the nanomolar range). Several factors may be involved in explaining this discrepancy, namely:

The difference may be due, in part, to the high protein binding of these compounds.

A concentration of drug equal to the pA₂ value may not be sufficient to decrease functional responses since it only shifts the concentration response curves 2-fold to the right – most likely a concentration of a least 10 times pA₂ would functionally inhibit relaxations to CGRP.

As nerve terminals releasing CGRP are located in the adventitia close to the media layer of the blood vessels, the concentration of telcagepant at the receptors may be substantially smaller than that at the lumen of the blood vessel, i.e. the plasma concentration. This phenomenon is unlikely to occur in vitro, where the antagonist can reach the CGRP receptors from both the luminal and abluminal sides.

The therapeutic effect of CGRP receptor antagonists could obviously also be mediated via other pathways than only inhibition of blood vessel dilatation induced by CGRP. Penetration of telcagepant through the blood–brain barrier may be necessary in addition to the peripheral blockade to achieve anti-migraine efficacy (31).

Arguments in favour of a neuronal mechanism are the lack of presynaptic CGRP receptors in the meninges, which suggests that exogenous CGRP is unlikely to modify the innervating sensory nerve fibres directly (32). This finding is also in agreement with in vivo data obtained in the rat, suggesting that an action of CGRP on the dura mater cannot account for the activation of peripheral afferents during migraine (33). In this study, the effects of CGRP in the meninges, including meningeal vasodilatation, were not sufficient to activate or sensitise meningeal nociceptors. Clearly, further studies are needed to resolve the therapeutic mechanisms involved of CGRP receptor antagonists.

Conclusions

Telcagepant antagonises relaxations induced by αCGRP with a potency that is consistent between human cerebral and meningeal arteries, without affecting the vascular tone per se. To predict potential vascular side effects, the effects of telcagepant obviously have to be investigated in more arteries, including the human coronary artery, as well as an in vivo model. Also, it remains to be demonstrated whether inhibition of vasodilatation by CGRP mediates the therapeutic action of telcagepant or that central penetration is also required. Our findings on the vascular properties of telcagepant provide insight into a possible site of action in the treatment of migraine.

Footnotes

Acknowledgement

Lars Edvinsson and Kayi Y. Chan contributed equally to this study.

References

Edvinsson

. Functional role of perivascular peptides in the control of cerebral circulation. Trends Neurosci 1985; 8: 126–131.

Uddman

Edvinsson

Ekman

Kingman

McCulloch

. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P. Neurosci Lett 1985; 62: 131–136.

Goadsby

Edvinsson

Ekman

. Release of vasoactive peptides in the extracerebral circulation of humans and the cat during activation of the trigeminovascular system. Ann Neurol 1988; 23: 193–196.

Goadsby

Edvinsson

. The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann Neurol 1993; 33: 48–56.

Goadsby

Edvinsson

Ekman

. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28: 183–187.

Juul

Edvinsson

Gisvold

Ekman

Brubakk

Fredriksen

. Calcitonin gene-related peptide-LI in subarachnoid haemorrhage in man. Signs of activation of the trigemino-cerebrovascular system? Br J Neurosurg 1990; 4: 171–179.

Juul

Hara

Gisvold

. Alterations in perivascular dilatory neuropeptides (CGRP, SP, VIP) in the external jugular vein and in the cerebrospinal fluid following subarachnoid haemorrhage in man. Acta Neurochir (Wien) 1995; 132: 32–41.

Edvinsson

Ekman

Jansen

Ottosson

Uddman

. Peptide-containing nerve fibers in human cerebral arteries: immunocytochemistry, radioimmunoassay, and in vitro pharmacology. Ann Neurol 1987; 21: 431–437.

Bell

McDermott

. Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev 1996; 48: 253–288.

10.

Gupta

Mehrotra

Villalon

. Characterisation of CGRP receptors in human and porcine isolated coronary arteries: evidence for CGRP receptor heterogeneity. Eur J Pharmacol 2006; 530: 107–116.

11.

Franco-Cereceda

Lundberg

. Actions of calcitonin gene-related peptide and tachykinins in relation to the contractile effects of capsaicin in the guinea-pig and rat heart in vitro . Naunyn Schmiedebergs Arch Pharmacol 1988; 337: 649–655.

12.

Saetrum Opgaard

Hasbak

de Vries

Saxena

Edvinsson

. Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae. Eur J Pharmacol 2000; 397: 373–382.

13.

Gupta

Mehrotra

Avezaat

Villalon

Saxena

Maassenvandenbrink

. Characterisation of CGRP receptors in the human isolated middle meningeal artery. Life Sci 2006; 79: 265–271.

14.

Jansen-Olesen

Mortensen

Edvinsson

. Calcitonin gene-related peptide is released from capsaicin-sensitive nerve fibres and induces vasodilatation of human cerebral arteries concomitant with activation of adenylyl cyclase. Cephalalgia 1996; 16: 310–316.

15.

Doods

Arndt

Rudolf

Just

. CGRP antagonists: unravelling the role of CGRP in migraine. Trends Pharmacol Sci 2007; 28: 580–587.

16.

Olesen

Diener

Husstedt

. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 2004; 350: 1104–1110.

17.

Paone

Shaw

Nguyen

. Potent, orally bioavailable calcitonin gene-related peptide receptor antagonists for the treatment of migraine: discovery of N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin- 1-yl)piperidine-1-carboxamide (MK-0974). J Med Chem 2007; 50: 5564–5567.

18.

Ferrari

Dodick

. Efficacy and tolerability of MK-0974 (telcagepant), a new oral antagonist of calcitonin gene-related peptide receptor, compared with zolmitriptan for acute migraine: a randomised, placebo-controlled, parallel-treatment trial. Lancet 2008; 372: 2115–2123.

19.

Mannix

Fan

. Randomized controlled trial of an oral CGRP receptor antagonist, MK-0974, in acute treatment of migraine. Neurology 2008; 70: 1304–1312.

20.

Hogestatt

Andersson

Edvinsson

. Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels. Acta Physiol Scand 1983; 117: 49–61.

21.

Mulvany

Halpern

. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 1977; 41: 19–26.

22.

Arunlakshana

Schild

. Some quantitative uses of drug antagonists. Br J Pharmacol Chemother 1959; 14: 48–58.

23.

Edvinsson

Goadsby

. Extracerebral manifestations in migraine. A peptidergic involvement? J Int Med 1990; 228: 299–304.

24.

Oliver

Wainwright

Edvinsson

Pickard

Hill

. Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature. J Cereb Blood Flow Metab 2002; 22: 620–629.

25.

Shen

Pittman

Buie

. Functional role of alpha-calcitonin gene-related peptide in the regulation of the cardiovascular system. J Pharmacol Exp Ther 2001; 298: 551–558.

26.

Edvinsson

Sams

Jansen-Olesen

. Characterisation of the effects of a non-peptide CGRP receptor antagonist in SK-N-MC cells and isolated human cerebral arteries. Eur J Pharmacol 2001; 415: 39–44.

27.

Edvinsson

Alm

Shaw

. Effect of the CGRP receptor antagonist BIBN4096BS in human cerebral, coronary and omental arteries and in SK-N-MC cells. Eur J Pharmacol 2002; 434: 49–53.

28.

Verheggen

Bumann

Kaumann

. BIBN4096BS is a potent competitive antagonist of the relaxant effects of alpha-CGRP on human temporal artery: comparison with CGRP(8-37). Br J Pharmacol 2002; 136: 120–126.

29.

Doods

Hallermayer

. Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist. Br J Pharmacol 2000; 129: 420–423.

30.

Salvatore

Hershey

Corcoran

. Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carbox amide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine. J Pharmacol Exp Ther 2008; 324: 416–421.

31.

Edvinsson

. CGRP blockers in migraine therapy: where do they act? Br J Pharmacol 2008; 155: 967–969.

32.

Lennerz

Ruhle

Ceppa

. Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: differences between peripheral and central CGRP receptor distribution. J Comp Neurol 2008; 507: 1277–1299.

33.

Levy

Burstein

Strassman

. Calcitonin gene-related peptide does not excite or sensitize meningeal nociceptors: implications for the pathophysiology of migraine. Ann Neurol 2005; 58: 698–705.