British Journal of Pharmacology (2008) 155, 1093–1103
`& 2008 Macmillan Publishers Limited All rights reserved 0007–1188/08 $32.00
`www.brjpharmacol.org
`
`RESEARCH PAPER
`
`CGRP function-blocking antibodies inhibit
`neurogenic vasodilatation without affecting heart
`rate or arterial blood pressure in the rat
`
`J Zeller1, KT Poulsen1, JE Sutton, YN Abdiche, S Collier, R Chopra, CA Garcia2, J Pons, A Rosenthal3
`and DL Shelton
`
`Rinat Laboratories, Biotherapeutics and Bioinnovation Center, Pfizer Inc., South San Francisco, CA, USA
`
`Background and purpose: Calcitonin gene-related peptide (CGRP) receptor antagonists effectively abort migraine headache
`and inhibit neurogenic vasodilatation in humans as well as rat models. Monoclonal antibodies typically have long half-lives,
`and we investigated whether or not function-blocking CGRP antibodies would inhibit neurogenic vasodilatation with a long
`duration of action and therefore be a possible approach to preventive therapy of migraine. During chronic treatment with anti-
`CGRP antibodies, we measured cardiovascular function, which might be a safety concern of CGRP inhibition.
`Experimental approach: We used two rat blood flow models that measure electrically stimulated vasodilatation in the skin or
`in the middle meningeal artery (MMA). These vasomotor responses are largely dependent on the neurogenic release of CGRP
`from sensory afferents. To assess cardiovascular function during chronic systemic anti-CGRP antibody treatment, we measured
`heart rate and blood pressure in conscious rats.
`Key results: Treatment with anti-CGRP antibodies inhibited skin vasodilatation or the increase in MMA diameter to a similar
`magnitude as treatment with CGRP receptor antagonists. Although CGRP antibody treatment had a slower onset of action
`than the CGRP receptor antagonists, the inhibition was still evident 1 week after dosing. Chronic treatment with anti-CGRP
`antibodies had no detectable effects on heart rate or blood pressure.
`Conclusions and implications: We showed for the first time that anti-CGRP antibodies exert a long lasting inhibition of
`neurogenic vasodilatation in two different rat models of arterial blood flow. We have provided strong preclinical evidence that
`anti-CGRP antibody may be a suitable drug candidate for the preventive treatment of migraine.
`British Journal of Pharmacology (2008) 155, 1093–1103; doi:10.1038/bjp.2008.334; published online 8 September 2008
`
`Keywords: antibody; CGRP; headache; migraine; neuropeptide; vasodilatation
`
`Abbreviations: CGRP, calcitonin gene-related peptide; Fab, antigen binding fragment; MABP, mean arterial blood pressure;
`MMA, middle meningeal artery; muMab, murine monoclonal antibody
`
`Introduction
`
`is a vasoactive
`Calctonin gene-related peptide (CGRP)
`neuropeptide and a key mediator in migraine headache
`(Arulmani et al., 2004). CGRP occurs in two isoforms, aCGRP
`(Amara et al., 1982; Rosenfeld et al., 1983) and bCGRP
`(Amara et al., 1985), and is expressed in the central and
`peripheral nervous system where it is localized in the
`majority of small- and medium-sized sensory afferents,
`
`Correspondence: Dr DL Shelton, Rinat Laboratories, Pfizer Inc., 230 East Grand
`Avenue, South San Francisco, CA 94080, USA.
`E-mail: dave.shelton@rinat.pfizer.com
`1These authors contributed equally to this work.
`2Current address: Arresto Biosciences, 3183 Porter Drive, Palo Alto, CA 94304,
`USA
`3Current address: MazorX Corporation, PO Box 610098, Redwood City, CA
`94061, USA
`Received 21 May 2008; revised 7 July 2008; accepted 16 July 2008; published
`online 8 September 2008
`
`(Edvinsson
`afferents
`trigeminal
`including perivascular
`et al., 1987b; Uddman et al., 1986, 1989). Upon afferent
`stimulation, CGRP is released from sensory nerve terminals
`(Holzer, 1998) contributing to neurogenic effects such as
`vasodilatation (Peroutka, 2005) and nociceptive transmis-
`sion (Storer et al., 2004). The functional system between
`trigeminal afferents and intracranial blood vessels has been
`termed the trigeminovascular system (Buzzi and Moskowitz,
`1992).
`Many lines of evidence suggest that, as well as dilating
`vessels, CGRP is involved in activating the trigeminovascular
`system in rats (Cumberbatch et al., 1999) and humans
`(Lassen et al., 2002; Petersen et al., 2005). In particular, the
`trigeminovascular system of migraineurs is more sensitive to
`exogenous CGRP (Lassen et al., 2002), and that during
`migraine and cluster headache attacks, CGRP is increased in
`
`1
`
`EX2130
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01426
`
`

`

`1094
`
`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`the venous outflow from the head, suggesting an endogen-
`ous source of CGRP (Goadsby et al., 1990; Goadsby and
`Edvinsson, 1994). This increased concentration of CGRP is
`normalized upon successful sumatriptan (5-HT1B/5-HT1D
`agonist) treatment of migraine symptoms (Edvinsson and
`Goadsby, 1994). A very important piece of evidence of the
`relevance of CGRP mechanisms in migraine has come from
`two-phase two clinical trials using different CGRP1 receptor
`antagonists, which showed these compounds to effectively
`relieve the pain of migraine in patients (Durham, 2004;
`Olesen et al., 2004; Ho et al., 2008). The combination of these
`results suggests that CGRP has an important function in
`migraine.
`if CGRP function-blocking
`to discover
`We sought
`antibodies could exert an effect on physiological CGRP
`mechanisms by using vasodilatation as an indicator of
`endogenous CGRP effects. A number of studies have been
`performed to investigate the vasoactive effects of CGRP.
`Exogenous CGRP is a potent vasodilator of cranial blood
`vessels (Edvinsson et al., 1987a). Endogenous CGRP released
`from sensory afferents in their innervation target zones also
`causes vasodilatation. Electrical stimulation of the saphe-
`nous nerve leads to locally increased blood flow in the skin
`of the dorsal medial part of the rat hind paw (Escott and
`Brain, 1993; Tan et al., 1995). In addition, this blood flow
`increase can be blocked with the CGRP receptor antagonist
`CGRP-(8-37) (Escott and Brain, 1993) or anti-CGRP antibody
`Fab fragments (antigen binding fragment) (Tan et al., 1995).
`These investigators were unable to demonstrate any effect
`of the IgG form of an anti-CGRP antibody on blood flow.
`A CGRP-dependent vasodilator response was also demon-
`strated more directly in a series of experiments by
`Williamson et al., 1997a; Williamson and Hargreaves,
`2001a). In this dural blood flow model, vasodilatation of a
`branch of the middle meningeal artery (MMA) can be
`observed visually in response to electrical field stimulation
`and this vasodilatation response was shown to be largely
`dependent on CGRP signalling (Williamson et al., 1997b).
`Several drugs that effectively abort or prevent migraine
`symptoms in humans also inhibit blood flow increase in the
`same or similar dural blood flow models (Williamson et al.,
`1997b, 2001b; Petersen et al., 2004; Akerman and Goadsby,
`2005). Hence, the blood flow increases after neurogenic
`stimulation (Williamson and Hargreaves, 2001a) can be used
`as an indicator for CGRP-mediated effects and these effects
`can have implications for migraine therapy.
`Given that CGRP is a potent vasodilator, many of the
`effects of CGRP are also associated with the cardio-
`vascular system (Brain and Grant, 2004). In rats (Ando
`et al., 1990) and humans (Lassen et al., 2002), intravenous
`administration of CGRP induces hypotension and is
`associated with decreased blood pressure and increased
`heart rate. Although intravenous injection of the CGRP1
`receptor antagonist BIBN4096BS into healthy pigs (Kapoor
`et al., 2003), primates (Doods et al., 2000) or humans
`(Olesen et al., 2004; Petersen et al., 2005) did not affect the
`heart rate or blood pressure, the long-term inhibition of
`CGRP, due to the long half-life of a function-blocking
`antibody in vivo, potentially bears a risk of cardiovascular
`effects.
`
`British Journal of Pharmacology (2008) 155 1093–1103
`
`In this study, we used a skin and a dural vasodilatation
`model in the rat to test the hypothesis that systemic treatment
`with two different CGRP function-blocking antibodies will
`result in reduced blood flow increases after electrical stimula-
`tion. Secondly, we used the skin vasodilatation model to test
`the hypothesis that a single dose of a CGRP function-blocking
`antibody will result in a long-lasting inhibition of blood flow
`increases after electrical stimulation. Thirdly, we used a
`telemetric rat model to test the hypothesis that chronic
`administration of a CGRP function-blocking antibody will not
`lead to a change in weight gain, motor activity, heart rate or
`blood pressure. Together our data provide strong preclinical
`evidence supporting the use of CGRP function-blocking
`antibodies for the preventive treatment of vascular headaches
`including migraine.
`
`Methods
`
`Animal handling
`All protocols involving animal handling were reviewed and
`approved by an ethics committee according to IACUC guidelines.
`
`Saphenous nerve assay
`Experiments were carried out as described earlier (Escott and
`Brain, 1993) with the following modifications. Sprague
`Dawley rats (200–400 g) were anaesthetized with 2% isoflur-
`1, i.v.) to
`ane and treated with bretylium tosylate (10 mg kg
`block sympathetic activity. The saphenous nerve of the left
`hind limb was exposed surgically, cut proximally and placed
`over platinum bipolar electrodes for stimulation. In between
`stimulations, the nerve was removed from the electrodes and
`was covered with plastic wrap to prevent it from drying. Skin
`blood flow was measured on the mediodorsal side of the
`hind paw using a skin probe connected to a laser Doppler
`flow metre. After a stable baseline flux (less than 5%
`variation) had been established, the distal end of the
`saphenous nerve was electrically stimulated with 60 pulses
`(2 Hz, 10 V, 1 ms, for 30 s) and repeated at 30 min intervals.
`All data were recorded using chart software. All compounds
`and controls were injected intravenously through the right
`femoral vein except in animals injected 1 and 7 days prior to
`nerve stimulation where test compounds were injected
`1,
`through the
`tail
`vein. CGRP-(8-37)
`(400 nmol kg
`1), a CGRP receptor antagonist, was injected
`1520 mg kg
`3–5 min before nerve stimulation in one group as a positive
`control for blocking CGRP-induced vasodilatation. Intra-
`group comparison was performed for each group using a
`one-way ANOVA (time) with repeated measures at each time
`point, followed by Dunnett’s multiple comparison test in
`case of significant time effect to compare each time value
`with the T0 value (Figures 2a and b). Inter-group comparison
`was performed with a non-parametric Kruskal–Wallis test
`followed by Dunn’s multiple comparison test (Figures 2c).
`
`Closed cranial window model
`Experiments were carried out as described earlier (Williamson
`et al., 1997a, b) with the following modifications: Sprague
`Dawley rats (300–400 g) were anaesthetized with pento-
`
`2
`
`

`

`1, i.p.). Anaesthesia was maintained with
`barbital (70 mg kg
`1 h
`1). The rats were tracheoto-
`i.v. pentobarbital (20 mg kg
`mized and breathing rate was maintained at 75 breaths per
`minute at a volume of 3.5 mL. The jugular vein of each rat
`was cannulated for delivery of all drugs. Blood pressure was
`monitored with a probe (Mikro-Tip catheter)
`threaded
`through the femoral artery into the abdominal aorta. After
`the head had been immobilized in a stereotactic apparatus,
`an incision along the midline was made and the skull was
`exposed. A 2 6 mm window in the left parietal area just
`lateral to the sagittal suture was made by thinning the bone
`with a dental drill and a 0.9 mm steel burr bit. Using a
`micromanipulator, a platinum bipolar electrode was lowered
`onto the surface and covered with heavy mineral oil. Lateral
`to the electrode window another window of 5 6 mm was
`created and filled with heavy mineral oil through which the
`diameter of a branch of
`the MMA was continuously
`monitored with a charge coupled device camera and a video
`dimension analyser. The rats were rested for no less than
`45 min after the preparation. Stimulations of 15 V, 10 Hz,
`0.5 ms and 30 s were performed every 30 min. All data were
`recorded using chart software. Intra-group comparison was
`performed using a one-way ANOVA (time) with repeated
`measures at each time point, followed by Dunnett’s multiple
`comparison test in case of significant time effect to compare
`each time value with the T0 value (Figures 2d).
`
`Rat telemetry
`Male Wistar rats were anaesthetized (sodium pentobarbital,
`1,
`i.p.). After a 5-cm midline incision on the
`50 mg kg
`abdomen, a DSI TA11PA-C40 (Data Sciences International,
`St Paul, MN, USA) implantable telemetric device was inserted
`into the peritoneal cavity with the catheter facing upstream
`into the descending aorta, below the renal arteries. The
`abdominal and skin incisions were closed. The animals were
`1, s.c.) and an
`given an injection of carprofen (7.5 mg kg
`1, i.m.) and returned
`injection of amoxicillin (100 mg kg
`individually to their cages. After 24 h, animals were again
`1, s.c.). One week later, the
`given amoxicillin (100 mg kg
`animals were placed individually within their cage on a RPC-
`1 or a RLA 2000 receiver (Data Sciences International) to
`record on computer mean, systolic and diastolic arterial
`blood pressure (BP, mm Hg), heart rate (HR, beats per
`minute), respiratory rate (r.p.m.), which is derived from
`pulse blood pressure, and motor activity (c.p.m.). All
`generated data were acquired using Data Sciences Interna-
`tional software. Recordings were taken in blocks of 2 min for
`2 h the day prior to the first administration (day 0). Then,
`recordings were taken in blocks of 2 min 1 h before and for
`48 h after test substance or vehicle administration on days 1,
`8 and 22. Cardiovascular effects were measured at time
`points 0, 30, 60, 90, 120, 150 min and 3, 4, 8, 12, 24, 36 and
`48 h after antibody treatment. Motor activity effects are
`presented per blocks of 1 h for the first 12 h and then for 1 h
`between the 23rd and 24th hour, between the 35th and 36th
`hour and between the 47th and 48th hour after administra-
`tion. Baseline recordings were taken again on day 35 (blocks
`of 2 min for 2 h). To validate the model, on day 36, all
`animals received an oral administration of sibutramine
`
`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`1095
`
`1 base) to induce hypertension. The absence of
`(15 mg kg
`heterogeneity between groups before the drug administra-
`tion of each 48-h test session (T0 values) was verified using a
`one-way ANOVA (group).
`Intra-group comparison was
`performed for the control group (days 1, 8 and 22) using a
`one-way ANOVA (time) with repeated measures at each time,
`followed by Dunnett’s t-tests in case of significant time effect
`to compare each time value with the T0 value (basal value
`before administration). Inter-group comparison was per-
`formed using a two-way ANOVA (group, time) with repeated
`measures at each time, followed by a one-way ANOVA
`(group) at each time in case of significant group time
`interaction, on days 1, 8 and 22. Intra-group comparison was
`also performed for the sibutramine-treated groups (day 36)
`using a one-way ANOVA (time) with repeated measures at
`each time point, followed by Dunnett’s t-tests in case of
`significant time effect to compare each time value with the
`T0 value (that is, basal value before administration). To
`confirm that anti-CGRP antibody was not eliminated and
`present
`throughout
`the study, blood samples from all
`animals were taken before the first dosing and at the end
`of each study period (days 0, 3, 10, 17, 24 and 36). The
`presence of free CGRP antibody in the serum was then
`determined as described below under
`serum analysis.
`Statistical analysis was performed on GraphPad Prism.
`
`Cell-based cAMP assay. A volume of 5 mL of 100 nM human
`or rat aCGRP in the presence of anti-CGRP antibody (0.5-
`3000 nM) was dispensed into a 384-well plate at 25 1C and
`incubated for 15 min. Human SK-N-MC or rat L6 cells from
`ATCC were dissociated with 2.5 mM EDTA, washed once and
`resuspended in stimulation buffer (20 mM HEPES, pH 7.4,
`146 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 500 mM
`3-isobutyl-1-methylxanthine); 30 000 cells in 10 mL stimula-
`tion buffer were added to each well. The plate was then
`incubated at 37 1C for 30 min before cells were lysed. A
`HitHunter cAMP XS assay kit was used to measure cAMP.
`Lysis buffer, enzyme donor (ED), enzyme acceptor (EA)
`reagents and substrate were added according to the manu-
`facturer’s instructions. Plates were analysed on a plate reader
`(Tecan, Ma¨nnedorf, Switzerland) detecting chemilumine-
`scence (1 s per well).
`
`Radioligand binding assay
`A binding assay was performed to measure the IC50 of anti-
`CGRP antibody in blocking human aCGRP from binding to
`the receptor as described earlier (Zimmermann et al., 1995;
`Doods et al., 2000). Membranes (25 mg) from SK-N-MC cells
`were incubated for 90 min at 25 1C in incubation buffer
`(50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1% BSA) containing
`10 pM human [125I]aCGRP in a total volume of 1 mL. To
`determine IC50, antibodies or unlabelled human aCGRP was
`added at varying concentrations in the incubation buffer
`and incubated at the same time with membranes and 10 pM
`human [125I]aCGRP. Incubation was terminated by filtration
`through a glass microfibre filter (GF/B, 1 mm), which had
`been blocked with 0.5% polyethylenimine. The protein-
`bound radioactivity was determined in a g-counter. Dose–
`response curves were plotted, and Ki values were determined
`
`British Journal of Pharmacology (2008) 155 1093–1103
`
`3
`
`

`

`1096
`
`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`using the following equation: Ki¼ IC50(1þ ([ligand]KD
`1)
`1,
`where the equilibrium dissociation constant KD¼ 8 pM for
`human aCGRP to CGRP1 receptor as present in SK-N-MC
`cells. The reported IC50 value (in terms of IgG molecules) was
`converted into binding sites (by multiplying it by 2) so that it
`could be compared with the affinities (KD) determined by
`Biacore/GE Healthcare, Piscataway, NJ, USA (see Table 1).
`
`affinity chromatography according to the manufacturer’s
`instructions (Qiagen, Valencia, CA, USA). Balb/C mice were
`first immunized with 50 mg ofDrosophila amnesiac coupled
`to KLH in CFA. As described for anti-CGRP antibodies, mice
`were boosted with KLH coupled in IFA. Splenocytes were
`fused and antibody-secreting clones were identified by ELISA
`as described above using Drosophila amnesiac-coated plates.
`
`Immunization and antibody screening, anti-CGRP antibodies
`Balb/C mice were first immunized with 50 mg of human
`aCGRP or bCGRP conjugated to KLH (keyhole limpet
`haemocyanin) in CFA (complete Freund’s adjuvant). After
`21 days, mice were boosted with 25 mg of human bCGRP (for
`mice first immunized with human aCGRP) or aCGRP (for
`mice first immunized with human bCGRP) conjugated to
`KLH in IFA (incomplete Freund’s adjuvant). Twenty-three
`days after the second immunization, a third immunization
`was performed with 25 mg of rat aCGRP conjugated to KLH in
`IFA. Ten days later, antibody titres were estimated using
`ELISA. Twenty-four days after the third immunization, a
`fourth immunization was performed with 25 mg of rat aCGRP
`conjugated to KLH in IFA. A final booster was performed
`with 100 mg soluble peptide (rat aCGRP) 32 days after the
`fourth immunization. Hybridomas were gained as described
`earlier (Kohler and Milstein, 1975, 1976) with the following
`modifications: splenocytes were obtained from the immu-
`nized mouse and fused with SP2 myeloma cells at a ratio of
`10:1, with polyethylene glycol 1500 (PEG-1500). The hybrids
`were plated out into 96-well plates in DMEM (Dulbecco’s
`modified Eagle’s medium) containing 15% foetal bovine
`serum, 10% hybridoma cloning factor, penicillin/streptomy-
`cin and grown for 2 days before selection was begun by
`adding hypoxanthine/aminopterin/thymidine (HAT). On
`day 14 after fusion, 100 mL supernatant from each well was
`transferred to a CGRP-coated ELISA plate, which had been
`coated overnight at 4 1C with 100 mL human or rat aCGRP
`1),
`diluted in phosphate-buffered saline (PBS; final 1 mg mL
`blocked with 0.5% albumin in PBS for 1 h at
`room
`temperature and washed four times with 200 mL per well of
`0.1% Tween 20 in PBS. After the application of cell super-
`natant or detection, antibody plates were incubated for 1 h
`and washing was repeated as above. Determination of
`antibody subtype class was done with subtype-specific
`secondary HRP rabbit anti-mouse IgG/g-specific or rabbit
`anti-mouse IgM.
`
`Antibody production and purification
`Hybridoma cells were cultured in DMEM, 10% foetal bovine
`serum containing penicillin/streptomycin, harvested and
`washed with DMEM and then injected intraperitoneally into
`pristane-primed balb/C mice at 8 106 cells per mL in
`0.5 mL. After 8–10 days, injected mice were anaesthetized
`and asphyxiated with CO2 and ascites fluid was removed
`with an 18-gauge needle connected to a syringe. Ascites fluid
`was diluted 1:2 with PBS, filtered and bound in batch mode
`to protein A resin before washing with PBS (10 times resin
`volume) and eluting with 0.1 M citric acid (pH 3). The eluate
`was neutralized with 1:10 volume 0.1 M Tris (pH 8.5) and
`dialyzed overnight in PBS 0.01% Tween 20.
`
`Analysis to determine anti-CGRP antibody concentration in serum
`samples
`Nunc Maxisorp plates were coated overnight at 4 1C with
`1) and
`100 mL of rat aCGRP diluted in PBS (final 1 mg mL
`processed as described above under anti-CGRP-antibody
`screening. Antibody standard (muMab 4901) or rat serum
`samples were diluted appropriately in 0.5% albumin in PBS
`and applied in duplicates. An HRP-conjugated goat
`anti-mouse IgG (Hþ L) (dilution: 1:10 000) was used for
`detection.
`
`Epitope mapping of anti-CGRP antibodies
`Nunc Maxisorp plates were coated overnight at 4 1C with
`100 mL of rat aCGRP, human aCGRP or human bCGRP
`fragments 1-13-COOH, 1-19-COOH, 19-27-COOH, 8-37-
`COOH, 1-36-COOH and 19-37-CONH2 diluted in PBS (final
`1) and processed as described above under anti-
`1 mg mL
`CGRP antibody screening. A constant concentration of
`1 (100 mL per well) murine monoclonal anti-
`111 ng mL
`bodies (muMab) 4901 or muMab 7E9 was applied. An HRP-
`conjugated goat anti-mouse IgG (Hþ L) (dilution: 1:10 000)
`was used for detection.
`
`Control antibody
`Histidine-tagged Drosophila amnesiac was transiently ex-
`pressed in HEK293 cells and purified from supernatants by
`
`Biacore assay
`Interaction analysis was conducted at 25 1C on a Biacore
`3000 system equipped with streptavidin-coated sensor chips
`
`Table 1 In vitro characterization of CGRP function blocking antibodies by Biacore (KD), cell-based cAMP blocking and radioligand binding
`
`Name
`
`KD rat
`25 1C (nM)
`
`cAMP blocking assay
`rat aCGRP
`
`KD human
`25 1C (nM)
`
`Binding assay,
`IC50 (nM), (in anti-CGRP binding sites)
`
`cAMP blocking assay
`human aCGRP
`
`MuMab 4901a
`MuMab 7E9
`
`1.0
`58
`
`Yes
`Yes
`
`17
`1.0
`
`125.9
`10.9
`
`Yes
`Yes
`
`Abbreviation: CGRP, calcitonin gene-related peptide.
`amuMab 4901 was identified earlier (Wong et al., 1993).
`
`British Journal of Pharmacology (2008) 155 1093–1103
`
`4
`
`

`

`using a standard Biacore running buffer. N-biotinylated
`human and rat aCGRPs were captured on individual flow
`cells at low levels (typically 100 response units) to provide
`the reaction surfaces, whereas an unmodified flow cell served
`as a reference channel. Purified Fab fragments of anti-CGRP
`antibodies were generated by digesting full-length IgGs with
`papain using an ImmunoPure Fab preparation kit according
`to the manufacturer’s instructions. Fabs were titrated over
`the chip using 1 mM as the top concentration of a two-fold
`dilution series. Association and dissociation phases were
`1 for 1 min and 5 min, respectively.
`monitored at 100 mL min
`Surfaces were regenerated with a mixture of 35% etha-
`nol þ 25 mM NaOHþ 0.5 M NaCl. Injections were duplicated
`to demonstrate that the assay was reproducible. The binding
`responses were double-referenced and fit globally to a simple
`model using BiaEvaluation v. 4.0 software. Affinities were
`deduced from the quotient of the kinetic rate constants
`(KD¼ koff kon
`1).
`
`Materials
`
`Isoflurane (Aerrane) was obtained from Baxter (Deerfield, IL,
`USA); the laser Doppler flow meter from Moor Instruments
`(Axminster, UK); pentobarbital from Ovation Pharmaceuti-
`cals (Deerfield, IL, USA); the Mikro-Tip catheter from Millar
`Instruments (Houston, TX, USA);
`the video dimension
`analyser from Living Systems (Burlington, VT, USA); chart
`software from AD Instruments (Colorado Springs, CO, USA);
`carprofen, Rimadyl; amoxicillin, Clamoxyl LA; sibutramine
`from Chempacific (Baltimore, MD, USA); the 384-well plate
`from Nunc (Rochester, NY, USA); EDTA and HEPES from
`Gibco BRL (Gaithersburg MD, USA). The following chemicals
`were all obtained from Sigma (St Louis, MO, USA): NaCl, KCl,
`CaCl2, MgCl2, KLH, 3-isobutyl-1-methylxanthine, Tris-HCl,
`MgCl2, polyethylenimine, NaOH, BSA, HAT, Tween 20,
`albumin, citric acid, Tris, pristine and bretylium tosylate.
`HitHunter cAMP XS assay kit was from DiscoveRx/GE
`Healthcare (Fremont, CA, USA) and the plate reader from
`Tecan (Ma¨nnedorf, Switzerland). Human [125I]aCGRP was
`from MDS Pharma Services (King of Prussia, PA, USA) and the
`glass microfibre filter from Perkin Elmer (Waltham, MA,
`USA). CGRP-(8-37), BCGRP, rat aCGRP, human aCGRP and
`human bCGRP fragments and N-biotinylated human and rat
`aCGRPs were all obtained from Global Peptide Services
`(Huntsville, AL, USA); CFA and incomplete Freund’s adju-
`vant form Difco (Franklin Lakes, NJ, USA); DMEM, PBS and
`penicillin/streptomycin from Gibco BRL;
`foetal bovine
`serum from Hyclone (Waltham, MA, USA); hybridoma
`cloning factor from Bioveris Corporation (Gaithersburg,
`MD, USA); CGRP-coated ELISA plate from Nunc. Subtype-
`specific secondary HRP-rabbit anti-mouse IgG/g-specific and
`rabbit anti-mouse IgM were obtained from Zymed/Invitro-
`gen (Gaithersburg, MD, USA); HRP-conjugated goat anti-
`mouse IgG from Jackson ImmunoResearch (West Grove,
`PA, USA); protein A resin from Mabselect/GE Healthcare
`(Piscataway, NJ, USA); HBS-P and Biacore 3000 system from
`GE Healthcare (Piscataway, NJ, USA); streptavidin-coated
`sensor chips from Biacore AB (Uppsala, Sweden); Immuno-
`Pure Fab preparation kit from Pierce (Rockford, IL, USA).
`
`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`1097
`
`MuMab 4901 anti-CGRP antibody was identified pre-
`viously (Wong et al., 1993) and received through a licensing
`agreement from UCLA (CA, USA).
`BIBN4096BS (C38H47Br2N9O5) was synthesized by standard
`techniques. The purity and molecular weight of
`the
`compound were determined by LC-MS (liquid chromatogra-
`phy-mass spectrometry). BIBN4096BS was dissolved in 0.1 N
`1 and then diluted in PBS at 1 mg mL
`1.
`HCl at 10 mg mL
`Aliquots were stored at 20 1C. CGRP1 receptor-specific
`antagonistic
`activity was
`determined
`experimentally
`(Ki¼ 34 pM) by CGRP binding assay as described above.
`All drug/molecular target nomenclature (for example,
`receptors and ion channels) conform with BJP’s Guide to
`Receptors and Channels (Alexander et al., 2008).
`
`Results
`
`Identification of anti-CGRP antibodies
`Previous work identified an anti-CGRP antibody (muMab
`4901) that blocks the function of rat aCGRP with high
`affinity (Plourde et al., 1993; Wong et al., 1993). To develop a
`CGRP function-blocking antibody for the preventive treat-
`ment of migraine, we intended to identify an anti-CGRP
`antibody that blocks the function of human CGRP with high
`affinity. Therefore, we fused spleen cells prepared from a
`mouse immunized with human and rat CGRPs with murine
`plasmacytoma cells and screened the supernatants of the
`resulting hybridoma cells by CGRP ELISA. We identified a
`hybridoma cell clone that secreted an antibody (muMab
`7E9) that bound aCGRP and bCGRP derived from human
`and rat. Affinities of muMab 7E9 and muMab 4901 were
`determined on a Biacore 3000 (Figures 1a–d and Table 1).
`Their affinities against rat (Figures 1a and c and Table 1) and
`human aCGRP (Figures 1b and d and Table 1) displayed
`species selectivity, in that muMab 4901 had the highest
`affinity to rat aCGRP (Figure 1a and Table 1), whereas
`muMab 7E9 had the highest affinity to human aCGRP
`(Figure 1d and Table 1). Both antibodies bound to a C-
`terminal epitope of CGRP as determined by CGRP-ELISA
`(Figures 1e and f).
`
`Identification of CGRP function-blocking antibodies
`To test both antibodies (muMab 7E9 and muMab 4901) for
`their function-blocking effect, we used cell lines expressing
`either rat (L6 cells) or human (SK-N-MC cells) CGRP1
`receptor. Rat or human aCGRP dose-dependently increased
`intracellular cAMP levels (data not shown). The blocking
`effect was determined using anti-CGRP antibodies at various
`concentrations (3000–0.5 nM) to antagonize the response to
`17 nM of rat or human aCGRP. We observed that both
`antibodies blocked the increase of cAMP induced by human
`(Table 1; data not shown) and rat aCGRP (Figure 1g and
`Table 1) in a dose-dependent manner.
`To characterize this biological inhibition further, both
`antibodies were tested in a sensitive-binding assay using
`human CGRP1 receptors from cell membrane extracts
`(SK-N-MC cells). The inhibition concentrations (IC50) were
`determined using 10 pM human [125I]aCGRP and varying
`concentrations of anti-CGRP antibody. Both antibodies
`
`British Journal of Pharmacology (2008) 155 1093–1103
`
`5
`
`

`

`1098
`
`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`muMab 4901/human αCGRP
`
`300
`
`150
`
`0
`
`100
`
`300
`
`600
`Time (s)
`
`900
`
`muMab 7E9/human αCGRP
`
`100
`
`300
`
`600
`Time (s)
`
`900
`
`muMab 7E9
`
`1-13 1-19 19-27 8-37 1-36 19-37 h1-37 r1-37
`
`muMab 7E9
`
`muMab 4901
`
`200
`
`100
`
`0
`
`1.5
`
`1.0
`
`0.5
`
`0
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`b
`
`Response (RU)
`
`d
`
`Response (RU)
`
`f
`
`OD450
`
`h
`
`% Inhibition
`
`muMab 4901/rat αCGRP
`
`0
`
`1000
`
`2000
`Time (s)
`
`3000
`
`muMab 7E9/rat αCGRP
`
`0
`
`250
`
`Time (s)
`
`500
`
`750
`
`muMab 4901
`
`1-13 1-19 19-27 8-37 1-36 19-37 h1-37 r1-37
`
`muMab 4901
`
`muMab 7E9
`
`400
`
`200
`
`0
`
`120
`
`60
`
`0
`
`1.5
`
`1.0
`
`0.5
`
`0
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`a
`
`Response (RU)
`
`c
`
`Response (RU)
`
`e
`
`OD450
`
`g
`
`% Inhibition
`
`-10
`
`-9
`
`-8
`-7
`-6
`anti-CGRP sites (log M)
`
`-5
`
`-10
`
`-9
`
`-8
`-7
`-6
`anti-CGRP sites (log M)
`
`-5
`
`Figure 1 Antibody characterization of muMab 4901 and muMab 7E9 by Biacore, CGRP-ELISA, cell-based cAMP assay and CGRP binding
`assay. (a–d) Binding kinetics of muMab 4901 and muMab 7E9 to human and rat aCGRP was determined by surface plasmon resonance using
`Biacore. The coloured lines represent the measured data and the black lines show the simulated global fits. (e, f) Epitope mapping of muMab
`4901 and muMab 7E9 by CGRP-ELISA. Both antibodies bind to a C-terminal epitope. Full-length human aCGRP, full-length rat aCGRP and
`CGRP fragments were used for binding. (g) Cell-based cAMP inhibition assay using 17 nM rat aCGRP with various concentrations of muMab
`4901 and muMab 7E9. (h) CGRP binding assay using 10 pM human [125I]-aCGRP with various concentrations of muMab 4901 and
`muMab 7E9.
`
`blocked the binding of human [125I]aCGRP in a dose-
`dependent manner (Figure 1h and Table 1). This shows that
`binding of these monoclonal antibodies (Mabs) to CGRP
`prevents its interaction with the CGRP1 receptor.
`
`Anti-CGRP antibody inhibits neurogenic vasodilatation
`Antidromic stimulation of the saphenous nerve produces a
`transient and significant increase in blood flow in the
`dorsomedial skin of the rat hind paw as measured by laser
`Doppler. This has been demonstrated to be largely a CGRP-
`dependent effect (Escott and Brain, 1993; Tan et al., 1995).
`
`British Journal of Pharmacology (2008) 155 1093–1103
`
`We used this skin blood flow model to test the efficacy of
`muMab 4901 and muMab 7E9 to block endogenously
`released CGRP in vivo. Changes in blood flow parameters
`were expressed as the area under the curve (change in
`arbitrary Doppler flux units multiplied by time). CGRP
`1, i.v.) was
`receptor antagonist CGRP-(8-37) (400 nmol kg
`used as a positive control. To determine the effect of CGRP-
`(8-37) or anti-CGRP antibody, prior to dosing for each
`animal, the baseline blood flow response was established
`with two saphenous nerve stimulations 30 min apart. On
`account its short half-life in vivo, rats were treated with
`CGRP-(8-37) 5 min before a third stimulation.
`In this
`
`6
`
`

`

`Anti-CGRP antibodies and neurogenic vasodilatation
`J Zeller et al
`
`1099
`
`paradigm, CGRP-(8-37) significantly blocked the blood flow
`response to electrical stimulation (Figure 2a). The inhibitory
`effect of CGRP-(8-37) had disappeared at
`the second
`stimulation 35 min after dosing.
`In a separate group, rats were treated with control IgG,
`muMab 7E9 or muMab 4901 after the blood flow response of
`the second stimulation had returned to baseline levels
`(approximately 10 min post-stim