The ATP sensitive potassium channel (KATP) is a novel target for migraine drug development.

Migraine is one of the leading causes of disability worldwide, affecting work and social life. It has been estimated that sales of migraine medicines will reach 12.9 billion USD in 2027. To reduce social impact, migraine treatments must improve, and the ATP-sensitive potassium (KATP) channel is a promising target because of the growing evidence of its implications in the pathogenesis of migraine. Strong human data show that opening of the KATP channel using levcromakalim is the most potent headache and migraine trigger ever tested as it induces headache in almost all healthy subjects and migraine attacks in 100% of migraine sufferers. This review will address the basics of the KATP channel together with clinical and preclinical data on migraine implications. We argue that KATP channel blocking, especially the Kir6.1/SUR2B subtype, may be a target for migraine drug development, however translational issues remain. There are no human data on the closure of the KATP channel, although blocking the channel is effective in animal models of migraine. We believe there is a good likelihood that an antagonist of the Kir6.1/SUR2B subtype of the KATP channel will be effective in the treatment of migraine. The side effects of such a blocker may be an issue for clinical use, but the risk is likely only moderate. Future clinical trials of a selective Kir6.1/SUR2B blocker will answer these questions.

Pharmacological Profiling of KATP Channel Modulators: An Outlook for New Treatment Opportunities for Migraine.

Migraine is a highly disabling pain disorder with huge socioeconomic and personal costs. It is genetically heterogenous leading to variability in response to current treatments and frequent lack of response. Thus, new treatment strategies are needed. A combination of preclinical and clinical data indicate that ATP-sensitive potassium (KATP) channel inhibitors could be novel and highly effective drugs in the treatment of migraine. The subtype Kir6.1/SUR2B is of particular interest and inhibitors specific for this cranio-vascular KATP channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize KATP channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different KATP subtypes is difficult to assess. Here, we characterize available KATP channel activators and inhibitors in fluorescence-based thallium-flux assays using HEK293 cells stably expressing human Kir6.1/SUR2B, Kir6.2/SUR1, and Kir6.2/SUR2A KATP channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the KATP channel subtype Kir6.1/SUR2B, whereas BMS-191095, NN414, and VU0071306 demonstrated preferred activation of the Kir6.2/SUR1 subtype. In the group of KATP channel blockers, only Rosiglitazone and PNU-37783A showed selective inhibition of the Kir6.1/SUR2B subtype. PNU-37783A was stopped in clinical development and Rosiglitazone has a low potency for the vascular KATP channel subtype. Therefore, development of novel selective KATP channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.

Role of PACAP in migraine: An alternative to CGRP?

Migraine is a widespread and debilitating neurological condition affecting more than a billion people worldwide. Thus, more effective migraine therapies are highly needed. In the last decade, two endogenous neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP), were identified to be implicated in migraine. Recently, introduction of monoclonal antibodies (mAbs) blocking the CGRP is the most important advance in migraine therapy for decades. However, 40% of patients are unresponsive to these new drugs. We believe that PACAP may be involved in these patients. Like CGRP, PACAP is located to sensory nerve fibers, it dilates cranial arteries, it causes migraine when infused into patients and it is a peptide that lends itself to antibody therapy. Also, recent studies suggest that the PACAP pathway is independent of the CGRP pathway. Understanding the signaling pathways of PACAP may therefore lead to identification of novel therapeutic targets of particular interest in patients unresponsive to anti-CGRP therapy. Accordingly, neutralizing mAb to PACAP is currently in clinical phase II development. The aim of the present review is, therefore, to give a thorough account of the existing data on PACAP, its receptors and its relation to migraine.

ATP-Sensitive Potassium Channels in Migraine: Translational Findings and Therapeutic Potential.

Globally, migraine is a leading cause of disability with a huge impact on both the work and private life of affected persons. To overcome the societal migraine burden, better treatment options are needed. Increasing evidence suggests that ATP-sensitive potassium (KATP) channels are involved in migraine pathophysiology. These channels are essential both in blood glucose regulation and cardiovascular homeostasis. Experimental infusion of the KATP channel opener levcromakalim to healthy volunteers and migraine patients induced headache and migraine attacks in 82-100% of participants. Thus, this is the most potent trigger of headache and migraine identified to date. Levcromakalim likely induces migraine via dilation of cranial arteries. However, other neuronal mechanisms are also proposed. Here, basic KATP channel distribution, physiology, and pharmacology are reviewed followed by thorough review of clinical and preclinical research on KATP channel involvement in migraine. KATP channel opening and blocking have been studied in a range of preclinical migraine models and, within recent years, strong evidence on the importance of their opening in migraine has been provided from human studies. Despite major advances, translational difficulties exist regarding the possible anti-migraine efficacy of KATP channel blockage. These are due to significant species differences in the potency and specificity of pharmacological tools targeting the various KATP channel subtypes.

Characterization of erenumab and rimegepant on calcitonin gene-related peptide induced responses in Xenopus Laevis oocytes expressing the calcitonin gene-related peptide receptor and the amylin-1 receptor.

BACKGROUND: The clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY1-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized. METHODS: The effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY1-R and their subunits. RESULTS: CGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY1-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY1-R and CTR. Inhibition potencies (pIC50 values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY1-R, respectively. Rimegepant inhibited CGRP induced currents with pIC50 values of 11.30 and 9.91 for CGRP-R and AMY1-R, respectively. CONCLUSION: Our results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY1-R with 32- and 25-times preference for the CGRP-R over the AMY1-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.

Ex vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents.

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

The PACAP pathway is independent of CGRP in mouse models of migraine: possible new drug target?

Calcitonin gene-related peptide (CGRP)-antagonizing drugs represent a major advance in migraine treatment. However, up to 50% of patients do not benefit from monoclonal antibodies against CGRP or its receptor. Here, we test the hypothesis that a closely related peptide, pituitary adenylate cyclase-activating peptide (PACAP-38), works independently of CGRP and thus might represent a new, alternative drug target. To understand differences in CGRP- and PACAP-mediated migraine pain, we used mouse models of provoked migraine-like pain based on multiple stimulations and subsequent measurement of tactile sensitivity response with von Frey filaments. Genetically modified mice lacking either functional CGRP receptors (Ramp1 knockout) or TRPA1 channels (Trpa1 knockout) were used together with CGRP-targeting antibodies and chemical inhibitors in wild-type mice (ntotal = 299). Ex vivo myograph studies were used to measure dilatory responses to CGRP and PACAP-38 in mouse carotid arteries. PACAP-38 provoked significant hypersensitivity and dilated the carotid arteries independently of CGRP. In contrast, glyceryl trinitrate-induced hypersensitivity is dependent on CGRP. Contrary to previous results with the migraine-inducing substances glyceryl trinitrate, cilostazol and levcromakalim, PACAP-38-induced hypersensitivity worked only partially through inhibition of ATP-sensitive potassium channels. Using multiple migraine-relevant models, these findings establish the PACAP-38 pathway as distinct from other migraine provoking pathways such as CGRP and glyceryl trinitrate. PACAP antagonism may therefore be a novel therapeutic target of particular interest in patients unresponsive to CGRP-antagonizing drugs.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models.

BACKGROUND: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. METHODS: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. RESULTS: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. CONCLUSION: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

Phase 1 study to access safety, tolerability, pharmacokinetics, and pharmacodynamics of kynurenine in healthy volunteers.

The kynurenine pathway (KP) is the main path for tryptophan metabolism, and it represents a multitude of potential sites for drug discovery in neuroscience, including pain, stroke, and epilepsy. L-kynurenine (LKYN), the first active metabolite in the pathway, emerges to be a prodrug targeting glutamate receptors. The safety, tolerability, pharmacokinetics, and pharmacodynamics of LKYN in humans have not been previously investigated. In an open-label, single ascending dose study, six participants received an intravenous infusion of 50, 100, and 150 µg/kg LKYN and new six participants received an intravenous infusion of 0.3, 0.5, 1, and 5 mg/kg LKYN. To compare the pharmacological effects between species, we investigated in vivo the vascular effects of LKYN in rats. In humans, LKYN was safe and well-tolerated at all dose levels examined. After infusion, LKYN plasma concentration increased significantly over time 3.23 ± 1.12 µg/mL (after 50 µg/kg), 4.04 ± 1.1 µg/mL (after 100 µg/kg), and 5.25 ± 1.01 µg/mL (after 150 µg/kg) (p ≤ 0.001). We observed no vascular changes after infusion compared with baseline. In rats, LKYN had no effect on HR and MAP and caused no dilation of dural and pial arteries. This first-in-human study of LKYN showed that LKYN was safe and well-tolerated after intravenous infusion up to 5 mg/kg over 20 minutes. The lack of change in LKYN metabolites in plasma suggests a relatively slow metabolism of LKYN and no or little feed-back effect of LKYN on its synthesis. The therapeutic potential of LKYN in stroke and epilepsy should be explored in future studies in humans.

Localization of TRPA1 channels and characterization of TRPA1 mediated responses in dural and pial arteries in vivo after intracarotid infusion of Na2S.

BACKGROUND: The Transient Receptor Potential Ankyrin 1 (TRPA1) channel might play a role in migraine. However, different mechanisms for this have been suggested. The purpose of our study was to investigate the localization and significance of TRPA1 channels in rat pial and dural arteries. METHODS: Immunofluorescence microscopy was used to localize TRPA1 channels in dural arteries, pial arteries, dura mater and trigeminal ganglion. The genuine closed cranial window model was used to examine the effect of Na2S, a donor of the TRPA1 channel opener H2S, on the diameter of pial and dural arteries. Further, we performed blocking experiments with TRPA1 antagonist HC-030031, calcitonin gene-related peptide (CGRP) receptor antagonist olcegepant and KCa3.1 channel blocker TRAM-34. RESULTS: TRPA1 channels were localized to the endothelium of both dural and pial arteries and in nerve fibers in dura mater. Further, we found TRPA1 expression in the membrane of trigeminal ganglia neuronal cells, some of them also staining for CGRP. Na2S caused dilation of both dural and pial arteries. In dural arteries, this was inhibited by HC-030031 and olcegepant. In pial arteries, the dilation was inhibited by TRAM-34, suggesting involvement of the KCa3.1 channel. CONCLUSION: Na2S causes a TRPA1- and CGRP-dependent dilation of dural arteries and a KCa3.1 channel-dependent dilation of pial arteries in rats.

ATP sensitive potassium (KATP) channel inhibition: A promising new drug target for migraine.

BACKGROUND: Recently, the adenosine triphosphate (ATP) sensitive potassium channel opener levcromakalim was shown to induce migraine attacks with a far higher incidence than any previous provoking agent such as calcitonin gene-related peptide. Here, we show efficacy of ATP sensitive potassium channel inhibitors in two validated rodent models of migraine. METHODS: In female spontaneous trigeminal allodynic rats, the sensitivity of the frontal region of the head was tested by an electronic von Frey filament device. In mice, cutaneous hypersensitivity was induced by repeated glyceryl trinitrate or levcromakalim injections over nine days, as measured with von Frey filaments in the hindpaw. Release of calcitonin gene-related peptide from dura mater and trigeminal ganglion was studied ex vivo. RESULTS: The ATP sensitive potassium channel inhibitor glibenclamide attenuated the spontaneous cephalic hypersensitivity in spontaneous trigeminal allodynic rats and glyceryl trinitrate-induced hypersensitivity of the hindpaw in mice. It also inhibited CGRP release from dura mater and the trigeminal ganglion isolated from spontaneous trigeminal allodynic rats. The hypersensitivity was also diminished by the structurally different ATP sensitive potassium channel inhibitor gliquidone. Mice injected with the ATP sensitive potassium channel opener levcromakalim developed a progressive hypersensitivity that was completely blocked by glibenclamide, confirming target engagement. CONCLUSION: The results suggest that ATP sensitive potassium channel inhibitors could be novel and highly effective drugs in the treatment of migraine.

Effect of TRPA1 activator allyl isothiocyanate (AITC) on rat dural and pial arteries.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channels may have a role in migraine as some substances known to cause headache activate the channel. In the craniovascular system such activation causes a calcitonin gene-related peptide (CGRP)-dependent increase in meningeal blood flow. TRPA1 channels in the endothelium of cerebral arteries cause vasodilation when activated. The headache preventive substance feverfew inhibits activation of TRPA1 channels. In this study we aim to compare and characterize the effect of the TRPA1 agonist allyl isothiocyanate (AITC) on the diameter of rat dural and pial arteries in vivo. METHODS: The genuine closed-cranial window technique in rats was used to examine changes in dural and pial artery diameter and mean arterial blood pressure (MABP) after intracarotid infusion of AITC. Blockade experiments were performed by intravenous infusion of olcegepant, HC-030031, sumatriptan or capsazepine immediately after infusion of AITC, in four different groups of rats. RESULTS: AITC caused a significant dilation of dural arteries, which was inhibited by HC-030031, olcegepant and sumatriptan, but not by capsazepine. In pial arteries AITC caused a significant dilation, which was not inhibited by any of the pre-treatments, suggesting a poor penetration of the blood-brain barrier or autoregulation due to dimethyl sulfoxide (DMSO) mediated decrease in MABP during HC-030031 infusion. AITC did not cause a significant change in MABP. CONCLUSION: AITC causes dilation of dural arteries via a mechanism dependent on CGRP and TRPA1 that is sensitive to sumatriptan. AITC causes a small but significant dilation of pial arteries.

PACAP-38 and PACAP(6-38) Degranulate Rat Meningeal Mast Cells via the Orphan MrgB3-Receptor.

Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating the involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study, we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor-expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than Pituitary Adenylate Cyclase Activating Peptide-27 (PACAP-27) in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor-expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed Pituitary Adenylate Cyclase Activating Peptide(6-38) [PACAP(6-38)] to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6-38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor.

Transcriptomic profiling of trigeminal nucleus caudalis and spinal cord dorsal horn.

The pain sensation system is highly conserved among species, thus animal models have been used to investigate relevant tissues. The focus for head-specific pain has been on the primary nociceptive neurons in the trigeminal pathway, i.e. trigeminal ganglia. The secondary nociceptive neurons of the trigeminal pathway, trigeminal nucleus caudalis (TNC), have not been assessed. We expect different gene expression profiles compared to the homologous spinal cord dorsal horn (SDH), as several signalling substances provoke head-specific pain but not peripheral pain. We aim to provide expression profiles of TNC and SDH, tissues highly relevant for pain- and migraine-studies. We extracted RNA from laser capture microdissected laminae I-V from TNC and SDH from six Wistar rats for RNA-Sequencing. We showed the expression profiles of genes involved in neural signal transmission and found that among all G protein-coupled receptors Gabbr1 was highest expressed in both tissues. Among the migraine-associated genes we showed that Cacna1a, where non-synonyms mutations can cause familial hemiplegic migraine, was highly expressed with a slightly lower expression in TNC than in SDH. To show the genetic differences between the two homologous systems we performed a differential expression analysis, revealing 1696 genes higher and 1895 genes lower expressed genes in TNC than in SDH, of which many were neuronal-related. The high number of differentially expressed genes shows the large genetic difference between the trigeminal and spinothalamic system. Our results contribute to the characterization of nociceptive pathways, which may help us understanding why several signalling molecules cause headache and no peripheral pain.

PACAP and its receptors in cranial arteries and mast cells.

BACKGROUND: In migraineurs pituitary adenylate cyclase activating peptide1-38 (PACAP1-38) is a potent migraine provoking substance and the accompanying long lasting flushing suggests degranulation of mast cells. Infusion of the closely related vasoactive intestinal peptide (VIP) either induces headache or flushing. This implicates the pituitary adenylate cyclase activating peptide type I receptor (PAC1) to be involved in the pathophysiology of PACAP1-38 provoked headaches. Here we review studies characterizing the effects of mainly PACAP but also of VIP on cerebral and meningeal arteries and mast cells. DISCUSSION: PACAP1-38, PACAP1-27 and VIP dilate cerebral and meningeal arteries from several species including man. In rat cerebral and meningeal arteries the dilation seems to be mediated preferably via vasoactive intestinal peptide receptor type 1 (VPAC1) receptors while, in human, middle meningeal artery dilation induced via vasoactive intestinal peptide receptor type 2 (VPAC2) receptors cannot be ruled out. PACAP1-38 is a strong degranulator of peritoneal and dural mast cells while PACAP1-27 and VIP only have weak effects. More detailed characterization studies suggest that mast cell degranulation is not mediated via the known receptors for PACAP1-38 but rather via a still unknown receptor coupled to phospholipase C. CONCLUSION: It is suggested that PACAP1-38 might induce migraine via degranulation of dural mast cells via a yet unknown receptor.

A unique inbred rat strain with sustained cephalic hypersensitivity as a model of chronic migraine-like pain.

Animal models of migraine-like pain enabling ongoing study of behaviour typically involve the systemic administration of chemical vasodilators or dural administration of inflammatory algogens. However, neither method mediates prolonged effects on behavior indicative of enduring pathophysiological changes occurring within dural or trigeminal pain circuits. We generated successive generations of a unique inbred rat strain, spontaneous trigeminal allodynia (STA) rats, previously reported to exhibit an episodic migraine-like behavioural phenotype. We show that both male and female STA rats display robust and sustained reductions in periorbital thresholds to cutaneous mechanical stimulation. Otherwise, the general behavior (e.g. locomotor, grooming) of these rats appeared normal. In female STA rats, the mechanical hypersensitivity was confined to the cephalic region, manifested after puberty through adolescence, and was sustained into adulthood recapitulating the clinical manifestation of migraine. We exploited this hitherto unidentified chronic phenotype to show that the migraine-specific drugs sumatriptan (5-HT1B/1D receptor agonist) and olcegepant (CGRP receptor antagonist) could completely reverse cephalic hypersensitivity using a within subject cross-over paradigm. Our findings indicate that STA rats actually possess a phenotype indicative of migraine chronicity which is exquisitely sensitive to migraine therapeutics. This unique strain could prove to be an invaluable resource in preclinical migraine drug discovery.

Cilostazol induces C-fos expression in the trigeminal nucleus caudalis and behavioural changes suggestive of headache with the migraine-like feature photophobia in female rats.

Introduction Research in development of new migraine therapeutics is hindered by the lack of suitable, predictive animal models. Cilostazol provokes headache in healthy humans and migraineurs by increasing intracellular cAMP levels. We aimed to investigate whether cilostazol could provoke headache-like behaviours and c-fos expression in rats. In order to evaluate the predictive validity of the model, we examined the response to the migraine specific drug sumatriptan. Methods The effect of cilostazol (125 mg/kg p.o.) in female Sprague Dawley rats was evaluated on a range of spontaneous behavioural parameters, light sensitivity and mechanical sensitivity thresholds. We also measured c-fos expression in the trigeminal nucleus caudalis. Results Cilostazol increased light sensitivity and grooming behaviour. These manifestations were not inhibited by sumatriptan. Cilostazol also induced c-fos expression in the trigeminal nucleus caudalis. Furthermore, trigeminal - but not hind paw hyperalgesia was observed. Conclusion The altered behaviours are suggestive of cilostazol induced headache with migraine-like features, but not specific. The presence of head specific hyperalgesia and the c-fos response in the trigeminal nucleus caudalis imply that the model involves trigeminal nociception. The model will be useful for studying mechanisms related to the cAMP pathway in headache, but its predictive properties appear to be more limited due to the lack of response to sumatriptan.

Selective cephalic upregulation of p-ERK, CamKII and p-CREB in response to glyceryl trinitrate infusion.

Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.

The KATP channel in migraine pathophysiology: a novel therapeutic target for migraine.

BACKGROUND: To review the distribution and function of KATP channels, describe the use of KATP channels openers in clinical trials and make the case that these channels may play a role in headache and migraine. DISCUSSION: KATP channels are widely present in the trigeminovascular system and play an important role in the regulation of tone in cerebral and meningeal arteries. Clinical trials using synthetic KATP channel openers report headache as a prevalent-side effect in non-migraine sufferers, indicating that KATP channel opening may cause headache, possibly due to vascular mechanisms. Whether KATP channel openers can provoke migraine in migraine sufferers is not known. CONCLUSION: We suggest that KATP channels may play an important role in migraine pathogenesis and could be a potential novel therapeutic anti-migraine target.

Animal models of pain and migraine in drug discovery.

Preclinical research activities in relation to pain typically involve the 'holy trinity' of nociceptive, inflammatory and neuropathic pain for purposes of target validation and defining target product profiles of novel analgesic compounds. For some reason it seems that headache or migraine are rarely considered as additional entities to explore. Frontline medications used in the treatment of, for example, inflammatory pain, neuropathic pain and migraine (NSAIDs versus pregabalin/duloxetine versus triptans) reveal distinct differences in pathophysiology that partially explain this approach. Nevertheless, for many patients enduring chronic pain, regardless of aetiology, high unmet needs remain. By focusing more on commonalities shared between neuropathic pain and headache disorders such as migraine, drug discovery efforts could be spread more efficiently across a larger indication area. Here, some of the most commonly used models and methods employed within 'pain and migraine' drug development will be presented. Recent advances within these disciplines suggest that, with the addition of a few extra carefully chosen ancillary models and/or endpoints, the relative value in terms of resources used, reciprocal flow of information and net worth of a 'typical' package could be increased substantially for the pain and migraine fields.