Dural Protein Extravasation Research Articles

Synthesis and pharmacological evaluation of optically pure, novel carbonyl guanidine derivatives as dual 5-HT2B and 5-HT7 receptor antagonists

A series of 9-disubstituted N-(9H-fluorene-2-carbonyl)guanidine derivatives have been discovered as potent and orally active dual 5-HT2B and 5-HT7 receptor antagonists. Upon screening several compounds, N-(diaminomethylene)-4′,5′-dihydro-3′H-spiro[fluorene-9,2′-furan]-2-carboxamide (17) exhibited potent affinity for both 5-HT2B (Ki=5.1nM) and 5-HT7 (Ki=1.7nM) receptors with high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Optical resolution of the intermediate carboxylic acid 16 via the formation of diastereomeric salts using chiral alkaloids gave the optically pure compounds (R)-17 and (S)-17. Both enantiomers suppressed 5-HT-induced dural protein extravasation in guinea pigs in a dose-dependent manner and the amount of leaked protein was suppressed to near normal levels when orally administrated at 10mg/kg. (R)-17 and (S)-17 were therefore selected as candidates for human clinical trials.

Synthesis and structure–activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists. Part 2

We previously reported that the novel dual 5-HT2B and 5-HT7 receptor antagonist N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (4) exerted a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs. To develop a synthetic strategy, we performed docking studies of lead compound 4 bound to 5-HT2B and 5-HT7 receptors, and observed that the carbonyl guanidine group forms a tight interaction network with an active center Asp (D135:5-HT2B, D162:5-HT7), Tyr (Y370:5-HT2B, Y374:5-HT7) and aromatic residue (W131:5-HT2B, F158:5-HT7). Based on molecular modeling results, we optimized the substituents at the 5- to 8-position and 9-position of the fluorene ring and identified N-(diaminomethylene)-9-hydroxy-9-methyl-9H-fluorene-2-carboxamide (24a) exhibits potent affinity for 5-HT2B (Ki=4.3nM) and 5-HT7 receptor (Ki=4.3nM) with high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Compound 24a reversed the hypothermic effect of 5-carboxamidotryptamine (5-CT) in mice and also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered at 30mg/kg. Compound 24a is therefore a promising candidate for a novel class of anti-migraine agent without any adverse effects.

Synthesis and structure–activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists

To identify potent dual 5-HT2B and 5-HT7 receptor antagonists, we synthesized a series of novel carbonyl guanidine derivatives and examined their structure–activity relationships. Among these compounds, N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (10) had a good in vitro profile, that is, potent affinity for human 5-HT2B and 5-HT7 receptor subtypes (Ki=1.8nM and Ki=17.6nM, respectively) and high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Compound 10 also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered.

Different trigemino-vascular responsiveness between adolescent and adult rats in a migraine model

Pediatric migraine displays different clinical features from adult migraine. Because the trigemino-vascular system (TGVS) plays a pivotal role in migraine pathophysiology, this study compared TGVS responses in a migraine model induced by intracisternal (i.c.) instillation of capsaicin in adolescent and adult rats. TGVS responses measured included c-Fos-protein-expressing neurons in the trigeminal cervical complex (TCC), calcitonin gene-related peptide (CGRP) expression in the trigeminal ganglia (TG) and dura mater, and dural protein extravasation. The formulas for estimating total numbers of activated TCC neurons were established based on the c-Fos-positive neuronal numbers in three sample sections, +0.6, -1.2 and -9 mm and +0.6, -0.6 and -6 mm, from the obex in adult and adolescent rats, respectively. After capsaicin instillation, adolescent rats had comparable TCC neurons activated as adult rats, but less TGVS peripheral responsiveness than adults, including CGRP immunoreactivity in the TG, and protein extravasation and CGRP depletion (inversely reflected by CGRP immunoreactivity) in the dura mater. Age-dependent differences in TGVS responsiveness in the i.c. capsaicin-induced migraine model of rats are reminiscent of less severe migraine in pediatric patients. This finding may provide new insight into the pathophysiology of migraine and guide the development of new anti-migraine drugs for children.

Central Action of Peripherally Applied Botulinum Toxin Type A on Pain and Dural Protein Extravasation in Rat Model of Trigeminal Neuropathy

BackgroundInfraorbital nerve constriction (IoNC) is an experimental model of trigeminal neuropathy. We investigated if IoNC is accompanied by dural extravasation and if botulinum toxin type A (BoNT/A) can reduce pain and dural extravasation in this model.Methodology/Principal FindingsRats which developed mechanical allodynia 14 days after the IoNC were injected with BoNT/A (3.5 U/kg) into vibrissal pad. Allodynia was tested by von Frey filaments and dural extravasation was measured as colorimetric absorbance of Evans blue - plasma protein complexes. Presence of dural extravasation was also examined in orofacial formalin-induced pain. Unilateral IoNC, as well as formalin injection, produced bilateral dural extravasation. Single unilateral BoNT/A injection bilaterally reduced IoNC induced dural extravasation, as well as allodynia (lasting more than 2 weeks). Similarly, BoNT/A reduced formalin-induced pain and dural extravasation. Effects of BoNT/A on pain and dural extravasation in IoNC model were dependent on axonal transport through sensory neurons, as evidenced by colchicine injections (5 mM, 2 µl) into the trigeminal ganglion completely preventing BoNT/A effects.Conclusions/SignificanceTwo different types of pain, IoNC and formalin, are accompanied by dural extravasation. The lasting effect of a unilateral injection of BoNT/A in experimental animals suggests that BoNT/A might have a long-term beneficial effect in craniofacial pain associated with dural neurogenic inflammation. Bilateral effects of BoNT/A and dependence on retrograde axonal transport suggest a central site of its action.

Putative 5-ht5 receptors: localization in the mouse CNS and lack of effect in the inhibition of dural protein extravasation.

Putative 5-ht5 receptor binding sites were visualized by in vitro autoradiography using [125I]LSD (in the presence of clozapine and spiperone) or [3H]5-carboxamidotryptamine (in the presence 8-OH-DPAT, GR127935 and spiperone). Under these conditions, no [3H]5-carboxamidotryptamine labeling was detected in the brain of mice lacking the gene encoding the putative 5-ht5a receptor (knockout mice), whereas intermediate densities of binding sites were seen in the olfactory bulb and neocortex of wild-type mice. [125I]LSD labeled the same areas as [3H]5-carboxamidotryptamine in wild-type mice. High densities of [125I]LSD binding sites were observed in the medial habenula of wild type and knockout mice. 5-CT competed for [125I]LSD binding sites with an affinity of 2 nM in the olfactory bulb and neocortex of wild-type mice and an affinity of 30 nM in the habenula of knockout mice, suggesting that habenular labeling might be accounted for by putative 5-ht5b receptors. In the presence of 5'-guanylylimidodiphosphate, 5-CT displaced [125I]LSD from putative 5-ht5a and 5-ht5b sites with a 6-times and 3-times lower affinity, respectively, suggesting that both receptor subtypes are coupled to G proteins in brain. We also studied the inhibitory effect of 5-CT on dural neurogenic inflammation in knockout mice. In wild type mice, 3 ng/kg 5-CT inhibited dural protein extravasation by 60%. A similar effect was observed in knockout mice, even in the presence of the 5-HT1B receptor antagonist GR127935. These results suggest that the inhibitory effects of 5-CT are not mediated by a site with the characteristics of the putative 5-ht5 receptor.

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.

Characterization of LY344864 as a pharmacological tool to study 5-HT 1F receptors: Binding affinities, brain penetration and activity in the neurogenic dural inflammation model of migraine

LY344864 is a selective receptor agonist with an affinity of 6 nM (K i) at the recently cloned 5-HT 1F receptor. It possesses little affinity for the 56 other serotonergic and non-serotonergic neuronal binding sites examined. When examined for its ability to inhibit forskolininduced cyclic AMP accumulation in cells stably transfected with human 5-HT 1F receptors, LY344864 was shown to be a full agonist producing an effect similar in magnitude to serotonin itself. After an intravenous dose of 1 mg/kg, rat plasma LY344864 levels declined with time whereas brain cortex levels remained relatively constant for the first 6 hours after injection. Oral and intravenous LY344864 administration potently inhibited dural protein extravasation caused by electrical stimulation of the trigeminal ganglion in rats. Taken together, these data demonstrate that LY344864 is a selective 5-HT 1F receptor agonist that can be used to explore both the in vitro and in vivo functions of this receptor.