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IBN4096BS antagonizes human-calcitonin gene related peptide–inducedeadache and extracerebral artery dilatation

Background and Objective: Calcitonin gene–related peptide (CGRP) plays a pivotal role in migraine pathogenesis.BIBN4096BS is the first CGRP receptor antagonist available for human studies, and its efficacy in the acutetreatment of migraine has been demonstrated. We investigated the ability of BIBN4096BS to inhibit human�CGRP (h-�CGRP)–induced headache and cerebral hemodynamic changes in healthy volunteers.Methods: Ten healthy volunteers completed this double-blind, placebo-controlled crossover study with2.5 mg BIBN4096BS and placebo as pretreatments before a 20-minute intravenous infusion of h-�CGRP(1.5 �g/min). Transcranial Doppler ultrasonography was used to measure blood flow velocity in the middlecerebral artery (MCA); regional and global cerebral blood flow (CBF) was measured by xenon 133 inhalationsingle-photon emission computed tomography. The temporal and radial artery diameter was measured byhigh-frequency ultrasound. Systemic hemodynamics, end-tidal partial pressure of carbon dioxide (PETCO2),and headache were monitored.Results: Of the 10 volunteers, 6 had a CGRP-induced headache during the in-hospital phase after placebopretreatment but none after BIBN4096BS (P � .031). BIBN4096BS did not affect changes in the diameterof the MCA or changes in CBF induced by h-�CGRP. Vasodilatation of the extracranial arteries was,however, significantly inhibited (P < .001 for temporal artery and P � .001 for radial artery).Conclusions: These results show that BIBN4096BS effectively prevents CGRP-induced headache and extra-cerebral vasodilatation but does not significantly affect the induced cerebral hemodynamic changes. (ClinPharmacol Ther 2005;77:202-13.)

Kenneth A. Petersen, MD, Lisbeth H. Lassen, PhD, Steffen Birk, PhD,
Lynna Lesko, PhD, and Jes Olesen, DMSc Glostrup, Denmark, and Ridgefield, Conn
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Calcitonin gene–related peptide (CGRP) is a neu-opeptide found in the perivascular nerve terminalsurrounding arteries.1 A measurable concentration ofGRP is circulating in the blood at rest,2 and CGRP

rom the Danish Headache Center, University of Copenhagen, andDepartment of Neurology, Glostrup University Hospital, Glostrup,and Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield.

oehringer Ingelheim sponsored the study and providedBIBN4096BS. The authors were independently responsible for thestudy design, data analysis, and manuscript. The technical equip-ment used was partly sponsored by the Villum Kann RasmussenFoundation, Toyota Foundation, and Simon Fougner HartmannFoundation. The Lundbeck Foundation funds the research of theDanish Headache Center.

eceived for publication June 29, 2004; accepted Oct 6, 2004.eprint requests: Kenneth A. Petersen, MD, Danish Headache Center,University of Copenhagen and Department of Neurology, GlostrupUniversity Hospital, KAS Glostrup, DK-2600 Glostrup, Denmark.

-mail: [email protected]/$30.00opyright © 2005 by the American Society for Clinical Pharmacologyand Therapeutics.

Boi:10.1016/j.clpt.2004.10.001

02

eceptors are localized throughout the body.3 Cerebralnd other cephalic arteries have a particularly richnnervation of CGRP-containing afferent trigeminalerve fibers, and these arteries, as studied in tissueaths, are particularly sensitive to CGRP.4 CGRP isound in an increased concentration in external jugularenous blood but not in blood from the cubital veinuring a migraine attack.5 After infusion in patientsith migraine, CGRP caused a migraine-like headache

nd in some a genuine migraine attack with associatedymptoms that were indistinguishable from the pa-ients’ normal migraine attacks.6 Peptide antagonists ofGRP receptors have been available for experimental

tudies for several years, but previously available com-ounds have not been tested for safety and are, there-ore, not suitable for human clinical studies. One CGRPeceptor antagonist, BIBN4096BS, has been developedith the purpose of treating acute migraine, and a phase

I study has provided proof of efficacy.7 Although
IBN4096BS potently interacts with the human CGRP

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CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(3):202-13 CGRP receptor antagonism and vascular headache 203

eceptor in vitro,8,9 no information is available on itsbility to inhibit CGRP changes in human volunteers atncreased levels of the peptide as seen during a mi-raine attack.5

We, therefore, decided to conduct a placebo-ontrolled, double-blind crossover experiment, inhich BIBN4096BS and placebo in random order weresed as pretreatment followed by infusion of humanCGRP (h-�CGRP). The aims of this study were toalidate the ability of h-�CGRP to provoke headachend to describe its effect on cerebral, extracerebral, andystemic circulatory parameters. Furthermore, we ana-yzed whether BIBN4096BS could partly or fully blockhe changes evoked by h-�CGRP. It was our hope thathe study could thus contribute to a better understand-ng of the role of CGRP in neurovascular headache andhe site of action of this new antimigraine compound.

ETHODSDesign and participants. This was a placebo-

ontrolled, double-blind crossover study that included1 healthy subjects (7 men and 4 women). One femalearticipant had claustrophobia that developed duringaseline single-photon emission computed tomographySPECT) scanning and was excluded before any trialedication was given. Ten participants completed both

reatment days. The participants were aged 24 to 31ears (mean, 26.5 years) and weighed 68 to 89.4 kgmean, 77.4 kg). The participants had no current orrevious cardiovascular, cerebrovascular, endocrine, oreurologic disorder, including no migraine, hypoten-ion, or hypertension. A frequency of tension-typeeadache of 4 d/mo or lower was accepted. On the dayf enrollment, physical and neurologic examination,lectrocardiography, and blood sampling were done.

The healthy volunteers were randomized to receiveither 2.5 mg BIBN4096BS or placebo (xylitol 5%) asn intravenously administered pretreatment of 10 min-tes’ duration. After a free interval of 10 minutes,.5 �g/min h-�CGRP was administered continuouslyor 20 minutes on both trial days. The 2 trial days wereeparated by at least 1 week.

Boehringer Ingelheim GmbH supplied BIBN4096BSnd performed the randomization and blinding, whichas balanced (ClinPro, version 6; Clinical Systems,

nc, Garden City, NY). The dose effective in the treat-ent of acute migraine attacks was used.7

Human-�CGRP was purchased from Clinalfa AG,äufelfingen, Switzerland. In a study performed previ-usly, we used a dose of 2 �g/min.6 This dose, how-ver, induced pronounced hypotension that in 2 patients
ecessitated premature termination of the infusion. In m
he current study we, therefore, used the lower dose of.5 �g/min.

All participants gave written informed consent be-ore randomization. The Ethical Committee of Copen-agen (KA00079gs) and the Danish Medicines Agency2612-1376) approved the study, which was conductedn accordance with the Helsinki II Declaration and theuidelines for Good Clinical Practice.10

Recording of adverse events. Every 15th minuterom time (T) zero (T0) (baseline) to T240 (end of studyeriod), the volunteers were questioned regarding theresence of adverse events (AEs) and rated headache.etween questionings, the participants self-reportedny changes that they might have. The intensity of theEs was graded as mild, moderate, or severe, and their

elationship to study medication was classified as re-ated or not related by the investigator. Headache in-ensity was scored on an 11-point verbal rating scaleith 0 indicating no headache; 1 indicating a feeling of

he occurrence of something unusual inside the head,ot necessarily actual pain; 5 indicating headache ofedium severity; and 10 indicating worst imaginable

eadache. Accompanying symptoms were recorded ac-ording to the International Headache Classification.11

uring the study period, the investigator recorded theEs. After discharge, the volunteers made an hourly

ecording of AEs up to 24 hours after the infusion oflacebo or BIBN4096BS.Cerebral blood flow measurements. Global and re-

ional cerebral blood flow (CBF) was measured withenon 133 inhalation and SPECT with a brain-edicated camera (Ceraspect; DSI, Waltham, Mass).he apparatus consisted of a stationary annular sodium

odide crystal and a fast-rotating collimator system.ach rotation took 10 seconds, thereby acquiring 1

rame in a 30-frame dynamic protocol of 133Xe inha-ation, with 3 background, 9 wash-in, and 18 wash-outrames by use of the Kanno-Lassen algorithm.12 Ahotoelectric window of 70 to 100 keV was used.Thirty-two slices were reconstructed in a 64 � 64atrix with each pixel measuring 0.33 � 0.33 cm by

se of a Butterworth one-dimensional filter (cutoff, 1.5;rder, 6). The 32 slices were reduced to sets of 8ransaxial slices generated by adding 4 slices togethero a total slice thickness of 1.32 cm. A correction by usef the Chang algorithm (�m � 0.05 cm) and nosertifact was performed. The output for each pixel washe inhibition constant (Ki) value, and flow values werestimated from these by use of the partition coefficient�) of 0.85 (gray matter).

A Datex Normocap 200 (Dameca, Roedovre, Den-
ark) was used for end-tidal partial pressure of carbon

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CLINICAL PHARMACOLOGY & THERAPEUTICS204 Petersen et al MARCH 2005

ioxide (PETCO2) measurements during the CBF acqui-itions. A Ceratronic XAS SM 32C (Randers, Den-ark) was used for the 133Xe administration. Eacheasurement lasted 5 minutes.Calculations of flow in the perfusion territories of theajor cerebral arteries were performed by fitting of

tandard vascular regions of interest on the 5 rostrallices at 3.6, 5.0, 6.3, 7.6, and 9 cm above the orbito-eatal line. Flow in the territory of the middle cerebral

rtery (MCA) (rCBFMCA) was calculated as a mean ofhe left and right side.

Transcranial Doppler and C-scan. Transcranialoppler (TCD) ultrasonography (2 MHz) (Multi-Dop; DWL, Sipplingen, Germany) was used for the mea-

urement of blood flow velocity. The recordings wereone simultaneously and bilaterally as previously de-cribed but with handheld probes.13 Along the MCA, axed point was found for the measurement. The fixedoint was chosen as close as possible to the bifurcationf the anterior cerebral artery and MCA. The same fixoint was used for each individual and for each record-ng, for which the signal was optimized. On the basis ofhe envelope curve (the spectral TCD curve), a timeveraged mean (Vmean) over approximately 4 cardiacycles or 4 seconds was calculated by the built-inoftware (version 7.40x of MDX TCD-7 software forulti-Dop X hardware, DWL). The final measure used

or each time point was an average of 4 cycles (VMCA).imultaneously with the TCD recording, a mask cov-ring the subject’s mouth and nose region was placedor the measurement of PETCO2 (Datex Normocap 200;ameca).A high-resolution ultrasound scanner, C-scan

Dermascan C, 20 MHz; bandwidth, 15 MHz) (Cortexechnology, Hadsund, Denmark),14 was used to mea-ure the diameter of the left temporal and left radialrtery. The diameter of the former was measured at theront branch of the superficial temporal artery and theatter at the wrist. To ensure that the repeated measure-ents with TCD and C-scan were performed in the

ame place, marks were drawn on the skin. After theast recording on the first trial day, the coordinates ofhe marks were kept for reuse on the following trialay.Pharmacokinetics. Plasma concentrations of

IBN4096BS were sampled at the following timeoints: T�10 (baseline), T9.5, T30, T60, and T180 on eachrial day in Vacutainer blood-collecting tubes with eth-lenediaminetetraacetic acid (K3 10-mL glasses;ecton Dickinson, Rutherford, NJ). Samples were

tored on ice for a maximum of 30 minutes before
entrifuged for 10 minutes (2000 rpm) at 4°C. The m
lasma was stored at �20°C until analyzed at Boehr-nger Ingelheim Pharma GmbH & Co KG (Biberach aner Riis, Germany). The plasma concentration ofGRP was determined twice, at baseline (T�10) and at

he end of the h-�CGRP infusion (T40).BIBN4096BS antibodies. BIBN4096BS was modi-

ed with succinic acid anhydride. This hapten wasovalently coupled to human serum albumin. Poly-lonal antibodies were produced by immunization of-month-old female New Zealand rabbits with the im-unogen in complete Freund’s adjuvant. After several

ooster immunizations, the antibodies were purifiedrom rabbit serum by use of protein A–SepharoseSepharose is a registered trademark of Amershamiosciences).BIBN4096BS analytic methods. The procedures

ere conducted in accordance with current interna-ional guidelines.15 In this competitive enzyme-linkedmmunosorbent assay, the biotinylated anti-IBN4096BS antibodies (immunoglobulin G fraction)ere bound to microtiter plates that were adsorptive-

oated with avidin. BIBN4096BS in the plasma sampleompeted with added horseradish peroxidase–labeledIBN4096BS reagent for binding sites on the solid-hase antibodies. After incubation, unboundIBN4096BS and plasma components were removedy washing. Antibody-bound enzyme activity was de-ected with a chromogenic substrate. The amount ofolored product formed was measured photometricallynd decreased with the increasing concentration ofIBN4096BS in the plasma sample. The BIBN4096BSoncentration corresponding to the measured opticalbsorbance was calculated via data fitting of the non-inear standard curve.

To compensate for slight variations in immuno-hemical reaction parameters (such as temperature andntibody binding capacity) between microplates, a stan-ard curve was included on each plate. All steps of thenzyme-linked immunosorbent assay were performedt 22°C � 1°C, which corresponded to room tempera-ure of the air-conditioned laboratory.

Assay precision as assessed from 886 triplicate de-erminations by construction of a precision profile was.1% coefficient of variance (CV) at the lower limit ofuantification, 2.7% CV at the upper limit of quantifi-ation, and 1.6% CV in the middle of the working range0.5 ng/mL).

Human-�CGRP analysis. The analysis of-�CGRP plasma concentrations was performed at theepartment of Clinical Physiology and Nuclear Medi-

ine, Glostrup Hospital (Glostrup, Denmark). The
ethod of analysis has been described in detail else-

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here.2 In this study only 100 �L of serum was used.he normal values were 85 � 35.4 pmol/L for womennd 88 � 36.2 pmol/L for men (Schifter S, oral com-unication, November 2002).Trial procedure. The healthy volunteers began the

tudy at 8 AM, headache-free. For the preceding 8 hours,hey had abstained from drinking coffee, tea, andaffeine-containing beverages and smoking tobaccond they had not taken any medication, except oralontraception. They rested in the supine positionhroughout the study period (T�20 to T180) Two intra-enous catheters (Optiva*2 [18 gauge]; Johnson &ohnson, Ethicon SpA, Pomezia, Italy) were insertednto the cubital veins, one for the administration ofuman �CGRP and BIBN4096BS and the other forlood sampling. The volunteers rested for at least 30inutes before baseline values of CBF, VMCA, tempo-

al and radial diameter, blood pressure (BP), heart rateHR), and electrocardiogram were recorded. The startf infusion of 2.5 mg BIBN4096BS or placebo wasesignated as time zero (T0). The infusion lasted 10inutes. At T20, a 20-minute infusion of h-�CGRP (1.5g/min) was initiated. Infusions were administered bytime- and volume-controlled infusion pump (Braun

erfusor; B. Braun Melsungen AG, Melsungen,ermany).All measurements, except the CBF measurements,

ere recorded quarterly for 3 hours (study period), andP, HR, electrocardiogram (Cardiofax; Nihon Kohdenorporation, Tokyo, Japan), AEs, and headache were

ecorded for an additional hour. BP and HR wereeasured every 5 minutes for the first hour and there-

fter every 15th minute with an automatically inflatinguff (Omega 1400, In Vivo Research Laboratories Inc,opiague, NY). In the observation period from T180 to

240, the participants were allowed to sit upright. ThreePECT scans were done as follows: at baseline, at T60,nd at T90. VMCA was measured immediately after eachPECT scan.The estimated perfusion (rCBFx) in the area of a

iven artery (x) is dependent on the mean blood flowelocity [Vmean(x)] and the cross-sectional area (��r2)f the artery. The following equation is valid for theegional CBF:

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� ��rCBF2(x) ⁄ Vmean2(x)� Vmean1(x)

⁄ rCBF1(x)� � 1� � 100

Diameter is the relative percentage change in diam- p

ter, Vmean1(x) is the mean blood velocity before infu-ion of drugs, and Vmean2(x) is the velocity at a relevantime point after the infusion; the same designation ispplied for rCBF.16,17

Statistics. Baseline was calculated as a mean of theeasurements at time points T�20 and T�10 in the

nalysis. Values are presented as means � SD. P .05as considered significant. All analyses were per-

ormed by use of SPSS statistical software, version 10.0SPSS Inc, Chicago, Ill).

For changes over time on each trial day, VMCA,lobal CBF, rCBFMCA, diameter of the temporal andadial artery, BP, and PETCO2 were analyzed by a uni-ariate ANOVA for the factors time and subject. If aignificant change was found, a post hoc analysisDunnett multiple comparisons test) was performed toocalize the change. To eliminate the risk of massignificance of measurements with numerous repeatedeasurements, 4 points of interest were chosen as

ollows: baseline, 45 minutes, 105 minutes, and 165inutes. Absolute values were used for the statistical

nalysis. For the comparison between BIBN4096BSnd placebo, a paired t test was performed for theollowing measurements: VMCA, global CBF,CBFMCA, diameter of the temporal and radial arteries,P, and PETCO2. The summary measure for the t testas the area under the curve (AUC) calculated onercentage changes from baseline.Immediate headache was defined as any headache

uring the first 60 minutes after the start of the-�CGRP infusion. Any headache occurring thereafteras referred to as delayed headache. Peak values and

rea under the curve for headache (AUCheadache) wereompared between the 2 trial days by use of theilcoxon signed rank test. The occurrence of headache

nd AEs on the 2 trial days was compared by use of thecNemar test.

ESULTSBaseline values. All baseline measurements of the

emodynamic responses are summarized in Table I.nly the baseline PETCO2 measured simultaneouslyith the TCD recordings showed a significant differ-

nce between study days (P � .03). The finding wasnterpreted as incidental and was not taken into accountn the processing of data.

Effect of BIBN4096BS on CGRP-induced head-che and other AEs. On placebo days, 5 participantsad an immediate headache and 3 had a delayed head-che; the maximum immediate headache score was 2nd the maximum delayed headache score was 1. No
articipants had an immediate headache but 1 had a

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elayed headache after BIBN4096BS pretreatment.he delayed headache occurred 6 hours after the infu-ion of BIBN4096BS, lasted 3 hours, and was scored 1.he effect of BIBN4096BS in preventing immediateeadache was significant (P � .034 for peak headachend P � .04 for AUCheadache) and in preventing theccurrence of any headache during the in-hospitalhase (P � .031, McNemar test).

After placebo pretreatment, h-�CGRP caused flush-ng in all participants and all but 1 had bilateral con-

able II. Effect of BIBN4096BS pretreatment on-�CGRP–induced symptoms

Symptom

Placeboplus

h-�CGRP(No.)

BIBN4096BS(2.5 mg) plus

h-�CGRP(No.)

P value(McNemar

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Flushing 10 0 P � .002Heat sensation 8 0 P � .008Palpitations 5 0 P � .063Conjunctival

injection9 0 P � .004

Headache 6 0 P � .031

The flushing and conjunctival injection was based on the investigators’bservations. Heat sensation and palpitation were reported and headache wasystematically scored. The data shown are from the entire in-hospital studyeriod.

able I. Baseline values of measured variables

Measured variable

Global CBF (mL · 100 g brain tissue�1 · min�1)rCBFMCA (mL · 100 g brain tissue�1 · min�1)PETCO2 (mm Hg)

CBFTCD

VMCA (cm/s)C-scan

Temporal (mm)Radial (mm)

Systolic blood pressure (mm Hg)Diastolic blood pressure (mm Hg)Mean arterial blood pressure (mm Hg)Heart rate (beats/min)Plasma CGRP (pmol/L)

h-�CGRP, Human �-calcitonin gene–related peptide; CBF, cerebral blood floartial pressure of carbon dioxide; TCD, transcranial Doppler; VMCA, middle ceptide.*Significant difference between baseline on placebo and BIBN4096BS pret

unctival injection. Eight experienced a sensation of 8

eat. Five reported palpitations. None of these CGRP-nduced changes were seen on days whenIBN4096BS was administered as pretreatment (Table

I). AEs that could possibly be assigned to the CGRPeceptor antagonist were located to the infusion site.

CBF. Global CBF increased significantly after-�CGRP on both study days (P � .007 after placebond P � .009 after BIBN4096BS pretreatment). Thencrease was measured 20 minutes after the h-�CGRPnfusion was stopped. No difference was found be-ween the 2 days (P � .42).

After h-�CGRP, rCBFMCA increased significantly onoth trial days (P � .003 and P � .01), again 20inutes after the h-�CGRP infusion. No significant

ifference was observed between the 2 study days (P �38). Data were not corrected for PETCO2, because noignificant changes were found on either day (P � .2nd P � .6) or between treatment days (P � .1).

TCD. VMCA did not vary significantly over timeP � .3 for placebo and P � .7 for BIBN4096BS), andetween the 2 trial days, no difference was seen (P �

74). On the basis of the rCBFMCA and VMCA measure-ents, the effect on the relative percentage diameter

hange of MCA can be estimated.16,18 As seen in TableII, a dilation of the MCA was found on both studyays. Compared with baseline, the dilation occurring onlacebo days reached significance at T60 (P � .005).his corresponded to a diameter increase of 9.3% �

acebo plus-�CGRP

BIBN4096BS(2.5 mg) plus

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value

6.7 � 10.8 45.6 � 10.5 .55.9 � 10.5 44.7 � 10.6 .3

39 � 3.5 39 � 4.1 .941 � 3.0 39 � 3.6 .03*78 � 17.0 74 � 15.3 .1

.26 � 0.4 1.29 � 0.3 .8

.76 � 0.5 2.63 � 0.4 .213 � 8 113 � 6 .964 � 8 64 � 5 .879 � 6 80 � 7 .554 � 8 53 � 4 .789 � 20.3 91 � 20.4 .5

CA, cerebral blood flow in territory of middle cerebral artery; PETCO2, end-tidaltery blood flow velocity (average of 4 cycles); CGRP, calcitonin gene–related

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n increase was seen as well, but it did not reachtatistical significance (P � .2). There was no differ-nce between placebo and BIBN4096BS pretreatmentP � .17). Data were not corrected for PETCO2, becauseo significant changes were found on the trial daysP � .4 on both days) or between treatment days (P �45).

C-scan. A significant change in temporal artery di-meter over time on placebo days was observed (P 001). This significance was seen at all time points afterhe infusion of h-�CGRP.

A significant diameter change was not seen whenIBN4096BS was infused as pretreatment (P � .7).he increase in temporal artery diameter was signifi-antly inhibited by BIBN4096BS (P .001). Theadial artery revealed a similar finding, with a signifi-ant change on placebo days (P � .01) and a nonsig-ificant response after BIBN4096BS pretreatmentP � .07). The difference between placebo andIBN4096BS was significant (P � .001) (Fig 1).Peripheral hemodynamics. Table IV summarizes

ata on systolic BP, diastolic BP, mean arterial BP, andR. No significant time-dependent changes were seen

n systolic, diastolic, or arterial mean BP on either trialay or between days. The HR increased significantly onlacebo days (P .001) at all time points comparedith baseline. This increase was not seen onIBN4096BS pretreatment days. The increase in HRas significantly inhibited by BIBN4096BS (P � .003).Pharmacokinetics of BIBN4096BS and h-�CGRP. The

ighest measured plasma concentration ofIBN4096BS occurred just before the end of infusiont 9.5 minutes after the start of the infusion, with aean of 170.4 � 25.4 ng/mL. The lowest plasma

able III. Effect of BIBN4096BS pretreatment on h-�

Time point

VMCA (cm/s)

Placeboplus CGRP

BIBN4096BSplus CGRP

Ppl

Baseline 78 � 17.0 74 � 15.360 min 75 � 15.5 75 � 12.9 9.

(19.90 min 77 � 14.3 74 � 15.5 3

(7.

Values are given as mean � SD. The percentage change in the mean diametahl et al.16 VMCA and rCBF on left and right sides analyzed separately show*Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P �†Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P �‡Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P �§Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P �

oncentration was measured 180 minutes after the start t

f the infusion, with a mean of 8.7 � 5.7 ng/mL. NoIBN4096BS was detected on placebo days (Fig 2).he pharmacokinetic parameters for BIBN4096BSave previously been published and are summarized.n the basis of administration of 5 and 10 mg intrave-ously for 10 minutes, BIBN4096BS had a total plasmalearance of 12 L/h and a terminal half-life of 2.5 hours.pproximately 15% of the dose was excreted un-

hanged in urine, with a mean renal clearance of 2 L/h.he geometric mean maximum plasma concentrationfter 2.5 mg of BIBN4096BS was 210 ng/mL.19

At the end of the CGRP administration, a significantifference between treatment days was found (P

001, paired t test); the CGRP concentration was 342 �8 pmol/L when placebo was administered as pretreat-ent and 442 � 70 pmol/L when BIBN4096BS was

iven (Fig 3). To elucidate whether this difference wasaused by a cross-reaction between BIBN4096BS andur CGRP analytic kit, blood samples from 3 healthyolunteers with the following concentrations ofIBN4096BS were analyzed for CGRP: sample 1 and, 0 ng/mL (control); sample 3, 1000 ng/mL; sample 4,00 ng/mL; sample 5, 250 ng/mL; and sample 6, 125g/mL. The analysis did not reveal any correlationetween the concentration of BIBN4096BS and CGRP.hus BIBN4096BS did not cross-react with CGRPetection in our analytic kit.

ISCUSSIONThis study has demonstrated that a specific CGRP

ntagonist, BIBN4096BS, can prevent CGRP-inducedymptoms such as headache, flushing, heat sensation,nd palpitations. It furthermore prevents a CGRP-nduced increase in HR and dilatation of the superficial

–induced cerebral hemodynamic changes

eterMCA (%) andreaMCA (%)

rCBFMCA

(mL · 100 g�1 · min�1)

BIBN4096BSplus CGRP

Placeboplus CGRP

BIBN4096BSplus CGRP

0 (0) 45.9 � 10.5 44.7 � 10.6

.9)3.8 � 5.4

(7.96 � 11.3)52.4 � 9.4† 49.1 � 12.1‡

)3.0 � 7.9

(6.8 � 16.3)49.3 � 12.7 47.9 � 10.1§

right sides) of the middle cerebral artery (MCA) was estimated according toresults.

CGRP

Diam A

lacebous CGRP

0 (0)3 � 8.1*95 � 17.4 � 7.54 � 15.1

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ffect on CGRP-induced increase in CBF and dilatationf the MCA.

able IV. Effect of BIBN4096BS pretreatment on h-�

Timepoint

Systolic blood pressure(mm Hg)

Diastolic blood p(mm Hg)

Placeboplus CGRP

BIBN4096BSplus CGRP

Placeboplus CGRP

BIBNplus

Baseline 113 � 8 113 � 6 64 � 8 6445 min 115 � 9 111 � 9 59 � 5 67105 min 112 � 11 110 � 10 62 � 6 66165 min 115 � 12 115 � 7 66 � 9 67

All values are given as mean � SD. Only time points selected for the stati*Significant difference between placebo and BIBN4096BS pretreatment (P

Fig 1. Diameter (in millimeters) of superficisignificant difference (P .05) between p�-calcitonin gene–related peptide (h-�CGRP)Temporal artery, placebo pretreatment; opensquares, temporal artery, 2.5-mg BIBN4096BIBN4096BS pretreatment (mean � SD).

Localization, function, and role of CGRP in mi- h

raine. CGRP is one of the most potent vasodilatorsnown.4 In the brain, immunohistochemical studies

–induced systemic hemodynamic changes

Mean blood pressure(mm Hg)

Heart rate(beats/min)

Placeboplus CGRP

BIBN4096BSplus CGRP

Placeboplus CGRP

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sis (ANOVA) are shown.ired t test).

ral artery and radial artery. Asterisk denotesand BIBN4096BS pretreatment on humanvasodilatation (paired t test). Open squares,

, radial artery, placebo pretreatment; closedeatment; solid circles, radial artery, 2.5-mg

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-fibers surrounding cerebral and extracerebral arteriesnd to the trigeminal ganglion cell bodies.20

CGRP is present in plasma from healthy volun-eers at rest.2 We have previously shown that infu-ion of the selective CGRP receptor antagonistIBN4096BS did not alter CBF or the diameter oferebral and peripheral arteries.21 Hence circulatingevels of CGRP do not seem to exert a tonic dilatorction in these vascular beds. During a migraine

Fig 2. Plasma concentration of BIBN4096BSas mean � SD.

Fig 3. Plasma concentration of calcitonin geTwo asterisks denote a significant differencpretreatments (mean � SD) (paired t test).

ttack5 and between attacks, CGRP is increased in h

enous blood,22 implicating a role of CGRP in mi-raine pathogenesis. During an attack, the plasmaevels were increased (2-2.5 times) compared withormal controls.5 The infusion of 1.5 �g/min of-�CGRP in the current study increased the plasmaoncentration approximately 3 to 4 times.

In healthy volunteers the infusion of CGRP has pre-iously been shown to induce a sensation of fullness inhe head or mild headache,23,24 corresponding to a

-minute infusion of 2.5 mg. Values are given

ed peptide (CGRP) on 2 different trial days..001) between BIBN4096BS and placebo

after 10

ne–relate (P

eadache score of 1 in the current study. Because the

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tudy was performed in healthy volunteers, a genuineigraine attack was not expected. Why a migraine

ttack is induced only in migraine patients and not inealthy volunteers is most likely a threshold phenom-non. Through use of the human experimental head-che model, previously performed studies have shownhat headache response and MCA changes in healthyolunteers compose a valid model for investigations ofigraine pathogenesis.25-27

Methods of pharmacologic studies of cephalic he-odynamics relevant to migraine. We have developed

he combination of methods applied in this study torovide the most extensive and precise description ofharmacologic effects on cephalic hemodynamics aselated to migraine. For CBF measurement, positronmission tomography (PET) and magnetic resonanceMR) techniques provide better spatial solution than33Xe SPECT, but this is of minor importance in phar-acologic studies because no major variation has been

escribed in the response of cortical CBF to drugs.28-30

ur pharmacologic experiments last several hours, andhe costs of blocking MR and especially PET equip-ent for half a day or more on at least 30 experimental

ays in 1 drug study are prohibitive in most centers.urthermore, studies of migraine patients in whom nau-ea and vomiting may develop are very difficult insidemagnet. The day-to-day CV of 133Xe SPECT mea-

urements is 8%.29 These figures are roughly equal tohose of PET and slightly better than MR-determinedBF.31 TCD measurements of velocity (Vmean) as

tand-alone measurements are difficult to interpret be-ause they depend both on the diameter of the MCAnd on CBF in the MCA territory (rCBFMCA), but theyre useful in combination with quantitative rCBF. TCDeasurements are most precise in the MCA, with a CV

f 26% between subjects, 20% between sides, 16%etween days, and 7% 5 minutes apart.13 In studies ofeadache, fixed probes and continuous measurementsannot be used because the probes cause local discom-ort and headache.32 Because of the relatively highntersubject and interobserver variability of TCD mea-urements, the current study was designed as a cross-ver study and the same trained investigator performedll measurements. Power calculations based on an SDf TCD measurements of 7.8 cm/s and allowing a 5%ype I and a 20% type II error showed that 7 subjectsere needed in a crossover design to detect a differencef 15 cm/s (change from baseline of approximately5%).13

An alternative to these measurements could be MRngiography, but MR angiography is difficult to time
ith CBF measurements and poses problems in relation h
o migraine induction. Its precision is not yet suffi-iently documented and may not be high enough toetect modest pharmacologic effects. In summary, theombination of methods applied in this study remainsreferable to other alternatives for a broad character-zation of the effects of messenger molecules and drugsn the cephalic circulation.

Hemodynamic effects of CGRP. CGRP increasesBF in dogs33 and striatal blood flow in rats.34 Inuinea pigs a pretreatment with urea was necessaryefore CGRP-induced CBF changes could be in-uced.35 The peptide is partly responsible for cerebralasodilatation in response to hypotension,36 postocclu-ive hyperemia,37 and cortical spreading depression.38

In humans h-�CGRP (0.6 �g/min for 3 hours)aused a significant decrease of approximatelymL · 100 g brain tissue�1 · min�1 in the left hemi-

phere CBF and a nonsignificant decrease of 5 mL ·00 g brain tissue�1 · min�1 in the right hemisphere inne study.39 The decrease was seen 30 minutes after thenitiation of the CGRP infusion but not after 2.5 hoursnd was attributed to a decrease in PETCO2. Design, areaf measurement, and method of administration ofGRP were different from those in this study, which

howed a slight but significant increase in global andegional CBF without changes in PETCO2.

In healthy volunteers (n � 5) long-term infusion ofGRP with a maximum dosage of 1.15 �g/min causednonsignificant decrease in the blood flow velocity in

he MCA (60 cm/s to 54 cm/s); CBF was not mea-ured.40 A similar decrease was seen with 0.6 �g/in.39 In patients studied after subarachnoidal hemor-

hage, VMCA decreased only on the side of the bleeding.t was suggested that preconstriction of the artery or aisruption of the blood-brain barrier was necessary forGRP to dilate MCA.41,42 In the current study VMCA

id not change after CGRP. We found, however, thatlobal CBF, as well as rCBFMCA, increased after CGRPdministration and calculated that CGRP increased theiameter of the MCA16 (Table III).

BIBN4096BS pretreatment did not prevent theGRP-induced increase in global CBF or rCBFMCA.urthermore, it did not prevent the CGRP-induced in-rease in MCA diameter. However, the study was notowered to rule out effects on MCA diameter, which isalculated from 2 observed values and, therefore, isore variable than the parameters alone.In healthy volunteers and patients with subarachnoi-

al hemorrhage, h-�CGRP did not affect systolic oriastolic BP. HR was increased significantly.24,41 In
ealthy male volunteers 1.18 �g/min induced a 30%

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ncrease in HR.43,44 In the current study we foundimilar effects of CGRP.

CGRP receptors and CGRP receptor blockade. TheGRP receptor can be subdivided into 2 subtypes,GRP1 and CGRP2; however, some discussion of thealidity of this classification exists.45 The CGRP1 re-eptor predominates in cerebral arteries, trigeminalanglion cell bodies, and perivascular nerve fibers.4,46

BIBN4096BS is the first CGRP receptor antagonistvailable for human clinical trials. BIBN4096BS hasigh specificity and affinity for the human CGRP re-eptor; in human SK-N-MC (neuroblastoma cell line ofuman origin) cells, it displayed an affinity of Ki of4.4 � 6.3 pmol/L, and for rat spleen, it demonstratedKi of 3.4 � 0.5 nmol/L.47 The antagonist is 10 timesore potent in blocking rat or h-�CGRP and h-�CGRP

esponses compared with the peptide antagonistGRP8-37. Furthermore, it was 10 times more potent in

nhibiting responses in rat atrium than in rat vas defer-ns.48 These results, together with the findings thatK-N-MC cell lines only express the CGRP1 recep-

or,49 indicate that BIBN4096BS preferably binds tohis receptor subtype. BIBN4096BS inhibits bothGRP-induced BP changes in rats and the neurogenic-

nduced increase in facial blood flow in marmosets.47,50

n isolated human middle cerebral, meningial, and tem-oral arteries, BIBN4096BS blocks the dilatatory re-ponse to CGRP.9,51

In our study, the CGRP plasma concentration wasound to be significantly higher on BIBN4096BS pre-reatment days than on placebo pretreatment days. Anxplanation would be a cross-reaction betweenIBN4096BS and CGRP, but this was ruled out. Thending can be explained by a complete blockade ofGRP receptors by the antagonist, resulting in a higheroncentration of circulating CGRP. This explanationemains hypothetic until confirmed by further studies.

Our study showed that BIBN4096BS completely in-ibits the effects of CGRP on the superficial temporalnd radial artery, as well as its effect on HR. In con-rast, it had no significant effect on the CGRP-mediatedBF and VMCA increase. These results are importantith regard to the understanding of the action of CGRPlockade in the treatment of acute migraine. In a phaseI proof-of-concept study in migraine patients,IBN4096BS showed a significant effect in abortingigraine attacks.7 In agreement with this clinical result,IBN4096BS completely prevented CGRP-inducedeadache in normal volunteers in our study.In summary, our study has demonstrated the potency

f the CGRP receptor antagonist BIBN4096BS in hu-
ans. Thus it prevented all symptoms and signs elicited
y h-�CGRP and caused no symptoms on its own.ore importantly, h-�CGRP–elicited headache was

ompletely prevented. Given that the compound inhib-ted all effects of h-�CGRP on extracerebral arteriesut had no effect on the induced increase in CBF or onhe calculated diameter of the MCA, it is suggested thatIBN4096BS prevents or treats headache predomi-antly in an extracerebral manner. Whether the effectakes place in the dura mater or in extracranial arteriesnd whether areas of the brain stem or hypothalamusevoid of a blood-brain barrier also play a role remaino be determined.

We thank Lene Elkjær and Kirsten Bruunsgaard for excellentechnical support and Dr Kirsten Kassøe for making everything workerfectly.

None of the authors at the Danish Headache Center receivedayment or honoraria. Lynna Lesko is an employee of Boehringerngelheim Pharmaceuticals Inc. However, she did not receive specialonoraria from the company and has no equity or other ownershipnterest.

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bibn4096bs antagonizes human α-calcitonin gene related peptide–induced headache and extracerebral...

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