Neurobiology of Disease 74 (2015) 137–143

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Evidence for orexinergic mechanisms in migraine Jan Hoffmann a,1, Weera Supronsinchai a,2, Simon Akerman a, Anna P. Andreou a,3, Christopher J. Winrow c, John Renger c, Richard Hargreaves c, Peter J. Goadsby a,b,⁎ a b c

Headache Group-Department of Neurology, University of California, San Francisco, San Francisco, CA, USA NIHR-Wellcome Trust Clinical Research Facility, King's College London, UK Neuroscience Department, Merck Research Laboratories, Sumneytown Pike, West Point, PA 19486, USA

a r t i c l e

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Article history: Received 21 August 2014 Revised 8 October 2014 Accepted 29 October 2014 Available online 4 November 2014 Keywords: Headache Migraine Sleep Orexin Dual Orexin Receptor Antagonists

a b s t r a c t Objective: To examine the effect of the orexinergic blockade with a dual orexin receptor antagonist (DORA) on experimental models of peripheral and central trigeminal as well as cortical activation relevant to migraine and migraine aura. Methods: In this study we used a precursor of suvorexant, a dual orexin receptor antagonist #12 (DORA-12) in established experimental in vivo models of dural trigeminovascular nociception in rat. Neurogenic dural vasodilation and electrophysiological recordings of second order trigeminocervical neurons were used to study trigeminal nociceptive mechanisms directly. KCl-evoked cortical spreading depression was also used as a surrogate for migraine aura. Results: Neurogenically-induced vasodilation of the middle meningeal artery, caused by nociceptive activation of peripheral afferent projections of the trigeminal nerve, was attenuated by intravenous DORA-12 (1 mg kg−1). Second-order trigeminocervical complex neuronal activity was significantly inhibited by intravenous DORA-12 (1 mg kg−1). DORA-12 significantly reduced susceptibility to KCl-evoked cortical spreading depression. Conclusion: The study provides the first direct evidence, that simultaneous antagonism on both orexin receptors is able to attenuate trigeminal nociceptive activity as well as to induce an elevation of the threshold for the induction of a cortical spreading depression (CSD). In the clinical context, these data imply that targeting the hypothalamic orexinergic system may offer an entirely novel mechanism for the preventive treatment of migraine with and without aura. © 2014 Elsevier Inc. All rights reserved.

Introduction Worldwide migraine is one of the most common and disabling neurological disorders (Goadsby et al., 2002; Lipton et al., 2001; Murray et al., 2012). While current preventive therapies are effective to some degree, in approximately 50% of patients, there is a significant need for novel, safer treatments (Goadsby and Sprenger, 2010). Besides the trigeminovascular system, where most therapeutic drugs are thought to act, specific brainstem and diencephalic nuclei, including the hypothalamus, are also thought to be essential elements in the

⁎ Corresponding author at: NIHR-Wellcome Trust Clinical Research Facility, King's College Hospital, London SE5 9PJ UK. E-mail address: [email protected] (P.J. Goadsby). Available online on ScienceDirect (www.sciencedirect.com). 1 Current address: Department of Neurology, Charité - Universitätsmedizin Berlin, Berlin, Germany. 2 Current address: Department of Physiology, Faculty of Dentistry, Chulalongkorn University, Bangkok Thailand. 3 Current address: Department of Anesthesia and Pain Management, Imperial College London, UK.

http://dx.doi.org/10.1016/j.nbd.2014.10.022 0969-9961/© 2014 Elsevier Inc. All rights reserved.

pathophysiology of migraine, and may represent novel target loci (Akerman et al., 2011). The hypothalamus has long been suspected to play a prominent role in migraine pathophysiology as many of the features and symptoms associated with the clinical syndrome, such as the circadian periodicity as well as premonitory and autonomic symptoms, are all regulated to some extent by the hypothalamus (Goadsby, 2009). From a neuroanatomical perspective, specific hypothalamic nuclei have known ascending and descending connections to pain processing structures within the brain, as well as to the dorsal horns of the spinal cord (Akerman et al., 2011; van den Pol, 1999), through which the hypothalamus may modulate nociceptive neurotransmission. These connections include orexin-containing neurons which originate in the posterior, lateral and paraventricular parts of the hypothalamus (Gotter et al., 2012a,b; Sakurai et al., 1998), areas known to modulate basal and dural-evoked nociceptive activation in the trigeminocervical complex (TCC) (Bartsch et al., 2004; Benjamin et al., 2004; Robert et al., 2013). The orexinergic system comprises the orexinergic neuropeptides, orexin A and B, which are exclusively produced in the hypothalamus and act via G-protein-coupled OX1 and OX2 receptors through projections to the prefrontal cortex, thalamus and other subcortical areas to promote

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arousal (Smart and Jerman, 2002) and exert modulating effects on nociceptive neurotransmission, thermoregulation, neuroendocrine and autonomic functions (Ferguson and Samson, 2003; Holland and Goadsby, 2007; Samson et al., 2005). The premonitory phase of a migraine attack may include symptoms such as yawning, food cravings and changes in wakefulness (Giffin et al., 2003), which are thought to be regulated to a significant extent by the hypothalamus and its orexinergic neurons. The same applies to the hypothalamic regulation of the sleep wake cycle (George and Singh, 2000). Orexins are required for normal wakefulness as the loss of orexinergic neurons induces narcolepsy (Siegel, 1999). In migraine, alterations in sleep pattern such as sleep deprivation have been identified as trigger factors for single attacks (Wober et al., 2007) and attack prevalence follows a diurnal variation with most migraine attacks occurring in the early morning hours (Alstadhaug et al., 2007). Taken together, current evidence indicates that the hypothalamus, and probably the orexinergic system, has substantial influence on migraine, especially during the early phases of a migraine attack (Maniyar et al., 2014). This concept is supported by imaging studies that demonstrate hypothalamic activations in the premonitory (Maniyar et al., 2014) and acute phases of the migraine attack (Denuelle et al., 2007, 2008). Several dual orexin receptor antagonists (DORAs), which simultaneously block the OX1 and OX2 receptors, have been developed, primarily for the treatment of insomnia, inducing and maintaining sleep with natural sleep architecture, without sedating side-effects (Gotter et al., 2012a), including suvorexant (MK-4305) (Herring et al., 2012). Based on the existing preclinical and clinical evidence, we hypothesized that a DORA treatment strategy may inhibit nociceptive trigeminal neurotransmission. To test this hypothesis, DORA-12 (Fig. 1) (Coleman et al., 2012; Gotter et al., 2012a), a precursor of the dual orexin receptor antagonist suvorexant, was studied in several established in vivo models of migraine known to be predictive of clinical efficacy to elucidate a potential effect in the treatment of migraine (Bergerot et al., 2006). Some data has been presented in preliminary form at the 65th annual meeting of the American Academy of Neurology (San Diego, CA, Hoffmann et al., 2013).

frame (Kopf Instruments, Tujunga, CA, USA) rats were ventilated (Ugo Basile, Comerio, VA, Italy) with oxygen-enriched air (2.5–3.5 ml per stroke, 80–100 strokes per minute). End-tidal CO2 was monitored (CapStar-100, CWE, Ardmore, PA, USA) and kept between 3.5 and 4.5%. Arterial blood pressure was continuously monitored using a transducer (DTX Plus DT-XX, Becton Dickinson, Sandy, UT, USA) connected to the cannula placed in the femoral artery. Arterial blood pressure and end-tidal CO2 were displayed and recorded on a personal computer using an online data analysis system (Power 1401plus) and Spike 5.2 software (Cambridge Electronic Design, Cambridge, UK). In all experiments a cranial window was created over the parietal cortex exposing the middle meningeal artery (MMA) for meningeal or cortical stimulation (Fig. 2). At the end of each experiment animals were euthanized by administration of a lethal dose of intravenously administered pentobarbital and phenytoin sodium (Euthasol®, Virbac AH, Inc., Fort Worth, TX, USA). Electrical stimulation and recording of the neurogenic dural vasodilation In this subset of experiments electrical stimulation with squarewave pulses (5 Hz, 1 ms duration, 100–200 μA) was performed for 10 s using a bipolar stimulating electrode (NE200, Rhodes Medical Instruments, Summerland, CA, USA) placed over the MMA on a closed cranial window (Fig. 2A). Electrical stimulation of the cranial window is thought to cause the prejunctional release of calcitonin gene-related peptide (CGRP) from the activated trigeminal nerve which in turn induces vasodilation (Williamson et al., 1997). The extent of the induced vasodilation was quantified using an intravital microscope (Microvision MV2100, Finlay Microvision, Warwickshire, UK) connected to a video dimension analyzer (VDA-10, Living Systems Instrumentation, St. Albans, VT, USA) and the output fed through a data acquisition system (Power 1401, Cambridge Electronic Design, Cambridge, UK) into a personal computer using Spike 5.2 software (Cambridge Electronic Design, Cambridge, UK) (Fig. 2A). Following 3 baseline stimulations DORA-12 (1 mg kg−1) or its vehicle (25% cyclodextrin in water for injection) were administered intravenously and stimulations were performed 5, 15, 30, 45 and 60 min after drug administration (Holland et al., 2012).

Materials and methods All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Francisco. Male Sprague-Dawley rats (n = 37, 300–350 g, Charles River Laboratories, Hollister, CA, USA) were anesthetized using pentobarbital (60 mg kg−1 i.p.; Nembutal, Lundbeck, Deerfield, IL, USA) for induction and propofol (20–25 mg kg−1 h−1 i.v.; Propoflo, Abbott, Abbott Park, IL, USA) for maintenance throughout the experiment. A femoral artery and both femoral veins were cannulated for monitoring of arterial blood pressure, continuous administration of the anesthetic and drug administration, respectively. A self-regulating homeothermic blanket system (Harvard Apparatus, Holliston, MA, USA) was used to maintain body temperature at 37 ± 0.5 °C. After fixation of the skull in a stereotaxic

Electrical stimulation and recording of neuronal activity in the trigeminocervical complex Following removal of the parietal bone within the cranial window, a bipolar stimulating electrode (NE200, Rhodes Medical Instruments, Summerland, CA, USA) connected to a stimulus isolation unit (SIU5A, Grass Instruments, Quincy, MA, USA) was placed on the intact dura mater above the middle meningeal artery for electrical stimulation of the perivascular afferents of the trigeminal nerve (Fig. 2B). Stimulation of primary trigeminal afferents was performed supramaximally with square wave pulses generated by a Grass S88 stimulator (Grass Instruments, Quincy, MA, USA, stimulation parameters: 10–18 V, 0.1–0.2 ms duration, 0.5 Hz, 20 sweeps).

Fig. 1. Dual Orexin Receptor Antagonist 12 (DORA-12). Fig. 1 depicts the structure, potency and selectivity of DORA-12. Ki values were determined from a radioligand assay, IC50 values were determined from a cell-based Ca2+ mobilization assay (FLIPR). (Gotter et al., 2012a,b).

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Fig. 2. A–C: Experimental setup. Fig. 2A illustrates the experimental setup for the assessment of the vessel diameter of the MMA using intravital microscopy. Neurogenic vasodilation is induced through an electrical stimulus applied with a stimulation electrode placed above the MMA and vessel diameter is assessed with a video camera connected to a video dimension analyzer. Fig. 2B illustrates the experimental setup for the recording of stimulus-evoked and spontaneous neuronal activity in the TCC. Electrical stimulation is performed on trigeminal dural afferents in the vicinity of the MMA. Recording of stimulus-induced neuronal transmission of wide-dynamic range neurons is performed in the TCC. Fig. 2C illustrates the experimental setup for the induction and recording of repetitive cortical spreading depressions. Repetitive CSD are induced by topical application of 3 mg solid KCl on the cortical surface. DC-shifts are recorded using a single barreled glass microelectrode placed at a depth of 500 μm into the cortical surface.

For extracellular recording of neuronal activity in the trigeminocervical complex a laminectomy was performed at the level of C1 and after removal of the dura and pia mater a tungsten electrode with a nominal impedance of 1 MΩ (World Precision Instruments, Sarasota, FL, USA) was introduced in the trigeminocervical complex in the vicinity of the dorsal root entry zone (Fig. 2B). Wide dynamic range neurons were identified by their responsiveness to innocuous brush, noxious pinch and electrical stimulation of primary trigeminal afferents surrounding the MMA. A piezo-electric motor-driven microelectrode positioner attached to a micromanipulator (Kopf 1760-61, Tujunga, CA, USA) was used to locate the optimal recording site allowing movements of the recording electrode in 5 μm steps. Electrical signals from the recording electrode were fed into a head-stage amplifier (NL100AK, Neurolog, Digitimer, Welwyn Garden City, Hertfordshire, UK) and passed to an AC preamplifier (Neurolog NL104, Digitimer, Welwyn Garden City, Hertfordshire, UK) set to a gain of × 2000 and using a 10 Hz low frequency cutoff filter to remove DC components. The signal was then bandpass filtered (Neurolog NL125/126, Digitimer, Welwyn Garden City, Hertfordshire, UK) from 300 Hz to 20 kHz and passed through a Hum Bug 60 Hz noise eliminator (Quest Scientific, Vancouver, BC, Canada) for removal of line interference before further amplification using an AC-DC amplifier (Neurolog NL106, Digitimer, Welwyn Garden City, Hertfordshire, UK) with a gain range of × 50–×100 (total gain used approx. 20,000–30,000 Hz). The obtained electrical signal was then fed through a gated amplitude discriminator (NL201) and a data acquisition system (Power 1401, Cambridge Electronic Design, Cambridge, UK) to a personal computer using Spike 5.2 software (Cambridge Electronic Design, Cambridge, UK). For the analysis of stimulus-evoked neuronal activity, post-stimulus histograms were constructed online. For the analysis of post-stimulus histograms a mean of 20 stimulations was calculated. Background activity was analyzed as cumulative rate histograms in which neuronal activity gated through the amplitude discriminator was collected into successive bins. To facilitate spike discrimination the signal was also fed to an oscilloscope and an audio amplifier (Neurolog NL120, Digitimer, Welwyn Garden City, Hertfordshire, UK) connected to a loudspeaker. Baseline values of the stimulus-evoked responses were obtained calculating the mean of 3 consecutive stimulation series prior to any drug administration. Following drug administration (DORA-12, 1 mg kg−1 or vehicle 25% cyclodextrin in water for injection) post-stimulus histograms were recorded at 5, 10, 15, 20, 25, 30, 45 and 60 min (Akerman et al., 2013).

Induction and recording of cortical spreading depression Repetitive cortical spreading depressions (CSD) were induced by topical application of 3 mg solid KCl onto the parietal cortex (Supornsilpchai et al., 2010). Cortical steady potentials (DC-shifts) were measured. For DC-shift recordings two craniotomies were made 7 mm posterior and 1 mm lateral to bregma for stimulation as well as 1 mm anterior and 1 mm lateral for recording. For recording a singlebarrel glass microelectrode containing an Ag/AgCl wire and filled with 4 mol/L NaCl was inserted to a depth of 500 μm into the cortical surface (Fig. 2C). The signal was amplified and passed through a 60 Hz noise eliminator (Humbug, Quest Scientific, Vancouver, BC, Canada) from there it passed to an analog-to-digital converter and an online analysis system (Power 1401, Cambridge Electronic Design, Cambridge, UK) to be finally displayed on a personal computer using Spike 5.2 software (Cambridge Electronic Design, Cambridge, UK). Following intravenous administration of DORA-12 (1 mg kg−1) or its vehicle (25% cyclodextrin in water for injection), repetitive CSDs were induced and the number of DC-shifts occurring within one hour were counted (Akerman and Goadsby, 2005; Supornsilpchai et al., 2010).

Statistical analyses Statistical analysis was performed using IBM SPSS 20.0 software. Data are expressed as percentages of baseline values (TCC recordings and intravital microscopy) or mean values (CSD recordings) with standard errors of the mean (± SEM). Data were normally distributed (Kolmogorov–Smirnov test). Treatment effects were analyzed using an ANOVA with repeated measures. Greenhouse–Geisser correction was used if the assumption of sphericity was violated (Field, 2000). Bonferroni correction was applied for multiple comparisons. Treatment effects at single time points were compared to baseline values using the paired t-test. The inhibition in relation to baseline was compared for each time point between both treatment groups using the independent t-test. Statistical significance was assessed at p b 0.05.

Results In all experiments temperature and end-tidal CO2 were kept at physiological levels. The intravenous administration of DORA-12 or vehicle had no significant effect on arterial blood pressure (data not shown).

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Neurogenic dural vasodilation A total of 12 animals were studied. Electrical stimulation of meningeal blood vessels induced a transient vasodilation which did not change after intravenous vehicle administration (n = 6). In contrast, neurogenic dural vasodilation was significantly attenuated by 17 ± 8% after intravenous administration of DORA-12 (1 mg kg−1) (F5,25 = 2.72; p b 0.05; n = 6) (Fig. 3A). Maximum inhibition was observed at 5 min (−17 ± 8%, DORA-12, n = 6 vs. 8 ± 6%, vehicle, n = 6; t10 = −2.38; p b 0.05). The results indicate an effect of DORA-12 on the peripheral dural projections of the trigeminovascular system.

Trigeminocervical complex (TCC) effects To test for a potential action of DORA-12 on central trigeminal neurons extracellular recordings of stimulus-evoked activity were performed in wide dynamic range neurons located in the vicinity of the dorsal root entry zone within the TCC. In this experimental setup 13 animals were studied. DORA-12 (1 mg kg−1) significantly attenuated stimulus-evoked neuronal activity in the TCC (F3.3,20.1 = 6.43; p b 0.01; n = 7) whereas its vehicle (cyclodextrin 25%, n = 6) had no effect. Maximum inhibition was recorded at 60 min (− 31 ± 6%, t6 = 5.53;

p b 0.01) (Fig. 3B). Stimulus-evoked neuronal activity did not recover within the observational period. In contrast to the stimulus-evoked responses, DORA-12 or its vehicle had no significant effect on background firing of wide-dynamic range neurons in the TCC. The results indicate an effect on second-order neurons of the trigeminovascular system which appears to be selective to nociceptive stimulus-evoked neuronal activity. Cortical spreading depression The action of DORA-12 on cortical spreading depression (CSD) was analyzed in order to obtain information related to a potential preventive action in migraine aura (Akerman and Goadsby, 2005). On average, the observed number of DC-shifts per hour was significantly attenuated from 7.5 ± 1.1 (vehicle, n = 6) to 3.7 ± 0.6 (n = 6; t10 = − 3.01; p b 0.05) after intravenous administration of DORA-12 (1 mg kg− 1) (Figs. 4A and B). The results suggest, that DORA-12 may be able to prevent migraine aura by elevating the threshold for the activation of cortical neurons and thereby preventing the induction of a CSD. Discussion Here we have demonstrated that a dual orexin 1 and 2 receptor antagonist, DORA-12, is able to attenuate neurogenic dural vasodilation and trigeminocervical complex activation. Moreover, DORA-12 has an inhibitory effect on KCl-stimulated cortical spreading depression. Taken together with previous observations (Bartsch et al., 2004; Holland et al., 2006) and the clinical features of migraine, these data suggest that orexinergic modulation may offer a potential novel approach to migraine preventive therapy. Orexins and the trigeminovascular system In the context of migraine, perhaps the most relevant finding is the observation that orexins can modulate trigeminal nociceptive transmission, either systemically, or via local anatomical structures, where the hypothalamus has proven to be an important location (Holland et al., 2005; Holland and Goadsby, 2007). It is known that chemical manipulation of descending projections from the posterior, lateral and paraventricular

Fig. 3. A and B: Neurogenic vasodilation and extracellular recording of neuronal activity in the TCC following electrical stimulation of the MMA. Neurogenic dural vasodilation is largely mediated by the stimulus-induced release of the vasodilatory calcitonin generelated peptide (CGRP). DORA-12 (1 mg kg−1) significantly inhibited neurogenic vasodilation of the MMA with a maximum inhibition at 5 min (Fig. 3A). DORA-12 (1 mg kg−1) significantly attenuated stimulus-induced neuronal activity in the TCC whereas its control had no effect. Maximum inhibition of stimulus-induced activity was recorded at 60 min (Fig. 3B).

Fig. 4. A and B: Recording of KCl-evoked cortical spreading depressions. Repetitive CSD's induced by topical application of 3 mg KCl were significantly reduced by the intravenous administration of DORA-12. The mean number of CSD's occurring in 1 h was reduced from 7.5 ± 1.1 (vehicle) to 3.7 ± 0.6 (DORA-12).

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hypothalamus is able to modulate trigeminovascular nociceptive transmission (Bartsch et al., 2004; Robert et al., 2013). These are areas known specifically to be involved in the production of orexins. However, orexinergic effects on trigeminovascular nociceptive processing are complex and not yet fully understood, and perhaps illustrate the need for a dual receptor approach to treatment, rather than targeting a single receptor. Bartsch et al. have demonstrated in an in vivo model of trigeminovascular nociception that orexin peptides in the posterior hypothalamus induce opposing effects in nociceptive neurotransmission within the TCC. While orexin A induced antinociceptive effects, orexin B, the more specific OX2 receptor agonist, was pronociceptive (Bartsch et al., 2004). The influence of orexins on the peripheral nervous system is less clear. While neurogenic vasodilation in the dura mater can be attenuated by orexin A, which seems to be mediated specifically by the OX1 receptor (Holland et al., 2005), in vitro models using orexin A, which activates the OX1 and OX2 receptors simultaneously, indicate a sensitizing effect (Ozcan et al., 2010). In addition to the described postsynaptic effects, orexins also act presynaptically thereby inducing the release of glutamate and, to a small extent, GABA. The fact that, despite having mainly excitatory effects, orexin may act on inhibitory interneurons, highlights the complexity of the system (Scammell and Winrow, 2011). Based on this complexity, the findings may not yet be generalized. Clinical implications The new data have clinical implications for the treatment of migraine and for our understanding of its pathophysiology. First, the hypothalamus is thought to influence many aspects of migraine. These effects range from the regulation of the circadian rhythm and the appearance of premonitory symptoms, to modulating effects of nociceptive neurotransmission, as well as being activated at the beginning (Maniyar et al., 2014) and during a migraine attack (Denuelle et al., 2007). The neuroanatomical basis for these effects are ascending and descending connections to the dorsal horns of the spinal cord as well as to multiple structures in the brain involved in nociceptive processing such as the rostroventromedial medulla (RVM), the periaqueductal gray (PAG) and the nucleus raphe magnus (NRM). These connections include corticolimbic structures that mediate the affective components of pain perception (Date et al., 1999; Marcus et al., 2001; Nambu et al., 1999; Peyron et al., 1998). A wide variety of neurotransmitters are involved in the functional communication of this network. Among these, and secondly from the perspective of the clinical implications of these data, the orexinergic system plays a prominent role. This includes the neuropeptides orexin A and B, which are thought to be key regulators in feeding, sleep and wakefulness (Chemelli et al., 1999; de Lecea et al., 1998; Sakurai et al., 1998). In general migraine is believed to involve some level of dysfunction in areas of the brainstem and diencephalon, including the hypothalamus, vlPAG, NRM and A11 hypothalamic nucleus (Akerman et al., 2011), and it seems clear this may affect release of orexins and cause an imbalance of their effects in a system which alters the processing of trigeminovascular nociceptive neurons resulting in headache. Clinical data suggest that elevated orexin levels are associated with the early stages of a migraine attack. Based on these findings and to circumvent the difficulties arising from opposing orexin effects depending on the site of action and dosage used, studying the orexinergic system with receptor antagonists may offer a more objective approach. In this context, the effect of simultaneous antagonism on both receptors has not been investigated previously in animal models of migraine. Considering the putative hypothalamic involvement in migraine based on its clinical features and the association between migraine and sleep, we studied a dual orexin-1 and -2 receptor antagonist (DORA-12) in animal models that are likely to predict efficacy in migraine. It is known from rodent sleep studies that DORA-12 is orally bioavailable with a plasma protein binding rate of 97%, has good brain penetrance and demonstrates orexin receptor occupancy (Cox et al., 2010; Gotter et al., 2012a,b). Our results

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show, that DORA-12 inhibited neurogenic dural vasodilation, nociceptive trigeminovascular activation in the trigeminocervical complex, and cortical spreading depression. The results therefore indicate that DORA-12 acts on peripheral and central elements of the trigeminovascular system, suggesting that in migraine, the facilitatory effects of orexinergic neurons on trigeminal nociceptive neurotransmission prevail. These preclinical findings are in line with clinical data showing that high orexin levels are associated with the early stages of migraine. The observation that it also acts on the cortex influencing the susceptibility for the induction of cortical spreading depression, suggests that besides of its effect on nociceptive neurotransmission, it may also be effective in attenuating migraine aura. However, our model does not allow conclusions on the exact site of action as DORA-12 was administered systemically. Rather these data suggest that during nociceptive activation of trigeminovascular pathways and cortical excitability a change in orexin peptide levels occurs, and a dual orexin receptor antagonist approach balances the effects of orexin A and B on these neuronal mechanisms to reduce nociceptive activation and cortical excitability. The precedence that a dual orexin receptor antagonist approach can be clinically effective is demonstrated with suvorexant, which is beneficial in the treatment of insomnia (Herring et al., 2012). Re-establishing a balance and control of orexin peptides can have significant therapeutic effects in a neurological condition. Furthermore, the fact that a DORA approach improves sleep quality may also add to its beneficial effect in migraineurs, patients known to have disturbed sleep as both a symptom and a trigger of migraine. In this context it may be speculated that they may also affect premonitory symptoms such as mood changes or food cravings as these are mediated to a large extent by the hypothalamus and its orexinergic neurons. Orexin biology Orexins are exclusively synthesized in the lateral, posterior and paraventricular nuclei of the hypothalamus (Gotter et al., 2012a; Sakurai, 2005), which interestingly represent specific areas of the hypothalamus that have functional connections with neurons of the TCC. Orexinergic neurons also project to a wide range of areas within the brain such as the cerebral cortex, thalamus, hypothalamus, brainstem, locus coeruleus and the raphe nucleus (Marcus et al., 2001; Peyron et al., 1998). Most is known about the role of orexins in the sleep– wake cycle, which has implications in migraine, where they stabilize wakefulness and decrease REM and NREM sleep time (Hagan et al., 1999). In this context, extracellular orexin levels are elevated during wakefulness and during sleep deprivation (Lee et al., 2005). While the OX2 receptor regulates arousal, the OX1 receptor is involved in gating between vigilance states (Cox et al., 2010; Gotter et al., 2012a). The relevance of the sleep regulating effect of the orexinergic system is highlighted by the observation that the lack of orexinergic neurons induces the clinical picture of narcolepsy (Peyron et al., 2000). In this regard, patients with narcolepsy show a higher prevalence of migraine (Dahmen et al., 1999); migraine attacks initiate mostly in the early morning hours (Alstadhaug et al., 2007) and sleep deprivation may trigger migraine attacks (Rasmussen, 1993; Wober et al., 2007). These observations suggest that high orexin levels may increase the susceptibility for the triggering of a migraine attack (Sarchielli et al., 2008). In addition, preclinical and clinical studies indicate low glucose levels induce prepro-orexin mRNA synthesis and as a consequence elevate orexin levels leading to an activation of orexinergic neurons (Scammell and Winrow, 2011). Stressful stimuli may also induce an increase in orexin levels (Boutrel et al., 2005; Scammell and Winrow, 2011). Since fasting and stress are suggested migraine triggers and may also appear as part of the premonitory symptoms of migraine attacks (Rasmussen, 1993; Wober et al., 2007), these findings further support the link between orexinergic mechanisms and migraine. The local effects of orexins in other areas of the brain that receive projections from orexinergic hypothalamic regions, on trigeminovascular nociceptive transmission, also demonstrate this complexity. We have

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shown previously, that microinjection of orexin A and orexin B in to the NRM, which is mainly an inhibitory structure, facilitates neuronal activity in the TCC, an effect that was mainly driven by OX2 receptors (Supronsinchai et al., 2013). The results demonstrate that OX2 receptors within the NRM are also implicated in the descending control of pain modulation, and most likely via a pro-nociceptive action. Orexins also affect trigeminovascular nociceptive processing when locally injected into the vlPAG (Holland et al., 2008) and A11 hypothalamic nucleus (Charbit et al., 2009). In each case orexin A inhibited trigeminovascular neuronal responses of both basal and noxious inputs. Orexin A and B and their receptors are clearly involved in modulating trigeminovascular nociceptive transmission in many areas of the brain. These data demonstrate the complexity of their influence and considering that distinct orexin dosages may also induce opposing effects (Siegel, 2004) adds to the complexity of the system and confirms that caution has to be taken in generalizing results from specific loci. Taken together, these data demonstrate the first example of a dual orexin receptor antagonist approach in preclinical models of migraine and indicate that this should be pursued further in preclinical models and clinical trials to elucidate whether they may offer a novel therapeutic option in the preventive management of migraine. Conflicts of interest Jan Hoffmann, Weera Supronsinchai, Simon Akerman and Anna Andreou have no disclosures. Peter J Goadsby has consulted for Merck on the potential for orexin mechanisms in migraine. Christopher J. Winrow, John Renger and Richard Hargreaves are employees of Merck. Source of funding Merck Research Labs. Acknowledgments Jan Hoffmann was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft — DFG) (HO4369/1-1). The authors would like to thank Philipp R. Holland for his valuable advice on the intravital microscopy experiments and his contribution on the drawings for the experimental setup. References Akerman, S., Goadsby, P.J., 2005. Topiramate inhibits cortical spreading depression in rat and cat: impact in migraine aura. NeuroReport 16, 1383–1387. Akerman, S., et al., 2011. Diencephalic and brainstem mechanisms in migraine. Nat. Rev. Neurosci. 12, 570–584. Akerman, S., et al., 2013. Endocannabinoids in the brainstem modulate dural trigeminovascular nociceptive traffic via CB1 and ‘triptan’ receptors: implications in migraine. J. Neurosci. 33, 14869–14877. Alstadhaug, K., et al., 2007. Insomnia and circadian variation of attacks in episodic migraine. Headache 47, 1181–1188. Bartsch, T., et al., 2004. Differential modulation of nociceptive dural input to [hypocretin] Orexin A and B receptor activation in the posterior hypothalamic area. Pain 109, 367–378. Benjamin, L., et al., 2004. Hypothalamic activation after stimulation of the superior sagittal sinus in the cat: a Fos study. Neurobiol. Dis. 16, 500–505. Bergerot, A., et al., 2006. Animal models of migraine. Looking at the component parts of a complex disorder. Eur. J. Neurosci. 24, 1517–1534. Boutrel, B., et al., 2005. Role for hypocretin in mediating stress-induced reinstatement of cocaine-seeking behavior. Proc. Natl. Acad. Sci. U. S. A. 102, 19168–19173. Charbit, A.R., et al., 2009. Neurons of the dopaminergic/CGRP A11 cell group modulate neuronal firing in the trigeminocervical complex: an electrophysiological and immunohistochemical study. J. Neurosci. 29, 12532–12541. Chemelli, R.M., et al., 1999. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437–451. Coleman, P.J., et al., 2012. Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties. ChemMedChem 7 (415–24), 337.

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