Neuropharmacology 40 (2001) 911–917

www.elsevier.com/locate/neuropharm

4991W93, a potent blocker of neurogenic plasma protein

extravasation, inhibits trigeminal neurons at 5-hydroxytryptamine
(5-HT1B/1D) agonist doses夽
R. James Storer a, Simon Akerman a, Helen E. Connor b, Peter J. Goadsby a,*

a
Institute of Neurology, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK
b
GlaxoWellcome Research and Development, Stevenage, UK

Received 14 September 2000; received in revised form 3 January 2001; accepted 5 January 2001

Abstract

Triptans share the pharmacological profile of being 5-hydroxytryptamine (5-HT1B/1D) agonists and having potent anti-migraine
activity. The conformationally restricted zolmitriptan analogue 4991W93 was developed as a potent, and at low doses, specific,
non-vasconstrictor inhibitor of neurogenic dural plasma protein extravasation. Here, we sought to study the effect of 4991W93 at
plasma protein extravasation blocking and at 5-HT1B/1D agonist doses. Nociceptive cells with firing latencies consistent with Aδ
fibres were recorded in the dorsal horn region of the trigeminal nucleus caudalis after electrical stimulation of the sagittal sinus.
Both evoked (13 units) and free running (6 units) activity in cells linked to sagittal sinus stimulation were inhibited by 4991W93
delivered microiontophoretically or by intravenous administration at 10 µg/kg or 100 µg/kg, but not 0.1 µg/kg. When applied
iontophoretically, 4991W93 did not appear to have an additive effect over a 5-HT1B/1D agonist effective concentration of zolmitriptan.
These data suggest that 4991W93 is only effective at modulating the trigeminocervical complex at 5-HT1B/1D agonist doses. To
account for neurogenic dural plasma protein extravasation blockade in animal studies, 4991W93 might have non-5-HT1B/1D-based
pharmacological targets that are yet to be described.  2001 Elsevier Science Ltd. All rights reserved.

Keywords: Migraine; Plasma protein extravasation; Trigeminal nucleus; Pharmacology; 4991W93

The pathophysiology of migraine involves trigeminal
innervation of the large cranial vessels and dura mater
that may produce pain when stimulated (Goadsby,
2001). Stimulation of intracranial structures produces
pain referred to areas innervated by the ophthalmic
division of the trigeminal nerve (Feindel et al., 1960;
Nichols et al., 1990; Wolff, 1963) but the detail of the
pain production in migraine has not yet been elucidated.
Electrical stimulation of the trigeminal ganglion leads to
a sterile neurogenic dural plasma protein extravasation
(PPE) (Markowitz et al., 1987) which has been impli-
cated in the pathophysiology of migraine and the action
of acute anti-migraine drugs (Moskowitz and Cutrer,
Abbreviations: 5-HT, 5-hydroxytryptamine; PPE, plasma protein
extravasation.
夽
Presented in part at the Xth International Headache Congress,
Barcelona, Spain, 24–26 June, 1999 (Storer et al., 1999)
* Corresponding author. Fax: +44-20-7813-0349.
E-mail address: peterg@ion.ucl.ac.uk (P.J. Goadsby).
1993). However, it is far from clear what the crucial
mechanism, or mechanisms of action of triptans might
be (Humphrey and Goadsby, 1994).
Current specific acute anti-migraine compounds, the
triptans, are all 5-HT1B/1D agonists (Goadsby, 2000).
These drugs represent a significant advance in migraine
management. 5-HT1B receptors are mainly localised on
cranial blood vessels (Longmore et al., 1997; Nilsson et
al., 1999a,b; Razzaque et al., 1999), and to a lesser
extent may also be found on the coronary vessels
(Nilsson et al., 1999a,b). The rare, but well described
and potentially serious vascular side effects due to the
existence of 5-HT1B receptors on the coronary vessels
has driven the search to find anti-migraine drugs without
vasoconstrictor effects (Ferrari, 1998). Because neuro-
genic dural PPE can be blocked in rodents by pre-junc-
tional non-vasoconstrictor effects of sumatriptan (Buzzi
and Moskowitz, 1990), PPE has been used to detect non-
vascular candidate anti-migraine compounds. CP122,288
is a conformationally restricted analogue of sumatriptan

0028-3908/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 8 - 3 9 0 8 ( 0 1 ) 0 0 0 1 4 - 4
912 R.J. Storer et al. / Neuropharmacology 40 (2001) 911–917
(Gupta et al., 1995), while 4991W93 is a confor-
mationally restricted analogue of zolmitriptan with a
similar 5-HT1B/1D binding affinity (Giles et al., 1999);
both are characterised by potent inhibition of neurogenic
plasma protein extravasation at doses without vasocon-
strictor effects (Giles et al., 1999; Lee and Moskowitz,
1993). In the rat and guinea pig pre-treatment with an
i.v. bolus dose of 4991W93 resulted in a potent and dose
dependent inhibition of neurogenic plasma protein
extravasation, with ED50s of 0.077 and 0.013 µg/kg,
respectively (Giles et al., 1999). These doses are
approximately 1000-fold more potent than sumatriptan
(Buzzi and Moskowitz, 1990) or zolmitriptan (Martin et
al., 1997) in inhibiting cranial neurogenic plasma protein
extravasation PPE.
Previously, it has been shown using c-Fos immunohis-
tochemistry in cat that non-vascular but PPE-effective
in rat doses of CP122,288 had no effect on nociceptive
processing in the trigemino-cervical complex (Goadsby
and Hoskin, 1999). Furthermore, 4991W93 only inhibits
release of calcitonin gene-related peptide (CGRP) in the
cat at 5-HT1B/1D agonist doses, such as 100 µg/kg
(Knight et al., 1999a,b). In this study we examined the
ability of 4991W93 to inhibit trigeminal neurons in our
well-established model of craniovascular nociception in
the cat (Goadsby, 1999). We explored a range of doses
with and without vasoconstrictor effects to determine
whether 5-HT1B/1D agonist activity is essential for modu-
lation of trigeminocervical complex activity linked to
sagittal sinus stimulation (SSS) in cat. We looked at the
effects of 4991W93 when it was delivered microionto-
phoretically to determine its effects on activity evoked
by SSS, spontaneous activity of cells linked to SSS, and
effect of 4991W93 on activity of cells linked to SSS
evoked by dl-homocysteate as compared with ergome-
trine and zolmitriptan.
1. Methods
All studies reported were carried out under a project
license issued by the Home Office of the United King-
dom under the Animals (Scientific Procedures) Act,
1986. Fifteen cats weighing 2.99±0.54 kg (mean±SD)
were anaesthetised with α-chloralose (60 mg/kg intrap-
eritoneally; Sigma, St Louis, MO) and prepared for
physiological monitoring. Halothane (May & Baker;
Rhone-Poulenc, Dagenham, UK/Fluothane, Zeneca,
Macclesfield, UK) (0.5–3%) was administered during
surgical procedures and then discontinued during experi-
mental protocols. A catheter was placed in the femoral
artery for continuous measurement of blood pressure and
heart rate and a second catheter placed in the femoral
vein allowed for fluid and drug administration. Sup-
plementary doses of α-chloralose in 2-hydroxypropyl-β-
cyclodextrin (RBI, Natick, MA) were given intra-
venously at a rate of 5–10 mg/kg/h (Storer et al., 1997).
The cats were intubated after local anaesthesia with lig-
nocaine hydrochloride (Intubeaze, Arnolds, Shrewsbury,
UK) and fixed in a stereotaxic frame (Kopf Instruments,
Tujunga, CA). A Jackson/Foley urethral catheter was
inserted to drain the bladder providing more stable con-
trol of blood pressure through control of bladder disten-
sion, and monitoring of urine output. Core temperature
was monitored and maintained between 37 and 39°C
using a rectal thermistor probe and a low noise emitting
homeothermic heater blanket system (Harvard Appar-
atus, Holliston, MA). The cat was ventilated (Harvard
Apparatus), end-tidal CO2 was maintained at 2–4% and
inspired oxygen continuously monitored (Datex-
Ohmeda, Helsinki, Finland). Blood gas parameters were
measured at intervals throughout the experiment and
kept within normal limits.
1.1. Surgery
A midline craniotomy (20-mm diameter) and C1/C2
laminectomy were performed allowing access to the
superior sagittal sinus (SSS) and the recording site in the
spinal cord. The dura and falx cerebri adjacent to the
sinus were dissected over approximately 15 mm
allowing its isolation. A small polyethylene sheet was
inserted under the sinus and tucked under the outlying
dura. The isolated sinus was then gently lifted onto a
pair of bipolar platinum hook electrodes. To prevent
dehydration of the dura mater and underlying brain, and
to provide electrical insulation to the cortex, a polypro-
plyene dam was sealed to the bone around the craini-
otomy with dental acrylic (Vertex, Zeist, The
Netherlands) and filled with paraffin oil. Possible arte-
facts from arterial pulsation and respiratory movement
were reduced by bilateral pneumothoraces held patent
with polypropylene tubes; and through immobilisation
of the spine by clamping the thoracic spinous process,
and clamping C1 transverse processes, and the C2 spin-
ous process to the stereotaxic frame.
1.2. Stimulation and recording
To activate primary trigeminal afferents, the SSS was
supramaximally stimulated with a Grass S88 stimulator
(20–28 V, 250 µs, 0.1–1.0 Hz) connected to a stimulus
isolation unit (SIU5A; Grass Instruments, West War-
wick, RI) after the animals had been paralysed with gal-
lamine triethiodide (Concord, Essex, UK) (10–15 mg/kg
i.v., supplemented with 5–10 mg/kg/h). Extracellular
recordings were made using glass coated tungsten elec-
trodes with typical tip lengths of 10–15 µm and an
impedance of approximately 500 k⍀ measured at 1 kHz
in 0.9% saline. After limited removal of the dura and
pia mater above the recording regions on the surface of
the spinal cord, the electrode was lowered into the cord
 R.J. Storer et al. / Neuropharmacology 40 (2001) 911–917
substance caudal to the C2 roots in the area of the dorsal
root entry zone. The electrode was advanced or retracted
in the cord substance using a hydraulic microdrive (Kopf
Instruments, Tujunga, CA). Tissue culture grade agar
(3% in saline; Sigma, St Louis, MO) was set over the
exposed cord after electrode insertion to reduce cardio-
vascularly related movements. The signal from the rec-
ording electrode was fed to a Neurolog preamplifier
(Digitimer, Herts, UK) and then via a window discrimin-
ator to an A/D converter in an 80486-based microcom-
puter. Amplifier bandwidth was usually 300 Hz to 10
kHz. When discriminating between somatic and axonal
recordings, an amplifier bandwidth of d.c. to 30 kHz
was used.
In order to record the response of single units to
stimulation, post-stimulus histograms were constructed
on-line and saved to disk. A delay line and averaging
routine were used to construct averaged action poten-
tials. Selected single-unit sites were marked for sub-
sequent identification by an electrolytic lesion made by
passing a 20–25 µA d.c. cathodal current through the
recording electrode for 20–30 s. At the end of the experi-
ment the animal was given a lethal dose of sodium
pentobarbital (400 mg), followed by 10% KCl (5 ml).
The section of spinal cord containing the recording sites
was removed, fixed with neutral buffered 10% formalin,
sectioned (40 µm), and stained with cresyl violet. The
recording sites were identified by reference to the
microdrive readings and the lesion sites.
1.3. Microiontophoresis
Seven barrelled glass pipettes incorporating a central
tungsten recording electrode (Hellier et al., 1990) were
used for microiontophoresis. The recording electrode
impedance was typically 400 k⍀ with an exposed tip
length of 12 µm; after filling, the iontophoretic pipette
barrels had impedances of 60–150 M⍀. A microiontoph-
oresis current generator (Dagan 6400, Dagan Corpor-
ation, Minneanapolis, MN) provided the current for
ejecting test substances from the barrels. Retaining and
balancing currents were used routinely (Bloom, 1974).
1.4. Materials
4991W93,4S-[3-(trans-3-dimethylamino-cyclobutyl)-
1H-indol-5-yl methyl] oxazolidin-2-one and zolmitriptan
were obtained from GlaxoWellcome R&D (Stevenage,
UK); 1.0 M l-glutamate, monosodium, pH 8.0 (Sigma);
1.0 M dl-homocysteate, sodium, pH 8.0 (ICN Pharma-
ceuticals, Thame, UK); ergometrine maleate (RBI, Nat-
ick, MA). Zolmitriptan, 4991W93 and ergometrine were
ionised as cations and retained in the iontophoretic bar-
rels with small negative currents (approximately ⫺5
nA). dl-Homocysteate and l-glutamate were ionised as
anions and retained with small positive currents
913
(approximately 5 nA). Ejection currents (2–200 nA) in
directions opposite to the retaining currents were used.
Current balancing was provided through a barrel con-
taining 1 M NaCl. 4991W93 was also prepared as an
acetate and administered intravenously at 0.1, 10 and
100 µg/kg.
1.5. Statistical analysis
Group data are expressed as mean±SEM unless other-
wise stated. The firing rates before and after iontophor-
etic application of 4991W93 in spontaneously firing and
SSS-evoked units were compared with a Student’s
paired t test (SPSS v9.0). The dose–response relationship
for 4991W93 inhibition of trigeminal firing was first
examined using a repeated measures analysis of variance
(SPSS v9.0). Significance was assessed at the p⬍0.05
level.
2. Results
Animals from which data are reported in this study
had cardio-respiratory parameters that were normal for
the anaesthetised cat; notably arterial blood pH
7.39±0.01 and pCO2 3.14±0.1 kPa (mean±SD, n=15).
Neurones responded to electrical stimulation of the
superior sagittal sinus with latencies typically 8–10 ms,
consistent with transmission from A–δ fibres. All units
studied had a probability of firing of ⱖ0.4 to superior
sagittal sinus (SSS) stimulation. Extracellular responses
from 19 neuronal soma in lamina I and II of the trigemi-
nocervical complex were recorded. Sodium or chloride
ions ejected from a barrel containing saline did not acti-
vate or suppress any units while all cells reported were
activated by dl-homocysteate or l-glutamate.
2.1. Iontophoretic application of 4991W93
Units responding to SSS stimulation (n=5) could be
reversibly inhibited by 4991W93 delivered microionto-
phoretically (ejection current 50 nA for 5 min) onto neu-
ronal soma by 43±12% (t4=3.5; p⬍0.03) Fig. 1). Spon-
taneously firing units in the trigeminocervical complex,
also linked to SSS stimulation (n=6), could be signifi-
cantly inhibited by 26±2% (t5=11.3; p⬍0.001) with
microiontophoretic application of 4991W93 (50 nA;
Fig. 2).
2.2. Iontophoretic application of 4991W93 —
comparison with other 5-HT1B/1D agonists
Spontaneously firing units in the trigeminocervical
complex, also linked to SSS stimulation (n=6), could be
activated by ejection of dl-homocysteate or l-glutamate.
The same units were inhibited by zolmitriptan, 4991W93
914 R.J. Storer et al. / Neuropharmacology 40 (2001) 911–917

Fig. 2. Reversible suppression of spontaneous (SSS)-linked neuronal

firing in the trigeminocervical complex by microiontophoretically
applied 4991W93 (40 nA for 10 s pulses, bars). Firing rate is plotted
for 1 s bins. Saline had no effect at 20 nA and dl-homocysteate (⫺2
nA) increased the firing rate (Figs. 3 and 4).

Fig. 1. Post-stimulus histograms illustrating typical reversible inhi-

bition of superior sagittal sinus (SSS)-evoked neuronal activity in the
trigeminocervical complex by 4991W93 applied microiontophoret-
ically to the cell soma. Panel (a) illustrates control activity with SSS
stimulation tightly linked; panel (b) demonstrates the effect of locally
applied 4991W93 (ejection current 50 nA for 5 min); panel (c) demon-
strates the recovery post-stimulus histogram 20 min after ejection of
4991W93 was ceased.

and ergometrine (Fig. 3). 4991W93 had no additional

effect on neuronal inhibition above that seen with either
zolmitriptan or ergometrine.

2.3. Intravenous administration of 4991W93

Considering all doses administered, units responding

to SSS stimulation (n=9) could be inhibited by i.v.
4991W93 (F1,8=26.3; p⬍0.001). After i.v. adminis-
tration of 4991W93 at 10 µg/kg (18±4%) (Fig. 4) and
100 µg/kg (29±2%) (8 units) inhibition was observed,
but not at 0.1 µg/kg (⫺6±4%) (4 units). 4991W93 had
no systematic effect on resting cardiovascular para-
meters.
3. Discussion
These data demonstrate that 4991W93 at 5-HT1B/1D
agonist doses can inhibit trigeminocervical neuronal
activity evoked by stimulation of the superior sagittal
Fig. 3. Reversible suppression of spontaneous superior sagittal sinus
(SSS)-linked neuronal firing in the trigeminocervical complex by
microiontophoretically applied zolmitriptan (100 nA), 4991W93 (100
nA), ergometrine (60 nA), saline (50 nA), dl-homocysteate (⫺3 nA).
sinus (SSS). Furthermore, the inhibitory effect of
4991W93 can be explained, at least in part, by direct
inhibition of neurones within the trigeminocervical com-
plex since a potent reversible inhibition was observed
after iontophoresis of 4991W93 onto both SSS-evoked
and spontaneously active neurones. However, there was
no additional effect of 4991W93 over zolmitriptan, nor
over a prototypic ergot-derivative, ergometrine, suggest-
ing that 4991W93’s central inhibitory effects can be
 R.J. Storer et al. / Neuropharmacology 40 (2001) 911–917
Fig. 4. Post-stimulus histograms illustrating the inhibition of (panel
(a) — control) superior sagittal sinus (SSS)-evoked trigeminocervical
complex activity (panel b) 20 min after 10 µg/kg i.v. 4991W93.
accounted for by 5-HT1B/1D agonist activity. Moreover,
4991W93 had no effect on central trigeminal activity
when administered in doses that potently inhibit neuro-
genic plasma protein extravasation in rodents, suggesting
that this mechanism does not play an important role in
the SSS stimulation model system in the cat.
In contrast to sumatriptan (Buzzi and Moskowitz,
1990) or zolmitriptan (Martin, 1997), which produce
vasoconstriction at the same doses required for inhibition
of neurogenic dural plasma protein extravasation,
4991W93 has only very limited vasoconstrictor activity
(Giles et al., 1999). No significant blood pressure
changes were produced in rats or guinea pigs following
administration of doses up to 30–100 µg/kg intra-
venously, although significant increases in blood press-
ure were observed with higher doses (ⱖ300 µg/kg).
There were no significant changes in ear skin vascular
conductance following the i.v. administration doses of
4991W93 up to 100 µg/kg, although small decreases (up
to 11±8%) were observed at higher doses (Honey, A.,
GlaxoWellcome R&D, unpublished data). 4991W93 is,
however, a potent inhibitor of neurogenic plasma protein
extravasation (Giles et al., 1999).
Each of zolmitriptan (Goadsby and Hoskin, 1996), riz-
atriptan (Cumberbatch et al., 1997) and naratriptan
(Cumberbatch et al., 1998; Goadsby and Knight, 1997)
modulates nociceptive neurotransmission in the trigemi-
nocervical complex, as does 4991W93 when adminis-
tered at 5-HT1B/1D agonist doses (Goadsby, 2000). Inhi-
bition of SSS-evoked activity in the trigeminocervical
complex in this study was only seen at doses which had
cranial vasoconstrictor effects (Giles et al., 1999).
915
4991W93 at 10 µg/kg is just sub-threshold for causing
cranial vasoconstriction in cats, but 100-fold the dose
required to maximally inhibit dural plasma protein
extravasation (PPE) in rodents. The 0.1 µg/kg dose was
ineffective at inhibiting SSS-evoked activity but is a
maximally effective PPE inhibiting dose in rodents
(Giles et al., 1999). Similarly, the conformationally
restricted sumatriptan analogue CP122,288 (Gupta et al.,
1995) is a potent inhibitor of neurogenic PPE in rodent
(Lee and Moskowitz, 1993). However, as with
4991W93, it does not block SSS-evoked trigeminocerv-
ical activity in the cat (Goadsby and Hoskin, 1999) at
low doses that are effective in the rodent PPE model.
These data are consistent with clinical observations that
4991W93 was not effective as an acute anti-migraine
compound at non-vasoconstrictor doses up to 2.5 mg
intravenously (Earl et al., 1999) and similarly, that
CP122,288 is ineffective in the treatment of acute mig-
raine (Roon et al., 2000). Whether contrast between the
rat data and the cat data/human outcome is due to a dis-
tinct receptor agonist difference, or intra-cellular events,
such as differences in coupling efficiency or variations
in second message utilisation, are not clear.
A reliable marker of trigeminovascular activation in
humans during trigeminal ganglion stimulation
(Goadsby et al., 1988) or primary neurovascular head-
ache, such as migraine (Goadsby et al., 1990) or cluster
headache (Fanciullacci et al., 1995; Goadsby and
Edvinsson, 1994), is elevation of calcitonin gene-related
peptide (CGRP) levels in plasma. Stimulation of the
superior sagittal sinus (SSS) leads to elevation of CGRP
levels in the cranial circulation of the cat (Zagami et
al., 1990). Effective anti-migraine compounds, such as
sumatriptan (Goadsby and Edvinsson, 1993) and zolmi-
triptan (Goadsby and Hoskin, 1996), block CGRP
release associated with SSS stimulation in the cat at
clinically relevant doses. In contrast, CP122,288, which
is clinically ineffective (Roon et al., 2000), does not
block CGRP release in the cat model at a dose relevant
to PPE (Knight et al., 1999a,b). Similarly, 4991W93
does not inhibit SSS-evoked CGRP release in the cat
unless it is administered in 5-HT1B/1D agonist doses
(Knight et al., 1999a,b).
SSS-evoked activity in the trigeminocervical complex
was also inhibited by 4991W93 applied directly on neu-
ronal cell bodies by microiontophoresis as previously
seen with the serotonin 5-HT1B/1D agonists sumatriptan
and the non-specific 5-HT1 agonist, ergometrine (Storer
and Goadsby, 1997). Each of these observations makes
clear the possibility that specific anti-migraine com-
pounds may act in the brain. The effect of 4991W93
administered intravenously was comparable to the pre-
viously published effects of zolmitriptan in the same
model of SSS stimulation in the cat (Goadsby and Hos-
kin, 1996). dl-Homocysteate or l-glutamate activated
and spontaneous activity of cells linked to SSS were also
916 R.J. Storer et al. / Neuropharmacology 40 (2001) 911–917
inhibited by microiontophoretically applied 4991W93.
The inhibitory effects of ionotophoretically applied
4991W93 could not be differentiated from those of sum-
atriptan. In this model no evidence for an additional non-
5-HT1B/1D effect of 4991W93 equivalent to the elusive
receptor mediating the inhibition of cranial neurogenic
PPE in rodents, where 4991W93 is extremely potent,
could be detected.
The data presented here suggest that 4991W93 acts as
an agonist at inhibitory 5-HT1B/1D receptors on neurones
in the trigeminocervical complex to reduce nociceptive
neurotransmission. Since microiontophoretic adminis-
tration is spatially selective and allows the delivery of
such small quantities of test compounds that the effects
seen are relatively short lasting and therefore reversible,
it is clear the actions of the compounds tested are within
the trigeminal nucleus and the effects are predictably
reversible. Given that CP122,288 and 4991W93 do not
inhibit sagittal sinus evoked trigeminal nucleus activity
after i.v. administration at doses that block neurogenic
plasma protein extravasation in the rodent, it is unlikely
that this process plays a role in trigeminal nociceptive
neurotransmission in the cat. It cannot be excluded that
an undetected PPE-like process occurs in the cat, but
since the positive evidence presented here adequately
accounts for the effects of 4991W93 in terms of 5-
HT1B/1D agonist activity, any putative PPE-like process
must be relatively insignificant in this model. Moreover,
since both CP122,288 and 4991W93 are without any
efficacy in migraine at doses devoid of 5-HT1B/1D recep-
tor activity, and given that the phenomenon of neuro-
genic inflammation has not been observed when directly
studied in migraine in humans (May et al., 1998), its
role may not be pivotal in migraine pathophysiology.
Our conclusions do not exclude a role for PPE in dural
sensitisation related to movement sensitivity that is such
a remarkable aspect of the clinical syndrome (Burstein
et al., 2000), but do suggest a more modest role for the
phenomenon than has been previously considered.
Acknowledgements
The authors thank Michele Lasalandra, Yolande
Knight and Paul Hammond for their assistance in con-
ducting the studies. Dr G. Martin’s insight-producing
comments during the formative stages of this project are
gratefully acknowledged. This work was supported by
GlaxoWellcome R&D and The Wellcome Trust. PJG is
a Wellcome Senior Research Fellow.
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