The role of CGRP in the pathophysiology of migraine and efficacy of CGRP receptor antagonists as acute antimigraine drugs

Pharmacology & Therapeutics 124 (2009) 309–323

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Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a

Associate editor: C.M. Villalón

The role of CGRP in the pathophysiology of migraine and efficacy of CGRP receptor antagonists as acute antimigraine drugs Carlos M. Villalón a,⁎, Jes Olesen b a b

Departamento de Farmacobiología, Cinvestav-Coapa, Czda. de los Tenorios 235, Col. Granjas-Coapa, Deleg. Tlalpan, C.P. 14330, México D.F., Mexico University of Copenhagen, Dept. of Neurology, Glostrup Hospital, Ndr. Ringvej 57, DK-2600 Glostrup, Copenhagen, Denmark

a r t i c l e

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Keywords: Antimigraine drug CGRP CGRP receptor antagonists Drug development Headache Migraine Olcegepant Telcagepant

a b s t r a c t Migraine is a highly prevalent neurovascular disorder that can be provoked by infusion of calcitonin generelated peptide (CGRP). CGRP, a neuropeptide released from activated trigeminal sensory nerves, dilates intracranial and extracranial blood vessels and centrally modulates vascular nociception. On this basis, it has been proposed that: (i) CGRP may play an important role in the pathophysiology of migraine; and (ii) blockade of CGRP receptors may abort migraine. With the advent of potent and selective CGRP receptor antagonists, the importance of CGRP in the pathophysiology of migraine and the therapeutic principle of CGRP receptor antagonism were clearly established. Indeed, both olcegepant (BIBN4096BS, given intravenously) and telcagepant (MK-0974, given orally) have been shown to be safe, well tolerated and effective acute antimigraine agents in phase I, phase II, and for telcagepant phase III, studies. However, recent data reported elevated liver transaminases when telcagepant was dosed twice daily for three months for the prevention of migraine rather than acutely. The potential for a specific acute antimigraine drug, without producing vasoconstriction or vascular side effects and with an efficacy comparable to triptans, is enormous. The present review will discuss the role of CGRP in the pathophysiology of migraine and the various treatment modalities that are currently available to target this neuropeptide. © 2009 Elsevier Inc. All rights reserved.

Contents 1. General introduction . . . . . . . . . . . . 2. Calcitonin gene-related peptide . . . . . . . 3. Calcitonin gene-related peptide and migraine . 4. Implications, future directions and conclusions Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AM, adrenomedullin; AMY, amylin; BIBN4096BS, (1-piperidinecarboxamide,N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3, 5dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-); cAMP, 3′–5′-cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CSD, cortical spreading depression; CT, calcitonin; Cys(ACM)2,7-hα-CGRP, [acetamidomethyl-cysteine2,7]-human-α-CGRP; DRG, dorsal root ganglia; ERK1/2, extracellular signal-regulated kinase ½; GTN, glyceryl trinitrate; ICHD-2, International Classification of Headache Disorders, Second Edition; iNOS, inducible form of nitric oxide synthase; i.v., intravenous route of administration; LY334370, (4-fluoro-N-[3-(1-methyl-4piperidinyl)-1H-indol-5-yl]-benzamide); MA, migraine with typical aura; MAPKs, mitogen-activated protein kinases; MO, migraine without aura; MK-0974, [N-[(3R,6S)-6-(2,3difluorophenyl)-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]; NO, nitric oxide; PACAP, pituitary adenylate activating peptide; PNU-142633, [(s)-3,4-dihydro-1-[2-[4-[4-aminocarbonyl)phenyl]-1-piperazinyl]ethyl]-N-methyl-1H-2-benzopyran-6-carboximide); RAMP, receptor activity-modifying protein; RCP, receptor component protein; rCBF, regional cerebral blood flow; SNP, sodium nitroprusside; TRPV1, transient receptor potential vanilloid type 1. ⁎ Corresponding author. E-mail address: [email protected] (C.M. Villalón). 0163-7258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2009.09.003

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1. General introduction Headache or cephalalgia is defined as pain in the head above the orbito-meatal line, sometimes including pain in the upper neck; some of the causes are benign while others are medical emergencies. It is one of the most common locations of pain in the body (Olesen et al., 2006). Headaches are classified and defined by explicit (operational) diagnostic criteria in the International Classification of Headache Disorders, Second Edition (ICHD-2) (Committee, 2004). There are three major categories of headaches: (i) primary headaches; (ii) secondary headaches; and (iii) cranial neuralgias, facial pain, and headaches not classifiable. Primary headaches (which include migraine, tension-type headache, cluster headache and other primary headaches) can be defined as independent disorders not caused by another disease or trauma (Committee 2004). Although primary headaches are not life-threatening per se, they cause a much decreased quality of life, suffering and carry a high personal and societal financial cost (Jensen & Stovner, 2008). Their pathophysiological bases are increasingly being understood, although much still remains to be investigated (Olesen et al., 2004). In contrast, secondary headaches are a symptom of an underlying disease or injury that needs to be diagnosed and treated (e.g. meningitis, encephalitis, hypertension, brain bleeding, brain tumor, dehydration, etc.); some of the causes of secondary headache may be potentially life-threatening. Finally, cranial neuralgias are a group of headaches that occur because of a sudden burst of activity in sensory cranial nerves, predominantly the trigeminal nerve (Olesen et al., 2006). In view of the above, it would be impossible for us to deal with all categories of headaches, their causes, symptoms and treatments in a single review. For this reason and because the implication of the neuropeptide α-calcitonin gene-related peptide (CGRP) is strongest for migraine, this review will focus on migraine, with particular emphasis on analysing: (i) the role of CGRP in migraine pathophysiology; and (ii) the efficacy of CGRP receptor antagonists as acute antimigraine drugs. A forthcoming book based on a symposium held in March 2009 in Copenhagen shall cover many aspects of CGRP involvement in migraine (Olesen, in press). Here we have reviewed all literature and present the facts in a more coherent and condensed form. 1.1. What is migraine? Migraine is a neurovascular disorder characterized by a severe, debilitating and throbbing unilateral headache associated with anorexia, nausea, vomiting, photophobia, phonophobia and/or diarrhoea (Goadsby et al., 2002b). Amongst other features, this disorder: (i) affects 12% of adults in occidental countries, with about 18% of women and 6% of men (Jensen & Stovner, 2008; Bigal & Lipton, 2009); (ii) represents an enormous socio-economic burden to the individual as well as to the society (Andlin-Sobocki et al., 2005); and (iii) has a profound negative effect on the patient's quality of life (Ruiz de Velasco et al., 2003). The International Headache Society (Committee, 2004) currently recognizes six variants of migraine, but the most common types are: (i) migraine with aura (starting with visual, sensory, speech or motor symptoms, i.e. aura, followed by headache; formerly called “classic” migraine); and (ii) migraine without aura (the head pain is similar to that of patients with aura, but it is not preceded by aura; formerly called “common” migraine). It is noteworthy that not all auras are followed by headache or migraine (i.e. aura without headache; Committee, 2004). 1.2. Pharmacotherapy of migraine The history of the treatment of headache in general, and migraine in particular, spans the millennia, from the Neanderthal era to the

Space Age (Edmeads, 1999). Rational treatment for this ancient complaint evolved slowly from a disease of supernatural causes to a neurovascular disorder (Villalón et al., 2003). With this long history, notwithstanding, it is extremely surprising that effective antimigraine drugs had been, until very recently, limited in number. In the last decades, there have been big steps in understanding migraine pathophysiology and in the development of antimigraine drugs (Olesen et al., 2006). Basically, antimigraine drugs can be divided into: (i) agents that abolish an individual migraine attack (acute antimigraine drugs; e.g. triptans, ergots, etc.); and (ii) agents aimed at its prevention (prophylactic drugs; e.g. beta-adrenoceptor blockers, antiepileptics, etc.). All patients need treatment to abolish attacks (acute treatment) while only patients with frequent attacks need, in addition to acute treatment, prophylactic treatment by drugs taken on a daily basis in order to reduce the number and/or severity of attacks (Olesen et al., 2006). Regarding acute antimigraine treatment, the triptans have represented a considerable advance (Olesen et al., 2001; Villalón et al., 2003) and did indeed change the lives of numerous sufferers (Goadsby et al., 2002b), but their vasoconstrictor side effects limit their use in patients with multiple vascular risk factors (Dahlöf, 2002). Other side effects, such as dizziness, nausea, fatigue, chest symptoms and paresthesias, prevent some patients from using triptans. Moreover, a number of patients do not respond well to the triptans. Thus, a considerable unmet need for better acute antimigraine treatments exists which could be fulfilled, as discussed below, with the recent advent of CGRP receptor antagonists; these drugs, which have shown an efficacy comparable to triptans, seem to have a better safety and tolerability profile (Ho et al., 2008a,b; 2009). 1.3. Pathophysiology of migraine Although theories of migraine have usually posited the cerebral blood vessels as the origin of migraine attacks (Villalón et al., 2003), other current hypotheses place the primary dysfunction in brain stem centres that regulate vascular tone and pain sensation (Goadsby, 2007; Durham, 2008; Link et al., 2008). Pain-sensitive structures such as the intracranial blood vessels and the meninges, especially the dura mater, are supplied with sensory nerve fibres (Pietrobon & Striessnig, 2003) by the ophthalmic ramus of the first branch of the trigeminal nerve. They arise from pseudounipolar neurons located in the trigeminal ganglion (Link et al., 2008), which project onto secondorder sensory neurons in the trigeminal nucleus caudalis in the brain stem and its related extensions down to the C2-level called the trigeminocervical complex (Goadsby, 2007). From this region, a signal is transmitted to the ventroposterior thalamus leading to activation in cortical areas, including frontal cortex, insulae and cingulate cortex, that results in the experience of pain (Goadsby, 2007). In addition, a migraine active region has been pointed out in the brain stem by using positron emission tomography (Weiller et al., 1995; Diener & May, 1996; Afridi et al., 2005). Thus, the trigemino-cerebrovascular system seems to be involved both at the brain stem level and at the level of perivascular nerve terminals (Olesen et al., 2009). As discussed later, CGRP is abundant in perivascular trigeminal nerve fibres and in the spinal trigeminal nucleus, but it is not known whether one or the other or both sites are important for the action of CGRP receptor blockers in migraine. 1.4. Clinical features of migraine Based on clinical features, four distinct phases of migraine can be discerned, but not all patients have all phases (Fig. 1). These phases are: (i) early warning symptoms, officially called premonitory symptoms (when present); (ii) the migraine aura (when present); (iii) the headache phase; and (iv) the postdrome phase after the

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Fig. 1. The four phases of migraine attacks. Symptoms typical of each phase are present. Note that not all patients have all phases. Many do not have premonitory symptoms (phase 1), patients without aura obviously have no aura and many have no postdromes (phase 4). Modified from Blau (1992) with permission.

headache has gone (Blau, 1991; Goadsby et al., 2002b; Committee, 2004; Olesen et al., 2006). 1.4.1. Aetiology and triggering of migraine Studies performed in twins suggest that the migraine risk is influenced by genetic and environmental factors (Ulrich et al., 1999; 2004). Indeed, patients with the common forms of migraine, namely, migraine with typical aura (MA) and migraine without aura (MO), have a complex inheritance, but their genes remain unknown (Russell & Olesen, 1993; Kirchmann et al., 2006). Although some migraine attacks can be explained by environmental factors such as drinking red wine, strong sun light or stress, the great majority of attacks remain unexplained. Our knowledge about the mechanisms of migraine has been greatly improved by human provocation experiments. Migraine sufferers, but not normal control subjects, develop a migraine-like headache or even attacks identical to spontaneous attacks after intravenous (i.v.) infusion of glyceryl trinitrate (GTN) (Fig. 2), which is a donor of nitric oxide (NO), histamine, CGRP, pituitary adenylate cyclase-activating polypeptide (PACAP) and prostacyclin (Lassen et al., 1995, 2002; Iversen et al., 1989; Wienecke et al., 2008; Schytz et al., 2009). Some patients with or without aura have early warning symptoms (called premonitory symptoms) which include tiredness, hyperactivity, craving for certain foods, yawning and feeling depressed. In fact, GTN-provoked attacks also have such symptoms

Fig. 2. Provocation of a migraine attack using i.v. infusion of GTN. Note immediate headache during and shortly after the infusion and a delayed headache hours later. The delayed headache is similar to the usual migraine attacks. Reproduced with permission, from Thomsen et al. (1994).

before the induction of migraine attacks (Giffin et al., 2003b; Afridi et al., 2004). The nature of the symptoms suggests hypothalamic activation, but no measurements of pathophysiological parameters have been made during this phase. 1.4.2. Aura phase One third of migraine patients have attacks with aura either exclusively or in combination with attacks without aura (Russell et al., 1995). Regional cerebral blood flow (rCBF) studies have shown that the aura, associated with occipitally decreased rCBF slowly spreading anteriorly, is caused by a cortical spreading depression (CSD), a well known and much investigated animal experimental phenomenon consisting of a depolarization of all cortical cellular elements that spreads slowly and does not cross to the other hemisphere (Olesen et al., 1982; Lauritzen, 1984). 1.4.3. Headache phase In MA patients the headache often starts while rCBF is reduced and continues as rCBF becomes increased (Olesen et al., 1982). In MO patients, rCBF is focally increased in the brain stem while cortical blood flow is normal except in areas activated by pain of any kind (Olesen et al., 1981; Weiller et al., 1995). Thus, no cortical pattern specific to migraine headache has been seen and the marked changes indicative of CSD seen in MA are completely absent. Brain and extracranial arteries may be slightly dilated on the pain side (Iversen et al., 1990; Thomsen et al., 1995) but, most recently, a study using magnetic resonance angiography found no difference in the diameter of the middle meningeal artery and the middle cerebral artery during and outside of induced migraine attacks (Schoonman et al., 2008). Hence, the degree and even the existence of vasodilatation during migraine have been questioned. It is striking, however, that all substances capable of inducing a migraine attack are vasodilators. Some of these substances, such as NO and CGRP, are active throughout the migraine attack because blocking their production or receptor is effective in treating spontaneous migraine attacks (Lassen et al., 1997; Ho et al., 2008a,b). Consistent with these findings, recent studies have shown that activation of CGRP receptors can increase the expression of the inducible form of NO synthase (iNOS) and stimulate NO release from trigeminal ganglion glial cells (Li et al., 2008; Vause & Durham, 2009). Hence, CGRP released from the cell bodies of trigeminal neurons could function to promote and maintain an inflammatory cycle within the ganglion that mediates peripheral sensitization; this mechanism may offer another plausible explanation of how NO and CGRP are involved in causing a migraine attack in some individuals.

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Furthermore, CGRP given by i.v. infusion can induce a migraine attack, but CGRP does not cross the blood-brain barrier. BIBN4096BS (olcegepant), a selective CGRP receptor antagonist (Doods et al., 2000), could not block the effect of CGRP release on pial arteries after electrical stimulation in the rat (Petersen et al., 2004), and CGRP in a perfusion system using isolated pial arteries dilated the artery only when given abluminally (Petersen et al., 2005c). Hence, vasodilatation and extracerebral mechanisms remain very important in migraine, but the vascular theory of migraine in its simple form has been outdated for decades. While migraine has been considered neurovascular in origin, particular interest has recently been directed towards possible central mechanisms such as hypothalamic and, more likely, brain stem activation (Weiller et al., 1995). Thus, some consider that vasodilatation by migraine-provoking agents may be a parallel phenomenon or a surrogate marker of the real migraine process. In fact, it has been proposed that there is no abnormal sensory input in migraine, and that the mechanism of pain is entirely central. In an extensive review we have recently argued against this rather radical view (Olesen et al., 2009). Another factor currently investigated is peripheral and central sensitization of nociceptive pathways in the head. Peripheral sensitization is presumed to happen around cranial arteries in trigeminal terminals, but precisely how this comes on remains unknown in MO, while CSD is presumed to cause it in MA, possibly via activation of matrix metalloproteinases and opening of the blood-brain barrier. This allows chemical changes in cortex during CSD to reach nociceptors in the dura mater (Gursoy-Ozdemir et al., 2004). Central sensitization occurs at the first synapse in the spinal trigeminal nucleus of the brain stem and at higher centres as well (Burstein, 2001; Levy et al., 2004). It is the combination of peripheral and central sensitization with cranial

vasodilatation that most investigators consider responsible for migraine pain. The triptans interact with this system at several levels (Fig. 3) producing: (i) stabilization of peripheral nerve endings inhibiting, among others, the release of CGRP; (ii) inhibition of signal transduction at the first synapse (Levy et al., 2004); and (iii) constriction of cranial and other blood vessels. Whether they have additional central effects that are important is currently debated. 1.4.4. Postdromes After pain relief some, but not all, patients still do not feel completely restored. They may feel depressed, slow thinking, lacking energy, “washed out”, etc. (Blau, 1991; Giffin et al., 2003b). It seems likely that some disturbance of neurotransmitter homeostasis remains, but nothing is known about the brain chemistry of this phenomenon. Drugs that not only treat migraine pain but also prevent the postdromal phase would certainly be preferred. 2. Calcitonin gene-related peptide 2.1. Introduction and discovery The calcitonin family of peptides consists of at least six members, namely, calcitonin, amylin, intermedin (adenomedullin-2), adrenomedullin and CGRP (two isoforms: α-CGRP and β-CGRP) (Juaneda et al., 2000; Poyner et al., 2002). CGRP is a 37-amino acid neuropeptide, which was first identified in 1983 in rats (Rosenfeld et al., 1983). The rat α-CGRP differs from rat β-CGRP by one amino acid and the human β-CGRP differs by three amino acids from homologous human α-CGRP (Wimalawansa, 1996); the α- and β-isoforms of CGRP are very similar in their biological activities (Poyner & Marshall, 2001). α-CGRP is widely

Fig. 3. The triptans (agonists at 5-HT1B/1D/1F receptors) have several possible sites of action in the peripheral and central nervous systems, as well as in cranial blood vessels. They inhibit the release of CGRP from peripheral nerve terminals, inhibit neurotransmission in the brain stem and constrict cranial arteries. Reproduced with permission, from Goadsby et al. (2002b).

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distributed in the central and peripheral nervous systems (Rosenfeld et al., 1983). β-CGRP is encoded by a different gene that is highly homologous to the calcitonin-CGRP gene and is primarily found in enteric nerves and in the pituitary gland (Sternini, 1992). While both CGRP isoforms are located in nerves, immunohistological studies have demonstrated that α-CGRP is preferentially expressed in sensory neurons and its concentration is 3- to 6-fold higher than that of β-CGRP (Mulderry et al., 1988). Importantly, α-CGRP has been reported to be the predominant isoform expressed in trigeminal ganglia neurons (Amara et al., 1985) and dilation of human cerebral arteries has been shown to be mainly mediated by α-CGRP (Jansen-Olesen et al., 1996). 2.2. Structure of α-calcitonin gene-related peptide and structure–activity relationships All species variants of α-CGRP have 37 amino acids, constituted as a single polypeptide chain (Poyner et al., 2002). In general, the structure of α-CGRP comprises: (i) an N-terminal disulfide bridge between positions 2 and 7 (Cys2 and Cys7); and (ii) a phenylalanylamide C-terminus in the regions of residues 28 and 30, and also in 32 and 34 (Conner et al., 2002). CGRP shares 50% homology in its sequence of amino acids with adrenomedullin and has some homology with amylin (for further details, see Arulmani et al., 2004a). The intact peptide is required for the full biological activity of a CGRP molecule. In this respect, Conner et al. (2002) have reported that: (i) the N-terminal loop is required for triggering the signal transduction and receptor activation; (ii) removal of the first seven amino acids engenders CGRP(8–37), which is an antagonist with high affinity for CGRP receptors; (iii) the amphipathic α-helix (residues 8– 18) plays an important role in the binding of the molecule to the receptor, and its deletion causes ~100-fold loss of affinity; (iv) the residues 19–27 are necessary as a spacer or hinge region, and removal of this segment causes a 10-fold decrease in the affinity of CGRP(8–37); and (v) the C-terminal region is a requisite for the peptide to acquire the right conformation in the interaction with its receptor. 2.3. Distribution and localization CGRP is widely distributed in the peripheral and central nervous systems (van Rossum et al., 1997) as well as in the cardiovascular, respiratory and gastrointestinal systems (Arulmani et al., 2004a). CGRP-containing neurons innervate all major organs and joints of the human body and thus modulate the functioning of the immune, respiratory, endocrine, gastrointestinal, musculoskeletal and cardiovascular systems (Poyner et al., 2002; Durham, 2008). In the cardiovascular system, CGRP-containing nerve fibres are more abundant around the arteries than around the veins (Bell & McDermott, 1996); in the arterial system, they are predominantly seen in the junction of the adventitia and media (Wimalawansa, 1996). Moreover, CGRP-containing nerve fibres are more abundant in atria than in ventricles; within the right atrium, they are localized in the sinoatrial node, the atrioventricular node and the specialised conduction system; whilst the myocardium is less densely innervated than the epicardium, endocardium or pericardium (Wimalawansa, 1996). In the periphery, CGRP-containing nerve fibres are associated with smooth muscles such as: (i) most parts of the gastrointestinal tract, including the excretory ducts of the parotid gland, over the epithelium of the fundic glands of stomach, endocrine cells of the duodenum and ileum and some myenteric ganglia; (ii) lungs; (iii) thyroid gland (close to C cells); (iv) splenic vein and sinusoids; (v) human skin; and (vi) pituitary gland (Hagner et al., 2002a,b,c). 2.4. Physiological functions of calcitonin gene-related peptide The wide distribution of CGRP-containing nerves and its receptors in the body suggests that CGRP plays an important physiological role

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(Durham, 2008; Link et al., 2008). For example, in the cardiovascular system, CGRP: (i) induces positive chronotropic and inotropic effects as well as an increase in coronary perfusion pressure and blood flow (Saetrum Opgaard et al., 2000; Kaygisiz et al., 2003); (ii) mediates cardioprotective effects through preconditioning induced by brief ischaemia (Li & Peng, 2002; Brzozowski et al., 2004); and (iii) produces vasodilatation in capacitance blood vessels and regulates vascular tone and angiogenesis (Wimalawansa, 1996). In the central nervous system, CGRP modulates the motor, sensory and integrative systems (van Rossum et al., 1997). Accordingly, CGRP: (i) modulates pain perception (van Rossum et al., 1997; Powell et al., 2000); and (ii) potentiates in general the excitatory actions by enhancing the release of substance P (Oku et al., 1987) as well as of excitatory amino acids from primary afferent fibres (Kangrga et al., 1990; van Rossum et al., 1997). Moreover, peripherally, CGRP: (i) modulates synaptic transmission on motoneurons (at the neuromuscular junction) by locally inhibiting the expression of acetyl cholinesterase, which terminates acetylcholine neurotransmission (Rossi et al., 2003); and (ii) induces degranulation and subsequent release of pro-inflammatory agents (e.g. histamine) from dural mast cells (Ottosson & Edvinsson, 1997). Most significantly, within the scope of the present review, the ability of CGRP to cause cranial vasodilatation and to facilitate nociception is important in the pathophysiology of migraine (Arulmani et al., 2004a; Hargreaves, 2007; Durham, 2008). More recent lines of evidence support an important role for CGRP in regulating the activity of neurons and satellite glial cells in trigeminal ganglia. For example, CGRP released from neuronal cell bodies in the trigeminal ganglion can: (i) increase its own synthesis within trigeminal neurons (Zhang et al., 2007); (ii) stimulate the release of NO and several pro-inflammatory cytokines from satellite glial cells (Thalakoti et al., 2007; Li et al., 2008); and (iii) have autocrine and paracrine functions (Thalakoti et al., 2007; Zhang et al., 2007). These cellular effects of CGRP are mediated via activation of the CGRP receptor. Since CGRP mediates key cellular events at multiple sites within the trigeminovascular system that are thought to be involved in the pathophysiology of migraine, selective CGRP receptor antagonists should be effective antimigraine drugs. 2.5. The calcitonin gene-related peptide receptor 2.5.1. Classification and pharmacological characterisation The classification of CGRP receptors has undergone a notable evolution (for references see Hay et al., 2008). Historically, heterogeneity among CGRP receptors was first detected in 1989, when it was shown that the truncated CGRP receptor antagonist, CGRP12–37, preferentially antagonized the chronotropic and ionotropic actions of CGRP on the guinea-pig atrium, but not its ability to inhibit the contraction of the electrically stimulated rat vas deferens. In contrast, the linear CGRP receptor agonist, [Cys(ACM)2,7] hα-CGRP, selectively activated CGRP receptors on the rat vas deferens (Dennis et al., 1989). Based on this evidence, it was proposed that the CGRP-induced responses are mediated by CGRP1 receptors in the guinea-pig atrium and by CGRP2 receptors in the rat vas deferens. Subsequent work with the antagonist CGRP8–37 confirmed these observations: CGRP1 receptors were classified as being potently antagonized by CGRP8–37, whereas CGRP2 receptors were less sensitive to this antagonist (Dennis et al., 1990). Consistent with these findings, in isolated large porcine coronary arteries, the CGRP receptors seemed to display the pharmacological profile of the CGRP2 subtype as they show a low affinity for CGRP8–37, whilst [Cys(ACM)2,7] hα-CGRP causes vasorelaxation (Waugh et al., 1999). With the advent the first selective non-peptide CGRP receptor antagonist, BIBN4096BS (olcegepant) (Doods et al., 2000), it was further shown that this antagonist displayed a 10-fold higher affinity for CGRP receptors in rat left atrium (CGRP1) as compared to rat vas deferens (CGRP2) (Wu et al., 2000). Using olcegepant, such a heterogeneity was

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also shown in several blood vessels including cerebral, cranial, coronary and omental arteries (e.g. Edvinsson et al., 2001; 2002; Moreno et al., 2002a,b; Verheggen et al., 2002; Wu et al., 2002; Hasbak et al., 2003; Gupta et al., 2006). This was considered good evidence that CGRP may act via more than one receptor when applied pharmacologically; however, the International Union of Pharmacology nomenclature subcommittee pointed out that the basic molecular structure of the CGRP2 subtype remained unclear and it was admitted that definite demonstration would come from its cloning and the development of highly selective agonists and antagonists (Poyner et al., 2002). In fact, other lines of evidence (particularly obtained from molecular biology studies) cast doubt upon CGRP receptor heterogeneity (for references see Arulmani et al., 2004a). 2.5.2. Basic structure of receptors for calcitonin gene-related peptide, adrenomedullin and amylin In general, the receptors for CGRP and adrenomedullin (AM) basically consist of: (i) a G-protein-coupled receptor, namely the calcitonin receptor-like receptor (CLR); and (ii) an accessory protein known as receptor activity-modifying protein (RAMP) (McLatchie et al., 1998). Hence CLR forms the basic receptor protein for CGRP and AM receptors (which means that CGRP and AM bind with CLR), but the receptor specificity is determined by a specific RAMP variant (McLatchie et al., 1998). The RAMPs (148–175 amino acids in size) are cleavable signal peptides, with a relatively large N-terminal extracellular domain, one transmembrane spanning domain and nine amino acid intracellular Cterminal domains (Fitzsimmons et al., 2003). The RAMPs have been localized in the endoplasmic reticulum and they are required to: (i) facilitate the intracellular translocation of the CLR-maturing protein and its insertion into plasma membranes (McLatchie et al., 1998); (ii) express CLR on the cell surface and thereby determine the relative affinity of this receptor for CGRP and AM (Foord & Marshall, 1999); and (iii) modulate the pharmacology of the given CLR by providing a mechanism whereby a cell changes its sensitivity from one receptor to another receptor (Mallee et al., 2002). Three RAMP variants have been identified in human tissues, namely RAMP1, RAMP2 and RAMP3 (Poyner et al., 2002). It is noteworthy that co-expression of CLR with RAMP1 forms a heterodimer that results in the CGRP receptor (Banerjee et al., 2006; see Fig. 4); moreover, RAMP1 is required for glycosylation and

transport of CLR to the plasma membrane (Foord & Marshall, 1999; Hay et al., 2006a,b). A third component of the CGRP receptor, the receptor component protein (RCP), is an accessory protein required for proper biological function as it is involved in coupling the receptor to downstream signalling pathways like the protein kinase A pathway (Evans et al., 2000). As discussed below, the CGRP receptor displays the pharmacological profile of the previously called “CGRP1 subtype”. In contrast, co-expression of CLR with either RAMP2 or RAMP3 gives receptors that preferentially bind AM (McLatchie et al., 1998); these are the AM1 and AM2 receptors (Poyner et al., 2002). Subsequently, Hay et al. (2003) reported that: (i) the AM2 receptor, in particular, can have significant affinity for CGRP and might be activated by this peptide at pharmacological concentrations; and (ii) CGRP8–37 can antagonize AM1 and AM2 receptors with estimated pA2 values in the range of 6.0 to 7.0, whereas olcegepant has no appreciable affinity for these receptors. Therefore, the AM2 receptor could be activated pharmacologically by CGRP and weakly antagonized by CGRP8–37; this is in fact the pharmacological profile of the previously called “CGRP2 subtype” (see below; Hay et al., 2003). Likewise, amylin (AMY) receptors can show significant affinity for CGRP. In particular, the AMY1(a) receptor (insert negative calcitonin receptor [CT(a)] plus RAMP1), at least in transfected cells, may potentially be activated by CGRP (Kuwasako et al., 2004; Hay et al., 2005). The AMY3(a) [CT(a)/RAMP3] receptor shows activation by CGRP similar to that of the AM2 receptor (Hay et al., 2005). Furthermore, both of these AMY receptor subtypes were shown to be weakly antagonized by CGRP8–37. Therefore, AMY1 and AMY3 receptors also have the pharmacological characteristics of a “CGRP2 receptor subtype”. The AMY1(a) receptors [but not the AMY3(a) receptors] show significant affinity for olcegepant (Hay et al., 2006a,b), but at ~150-fold lower than that seen at “CGRP1 receptors”. 2.5.3. Are the so called calcitonin gene-related peptide 1 and calcitonin gene-related peptide 2 receptor subtypes really different proteins that represent independent receptors? The above lines of evidence (see Section 2.5.2), taken together, indicate that there are clear molecular correlates for CGRP receptors identified pharmacologically, with no support for heterogeneity. So after the cloning of CLR and RAMPs, the International Union of Pharmacology nomenclature subcommittee for CGRP receptors (Hay et al., 2008) has concluded that: (i) the CGRP1 receptor corresponds to the CLR/RAMP1 complex; and (ii) the pharmacological profile of the CGRP2 receptor can be generated by the AMY1 receptor and, to a lesser extent, by the AMY3 and AM2 receptors. Therefore, it is recommended that the “CGRP1” receptor should now be called the “CGRP” receptor and the term “CGRP2” receptor should not be used (Hay et al., 2008). 2.5.4. General distribution and binding In general, CGRP receptors are widely distributed in the peripheral and central nervous systems (Arulmani et al., 2004a; Durham, 2008; Link et al., 2008) as well as in the cardiovascular, gastrointestinal, respiratory, endocrine, musculoskeletal and trigeminovascular systems (Arulmani et al., 2004a; Hagner et al., 2002a,b,c; Rossi et al., 2003). In addition, the co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons (Ma et al., 2003).

Fig. 4. Postulated interactions of specific CGRP domains with the CLR–RAMP1 heterodimer. Co-expression of the CLR with RAMP1 forms a mature CGRP receptor on the cell membrane surface. The characteristically long extracellular domains of the CLR–RAMP heterodimer create a high affinity binding pocket important for docking the C-terminal phenylalaninamide of CGRP. This interaction represents the CGRP Nterminus domain, essential for biological activity, to a “classical” agonist binding pocket formed in part by the transmembrane α-helices of the CLR–RAMP1 heterodimer. Reproduced with permission, from Banerjee et al. (2006).

2.5.4.1. Nervous system Both high and low affinity binding sites for CGRP have been reported in the central nervous system (Morara et al., 2000; Segond von Banchet et al., 2002). In dorsal root ganglia (DRG) and other neurons, CGRP receptors co-exist with receptors for other neurotransmitters and neuromodulators, such as substance P, noradrenaline, neuropeptide Y, vasoactive intestinal peptide, etc. (van Rossum et al., 1997; Ohtori et al., 2002). Moreover, in the nonadrenergic and noncholinergic fibres, CGRP receptors co-exist with receptors for

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tachykinins and substance P (Wiesenfeld-Hallin et al., 1984; Ursell et al., 1991). CGRP receptors are also found in Schwann cells in the periphery, in arterial smooth muscle cells and in mononuclear cells including mast cells (Oliver et al., 1999; Ma et al., 2003). 2.5.4.2. Cardiovascular system The highest density of CGRP binding sites is present in the heart and in blood vessels (Wimalawansa, 1996). Regardless of the species, the density of the CGRP binding sites in atria invariably exceeds that of ventricles (Chang et al., 2001). Autoradiographic studies in the hearts of rats (Chang et al., 2001), guinea pigs and humans (Coupe et al., 1990) have shown the highest density of CGRP binding sites in the coronary arteries, coronary veins and in the heart valves, while a lower density is found in the coronary arterioles and endocardium (Wimalawansa, 1996). Hence CGRP receptors play a role in regulating blood flow as well as inotropic and chronotropic effects in the heart (Saetrum Opgaard et al., 2000; Poyner et al., 2002). 2.5.5. Signal transduction mechanisms In general, activation of CGRP receptors couples to increases in cAMP and cGMP levels in a number of different cell types (Fiscus et al., 1991; Cheng et al., 1995; Wimalawansa, 1996; Poyner et al., 2002). However, CGRP receptors have also been reported to couple to activation of mitogen-activated protein kinases (MAPKs) (Parameswaran et al., 2000; Schaeffer et al., 2003; Vause & Durham, 2009). MAPKs are important signal transducing enzymes that connect activation of cell surface receptors to key regulatory events within the cell via a series of reversible phosphorylation events (Seger & Krebs 1995; Chang & Karin 2001). At least four distinctly regulated groups of MAPKs are present in mammalian cells, namely, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinases, p38 proteins and extracellular signal-regulated kinase 5 that are activated by specific MAPKs (Schaeffer & Weber 1999; Widmann et al., 1999; Chang & Karin 2001). For example, CGRP has been shown in smooth muscle cells to upregulate the activity of ERK1/2 and p38 proteins (Schaeffer et al., 2003). In addition, the cellular effects of CGRP on DRG neurons (Anderson & Seybold, 2004) and in keratinocytes (Yu et al., 2006) were shown to involve the ERK1/2 MAPK signalling pathway. Importantly, many of the agents implicated in the initiation or maintenance of inflammation and pain have been shown to directly activate MAPK cellular signalling cascades in neurons and glial cells (Ji, 2004a,b; Obata & Noguchi 2004). Moreover, CGRP stimulation of iNOS and NO release involves activation of several MAPKs via activation of the CGRP receptor (Vause & Durham, 2009). Thus, CGRP receptor activation on satellite glial cells would be expected to lead to increased expression of not only iNOS, but also induction of other pro-inflammatory genes such as cytokines and interleukins that are known to be regulated by MAPKs and are expressed by satellite glial cells (Kaminska 2005; Schindler et al., 2007). Interestingly, in animal models of tissue injury and inflammation, the active levels of the MAPKs ERK1/2 and p38, which are found to be increased in response to CGRP (Vause & Durham, 2009), play a role in the development and maintenance of peripheral sensitization (Ji, 2004a,b). As far as vascular signal transduction mechanisms are concerned, the CGRP-induced vasodilator responses are mediated by both endothelium-dependent and endothelium-independent mechanisms (Wimalawansa, 1996). In the endothelium-dependent pathway (e.g. thoracic aorta, pulmonary and renal arteries in rats as well as brachial artery in humans), CGRP activates adenylyl cyclase in the endothelium thereby increasing cyclic adenosine monophosphate (cAMP) levels. This activates the enzyme NO synthase which, in turn, increases the level of NO. NO acts on the smooth muscle cells by activating guanylyl cyclase with an ensuing production of cyclic guanosine monophosphate (cGMP) leading to smooth muscle relaxation (Wimalawansa, 1996; de Hoon et al., 2003). In the endothelium-independent pathway (e.g. rat and human cranial

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arteries), CGRP bypasses the endothelium either by diffusion in arteries without a blood-brain barrier or released from perivascular nerve endings to the abluminal side of the artery and directly binds to CGRP receptors on the smooth muscle cells, activating adenylyl cyclase; this, in turn, increases cAMP levels leading to vascular relaxation (Wimalawansa, 1996). Interestingly, some blood vessels such as the rat basilar and superior mesenteric arteries show both endothelium-dependent and endothelium-independent mediated mechanisms (Wimalawansa, 1996). CGRP-induced increase in cAMP activates protein kinase A that phosphorylates and thereby opens K+ ATP channels in rabbit arterial smooth muscle (van Rossum et al., 1997). In rat dural arteries the effect of CGRP was significantly inhibited by the KATP channel inhibitor, glibenclamide (Gozalov et al., 2008). Evidently, CGRP-induced vasodilator responses involve multiple second messengers, including cAMP, NO-cGMP and K+ channels (Kitazono et al., 1993; Hong et al., 1996; van Rossum et al., 1997). Furthermore, in cultured trigeminal ganglion cells, activation of CGRP receptors increases cAMP, which in turn leads to elevated CGRP promoter activity and increased CGRP mRNA levels (Zhang et al., 2007). These results point to an auto-activation of CGRP expression and indicate that elevated CGRP release in the trigeminal system may create a self-sustaining feedback loop. 2.6. Therapeutic potentials of CGRP receptor ligands 2.6.1. Calcitonin gene-related peptide receptor agonists Since CGRP is a potent vasodilator agent (Brain et al., 1985), it is tempting to suggest that CGRP receptor agonists could be of use in the treatment of several cardiovascular pathologies including coronary heart disease, myocardial ischaemia (CGRP relieves arterial vasospasm) and hypertension (for references see Arulmani et al., 2004a). Despite these interesting possibilities, there are currently no proven clinical indications for the use of CGRP receptor agonists. 2.6.2. Calcitonin gene-related peptide receptor antagonists Several lines of evidence have shown that an inappropriate release of CGRP is a potential causative factor in several diseases including, amongst others: (i) migraine (discussed below); (ii) inflammation (as meningitis); (iii) cardiogenic shock associated with sepsis; and (iv) thermal injury (Wimalawansa, 1996; Hoffmann et al., 2002; Arulmani et al., 2004a). Moreover, since CGRP and substance P containing nerve fibres are abundantly seen in atopic dermatitis and nummular eczema, CGRP receptor antagonists may dampen the associated inflammatory response, neurogenic inflammation and/or pain transmission (Jarvikallio et al., 2003). 3. Calcitonin gene-related peptide and migraine Since the discovery and identification of CGRP (Rosenfeld et al., 1983), its involvement in migraine has been gradually clarified and this has led to the development of the first CGRP receptor antagonist effective as an antimigraine drug. A brief overview of this exciting journey shall be given here followed by a more extensive account of the evidence in subsequent sections. Almost immediately after the discovery of CGRP, Uddman et al. (1985) studied its presence in cranial blood vessels and found that it was localized in the trigeminal perivascular nerves. Moreover, McCulloch et al. (1986) demonstrated the presence of a protective cerebrovascular reflex using CGRP as its transmitter. Subsequently, Goadsby et al. (1990) showed that during migraine headache plasma concentrations of CGRP, but not of other neuropeptides, are elevated in the external jugular venous blood (i.e. the blood draining the extracerebral tissues including the dura mater). Together with the basic science findings, this created interest in CGRP in the pharmaceutical companies although, subsequently, it has been difficult to reproduce the increase in CGRP (Tvedskov et al., 2005).

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The pharmaceutical company Boehringer Ingelheim developed a non-peptide CGRP receptor antagonist, BIBN4096BS, later called olcegepant (Doods, 2001) which was considered, for the above reasons, to be possibly effective in the treatment of migraine. Greatly supporting the decision to move this compound into clinical development was the finding that infusion of CGRP in migraine sufferers caused a migraine-like headache (Lassen et al., 1998, 2002). Finally, the proof of the principle of CGRP antagonism for acute migraine treatment was delivered in a phase II clinical trial of olcegepant (Olesen et al., 2004). This compound could only be given i.v., but subsequently Merck Research Laboratories developed an orally bioavailable agent, telcagepant, which was effective as a tablet (Ho et al., 2008a,b; 2009). 3.1. Calcitonin gene-related peptide and its role in migraine pathophysiology In previous sections we have pointed out the general localization of CGRP in the body (see Section 2.3) and its receptors (see Section 2.5.4). Now this section will make particular reference to the sources of cerebrovascular CGRP, specific sites of action and mechanisms of CGRP that may be relevant during migraine. 3.1.1. Cerebrovascular sources of calcitonin gene-related peptide Six main sources of cerebrovascular CGRP are particularly relevant to migraine, namely, the spinal trigeminal nucleus, the trigeminal ganglion, its ophthalmic and maxillary branches supplying perivascular nerve terminals, the internal carotid miniganglia, the cervical roots 1–3 and the parasympathetic supply of the cranial blood vessels (Edvinsson et al., 1987; Suzuki et al., 1989). 3.1.2. Localization of calcitonin gene-related peptide receptors relevant to migraine There are at least four sites of action of CGRP that may be relevant in the pathophysiology of migraine (Arulmani et al., 2004a; Durham, 2008; Link et al., 2008), namely: (i) cerebral and extracerebral blood vessels, where activation of CGRP receptors produces a vasodilator response that can be blocked by olcegepant (Edvinsson et al., 2002; Moreno et al., 2002a,b; Oliver et al., 2002; Kapoor et al., 2003a; Petersen et al., 2005b,c); (ii) dural mast cells, from which activation of CGRP receptors can release cytokines and inflammatory agents during neurogenic inflammation (Theoharides et al., 2005); (iii) secondorder sensory neurons within brain stem trigeminal nuclei, on which postsynaptic CGRP receptors can also be blocked by olcegepant (Storer et al., 2004; Fischer et al., 2005; Levy et al., 2005); and (iv) trigeminal ganglion, where CGRP increases its own synthesis (Zhang et al., 2007) and stimulates the release of NO and several proinflammatory cytokines (Thalakoti et al., 2007; Li et al., 2008).

Consistent with the above findings, CLR and RAMP1 proteins (which demonstrate functional CGRP receptors) have been shown to be highly expressed in several tissues/cells, including: (i) the human cerebral vasculature (Moreno et al., 2002a,b; Oliver et al., 2002); (ii) cranial dura mater, dural mast cells, trigeminal ganglion and presynaptic nerve terminals in the spinal trigeminal nucleus (Lennerz et al., 2008); (iii) neurons and glia in the peripheral and central nervous systems, such as second-order neurons and astrocytes (Levy et al., 2004; Morara et al., 2008); and (iv) Schwann cells (Lennerz et al., 2008) and trigeminal ganglion glial cells (Li et al., 2008). Furthermore, Zhang et al. (2007) have demonstrated that RAMP1 levels are functionally rate limiting for the actions of CGRP in trigeminal ganglia; hence, elevated neuronal RAMP1 could potentially sensitize the trigeminal ganglia of individuals to the actions of CGRP. These actions would include increased CGRP synthesis and increased neurogenic inflammation, which could potentially help sustain and intensify the nociceptive actions of CGRP in migraine.

3.1.3. Functional role of cranial calcitonin gene-related peptide and its receptors As discussed in previous sections, i.v. CGRP causes a migraine-like headache (Fig. 5). It potently dilates cerebral and extracerebral arteries, but not veins (McCulloch et al., 1986), in agreement with the absence of CGRP receptors on cerebral veins (Lennerz et al., 2008). After electrical stimulation, the nerve terminals release CGRP and this dilates the arteries and arterioles (Petersen et al., 2004). However, in contrast to these rather non-physiological stimuli, no spontaneous efferent activity has ever been recorded in sensory nerves anywhere in the body. Thus, any physiological or pathophysiological stimulus to release CGRP from nerve terminals remains unknown. Possibly, efferent activity in parasympathetic nerves and release of NO or other neurotransmitters can have this effect. Certainly, activation of certain ion channels such as the transient receptor potential vanilloid type 1 (TRPV1) can do it, but whether this happens in vivo is unknown. In studies of CGRP application to the rat dura mater, no sensitizing effect could be shown on neurons with a dural input (Levy et al., 2005). Furthermore, CGRP caused no pain alone or in combination with bradykinin in a human skin model (Pedersen-Bjergaard et al., 1991) and CGRP receptors were not present in peripheral trigeminal nerve terminals (Lennerz et al., 2008). These studies indicate that a peripheral role of CGRP in headache, as in nociception in general, may be unlikely. Moreover, the CGRP receptor antagonist olcegepant may inhibit central trigeminocervical neurons in vivo in the cat (Storer et al., 2004). However, it still remains to be explained how infused CGRP can cause a migraine attack as discussed above.

Fig. 5. The individual headache scores after i.v. infusion of either CGRP (left panel) or placebo (right panel) to migraine patients. Pain is scored on a 0–10 scale on the ordinate and time in hours on the abscissa. Based on data from Lassen et al. (2002).

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CGRP is present in normal human plasma, but its role is unknown. It could provide a constant dilatory effect on human arteries. Likewise, there could perhaps be a tonic effect of CGRP that might leak out from periarterial nerve terminals. If this were the case, blockade of CGRP receptors could involve a risk of vasoconstriction or vasospasm. However, experiments using a high dose of olcegepant showed that there was absolutely no effect of this CGRP receptor antagonist on blood pressure, heart rate or cerebral blood flow in healthy human volunteers (Fig. 6). This finding strongly suggested that CGRP receptor antagonists would be safe from a cardiovascular point of view (Petersen et al., 2005a). 3.1.4. Calcitonin gene-related peptide and nitric oxide Within the context of this review, NO is an important signalling molecule involved in: (i) the development and maintenance of inflammation and pain (Guzik et al., 2003; Naik et al., 2006); (ii) the synthesis and release of CGRP from trigeminal ganglion neurons (Bowen et al., 2006); and (iii) the pathophysiology of migraine (Olesen et al., 1994; Olesen, 2008). In fact, both NO and CGRP cause an immediate headache and a delayed migraine-like headache in migraine sufferers with almost exactly the same characteristics (Figs. 2 and 5). In addition, inhibitors of NO production (Lassen et al., 1997; Olesen, 2008) and CGRP receptor antagonists (Olesen et al., 2004; Ho et al., 2008a,b, 2009) are effective in treating spontaneous migraine attacks. Hence there could be some sort of common final pathway between the actions of these two molecules in migraine. This interaction has previously been discussed (Olesen, 2008), but new important data are now available. A slow i.v. infusion of the NO donor, sodium nitroprusside (SNP), caused a delayed neuronal activity in the spinal trigeminal nucleus of the rat, and this could be reversed by infusion of olcegepant (Koulchitsky et al., 2004, 2009) (Fig. 7). These and many other animal studies indicate that NO may cause migraine by releasing CGRP from central sites. However, a recent human study showed that pretreatment with a high dose of olcegepant could not block NO-induced migraine in migraine patients. This would either indicate that NO does not work via CGRP release or that olcegepant has no access to the site of CGRP release (Tvedskov et al., in press). 3.1.5. Is calcitonin gene-related peptide released during a migraine attack? Stimulation of the trigeminal ganglion in cats and humans results in elevations in CGRP and substance P levels in the cranial circulation (Goadsby et al., 1988). During spontaneous attacks of migraine, CGRP has been found to be increased in two studies (Goadsby et al., 1990; Gallai et al., 1995). In the first study, the increase was only seen in the external jugular vein, but not in the cubital vein, indicating that CGRP was released from the head (probably from an extracranial site). The other study found an increase in both cranial and systemic venous

Fig. 6. Olcegepantn (BIBN4096BS) has no effect on global or regional cerebral blood flow (rCBF) in the territory of the middle cerebral artery (MCA) as shown in the figure. It also did not affect systemic blood pressure, heart rate or velocity of blood in the MCA (not shown). Reproduced with permission, from Petersen et al. (2005a).

Fig. 7. Olcegepant (BIBN4096BS) can block the increase in firing of neurons with dural input elicited by the NO donor, sodium nitroprusside (SNP). Reproduced with permission, from Koulchitsky et al. (2004).

blood, suggesting no specific cranial release. In a newer study, patients served as their own controls comparing CGRP levels during and outside of attack. This is a much stronger design because it eliminates between-patient differences. The study used the same assay as the above study plus a much better validated and more specific assay. There was no sign of increased CGRP during an attack neither in the external jugular venous blood nor in cubital fossa blood with any of the assays (Tvedskov et al., 2005) (Fig. 8). Other studies have indirectly supported an increase. Thus, CGRP concentration decreased after treatment of migraine attacks with a triptan (Goadsby & Edvinsson, 1991), and the same was observed in humans where migraine-like attacks had been induced by nitroglycerin (Juhasz et al., 2003). The latter study suffered, however, from post hoc change of efficacy parameters. Seemingly, most evidence is in favour of an increase in CGRP during a migraine attack, but the methodologically best study was negative. More studies are therefore needed. 3.1.6. Is inhibition of calcitonin gene-related peptide release a therapeutic option? The development of antimigraine agents with no cardiovascular side effects, but capable of inhibiting trigeminal CGRP release would avoid the vasoconstrictor action of the triptans and might represent a major improvement over current treatments (Ramadan & Buchanan, 2006). Several novel approaches have been proposed, which include: (i) selective agonists at 5-HT1D receptors, such as PNU-142633 [(s)-3,4-dihydro-1-[2[4-[4-aminocarbonyl)phenyl]-1-piperazinyl]ethyl]-N-methyl-1H-2-benzopyran-6-carboximide) (McCall et al., 2002); and (ii) selective 5-HT1F receptor agonists such as LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]-benzamide) (Ramadan et al., 2003). Unlike the triptans (5-HT1B/1D/1F receptor agonists), 5-HT1D and 5-HT1F receptor agonists are devoid of contractile effects on coronary and cerebral blood vessels (Bouchelet et al., 2000; McCall et al., 2002); in addition, these receptors may have presynaptic actions, such as inhibition of CGRP release and central nociception (Shepheard et al., 1999; Ramadan et al., 2003). However, PNU-142633 proved to be ineffective in the acute treatment of migraine (GómezMancilla et al., 2001), whilst LY334370 did show some efficacy when

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Fig. 8. Most studies show that CGRP is increased in external jugular venous blood during a migraine attack. However, the most recent study compared CGRP during and outside an attack in each patient, a much stronger design than a group comparison. Using two different assays, none of them showed any increase in CGRP levels. Reproduced with permission, from Tvedskov et al. (2005).

used in doses which may have interacted with 5-HT1B receptors (Goldstein et al., 2001; Ramadan et al., 2003). Though clinical studies have shown that LY334370 is effective in treating migraine headaches without coronary side effects (Ramadan et al., 2003), the high dose of 5-HT1F receptor agonists clouds the outcomes (Goldstein et al., 2001). Clearly, more studies on the role of the 5-HT1F receptor in migraine are warranted (Goadsby & Classey, 2003). Likewise, several lines of evidence have shown that agonists at α2-adrenoceptors and adenosine A1 receptors inhibit CGRP release and trigeminal nociception (Goadsby et al., 2002b; Willems et al., 2003). Indeed, in vivo studies have shown that a selective adenosine A1 receptor agonist, GR79236 (N-[(2-methylphenyl)methyl]adenosine (metrifudil), 2-(phenylamino) adenosine), inhibits: (i) neurogenic vasodilatation in rats (Humphrey et al., 2001); (ii) trigeminal nociception as well as CGRP release in cats (Goadsby et al., 2002a); and (iii) trigeminal nociception in humans (Giffin et al., 2003a). Taken together, these findings suggest that GR79236 may have antimigraine potential (Goadsby et al., 2002b). In fact, results from pilot clinical studies have reported that GR79236 has antimigraine action, probably due to an inhibitory effect on nociceptive trigeminal neurons (Humphrey et al., 2001), but more clinical studies are indispensable. Stimulation of presynaptic α2-adrenoceptors mediates antinociceptive effects and inhibits the expression and release of CGRP (Supowit et al., 1998; Shi et al., 2000; Hargreaves et al., 2003). Hence, selective agonists of α2-adrenoceptor subtypes (not yet available) may also have potential antimigraine usefulness; however, BHT933 (an α2-adrenoceptor agonist) also produced carotid vasoconstriction (Willems et al., 2003). Other inhibitors of CGRP release may include antagonists (capsazepine) at capsaicin vanilloid receptors as well as agonists at cannabinoid receptors (anandamide). These agents seem to have potential antimigraine properties in preclinical studies (Akerman et al., 2003; 2004).

In isolated human cerebral and extracerebral arteries, olcegepant produced a selective antagonism of α-CGRP-induced vasorelaxation (Edvinsson et al., 2002; Moreno et al., 2002a,b; Verheggen et al., 2002). In the in vivo (preclinical) animal models of antimigraine activity, olcegepant attenuated: (i) the vasodilatation induced by trigeminal stimulation in marmosets (Doods et al., 2000); (ii) the capsaicininduced porcine carotid vasodilator responses (mediated by release of CGRP), including carotid arteriovenous anastomotic dilatation (Kapoor et al., 2003a); (iii) α-CGRP-induced porcine carotid vasodilatation and arterial-jugular venous oxygen saturation difference (Kapoor et al., 2003b); and (iv) α-CGRP-induced dilatation of the rat middle meningeal artery (Petersen et al., 2004). In addition to its antimigraine potential, olcegepant had no effect on baseline systemic and regional haemodynamics in porcine (Kapoor et al., 2003b) and rat (Arulmani et al., 2004b) cardiovascular models, reinforcing its cardiovascular safety. These findings suggested that olcegepant could be developed as an effective antimigraine compound with little cardiovascular side effects. In clinical trials, in a single-centre, double-blind (within dose levels), placebo-controlled, randomized, single-rising dose design, 41 volunteers received olcegepant, whilst 14 were given placebo (Iovino et al., 2004). Sixteen adverse events were seen in 8 of the 41 subjects treated with olcegepant, compared with adverse events in 4/14 treated with placebo. Two-thirds of the adverse events occurred at the highest dose, 10 mg. Adverse events were transient and included mild paresthesias, flushing, feeling of facial warmth, head and body crawling sensations, numb and cold feelings in hand and forearm infusion site, stabbing in throat and head, and congestion in the head. “No clinically relevant” drug-induced changes in blood pressure, pulse rate, respiratory rate, EKG, lab tests, or forearm blood flow were seen (Iovino et al., 2004). In a study of normal volunteers, there was no

3.2. Efficacy of calcitonin gene-related peptide receptor antagonists in the acute treatment of migraine 3.2.1. Olcegepant Doods et al. (2000) and Rudolf et al. (2005) used high-throughput screening to find small molecule CGRP receptor antagonists, and number 19 (first called BIBN4096, and later named olcegepant; see Fig. 9), was selected for preclinical studies and clinical trials.

Fig. 9. Chemical formula of olcegepant.

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effect of olcegepant 2.5 mg or 10 mg on blood pressure, heart rate, rCBF or transcranial Doppler signal from the middle cerebral artery (Petersen et al., 2005a). In another clinical trial of olcegepant published by Petersen et al. (2005b), 10 participants in a doubleblind placebo-controlled crossover study received either olcegepant (2.5 mg, i.v.) or placebo as pretreatment. Then, they were given hαCGRP. Six of 10 placebo subjects, but none of the olcegepant patients, experienced an h-αCGRP-induced headache. Also the hemodynamic effects of CGRP were completely blocked by olcegepant. These researchers concluded that olcegepant effectively prevented CGRP-induced headache and haemodynamic responses. A large phase IIB trial on i.v. olcegepant provided the first proof of the concept that CGRP receptor antagonists are effective in the acute treatment of migraine (Olesen et al., 2004). This was an international, multicentre, double-blind, randomized proof of concept clinical trial in which 126 migraine patients received placebo or 1 of 6 doses of olcegepant i.v. at 16 centres in Denmark, Germany, The Netherlands and the UK. Groups of 6 received olcegepant or placebo, starting with 1 mg olcegepant. If 3/4 in the active group responded, the dose was reduced; otherwise, the dose was increased. The optimal dose was established when there was a response in ≥4 groups in ≥3/4 treated and ≥20 were treated with the selected dose, while at least 18 had been treated with placebo. The best dose found using this novel method was 2.5 mg i.v. olcegepant. Pain-free response rate from 2.5mg olcegepant was 44% at 2 h and 56% at 4 h (placebo pain-free responses were 2% and 10%). Sustained response rate was 47% (placebo 15%). Nausea, phonophobia and photophobia all improved in parallel with pain response. Rate of recurrence was 19% (placebo 46%). Adverse event rate was 20% for the olcegepant group and 12% in the placebo group. The most common adverse event was mild paresthesias, seen in 7 patients (8%) who received active drug, none on placebo. Nausea occurred in 2% of both active and treatment groups; headache, dry mouth, and “abnormal vision” occurred in 2% of the olcegepant group, but none with placebo. Thus, proof of concept for a CGRP receptor antagonist in the acute treatment of migraine was established, and compelling evidence for a role of CGRP in migraine pathophysiology was obtained (Olesen et al., 2004). However, this study minimized the number of participants and, therefore, with a small number of treated patients it is difficult to compare the magnitude of clinical response to previous studies with triptans. Notwithstanding, this study suggested that CGRP receptor antagonists are a class of substances that might compete with or supplement the triptans for the acute treatment of migraine. Olcegepant can only be given i.v. and therefore could not be developed as a drug for migraine attacks, but the study stimulated other companies to search for more suitable CGRP receptor antagonists. 3.2.2. Telcagepant Merck Research Laboratories took the lead with a research program aimed at identifying orally bioavailable CGRP receptor antagonists that would be suitable for the treatment of migraine (Williams et al., 2006). This program led to the identification of a novel benzodiazepine CGRP receptor antagonist by high-throughput screening (Williams et al., 2006). Subsequent lead optimization led to the identification of the potent and orally bioavailable CGRP receptor antagonist MK-0974 (Paone et al., 2007; see Fig. 10). This compound, later named telcagepant, displayed good oral bioavailability in rats (20%) and dogs (35%). In rats, clearance was low (9.4 l/min kg) with a moderate i.v. half-life (1.6 h) and a short oral Tmax (0.67 h). In dogs, clearance was moderate at 17 ml/min kg (Paone et al., 2007). Human pharmacokinetic data have not yet been published. Moreover, the pharmacological characterization of telcagepant (Salvatore et al., 2008) showed that this drug: (i) displays equal affinity for native and cloned CGRP receptors as determined by radioligand binding experiments; (ii) competitively antagonizes CGRP-induced cAMP accumulation in cells expressing the recombi-

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Fig. 10. Chemical formula of telcagepant.

nant human CGRP receptor; (iii) is highly selective for the CGRP receptor versus the closely related human AM receptors, CLR/RAMP2 and CLR/RAMP3; (iv) has no significant activity (IC50 > 10 µM) in a screen of 166 enzyme and binding assays, including the human calcitonin receptor; (v) displays a marked species-selectivity (as shown for olcegepant; Doods et al., 2000; Rudolf et al., 2005), exhibiting 1500-fold higher affinity for the human and rhesus CGRP receptors compared to the rat and dog CGRP receptors; and (vi) produces a concentration-dependent inhibition of capsaicininduced dermal vasodilatation in the rhesus forearm, which results from endogenous CGRP release via activation of TRPV1 receptors (Akerman et al., 2003). It is noteworthy that this rhesus forearm model was translated to the clinical setting and served as a valuable tool for dose selection for the clinical development of telcagepant (Sinclair et al., 2007). Subsequently, Ho et al. (2008b) carried out a multicentre, randomized, double-blind parallel group study with an adaptive dose-ranging design in 2 stages, in which 420 patients were treated orally with telcagepant in the dosages 25, 50, 100, 200, 300, 400, or 600 mg, with rizatriptan 10 mg, or with placebo. The dosages of 200 mg or below of telcagepant were eliminated in the adaptive doseranging procedure after 192 patients had been treated in the first step. For the remaining 3 dosages, there was a significantly better effect of telcagepant for the primary outcome parameter (p = 0.015). For the different dosages (with patient numbers) pain relief was as follows: 300 mg (n = 38) 68.1%, 400 mg (n = 45) 48.2%, 600 mg (n = 40) 67.5%, rizatriptan 10 mg (n = 34) 69.5%, and placebo (n = 115) 46.3%. Adverse events in the telcagepant 300–600 mg groups were nausea, dizziness, and somnolence; all of them were mild and only nausea was more frequent compared with the placebo group (Ho et al., 2008b). More recently, Ho et al. (2008a) performed a randomized, paralleltreatment, placebo-controlled, double-blind, trial at 81 sites in Europe and the U.S.A., in which adults with migraine treated moderate or severe attacks with either oral telcagepant 150 mg or 300 mg, zolmitriptan 5 mg, or placebo. For this purpose, 1380 patients were randomly assigned to receive telcagepant 150 mg (n = 333) or 300 mg (354), zolmitriptan (345), or placebo (348). Telcagepant 300 mg was more effective than placebo for pain freedom (95 [27%] of 353 patients versus 33 [10%] of 343 [p < 0.0001]), pain relief (194 [55%] of 353 versus 95 [28%] of 343 [p < 0.0001]), and absences of phonophobia (204 [58%] of 353 versus 126 [37%] of 342 [p < 0.0001]), photophobia (180 [51%] of 353 versus 99 [29%] of 342 [p < 0.0001]), and nausea (229 [65%] of 352 versus 189 [55%] of 342 [p = 0.0061]). Efficacies of telcagepant 300 mg and zolmitriptan 5 mg were practically the same and both were more effective than telcagepant 150 mg. Adverse events were recorded for 31% taking telcagepant 150 mg, 37% taking telcagepant 300 mg, 51% taking zolmitriptan

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5 mg, and 32% taking placebo. With these findings, it is clear that telcagepant 300 mg is effective for the acute treatment for migraine with an efficacy comparable to that of zolmitriptan 5 mg, but with fewer associated adverse effects (Ho et al., 2008a). Regarding potential adverse effects by telcagepant, Van der Schueren et al. (2008) have analysed the effect of this drug on the haemodynamic responses to sublingual GTN of 22 healthy males. The subjects received telcagepant 500 mg or placebo, followed after 90 min by 0.4 mg sublingual GTN. Central augmentation index, a measure of arterial stiffness and brachial artery diameter, was measured at multiple times post dosing, and there was no alteration in GTN effects, specifically in its vasodilator responses; this suggests that vasodilatation by NO is unlikely to be affected by blockade of CGRP receptors. While the side effect profile with intermittent dosing as required in the acute treatment of migraine looks excellent, a press release has recently informed that Merck Research Laboratories do not any longer expect to file an application to the FDA for telcagepant in 2009. This decision was based on findings from a phase IIA exploratory study in which a small number of patients taking telcagepant twice daily for three months for the prevention of migraine had marked elevations in liver transaminases (Tepper & Cleves, 2009). This daily intake was different from the intermittent intake relevant to the acute treatment of migraine. The company is currently conducting an analysis of the way forward for the compound. It is not known whether the liver toxicity is related to the particular compound or whether it is a class effect, most likely the former. Until more information is available, if telcagepant-induced hepatic toxicity is not observed in ongoing/ future trials of its acute use, then this agent may offer an alternative to triptan therapy for the treatment of migraine. 4. Implications, future directions and conclusions A number of other CGRP receptor antagonists are at various stages of preclinical or clinical development, but little is in the public domain about these compounds. A recent review discusses the available evidence (Davis & Xu, 2008). It can be expected that a number of compounds will be developed to compete with telcagepant. A large body of evidence indicates that CGRP plays an important role in the pathophysiology of migraine and that small molecule CGRP receptor antagonists (i.e. olcegepant and telcagepant) possess acute antimigraine properties with an efficacy similar to that of triptans. An important advantage of CGRP receptor antagonists over the triptans (which may provoke vasoconstrictor or vascular side effects) is the absence of vasoconstrictor activity, possibly allowing the treatment of migraineurs with cardiovascular risk factors. Moreover, CGRP receptor antagonists have no central side effects such as sedation or dizziness, which often pose a problem in the use of triptans. The prolonged antimigraine action of olcegepant and telcagepant may suggest not only less rebound headache, but perhaps also a prophylactic possibility. Notwithstanding: (i) olcegepant could not be further developed as an antimigraine medication because it can only be given i.v., a therapeutic limitation that led to the development of the orally bioavailable agent, telcagepant; and (ii) telcagepant taken twice daily for three months for the prophylactic treatment of migraine (not in acute treatment) produced hepatic toxicity. Regarding future developments, the acute and prophylactic antimigraine efficacy of other oral CGRP receptor antagonists devoid of hepatotoxic properties is waited with great interest. Moreover, it would be interesting to confirm whether a molecule that binds directly to CGRP and blocks its binding to CGRP receptors (e.g. NOXC89; Juhl et al., 2007) or specific monoclonal antibodies against human CGRP (Edvinsson et al., 2007) have antimigraine properties. Clearly, further investigation to understand migraine pathophysiology and the development of better treatments will continue in the emerging post-triptan era.

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