Inhibitory effect of BIBN4096BS on cephalic vasodilatation induced by CGRP or transcranial electrical stimulation in the rat

Calcitonin gene-related peptide (CGRP) is believed to play a pivotal role in the pathogenesis of migraine via activation of CGRP receptors in the trigeminovascular system. The CGRP receptor antagonist, BIBN4096BS, has proven efficacy in the acute treatment of migraine attacks and represents a new therapeutic principle. We used an improved closed cranial window model to measure changes of the middle meningeal artery (MMA) and cortical pial artery/arteriole diameter (PA) and changes in local cortical cerebral blood flow (LCBF(Flux)) in anaesthetised artificially ventilated rats. The ability of BIBN4096BS (i.v.) to prevent the vasodilatatory actions of rat-alphaCGRP, betaCGRP and endogenously released CGRP following transcranial electrical stimulation (TES) was investigated. BIBN4096BS was per se without vasoactive effect on any of the measured variables and significantly inhibited the hypotension induced by both types of CGRP (P < 0.001). The alphaCGRP induced MMA dilatation was reduced from 97.4 +/- 14 to 2.1 +/- 1.3% (P < 0.001) and the betaCGRP induced dilatation was fully blocked by BIBN4096BS. ID(50) was 5.4 +/- 1.6 microg kg(-1) for alphaCGRP and 16.3 +/- 1.6 microg kg(-1) for betaCGRP. Transcranial electrical stimulation induced a 119.1 +/- 6.9% increase in MMA diameter. BIBN4096BS (333 microg kg(-1)) attenuated this increase (19.8 +/- 2.1%) (P < 0.001). Systemic CGRP and TES induced an increase in PA diameter that was not significantly inhibited by BIBN4096BS. The CGRP induced increase in LCBF(Flux) was similar not prevented by the antagonist. We suggest that systemic BIBN4096BS exerts its inhibitory action mainly on large dural blood vessels (MMA).     

Effect of the CGRP receptor antagonist BIBN4096BS in human cerebral, coronary and omental arteries and in SK-N-MC cells.

Several lines of evidence suggest that a calcitonin-gene related peptide (CGRP) receptor antagonist may serve as a novel abortive migraine treatment. Here we present data on a human cell line and isolated human vessels for such an antagonist, BIBN4096BS. On SK-N-MC membranes, radiolabelled CGRP was displaced by both CGRP-(8-37) and BIBN4096BS, yielding pK(i) values of 8.5 and 11.4, respectively. Functional studies with SK-N-MC cells demonstrated that CGRP-induced cAMP production was antagonised by both CGRP-(8-37) and BIBN4096BS with pA(2) values of 7.8 and 11.2, respectively. Isolated human cerebral, coronary, and omental arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation that was antagonized by both CGRP-(8-37) and BIBN4096BS in a competitive manner. CGRP was a weaker agonist on coronary arteries as compared to intracranial arteries; however, BIBN4096BS was an equally effective antagonist. In human omental arteries, CGRP did not induce relaxation. BIBN4096 had a pA(2) value of 10.1 in cerebral and 10.4 in coronary arteries. The results of clinical trials with BIBN4096BS for acute migraine attacks are awaited with great interest.     

Effect of two novel CGRP-binding compounds in a closed cranial window rat model

We investigated the in vivo effects of two novel calcitonin gene-related peptide (CGRP) binding molecules in the genuine closed cranial window model in the rat. The RNA-Spiegelmer (NOX-C89) and the monoclonal CGRP antibody are CGRP scavengers and might be used as an alternative to CGRP-receptor antagonists in the treatment of migraine. Rats were anaesthetized and a closed cranial window established. Changes in dural and pial artery diameter and mean arterial blood pressure were measured simultaneously. Infusion of the RNA-Spiegelmer or the CGRP antibody alone had no effect on the arteries or the mean arterial blood pressure. We then used a bolus of 0.3 microg/kg CGRP (n=6) or electrical stimulation (25 V, 5 Hz, 1 ms pulse width and of 10 s of duration) (n=6) to induce dilatation of dural and pial arteries (mediated via CGRP-receptors). Pre-treatment with the RNA-Spiegelmer inhibited CGRP-induced vasodilatation of the dural artery (from 38+/-17% to 7+/-3%) and the pial artery (from 14+/-1% to 3+/-2%) (P<0.05). The RNA-Spiegelmer, however, did not significantly inhibit dilatation induced by electrical stimulation (P>0.05). The CGRP antibody caused a significant reduction of the dural artery diameter caused by intravenous CGRP-infusion (from 23+/-5% to 12+/-3%) (P<0.05), but did not inhibit dilatation caused by electrical stimulation (P>0.05). In conclusion, the CGRP scavengers effectively inhibited the effect of circulating CGRP but do not modify the effect of electrical stimulation and the consequent liberation of CGRP from perivascular sensory nerve fibres.     

Inhibition by adrenomedullin of amine release from adrenergic nerves in dog mesenteric arteries

Adrenomedullin and calcitonin gene-related peptide (CGRP) inhibited the pressor response to transmural electrical stimulation in perfused isolated canine mesenteric arteries. The response was abolished by treatment with either prazosin or tetrodotoxin. Adrenomedullin-(22-52), an adrenomedullin receptor antagonist, reduced the inhibitory effect of adrenomedullin (10(-10) to 10(-8) mol/l), but did not alter the action of CGRP. CGRP-(8-37), a CGRP(1) receptor antagonist, did not affect the inhibition induced by adrenomedullin, but reversed the CGRP-induced inhibition. In helical strips of the arteries, adrenomedullin (up to 10(-8) mol/l) did not influence the contraction induced by noradrenaline, whereas CGRP attenuated the response. Adrenomedullin decreased the release of noradrenaline from adrenergic nerves elicited by transmural electrical stimulation, but CGRP had no effect. Adrenomedullin-(22-52) reversed the decrease in noradrenaline release induced by adrenomedullin. The adrenomedullin-induced relaxation of vascular strips precontracted with prostaglandin F(2alpha) was suppressed by CGRP-(8-37) but was unaffected by adrenomedullin-(22-52). These findings suggest that adrenomedullin impairs noradrenaline release from adrenergic nerves by acting on adrenomedullin receptors located in the nerve terminals, whereas arterial relaxation caused by adrenomedullin and CGRP is due to activation of CGRP(1) receptors in vascular smooth muscle.     

Effects of BIBN4096BS on cardiac output distribution and on CGRP-induced carotid haemodynamic responses in the pig

Calcitonin gene related peptide (CGRP) seems to be involved in the pathogenesis of migraine, since plasma CGRP levels increase during the headache phase. In the present study, we investigated the effects of a novel CGRP receptor antagonist, BIBN4096BS (1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-), on the regional cardiac output distribution and on the carotid haemodynamic changes induced by alpha-CGRP in anaesthetised pigs. Treatment with BIBN4096BS (100, 300 and 1000 microg kg(-1), i.v.) did not affect the heart rate, mean arterial blood pressure or systemic vascular conductance, but a small decrease in cardiac output was noticed; the latter was, however, not significantly different from that in vehicle-treated animals. The highest dose of BIBN4096BS moderately decreased vascular conductance in the lungs, kidneys, spleen and adrenals. Vascular conductance in other tissues including the brain, heart, gastrointestinal system, skin and skeletal muscles remained unchanged. Intracarotid artery infusions of alpha-CGRP (10, 30 and 100 pmol kg(-1) min(-1) during 3 min) increased the total carotid blood flow and conductance, but decreased the arterial blood pressure. These responses were dose-dependently blocked by BIBN4096BS. The above results show that BIBN4096BS is a CGRP receptor antagonist in the porcine carotid and systemic circulations, but the endogenous CGRP does not seem to play an important physiological role in regulating basal vascular tone. These findings suggest that BIBN4096BS may have therapeutic usefulness in migraine.     

A comparison of the actions of BIBN4096BS and CGRP(8-37) on CGRP and adrenomedullin receptors expressed on SK-N-MC,L6, Col 29 and Rat 2 cells

1. The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. 2. On human SK-N-MC cells (expressing human calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA(2) of 9.95 although the slope of the Schild plot (1.37 +/- 0.16) was significantly greater than 1. 3. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA(2) of 9.25 and a Schild slope of 0.89 +/- 0.05, significantly less than 1. 4. On human Colony (Col) 29 cells, CGRP(8-37) had a significantly lower pA(2) than on SK-N-MC cells (7.34 +/- 0.19 (n = 7) compared to 8.35 +/- 0.18, (n = 6)). BIBN4096BS had a pA(2) of 9.98 and a Schild plot slope of 0.86 +/- 0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK-N-MC cells. 5. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 microM. CGRP(8-37) had a pA(2) of 6.72 against adrenomedullin. 6. BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK-N-MC cells and the CGRP-responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent 'non-competitive' behaviour. At concentrations of up to 10 micro M, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP(8-37).     

The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in isolated rat hearts

Brief coronary artery occlusion can protect the heart against damage during subsequent prolonged coronary artery occlusion; ischemic preconditioning. The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning is investigated in isolated perfused rat hearts, by measuring CGRP release during ischemic preconditioning and mimicking this by exogenous CGRP infusion, either in the absence or presence of the CGRP antagonist BIBN4096BS. CGRP increased left ventricular pressure and coronary flow in a concentration dependent manner, which was effectively antagonized by BIBN4096BS. Rat hearts (n=36) were subjected to 45 min coronary artery occlusion and 180 min reperfusion, which was preceded by: (1) sham pretreatment, (2) BIBN4096BS infusion (1 microM), (3) preconditioning by 15 min coronary artery occlusion and10 min reperfusion, (4) as 3, but with BIBN4096BS, (5) 15 min CGRP infusion (5 nM) and 10 min washout, (6) as 5, but with BIBN4096BS. Cardiac protection was assessed by reactive hyperaemia, creatine kinase release, infarct size related to the area at risk (%), and left ventricular pressure recovery. Preconditioning increased CGRP release into the coronary effluent from 88+/-13 to 154+/-32 pg/min/g, and significantly protected the hearts by decreasing reactive hyperaemia (35%), reducing creatine kinase release (53%), limiting infarct size (48%), and improving left ventricular pressure recovery (36%). Exogenous CGRP induced preconditioning-like cardioprotection. BIBN completely abolished the cardioprotection induced by preconditioning as well as by exogenous CGRP. In conclusion, since cardioprotection of preconditioning-induced CGRP release can be mimicked by exogenous CGRP, and both can be blocked by a CGRP antagonist, results indicate an important role for CGRP in ischemic preconditioning.     

Characterisation of CGRP receptors in human and porcine isolated coronary arteries: evidence for CGRP receptor heterogeneity

This study sets out to characterise calcitonin gene-related peptide (CGRP) receptors in human and porcine isolated proximal and distal coronary arteries using BIBN4096BS. Human (h)-alphaCGRP induced relaxations that were blocked by BIBN4096BS in all arteries studied. In contrast to the other vessels, the Schild plot slope in the human distal coronary artery segments (0.68 +/- 0.07) was significantly less than unity and BIBN4096BS potently blocked these responses (pK(b) (10 nM): 9.29 +/- 0.34, n = 5). In the same preparation, h-alphaCGRP(8-37) behaved as a weak antagonist of h-alphaCGRP-induced relaxations (pK(b) (3 microM): 6.28 +/- 0.17, n = 4), with also a Schild plot slope smaller than unity. The linear agonists, [ethylamide-Cys(2,7)]-h-alphaCGRP ([Cys(Et)(2,7)]-h-alphaCGRP) and [acetimidomethyl-Cys(2,7)]-h-alphaCGRP ([Cys(Acm)(2,7)]-h-alphaCGRP), had a high potency (pEC(50): 8.21 +/- 0.25 and 7.25 +/- 0.14, respectively), suggesting the presence of CGRP(2) receptors, while the potent blockade by BIBN4096BS (pK(b) (10 nM): 10.13 +/- 0.29 and 9.95 +/- 0.11, respectively) points to the presence of CGRP(1) receptors. Using RT-PCR, mRNAs encoding for the essential components for functional CGRP(1) receptors were demonstrated in both human proximal and distal coronary artery. Further, h-alphaCGRP (100 nM) increased cAMP levels, and this was attenuated by BIBN4096BS (1 microM). The above results demonstrate the presence of CGRP(1) receptors in all coronary artery segments investigated, but the human distal coronary artery segments seem to have an additional population of CGRP receptors not complying with the currently classified CGRP(1) or CGRP(2) receptors.     

Effects of the CGRP receptor antagonist BIBN4096BS on capsaicin-induced carotid haemodynamic changes in anaesthetised pigs

1. Calcitonin gene-related peptide (CGRP), a potent vasodilator released from capsaicin-sensitive trigeminal sensory nerves, seems to be involved in the pathogenesis of migraine. Hence, CGRP receptor antagonists may serve as a novel treatment for migraine. This study was therefore designed to investigate the effects of BIBN4096BS (100, 300 and 1000 microg kg-1, i.v.), a potent and selective CGRP receptor antagonist, on capsaicin-induced carotid haemodynamic changes in anaesthetised pigs. Both vagosympathetic trunks were cut and phenylephrine was infused into the carotid artery (i.c.) to support carotid vascular tone. 2. Infusions of capsaicin (0.3, 1, 3 and 10 microg kg-1 min-1, i.c.) did not alter the heart rate, but dose-dependently increased the mean arterial blood pressure. This moderate hypertensive effect was not modified by BIBN4096BS. 3. Capsaicin infusion (10 microg kg-1 min-1, i.c.) increased total carotid, arteriovenous anastomotic and tissue blood flows and conductances as well as carotid pulsations, but decreased the difference between arterial and jugular venous oxygen saturations. These responses to capsaicin were dose-dependently blocked by BIBN4096BS. 4. Capsaicin infusion (10 microg kg-1 min-1, i.c.) more than doubled the jugular venous plasma concentration of CGRP. This effect was not blocked, but rather increased, by BIBN4096BS. 5. The above results show that BIBN4096BS behaves as a potent antagonist of capsaicin-induced carotid haemodynamic changes that are mediated via the release of CGRP. Therefore, this compound may prove effective in the treatment of migraine.     

Involvement of imidazoline receptors in the centrally acting muscle-relaxant effects of tizanidine

BIBN4096BS [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] is a selective calcitonin gene-related peptide (CGRP) receptor antagonist with a picomolar affinity to the CGRP receptor in human neuroblastoma SK-N-MC cells. Here, we describe the characterisation of the binding properties of the tritiated radioanalogue of BIBN4096BS in SK-N-MC cells as well as in marmoset tissue. [(3)H]BIBN4096BS showed reversible and saturable binding to SK-N-MC cells with a K(D) of 0.045 nM. In competition experiments, [3(H)]BIBN4096BS is concentration-dependently displaced from SK-N-MC cell membranes by BIBN4096BS as well as by the endogenous ligand CGRP and its analogues with the rank order of affinity BIBN4096BS>human alpha-CGRP=human beta-CGRP>[Cys(Et)(2,7)]human alpha-CGRP>adrenomedullin (high affinity site)=human alpha-CGRP-(8-37)=human beta-CGRP-(8-37)>calcitonin=amylin. In the marmoset cortex, saturable [(3)H]BIBN4096BS binding was observed with a K(D) of 0.077 nM. CGRP showed biphasic competition of [(3)H]BIBN4096BS binding, whilst BIBN4096BS monophasically displaced its radioanalogue with a K(i) of 0.099 nM. These data, using [(3)H]BIBN4096BS, confirm the high affinity of this novel antagonist for the primate CGRP receptor and demonstrate furthermore that this radioligand is a useful tool to study CGRP receptor pharmacology.     

The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS reduces neurogenic increases in dural blood flow

In an in vivo preparation of the exposed rat cranial dura mater electrical field stimulation causes increases in blood flow that are mainly due to the vasodilatory effect of calcitonin gene-related peptide (CGRP) released from meningeal afferents. In this preparation the effect of BIBN4096BS, a non-peptide competitive antagonist of CGRP receptors, was examined. Additionally, in an in vitro preparation of the hemisected rat skull the effect of BIBN4096BS on CGRP release stimulated by activation of meningeal afferents was analysed. Injection of BIBN4096BS at cumulative doses of 300 microg/kg and 900 microg/kg caused dose-dependent inhibition of the electrically evoked blood flow increases. The basal blood flow and vital parameters were not significantly changed by any dose. In the hemisected skull BIBN4096BS at 10(-6) M did not alter the CGRP release evoked by depolarizing K(+) concentrations or antidromic electrical stimulation of the trigeminal ganglion. We conclude that neurogenic increases in dural blood flow are reduced by BIBN4096BS without changing basal vascular parameters. This peripheral effect may be important with regard to CGRP receptor inhibition as an antimigraine strategy.     

Efficacy of the non-peptide CGRP receptor antagonist BIBN4096BS in blocking CGRP-induced dilations in human and bovine cerebral arteries: potential implications in acute migraine treatment

Calcitonin gene-related peptide (CGRP) is a potent vasodilator in brain vessels and it has been implicated in the pathogenesis of migraine headache. Blocking post-junctional CGRP receptors, mediators of trigeminal-induced vasodilation, has been suggested as a potential antimigraine strategy. In this study, we tested the ability of a new non-peptide CGRP receptor antagonist, BIBN4096BS, to inhibit the CGRP-induced dilation in human and/or bovine brain vessels and compared it to that of the antagonist alpha-CGRP(8-37). BIBN4096BS and alpha-CGRP(8-37) both blocked the alpha-CGRP-induced dilation in bovine middle artery segments with respective potency (pK(B) values) of 6.3 and 7.8. In human pial vessels, BIBN4096BS was particularly potent. When tested at 10(-14)-10(-9) M concentrations, it induced a rightward shift in the alpha-CGRP concentration-response curve and yielded a biphasic Schild plot suggesting interaction with more than one receptor population, as was also indicated by the significant best fit of the alpha-CGRP-induced dilation in human brain vessels with a two receptor site interaction. Schild plot analysis in the linear portion of the BIBN4096BS inhibition curve revealed interaction with one high affinity site (pA(2) value approximately 14). In bovine vessels, both alpha-CGRP(8-37) and BIBN4096BS concentration-dependently reversed a pre-established CGRP-induced dilation ( approximately 59 and 85%, respectively), BIBN4096BS being approximately tenfold more potent than alpha-CGRP(8-37) (respective pIC(50) values of 7.5 and 6.75). In human middle cerebral and middle meningeal arteries, BIBN4096BS reversed the alpha-CGRP-induced dilation (> or =70%) by interaction with two different receptor populations: it exhibited a high affinity for one population (pIC(50) value approximately 13) and a lower affinity for the other (pIC(50) value approximately 8). The present data demonstrate that BIBN4096BS is a very potent antagonist that could, depending on its bioavailability and in vivo affinity, be of potential benefit in the acute treatment of migraine headache by blocking and/or reversing the CGRP-mediated dilation of intracranial vessels induced by activation of trigeminovascular afferents.     

Adrenomedullin receptor binding sites in rat brain and peripheral tissues

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.     

Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist

Calcitonin gene-related peptide (CGRP) is one of the most potent endogenous vasodilators known. This peptide is increased during migraine attacks and has been implicated in the pathogenesis of migraine headache. Here we report on the first small molecule selective CGRP antagonist: BIBN4096BS. In vitro, this compound is extremely potent at primate CGRP receptors exhibiting an affinity (Ki) for human CGRP receptors of 14.4 +/- 6.3 (n = 4) pM. In an in vivo model, BIBN4096BS in doses between 1 and 30 micrograms kg-1 (i.v.) inhibited the effects of CGRP, released by stimulation of the trigeminal ganglion, on facial blood flow in marmoset monkeys. It is concluded that BIBN4096BS is a potent and selective CGRP antagonist.     

BIBN4096BS is a potent competitive antagonist of the relaxant effects of alpha-CGRP on human temporal artery: comparison with CGRP(8-37)

Release of CGRP during migraine may produce harmful dilatation of cranial arteries, thereby possibly causing pain. We have compared the antagonism by BIBN4096BS and CGRP(8-37) of the relaxant effects of alpha-CGRP on rings of human temporal artery. alpha-CGRP relaxed the arteries precontracted with 9 - 24 mM KCl (-logEC50=9.4) nearly as efficaciously as sodium nitroprusside (10 microM). BIBN4096BS (0.1 - 100 nM) antagonized the effects of alpha-CGRP in surmountable manner with slopes of Schild-plots not different from unity. -LogKB values of 10.1 and 10.4 were estimated for BIBN4096BS when administered before or during the KCl-contracture respectively. BIBN4096BS (1 microM) did not modify the relaxant effects of papaverine and sodium nitroprusside. CGRP(8-37) (1 - 10 microM) antagonized the effects of alpha-CGRP in a surmountable manner with slopes of Schild-plots not different from unity. -LogKB values of 6.6 and 6.7 were estimated for CGRP(8-37) administered before or during the KCl-contracture respectively. The high affinity of BIBN4096BS for CGRP receptors of human temporal artery makes it an excellent tool to explore the hypothesis of CGRP-evoked cerebral vasodilation in migraine.     

Effects of the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS on alpha-CGRP-induced regional haemodynamic changes in anaesthetised rats

Several studies suggest that a calcitonin gene-related peptide (CGRP) receptor antagonist may have antimigraine properties, most probably via the inhibition of CGRP-induced cranial vasodilatation. We recently showed that the novel selective CGRP receptor antagonist, BIBN4096BS (1-piperidinecarboxamide, -N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [[R-(R,(R*,S*)]), attenuated the CGRP-induced porcine carotid vasodilatation in a model predictive of antimigraine activity. In order to evaluate the potential safety of BIBN4096BS in migraine therapy, this study was designed to investigate the effects of intravenous BIBN4096BS on alpha-CGRP-induced systemic and regional haemodynamic changes in anaesthetised rats, using radioactive microspheres. In vehicle-pretreated animals, consecutive intravenous infusions of alpha-CGRP (0.25, 0.5 and 1 microg kg(-1) min.(-1)) dose-dependently decreased mean arterial blood pressure with an accompanying increase in heart rate and systemic vascular conductance whereas cardiac output remained unchanged. Alpha-CGRP also increased the vascular conductance to the heart, brain, gastrointestinal tract, adrenals, skeletal muscles and skin, whilst that to the kidneys, spleen, mesentery/pancreas and liver remained unaltered. The above systemic and regional haemodynamic responses to alpha-CGRP were clearly attenuated in BIBN4096BS (3 mg kg(-1) intravenously)-pretreated animals. These results indicate that exogenously administered alpha-CGRP dilates regional vascular beds via CGRP receptors on the basis of the antagonism produced by BIBN4096BS. Moreover, the fact that BIBN4096BS did not alter baseline haemodynamics suggests that endogenously produced CGRP does not play an important role in regulating the systemic and regional haemodynamics under resting conditions.     

The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS blocks CGRP and adrenomedullin vasoactive responses in the microvasculature

Calcitonin gene-related peptide (CGRP) is a potent microvascular dilator neuropeptide that is considered to play an essential role in neurogenic vasodilatation and in maintaining functional integrity in peripheral tissues. We have examined the effect of the nonpeptide CGRP antagonist BIBN4096BS on responses to CGRP and the structurally related peptide adrenomedullin, AM, in murine isolated aorta and mesentery preparations, and in the cutaneous microvasculature in vivo. We show for the first time that BIBN4096BS is an effective antagonist of CGRP and AM responses in the murine mesenteric and cutaneous microvasculature, and of CGRP in the murine aorta. After local administration, BIBN4096BS selectively inhibits the potentiation of microvascular permeability in the cutaneous microvasculature by CGRP and AM, with no effect on responses induced by other microvascular vasodilators. BIBN4096BS reversed both newly developed and established vasoactive responses induced by CGRP. The ability of CGRP to potentiate plasma extravasation was lost when coinjected with compound 48/80 (where mast cells would be activated to release proteases), but regained when soybean trypsin inhibitor was coinjected with compound 48/80. These results demonstrate that BIBN4096BS is a selective antagonist of responses induced by CGRP and AM in the mouse microvasculature, and CGRP in the mouse aorta. The ability of BIBN4096BS to block an established CGRP microvascular vasodilatation indicates that the sustained vasodilator activity of CGRP is due to the retention of the active intact peptide and the continued involvement of the CGRP receptor.     

Determinants of 1-piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) affinity for calcitonin gene-related peptide and amylin receptors--the role of receptor activity modifying protein 1

1-Piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS), a calcitonin gene-related peptide (CGRP) receptor antagonist, can alleviate the symptoms of migraine and is highly selective for CGRP over adrenomedullin (AM) receptors. These receptors are heterodimers of the calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs), with the pharmacological properties determined by the RAMP subunit. BIBN4096BS-sensitive CGRP(1) receptors are CL/RAMP1, whereas BIBN4096BS-insensitive AM receptors are CL/RAMP2 or CL/RAMP3 (AM(1) and AM(2), respectively), implicating RAMP1 in conferring BIB-N4096BS sensitivity. Because calcitonin receptors [CT((a))] also interact with RAMP1 [AMY(1(a)) receptors], BIBN4096BS could also have affinity for these receptors. To test this, receptors were transfected into COS-7 cells and agonist-stimulated cAMP levels measured in the presence and absence of antagonists. We found that AMY(1(a)) receptors were approximately 150-fold less sensitive to BIBN4096BS antagonism than CGRP(1) receptors. In contrast, AMY(3(a)) [CT((a))/RAMP3] or AM(2) receptors were not sensitive to BIBN4096BS antagonism. We investigated Trp74 in RAMP1, a residue implicated in the species selectivity of BIBN4096BS. BIBN4096BS affinity was reduced at AMY(1(a)) and CGRP(1) receptors when this residue was mutated to lysine or alanine. The equivalent residue in RAMP3, Glu74, when mutated to tryptophan (E74W), induced BIBN4096BS sensitivity at AM(2) and AMY(3(a)) receptors. It is interesting that a selective reduction in AM potency was observed at E74W AM(2) receptors, implicating this residue in AM interactions with this receptor. These data support the importance of Trp74 in RAMP1 in the interaction of BIBN4096BS with CGRP(1) and AMY(1(a)) receptors and identified Glu74 in RAMP3 as the first amino acid in RAMP important for agonist interactions with calcitonin-family receptors.     

Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)(2, 7)]hCGRP-preferring receptor

The present study was performed in order to characterise calcitonin gene-related peptide (CGRP) receptor subtypes in rat left atrium and vas deferens by using [R-(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide (BIBN4096BS), a novel CGRP receptor antagonist. When CGRP was used as an agonist, BIBN4096BS exhibited an almost 10-fold higher affinity for CGRP receptors in rat left atrium compared to those in the vas deferens, indicating that CGRP acts through different CGRP receptor subtypes in these two tissues. In addition, BIBN4096BS was almost 10-fold more potent in antagonizing [Cys(Et)^2,7]hCGRP and human adrenomedullin-induced responses than CGRP-induced responses in rat vas deferens. This might indicate receptor heterogeneity in rat vas deferens. Accordingly, the present work provides first experimental evidence that the rat vas deferens contains two CGRP-like receptor subtypes. Namely, the CGRP₂ receptor and a “novel” receptor that possesses low efficacy for CGRP and that is selectively stimulated by [Cys(Et)^2,7]hCGRP or adrenomedullin and which can be blocked with high affinity by BIBN4096BS.