BIB \4096BS antagonizes human
`a- calcitonin gene related peptide- induced
`headache 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 acute
`treatment of migraine has been demonstrated. We investigated the ability of BIBN4096BS to inhibit human
`aCGRP (h- aCGRP)- induced headache and cerebral hemodynamic changes in healthy volunteers.
`Methods: Ten healthy volunteers completed this double -blind, placebo - controlled crossover study with
`2.5 mg BIBN4096BS and placebo as pretreatments before a 20- minute intravenous infusion of h -aCGRP
`(1.5 p.g /min). Transcranial Doppler ultrasonography was used to measure blood flow velocity in the middle
`cerebral artery (MCA); regional and global cerebral blood flow (CBF) was measured by xenon 133 inhalation
`single- photon emission computed tomography. The temporal and radial artery diameter was measured by
`high -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 placebo
`pretreatment but none after BIBN4096BS (P = .031). BIBN4096BS did not affect changes in the diameter
`of the MCA or changes in CBF induced by h- aCGRP. 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. (Clin
`Pharmacol 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
`
`Calcitonin gene -related peptide (CGRP) is a neu-
`ropeptide found in the perivascular nerve terminals
`surrounding arteries.' A measurable concentration of
`CGRP is circulating in the blood at rest,2 and CGRP
`
`From the Danish Headache Center, University of Copenhagen, and
`Department of Neurology, Glostrup University Hospital, Glostrup,
`and Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield.
`Boehringer
`Ingelheim
`provided
`sponsored
`study
`the
`and
`BIBN4096BS. The authors were independently responsible for the
`study design, data analysis, and manuscript. The technical equip-
`ment used was partly sponsored by the Villum Kann Rasmussen
`Foundation, Toyota Foundation, and Simon Fougner Hartmann
`Foundation. The Lundbeck Foundation funds the research of the
`Danish Headache Center.
`Received for publication June 29, 2004; accepted Oct 6, 2004.
`Reprint requests: Kenneth A. Petersen, MD, Danish Headache Center,
`University of Copenhagen and Department of Neurology, Glostrup
`University Hospital, KAS Glostrup, DK -2600 Glostrup, Denmark.
`E -mail: kapetersen. @dadlnet.dk
`0009 -9236/$30.00
`Copyright © 2005 by the American Society for Clinical Pharmacology
`and Therapeutics.
`doi:10.1016 /j.clpt.2004.10.001
`
`202
`
`receptors are localized throughout the body. Cerebral
`and other cephalic arteries have a particularly rich
`innervation of CGRP- containing afferent trigeminal
`nerve fibers, and these arteries, as studied in tissue
`baths, are particularly sensitive to CGRP.4 CGRP is
`found in an increased concentration in external jugular
`venous blood but not in blood from the cubital vein
`during a migraine attack.5 After infusion in patients
`with migraine, CGRP caused a migraine -like headache
`and in some a genuine migraine attack with associated
`symptoms that were indistinguishable from the pa-
`tients' normal migraine attacks.6 Peptide antagonists of
`CGRP receptors have been available for experimental
`studies for several years, but previously available com-
`pounds have not been tested for safety and are, there-
`fore, not suitable for human clinical studies. One CGRP
`receptor antagonist, BIBN4096BS, has been developed
`with the purpose of treating acute migraine, and a phase
`II study has provided proof of efficacy.' Although
`BIBN4096BS potently interacts with the human CGRP
`
`Lilly Exhibit 1333
`Eli Lilly & Co. v. Teva
`Pharms. Int'l GMBH
`
`

`

`CLINICAL. PHARMACOLOGY & THERAPEUTICS
`2005;77(3)102-13
`
`CGRP receptor antagonism and vascular headache 203
`
`receptor in vitro,$" no information is available on its
`ability to inhibit CGRP changes in human volunteers at
`increased levels of the peptide as seen during a mi-
`graine attack.5
`to conduct a placebo -
`We,
`therefore, decided
`controlled, double -blind crossover experiment,
`in
`which BIBN4096BS and placebo in random order were
`used as pretreatment followed by infusion of human
`aCGRP (h- aCGRP). The aims of this study were to
`validate the ability of h -aCGRP to provoke headache
`and to describe its effect on cerebral, extracerebral, and
`systemic circulatory parameters. Furthermore, we ana-
`lyzed whether BIBN4096BS could partly or fully block
`the changes evoked by h- aCGRP. It was our hope that
`the study could thus contribute to a better understand-
`ing of the role of CGRP in neurovascular headache and
`the site of action of this new antimigraine compound.
`
`METHODS
`Design and participants. This was a placebo -
`controlled, double -blind crossover study that included
`11 healthy subjects (7 men and 4 women). One female
`participant had claustrophobia that developed during
`baseline single -photon emission computed tomography
`(SPECT) scanning and was excluded before any trial
`medication was given. Ten participants completed both
`treatment days. The participants were aged 24 to 31
`years (mean, 26.5 years) and weighed 68 to 89.4 kg
`(mean, 77.4 kg). The participants had no current or
`previous cardiovascular, cerebrovascular, endocrine, or
`neurologic disorder, including no migraine, hypoten-
`sion, or hypertension. A frequency of tension -type
`headache of 4 d/mo or lower was accepted. On the day
`of enrollment, physical and neurologic examination,
`electrocardiography, and blood sampling were done.
`The healthy volunteers were randomized to receive
`either 2.5 mg BIBN4096BS or placebo (xylitol 5 %) as
`an intravenously administered pretreatment of 10 min-
`utes' duration. After a free interval of 10 minutes,
`1.5 p g /min h -aCGRP was administered continuously
`for 20 minutes on both trial days. The 2 trial days were
`separated by at least 1 week.
`Boehringer Ingelheim GmbH supplied BIBN4096BS
`and performed the randomization and blinding, which
`was balanced (ClinPro, version 6; Clinical Systems,
`Inc, Garden City, NY). The dose effective in the treat-
`ment of acute migraine attacks was used.'
`Human -aCGRP was purchased from Clinalfa AG,
`Läufelfingen, Switzerland. In a study performed previ-
`ously, we used a dose of 21..t.g /min.6 This dose, how-
`ever, induced pronounced hypotension that in 2 patients
`necessitated premature termination of the infusion. In
`
`the current study we, therefore, used the lower dose of
`1.5 µg /min.
`All participants gave written informed consent be-
`fore randomization. The Ethical Committee of Copen-
`hagen (KA00079gs) and the Danish Medicines Agency
`(2612 -1376) approved the study, which was conducted
`in accordance with the Helsinki II Declaration and the
`Guidelines for Good Clinical Practice.10
`Recording of adverse events. Every 15th minute
`from time (T) zero (T0) (baseline) to T240 (end of study
`period), the volunteers were questioned regarding the
`presence of adverse events (AEs) and rated headache.
`Between questionings, the participants self -reported
`any changes that they might have. The intensity of the
`AEs was graded as mild, moderate, or severe, and their
`relationship to study medication was classified as re-
`lated or not related by the investigator. Headache in-
`tensity was scored on an 11 -point verbal rating scale
`with 0 indicating no headache; 1 indicating a feeling of
`the occurrence of something unusual inside the head,
`not necessarily actual pain; 5 indicating headache of
`medium severity; and 10 indicating worst imaginable
`headache. Accompanying symptoms were recorded ac-
`cording to the International Headache Classification.'I
`During the study period, the investigator recorded the
`AEs. After discharge, the volunteers made an hourly
`recording of AEs up to 24 hours after the infusion of
`placebo or BIBN4096BS.
`Cerebral blood flow measurements. Global and re-
`gional cerebral blood flow (CBF) was measured with
`inhalation and SPECT with a brain -
`xenon 133
`dedicated camera (Ceraspect; DSI, Waltham, Mass).
`The apparatus consisted of a stationary annular sodium
`iodide crystal and a fast -rotating collimator system.
`Each rotation took 10 seconds, thereby acquiring 1
`frame in a 30 -frame dynamic protocol of 133Xe inha-
`lation, with 3 background, 9 wash -in, and 18 wash -out
`frames by use of the Kanno- Lassen algorithm.' 2 A
`photoelectric window of 70 to 100 keV was used.
`Thirty -two slices were reconstructed in a 64 X 64
`matrix with each pixel measuring 0.33 X 0.33 cm by
`use of a Butterworth one -dimensional filter (cutoff, 1.5;
`order, 6). The 32 slices were reduced to sets of 8
`transaxial slices generated by adding 4 slices together
`to a total slice thickness of 1.32 cm. A correction by use
`of the Chang algorithm (µm = 0.05 cm) and nose
`artifact was performed. The output for each pixel was
`the inhibition constant (K1) value, and flow values were
`estimated from these by use of the partition coefficient
`(A) of 0.85 (gray matter).
`A Datex Normocap 200 (Dameca, Roedovre, Den-
`mark) was used for end -tidal partial pressure of carbon
`
`

`

`204 Petersen et al
`
`CLINICAL PHARMACOLOGY & THERAPEUTICS
`MARCH 2005
`
`dioxide (PETc02) measurements during the CBF acqui-
`sitions. A Ceratronic XAS SM 32C (Randers, Den-
`33Xe administration. Each
`mark) was used for the
`'
`measurement lasted 5 minutes.
`Calculations of flow in the perfusion territories of the
`major cerebral arteries were performed by fitting of
`standard vascular regions of interest on the 5 rostral
`slices at 3.6, 5.0, 6.3, 7.6, and 9 cm above the orbito-
`meatal line. Flow in the territory of the middle cerebral
`artery (MCA) (rCBFMCA) was calculated as a mean of
`the left and right side.
`Transcranial Doppler and C -scan. Transcranial
`Doppler (TCD) ultrasonography (2 MHz) (Multi -Dop
`X; DWL, Sipplingen, Germany) was used for the mea-
`surement of blood flow velocity. The recordings were
`done simultaneously and bilaterally as previously de-
`scribed but with handheld probes.13 Along the MCA, a
`fixed point was found for the measurement. The fixed
`point was chosen as close as possible to the bifurcation
`of the anterior cerebral artery and MCA. The same fix
`point was used for each individual and for each record-
`ing, for which the signal was optimized. On the basis of
`the envelope curve (the spectral TCD curve), a time
`averaged mean (Vmcan) over approximately 4 cardiac
`cycles or 4 seconds was calculated by the built -in
`software (version 7.40x of MDX TCD -7 software for
`Multi -Dop X hardware, DWL). The final measure used
`for each time point was an average of 4 cycles (YMCA).
`Simultaneously with the TCD recording, a mask cov-
`ering the subject's mouth and nose region was placed
`for the measurement of PErco2 (Datex Normocap 200;
`Dameca).
`A high -resolution ultrasound
`scanner, C -scan
`(Dermascan C, 20 MHz; bandwidth, 15 MHz) (Cortex
`Technology, Hadsund, Denmark),14 was used to mea-
`sure the diameter of the left temporal and left radial
`artery. The diameter of the former was measured at the
`front branch of the superficial temporal artery and the
`latter at the wrist. To ensure that the repeated measure-
`ments with TCD and C -scan were performed in the
`same place, marks were drawn on the skin. After the
`last recording on the first trial day, the coordinates of
`the marks were kept for reuse on the following trial
`day.
`Pharmacokinetics. Plasma
`concentrations
`of
`BIBN4096BS were sampled at the following time
`points: T_ Io (baseline), T9.5, T30, T60, and T180 on each
`trial day in Vacutainer blood -collecting tubes with eth-
`ylenediaminetetraacetic acid
`10 -mL glasses;
`(K3
`Becton Dickinson, Rutherford, NJ). Samples were
`stored on ice for a maximum of 30 minutes before
`centrifuged for 10 minutes (2000 rpm) at 4 °C. The
`
`plasma was stored at -20 °C until analyzed at Boehr-
`inger Ingelheim Pharma GmbH & Co KG (Biberach an
`der Rii.s, Germany). The plasma concentration of
`CGRP was determined twice, at baseline (T_Io) and at
`the end of the h -aCGRP infusion (T40).
`BIBN4096BS antibodies. BIBN4096BS was modi-
`fied with succinic acid anhydride. This hapten was
`covalently coupled to human serum albumin. Poly -
`clonal antibodies were produced by immunization of
`3- month -old female New Zealand rabbits with the im-
`munogen in complete Freund's adjuvant. After several
`booster immunizations, the antibodies were purified
`from rabbit serum by use of protein A- Sepharose
`(Sepharose is a registered trademark of Amersham
`Biosciences).
`BIBN4096BS analytic methods. The procedures
`were conducted in accordance with current interna-
`tional guidelines.15 In this competitive enzyme -linked
`immunosorbent
`assay,
`the
`biotinylated
`anti -
`BIBN4096BS antibodies (immunoglobulin G fraction)
`were bound to microtiter plates that were adsorptive -
`coated with avidin. BIBN4096BS in the plasma sample
`competed with added horseradish peroxidase- labeled
`BIBN4096BS reagent for binding sites on the solid -
`phase
`incubation,
`antibodies. After
`unbound
`BIBN4096BS and plasma components were removed
`by washing. Antibody -bound enzyme activity was de-
`tected with a chromogenic substrate. The amount of
`colored product formed was measured photometrically
`and decreased with the increasing concentration of
`BIBN4096BS in the plasma sample. The BIBN4096BS
`concentration corresponding to the measured optical
`absorbance was calculated via data fitting of the non-
`linear standard curve.
`To compensate for slight variations in immuno-
`chemical reaction parameters (such as temperature and
`antibody binding capacity) between microplates, a stan-
`dard curve was included on each plate. All steps of the
`enzyme -linked immunosorbent assay were performed
`at 22 °C ± 1°C, which corresponded to room tempera-
`ture of the air -conditioned laboratory.
`Assay precision as assessed from 886 triplicate de-
`terminations by construction of a precision profile was
`9.1% coefficient of variance (CV) at the lower limit of
`quantification, 2.7% CV at the upper limit of quantifi-
`cation, and 1.6% CV in the middle of the working range
`(0.5 ng /mL).
`Human -aCGRP
`of
`analysis. The
`analysis
`h -aCGRP plasma concentrations was performed at the
`Department of Clinical Physiology and Nuclear Medi-
`cine, Glostrup Hospital (Glostrup, Denmark). The
`method of analysis has been described in detail else-
`
`

`

`CLINICAL PHARMACOLOGY & THERAPEUTICS
`2005;77(3002-13
`
`CGRP receptor antagonism and vascular headache 205
`
`where.2 In this study only 100 pL of serum was used.
`The normal values were 85 ± 35.4 pmol/L for women
`and 88 ± 36.2 pmol/L for men (Schifter S, oral com-
`munication, November 2002).
`Trial procedure. The healthy volunteers began the
`study at 8 AM, headache -free. For the preceding 8 hours,
`they had abstained from drinking coffee, tea, and
`caffeine -containing beverages and smoking tobacco
`and they had not taken any medication, except oral
`the supine position
`contraception. They rested
`in
`throughout the study period (T_20 to T180) Two intra-
`venous catheters (Optiva *2 [ 18 gauge]; Johnson &
`Johnson, Ethicon SpA, Pomezia, Italy) were inserted
`into the cubital veins, one for the administration of
`human aCGRP and BIBN4096BS and the other for
`blood sampling. The volunteers rested for at least 30
`minutes before baseline values of CBF, VMCA, tempo-
`ral and radial diameter, blood pressure (BP), heart rate
`(HR), and electrocardiogram were recorded. The start
`of infusion of 2.5 mg BIBN4096BS or placebo was
`designated as time zero (T0). The infusion lasted 10
`minutes. At T20, a 20- minute infusion of h -aCGRP (1.5
`pg/min) was initiated. Infusions were administered by
`a time- and volume -controlled infusion pump (Braun
`perfusor; B. Braun Melsungen AG, Melsungen,
`Germany).
`All measurements, except the CBF measurements,
`were recorded quarterly for 3 hours (study period), and
`BP, HR, electrocardiogram (Cardiofax; Nihon Kohden
`Corporation, Tokyo, Japan), AEs, and headache were
`recorded for an additional hour. BP and HR were
`measured every 5 minutes for the first hour and there-
`after every 1 5th minute with an automatically inflating
`cuff (Omega 1400, In Vivo Research Laboratories Inc,
`Copiague, NY). In the observation period from T180 to
`T240, the participants were allowed to sit upright. Three
`SPECT scans were done as follows: at baseline, at TG0,
`and at T90. VMCA was measured immediately after each
`SPECT scan.
`The estimated perfusion (rCBFx) in the area of a
`given artery (x) is dependent on the mean blood flow
`velocity [Vmean(x)] and the cross -sectional area (7rXr2)
`of the artery. The following equation is valid for the
`regional CBF:
`
`rCBF(x) = Vmean(x)
`
`12
`
`Hence,
`
`Diameter
`/rCBF2(x) /Vmean,(x, X Vmean1w/rCBFI(x)] - 1) X 100
`= ([
`Diameter is the relative percentage change in diam-
`
`eter, Veaan I (x) is the mean blood velocity before infu-
`sion of drugs, and Vmean2(x) is the velocity at a relevant
`time point after the infusion; the same designation is
`applied for rCBF. I6' 17
`Statistics. Baseline was calculated as a mean of the
`measurements at time points T_20 and T_10 in the
`analysis. Values are presented as means ± SD. P < .05
`was considered significant. All analyses were per-
`formed by use of SPSS statistical software, version 10.0
`(SPSS Inc, Chicago, Ill).
`For changes over time on each trial day, YMCA,
`global CBF, rCBFMCA, diameter of the temporal and
`radial artery, BP, and PETCO2 were analyzed by a uni-
`variate ANOVA for the factors time and subject. If a
`significant change was found, a post hoc analysis
`(Dunnett multiple comparisons test) was performed to
`localize the change. To eliminate the risk of mass
`significance of measurements with numerous repeated
`measurements, 4 points of interest were chosen as
`follows: baseline, 45 minutes, 105 minutes, and 165
`minutes. Absolute values were used for the statistical
`analysis. For the comparison between BIBN4096BS
`and placebo, a paired t test was performed for the
`global CBF,
`following measurements: YMCA,
`rCBFMCA, diameter of the temporal and radial arteries,
`BP, and PETco2. The summary measure for the t test
`was the area under the curve (AUC) calculated on
`percentage changes from baseline.
`Immediate headache was defined as any headache
`during the first 60 minutes after the start of the
`h -aCGRP infusion. Any headache occurring thereafter
`was referred to as delayed headache. Peak values and
`area under the curve for headache (AUCheadache) were
`compared between the 2 trial days by use of the
`Wilcoxon signed rank test. The occurrence of headache
`and AEs on the 2 trial days was compared by use of the
`McNemar test.
`
`RESULTS
`Baseline values. All baseline measurements of the
`hemodynamic responses are summarized in Table I.
`Only the baseline PETCO2 measured simultaneously
`with the TCD recordings showed a significant differ-
`ence between study days (P = .03). The finding was
`interpreted as incidental and was not taken into account
`in the processing of data.
`Effect of BIBN4096BS on CGRP -induced head-
`ache and other AEs. On placebo days, 5 participants
`had an immediate headache and 3 had a delayed head-
`ache; the maximum immediate headache score was 2
`and the maximum delayed headache score was I. No
`participants had an immediate headache but I had a
`
`

`

`206 Petersen et al
`
`CLINICAL PHARMACOLOGY & THERAPEUTICS
`MARCH 2005
`
`Placebo plus
`h -aCGRP
`
`46.7 ± 10.8
`45.9 ± 10.5
`
`39 ± 3.5
`41 ± 3.0
`78 ± 17.0
`
`BIBN4096BS
`(2.5 mg) plus
`h -aCGRP
`45.6 ± 10.5
`44.7± 10.6
`
`39 ± 4.1
`39 ± 3.6
`74 ± 15.3
`
`P
`value
`
`.5
`.3
`
`.9
`.03*
`.1
`
`Table I. Baseline values of measured variables
`
`Measured variable
`
`Global CBF (mL 100 g brain tissue-1 min-1)
`rCBFMCA (mL 100 g brain tissue -' min-1)
`PETCO, (mm Hg)
`CBF
`TCD
`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)
`
`1.26 ± 0.4
`2.76 ± 0.5
`113 ± 8
`64-8
`79 ±6
`54-!-8
`89 ± 20.3
`h- aCGRP, Human a- calcitonin gene -related peptide; CBF, cerebral blood flow; rCBFMCA, cerebral blood flow in territory of middle cerebral artery; PETco2, end -tidal
`partial pressure of carbon dioxide; TCD, transcranial Doppler; VMÇA, middle cerebral artery blood flow velocity (average of 4 cycles); CGRP, calcitonin gene- related
`peptide.
`*Significant difference between baseline on placebo and BIBN4096BS pretreatment days (paired t test).
`
`1.29 ± 0.3
`2.63 ± 0.4
`113 ± 6
`64 ± 5
`80 ± 7
`53 ± 4
`91 -!-20.4
`
`.8
`.2
`.9
`.8
`.5
`.7
`.5
`
`Table II. Effect of BIBN4096BS pretreatment on
`h- aCGRP- induced symptoms
`Placebo
`BIBN4096BS
`plus
`(2.5 mg) plus
`h -aCGRP
`h -aCGRP
`(No.)
`(No.)
`
`P value
`(McNemar
`test)
`
`Symptom
`
`Flushing
`Heat sensation
`Palpitations
`Conjunctival
`injection
`Headache
`
`10
`
`8
`
`5
`9
`
`6
`
`0
`0
`0
`0
`
`0
`
`P = .002
`P=.008
`P = .063
`P=.004
`
`P = .031
`
`The flushing and conjunctival injection was based on the investigators'
`observations. Heat sensation and palpitation were reported and headache was
`systematically scored. The data shown are from the entire in- hospital study
`period.
`
`delayed headache after BIBN4096BS pretreatment.
`The delayed headache occurred 6 hours after the infu-
`sion of BIBN4096BS, lasted 3 hours, and was scored 1.
`The effect of BIBN4096BS in preventing immediate
`headache was significant (P = .034 for peak headache
`and P = .04 for AUCheadache) and in preventing the
`occurrence of any headache during the in- hospital
`phase (P = .031, McNemar test).
`After placebo pretreatment, h -aCGRP caused flush-
`ing in all participants and all but 1 had bilateral con-
`junctival injection. Eight experienced a sensation of
`
`heat. Five reported palpitations. None of these CGRP-
`changes were
`days when
`induced
`seen
`on
`BIBN4096BS was administered as pretreatment (Table
`II). AEs that could possibly be assigned to the CGRP
`receptor antagonist were located to the infusion site.
`increased significantly after
`CBF. Global CBF
`h -aCGRP on both study days (P = .007 after placebo
`and P = .009 after BIBN4096BS pretreatment). The
`increase was measured 20 minutes after the h -aCGRP
`infusion was stopped. No difference was found be-
`tween the 2 days (P = .42).
`After h- aCGRP, rCBFMCA increased significantly on
`.003 and P =
`both trial days (P =
`.01), again 20
`minutes after the h -aCGRP infusion. No significant
`difference was observed between the 2 study days (P =
`.38). Data were not corrected for PETco2, because no
`significant changes were found on either day (P = .2
`and P = .6) or between treatment days (P = .1).
`TCD. YMCA did not vary significantly over time
`(P = .3 for placebo and P = .7 for BIBN4096BS), and
`between the 2 trial days, no difference was seen (P =
`.74). On the basis of the rCBFMCA and YMCA measure-
`ments, the effect on the relative percentage diameter
`change of MCA can be estimated.' 6'18 As seen in Table
`III, a dilation of the MCA was found on both study
`days. Compared with baseline, the dilation occurring on
`placebo days reached significance at TG0 (P = .005).
`This corresponded to a diameter increase of 9.3% ±
`8.1 %. When BIBN4096BS was given as pretreatment,
`
`

`

`CLINICAL. PHARMACOLOGY & THERAPEUTICS
`2005;77(3002-13
`
`CGRP receptor antagonism and vascular headache 207
`
`Table III. Effect of BIBN4096BS pretreatment on h- aCGRP- induced cerebral hemodynamic changes
`DiametermcA (%) and
`rCBFMCA
`(mL 100 g -' min -1)
`A AreaMCA (%)
`
`VMCA (cm/s)
`
`BIBN4096BS
`plus CGRP
`
`Placebo
`plus CGRP
`
`BIBN4096BS
`pins CGRP
`
`Placebo
`plus CGRP
`
`BIBN4096BS
`plus CGRP
`
`Time point
`
`Baseline
`60 min
`
`Placebo
`plus CGRP
`78 ± 17.0
`75 ± 15.5
`
`74 ± 15.3
`75 ± 12.9
`
`90 min
`
`77 ± 14.3
`
`74-!- 15.5
`
`0 (0)
`0 (0)
`9.3 ± 8.1*
`3.8 ± 5.4
`(7.96 ± 11.3)
`(19.95 ± 17.9)
`3.0 ± 7.9
`3.4 -!- 7.5
`(7.4 ± 15.1)
`(6.8 ± 16.3)
`Values are given as mean ± SD. The percentage change in the mean diameter (left and right sides) of the middle cerebral artery (MCA) was estimated according to
`Dahl et a1.16 VMCA and rCBF on left and right sides analyzed separately showed similar results.
``Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P = .003.
`tDifference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P = .002.
`Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P = .006.
`§Difference from baseline on 2 trial days (ANOVA, Dunnett post hoc): P = .046.
`
`45.9 ± 10.5
`52.4 ± 9.4t
`
`44.7 ± 10.6
`49.1 ± 12.11:
`
`49.3 ± 12.7
`
`47.9 ± 10.1§
`
`an increase was seen as well, but it did not reach
`statistical significance (P = .2). There was no differ-
`ence between placebo and BIBN4096BS pretreatment
`(P = .17). Data were not corrected for PETCO2, because
`no significant changes were found on the trial days
`(P = .4 on both days) or between treatment days (P =
`.45).
`C -scan. A significant change in temporal artery di-
`ameter over time on placebo days was observed (P <
`.001). This significance was seen at all time points after
`the infusion of h- aCGRP.
`A significant diameter change was not seen when
`BIBN4096BS was infused as pretreatment (P = .7).
`The increase in temporal artery diameter was signifi-
`cantly inhibited by BIBN4096BS (P < .001). The
`radial artery revealed a similar finding, with a signifi-
`cant change on placebo days (P = .01) and a nonsig-
`nificant response after BIBN4096BS pretreatment
`.07). The difference between placebo and
`(P =
`BIBN4096BS was significant (P = .001) (Fig 1).
`Peripheral hemodynamics. Table IV summarizes
`data on systolic BP, diastolic BP, mean arterial BP, and
`HR. No significant time- dependent changes were seen
`in systolic, diastolic, or arterial mean BP on either trial
`day or between days. The HR increased significantly on
`placebo days (P < .001) at all time points compared
`increase was not
`seen on
`with baseline. This
`BIBN4096BS pretreatment days. The increase in HR
`was significantly inhibited by BIBN4096BS (P = .003).
`Pha macokinetics of BIBN4096BS and h- aCGRP. The
`highest measured
`concentration
`plasma
`of
`BIBN4096BS occurred just before the end of infusion
`at 9.5 minutes after the start of the infusion, with a
`mean of 170.4 -!- 25.4 ng /mL. The lowest plasma
`concentration was measured 180 minutes after the start
`
`of the infusion, with a mean of 8.7 ± 5.7 ng /mL. No
`BIBN4096BS was detected on placebo days (Fig 2).
`The pharmacokinetic parameters for BIBN4096BS
`have previously been published and are summarized.
`On the basis of administration of 5 and 10 mg intrave-
`nously for 10 minutes, BIBN4096BS had a total plasma
`clearance of 12 L/h and a terminal half -life of 2.5 hours.
`Approximately 15% of the dose was excreted un-
`changed in urine, with a mean renal clearance of 2 L /h.
`The geometric mean maximum plasma concentration
`after 2.5 mg of BIBN4096BS was 210 ng /mL.19
`At the end of the CGRP administration, a significant
`difference between treatment days was found (P <
`.001, paired t test); the CGRP concentration was 342 ±
`68 pmol/L when placebo was administered as pretreat-
`ment and 442 ± 70 pmol/L when BIBN4096BS was
`given (Fig 3). To elucidate whether this difference was
`caused by a cross -reaction between BIBN4096BS and
`our CGRP analytic kit, blood samples from 3 healthy
`following concentrations of
`volunteers with
`the
`BIBN4096BS were analyzed for CGRP: sample 1 and
`2, 0 ng /mL (control); sample 3, 1000 ng /mL; sample 4,
`500 ng /mL; sample 5, 250 ng /mL; and sample 6, 125
`ng /mL. The analysis did not reveal any correlation
`between the concentration of BIBN4096BS and CGRP.
`Thus BIBN4096BS did not cross -react with CGRP
`detection in our analytic kit.
`
`DISCUSSION
`This study has demonstrated that a specific CGRP
`antagonist, BIBN4096BS, can prevent CGRP- induced
`symptoms such as headache, flushing, heat sensation,
`and palpitations. It furthermore prevents a CGRP -
`induced increase in HR and dilatation of the superficial
`temporal and radial artery, whereas it has no significant
`
`

`

`208 Petersen et al
`
`CLINICAL PHARMACOLOGY & THERAPEUTICS
`MARCH 2005
`
`3.25 -
`
`3.00-
`
`2.75
`
`2.50-
`
`2.25-
`
`2.00-
`
`1.75-
`
`I .50-
`
`1.25
`
`1.00 -
`
`0.75 -
`
`0.50 -
`
`0.25-
`
`*
`
`*
`
`---
`
`10 minutes infusion of Placebo or BIBN4096BS
`
`20 minutes infusion of h -a CGRP (1.5µg min-1)
`
`0.00
`Baseline
`
`i
`
`i
`
`i
`
`i
`
`15
`
`60
`
`i
`
`75
`
`90
`
`r
`
`105
`
`120
`
`i
`
`135
`
`150
`
`r
`165
`
`i
`180
`
`Time (Minutes)
`
`Fig 1. Diameter (in millimeters) of superficial temporal artery and radial artery. Asterisk denotes
`significant difference (P < .05) between placebo and BIBN4096BS pretreatment on human
`a- calcitonin gene- related peptide (h- aCGRP)- induced vasodilatation (paired t test). Open squares,
`Temporal artery, placebo pretreatment; open circles, radial artery, placebo pretreatment; closed
`squares, temporal artery, 2.5 -mg BIBN4096BS pretreatment; solid circles, radial artery, 2.5 -mg
`BIBN4096BS pretreatment (mean ± SD).
`
`Table IV. Effect of BIBN4096BS pretreatment on h- aCGRP- induced systemic hemodynamic changes
`Systolic blood pressure
`Diastolic blood pressure
`Mean blood pressure
`Heart rate
`(mm Hg)
`(beats /Hain)
`(mm Hg)
`(nun Hg)
`
`Time
`point
`
`Placebo
`plus CGRP
`
`BIBN4096BS
`plus CGRP
`
`Placebo
`plus CGRP
`
`Placebo BIBN4096BS
`BIBN4096BS
`plus CGRP plus CGRP plus CGRP
`
`Placebo
`plus CGRP
`
`BIBN4096BS
`plus CGRP
`
`80 ± 7
`64 ± 5
`64 ± 8
`113 ± 6
`113 ± 8
`Baseline
`79 ± 6
`111 ± 9
`115±9
`67 ± 7
`59 ± 5
`45 min
`81 ± 7
`80 ± 6
`83 ± 9
`110± 10
`66 ± 7
`62 ± 6
`112 ± 11
`105 min
`80 ± 7
`115 ± 12
`67 ± 9
`66 ± 9
`115 ± 7
`165 min
`80 ± 7
`81 ± 10
`All values are given as mean ± SD. Only time points selected for the statistical analysis (ANOVA) are shown.
`*Significant difference between placebo and BIBN4096BS pretreatment (P < .005, paired t test).
`
`54 ± 8
`72 ± 8*
`61 ± 8*
`60 ± 8
`
`53 ± 4
`57 ± 10
`54 ± 5
`57 ± 6
`
`effect on CGRP- induced increase in CBF and dilatation
`of the MCA.
`Localization, function, and role of CGRP in mi-
`
`graine. CGRP is one of the most potent vasodilators
`known.4 In the brain, immunohistochemical studies
`located
`the peptide
`have
`to perivascular sensory
`
`

`

`CLINICAL PHARMACOLOGY &'rHERAPEUr1CS
`2005;77( 31:202-13
`
`CGRP receptor antagonism and vascular headache 209
`
`200 -
`
`180-
`
`160-
`
`140-
`
`120-
`
`100-
`
`80-
`
`60-
`
`40-
`20-
`0-
`
`C v
`N
`m
`
`Z
`m
`m
`
`0
`
`1
`
`'
`
`10 20 30 40 50 60 70 80 90 100110120130140150,;() 17010
`Time (minutes)
`
`<
`
`<
`
`i
`
`Fig 2. Plasma concentration of BIBN4O96BS after 10- minute infusion of 2.5 mg. Values are given
`as mean ± SD.
`
`Baseline
`
`End of CGRP infusion
`
`Plaxbo pno-trealnait
`
`EffiN409612S 25ngpro-tieatriad
`
`Fig 3. Plasma concentration of calcitonin gene -related peptide (CGRP) on 2 different trial days.
`Two asterisks denote a significant difference (P < .001) between BIBN4O96BS and placebo
`pretreatments (mean ± SD) (paired t test).
`
`C -fibers surrounding cerebral and extracerebral arteries
`and to the trigeminal ganglion cell bodies.20
`CGRP is present in plasma from healthy volun-
`teers at rest.2 We have previously shown that infu-
`sion of the selective CGRP receptor antagonist
`BIBN4096BS did not alter CBF or the diameter of
`cerebral and peripheral arteries.21 Hence circulating
`levels of CGRP do not seem to exert a tonic dilator
`action in these vascular beds. During a migraine
`attacks and between attacks, CGRP is increased in
`
`venous blood,22 implicating a role of CGRP in mi-
`graine pathogenesis. During an attack, the plasma
`levels were increased (2 -2.5 times) compared with
`normal controls.5 The infusion of 1.5 Rg /min of
`h -aCGRP in the current study increased the plasma
`concentration approximately 3 to 4 times.
`In healthy volunteers the infusion of CGRP has pre-
`viously been shown to induce a sensation of fullness in
`the head or mild headache,23'2