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Abstract

Background

The release of calcitonin gene-related peptide (CGRP) from trigeminal nerves plays a central role in the pathophysiology of migraine and clinical evidence shows an antimigraine effect for CGRP receptor antagonists. Systemic administration of nitroglycerin (NTG), a nitrovasodilator, consistently provokes spontaneous-like migraine attacks in migraine sufferers; in the rat, systemic NTG induces a condition of hyperalgesia, probably through the activation of cerebral/spinal structures involved in nociceptive transmission.

Aim

The aim of this article is to test the analgesic effect of the CGRP receptor antagonist MK-8825 in two animal models of pain that may be relevant for migraine: the tail flick test and the formalin test performed during NTG-induced hyperalgesia.

Results

MK-8825 showed analgesic activity when administered alone at both the tail flick test and the formalin test. Furthermore, the CGRP antagonist proved effective in counteracting NTG-induced hyperalgesia in both tests. MK-8825 indeed reduced the nociceptive behavior when administered either simultaneously or prior to (30–60 minutes before) NTG.

Conclusion

These data suggest that MK-8825 may represent a potential therapeutic tool for the treatment of migraine.

Keywords

Nitroglycerin hyperalgesia migraine CGRP receptor antagonist

Introduction

The release of calcitonin gene-related peptide (CGRP) from trigeminal nerves plays a central role in the pathophysiology of migraine. CGRP was demonstrated to have a causative role in migraine, since the infusion of CGRP induces migraine-like headaches in susceptible subjects (1). CGRP is a potent dilator of cerebral and dural vessels and it is involved in the transmission of nociceptive information from intracranial vessels to the central nervous system (CNS) (1 –3). Serum levels of CGRP in the external jugular vein are elevated in patients during several types of vascular headaches, including migraines (with or without aura) and cluster headaches, while they normalize concomitantly with pain relief (4,5).

These observations, along with other evidence, have prompted the possibility that CGRP receptor antagonism represents a potential target for the treatment of migraine. Clinical studies show that antagonists of CGRP receptor display antimigraine effects similar to those reported for triptans (2,3). Microinjection of olcegepant (BIBN4096BS), a CGRP receptor antagonist, into the nucleus trigeminalis caudalis (TNC) reduces the firing of central trigeminal neurons evoked by the microiontophoresic application of glutamate or by electrical stimulation of the superior sagittal sinus (6). Furthermore, Fischer et al. (7) demonstrated that systemic infusion of olcegepant decreases both spontaneous and heat-evoked activity (firing rate) in the TNC, probably via the inhibition of CGRP release from trigeminal endings located in the wall of dural arteries. By contrast, topical application of olcegepant to the dura was ineffective, which leaves unanswered the question regarding the actual site of CGRP receptor inhibition, also in consideration of the limited ability of the drug to cross the blood-brain barrier (8). Two other CGRP receptor antagonists, telcagepant and MK-3207, have been studied for their potential use in migraine treatment, but their development has been discontinued because of safety concerns (9,10).

More recently, another CGRP antagonist has become available, MK-8825, an analog of MK-3207 (11), characterized by an increased unbound fraction in rat plasma, an enhanced aqueous solubility and, most importantly, a higher potency in vivo in the rat (10). The present study is aimed at evaluating the potential analgesic effect of MK-8825 in animal models of hyperalgesia associated with nitroglycerin (NTG) administration. These models have been tested over the years with different drugs and are considered reliable animal models for migraine (12 –15), since it is known that NTG induces delayed migraine-like headache in migraineurs (16).

Materials and methods

Adult male Sprague-Dawley rats (weight 250–270 g) were evaluated in the present experiments. The principles of the Helsinki Declaration and the International Association for the Study of Pain (IASP) guidelines for pain research in animals were rigorously applied (17). Animals were housed in plastic boxes in groups of two with water and food available ad libitum and kept on a 12:12-hour light-dark cycle at the Centralized Animal Facility of the University of Pavia. All the rats were acclimatized to the test chamber before testing began.

Drugs

MK-8825 (MSD Company), suspended in saline, was injected intraperitoneally (i.p.) at a dose of 100 mg/kg in 2 ml/kg of 100% saline vehicle.

NTG (Astra Company, Italy), dissolved in saline alcohol and propylene glycol, was injected i.p. at a dose of 10 mg/kg.

For the formalin test (FT), a 100 µl volume of 1% formalin (formaldehyde diluted in 0.9% saline) was injected intraplantarly.

Rats were randomly divided into groups of 4 to 10 animals each, and underwent the following experimental protocol. Rats were assigned to one of the treatment groups according to a randomization list whose codes were unblinded only after study completion. Therefore, the researchers who performed the behavioral testing (RG or SM) were blind to treatments.

Experiment I

Rats in this experiment were tested for latency of reflex tail withdrawal (tail flick test (TFT)) from a high-intensity light beam, a measure of physiological phasic pain. The test was performed with a commercially available instrument (Ugo Basile) that allows automatic recording of the TFT from radiant heat. Radiant heat is generated from an infrared source (50 W bulb) and energy level was fixed to 5. Latency at each evaluation was calculated as the mean of four measurements in four different parts of the tail. A cut-off limit of exposure corresponding to 20 seconds was set to prevent tissue damage.

Each animal was placed on the recording platform of the instrument where it was kept under slight, painless restraint, with its tail positioned on the radiant heat window. The movement of the tail away from the window allowed the beam of light to hit a sensor that automatically registered the time elapsed from the tail positioning.

The experimental plan for the TFT was performed according to the following treatment schedule:

Control group (n = 9): latency at the TFT was measured before and at 30, 60 and 90 minutes after the injection of saline.

MK-8825 group (n = 6): latency at the TFT was measured before and at 30, 60 and 90 minutes after the injection of MK-8825.

NTG group (n = 10): latency at TFT was measured before and four hours after NTG administration.

Control for NTG group (n = 10): latency at TFT was measured before and four hours after saline administration.

NTG + MK-8825 group (n = 4): latency at the TFT was measured at baseline. Subsequently rats were injected with NTG and, 3.5 hours later, with MK-8825. TFT was performed four hours after NTG administration (that is, 30 minutes after MK-8825 administration).

MK-8825 (30′) + NTG group (n = 5): MK-8825 administration 30 minutes before NTG injection. TFT evaluation was performed at baseline and four hours after NTG administration.

MK-8825 (60′) + NTG group (n = 9): MK-8825 administration 60 minutes before NTG injection. TFT evaluation was performed at baseline and four hours after NTG administration.

MK-8825/NTG group (n = 5): MK-8825 and NTG were administered simultaneously. TFT evaluation was performed at baseline and four hours after drugs administration.

Experiment II

Rats in this experiment underwent the formalin test (FT) for the evaluation of inflammatory tonic pain.

Formalin (1%) was injected intraplantarly into the center of the plantar surface of the left hind paw with slight restraint. The rat was then repositioned in the box, the clock was started and pain response was recorded for a period of one hour. Pain behavior was readily discriminated as flinching/shakes, characterized as rapid and brief withdrawal or flexion movements of the injected paw. The pain-related behavior was quantified by counting the total number of flinches and shakes occurring for one-minute periods from one to five minutes (Phase I) and, then for one-minute periods at five-minute intervals during the period from 10 to 60 minutes (Phase II) after formalin injection. Phase I is generally considered the result of chemical activation of nociceptors, while Phase II reflects the inflammatory reaction and central processing.

The experimental plan for the FT was performed according to the following treatment schedule:

Control group (n = 9): saline followed four hours later by the FT.

MK-8825 (30′) group (n = 4): MK-8825 administration 30 minutes before saline injection (10 mg/kg, i.p.). FT four hours after saline injection.

NTG group (n = 10): NTG administration followed four hours later by the FT.

MK-8825 (30′) + NTG group (n = 4): MK-8825 administration 30 minutes before NTG. FT was performed four hours after NTG administration.

MK-8825 (60′) + NTG group (n = 4): MK-8825 administration 60 minutes before NTG. FT was performed four hours after NTG administration.

MK-8825/NTG group (n = 5) = MK-8825 and NTG were administered simultaneously. FT was performed four hours after drug administration.

Statistical evaluation

The effects of treatments on the latency of the TFT were evaluated by means of the Wilcoxon rank-sum test (baseline vs post-treatment) or the paired Student’s t test (for groups with n ≥ 10). The Kruskal-Wallis test followed by Dunn’s Multiple Comparison test was used when more than two groups were compared. A probability level of less than 5% was regarded as significant.

For the FT, the total number of flinches/shakes evoked by formalin injection was counted separately for Phase I and for Phase II, as described above. Differences between groups were analyzed by the nonparametric one-way analysis of variance (Mann-Whitney test or Kruskal-Wallis test) followed by Dunn’s Multiple Comparison test when more than two groups were compared. A probability level of less than 5% was regarded as significant.

Results I

TFT

MK-8825 injection into naïve (untreated) rats induced analgesia recorded as a significant increase in the latency at 30 and 60 minutes, as compared to the baseline value (Figure 1(a)). No significant variation of latency was observed following saline administration (data not shown). In agreement with our previous reports (13), NTG induced a hyperalgesic response at the TFT, as suggested by the significant decrease in the latency four hours after its administration (Figure 1(b)), while no significant variation of latency was observed following saline administration four hours later. When MK-8825 was administered 3.5 hours after NTG injection, i.e. 30 minutes before the test, we did not detect any significant analgesic/antihyperalgesic effect (Figure 1(c)). On the contrary, when MK-8825 was administered either simultaneously or prior to NTG (both at 30 and 60′), it did show a significant antihyperalgesic effect (Figure 1(c)).

Figure 1.

Activity of MK-885 at the tail flick test.

FT

In the control group, the injection of formalin resulted in a highly reliable, typical, biphasic pattern of flinches/shakes of the injected paw, characterized by an initial acute phase of nociception within the first 5 minutes (Phase I), followed by a prolonged tonic response from 15 to 60 minutes after the formalin injection (Phase II), in agreement with previous reports (13,15). As expected, NTG administration significantly increased the total number of flinches/shakes in Phase II of the FT, when compared to the control group (Figure 2(a)). MK-8825 pretreatment significantly inhibited the nociceptive behavior induced by the formalin injection during Phase II of the test, in baseline condition (Figure 2(b)). MK-8825 injected either simultaneously or 30 or 60 minutes before NTG reduced the total number of flinches/shakes during Phase II of the test (Figure 2(c)).

Figure 2.

Activity of MK-885 at the formalin test.

Discussion

The mechanism by which a migraine attack initiates is not well understood. A dysfunction in the CNS leading to the release of inflammatory mediators has been proposed to cause sensitization and activation of trigeminal nerves (18). Activation of nociceptive neurons located in the trigeminal ganglion and their subsequent release of CGRP seem to be implicated in migraine pathology (19). CGRP is involved in the control of neuronal activity of the TNC, a relay station that receives nociceptive afferent inputs originating from trigeminally innervated areas, including the dural afferents. In the dura, CGRP causes dilation of arterial vessels and secretion of mediators of inflammation, which amplify vasodilation and activate specific primary afferents (5). Given the multiple receptor sites in the trigeminovascular system, in the dura, in the trigeminal ganglion and central/afferent projections in the TNC, the inhibition of CGRP receptor activation represents a promising therapeutic target for migraine headaches.

Several lines of evidence suggest the existence of a condition of trigeminal sensitization in migraineurs that results in hyperalgesia, allodynia and cognitive dysfunction during and between episodes (20,21). These findings may lend credence to the placement of migraine among the conditions characterized by central sensitization (22). In keeping with this hypothesis, spatial changes in pressure pain hypersensitivity, probably related to central sensitization of spinal tract neurons, have been described in unilateral migraine (23).

Nitric oxide (NO) is involved in the development and maintenance of hyperalgesia (24), in the periphery and probably also at central sites. Indeed, activation of neuronal nitric oxide synthase (NOS) may induce spinal (and/or supraspinal) synaptic plasticity with a prolonged increase in synaptic length (25). NTG is known to induce spontaneous-like headache attacks in migraine sufferers (16), probably as a consequence of sensitization phenomena (26). In a previous report, our group showed that NTG administration to migraineurs is associated with a significant facilitation in temporal summation of pain (reduced temporal summation threshold and increased painful sensation) 60', 120′ and 180′ after NTG intake when compared to baseline, to placebo condition and to controls. This finding suggests that migraineurs bear a susceptibility to developing migraine attack after NTG administration as a specific trait linked to a supersensitivity of the pain system to NTG (27). Quite in analogy, in the rat, NTG induces a condition of hyperalgesia that is detectable at the FT and at the TFT, as an increase of nocifensive behavior (13,15).

CGRP is released into dorsal horn neurons following painful stimuli (28), and lines of evidence indicate that CGRP may contribute to the generation of hyperalgesia (29,30). Indeed, alpha-CGRP null mice do not develop hyperalgesic responses to inflammatory insults (31).

In the present study, we have shown that inhibition of CGRP receptor, by means of MK-8825 administration, induces analgesia at both the TFT and the FT. These findings are in line with the observation that blockade of CGRP receptor with the peptide antagonist CGRP8-37 induces antinociception in animal models of inflammatory or central neuropathic pain (32). The novelty and specificity of our findings reside in the demonstration that MK-8825 is also capable of counteracting NTG-induced hyperalgesia. Previous studies showed that NTG activates neurons in the TNC (12), one of the proposed sites for the antinociceptive action of CGRP receptor antagonists (33). Accordingly, in a previous study we have shown a decrease in the density of CGRP-immunoreactivity (ir) fibers in laminae I and II of the TNC (lower brainstem and cervical spinal cord) associated with a decrease in the number of CGRP-positive varicosities in the same area following NTG administration (34), in agreement with the findings obtained by Pardutz et al. (35). These considerations seem to suggest a possible central site of action for the described antihyperalgesic effect of MK-8825. However, little is known about the capability of MK-8825 to cross the blood-brain barrier. In addition, MK-8825 effect might take place in the periphery. NTG induces vasorelaxation of blood vessel via multiple pathways (36), which also include CGRP release, as suggested by the increase in CGRP plasma levels following NTG administration (37). This effect may also take place at the trigeminovascular endings, as suggested by Bellamy et al. (38), who demonstrated that NO can directly stimulate the release of CGRP from trigeminal ganglia neurons. Therefore, MK-8825 probably may also act peripherally to block NTG-induced hyperalgesia from the dura by inhibiting CGRP release from peripheral terminals, including the trigeminovascular endings in the wall of meningeal vessels (33).

Taken together, these findings allow us to hypothesize that CGRP receptor blockade may interfere with the mechanisms associated with NTG-induced hyperalgesia at two levels: central and peripheral (Figure 3).

Figure 3.

Putative targets of MK-8825 (in red) within nitroglycerin (NTG)-induced hyperalgesia: direct and indirect mechanisms and possible mediators. Following systemic administration, NTG reaches the blood vessel wall, where it may induce vasorelaxation both directly, via the local formation of nitric oxide (NO), and indirectly, via its interaction with calcitonin gene-related peptide (CGRP) release from nerve terminals. NTG is also likely to reach the brain compartment, where it may exert a direct hyperalgesic effect via the formation of NO and CGRP release. NTG might induce hyperalgesia also indirectly, via the interaction with the trigeminovascular system. MK-8825 may act peripherally to block NTG-induced hyperalgesia from dura or via inhibition of CGRP receptors in the nucleus trigeminalis caudalis (TNC).

The time window that we have observed for the antihyperalgesic effect of MK-8825 during NTG-induced hyperalgesia in the TFT seems to offer further insights for interpreting our data. Indeed, MK-8825 reduces hyperalgesia when it is administered only before or simultaneously to NTG injection, while it failed to counteract NTG-induced hyperalgesia also at the FT when administered after NTG injection (data not shown). This suggests that MK-8825 administration is capable of interfering with the steps that are upstream as regards NO-mediated sensitization. Lambert et al. (39) have shown that infusion of NTG induces a rapid and short-lasting activation of second-order trigeminal neurons, a phenomenon that is likely to represent one of the first events that lead to NTG-hyperalgesia, a condition that fully develops in two to four hours, as suggested by ample experimental and clinical data (13,15,27). Therefore, the effective administration schedule for MK-8825 (before or simultaneously with NTG) seems to suggest that MK-8825 is likely to block CGRP release peripherally, at the level of trigeminovascular endings in the meninges.

Limitation of the study and future directions

The model adopted in this study is based on the stimulation of sensorial areas that are outside of the trigeminal distribution, which may limit the applicability of our findings to migraine. However, in a previous report we showed that plantar injection of formalin in rats induced a significant decrease in CGRP-ipsilateral to the injection side also in the TNC, while systemic NTG administration induced a reduction in CGRP-ir in the TNC, but not in the lumbar dorsal horns (34). This piece of evidence suggests that the NTG-potentiated FT may be relevant for investigating migraine circuitry. Though so far unexplained, the observation that a nociceptive stimulus delivered at the paw level is associated with TNC activation is further reported by the study of Han et al. (40), which showed that formalin injection in the paw induces Fos expression in TNC and in several other brainstem areas (i.e. locus coeruleus) known to be involved in the modulation of migraine pain (41). Further studies using more area-specific animal models (i.e. orofacial FT) are awaited for confirming and building on the present findings.

Conclusion

The analgesic and antihyperalgesic effect shown by MK-8825 in this study prompts the possibility of taking it into consideration as a future antimigraine drug.

Article highlights

Systemic nitroglycerin (NTG) induces a condition of hyperalgesia through the activation of cerebral/spinal structures involved in pain transmission.

MK-8825, a calcitonin gene-related peptide (CGRP) receptor antagonist, is effective in counteracting NTG-induced hyperalgesia, both in the tail flick and formalin tests.

MK-8825 may represent a potential therapeutic approach for the treatment of migraine by interacting with upstream actions in the cascade of events that mediate the attack.

Footnotes

Funding

This work was supported by a grant from Merck & Co (grant number IIS37408).

Conflict of interest

None declared.

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Effects of CGRP receptor antagonism in nitroglycerin-induced hyperalgesia

Abstract

Background

Aim

Results

Conclusion

Keywords

Introduction

Materials and methods

Drugs

Experiment I

Experiment II

Statistical evaluation

Results I

TFT

FT

Discussion

Limitation of the study and future directions

Conclusion

Article highlights

Footnotes

Funding

Conflict of interest

References