European Journal of Pharmacology 475 (2003) 69 – 77
`
`www.elsevier.com/locate/ejphar
`
`Effects of BIBN4096BS on cardiac output distribution and on
`CGRP-induced carotid haemodynamic responses in the pig
`
`Kapil Kapoor a, Udayasankar Arulmani a, Jan P.C. Heiligers a, Edwin W. Willems a,
`Henri Doods b, Carlos M. Villalo´n c, Pramod R. Saxena a,*
`
`a Department of Pharmacology, Erasmus MC, University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands
`b Boehringer Ingelheim Pharma KG, Biberach, Germany
`c Departamento de Farmacobiologı´a, CINVESTAV-IPN, Czda. de los Tenorios 235, Col. Granjas-Coapa, 14330 Mexico D.F., Mexico
`
`Received 17 April 2003; received in revised form 3 July 2003; accepted 8 July 2003
`
`Abstract
`
`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 a-CGRP in anaesthetised pigs. Treatment with BIBN4096BS (100, 300 and 1000 Ag 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 a-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.
`D 2003 Elsevier B.V. All rigths reserved.
`
`Keywords: Arteriovenous anastomosis; BIBN4096BS; Carotid vasodilatation; Cardiac output; CGRP (calcitonin gene-related peptide); a-CGRP human;
`Migraine; (Pig)
`
`1. Introduction
`
`Calcitonin gene related peptide (CGRP), a 37 amino acid
`neuropeptide generated by alternative splicing of the calci-
`tonin gene (Amara et al., 1982), is widely distributed in the
`body, including in trigeminal sensory nerve fibres innervat-
`ing central and peripheral blood vessels, where it is co-
`localised with other vasoactive neuropeptides, such as
`substance P and neurokinin A (Gulbenkian et al., 1995,
`2001). CGRP is a potent vasodilator agent in a wide variety
`of tissues (Brain et al., 1985; Juaneda et al., 2000; Poyner
`
`* Corresponding author. Tel.: +31-10-408-75-37; fax: +31-10-408-94-
`
`58.
`
`E-mail address: p.saxena@erasmusmc.nl (P.R. Saxena).
`URL: http://www.eur.nl/fgg/pharm/.
`
`0014-2999/$ - see front matter D 2003 Elsevier B.V. All rigths reserved.
`doi:10.1016/S0014-2999(03)02082-X
`
`and Marshall, 2001; Van Rossum et al., 1997) and, although
`exogenous a-CGRP has potent systemic and regional hae-
`modynamic effects (Gardiner et al., 1990), the physiological
`role of endogenous CGRP is not clear (Shen et al., 2001).
`This is mainly due to the unavailability of potent and
`selective CGRP receptor antagonists; the most widely used
`CGRP receptor antagonist thus far, CGRP-(8 – 37) is not
`very potent and displays partial agonist properties (Waugh et
`al., 1999; Wisskirchen et al., 1998). Clearly, the advent of
`‘silent’, selective and potent non-peptide CGRP receptor
`antagonists would be valuable in this regard.
`Interestingly, CGRP has been implicated in the patho-
`genesis of migraine (Ashina et al., 2000; Durham and
`Russo, 2002; Edvinsson, 2001; Goadsby et al., 1990), and
`it can mediate neurogenic dilatation of cranial blood vessels
`as well as sensory nerve transmission between the first
`
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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`and second order afferent input from these vessels during
`migraine headache (Goadsby et al., 2002; Gulbenkian et al.,
`2001; Smith et al., 2002; Williamson and Hargreaves,
`2001). Significantly, plasma levels of CGRP, but not of
`other neurotransmitter (e.g. neuropeptide Y, vasoactive
`intestinal peptide or substance P), are elevated during
`migraine and, after sumatriptan, these levels are normalised
`paralleling the resolution of headache (Goadsby, 1999;
`Goadsby et al., 1990). Therefore, inhibition of a-CGRP
`release or blockade of a-CGRP-induced vasodilatation may
`be a novel approach in the management of acute migraine
`headache.
`Doods et al. (2000) have recently described a small
`molecule CGRP receptor antagonist, BIBN4096BS (1-
`piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-py-
`ridinyl ) - 1 - pipera - zinyl ] carbonyl ] pentyl ]
`amino] - 1 - [ ( 3,
`5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-
`dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-), which
`possesses over 200 fold higher affinity for human (SK-N-
`MC cells; Ki: 14 pM) than for rat (spleen; Ki: 3.4 nM) CGRP
`receptors. BIBN4096BS as well as the endogenous ligand
`CGRP and its analogues concentration dependently displaces
`[3H]BIBN4096BS from SK-N-MC cell membranes with the
`rank order of affinity: BIBN4096BS>human a-CGRP = hu-
`man h-CGRP>[Cys(Et)2,7] human a-CGRP>adrenomedul-
`lin (high affinity site) = human a-CGRP8 – 37 = human h-
`CGRP8 – 37Hcalcitonin = amylin (Schindler and Doods,
`2002). The compound inhibits vasodilatation evoked by
`trigeminal ganglion stimulation in marmosets (Doods et al.,
`2000) and by CGRP in several human isolated blood vessels
`(Edvinsson et al., 2002; Moreno et al., 2002; Verheggen et al.,
`2002). The purpose of the present study in anaesthetised
`pigs was to investigate the effects of BIBN4096BS on: (i)
`the complete distribution of cardiac output to assess the
`potential role of endogenous CGRP in regulating basal
`vascular tone and thereby the cardiovascular safety of
`BIBN4096BS, and (ii) the haemodynamic responses pro-
`duced by intracarotid arterial (i.c.) infusion of a-CGRP in a
`model predictive of antimigraine activity (De Vries et al.,
`1999; Saxena, 1995).
`
`2. Materials and methods
`
`2.1. General
`
`After an overnight fast, 25 domestic pigs (Yorkshire x
`Landrace, females, 10 – 14 kg) were sedated with intramus-
`cular injections of azaperone (120 mg) and midazolam
`hydrochloride (10 mg) and then anaesthetised with sodium
`pentobarbital (600 mg, i.v.). After tracheal intubation, the
`animals were connected to a respirator
`(BEAR 2E,
`BeMeds, Baar, Switzerland) for intermittent positive pres-
`sure ventilation with a mixture of room air and oxygen.
`Respiratory rate,
`tidal volume and oxygen supply were
`adjusted to keep arterial blood gas values within physio-
`
`logical limits (pH: 7.35 – 7.48; pCO2: 35 – 48 mm Hg; pO2:
`100 – 120 mm Hg). Anaesthesia was maintained with a
`continuous i.v. infusion of sodium pentobarbital (12 – 20
`mg kg 1 h 1). Heart rate was measured with a tachograph
`(CRW, Erasmus University, Rotterdam, The Netherlands)
`triggered by electrocardiogram signals. A catheter was
`placed in the inferior vena cava via the right femoral vein
`for the administration of vehicle and BIBN4096BS. An-
`other catheter was placed in the aortic arch via the left
`femoral artery for the measurement of arterial blood
`pressure (Combitrans disposable pressure transducer; Braun
`Melsungen, Germany) and arterial blood withdrawal for the
`measurement of blood gases (ABL-510; Radiometer,
`Copenhagen, Denmark). During the experiment, body
`temperature was kept around 37 jC and the animal was
`continuously infused with physiological saline to compen-
`sate for fluid losses.
`Heart rate and systolic, diastolic and mean arterial blood
`pressure as well as the pulsatile and mean carotid artery
`blood flows (see later) were continuously monitored on a
`polygraph (CRW).
`
`2.2. Cardiac output and its distribution
`
`Cardiac output was measured by the thermodilution
`method using a 6F Swan – Ganz catheter (Braun Melsun-
`gen) introduced into the pulmonary artery via the left
`femoral vein.
`The distribution of cardiac output was determined with
`15.5 F 0.1 (S.D.) Am diameter microspheres labelled with
`141Ce, 113Sn, 103Ru, 95Nb or 46Sc (NEN Dupont, Boston,
`USA). For each measurement, a suspension of about
`1,000,000 microspheres, labelled with one of the isotopes,
`was injected into the left ventricle via a catheter guided by
`way of the left carotid artery. Starting 15 s before micro-
`sphere injection and lasting 70 s, a reference arterial blood
`sample was withdrawn (Withdrawal pump, Harvard Appa-
`ratus, Southnatick, Mass, USA; rate: 6 ml min 1) via a
`catheter placed into the right femoral artery. An infusion of
`the corresponding volume of Haemaccel compensated blood
`loss during this procedure.
`At the end of the experiment, the animal was killed
`using an overdose of pentobarbital. Subsequently, a number
`of tissues (lungs, kidneys, heart, stomach, small intestine,
`spleen, liver, adrenals, brain, skin and skeletal muscles)
`were dissected out, weighed and put
`into vials. The
`radioactivity in these vials was counted for 5 min in a
`g-scintillation counter (Packard, Minaxi autogamma 5000)
`using suitable windows for the discrimination of the
`different isotopes (141Ce: 120 – 167 KeV, 113Sn: 355 – 435
`KeV, 103Ru: 450 – 548 KeV, 95Nb: 706 – 829 KeV and 46Sc:
`830 – 965 KeV). All data were processed by a set of
`specially designed computer programs (Saxena et al.,
`1980) using a personal computer. Tissue blood flows were
`calculated by multiplying the ratio of tissue and reference
`blood sample radioactivities by the blood withdrawal rate
`
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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`71
`
`(6 ml min 1) and normalised to 100 g of tissue weight.
`Systemic and tissue vascular conductances were calculated
`by dividing cardiac output (ml min 1) and tissue blood
`flows (ml.min 1/100 g tissue), respectively, by mean
`arterial blood pressure (mm Hg). Radioactivity in the lungs
`mainly represents peripheral arteriovenous anastomotic
`blood flow (the non-nutrient part of the cardiac output),
`although a small part (1 – 1.5% of cardiac output) is derived
`from the bronchial arteries (Baile et al., 1982).
`
`2.3. Carotid haemodynamic responses to CGRP
`
`Both common carotid arteries and the external jugular
`veins were dissected free and the accompanying vagosym-
`pathetic trunks were cut between two ligatures in order to
`prevent a possible influence of CGRP via baroreceptor
`reflexes. Pulsatile and mean blood flows were measured
`in the right common carotid artery with a flow probe
`(internal diameter: 2.5 mm) connected to a sine-wave
`electromagnetic flow meter (Transflow 601-system, Skalar,
`Delft, The Netherlands). The amplitude of carotid blood
`flow signals provided an index of carotid flow pulse.
`Carotid vascular conductance was calculated by dividing
`the carotid blood flow (ml min 1) by the mean arterial
`blood pressure (mm Hg).
`The right external
`jugular vein was catheterised for
`obtaining jugular venous blood samples to determine blood
`gases. Two hub-less needles, connected to polyethylene
`tubes, were inserted into the right common carotid artery
`and used for intracarotid (i.c.) infusions of phenylephrine
`(a1-adrenoceptor agonist) and a-CGRP, respectively. It
`should be noted that under pentobarbital anaesthesia, ca-
`rotid arteriovenous anastomoses are dilated (Den Boer et
`al., 1993) and, therefore, to elicit vasodilator responses to
`CGRP, a continuous infusion of phenylephrine was used
`throughout the experiment. We have previously reported
`that phenylephrine decreases total carotid blood flow and
`conductance exclusively due to constriction of carotid
`arteriovenous anastomoses (Willems et al., 1999), resulting
`in an increase in the difference between arterial and jugular
`venous oxygen saturations (A-V SO2 difference) (Saxena,
`1987).
`
`2.4. Experimental protocols
`
`In the case of cardiac output distribution experiments
`(n = 12), baseline values of heart rate, mean arterial blood
`pressure, cardiac output and its distribution to the various
`tissues (see above) were determined after a stabilisation
`period of at least 90 min. The animals were then divided
`into two groups (n = 6 each) receiving three i.v. infusions
`(rate: 0.5 ml min 1) of either BIBN4096BS (100, 300 and
`1000 Ag kg 1) or its vehicle (5 ml of acidified distilled
`water); each dose was given over 10 min with an inter-
`vening period of 10 min before the next dose. At the end
`of each infusion,
`the above-mentioned haemodynamic
`
`variables were collated again. Lastly, the final measure-
`ments were made 40 min after the third dose of vehicle or
`BIBN4096BS (recovery).
`In the case of the carotid artery experiments (n = 13),
`phenylephrine (10 Ag kg 1 min 1 for 10 min, followed by
`3 – 6 Ag kg 1 min 1 throughout the rest of the experiment)
`was infused into the right common carotid artery to maintain
`carotid blood flow at a constant low level. After a stabilisa-
`tion period of at least 90 min, values of heart rate, arterial
`blood pressure,
`total carotid blood flow and A-V SO2
`difference were collated. The animal was then given three
`sequential i.c. infusions (rate: 0.083 – 1 ml min 1, depend-
`ing on the weight of the animal) of CGRP (10, 30 and 100
`pmol kg 1 min 1) for 3 min and the above variables
`(except the A-V SO2 difference, which was determined
`only after the highest dose) were collated again. After the
`highest dose of a-CGRP, a recovery period of 20 min was
`allowed to elapse when all haemodynamic parameters
`returned to baseline levels. At this point, the animals were
`divided into two groups receiving three i.v. infusions (rate:
`0.5 ml min 1) of either BIBN4096BS (100, 300 and 1000
`Ag kg 1; n = 7) or its vehicle (5 ml of acidified distilled
`water; n = 6); each dose was given over a period of 10 min
`with an intervening period of about 10 min before the
`next dose. Ten minutes after each treatment, the values of
`mean arterial blood pressure, heart rate, total carotid blood
`flow and A-V SO2 difference were collated. CGRP was
`infused as above after each treatment and data were colla-
`ted again.
`It may be mentioned that the vehicle of a-CGRP (dis-
`tilled water) was devoid of any systemic and carotid
`haemodynamic responses (data not shown).
`
`2.5. Data presentation and statistical analysis
`
`All data have been expressed as mean F S.E.M., unless
`stated otherwise. The significance of changes from baseline
`values within one group (vehicle or BIBN4096BS) was
`evaluated with Duncan’s new multiple range test, once
`an analysis of variance (randomised block design) had
`revealed that the samples represented different populations
`(Saxena et al., 1980; Steel and Torrie, 1980). The differen-
`ces in baseline haemodynamic values and percent change
`(from baseline values) in haemodynamic variables by
`corresponding doses of the vehicle and BIBN4096BS (be-
`tween group comparisons) were evaluated by Student’s
`unpaired t-test. Student’s unpaired t-test was also applied
`to compare the changes in the effects of CGRP observed
`after different corresponding doses of the vehicle and
`BIBN4096BS. Statistical significance was accepted at
`P < 0.05 (two-tailed).
`
`2.6. Ethical approval
`
`The Ethics Committee of the Erasmus MC, Rotterdam,
`dealing with the use of animals in scientific experiments,
`
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`

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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`approved investigation protocols, which adhere to EEC
`guidelines.
`
`2.7. Compounds
`
`The following compounds were used: azaperone
`(StresnilR; Janssen Pharmaceuticals, Beerse, Belgium),
`BIBN4096BS and human a-CGRP (Boehringer Ingelheim
`Pharma, Biberach, Germany); heparin sodium (to prevent
`blood clotting in catheters; Leo Pharmaceutical Products,
`Weesp, The Netherlands), midazolam hydrochloride
`(DormicumR; Hoffmann La Roche b.v., Mijdrecht, The
`Netherlands), phenylephrine hydrochloride (Sigma-Aldrich
`Chemie b.v., Zwijndrecht, The Netherlands) and sodium
`pentobarbital (Sanofi Sante b.v., Maasluis, The Netherlands).
`Phenylephrine and a-CGRP were dissolved in distilled
`water, while BIBN4096BS was initially dissolved in 0.5 ml
`of 1 N HCl and subsequently diluted with 4 ml of distilled
`water, and then adjusted to pH 6.5 with 1 N NaOH.
`
`3. Results
`
`3.1. Effect of BIBN4096BS on cardiac output and its
`distribution
`
`3.1.1. Baseline values
`Baseline values of heart rate, mean arterial blood pres-
`sure, cardiac output (expressed as cardiac index) and sys-
`temic vascular conductance in anaesthetised pigs (n = 12)
`were: 108 F 3 beats min 1, 102 F 2 mm Hg, 133 F 4 ml
`min 1 kg 1 and 1491 F 54 ml min 1 mm Hg 1, respec-
`tively. Baseline values of regional vascular conductances (ml
`
`Fig. 1. Heart rate (HR), mean arterial blood pressure (MAP), cardiac index
`(CI) and systemic vascular conductance (SVC) measured at baseline, after
`i.v.
`treatments with either vehicle (V,
`three times 5 ml; n = 6) or
`BIBN4096BS (BIBN; 100, 300 and 1000 Ag kg 1; n = 7) and after
`40 min of recovery. All values are presented as mean F S.E.M. *P < 0.05
`vs. baseline. The changes after BIBN4096BS are not significantly different
`from those in the corresponding vehicle group.
`
`i.v.
`Fig. 2. Regional vascular conductances at baseline (Bas), after
`treatments with either vehicle (V, three times 5 ml; n = 6) or BIBN4096BS
`(100, 300 and 1000 Ag kg 1, i.v.; n = 6) and after 40 min of recovery (Rec).
`All values are presented as mean F S.E.M. *P < 0.05 vs. baseline. #P < 0.05
`vs. the corresponding change in animals treated with vehicle.
`
`min 1 mm Hg 1/100-g tissue) were: brain, 31 F 3; heart,
`104 F 10; liver, 34 F 8; stomach, 24 F 2; lungs (mainly
`systemic arteriovenous anastomoses), 229 F 37; adrenals,
`138 F 10; kidneys, 263 F 14; spleen, 126 F 15; skeletal
`muscles, 3.3 F 0.3; and skin, 11 F 2.
`
`3.1.2. Systemic and regional haemodynamic changes
`Systemic haemodynamic values collated at baseline,
`after vehicle or BIBN4096BS (100, 300 and 1000 Ag
`kg 1, i.v.) and after a 40-min recovery period, are shown
`in Fig. 1. There were no statistically significant differences
`( P>0.05) in baseline values in the vehicle and BIBN4096BS
`groups. Except for small decreases in heart rate by the
`vehicle (maximum change: 4 F 1%) and cardiac index by
`BIBN4096BS (maximum change: 19 F 8%), no other
`changes were observed. The changes in cardiac index by
`BIBN4096BS did not differ significantly ( P>0.05) from
`those in the vehicle-treated animals (maximum change:
`7 F 3%).
`Fig. 2 presents the regional vascular conductances in a
`number of tissues in animals treated with either vehicle or
`BIBN4096BS (100, 300 and 1000 Ag kg 1, i.v.). Baseline
`values in the two groups were not significantly different
`( P>0.05) in any of the tissues, including the liver, lungs and
`skin. Apart from decreases in liver conductance, no other
`
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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`73
`
`changes in regional vascular conductances were noticed in
`the vehicle-treated group. BIBN4096BS produced small
`decreases in vascular conductance to liver, and with the
`highest dose (1000 Ag kg 1) in lungs, adrenals, kidneys and
`spleen. Only the latter changes were significant when
`compared with the corresponding changes in the vehicle-
`treated animals.
`
`3.2. Effect of BIBN4096BS on the haemodynamic responses
`to i.c. infusions of a-CGRP
`
`3.2.1. Baseline values
`Baseline values in anaesthetised pigs (n = 13) were: heart
`rate, 129 F 5 beats min 1; mean arterial blood pressure,
`122 F 4 mm Hg; carotid flow pulse, 1.7 F 0.1 arbitrary units
`total carotid blood flow, 67 F 7 ml min 1;
`total
`(a.u.);
`carotid vascular conductance, 56 F 5 10 2 ml min 1 mm
`Hg 1; and A-V SO2 difference, 26 F 3%. Baseline values
`in the two groups of animals (vehicle and BIBN4096BS)
`did not differ significantly.
`
`3.2.2. Systemic and carotid haemodynamic responses
`Fig. 3 shows the original tracings illustrating the sys-
`temic (blood pressure and heart rate) and carotid (flow
`pulse and total carotid blood flow) haemodynamic
`responses in anaesthetised pigs obtained with a-CGRP
`(10, 30 and 100 pmol kg 1 min 1, i.c.) before and after
`i.v. treatments with three doses of vehicle (5 ml each time;
`upper panel) or BIBN4096BS (100, 300 and 1000 Ag kg 1;
`lower panel). The infusions of a-CGRP did not affect the
`
`heart rate, but decreased the arterial blood pressure and
`increased the carotid flow pulse and blood flow. These
`changes were accompanied by redness of head skin and
`ears on the side of infusion (not shown in the figure). The
`effects of a-CGRP were clearly attenuated in the animals
`receiving BIBN4096BS, but not in the ones treated with
`vehicle.
`The effects of a-CGRP (10, 30 and 100 pmol kg 1
`min 1, i.c.) in the animals treated with vehicle or BIBN
`4096BS (100, 300 and 1000 Ag kg 1, i.v.) were quantified as
`percent changes from baseline values (Fig. 4). In both groups,
`infusions of a-CGRP before treatments with vehicle or
`BIBN4096 (control infusions) produced dose-dependent
`decreases in mean arterial blood pressure and increases in
`total carotid blood flow (data not shown) and conductance;
`heart rate was not affected (data not shown). These responses
`to a-CGRP remained unaffected after vehicle, but, in con-
`trast, were dose-dependently antagonised by BIBN4096BS
`(Fig. 4).
`As shown in Fig. 5, infusions of a-CGRP (100 pmol
`kg 1 min 1,
`i.c.) clearly increased carotid blood flow
`(depicted as the maximum changes) and carotid blood flow
`pulsations (compare baseline and control values). While
`there was little change in animals treated with vehicle,
`BIBN4096BS (100, 300 and 1000 Ag kg 1, i.v.) dose-
`dependently antagonised the responses to a-CGRP.
`
`3.2.3. Changes in the A-V SO2 difference
`a-CGRP (100 pmol kg 1 min 1,
`i.c.) produced a
`significant reduction in the A-V SO2 difference in both
`
`Fig. 3. Original tracings from experiments in anaesthetised pigs illustrating systemic and carotid haemodynamic responses to infusions of a-CGRP (.; 10, 30
`or 100 pmol kg 1 min 1, i.c.) given before and after i.v. treatments with either vehicle (three times 5 ml; upper panel) or BIBN4096BS (BIBN, 100, 300 and
`1000 Ag kg 1; lower panel). BP; systolic and diastolic arterial blood pressures; MAP, mean arterial blood pressure; HR, heart rate; FP, carotid blood flow pulse;
`TCBF, total carotid blood flow.
`
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`

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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`Fig. 4. Changes in mean arterial blood pressure and total carotid vascular
`conductance from baseline values by i.c. infusion of a-CGRP in ana-
`esthetised pigs given before (Control) and after i.v. treatments with vehicle
`(three times 5 ml; n = 6) or BIBN4096BS (100, 300 and 1000 Ag kg 1,
`n = 7). All values are expressed as mean F S.E.M. The two highest doses of
`a-CGRP significantly decreased the mean arterial blood pressure and
`increased the total carotid vascular conductance (significance not shown for
`the sake of clarity). These effects of a-CGRP were dose-dependently
`antagonised by BIBN4096BS. #P < 0.05 vs. response after the correspond-
`ing volume of vehicle.
`
`Fig. 5. Maximum carotid blood flow changes and carotid blood flow
`pulsations measured at baseline and following infusions of a-CGRP
`(100 pmol kg 1 min 1, i.c.) given in anaesthetised pigs before (Control)
`and after i.v.
`treatments with vehicle (V, 5 ml
`three times; n = 6) or
`BIBN4096BS (100, 300 and 1000 Ag kg 1, n = 7). All values are expressed
`as mean F S.E.M. a.u., Arbitrary units. *P < 0.05 vs. baseline values;
`#P < 0.05 vs. response after the corresponding volume of vehicle.
`
`Fig. 6. Differences between arterial and jugular venous oxygen saturations
`(A V SO2 difference) measured at baseline and after infusions of a-CGRP
`(100 pmol kg 1 min 1, i.c.) given in anaesthetised pigs before (Control)
`and after i.v.
`treatments with vehicle (V, 5 ml
`three times; n = 6) or
`BIBN4096BS (100, 300 and 1000 Ag kg 1, n = 7). All values are expressed
`as mean F S.E.M. *P < 0.05 vs. baseline values; #P < 0.05 vs. response
`after the corresponding volume of vehicle.
`
`groups of animals (Fig. 6; compare baseline and control
`values). The response to CGRP remained largely unaffec-
`ted after treatments with vehicle, but BIBN4096BS (100,
`300 and 1000 Ag.kg 1, i.v.) dose-dependently blocked the
`reduction in the A-V SO2 difference by a-CGRP. In fact, the
`CGRP-induced decrease in the A-V SO2 difference was
`enhanced after the highest dose of BIBN4096BS (Fig. 6).
`
`4. Discussion
`
`4.1. General
`
`Undoubtedly, a remarkable progress has been achieved in
`acute antimigraine therapy (De Vries et al., 1996). Notwith-
`standing, the exact pathophysiological mechanisms under-
`lying migraine remain unclear. There is, however, evidence
`supporting the involvement of the trigeminovascular system
`in migraine pathophysiology (Goadsby, 1997, 1999; Har-
`greaves et al., 1999; Williamson and Hargreaves, 2001).
`Thus, activation of the trigeminovascular system leads to
`neuropeptide release, including that of CGRP, and neuro-
`genic dural vasodilatation (Williamson and Hargreaves,
`2001). Of particular relevance is the finding that plasma
`concentration of CGRP is elevated during the headache
`phase of migraine, and this is normalised after treatment
`with sumatriptan (Goadsby, 1997, 1999; Goadsby et al.,
`1990). Hence, it is reasonable to assume that a potent CGRP
`receptor antagonist, such as BIBN4096BS (Doods et al.,
`2000), might be useful in migraine therapy. BIBN4096BS
`behaves as a ‘silent’ competitive antagonist at CGRP
`receptors mediating relaxation of human temporal, cranial
`
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`
`75
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`and coronary arteries (Edvinsson et al., 2002; Moreno et
`al., 2002; Verheggen et al., 2002). The present study in
`anaesthetised pigs was designed: (i) to analyse, using
`BIBN4096BS, the potential role of endogenous CGRP in
`regulating vascular tone in vivo; and (ii) to investigate the
`effects of BIBN4096BS on the systemic and carotid hae-
`modynamic responses produced by a-CGRP.
`
`4.2. Systemic and regional haemodynamic effects of
`BIBN4096BS
`
`It is well known that CGRP-immunoreactive nerve fibres
`are widely distributed in the cardiovascular system, with a
`higher preponderance in arteries than in veins (Bell and
`McDermott, 1996). CGRP decreases blood pressure and
`has positive inotropic and chronotropic effects on the heart
`(Wimalawansa, 1996), which are mainly mediated via
`CGRP1 receptors (Bell and McDermott, 1996; Saetrum
`Opgaard et al., 1999, 2000). Though CGRP has diverse
`biological actions within the cardiovascular system, our
`experiments showing few systemic haemodynamic changes
`with BIBN4096BS do not support a major role for CGRP in
`the regulation of cardiovascular function in anaesthetised pig.
`As far as regional haemodynamics is concerned, a
`moderate decrease (compared to vehicle) in vascular con-
`ductances in the lungs, adrenals, kidneys and spleen was
`observed with the highest dose (1000 Ag kg 1) of
`BIBN4096BS (Fig. 2). Similarly, renal vasoconstriction
`was noticed in conscious rats with a high (300 nmol kg 1
`min 1), but not with a low (30 nmol kg 1 min 1) dose
`of CGRP-(8 – 37) (Gardiner et al., 1990). Since both
`BIBN4096BS and CGRP8 – 37 caused renal changes only
`in doses that were considerably higher than those needed for
`CGRP antagonism,
`it does not appear that endogenous
`CGRP regulates renal vascular tone. Also, Shen et al.
`(2001) recently reported that 30 Ag kg 1 min 1 ( f 10
`nmol kg 1 min 1) of CGRP-(8 – 37), which antagonised
`CGRP-induced haemodynamic responses, caused little re-
`gional haemodynamic effects in conscious dogs as well as
`anaesthetised rats,
`thereby not supporting an important
`physiological role for endogenous CGRP in regulating
`vascular tone. Although we cannot rule out the involvement
`of CGRP in certain other circumstances, for example,
`cardiac preconditioning or coronary artery disease (Lu et
`al., 1999; Peng et al., 2000; Wu et al., 2001), the present
`results imply cardiovascular safety of BIBN4096BS. Nev-
`ertheless, one will have to explore the role of CGRP in
`cardiovascular pathophysiology before establishing whether
`or not CGRP receptor antagonists are completely safe in
`patients afflicted with cardiovascular disorders.
`
`4.3. CGRP-induced haemodynamic responses and antago-
`nism by BIBN4096BS
`
`Activation of CGRP receptors elicits dilatation in differ-
`ent vascular beds in several species (Gardiner et al., 1990;
`
`Shen et al., 2001; Van Gelderen et al., 1995). Consistent
`with these studies, our experiments show that i.c. infusions
`of a-CGRP produced a marked vasodilatation in porcine
`carotid circulation, with accompanying fall in arterial blood
`pressure. The fact
`that
`the animals were systematically
`vagosympathectomised may explain why the hypotension
`was not accompanied by a baroreflex-mediated tachycardia,
`as reported earlier (Van Gelderen et al., 1995). Interestingly,
`the ipsilateral skin redness, together with the marked de-
`indicates that
`crease in A-V SO2 difference by CGRP,
`porcine carotid arteriovenous anastomoses dilated in re-
`sponse to a-CGRP (Saxena, 1987). However, we previously
`reported that i.c. infusions of a-CGRP failed to increase
`porcine arteriovenous anastomotic blood flow, despite a
`marked increase in the total carotid and capillary blood
`flows (Van Gelderen et al., 1995). Admittedly, arteriovenous
`anastomotic blood flow was not directly measured in these
`experiments, but we have recently observed that i.c. infu-
`sions of capsaicin, which released CGRP, did increase
`carotid arteriovenous anastomotic blood flow with a con-
`comitant decrease in the A-V SO2 difference (Kapoor et al.,
`2003). Thus, it appears that the discrepancy between the two
`investigations may be due to different anaesthetic regimens
`employed (pentobarbital and fentanyl/thiopental, respective-
`ly) and, particularly, the use of phenylephrine in the present
`experiments. Phenylephrine potently constricts arteriove-
`nous anastomoses (Willems et al., 1999).
`In the present experimental study in anaesthetised pigs,
`BIBN4096BS proved to be an effective antagonist at the
`CGRP receptors mediating the systemic (hypotension) as
`well as the carotid (increased blood flow, pulsations and
`skin redness) haemodynamic responses to a-CGRP. The fact
`that BIBN4096BS also abolished a-CGRP-induced
`decreases in the A-V SO2 difference suggests its action on
`carotid arteriovenous anastomoses; for further considera-
`tions, see Saxena (1987). Interestingly, BIBN4096BS also
`antagonised the capsaicin-induced increases in carotid arte-
`riovenous anastomotic blood flow as well as decreases in
`the A-V SO2 difference, but not the plasma CGRP concen-
`trations (Kapoor et al., 2003).
`One cannot be certain about the nature of CGRP recep-
`tors that mediate porcine carotid vascular responses, but
`cardiac inotropic and vasodilator responses are mediated
`predominantly by CGRP1 receptors (Saetrum Opgaard et al.,
`1999), where BIBN4096BS has a very high affinity (Doods
`et al., 2000; Poyner and Marshall, 2001).
`
`4.4. Potential therapeutic efficacy of BIBN4096BS in the
`treatment of migraine
`
`Considering that plasma CGRP levels are elevated
`during the headache phase of migraine (Goadsby, 1997)
`and that BIBN4096BS dose-dependently blocked a-CGRP-
`induced carotid haemodynamic responses, it is likely that
`BIBN4096BS may be effective in migraine. The compound
`is presently under clinical investigation for the acute treat-
`
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`

`76
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`K. Kapoor et al. / European Journal of Pharmacology 475 (2003) 69–77
`
`ment of migraine and the results are awaited with great
`interest.
`In conclusion, our study clearly demonstrates that
`BIBN4096BS is an effective antagonist at vascular CGRP
`receptors in anaesthetised pigs, but has little haemodynamic
`effects of its own, a finding that negates a major physio-
`logical role for CGRP in cardiovascular regulation. The
`potent blockade of the carotid haemodynamic effects of
`CGRP does suggest that BIBN4096BS may be effective in
`migraine treatment.
`
`Acknowledgements
`
`This study was partly supported from funds obtained
`from Boehringer Ingelheim Pharma (Biberach, Germany)
`for a contract research project with Erasmus Pharma.
`
`References
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`Alternative RNA processing in calcitonin gene express