Sara L. Shepheard

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.

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 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.

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.

Role of opioid receptors in neurogenic dural vasodilation and sensitization of trigeminal neurones in anaesthetized rats

Migraine headache is thought to be caused by a distension of meningeal blood vessels, the activation of trigeminal sensory neurones and the the development of a central sensitization within the trigeminal nucleus caudalis (TNC). It has been proposed that clinically effective 5‐HT1B/1D agonists act peripherally to inhibit the release of calcitonin gene‐related peptide (CGRP) and neurogenic dural vasodilation, and to attenuate nociceptive neurotransmission within the TNC. Since opioids are also effective anti‐migraine agents the present studies investigated the role of opioids within the trigemino‐vascular system in anaesthetised rats. Electrical stimulation of the dura mater evoked neurogenic dural vasodilation which was significantly inhibited by morphine (1 mg kg−1) the selective μ‐opioid agonist DAGO (10 μg kg−1) and the mixed agonist/antagonist butorphanol (1 mg kg−1) but not by the κ‐ and δ‐opioid agonists (±) U50488H (100 μg kg−1) and DPDPE (1 mg kg−1). Morphine had no effect on CGRP‐evoked dural vasodilation. In electrophysiological studies morphine (1 – 10 mg kg−1) significantly attenuated brainstem neuronal activity in response to electrical stimulation of the dura by 65% at 10 mg kg−1. Morphine (3 mg kg−1) also inhibited the TNC neuronal sensitization following CGRP‐evoked dilation. The present studies have demonstrated that opioids block the nociceptive neurotransmission within the trigeminal nucleus caudalis and in addition inhibit neurogenic dural vasodilation via an action on μ‐opioid receptors located on trigeminal sensory fibres innervating dural blood vessels. These peripheral and central actions are similar to those of the ‘triptan’ 5‐HT1B/1D agonists and could account for the anti‐migraine actions of opioids.

L-733,060, a novel tachykinin NK1 receptor antagonist; effects in [Ca2+]i mobilisation, cardiovascular and dural extravasation assays

This study investigated the properties of a novel piperidine ether-based tachykinin NK1 receptor antagonist L-733,060, ((2S,3S)-3-((3,5-bis(trifluoromethyl)phenyl)methyloxy)-2-phenyl piperidine and its 2R,3R-enantiomer L-733,061 on [Ca2+]i mobilisation in Chinese hamster ovary cells transfected with human tachykinin NK1 receptors, compared to their effects in rodent cardiovascular and neurogenic plasma extravasation assays. Using FURA-2-imaging techniques, L-733,060 inhibited substance P-induced [Ca2+]i mobilisation with an estimated affinity of 0.8 nM whereas L-733,061 (30-300 nM) did not. No significant effects of L-733,060 were observed on mean arterial blood pressure or heart rate in conscious or anaesthetised rats at doses of < 3000 micrograms kg-1 i.v. L-733,060 also stereoselectively inhibited neurogenic plasma extravasation in rat dura produced by electrical stimulation of trigeminal nerves with an ID50 of 212 +/- 19 micrograms kg-1 i.v. Thus, L-733,060 is a novel antagonist of human tachykinin NK1 receptors which stereoselectively inhibits neurogenic plasma extravasation at doses that do not cause adverse cardiovascular effects.

Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.

The chemical evolution of N,N-dimethyl-2-[5-(1,2,4-triazol-4-yl)-1H-indol-3-yl]ethylamine (L-741,604) and analogues: Potent and selective agonists for 5-HT1D receptors☆

Optimisation of a series of 5-(heterocyclyl)tryptamines led to the identification of the symmetrically substituted, N-4 linked 1,2,4-triazole as the best indole C-5 substituent for 5-HT1D receptor affinity and selectivity. The triazole (8) is the most potent and selective, orally bioavailable, 5-HT1D receptor agonist identified to date, showing an order of magnitude greater potency than the clinical compound sumatriptan with improved subtype selectivity.