Dural Plasma Protein Extravasation Research Articles

Design, synthesis and biological evaluation of pyridinylmethylenepiperidine derivatives as potent 5-HT1F receptor agonists for migraine therapy.

Migraine is a common neurovascular disease which has been classified as the sixth most disabling disorder. Current migraine therapy was triptans, however, riptans can cause contraction of blood vessels. Therefore, novel drugs without cardiovascular effects emerged, such as CGRP and selective 5-HT1F receptor agonists. In this work, a series of pyridinylmethylenepiperidine derivatives were designed, synthesized and evaluated for their 5-HT1F receptor agonist activity. The results invitro showed that compound C1-C6 displayed potent agonist activities compared with positive drug lasmiditan. Pharmacokinetic properties in rat indicated that 2,4,6-trifluoro-N-(6-(fluoro(1-methylpiperidin-4-ylidene)methyl)pyridin-2-yl)benzamide (C5) possessed high AUC and good bioavailability. In two rodent models of migraine, C5 significantly inhibited dural plasma protein extravasation and c-fos expression in the trigeminal nucleus caudalis. Moreover, C5 showed no effect on vasoconstriction. Through these studies, we identified C5 as a potent 5-HT1F receptor agonist for migraine therapy.

Role of central versus peripheral opioid system in antinociceptive and anti-inflammatory effect of botulinum toxin type A in trigeminal region.

Although botulinum toxin type A (BT-A) is approved for chronic migraine treatment, its site and mechanism of action are still elusive. Recently our group discovered that suppression of CGRP release from dural nerve endings might account for antimigraine action of pericranially injected BT-A. We demonstrated that central antinociceptive effect of BT-A in sciatic region involves endogenous opioid system as well. Here we investigated possible interaction of BT-A with endogenous opioid system within the trigeminal region. In orofacial formalin test we investigated the influence of centrally acting opioid antagonist naltrexone (2mg/kg, s.c.) versus peripherally acting methylnaltrexone (2mg/kg, s.c.) on BT-A's (5 U/kg, s.c. into whisker pad) or morphine's (6mg/kg, s.c.) antinociceptive effect and the effect on dural neurogenic inflammation (DNI). DNI was assessed by Evans blue-plasma protein extravasation. Naltrexone abolished the effect of BT-A on pain and dural plasma protein extravasation, whereas peripherally acting methylnaltrexone did not change either BT-A's effect on pain or its effect on dural extravasation. Naltrexone abolished the antinociceptive and anti-inflammatory effects of morphine, as well. However, methylnaltrexone decreased the antinociceptive effect of morphine only partially in the second phase of the test and had no significant effect on morphine-mediated reduction in DNI. Morphine acts on pain in trigeminal region both peripherally and centrally, whereas the effect on dural plasma protein extravasation seems to be only centrally mediated. However, the interaction of BT-A with endogenous opioid system, with consequent inhibition of nociceptive transmission as well as the DNI, occurs primarily centrally. Botulinum toxin type A (BT-A)'s axonal transport and potential transcytosis suggest that its antinociceptive effect might involve diverse neurotransmitters at different sites of trigeminal system. Here we discovered that the reduction in pain and accompanying DNI involves the interaction of BT-A with central endogenous opioid system (probably at the level of trigeminal nucleus caudalis).

Discovery of dual positive allosteric modulators (PAMs) of the metabotropic glutamate 2 receptor and CysLT1 antagonists for treating migraine headache

Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu2 receptor. The hydroxyacetophenone scaffold is a second series of mGlu2 PAMs we have identified. This series of molecules are potent mGlu2 potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.

Hypoxia facilitates neurogenic dural plasma protein extravasation in mice: a novel animal model for migraine pathophysiology.

Migraine animal models generally mimic the onset of attacks and acute treatment processes. A guinea pig model used the application of meta-chlorophenylpiperazine (mCPP) to trigger immediate dural plasma protein extravasation (PPE) mediated by 5-HT2B receptors. This model has predictive value for antimigraine drugs but cannot explain the delayed onset of efficacy of 5-HT2B receptor antagonists when clinically used for migraine prophylaxis. We found that mCPP failed to induce dural PPE in mice. Considering the role 5-HT2B receptors play in hypoxia-induced pulmonary vessel muscularization, we were encouraged to keep mice under hypoxic conditions and tested whether this treatment will render them susceptible to mCPP-induced dural PPE. Following four-week of hypoxia, PPE, associated with increased transendothelial transport, was induced by mCPP. The effect was blocked by sumatriptan. Chronic application of 5-HT2B receptor or nitric oxide synthase blockers during hypoxia prevented the development of susceptibility. Here we present a migraine model that distinguishes between a migraine-like state (hypoxic mice) and normal, normoxic mice and mimics processes that are related to chronic activation of 5-HT2B receptors under hypoxia. It seems striking, that chronic endogenous activation of 5-HT2B receptors is crucial for the sensitization since 5-HT2B receptor antagonists have strong, albeit delayed migraine prophylactic efficacy.

BF-1 – A novel selective 5-HT2B receptor antagonist blocking neurogenic dural plasma protein extravasation in guinea pigs

Serotonin 5-HT2B receptor antagonists have been proposed as migraine prophylactic drugs, but previously available 5-HT2B receptor antagonists displayed multiple monoaminergic side effects and had to be withdrawn from the market. Here, we set out to identify a novel antagonist with high affinity and selectivity towards 5-HT2B receptors. To test the affinity of new compounds towards various receptors, we generated a broad series of cells functionally coupling human monoaminergic receptors to luciferase. Using the cell lines we revealed pimethixene (1-methyl-4-(9H-thioxanthen-9-ylidene)piperidine) as highly potent, albeit non-selective 5-HT2B receptor antagonist and optimized its chemical structure to create highly potent and selective 5-HT2B receptor antagonists. We selected the methoxythioxanthene BF-1 for further analysis. In comparison to pimethixene, it lacked high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2 as well as muscarinic M1 and M2 receptors. BF-1 was tested as potential migraine prophylactic drug by blocking meta-chlorophenylpiperazine, (mCPP) or BW723C86 (5-((thiophen-2-yl)methoxy)-α-methyltryptamine) induced neurogenic dural plasma protein extravasation in a guinea pig model that may resemble a migraine attack. BF-1 was significantly more potent in this assay compared to the well know non-selective 5-HT2B antagonists, methysergide ((6aR,9R)-N-[(2S)-1-Hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide) or pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-thiophene). Therefore, we propose BF-1 as a new compound that may be developed for prophylactic migraine treatment without the typical monoaminergic side effects.

EHMTI-0219. Quantitative histological examinations of inter- and intraindividual differences in the dural vasculature of the mouse

To examine both migraine and pulmonary hypertension (PH) we developed a hypoxic mouse model. Under hypoxic conditions (four weeks at 10% O2) mice develop typical symptoms of PH, characterized by an increased muscularization of arterial blood vessels in the lung. The vessel remodeling can be prevented by the chronic application of 5-HT2B receptor antagonists. We found that hypoxic mice also develop increased sensitivity towards the 5-HT2 receptor agonist meta-Chlorophenylpiperazine (mCPP). In contrast to the control group (normoxic mice kept at 20% O2) at low doses of 1 µg / kg body weight, mCPP triggers plasma protein extravasation (PPE) in the dura mater in hypoxic mice. Dural PPE is a quantifiable indicator of migraine-like events in animal models.

EHMTI-0092. Activation of the 5-HT2B receptor induces dural plasma protein extravasation in a mouse migraine model

Migraine attacks originate in the meninges which are densely innervated by trigeminal nerve fibers. Upon stimulation, endothelial cells of dural blood vessels secrete nitric oxide and as a consequence, neuropeptides are released from trigeminal nerve fibers. This, in turn, leads to meningeal plasmaproteinextravasation (PPE) and vasodilation. Inflammatory components such as PPE and vasodilation serve as indicators for migraine attacks in animal models. A guinea pig migraine model was described in which the partial 5-HT2B/2C receptor agonist meta-Chlorphenylpiperazine (mCPP) is applied to induce PPE in the dura mater. We have transferred this model to mice.

Investigation of 5-HT2B receptor induced dural plasma protein extravasation in a mouse migraine model

Migraine attacks originate in the meninges which are densely innervated by trigeminal nerve fibers. Stimulated endothelial cells of dural blood vessels secrete nitric oxide, where neuropeptides are released from trigeminal nerve fibers. This, in turn, leads to meningeal Plasma Protein Extravasation (PPE), serving as an established indicator for migraine attacks in animal models. In our mouse migraine model, we sensitized mice against the 5-HT2B receptor agonist meta-chlorphenylpiperazine (mCPP). Mice kept under hypoxic conditions for four weeks displayed significantly elevated PPEs in the dura mater upon mCPP injection. Tissue accumulation of the tracer Evans Blue was measured to quantify the extent of the PPE. In this study, several histological tracers were employed to identify the part of the vasculature where PPE occurs and the cellular mechanism associated with it. Injections of BSA-FITC (FITC-linked bovine serum albumin) verified the mCPP induced PPE in the dura mater. After leaving the blood vessels, the tracer was incorporated into perivascular, CD68-positive macrophages. With electron microscopic studies using the tracer HRP (horse radish peroxidase) we demonstrated that the PPE is associated with increased transcytotic transport. After 5-HT2B receptor activation, HRP escapes from capillaries and venules of hypoxic mice via an increased transcytotic transport in the endothelium. The hypoxic treatment itself increases transcytosis in arterioles, which may be indicative of a proinflammatory state of the endothelium. HRP was also detectable in intercellular clefts, but was always retained at tight junctions In summary, we demonstrated that in mice hypoxic treatment induces dural PPE via increased transcytotis at arterioles and that this is elevated to capillaries and venules after mCPP-injection.

Investigation of 5-HT2B receptor induced dural plasma protein extravasation in a mouse migraine model

Investigation of 5-HT2B receptor induced dural plasma protein extravasation in a mouse migraine model

Preclinical pharmacological profile of the selective 5-HT1F receptor agonist lasmiditan

Lasmiditan (also known as COL-144 and LY573144; 2,4,6-trifluoro-N-[6-[(1-methylpiperidin-4-yl)carbonyl]pyridin-2yl]benzamide) is a high-affinity, highly selective serotonin (5-HT) 5-HT(1F) receptor agonist. In vitro binding studies show a K(i) value of 2.21 nM at the 5-HT(1F) receptor, compared with K(i) values of 1043 nM and 1357 nM at the 5-HT(1B) and 5-HT(1D) receptors, respectively, a selectivity ratio greater than 470-fold. Lasmiditan showed higher selectivity for the 5-HT(1F) receptor relative to other 5-HT(1) receptor subtypes than the first generation 5-HT(1F) receptor agonist LY334370. Unlike the 5-HT(1B/1D) receptor agonist sumatriptan, lasmiditan did not contract rabbit saphenous vein rings, a surrogate assay for human coronary artery constriction, at concentrations up to 100 µM. In two rodent models of migraine, oral administration of lasmiditan potently inhibited markers associated with electrical stimulation of the trigeminal ganglion (dural plasma protein extravasation, and induction of the immediate early gene c-Fos in the trigeminal nucleus caudalis). Lasmiditan presents a unique pyridinoyl-piperidine scaffold not found in any other antimigraine class. Its chemical structure and pharmacological profile clearly distinguish it from the triptans. The potency and selectivity of lasmiditan make it ideally suited to definitively test the involvement of 5-HT(1F) receptors in migraine headache therapy.

Pharmacological Characterization of the Competitive GLUK5 Receptor Antagonist Decahydroisoquinoline LY466195 in Vitro and in Vivo

The excitatory neurotransmitter glutamate has been implicated in both migraine and persistent pain. The identification of the kainate receptor GLU(K5) in dorsal root ganglia, the dorsal horn, and trigeminal ganglia makes it a target of interest for these indications. We examined the in vitro and in vivo pharmacology of the competitive GLU(K5)-selective kainate receptor antagonist LY466195 [(3S,4aR,6S,8aR)-6-[[(2S)-2-carboxy-4,4-difluoro-1-pyrrolidinyl]-methyl]decahydro-3-isoquinolinecarboxylic acid)], the most potent GLU(K5) antagonist described to date. Comparisons were made to the competitive GLU(K5)/alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahydroisoquinoline-3-carboxylic acid], other decahydroisoquinoline GLU(K5) receptor antagonists, and the noncompetitive AMPA receptor antagonist LY300168 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodi-azepine]. When characterized electrophysiologically in rat dorsal root ganglion neurons, LY466195 antagonized kainate (30 microM)-induced currents with an IC50 value of 0.045 +/- 0.011 microM. In HEK293 cells transfected with GLU(K5), GLU(K2)/GLU(K5), or GLU(K5)/GLU(K6) receptors, LY466195 produced IC50 values of 0.08 +/- 0.02, 0.34 +/- 0.17, and 0.07 +/- 0.02 microM, respectively. LY466195 was efficacious in a dural plasma protein extravasation (PPE) model of migraine with an ID100 value of 100 microg/kg i.v. LY466195 was also efficacious in the c-fos migraine model, with a dose of 1 microg/kg i.v. significantly reducing the number of Fos-positive cells in the rat nucleus caudalis after electrical stimulation of the trigeminal ganglion. Furthermore, LY466195 showed no contractile activity in the rabbit saphenous vein in vitro. The diethyl ester prodrug of LY466195 was also efficacious in the same PPE and c-fos models after oral administration at doses of 10 and 100 microg/kg, respectively while having no N-methyl-D-aspartate antagonist-like behavioral effects at oral doses up to 100 mg/kg.

Effect of Hyperoxia on Neurogenic Plasma Protein Extravasation in the Rat Dura Mater

We aimed to study the effect of normobaric hyperoxia on neurogenic inflammation of the rat dura mater. Inhalation of 100% oxygen is a first-line therapy for the treatment of acute cluster headache (CH). However, the mechanisms underlying the antinociceptive effect of oxygen are poorly understood. Sumatriptan, which is also effective in aborting CH attacks, is known to inhibit neurogenic inflammation of the dura mater. We hypothesized that hyperoxia reduces dural plasma protein extravasation in the model of electrically stimulating the rat trigeminal ganglion. Unilateral stimulation of the trigeminal ganglion was performed in anesthetized male Sprague-Dawley rats. We assessed plasma protein extravasation (PPE) in the ipsilateral dura mater under normoxic (group 1) and hyperoxic conditions (group 2: pO(2) 200 mmHg; group 3: pO(2) 300 mmHg; group 4: pO(2) 400 mmHg). The study results were compared to the effect of sumatriptan (300 microg/kg) on dural PPE. Under normoxic conditions, the calculated extravasation ratio was 1.72 +/- 0.2. Hyperoxic treatment (groups 2, 3, 4) significantly attenuated dural PPE. At oxygen levels of 400 mmHg, the PPE ratio was 1.14 +/- 0.2 (P < .01). After IV application of sumatriptan (300 microg/kg), PPE was nearly abolished (PPE ratio: 1.06 +/-0.17). Our findings demonstrate that hyperoxia is able to inhibit dural PPE. Hyperoxia may play an anti-inflammatory role in neurogenic inflammation, but further studies are needed to clarify whether this effect is either caused by prejunctional mechanisms or by modulation of the vascular permeability at postcapillary venules.

3H]LY334370, a novel radioligand for the 5-HT1F receptor. I. In vitro characterization of binding properties

[(3)H]LY334370 was developed as a radioligand to study the characteristics of this compound's interaction with the 5-HT(1F) receptor. Monovalent or divalent cations did not enhance the binding of [(3)H]LY334370 to the cloned human 5-HT(1F) receptor. In the presence of MgCl(2), the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [(3)H]LY334370 had high affinity for the cloned human 5-HT(1F) receptor (K(d)=0.446 nM) and the 5-HT(1F) receptor in rat brain (K(d)=0.388 nM). The expression density of 5-HT(1F) receptors, as determined by binding to homogenates of cortical regions from rat, was low (B(max)=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding and the pEC(50) for stimulation of [(35)S]GTPgammaS binding to homogenates of cells expressing the cloned human 5-HT(1F) receptor. In addition, there was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding to the cloned human 5-HT(1F) receptor and the pID(50) for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [(3)H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT(1F) receptor in rat brain or in cells transformed with the human 5-HT(1F) receptor.

Glutamate Receptor Antagonists

GLUR5 Antagonists as Novel Migraine TherapiesBACKGROUND: L‐glutamate activates neurons in the trigeminal nucleus caudalis (TNC). Furthermore, non‐NMDA ionotropic glutamate receptors such as GluR5 are expressed in rat trigeminal ganglion (TG), and GluR5 receptors play an important role in central sensitization (CS). CS is implicated in, and the trigeminal system is at the core of, migraine. Therefore, we hypothesized that GluR5 receptor antagonists would be effective in preclinical models of migraine.OBJECTIVES: 1) To test the potency and efficacy of GluR5 antagonists with varying degrees of affinity and selectivity for the GluR5/kainate receptor in the dural plasma protein extravasation (PPE) and the c‐fos models of migraine. 2) To test these GluR5 receptor antagonists for their ability to contract the rabbit saphenous vein (RSV) in vitro.METHODS: GluR5 antagonists were synthesized in the Lilly Research Laboratories. Ligand binding studies using recombinant human non‐NMDA receptors expressed in HEK293 cell membranes were used to determine compound affinity. We evaluated parenterally administered compounds in the PPE and the c‐fos models induced by electrical stimulation of male rats TG. Fos protein in the TNC was measured in the c‐fos experiment. Ring preparations of RSV were suspended in tissue baths and isometric contractions were recorded following incremental increases in the concentration of the test compound. Sumatriptan was used as a positive control for the RSV studies.RESULTS: LY293558, LY382884 and LY435735 dose‐dependently blocked protein extravasation in the PPE model. The rank order of potency correlated with affinity for GluR5. In contrast, LY300168, a potent and selective AMPA antagonist, did not block protein extravasation, even at relatively high doses. LY293558 and LY435735 also blocked c‐fos expression in the TNC. LY293558 and LY382884 did not contract the tissue in the RSV assay at concentrations up to 100 mM.CONCLUSIONS: The inhibitory effects of GluR5 antagonists in the PPE and c‐fos preclinical models of migraine suggest a novel role for GluR5 antagonism in migraine therapy. The lack of efficacy of the selective AMPA antagonist LY300168 in PPE does not support a role for this mechanism in migraine. The lack of activity of these compounds in the RSV provides a therapeutic advantage of GluR5 antagonists over current vasoactive migraine therapies. These pharmacological properties of GluR5 antagonists suggest that they could be used in the treatment of acute migraine without a cardiovascular liability.

5-HT1F receptor agonists in acute migraine treatment: a hypothesis.

Serotonin-1F receptor (5-HT1F) agonists may relieve acute migraine without vasoconstriction. We conducted a review of preclinical and clinical data that assessed the potential link between migraine and 5-HT1F activation. (i) A high correlation exists between the potency of various 5-HT1 receptor agonists in the guinea pig dural plasma protein extravasation assay and their 5-HT1F receptor binding affinity. (ii) 5-HT1F receptors are on the trigeminal system, and may participate in blocking migraine pain transmission through the trigeminal ganglion and nucleus caudalis. (iii) 5-HT1F receptors are located on glutamate-containing neurones and their activation might inhibit glutamate release; glutamate excess may play a role in migraine. (iv) Selective 5-HT1F receptor agonists (LY334370; LY344864) are effective in preclinical migraine models and are non-vasoconstrictive. (v) LY334370 is effective in acute migraine, and does not cause any symptoms/signs of coronary vasoconstriction. Preclinical experiments and clinical observations argue for a role of selective 5-HT1F agonists in migraine.

Ethyl (3S,4aR,6S,8aR)-6-(4-ethoxycar- bonylimidazol-1-ylmethyl)decahydroiso-quinoline-3-carboxylic ester: a prodrug of a GluR5 kainate receptor antagonist active in two animal models of acute migraine.

Amino diacid 3, a highly selective competitive GluR5 kainate receptor antagonist, exhibited high GluR5 receptor affinity and selectivity over other glutamate receptors. Its diethyl ester prodrug 4 was orally active in two models of migraine: the neurogenic dural plasma protein extravasation model and the nucleus caudalis c-fos expression model. These data suggest that a GluR5 kainate receptor antagonist might be an efficacious antimigraine therapy with a novel mechanism of action.

The effect of adrenergic compounds on neurogenic dural vasodilatation

The pharmacology of neurogenic trigeminovascular vasodilator responses in the dura mater is of interest for understanding the pathophysiology of migraine and to develop new therapies for this disabling common condition. Aminergic mechanisms have been implicated in migraine through direct study of amines in patients, and by inference from the pharmacology of many effective anti-migraine compounds, particularly preventative agents. This study used intravital microscopy to assess the role of aminergic transmission in neurogenic dural vasodilatation (NDV) by measuring directly the diameter of dural arteries in sodium pentobarbitone anaesthetised rats. Electrical stimulation of a closed cranial window produces, by local depolarisation of nerves, dural vessel dilation that is monitored continuously on-line using video-microscopy and a video dimension analyser. This dural vasodilatation was not affected by pre-treatment with an α 1-adrenoceptor agonist (phenylephrine, 1 and 5 μg/kg), or antagonist (corynanthine, 1 and 2 mg/kg), nor by an α 2-adrenoceptor agonist (UK14,304, 5 μg/kg) or antagonist (yohimbine, 1 and 3 mg/kg). Similarly, we saw no effect of β-adrenoceptor blockade (propranolol, 1 and 3 mg/kg). The lack of an inhibitory effect of UK14,304 the model of neurogenic dural vasodilation contrasts with its effect in neurogenic dural plasma protein extravasation model. The lack of inhibition of β-adrenoceptor antagonists in the neurogenic vasodilatation model contrasts with their usefulness as migraine prophylactics, and suggests that their mechanism of action in migraine is unlikely to be through sensory trigeminal fibre terminals at the neurovascular junction. Moreover, the data indicate that the adrenergic system does not play a significant role in neurogenic dural vasodilation.

4991W93, a potent blocker of neurogenic plasma protein extravasation, inhibits trigeminal neurons at 5-hydroxytryptamine (5-HT 1B/1D) agonist doses

Triptans share the pharmacological profile of being 5-hydroxytryptamine (5-HT 1B/1D) agonists and having potent anti-migraine activity. The conformationally restricted zolmitriptan analogue 4991W93 was developed as a potent, and at low doses, specific, non-vasconstrictor inhibitor of neurogenic dural plasma protein extravasation. Here, we sought to study the effect of 4991W93 at plasma protein extravasation blocking and at 5-HT 1B/1D agonist doses. Nociceptive cells with firing latencies consistent with Aδ fibres were recorded in the dorsal horn region of the trigeminal nucleus caudalis after electrical stimulation of the sagittal sinus. Both evoked (13 units) and free running (6 units) activity in cells linked to sagittal sinus stimulation were inhibited by 4991W93 delivered microiontophoretically or by intravenous administration at 10 μg/kg or 100 μg/kg, but not 0.1 μg/kg. When applied iontophoretically, 4991W93 did not appear to have an additive effect over a 5-HT 1B/1D agonist effective concentration of zolmitriptan. These data suggest that 4991W93 is only effective at modulating the trigeminocervical complex at 5-HT 1B/1D agonist doses. To account for neurogenic dural plasma protein extravasation blockade in animal studies, 4991W93 might have non-5-HT 1B/1D-based pharmacological targets that are yet to be described.

Human 5-HT 1F receptor-stimulated [ [formula omitted]]GTP γS binding: correlation with inhibition of guinea pig dural plasma protein extravasation

To determine the potency and efficacy of 5-HT 1F receptor ligands, a [ 35 S ]GTP γS binding assay was developed and optimized for the human 5-HT 1F receptor. Compounds which are known to be effective in the abortive treatment of migraine were tested for efficacy and potency in this assay. Naratriptan, sumatriptan, zolmitriptan, and rizatriptan all had agonist activity. The 5-HT 1F receptor ligand LY334370 (4-fluoro- N-[3-(1-methyl-4-piperidinyl)-1 H-indol-5-yl]-benzamide) was the most potent compound tested with an EC 50 of 2.13±0.15 nM. LY302148 (5-fluoro-3-[1-[2-(1-methyl-1 H-pyrazol-4-yl)ethyl]-4-piperidinyl]-1 H-indole), methysergide, LY306258 (3-dimethylamino-2,3,4,9-tetrahydro-1 H-carbazol-6-ol), dihydroergotamine (DHE), L-694,247 and CP-122,288 were also investigated for potency and efficacy. There was a statistically significant correlation between the pEC 50 for the stimulation of [ 35 S ]GTP γS binding and the pID 50 for the inhibition of trigeminal nerve-stimulated dural plasma protein extravasation in the guinea pig. In the course of these studies, it was found that the purportedly selective 5-HT 1D receptor antagonist GR127935 inhibited 5-HT 1F receptor-stimulated [ 35 S ]GTP γS binding with a K i of 39.6±9.5 nM. These studies demonstrate that 5-HT 1F receptor-mediated stimulation of [ 35 S ]GTP γS binding in a clonal cell system is a reproducible, high throughput assay that is predictive of an in vivo model of 5-HT 1F receptor activation.

A fluorescence-based method for assessing dural protein extravasation induced by trigeminal ganglion stimulation

Neurogenic dural inflammation has been proposed as a source of pain during migraine. Unilateral electrical stimulation of the trigeminal ganglion causes the ipsilateral release of inflammatory neuropeptides and subsequent dural plasma protein extravasation, a component of neurogenic inflammation. We measured the amount of protein leaking into the dural tissue of guinea pigs following trigeminal ganglion stimulation by exploiting the complexation reaction of endogenous proteins with the fluorescent dye Evans Blue, instead of utilizing exogenous radiolabeled albumin as commonly done in the literature. The amount of Evans Blue trapped in dural tissue following electrical stimulation of the trigeminal ganglion was measured using a fluorescence microscope equipped with a spectrophotometer. This method utilized multiple measurements on each dura sample which resulted in very precise values using a small number of animals per point ( n=3). Sumatriptan and CP-122,288 were found to dose-dependently prevent neurogenic dural extravasation. The potencies of CP-122,288 and sumatriptan were found to be similar to those reported in the literature when similar experimental protocols were used.