David J Williamson

Comparison of the effects of sumatriptan and the NK1 antagonist CP-99,994 on plasma extravasation in dura mater and c-fos mRNA expression in trigeminal nucleus caudalis of rats

Dural plasma extravasation produced by electrical stimulation of the trigeminal ganglion was measured in rats and the concomitant expression of c-fos mRNA produced in the trigeminal nucleus caudalis (NtV) was measured using in situ hybridization techniques. The non-peptide NK1 receptor selective antagonist CP-99,994 (1-3000 micrograms kg-1) and the 5HT1D receptor agonist sumatriptan (1-1000 micrograms kg-1) reduced dural plasma extravasation dose-dependently with ID50S of 52 micrograms kg-1 and 30 micrograms kg-1 respectively. CP-99,994 (1000 micrograms kg-1). a compound known to have good brain penetration, decreased c-fos mRNA expression in the NtV by 37 +/- 7% without disruption of the blood brain barrier (BBB). Sumatriptan (1000 micrograms kg-1), known to be poorly brain penetrant, had no significant effect on c-fos mRNA expression in the NtV unless the BBB was disrupted by infusion of a hyperosmolar mannitol solution after which sumatriptan decreased c-fos mRNA expression by 65 +/- 11%. The results suggest that brain penetrant NK1 receptor antagonists may have anti-migraine effects peripherally through blockade of dural extravasation and centrally by inhibition of nociceptive pathways. Furthermore the data indicates that the anti-migraine action of sumatriptan must be predominantly peripherally mediated, be it via inhibition of plasma extravasation or direct vasoconstriction, since it had little effect on the activation of neurones in the NtV unless the BBB was disrupted.

Intravital microscope studies on the effects of neurokinin agonists and calcitonin gene‐related peptide on dural vessel diameter in the anaesthetized rat

This study describes a novel intravital microscope technique for direct measurement of dural blood vessel diameter through a closed cranial window in anaesthetized rats. This technique avoids removal of the skull which can lead to problems o altered vessel reactivity and brain swelling that are encountered with open cranial window techniques. Substance P and calcitonin gene-related (CGRP) evoked increases in dura vessel diameter, which were abolished by the NK1 receptor antagonist, RP67580 and the CGRP receptor antagonist, human-CGRP(8–31) respectively. Neurokinin A produced increases in dural vessel diameter which were unaffected by the NK2 receptor antagonist SR 48968 but were blocked by RP67580, suggesting that neurokinin A can act through NK1 receptors to produce dural vasodilation in rats. The NK3 receptor agonist, senktide, had no effects on dural vessel diameter. All drugs were administered intravenously. In humans, vasodilation within the meningeal vasculature has been implicated in the pathogenesis of migraine, the present experiments indicate that substance P or neurokinin A (both acting through NK1 receptors) or CGRP may be responsible.

Nitric oxide synthase inhibitors can antagonize neurogenic and calcitonin gene‐related peptide induced dilation of dural meningeal vessels

The detailed pathophysiology of migraine is beginning to be understood and is likely to involve activation of trigeminovascular afferents. Clinically effective anti‐migraine compounds are believed to have actions that include peripheral inhibition of calcitonin gene‐related peptide (CGRP) release from trigeminal neurones, or preventing dural vessel dilation, or both. CGRP antagonists can block both neurogenic and CGRP‐induced dural vessel dilation. Nitric oxide (NO) can induce headache in migraine patients and often triggers a delayed migraine. The initial headache is thought to be caused via a direct action of the NO–cGMP pathway that causes vasodilation by vascular smooth muscle relaxation, while the delayed headache is likely to be a result of triggering trigeminovascular activation. Nitric oxide synthase (NOS) inhibitors are effective in the treatment of acute migraine. The present studies used intravital microscopy to examine the effects of specific NOS inhibitors on neurogenic dural vasodilation (NDV) and CGRP‐induced dilation. The non‐specific and neuronal NOS (nNOS) inhibitors were able to partially inhibit NDV, while the non‐specific and endothelial NOS (eNOS) inhibitors were able to partially inhibit the CGRP induced dilation. There was no effect of the inducible NOS (iNOS) inhibitor. The data suggest that the delayed headache response triggered by NO donors in humans may be due, in part, to increased nNOS activity in the trigeminal system that causes CGRP release and dural vessel dilation. Further, eNOS activity in the endothelium causes NO production and smooth muscle relaxation by direct activation of the NO–cGMP pathway, and may be involved in the initial headache response.

The novel anti-migraine agent rizatriptan inhibits neurogenic dural vasodilation and extravasation

These studies in anaesthetised rats showed, using intravital microscopy, that the novel anti-migraine agent, rizatriptan, significantly reduced electrically stimulated dural vasodilation but had no effect on increases in dural vessel diameter produced by exogenous substance P or calcitonin gene-related peptide (CGRP). Rizatriptan also significantly inhibited dural plasma protein extravasation produced by high intensity electrical stimulation of the trigeminal ganglion. We suggest that rizatriptan inhibits the release of sensory neuropeptides from perivascular trigeminal nerves to prevent neurogenic vasodilation and extravasation in the dura mater. These prejunctional inhibitory effects may be involved in the anti-migraine action of rizatriptan.

The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Dural vasodilation causes a sensitization of rat caudal trigeminal neurones in vivo that is blocked by a 5-HT1B/1D agonist

Migraine headache pain is thought to result from an abnormal distention of intracranial, extracerebral blood vessels and the consequent activation of the trigeminal nervous system. Migraine is also often accompanied by extracranial sensory disturbances from facial tissues. These experiments investigate whether meningeal dilation produces central sensitization of neurones that receive convergent input from the face. Single unit extracellular activity was recorded from the trigeminal nucleus caudalis of anaesthetized rats in response to either noxious stimulation of the dura mater, innocuous stimulation of the vibrissae or to a transient dilation of the meningeal vascular bed. Rat α‐CGRP (calcitonin gene‐related peptide; 1 μg kg−1, i.v.) caused a dilation of the middle meningeal artery and facilitated vibrissal responses by 36±7%. The 5‐HT1B/1D agonist, L‐741,604 (3 mg kg−1, i.v.), inhibited responses to noxious stimulation of the dura mater (16±7% of control) and, in a separate group of animals, blocked the CGRP‐evoked facilitation of vibrissal responses. L‐741,604 (3 mg kg−1, i.v.) also inhibited responses to innocuous stimulation of the vibrissa (14±10% of control) with neurones that received convergent input from the face and from the dura mater, but not with cells that received input only from the face (70±12% of control). These data show that dilation of meningeal blood vessels causes a sensitization of central trigeminal neurones and a facilitation of facial sensory processing which was blocked by activation of pre‐synaptic 5‐HT1B/1D receptors. Sustained dural blood vessel dilation during migraine may cause a sensitization of trigeminal neurones. This may underlie some of the symptoms of migraine, such as the headache pain and the extracranial allodynia. Inhibition of this central sensitization may therefore offer a novel strategy for the development of acute and/or prophylactic anti‐migraine therapies.

The non-peptide neurokinin1 receptor antagonist, RP 67580, blocks neurogenic plasma extravasation in the dura mater of rats.

A non‐peptide neurokinin1 (NK1) receptor antagonist, RP 67580, that is selective for the rodent subtype of the NK1 receptor, dose‐dependently reduced plasma extravasation in the dura mater produced by electrical stimulation of the trigeminal ganglion in rats, with an ID50 of 0.6 μg kg−1. Its enantiomer RP 68651 was some 400 fold less active. The results indicate that neurogenic plasma extravasation within the dura mater is NK1 receptor‐mediated and suggest that NK1 receptor antagonists may have a role as antimigraine agents.

Voltage-dependent calcium channels are involved in neurogenic dural vasodilatation via a presynaptic transmitter release mechanism

A missense mutation of the CACNA1A gene that encodes the α1A subunit of the voltage‐dependent P/Q‐type calcium channel has been discovered in patients suffering from familial hemiplegic migraine. This suggested that calcium channelopathies may be involved in migraine more broadly, and established the importance of genetic mechanisms in migraine. Channelopathies share many clinical characteristics with migraine, and thus exploring calcium channel functions in the trigeminovascular system may give insights into migraine pathophysiology. It is also known that drugs blocking the P/Q‐ and N‐type calcium channels have been successful in other animal models of trigeminovascular activation and head pain. In the present study, we used intravital microscopy to examine the effects of specific calcium channel blockers on neurogenic dural vasodilatation and calcitonin gene‐related peptide (CGRP)‐induced dilation. The L‐type voltage‐dependent calcium channel blocker calciseptine significantly attenuated (20 μg kg−1, n=7) the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation. The P/Q‐type voltage‐dependent calcium channel blocker ω‐agatoxin‐IVA (20 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation. The N‐type voltage‐dependent calcium channel blocker ω‐conotoxin‐GVIA (20 μg kg−1, n=8 and 40 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation. It is thought that the P/Q‐, N‐ and L‐type calcium channels all exist presynaptically on trigeminovascular neurons, and blockade of these channels prevents CGRP release, and, therefore, dural blood vessel dilation. These data suggest that the P/Q‐, N‐ and L‐type calcium channels may be involved in trigeminovascular nociception.

The anti-migraine 5-HT1B/1D agonist rizatriptan inhibits neurogenic dural vasodilation in anaesthetized guinea-pigs

These studies investigated the pharmacology of neurogenic dural vasodilation in anaesthetized guinea‐pigs. Following introduction of a closed cranial window the meningeal (dural) blood vessels were visualized using intravital microscopy and the diameter constantly measured using a video dimension analyser. Dural blood vessels were constricted with endothelin‐1 (3 μg kg−1, i.v.) prior to dilation of the dural blood vessels with calcitonin gene‐related peptide (CGRP; 1 μg kg−1, i.v.) or local electrical stimulation (up to 300 μA) of the dura mater. In guinea‐pigs pre‐treated with the CGRP receptor antagonist CGRP(8‐37) (0.3 mg kg−1, i.v.) the dilator response to electrical stimulation was inhibited by 85% indicating an important role of CGRP in neurogenic dural vasodilation in this species. Neurogenic dural vasodilation was also blocked by the 5‐HT1B/1D agonist rizatriptan (100 μg kg−1) with estimated plasma levels commensurate with concentrations required for anti‐migraine efficacy in patients. Rizatriptan did not reverse the dural dilation evoked by CGRP indicating an action on presynaptic receptors located on trigeminal sensory fibres innervating dural blood vessels. In addition, neurogenic dural vasodilation was also blocked by the selective 5‐HT1D agonist PNU‐142633 (100 μg kg−1) but not by the 5‐HT1F agonist LY334370 (3 mg kg−1) suggesting that rizatriptan blocks neurogenic vasodilation via an action on 5‐HT1D receptors located on perivascular trigeminal nerves to inhibit CGRP release. This mechanism may underlie one of the anti‐migraine actions of the triptan class exemplified by rizatriptan and suggests that the guinea‐pig is an appropriate species in which to investigate the pharmacology of neurogenic dural vasodilation.

The effect of anti-migraine compounds on nitric oxide-induced dilation of dural meningeal vessels

Migraine is characteristically accompanied by a throbbing quality of head pain thought to involve trigeminovascular afferents. Administration of nitric oxide (NO) donors provides the most reliable model of migraine induction in humans. The present studies used intravital microscopy to monitor the effect of local meningeal nerve stimulation and NO on dural blood vessels and any modulation of that effect by anti-migraine compounds. NO caused an immediate and reproducible dilation of meningeal blood vessels that was partially blocked by sumatriptan and indomethacin, while flunarizine and histamine H(1) and H(2) receptor antagonists were unable to block the dilation. Indomethacin also inhibited the neurogenic dilation while flunarizine did not. The present studies demonstrate that NO is unlikely to interact with histamine to produce its dilatory response. Sumatriptan and indomethacin inhibit the NO response by inhibiting trigeminal activation and calcitonin gene-related peptide (CGRP) release. Flunarizine does not modify either the neurogenic vasodilator response or the NO meningeal dilator response at least acutely.