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.

5-Hydroxytryptamine1B receptor-mediated contraction of rabbit saphenous vein and basilar artery: role of vascular endothelium.

This study characterizes the sumatriptan-sensitive [5-hydroxytryptamine (5-HT)(1B/1D)] receptor in rabbit saphenous vein and basilar artery. (S)-(-)-1-[2-[4-(4-Methoxy-phenyl)-piperazin-1-yl]-ethyl]isochroman-6-carboxylic acid methylamide (PNU-109291), a 5-HT(1D) subtype-selective agonist (human K(i) = 2.5 +/- 0.07 nM), did not contract either tissue, whereas o-methoxyphenylpiperazide derivative 4F (MPPA-4F), a 5-HT(1B) subtype-selective antagonist (human K(i) = 4.6 +/- 0.6 nM) potently inhibited sumatriptan-induced contraction in the saphenous vein and basilar artery. These results suggested that sumatriptan-induced contraction was mediated via the 5-HT(1B) receptor in these blood vessels. 5-HT(1B) receptor-mediated contraction was then compared in endothelium-intact and denuded vessels to evaluate the role of the endothelium in regulating sumatriptan-induced contractility in these tissues. The presence of an intact endothelium inhibited 5-HT(1B)-induced contraction in both tissues. Endothelial denudation or nitric-oxide synthase inhibition with N(omega) nitro-L-arginine methyl ester (L-NAME) (100 microM) increased the efficacy and potency of sumatriptan in the saphenous vein and basilar artery. Surprisingly, in endothelial-denuded vascular tissues, L-NAME (100 microM) also significantly increased the maximal 5-HT(1B) receptor-induced contraction in both tissues, with no effect on potency of sumatriptan. The effect of L-NAME after endothelial denudation may reflect the presence of a low density of residual endothelial cells as estimated by CD31 antibody staining combined with the modulating effect of nitric oxide released from nonendothelial cells in vascular tissue. Endothelial modulation was specific to 5-HT(1B) receptors because removal of the endothelium did not significantly alter contraction to norepinephrine, histamine, prostaglandin, or potassium chloride in the saphenous vein or basilar artery.

Novel potent 5-HT(1F) receptor agonists: structure-activity studies of a series of substituted N-[3-(1-methyl-4-piperidinyl)-1H-pyrrolo[3,2-b]pyridin-5-yl]amides.

Compound 1a (LY334370), a selective 5-HT(1F) receptor agonist (SSOFRA), inhibited dural inflammation in the neurogenic plasma protein extravasation model of migraine and demonstrated clinical efficacy for the acute treatment of migraine. Although 1a was greater than 100-fold selective over both the 5-HT(1B) and 5-HT(1D) receptors, it exhibited appreciable 5-HT(1A) receptor affinity. Described here is the synthesis and evaluation of a series of pyrrolo[2,3-c]pyridine and pyrrolo[3,2-b]pyridine (2a and 3a) as well as pyrrolo[3,2-d]pyrimidine (4a) analogues of 1a, compounds prepared in an effort to identify SSOFRAs with improved selectivity over other 5-HT(1) receptor subtypes. The pyrrolo[3,2-b]pyridine analogue 3a showed high 5-HT(1F) receptor affinity but offered no improvement in selectivity compared to 1a. However, the C-5 acetamide derivative, 3b, was greater than 100-fold selective over the 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors. SAR studies of this series determined that alkylamides in particular exhibited high selectivity for the 5-HT(1F) receptor. Replacement at C-5 with other substituents decreased affinity or selectivity. These SAR studies identified SSOFRAs that demonstrated oral activity in the neurogenic plasma protein extravasation model, a model indicative of antimigraine activity.

Amplification of sumatriptan-induced contraction in rabbit saphenous vein but not in basilar artery.

The modulation of serotonin (5-HT(1B/1D)) receptor-induced vascular contractility by histamine and U-46619 was compared in the rabbit basilar artery and saphenous vein. In the saphenous vein, histamine (5 x 10(-7) M) significantly increased the potency (from pEC(50) of 6.0 to 6.8) and efficacy (from 52.3% to 88.2%) of sumatriptan. Likewise, U-46619 (5 x 10(-9) M) also increased the potency (from pEC(50) of 5.9 to 6.6) and efficacy (from 51.8% to 92.1%) of sumatriptan in the saphenous vein. In contrast, equieffective concentrations of histamine (10(-7) M) and U-46619 (3 x 10(-9) M) failed to amplify contraction to sumatriptan in the basilar artery. Contraction to sumatriptan was inhibited by nitrendipine (10(-7) M) in the basilar artery but not in the saphenous vein, suggesting that different contractile signaling mechanisms are operating in these tissues. Furthermore, U-46619- and thrombin-induced contractility in the basilar artery were also not amplified by histamine, suggesting that lack of amplification of contraction in the basilar artery was not restricted to sumatriptan but was rather a characteristic of this cerebral vessel. These data suggest that in the in vivo plasma milieu sumatriptan will more markedly contract the peripheral saphenous vein than the basilar artery, a cerebral blood vessel.

Lack of sumatriptan-induced aortic contraction or relaxation: 5-HT1B receptor protein detected in endothelium and smooth muscle of vasa vasorum but not aorta.

Since 5-HT1B receptor mRNA was reported in rat aorta, and 5-HT1B receptor activation has been linked to vascular contraction, we explored sumatriptan-induced contractility and immunohistochemical density of 5-HT1B receptor protein in rat aorta. Sumatriptan (up to 10(-4) M), a 5-HT1B/1D receptor agonist, did not contract the endothelial intact or denuded rat aorta, even in the presence of L-NAME or after induction of modest tone with PGF2 alpha (10(-6) M). Sumatriptan also did not relax aortic preparations precontract with PGF2 alpha (3 x 10(-6) M) or phenylephrine (3 x 10(-7) M). Using a polyclonal antibody directed against the 5-HT1B receptor, minimal 5-HT1B receptor protein was detected in either the endothelium or smooth muscle of the rat aorta. However, dense 5-HT1B receptor protein was found in the vascular smooth muscle of the vasa vasorum supplying the aorta. Thus, the 5-HT1B receptor mRNA detected in tissue extracts of the rat aorta most likely reflects 5-HT1B receptor expression in the arterioles of the vasa vasorum. These studies support the link between the 5-HT1B receptor and vascular contraction in that the aorta with low density of 5-HT1B receptors lacked responses to sumatriptan, an agonist thought to contract blood vessels via 5-HT1B/1D receptors. Furthermore, caution must be observed when using 5-HT1B receptor mRNA to suggest receptor protein in tissues since this RT-PCR product may be derived predominantly from small blood vessels.