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.

No additive effect of combining sumatriptan and olcegepant in the GTN mouse model of migraine.

INTRODUCTION: Despite recent advances in migraine treatment there is a need for therapies with higher clinical efficacy and/or fewer side effects. Triptans (5-HT1B/1D/1F agonists) are essential in the present treatment regime and gepants (CGRP-receptor antagonists) are recognized as effective in acute migraine treatment. Triptans and gepants have different mechanisms of action and here we tested the hypothesis that a combination of these drugs (sumatriptan and olcegepant) would result in an additive effect. METHODS: Using the validated glyceryl trinitrate mouse model of migraine, we initially tested dose-response relationships of sumatriptan (0.1, 0.3, and 0.6 mg/kg IP) and olcegepant (0.25, 0.50, and 1.0 mg/kg IP) to find suitable high and low doses. Subsequently, we performed a combination study of the two drugs with a low and a high dose. All experiments were vehicle (placebo) controlled and blinded. RESULTS: Sumatriptan significantly reduced glyceryl trinitrate-induced allodynia (F(4,54) = 13.51, p < 0.0001) at all doses. Olcegepant also reduced glyceryl trinitrate-induced allodynia (F(4,53) = 16.11, p < 0.0001) with the two higher doses being significantly effective. Combining 0.50 mg/kg olcegepant with 0.1 or 0.6 mg/kg sumatriptan did not have any improved effect compared to either drug alone (p > 0.50 on all days) in our mouse model. CONCLUSION: Combining olcegepant and sumatriptan did not have an additive effect compared to single-drug treatment in this study. Triptan-gepant combinations will therefore most likely not improve migraine treatment. Nevertheless, further studies are necessary, and combinations should also be examined in patients with migraine.

CGRP-dependent signalling pathways involved in mouse models of GTN- cilostazol- and levcromakalim-induced migraine.

BACKGROUND: Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. METHODS: In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. RESULTS: Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. CONCLUSION: The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine mechanisms.

No central action of CGRP antagonising drugs in the GTN mouse model of migraine.

INTRODUCTION: Clinically, calcitonin gene-related peptide antagonising drugs are recognized as effective in migraine treatment, but their site of action is debated. Only a small fraction of these compounds pass the blood-brain barrier and accesses the central nervous system. Regardless, it has been argued that the central nervous system is the site of action. Here, we test this hypothesis by bypassing the blood-brain barrier through intracerebroventricular injection of calcitonin gene-related peptide antagonising drugs. METHODS: We used the glyceryl trinitrate (GTN) mouse model, which is well validated by its response to specific migraine drugs. The calcitonin gene-related peptide receptor antagonist olcegepant and the calcitonin gene-related peptide monoclonal antibody ALD405 were administered either intraperitoneally or intracerebroventricularly. The outcome measure was cutaneous mechanical allodynia. RESULTS: Mice given olcegepant intraperitoneally + GTN on day 1 had a mean 50% withdrawal threshold of 1.2 g in contrast to mice receiving placebo + GTN, which had a threshold of 0.3 g (p < 0.001). Similarly, in the ALD405 + GTN group, mice had thresholds of 1.2 g versus 0.2 g in the placebo + GTN group (p < 0.001). However, both drugs were ineffective when delivered intracerebroventricularly, as control and active groups had identical mechanical sensitivity thresholds, 0.2 g versus 0.1 g and 0.1 g versus 0.1 g for olcegepant and ALD405, respectively (p > 0.99 in both cases). DISCUSSION: The site of action of olcegepant and of the monoclonal antibody ALD405 is outside the blood-brain barrier in this mouse model of migraine. It is likely that these results can be generalised to all gepants and all antibodies and that the results are relevant for human migraine.

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.

Targeting CGRP via receptor antagonism and antibody neutralisation in two distinct rodent models of migraine-like pain.

INTRODUCTION: Rodent disease models can play an indispensable role in drug development. Confirming that translationally-relevant disease mechanisms are engaged in such models is a crucial facet of this process. Accordingly, we have validated the role of calcitonin gene-related peptide signaling in a mouse model of glyceryl trinitrate-provoked migraine-like pain and a spontaneous rat model of migraine-like pain by assessing their pharmacological responsiveness to the small molecule calcitonin gene-related peptide receptor antagonist olcegepant, and the humanised monoclonal calcitonin gene-related peptide antibody ALD405. METHODS: Cutaneous sensitivity to hind paw, and periorbital mechanical stimulation were used as surrogate markers of activation of relevant pain pathways in each respective model. Separate experiments were performed to identify the time-course of treatment response to olcegepant (1 mg/kg i.p.) and ALD405 (10 mg/kg i.p.). RESULTS: Olcegepant and ALD405 significantly alleviated cutaneous mechanical hypersensitivity in both models compared with corresponding control treatments (saline and IgG control antibody respectively). As expected, the duration of anti-nociceptive action obtained with ALD405 was considerably longer than that associated with olcegepant. Surprisingly, in the spontaneous rat model the onset of action of ALD405 occurred within just 4 hours after administration. DISCUSSION: The current data clearly show that calcitonin gene-related peptide-mediated signaling is critically involved in the manifestation of cutaneous hypersensitivity in distinct rodent models of migraine-like pain and emphasise their translational relevance. Moreover, the unexpected rapidity of onset observed for ALD405 supports i) a probable site of action outside the blood-brain barrier, and ii) a potential clinical utility of specific monoclonal calcitonin gene-related peptide antibodies in the abortive treatment of migraine.

CGRP infusion in unanesthetized rats increases expression of c-Fos in the nucleus tractus solitarius and caudal ventrolateral medulla, but not in the trigeminal nucleus caudalis.

BACKGROUND AND AIMS: Calcitonin gene-related peptide (CGRP) and glyceryl trinitrate (GTN) infusion in migraineurs provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective in the treatment of acute migraine. We hypothesized that CGRP infusion would increase molecular markers of neuronal activation in migraine-relevant tissues of the rat. METHODS: CGRP was infused intravenously (i.v.) in freely moving rats to circumvent factors like anesthesia, acute surgery and severe hypotension, the three confounding factors for c-Fos expression. The trigeminal nucleus caudalis (TNC) was isolated at different time points after CGRP infusion. The level of c-Fos mRNA and protein expression in TNC were analyzed by qPCR and immunohistochemistry. c-Fos-stained nuclei were also counted in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM), integrative sites in the brain stem for processing cardiovascular signals. We also investigated Zif268 protein expression (another immediate early gene) in TNC. The protein expression of p-ERK, p-CREB and c-Fos was analyzed in dura mater, trigeminal ganglion (TG) and TNC samples using Western blot. RESULTS: CGRP infusion caused a significant dose-dependent fall in mean arterial blood pressure. No significant activation of c-Fos in the TNC at mRNA and protein levels was observed after CGRP infusion. A significant increase in c-Fos protein was observed in the NTS and CVLM in the brain stem. Zif268 expression in the TNC was also not changed after CGRP infusion. p-ERK was increased in the dura mater 30 minutes after CGRP infusion. CONCLUSION: CGRP infusion increased the early expression of p-ERK in the dura mater but did not increase c-Fos and Zif268 expression in the TNC. The rats may, thus, differ from migraine patients, in whom infusion of CGRP caused headache and a delayed migraine attack. The rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing.

Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor.

BACKGROUND AND PURPOSE: The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. EXPERIMENTAL APPROACH: Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. KEY RESULTS: PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.

Xenobiotic Exposure and Migraine-Associated Signaling: A Multimethod Experimental Study Exploring Cellular Assays in Combination with Ex Vivo and In Vivo Mouse Models.

BACKGROUND: Mechanisms for how environmental chemicals might influence pain has received little attention. Epidemiological studies suggest that environmental factors such as pollutants might play a role in migraine prevalence. Potential targets for pollutants are the transient receptor potential (TRP) channels ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which on activation release pain-inducing neuropeptide calcitonin gene-related peptide (CGRP). OBJECTIVE: In this study, we aimed to examine the hypothesis that environmental pollutants via TRP channel signaling and subsequent CGRP release trigger migraine signaling and pain. METHODS: A calcium imaging-based screen of environmental chemicals was used to investigate activation of migraine pain-associated TRP channels TRPA1 and TRPV1. Based on this screen, whole-cell patch clamp and in silico docking were performed for the pesticide pentachlorophenol (PCP) as proof of concept. Subsequently, PCP-mediated release of CGRP and vasodilatory responses of cerebral arteries were investigated. Finally, we tested whether PCP could induce a TRPA1-dependent induction of cutaneous hypersensitivity in vivo in mice as a model of migraine-like pain. RESULTS: A total of 16 out of the 52 screened environmental chemicals activated TRPA1 at 10 or 100µM. None of the investigated compounds activated TRPV1. Using PCP as a model of chemical interaction with TRPA1, in silico molecular modeling suggested that PCP is stabilized in a lipid-binding pocket of TRPA1 in comparison with TRPV1. In vitro, ex vivo, and in vivo experiments showed that PCP induced calcium influx in neurons and resulted in a TRPA1-dependent CGRP release from the brainstem and dilation of cerebral arteries. In a mouse model of migraine-like pain, PCP induced a TRPA1-dependent increased pain response (Ntotal=144). DISCUSSION: Here we show that multiple environmental pollutants interact with the TRPA1-CGRP migraine pain pathway. The data provide valuable insights into how environmental chemicals can interact with neurobiology and provide a potential mechanism for putative increases in migraine prevalence over the last decades. https://doi.org/10.1289/EHP12413.

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.

Infusion of low dose glyceryl trinitrate has no consistent effect on burrowing behavior, running wheel activity and light sensitivity in female rats.

INTRODUCTION: Glyceryl trinitrate induces headache during infusion to man and migraine patients develop an additional migraine attack a few hours after the infusion. Recently, we have moved this model into rat with the intention of developing an animal model predictive of migraine therapy. In the current paper we have studied the effect of glyceryl trinitrate infusion on three different rat behaviors. METHODS: The stability of burrowing behavior, running wheel activity and light sensitivity towards repeated testing was evaluated also with respect to estrous cycle. Finally, the effect of glyceryl trinitrate on these behaviors in female rats was observed. RESULTS: Burrowing behavior and running wheel activity were stable in the individual rat between experiments. The burrowing behavior was significantly affected by the stage of estrous cycle. The other assays were stable throughout the cycle. None of the three behavioral tests were altered by glyceryl trinitrate infusion. In the light-dark box, some batches of rats showed light sensitivity after treatment with glyceryl trinitrate but it could not be repeated in other batches of rats. DISCUSSION: We have investigated the stability towards repeated testing and the effect of i.v. glyceryl trinitrate infusion to awake rats in three behavioral assays. Of the assays evaluated, only light sensitivity was capable of detecting changes after glyceryl trinitrate infusion but, this was not repeatable. Thus, the infusion of a low dose glyceryl trinitrate to concious rats together with the chosen behavioral tests is not a robust setup for studying immediate GTN induced headache behavior in rats.