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Examining the binding properties of MK-0974: A CGRP receptor antagonist for the acute treatment of migraine
European Journal of Pharmacology 602 (2009) 250–254
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Examining the binding properties of MK-0974: A CGRP receptor antagonist for the acute treatment of migraine☆ Eric L. Moore a,⁎, Christopher S. Burgey b, Daniel V. Paone b, Anthony W. Shaw b, Yui S. Tang c, Stefanie A. Kane a, Christopher A. Salvatore a a b c
Merck Research Laboratories, Department of Pain Research, West Point, PA 19486, USA Merck Research Laboratories, Department of Medicinal Chemistry, West Point, PA 19486, USA Merck Research Laboratories, DMPK Global Technologies, Rahway, NJ 07065, USA
a r t i c l e
i n f o
Article history: Received 27 August 2008 Received in revised form 6 November 2008 Accepted 24 November 2008 Available online 3 December 2008 Keywords: Migraine CGRP Telcagepant Radioligand MK-0974
a b s t r a c t Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent and selective antagonist of the human and rhesus CGRP receptors and is currently in Phase III clinical studies for the acute treatment of migraine. The pharmacology of MK-0974 has been studied extensively, but there has not been a thorough characterization of its binding properties. Here, we characterize the binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC) membranes and rhesus cerebellum. [3H]MK-0974 displayed reversible and saturable binding to both SK-N-MC membranes and rhesus cerebellum with a KD of 1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor displaced [3H]MK-0974 in a concentrationdependent manner in competition binding experiments. Both CGRP and adrenomedullin demonstrated biphasic competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced [3H]MK-0974 in a monophasic fashion. In competitive binding studies with [3H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPγS. In kinetic binding experiments, the off-rate of [3H]MK-0974 was determined to be 0.51 min− 1 with a half-life of 1.3 min. In conclusion, the radioligand [3H]MK-0974 has proven to be a useful tool for studying the binding characteristics of MK-0974 and has broadened our understanding of this promising molecule. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide that is produced by alternative splicing of the calcitonin gene (Amara et al., 1982). This peptide exerts its biological effects by binding to a receptor consisting of the G-protein-coupled receptor (GPCR), calcitonin receptor-like receptor (CL receptor) and a single transmembrane protein designated receptor activity-modifying protein (RAMP) 1 (McLatchie et al., 1998). A third protein known as receptor component protein (RCP) is located intracellularly and is required for signal transduction (Evans et al., 2000). In addition to the CGRP receptor, calcitonin receptor-like receptor is also able to form high affinity adrenomedullin receptors by dimerization with either receptor activity-modifying protein 2 (RAMP2) or receptor activity-modifying protein 3 (RAMP3) (McLatchie et al., 1998).
☆ Authors are employees of Merck & Co., Inc. and potentially own stock and/or hold stock options in the Company. ⁎ Corresponding author. Merck Research Laboratories, WP26A-2000, West Point, PA 19486, USA. Tel.: +1 215 652 2042; fax: +1 215 993 1401. E-mail address: [email protected] (E.L. Moore). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.050
CGRP is widely expressed and exhibits a range of biological functions with the most pronounced being vasodilation. CGRP is the most powerful of the vasodilator transmitters (Brain et al., 1985) and its vasoactive effects have been demonstrated in a variety of blood vessels including those of the cerebral vasculature (Jansen et al., 1992). CGRP-expressing trigeminal nerve endings have been shown to innervate these cerebral blood vessels (Uddman et al., 1985). Multiple lines of evidence point to a possible role for CGRP in migraine headache. CGRP levels in cranial circulation are increased during migraine attacks (Goadsby et al., 1990) and successful treatment of migraine pain with a triptan normalizes CGRP levels (Goadsby and Edvinsson, 1993). In addition, intravenous administration of CGRP to migraineurs results in migraine-like symptoms in some patients (Lassen et al., 1998). More recently, the potent CGRP receptor antagonist olcegepant (BIBN4096BS) demonstrated clinical proof-ofconcept for the acute treatment of migraine with intravenous dosing (Olesen et al., 2004). This body of evidence implicating CGRP in the pathophysiology of migraine led us to initiate our own research program to discover orally bioavailable small-molecule CGRP receptor antagonists that would be suitable for the treatment of migraine. The current standard of care for the treatment of migraine are the 5HT1B/1D receptor agonists which
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new class of migraine therapy devoid of these cardiovascular liabilities would be a significant advance in migraine care. We have recently described the identification and pharmacological characterization of MK-0974 (Paone et al., 2007; Salvatore et al., 2008), which is the first orally bioavailable compound in this class to show clinical proof-ofefficacy comparable to triptans in a Phase 2 study (Ho et al., 2008a, 2008b). The purpose of the study presented here is to characterize the binding properties of the tritiated radioanalog [3H]MK-0974 to human and monkey CGRP receptors. 2. Materials and methods 2.1. Membranes and rhesus cerebellum preparation SK-N-MC membranes were purchased from Receptor Biology, Inc (Beltsville, MD). Rhesus cerebellum tissue was disrupted in a laboratory homogenizer in cold buffer containing 10 mM HEPES and 5 mM MgCl2. This homogenate was used directly in the binding assays. 2.2. [3H]MK-0974 receptor binding assays
Fig. 1. Chemical structures of A) [3H]MK-0974 and B) Compound 3.
form the triptan class of anti-migraine drugs (Dodick et al., 2004). While adequately safe when used as directed, the triptans are direct vasoconstrictors and are therefore contraindicated for patients with cardiovascular disease (Goadsby et al., 2002). The development of a
Binding assays were performed by combining [3H]MK-0974, 500 nM Compound 3 (Hershey et al., 2005, Fig. 1B) for non-specific binding, and either 50 μg/well SK-N-MC membrane or 1.5 mg/well rhesus cerebellum homogenate. The assay mixtures were incubated for various times at room temperature in binding buffer (10 mM HEPES, 5 mM MgCl2, 0.2% BSA) in a total volume of 1 ml. Free radioligand was separated from membrane-bound radioligand by filtration through polyethylene imine (0.5%) treated GF/B glass fiber
Fig. 2. Saturation binding curves for [3H]MK-0974 on A, 50 μg/well SK-N-MC membranes or B, 1.5 mg/well rhesus cerebellum homogenate. Specific binding was determined by subtracting non-specific binding from the total binding curve. Symbols and error bars represent mean and standard deviation from 5 separate titrations. Panel C shows the association kinetics of [3H]MK-0974 binding to SK-N-MC membranes at room temperature. 1.5 nM [3H]MK-0974 was added to 50 μg/well SK-N-MC membranes and binding was monitored over a period of 10 min. This is a representative graph of three separate experiments. Symbols and error bars represent the mean and standard deviation of 5 points. Panel D shows the dissociation kinetics of [3H]MK-0974 binding to SK-N-MC membranes. 1.5 nM [3H]MK-0974 was added to 50 μg/well SK-N-MC membranes and incubated 3 h at room temperature. Dissociation was initiated by the addition of 500 nM compound 3 and monitored over a period of 20 min. Symbols and error bars represent the mean and standard deviation from two separate experiments.
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filters using a cell harvester. Radioactivity was determined using a Topcount scintillation counter from Perkin-Elmer (Waltham, MA). 2.3. [3H]MK-0974 saturation binding Saturation binding assays were performed by combining increasing concentrations of [3H]MK-0974, 500 nM Compound 3 for nonspecific binding, and either 50 μg/well SK-N-MC membrane or 1.5 mg/ well rhesus cerebellum homogenate. The mixtures were incubated overnight (18 h) at room temperature in binding buffer in a total volume of 1 ml. 2.4. [3H]MK-0974 competition binding Competition binding assays were performed by combining increasing concentrations of reference compounds, 500 nM Compound 3 for non-specific binding, 1.5 nM [3H]MK-0974, and either 50 μg/well SK-N-MC membrane or 1.5 mg/well rhesus cerebellum. The mixtures were incubated for 3 h at room temperature in binding buffer in a total volume of 1 ml.
2.5. [3H]MK-0974 association kinetics Association kinetic assays were performed by combining 1.5 nM [3H] MK-0974 with 50 μg/well SK-N-MC membranes in binding buffer and incubating at room temperature for various times from 30 s to 10 min. 2.6. [3H]MK-0974 dissociation kinetics Dissociation kinetic assays were performed by combining 1.5 nM [3H]MK-0974 with 50 μg/well SK-N-MC membranes in binding buffer and incubating at room temperature for 3 h. At that point, 500 nM Compound 3 was added and dissociation was monitored for various intervals from 0.5 to 20 min. 3. Results 3.1. Saturation binding studies Saturation binding experiments using [3H]MK-0974 (Fig. 1A) were carried out on SK-N-MC membranes and rhesus cerebellum
Fig. 3. Competition binding studies. Displacement of 1.5 nM [3H]MK-0974 from 50 μg/well SK-N-MC membranes by A, α-CGRP, B, β-CGRP, C, adrenomedullin, D, α-CGRP(8-37), E, unlabelled MK-0974. Symbols and error bars represent the mean and standard deviations from 3–5 separate experiments. Panel F shows displacement of 1.5 nM [3H]MK-0974 from SK-N-MC by α-CGRP in the presence or absence of 10 μM GTP. Symbols and error bars represent the mean and standard deviations from 2 separate experiments with 4 points each.
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homogenate to determine the KD and Bmax. [3H]MK-0974 displayed saturable binding to SK-N-MC membranes with a KD of 1.9 nM and Bmax of 479 fmol/mg protein (Fig. 2A). [3H]MK-0974 also displayed saturable binding to rhesus cerebellum homogenate with a KD of 1.3 nM and Bmax of 20 fmol/mg (Fig. 2B). 3.2. Kinetic binding studies Kinetic binding experiments were performed to determine the on and off-rates for [3H]MK-0974 on SK-N-MC membranes. In the association assay, [3H]MK-0974 reached saturation very quickly with an apparent kon of 1.01 × 109 M− 1 min− 1 (Fig. 2C). For the dissociation of [3H]MK-0974, the koff was calculated as 0.51 min− 1 with a t1/2 of 1.3 min (Fig. 2D). 3.3. Competition binding studies Several CGRP receptor agonists and antagonists were evaluated in competition binding studies to determine their potencies in displacing [3H]MK-0974 from SK-N-MC membranes. The peptide agonists α-CGRP, β-CGRP, and adrenomedullin all displayed biphasic competition curves with a high and low-affinity site while the peptide antagonist α-CGRP(8-37) displayed monophasic competition (Fig. 3, A–D). The addition of 10 μM GTP to the binding mixture shifted most of the binding from the high-affinity site to the low-affinity site for α-CGRP (Fig. 3F). None of these peptides were able to completely displace [3H]MK-0974 while unlabelled MK-0974 completely displaced [3H]MK-0974 with a K i of 1.0 nM (Fig. 3E). Amylin and calcitonin were unable to displace [3H]MK-0974 at concentrations up to 10 μM (data not shown). Human α-CGRP and MK-0974 were also evaluated in competition binding assays on rhesus cerebellum homogenate and displayed similar profiles (Fig. 4, panels A and B).
Fig. 4. Displacement of 1.5 nM [3H]MK-0974 from rhesus cerebellum homogenate by A, α-CGRP, and B, unlabelled MK-0974. Symbols and error bars represent the mean and standard deviations from 2 separate experiments with 8 points each.
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4. Discussion In this study, we have characterized the binding properties of a tritiated radioanalog of MK-0974, a novel CGRP receptor antagonist currently in Phase III studies for the acute treatment of migraine. A preliminary report recently indicated that MK-0974 demonstrated efficacy in a Phase III clinical trial (Ho et al., 2008a,b). Binding was assessed on membranes from the SK-N-MC cell line which constitutively expresses the human CGRP receptor (Muff et al., 1992; Semark et al., 1992) as well as cerebellum from rhesus monkey. We found that binding was saturable and equilibrium was reached within 10 min. Saturation binding studies showed that [3H]MK0974 has an apparent KD of 1.9 nM and 1.3 nM on SK-N-MC membranes and rhesus cerebellum homogenate, respectively. These values correlate well with the Ki values for unlabelled MK-0974 in competition binding studies using [125I]CGRP as the radioligand (Salvatore et al., 2008). In competition binding studies, α-CGRP and β-CGRP displaced [3H] MK-0974 with roughly equal potency while adrenomedullin was significantly less potent. All three of these peptide agonists displayed biphasic competition curves with a high and low-affinity site. By contrast, the antagonists CGRP(8-37) and unlabelled MK-0974 also displaced the radioligand but with monophasic curves. Biphasic competition curves for agonists are commonly observed in radioligand binding assays for G-protein-coupled-receptors using brokencell preparations as GTP can be limiting in these preparations, resulting in accumulation of the G-protein bound conformation of the receptor and the appearance of a high affinity site (Kenakin, 2006). To determine if this was the case in these experiments, α-CGRP competition binding was performed in the presence of 10 μM GTP and indeed, the majority of the high-affinity binding was shifted to the low-affinity site. Interestingly, none of the peptide agonists or antagonists were able to completely displace [3H]MK-0974. In contrast, the non-peptide antagonists Compound 3 and MK-0974 were able to completely displace the radioligand. This observation will be discussed more below. Competition studies for α-CGRP and MK0974 on rhesus cerebellum resulted in profiles similar those seen with SK-N-MC membranes. The association of [3H]MK-0974 to SK-N-MC membranes was very fast with equilibrium reached before 10 min with an on-rate of 1.01 × 109 M− 1 min− 1. [3H]MK-0974 binding was time-dependently reversed by addition of excess Compound 3 to the binding reaction. The dissociation was also very fast with a koff of 0.51 min− 1 and a t1/2 of 1.3 min. The kinetics of binding for this compound to SK-N-MC membranes are much faster than what has been previously described for either [125I]CGRP or [3H]BIBN4096BS (Muff et al., 1992; Shindler and Doods, 2002). Calcitonin receptor-like receptor is a member of the family B Gprotein-coupled-receptors which are characterized by having peptide agonists which bind to large N-terminal extracellular domains. It has proven difficult to discover small-molecule antagonists for family B Gprotein-coupled-receptors in part because the natural peptide ligands have extensive binding sites. There is some discussion centered on whether antagonists of these receptors can be strictly competitive or must have an allosteric mechanism meaning the antagonism does not result from direct steric hindrance of agonist binding (May et al., 2007). There are conflicting data in the literature about whether the CGRP receptor antagonist BIBN4096BS is a competitive or allosteric inhibitor. On one hand, Schild analyses of functional assays measuring cAMP have shown that increasing concentrations of BIBN4096BS causes rightward parallel shifts in the dose–response curves for αCGRP which is characteristic of competitive antagonism (Edvinsson et al., 2002; Hay et al., 2002). However, α-CGRP is unable to completely displace the radioanalog [3H]BIBN4096BS which could indicate allosteric inhibition (Shindler and Doods, 2002). In addition, BIBN4096BS appears to act allosterically at the amylin 1 receptor as it accelerates the dissociation of [125I]-Amylin (Hay et al., 2006). We have previously shown that increasing concentrations of MK-0974
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causes parallel shifts to the right in the CGRP dose–response curves in a cAMP functional assay and the dose–ratio plot of this data displays a straight line with a slope of 1 and a pA2 of 8.9 (Salvatore et al., 2008). No attenuation of this effect was observed at concentrations of antagonist up to 1000 fold over its KB. Also, the addition of excess unlabelled MK-0974 did not alter the dissociation kinetics of [125I]CGRP (data not shown). These behaviors are consistent with competitive antagonism. However, we also noted that none of the peptides we tested were able to fully displace [3H]MK-0974 in competition binding studies which could be an indicator of allosteric inhibition. These data make it difficult to determine conclusively whether MK-0974 is a strictly competitive antagonist or an allosteric one. In summary, we have characterized the binding properties of a tritiated version of the CGRP receptor antagonist MK-0974 on the SKN-MC neuroblastoma cell line as well as rhesus cerebellum. We showed that this radioligand binds with high affinity to the CGRP receptor and displays fast association and dissociation kinetics. Tritiated MK-0974 provides a useful tool for elucidating the binding properties of this promising molecule. References Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M., 1982. Alternative RNA processing in calcitonin gene expression generates mRNA encoding different polypeptide products. Nature 298, 240–244. Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R., MacIntyre, I., 1985. Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–56. Dodick, D., Lipton, R.B., Martin, V., Papademetriou, V., Rosamond, W., VanDenBrink, A.M., Loutfi, H., Welch, K.M., Goadsby, P.J., Hahn, S., Hutchinson, S., Matchar, D., Silberstein, S., Smith, T.R., Purdy, A., Saiers, J., 2004. Consensus statement: cardiovascular safety profile of triptans (5-HT1B/1D agonists) in the acute treatment of migraine. Headache 44, 414–425. Edvinsson, L., Alm, R., Shaw, D., Rutledge, R.Z., Koblan, K.S., Longmore, J., Kane, S.A., 2002. Effect of the CGRP receptor antagonist BIBN4096BS in human cerebral, coronary and omental arteries and in SK-N-MC cells. Eur. J. Pharmacol. 434, 49–53. Evans, B.N., Rosenblatt, M.I., Mnayer, L.O., Oliver, K.R., Dickerson, I.M., 2000. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J. Biol. Chem. 275, 31438–31443. Goadsby, P.J., Edvinsson, L., 1993. The trigeminal vascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann. Neurol. 33, 48–56. Goadsby, P.J., Edvinsson, L., Ekman, R., 1990. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann. Neurol. 28, 183–187. Goadsby, P.J., Lipton, R.B., Ferrari, M.D., 2002. Migraine — current understanding and treatment. N. Engl. J. Med. 346, 257–270. Hay, D.L., Howitt, S.G., Conner, A.C., Doods, H., Schindler, M., Poyner, D.R., 2002. A comparison of the actions of BIBN4096BS and CGRP8-37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col29 and Rat 2 cells. Br. J. Pharmacol. 137, 80–86. Hay, D.L., Christopoulos, G., Christopoulos, A., Sexton, P.M., 2006. Determinants of 1Piperidinecarboxamide, N-[2-[[5-Amino-/-[[4-(4-pyridinyl)-/-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096) affinity for calcitonin gene-related
peptide and amylin receptors — the role of receptor activity modifying protein 1. Mol. Pharmacol. 70, 1984–1991. Hershey, J.C., Corcoran, H.A., Baskin, E.P., Salvatore, C.A., Mosser, S., Williams, T.M., Koblan, K.S., Hargreaves, R.J., Kane, S.A., 2005. Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay. Regul. Pept. 127, 71–77. Ho, T.W., Mannix, L.K., Fan, X., Assaid, C., Furtek, C., Jones, C.J., Lines, C.R., Rapoport, A.M., On behalf of the MK-0974 Protocol 004 study group, 2008a. Randomized controlled trial of an oral CGRP antagonist, MK-0974, in acute treatment of migraine. Neurology 70, 1304–1312. Ho, T.W., Ferrari, M.D., Dodick, D.W., Galet, V., Kost, J., Fan, X., Leibensperger, H., Froman, S., Assaid, C., Koppen, H., Winner, P., 2008b. Acute antimigraine efficacy and tolerability of the novel oral CGRP receptor antagonist MK-0974: a phase III clinical trial versus placebo and zolmitriptan. Headache 48, S7–S8. Jansen, I., Uddman, R., Ekman, R., Olesen, J., Ottosson, A., Edvinsson, L., 1992. Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries. Peptides 13, 527–536. Kenakin, T.P., 2006. A Pharmacology Primer, Second edition. Elsevier, Massachusetts. Lassen, L.H., Jacobsen, V.B., Peterson, P., Sperling, B., Iverson, H.K., Olesen, J., 1998. Human calcitonin gene-related peptide (hCGRP)-induced headache in migraineurs. Eur. J. Neurol. 5, S63. May, L.T., Leach, K., Sexton, P.M., Christopoulos, R., 2007. Allosteric modulation of G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 47, 1–51. McLatchie, L.M., Fraser, N.J., Main, M.J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M.G., Foord, S.M.,1998. RAMPs regulate the transport and ligand specificity of the calcitoninreceptor-like receptor. Nature 393, 333–339. Muff, R., Stangl, D., Born, W., Fischer, J., 1992. Comparison of a calcitonin gene-related peptide receptor in a human neuroblastoma cell line (SK-N-MC) and a calcitonin receptor in a human breast carcinoma cell line (T47D). Ann. N.Y. Acad. Sci. 657, 106–116. Olesen, J., Diener, H.C., Husstedt, I.W., Goadsby, P.J., Hall, D., Meier, U., Pollentier, S., Lesko, L.M., 2004. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N. Engl. J. Med. 350, 1104–1110. Paone, D.V., Shaw, A.W., Nguyen, D.N., Burgey, C.S., Deng, J.Z., Kane, S.A., Koblan, K.S., Salvatore, C.A., Mosser, S.D., Johnston, V.K., Wong, B.K., Miller-Stein, C.M., Hershey, J.C., Graham, S.L., Vacca, J.P., Williams, T.M., 2007. 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-(2oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide (MK0974). J. Med. Chem. 50, 5564–5567. Salvatore, C., Hershey, J., Corcoran, H, Fay, J., Johnston, V., Moore, E., Mosser, S., Burgey, C., Paone, D., Shaw, A., Graham, S., Vacca, J., Williams, T., Koblan, K., Kane, S., 2008. Pharmacological Characterization of MK-0974 [N-[(3R,6S)-6-(2,3-Difluorophenyl)-2oxo-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], a potent and orally active calcitonin generelated peptide receptor antagonist for the treatment of migraine. J. Pharmacol. Exp. Ther. 324, 416–421. Semark, J.E., Middlemiss, D.N., Hutson, P.H., 1992. Comparison of calcitonin gene-related peptide receptors in rat brain and a human neuroblastoma cell-line SK-N-MC. Mol. Neuropharmacol. 2, 311–317. Shindler, M., Doods, H.N., 2002. Binding properties of the novel, non-peptide CGRP receptor antagonist radioligand, [3H]BIBN4096BS. Eur. J. Pharmacol. 442, 187–193. Uddman, R., Edvinsson, L., Ekman, R., Kingman, T., McCulloch, J., 1985. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P. Neurosci. Lett. 62, 131–136.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Examining the binding properties of MK-0974: A CGRP receptor antagonist for the acute treatment of migraine☆ Eric L. Moore a,⁎, Christopher S. Burgey b, Daniel V. Paone b, Anthony W. Shaw b, Yui S. Tang c, Stefanie A. Kane a, Christopher A. Salvatore a a b c
Merck Research Laboratories, Department of Pain Research, West Point, PA 19486, USA Merck Research Laboratories, Department of Medicinal Chemistry, West Point, PA 19486, USA Merck Research Laboratories, DMPK Global Technologies, Rahway, NJ 07065, USA
a r t i c l e
i n f o
Article history: Received 27 August 2008 Received in revised form 6 November 2008 Accepted 24 November 2008 Available online 3 December 2008 Keywords: Migraine CGRP Telcagepant Radioligand MK-0974
a b s t r a c t Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent and selective antagonist of the human and rhesus CGRP receptors and is currently in Phase III clinical studies for the acute treatment of migraine. The pharmacology of MK-0974 has been studied extensively, but there has not been a thorough characterization of its binding properties. Here, we characterize the binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC) membranes and rhesus cerebellum. [3H]MK-0974 displayed reversible and saturable binding to both SK-N-MC membranes and rhesus cerebellum with a KD of 1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor displaced [3H]MK-0974 in a concentrationdependent manner in competition binding experiments. Both CGRP and adrenomedullin demonstrated biphasic competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced [3H]MK-0974 in a monophasic fashion. In competitive binding studies with [3H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPγS. In kinetic binding experiments, the off-rate of [3H]MK-0974 was determined to be 0.51 min− 1 with a half-life of 1.3 min. In conclusion, the radioligand [3H]MK-0974 has proven to be a useful tool for studying the binding characteristics of MK-0974 and has broadened our understanding of this promising molecule. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide that is produced by alternative splicing of the calcitonin gene (Amara et al., 1982). This peptide exerts its biological effects by binding to a receptor consisting of the G-protein-coupled receptor (GPCR), calcitonin receptor-like receptor (CL receptor) and a single transmembrane protein designated receptor activity-modifying protein (RAMP) 1 (McLatchie et al., 1998). A third protein known as receptor component protein (RCP) is located intracellularly and is required for signal transduction (Evans et al., 2000). In addition to the CGRP receptor, calcitonin receptor-like receptor is also able to form high affinity adrenomedullin receptors by dimerization with either receptor activity-modifying protein 2 (RAMP2) or receptor activity-modifying protein 3 (RAMP3) (McLatchie et al., 1998).
☆ Authors are employees of Merck & Co., Inc. and potentially own stock and/or hold stock options in the Company. ⁎ Corresponding author. Merck Research Laboratories, WP26A-2000, West Point, PA 19486, USA. Tel.: +1 215 652 2042; fax: +1 215 993 1401. E-mail address: [email protected] (E.L. Moore). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.050
CGRP is widely expressed and exhibits a range of biological functions with the most pronounced being vasodilation. CGRP is the most powerful of the vasodilator transmitters (Brain et al., 1985) and its vasoactive effects have been demonstrated in a variety of blood vessels including those of the cerebral vasculature (Jansen et al., 1992). CGRP-expressing trigeminal nerve endings have been shown to innervate these cerebral blood vessels (Uddman et al., 1985). Multiple lines of evidence point to a possible role for CGRP in migraine headache. CGRP levels in cranial circulation are increased during migraine attacks (Goadsby et al., 1990) and successful treatment of migraine pain with a triptan normalizes CGRP levels (Goadsby and Edvinsson, 1993). In addition, intravenous administration of CGRP to migraineurs results in migraine-like symptoms in some patients (Lassen et al., 1998). More recently, the potent CGRP receptor antagonist olcegepant (BIBN4096BS) demonstrated clinical proof-ofconcept for the acute treatment of migraine with intravenous dosing (Olesen et al., 2004). This body of evidence implicating CGRP in the pathophysiology of migraine led us to initiate our own research program to discover orally bioavailable small-molecule CGRP receptor antagonists that would be suitable for the treatment of migraine. The current standard of care for the treatment of migraine are the 5HT1B/1D receptor agonists which
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new class of migraine therapy devoid of these cardiovascular liabilities would be a significant advance in migraine care. We have recently described the identification and pharmacological characterization of MK-0974 (Paone et al., 2007; Salvatore et al., 2008), which is the first orally bioavailable compound in this class to show clinical proof-ofefficacy comparable to triptans in a Phase 2 study (Ho et al., 2008a, 2008b). The purpose of the study presented here is to characterize the binding properties of the tritiated radioanalog [3H]MK-0974 to human and monkey CGRP receptors. 2. Materials and methods 2.1. Membranes and rhesus cerebellum preparation SK-N-MC membranes were purchased from Receptor Biology, Inc (Beltsville, MD). Rhesus cerebellum tissue was disrupted in a laboratory homogenizer in cold buffer containing 10 mM HEPES and 5 mM MgCl2. This homogenate was used directly in the binding assays. 2.2. [3H]MK-0974 receptor binding assays
Fig. 1. Chemical structures of A) [3H]MK-0974 and B) Compound 3.
form the triptan class of anti-migraine drugs (Dodick et al., 2004). While adequately safe when used as directed, the triptans are direct vasoconstrictors and are therefore contraindicated for patients with cardiovascular disease (Goadsby et al., 2002). The development of a
Binding assays were performed by combining [3H]MK-0974, 500 nM Compound 3 (Hershey et al., 2005, Fig. 1B) for non-specific binding, and either 50 μg/well SK-N-MC membrane or 1.5 mg/well rhesus cerebellum homogenate. The assay mixtures were incubated for various times at room temperature in binding buffer (10 mM HEPES, 5 mM MgCl2, 0.2% BSA) in a total volume of 1 ml. Free radioligand was separated from membrane-bound radioligand by filtration through polyethylene imine (0.5%) treated GF/B glass fiber
Fig. 2. Saturation binding curves for [3H]MK-0974 on A, 50 μg/well SK-N-MC membranes or B, 1.5 mg/well rhesus cerebellum homogenate. Specific binding was determined by subtracting non-specific binding from the total binding curve. Symbols and error bars represent mean and standard deviation from 5 separate titrations. Panel C shows the association kinetics of [3H]MK-0974 binding to SK-N-MC membranes at room temperature. 1.5 nM [3H]MK-0974 was added to 50 μg/well SK-N-MC membranes and binding was monitored over a period of 10 min. This is a representative graph of three separate experiments. Symbols and error bars represent the mean and standard deviation of 5 points. Panel D shows the dissociation kinetics of [3H]MK-0974 binding to SK-N-MC membranes. 1.5 nM [3H]MK-0974 was added to 50 μg/well SK-N-MC membranes and incubated 3 h at room temperature. Dissociation was initiated by the addition of 500 nM compound 3 and monitored over a period of 20 min. Symbols and error bars represent the mean and standard deviation from two separate experiments.
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filters using a cell harvester. Radioactivity was determined using a Topcount scintillation counter from Perkin-Elmer (Waltham, MA). 2.3. [3H]MK-0974 saturation binding Saturation binding assays were performed by combining increasing concentrations of [3H]MK-0974, 500 nM Compound 3 for nonspecific binding, and either 50 μg/well SK-N-MC membrane or 1.5 mg/ well rhesus cerebellum homogenate. The mixtures were incubated overnight (18 h) at room temperature in binding buffer in a total volume of 1 ml. 2.4. [3H]MK-0974 competition binding Competition binding assays were performed by combining increasing concentrations of reference compounds, 500 nM Compound 3 for non-specific binding, 1.5 nM [3H]MK-0974, and either 50 μg/well SK-N-MC membrane or 1.5 mg/well rhesus cerebellum. The mixtures were incubated for 3 h at room temperature in binding buffer in a total volume of 1 ml.
2.5. [3H]MK-0974 association kinetics Association kinetic assays were performed by combining 1.5 nM [3H] MK-0974 with 50 μg/well SK-N-MC membranes in binding buffer and incubating at room temperature for various times from 30 s to 10 min. 2.6. [3H]MK-0974 dissociation kinetics Dissociation kinetic assays were performed by combining 1.5 nM [3H]MK-0974 with 50 μg/well SK-N-MC membranes in binding buffer and incubating at room temperature for 3 h. At that point, 500 nM Compound 3 was added and dissociation was monitored for various intervals from 0.5 to 20 min. 3. Results 3.1. Saturation binding studies Saturation binding experiments using [3H]MK-0974 (Fig. 1A) were carried out on SK-N-MC membranes and rhesus cerebellum
Fig. 3. Competition binding studies. Displacement of 1.5 nM [3H]MK-0974 from 50 μg/well SK-N-MC membranes by A, α-CGRP, B, β-CGRP, C, adrenomedullin, D, α-CGRP(8-37), E, unlabelled MK-0974. Symbols and error bars represent the mean and standard deviations from 3–5 separate experiments. Panel F shows displacement of 1.5 nM [3H]MK-0974 from SK-N-MC by α-CGRP in the presence or absence of 10 μM GTP. Symbols and error bars represent the mean and standard deviations from 2 separate experiments with 4 points each.
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homogenate to determine the KD and Bmax. [3H]MK-0974 displayed saturable binding to SK-N-MC membranes with a KD of 1.9 nM and Bmax of 479 fmol/mg protein (Fig. 2A). [3H]MK-0974 also displayed saturable binding to rhesus cerebellum homogenate with a KD of 1.3 nM and Bmax of 20 fmol/mg (Fig. 2B). 3.2. Kinetic binding studies Kinetic binding experiments were performed to determine the on and off-rates for [3H]MK-0974 on SK-N-MC membranes. In the association assay, [3H]MK-0974 reached saturation very quickly with an apparent kon of 1.01 × 109 M− 1 min− 1 (Fig. 2C). For the dissociation of [3H]MK-0974, the koff was calculated as 0.51 min− 1 with a t1/2 of 1.3 min (Fig. 2D). 3.3. Competition binding studies Several CGRP receptor agonists and antagonists were evaluated in competition binding studies to determine their potencies in displacing [3H]MK-0974 from SK-N-MC membranes. The peptide agonists α-CGRP, β-CGRP, and adrenomedullin all displayed biphasic competition curves with a high and low-affinity site while the peptide antagonist α-CGRP(8-37) displayed monophasic competition (Fig. 3, A–D). The addition of 10 μM GTP to the binding mixture shifted most of the binding from the high-affinity site to the low-affinity site for α-CGRP (Fig. 3F). None of these peptides were able to completely displace [3H]MK-0974 while unlabelled MK-0974 completely displaced [3H]MK-0974 with a K i of 1.0 nM (Fig. 3E). Amylin and calcitonin were unable to displace [3H]MK-0974 at concentrations up to 10 μM (data not shown). Human α-CGRP and MK-0974 were also evaluated in competition binding assays on rhesus cerebellum homogenate and displayed similar profiles (Fig. 4, panels A and B).
Fig. 4. Displacement of 1.5 nM [3H]MK-0974 from rhesus cerebellum homogenate by A, α-CGRP, and B, unlabelled MK-0974. Symbols and error bars represent the mean and standard deviations from 2 separate experiments with 8 points each.
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4. Discussion In this study, we have characterized the binding properties of a tritiated radioanalog of MK-0974, a novel CGRP receptor antagonist currently in Phase III studies for the acute treatment of migraine. A preliminary report recently indicated that MK-0974 demonstrated efficacy in a Phase III clinical trial (Ho et al., 2008a,b). Binding was assessed on membranes from the SK-N-MC cell line which constitutively expresses the human CGRP receptor (Muff et al., 1992; Semark et al., 1992) as well as cerebellum from rhesus monkey. We found that binding was saturable and equilibrium was reached within 10 min. Saturation binding studies showed that [3H]MK0974 has an apparent KD of 1.9 nM and 1.3 nM on SK-N-MC membranes and rhesus cerebellum homogenate, respectively. These values correlate well with the Ki values for unlabelled MK-0974 in competition binding studies using [125I]CGRP as the radioligand (Salvatore et al., 2008). In competition binding studies, α-CGRP and β-CGRP displaced [3H] MK-0974 with roughly equal potency while adrenomedullin was significantly less potent. All three of these peptide agonists displayed biphasic competition curves with a high and low-affinity site. By contrast, the antagonists CGRP(8-37) and unlabelled MK-0974 also displaced the radioligand but with monophasic curves. Biphasic competition curves for agonists are commonly observed in radioligand binding assays for G-protein-coupled-receptors using brokencell preparations as GTP can be limiting in these preparations, resulting in accumulation of the G-protein bound conformation of the receptor and the appearance of a high affinity site (Kenakin, 2006). To determine if this was the case in these experiments, α-CGRP competition binding was performed in the presence of 10 μM GTP and indeed, the majority of the high-affinity binding was shifted to the low-affinity site. Interestingly, none of the peptide agonists or antagonists were able to completely displace [3H]MK-0974. In contrast, the non-peptide antagonists Compound 3 and MK-0974 were able to completely displace the radioligand. This observation will be discussed more below. Competition studies for α-CGRP and MK0974 on rhesus cerebellum resulted in profiles similar those seen with SK-N-MC membranes. The association of [3H]MK-0974 to SK-N-MC membranes was very fast with equilibrium reached before 10 min with an on-rate of 1.01 × 109 M− 1 min− 1. [3H]MK-0974 binding was time-dependently reversed by addition of excess Compound 3 to the binding reaction. The dissociation was also very fast with a koff of 0.51 min− 1 and a t1/2 of 1.3 min. The kinetics of binding for this compound to SK-N-MC membranes are much faster than what has been previously described for either [125I]CGRP or [3H]BIBN4096BS (Muff et al., 1992; Shindler and Doods, 2002). Calcitonin receptor-like receptor is a member of the family B Gprotein-coupled-receptors which are characterized by having peptide agonists which bind to large N-terminal extracellular domains. It has proven difficult to discover small-molecule antagonists for family B Gprotein-coupled-receptors in part because the natural peptide ligands have extensive binding sites. There is some discussion centered on whether antagonists of these receptors can be strictly competitive or must have an allosteric mechanism meaning the antagonism does not result from direct steric hindrance of agonist binding (May et al., 2007). There are conflicting data in the literature about whether the CGRP receptor antagonist BIBN4096BS is a competitive or allosteric inhibitor. On one hand, Schild analyses of functional assays measuring cAMP have shown that increasing concentrations of BIBN4096BS causes rightward parallel shifts in the dose–response curves for αCGRP which is characteristic of competitive antagonism (Edvinsson et al., 2002; Hay et al., 2002). However, α-CGRP is unable to completely displace the radioanalog [3H]BIBN4096BS which could indicate allosteric inhibition (Shindler and Doods, 2002). In addition, BIBN4096BS appears to act allosterically at the amylin 1 receptor as it accelerates the dissociation of [125I]-Amylin (Hay et al., 2006). We have previously shown that increasing concentrations of MK-0974
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causes parallel shifts to the right in the CGRP dose–response curves in a cAMP functional assay and the dose–ratio plot of this data displays a straight line with a slope of 1 and a pA2 of 8.9 (Salvatore et al., 2008). No attenuation of this effect was observed at concentrations of antagonist up to 1000 fold over its KB. Also, the addition of excess unlabelled MK-0974 did not alter the dissociation kinetics of [125I]CGRP (data not shown). These behaviors are consistent with competitive antagonism. However, we also noted that none of the peptides we tested were able to fully displace [3H]MK-0974 in competition binding studies which could be an indicator of allosteric inhibition. These data make it difficult to determine conclusively whether MK-0974 is a strictly competitive antagonist or an allosteric one. In summary, we have characterized the binding properties of a tritiated version of the CGRP receptor antagonist MK-0974 on the SKN-MC neuroblastoma cell line as well as rhesus cerebellum. We showed that this radioligand binds with high affinity to the CGRP receptor and displays fast association and dissociation kinetics. Tritiated MK-0974 provides a useful tool for elucidating the binding properties of this promising molecule. References Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M., 1982. Alternative RNA processing in calcitonin gene expression generates mRNA encoding different polypeptide products. Nature 298, 240–244. Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R., MacIntyre, I., 1985. Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–56. Dodick, D., Lipton, R.B., Martin, V., Papademetriou, V., Rosamond, W., VanDenBrink, A.M., Loutfi, H., Welch, K.M., Goadsby, P.J., Hahn, S., Hutchinson, S., Matchar, D., Silberstein, S., Smith, T.R., Purdy, A., Saiers, J., 2004. Consensus statement: cardiovascular safety profile of triptans (5-HT1B/1D agonists) in the acute treatment of migraine. Headache 44, 414–425. Edvinsson, L., Alm, R., Shaw, D., Rutledge, R.Z., Koblan, K.S., Longmore, J., Kane, S.A., 2002. Effect of the CGRP receptor antagonist BIBN4096BS in human cerebral, coronary and omental arteries and in SK-N-MC cells. Eur. J. Pharmacol. 434, 49–53. Evans, B.N., Rosenblatt, M.I., Mnayer, L.O., Oliver, K.R., Dickerson, I.M., 2000. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J. Biol. Chem. 275, 31438–31443. Goadsby, P.J., Edvinsson, L., 1993. The trigeminal vascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann. Neurol. 33, 48–56. Goadsby, P.J., Edvinsson, L., Ekman, R., 1990. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann. Neurol. 28, 183–187. Goadsby, P.J., Lipton, R.B., Ferrari, M.D., 2002. Migraine — current understanding and treatment. N. Engl. J. Med. 346, 257–270. Hay, D.L., Howitt, S.G., Conner, A.C., Doods, H., Schindler, M., Poyner, D.R., 2002. A comparison of the actions of BIBN4096BS and CGRP8-37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col29 and Rat 2 cells. Br. J. Pharmacol. 137, 80–86. Hay, D.L., Christopoulos, G., Christopoulos, A., Sexton, P.M., 2006. Determinants of 1Piperidinecarboxamide, N-[2-[[5-Amino-/-[[4-(4-pyridinyl)-/-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096) affinity for calcitonin gene-related
peptide and amylin receptors — the role of receptor activity modifying protein 1. Mol. Pharmacol. 70, 1984–1991. Hershey, J.C., Corcoran, H.A., Baskin, E.P., Salvatore, C.A., Mosser, S., Williams, T.M., Koblan, K.S., Hargreaves, R.J., Kane, S.A., 2005. Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay. Regul. Pept. 127, 71–77. Ho, T.W., Mannix, L.K., Fan, X., Assaid, C., Furtek, C., Jones, C.J., Lines, C.R., Rapoport, A.M., On behalf of the MK-0974 Protocol 004 study group, 2008a. Randomized controlled trial of an oral CGRP antagonist, MK-0974, in acute treatment of migraine. Neurology 70, 1304–1312. Ho, T.W., Ferrari, M.D., Dodick, D.W., Galet, V., Kost, J., Fan, X., Leibensperger, H., Froman, S., Assaid, C., Koppen, H., Winner, P., 2008b. Acute antimigraine efficacy and tolerability of the novel oral CGRP receptor antagonist MK-0974: a phase III clinical trial versus placebo and zolmitriptan. Headache 48, S7–S8. Jansen, I., Uddman, R., Ekman, R., Olesen, J., Ottosson, A., Edvinsson, L., 1992. Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries. Peptides 13, 527–536. Kenakin, T.P., 2006. A Pharmacology Primer, Second edition. Elsevier, Massachusetts. Lassen, L.H., Jacobsen, V.B., Peterson, P., Sperling, B., Iverson, H.K., Olesen, J., 1998. Human calcitonin gene-related peptide (hCGRP)-induced headache in migraineurs. Eur. J. Neurol. 5, S63. May, L.T., Leach, K., Sexton, P.M., Christopoulos, R., 2007. Allosteric modulation of G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 47, 1–51. McLatchie, L.M., Fraser, N.J., Main, M.J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M.G., Foord, S.M.,1998. RAMPs regulate the transport and ligand specificity of the calcitoninreceptor-like receptor. Nature 393, 333–339. Muff, R., Stangl, D., Born, W., Fischer, J., 1992. Comparison of a calcitonin gene-related peptide receptor in a human neuroblastoma cell line (SK-N-MC) and a calcitonin receptor in a human breast carcinoma cell line (T47D). Ann. N.Y. Acad. Sci. 657, 106–116. Olesen, J., Diener, H.C., Husstedt, I.W., Goadsby, P.J., Hall, D., Meier, U., Pollentier, S., Lesko, L.M., 2004. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N. Engl. J. Med. 350, 1104–1110. Paone, D.V., Shaw, A.W., Nguyen, D.N., Burgey, C.S., Deng, J.Z., Kane, S.A., Koblan, K.S., Salvatore, C.A., Mosser, S.D., Johnston, V.K., Wong, B.K., Miller-Stein, C.M., Hershey, J.C., Graham, S.L., Vacca, J.P., Williams, T.M., 2007. 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-(2oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide (MK0974). J. Med. Chem. 50, 5564–5567. Salvatore, C., Hershey, J., Corcoran, H, Fay, J., Johnston, V., Moore, E., Mosser, S., Burgey, C., Paone, D., Shaw, A., Graham, S., Vacca, J., Williams, T., Koblan, K., Kane, S., 2008. Pharmacological Characterization of MK-0974 [N-[(3R,6S)-6-(2,3-Difluorophenyl)-2oxo-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], a potent and orally active calcitonin generelated peptide receptor antagonist for the treatment of migraine. J. Pharmacol. Exp. Ther. 324, 416–421. Semark, J.E., Middlemiss, D.N., Hutson, P.H., 1992. Comparison of calcitonin gene-related peptide receptors in rat brain and a human neuroblastoma cell-line SK-N-MC. Mol. Neuropharmacol. 2, 311–317. Shindler, M., Doods, H.N., 2002. Binding properties of the novel, non-peptide CGRP receptor antagonist radioligand, [3H]BIBN4096BS. Eur. J. Pharmacol. 442, 187–193. Uddman, R., Edvinsson, L., Ekman, R., Kingman, T., McCulloch, J., 1985. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P. Neurosci. Lett. 62, 131–136.