Design, synthesis and evaluation in an LPS rodent model of neuroinflammation of a novel 18F-labelled PET tracer targeting P2X7

Article abstract of DOI:10.1186/s13550-017-0275-2

Background: The P2X7 receptor has been shown to play a fundamental role in the initiation and sustenance of the inflammatory cascade. The development of a novel fluorine-18 PET tracer superior and with a longer half-life to those currently available is a

Full text of DOI:10.1186/s13550-017-0275-2

Fantoni et al. EJNMMI Research (2017) 7:31
DOI 10.1186/s13550-017-0275-2
ORIGINAL RESEARCH
Open Access
Design, synthesis and evaluation in an LPS
rodent model of neuroinflammation of a
novel 18F-labelled PET tracer targeting P2X7
Enrico Raffaele Fantoni1, Diego Dal Ben2, Simonetta Falzoni3, Francesco Di Virgilio3, Simon Lovestone4
and Antony Gee1*
Abstract
Background: The P2X7 receptor has been shown to play a fundamental role in the initiation and sustenance of
the inflammatory cascade. The development of a novel fluorine-18 PET tracer superior and with a longer half-life
to those currently available is a promising step towards harnessing the therapeutic and diagnostic potential
offered by this target. Inspired by the known antagonist A-804598, the present study outlines the design via
molecular docking, synthesis and biological evaluation of the novel P2X7 tracer [18F]EFB. The tracer was
radiolabelled via a three-step procedure, in vitro binding assessed in P2X7-transfected HEK293 and in B16 cells by
calcium influx assays and an initial preclinical evaluation was performed in a lipopolysaccharide (LPS)-injected rat
model of neuroinflammation.
Results: The novel tracer [18F]EFB was synthesised in 210 min in 3–5% decay-corrected radiochemical yield (DC RCY),
>99% radiochemical purity (RCP) and >300 GBq/μmol and fully characterised. Functional assays showed that the
compound binds with nM Ki to human, rat and mouse P2X7 receptors. In vivo, [18F]EFB displayed a desirable distribution
profile, and while it showed low blood–brain barrier penetration, brain uptake was quantifiable and displayed significantly
higher mean longitudinal uptake in inflamed versus control rat CNS regions.
Conclusions: [18F]EFB demonstrates strong in vitro affinity to human and rodent P2X7 and limited yet quantifiable BBB
penetration. Considering the initial promising in vivo data in an LPS rat model with elevated P2X7 expression, this work
constitutes an important step in the development of a radiotracer useful for the diagnosis and monitoring of clinical
disorders with associated neuroinflammatory processes.
Keywords: P2X7, F-18, LPS, EFB, Radiosynthesis, Molecular modelling
Background
enable further understanding of the role of this import-
The P2X7 receptor has been identified as a key player in ant receptor, as well as providing a measurable outcome
the neuroinflammatory cascade leading to the onset and for clinical trials and providing a new avenue for the
progression of a wide range of CNS conditions [1]. For diagnosis of neuroinflammation.
example, in Alzheimer’s disease, it is crucial in mediating
Over the last decade, a number of PET tracers have
the secretion of interleukin-1β following beta-amyloid been developed and evaluated (Fig. 1). Neither [11C]A-
stimulation [2]. Likewise, neuropathic pain is directly re- 740003 nor [11C]SMW64-D16 showed appreciable brain
lated to P2X7’s action on IL-1β maturation [3] and ca- uptake; however, both succeeded in recognising the site of
thepsin release [4]. Therefore, the development of a inflammation in two different rodent models when exam-
non-invasive selective PET ligand targeting P2X7 would ined by in vitro autoradiography [5, 6]. [11C]GSK1482160,
however, shows greater promise due to its efficient radio-
* Correspondence: antony.gee@kcl.ac.uk
synthesis and strong P2X7 selectivity and blood–brain
1Department of Imaging Sciences and Biomedical Engineering, King’s
barrier (BBB) permeability, but its preclinical evaluation is
College London, St Thomas’ Hospital, 4th floor Lambeth Wing, SE1 7EH
still in progress [7].
London, UK
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.

Fantoni et al. EJNMMI Research (2017) 7:31
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Fig. 1 Chemical structures of A-804598 and of the P2X7 PET tracers described to date in the literature, including our novel P2X7 ligand [18F]EFB
This study presents the novel fluorine-18-labelled PET Chemistry
tracer [18F]EFB 2-cyano-1-(4-[18F]fluorobenzyl)-3-(quino- Hereafter, the synthesis of precursor 6 and of the reference
lin-5-yl)guanidine developed from the known blood–brain compound EFB is briefly described (Fig. 2). Full com-
barrier permeable P2X7 antagonist A-804598 [8, 9]. Cya- pound characterisation is described in Additional file 1,
noguanidines have been investigated at length due to their together with the synthesis of compounds 4 and 2.
strong pharmacological activity and affinity for P2X7
For the synthesis of methyl N′-cyano-N-5-quinolinylcar-
across a number of analogues. This includes the discovery bamimidothioate (6), a mixture of 5-isothiocyanatoquinoline
that an N-quinoline substituent results in higher binding (2, 6.38 g, 34.3 mmol) and sodium hydrogencyanamide
affinity compounds compared with analogues with smaller (2.19 g, 34.3 mmol) in dimethylformamide (30 mL) was
or less hydrophilic aromatic rings [10, 11], and that a 1- stirred at room temperature for 1 h. Methyl iodide
carbon guanidine-phenyl linker is more desirable than (2.13 mL, 34.3 mmol) was added at 0 °C, and the reaction
longer and more functionalised analogues [12–14]. In- was stirred at room temperature for 2 h. The reaction was
stead, the cyanoguanidine core is thought to provide rigid- poured into water and stirred further for 20 min. The or-
ity to the molecular structure by hydrogen bonding with ange precipitate was filtered and washed with water. Further
the receptor-binding pocket [15]. Hence, compound EFB purification was performed by flash chromatography on
is ideally placed to display an optimal fit into the binding SiO2 (EtOAc:CH2Cl2, 1:1) to obtain methyl N′-cyano-N-
pocket while featuring a fluorine-18 label. The work pre- (quinolin-5-yl)carbamimidothioate (compound 6) as an or-
sented here includes a molecular docking analysis of the ange solid in 70.0% yield (5.77 g).
structure–affinity relationship (SAR) between the receptor
In line with previously published procedures [8], for
ATP-binding pocket and both the benzyl-desmethylated the synthesis of 2-cyano-1-(4-fluorobenzyl)-3-(quino-
derivative of A-804598 and compound EFB. The synthesis lin-5-yl)guanidine (EFB), a mixture of 0.62 mL 4-
and radiolabelling of all compounds required for the syn- fluorobenzyl amine 3 (5.36 mmol, Alfa Aesar) and
thesis of [18F]EFB are also described. Finally, the biological 1.12 mL triethylamine (8.04 mmol) was added to com-
evaluation of [18F]EFB in P2X7-transfected cells and in an pound 2 (1 g, 5.36 mmol) in 25 mL anhydrous tetra-
intraperitoneal lipopolysaccharide (LPS) preclinical model hydrofuran (THF) and stirred for 1 h at room
of inflammation is presented.
temperature. Following the addition of mercury acetate
(5.36 mmol) and sodium hydrogen cyanamide
(16.1 mmol), the mixture was stirred for 24 h until dark
black. The mixture was filtered under reduced pressure
through a short pad of celite and washed with acetone
Methods
Molecular modelling
All molecular modelling studies were performed on a (30 mL). The filtrate was concentrated by rotary evap-
Core i7 CPU (PIV 2.20 GHZ) PC workstation. Hom- oration and purified by flash chromatography (SiO2, 8–
ology modelling studies of the human P2X7 (hP2X7) 10% MeOH in Et2O, then 0–25% EtOAc in 10% MeOH
were carried out using the closed state zebrafish in Et2O). The product was then dried by rotary evapor-
P2X4 (zP2X4) receptor as a template, as described in ation and recrystallized from acetone as a white powder
Dal Ben [15]. Molecular Operating Environment in 8% yield (140 mg).
(MOE by C.C.G., Montreal, CA, version 2012.10)
suite was employed for this task. The ligand struc- Radiochemistry
tures were docked into the binding site of the P2X7 Chromatography
receptor using the AutoDock tool (PyrX interface) by Chemical and radiochemical purities were assessed by
the Scripps Institute [16, 17]. All docking conformations analytical radio- and UV-HPLC as outlined in Table 1.
were then imported in MOE, and the partial charges were The chemical identity of [18F]EFB was confirmed by com-
assigned by semi-empirical RHF/AM1 calculations using parison with its respective standard reference compound,
the MOPAC package [18] implemented in MOE. Further while intermediate radioactive species were confirmed by
details are provided in Additional file 1.
thin-layer chromatography (TLC). The molar radioactivity

Fantoni et al. EJNMMI Research (2017) 7:31
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Fig. 2 Radiosynthetic route to [18F]EFB. a [18F]KF.K222, 15 min, 110 °C in DMSO followed by tC18 Sep-Pak SPE. b 2:1 w/w NaBH4/Co(OAc)2 in 2 mL:1 mL
THF/H2O, 5 min followed by Strata-X-CW purification in acidic media. c Excess triethylamine, THF, 140 °C, HPLC purification and concentration on a light
C18 Sep-Pak SPE cartridge
was determined via a standard UV curve with a sample of
the product in its final formulation. All radiochemical [18F]5 in THF was transferred without further purification
For the synthesis of 4-fluorobenzyl amine ([18F]3), 2 mL
yields (RCY) are reported as decay-corrected values.
into a reaction vial containing 10 mg NaBH4 and 5 mg
Co(OAc)2. To this, 1 mL water was added and the mixture
was mixed and allowed to react at RT for 3 min. Next, the
mixture was diluted in 40 mL pH 6.2 phosphate buffer
Radiosynthesis
[18F]Fluoride was produced using an RDS112 cyclotron (6.7:1 1 M NaH2PO4:1 M Na2HPO4, I = 9 mM) so as to
at King’s College London PET Centre by the 18O(p,n)18F limit the organic solvent to 5%. The mixture was filtered
reaction via proton irradiation of enriched (95%) 18O (0.2-μm pore Whatman Spartan filters) and loaded on a
water. 1–1.5 GBq of aqueous [18F]fluoride were trapped Phenomenex Strata-X-CW SPE cartridge (8B-S035-FBJ).
in a Sep-Pak Accell Plus QMA cartridge (WAT023525) Following a 5 mL acetonitrile wash, a purple band charac-
pre-activated with 5 mL 1 N NaOH, 10 mL water and teristic of compound [18F]3 formed in the cartridge and
5 mL 1 N K2CO3 followed by 10 mL water. The fluoride could be entirely eluted in the second of three aliquots of
was released with 0.65 mL 30 mM:15 mM Kryptofix 0.6–0.7 mL 5% TFA acetonitrile in 45–55% RCY (22–34%
222/potassium carbonate dissolved in 85:15 acetonitrile/ overall RCY) and >93% RCP. Radio-TLC (SiO2, nBuOH/
water. The solution was dried three times at 90 °C for HOAc–H2O (4:1:1), Rf = 0.4; lit.[19] 0.4).
5 min in a Wheaton V vial and under a flow of nitrogen.
Finally, for the synthesis of [18F]EFB, 0.4–0.7 mL of [18F]3
For the synthesis of 4-fluorobenzonitrile ([18F]5), the in 5% TFA acetonitrile were neutralised with 2 eq. triethyla-
method reported by Koslowsky [19] was followed with mine (110–112 μL) and added to 11 μmol of compound 6
some modifications. To dried [18F]fluoride (1–1.5 GBq), (3.6 mg) in a sealed vessel. The reaction vessel was heated
4-cyano-N,N,N-trimethylanilinium trifluoromethansulfo- at 160 °C for 15 min, cooled to 50 °C and the solvent was
nate 1 (2 mg) in DMSO (0.3 mL) was added and reacted concentrated at 50 °C under a stream of nitrogen for 7–
for 15 min at 110 °C. Once the reaction was completed, 10 min. Next, the crude mixture was diluted in 2 mL water
the mixture was diluted with water (30 mL) and passed ensuring <15% acetonitrile was present and injected in an
through a Waters Sep-Pak tC18 Plus Long SPE cartridge HPLC semi-prep C18 column to provide isolation of the
(WAT036800). The cartridge was washed with add- pure product in >99% RCP and >300 GBq/μmol molar
itional water (5 mL) before elution of compound [18F]5 radioactivity. The product was diluted to 30–50 mL in
in 2 mL THF in 40–60% RCY and >95% radiochemical water and passed through a light C18 Short Sep-Pak SPE
purity (RCP). Radio-TLC (SiO2, petroleum ether/ethyl cartridge (WAT023501) for concentration in 200–500 μL
acetate (1:1), Rf 0.7; lit. [19] 0.7).
ethanol. The resulting product was obtained in high purity
Table 1 HPLC methods
Method
EFB Rt
(min)
Flow rate
(mL/min)
Column
Mobile phase gradient
Mobile phase
UV (nm)
254
A
51’10”
3
Agilent eclipse XDB C18,
9.4 × 250 mm, 5 μm, 100 Å
15% B for 5 min; 15–35% B for 1 h; 35% B for
2 min; 35–20% B for 2 min; 20% B for 1 min
A: H2O
B: MeCN
A: H2O
B
19’10”
1
Kinetex Phenomenex XB-C18 with
guard, 4.6 × 150 mm, 5 μm, 100 Å
30 s at 20% B; 23.5 min 20–33.5% B; 3 min
33.5–70% B; 2 min 70% B; 1 min 70–20% B
300
B: MeCN

Fantoni et al. EJNMMI Research (2017) 7:31
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and 4.0% overall RCY in 3 h and 30 min from start of syn- Preclinical
thesis. Purity tests were performed with HPLC method B Animals
(Table 1). A stability test performed in 0.05% DMSO at Male Sprague–Dawley rats of 240–270 g were bought
37 °C for 2 h indicated no radioactive by-product forma- from Charles River Laboratories International, Inc., and
tion and high retention of its UV-active fingerprint used within 90 days. The animals were housed with a
(254 nm, Additional file 1: Figure S1).
week of acclimatisation before procedures were done
under pathogen-free conditions in a temperature- and
humidity-controlled environment and given access to
food and water ad libitum. All experiments were carried
out in the Rayne Institute, St. Thomas’ Hospital, King’s
College London, between 2015 and 2016 and according
to the Animals Act 1986 and with the approval of the
King’s College research ethics committee.
Optimisation of the reaction conditions
Iterative optimisation steps were performed with the use
of 100–150 MBq starting [18F]fluoride and were aimed at
improving the radiochemical yield of each step of the
radiosynthesis. The influence of the following parameters
was explored by TLC (compounds [18F]5 and [18F]3) and
HPLC analysis ([18F]EFB) of 50 μL reaction mixture sam-
ples filtered and diluted to 200 μL in water:
Injections
Twenty-four hours prior to scanning, all rats were pre-
treated by an intraperitoneal injection of 0.5 mg/kg LPS
from Escherichia coli (0111:B4 L2630, Sigma Aldrich) at
0.5 mg/mL or with 1 mL/kg sterile saline solution and
weighed before the start of the PET imaging procedure.
Then, the saline-dissolved reformulated radiotracer was
injected under isoflurane anaesthesia in 0.2–5 MBq, cor-
responding to an administered dose of 0.5–16 ng/kg,
and >300 GBq/μmol molar radioactivity via tail vein can-
ꢀ Temperature
ꢀ Precursor concentration
ꢀ Time
ꢀ Purification technique
ꢀ Catalytic base concentration
In vitro affinity assays
The materials and methods employed in this section nulation, followed by a 0.15 mL saline flush. Details on
are described in full in Cabrini 2005 [20] and in Add- anaesthesia and PET/CT acquisition and analysis are re-
itional file 1. Briefly, HEK293 were transfected with ported in Additional file 1. Immediately after imaging,
the calcium phosphate method described in Rizzuto the animals were culled by terminal anaesthesia and cer-
[21] and Morelli [22]. Stably P2X7-transfected, single- vical dislocation and dissected. Organs and body fluids
cell-derived clones were obtained by limiting dilu- were collected for gamma counting (LKB Wallac 1282)
tions. Transfected cells were then kept under selec- and biodistribution analysis.
tion in the presence of 0.2 mg/ml G418 sulfate.
Instead, B16 cells natively expressed the mouse P2X7 Statistical analysis
receptor. Experiments were performed in a Ca2+-con- Data are expressed as mean standard error mean
taining (1 mM) saline solution (150 mM NaCl, 5 mM (SEM) from three independent replicates, unless other-
KCl, 1 mM MgCl, 5.5 mM glucose, 20 mM Hepes, wise indicated. Statistical analysis of the %ID/g variation
pH 7.4) at 37 °C with a PerkinElmer fluorimeter. in the PET scans and biodistributions were performed
Cells were loaded with 1 μM of the fluorescent indi- with GraphPad Prism 5.0 (San Diego, USA) as a para-
cator Fura-2/AM for 30 min in the presence of metric two-way ANOVA test with Bonferroni post-test,
250 μM sulfinpyrazone to reduce excretion of intra- while the mean longitudinal percentage injected dose
cellular Fura-2. Cells were then rinsed and resus- per gram (%ID/g) and the post-LPS treatment weight
pended in saline solution at a final concentration of variation were analysed with a Student’s t test. The aster-
500,000/ml [23]. Excitation ratio and emission wave- isk indicates P < 0.01; **P < 0.005; ***P < 0.001. Where
lengths were 340/380 and 505 nm, respectively. The unspecified, no significance was found.
calcium ionophore ionomycin (1 μM) was added at
the end of each time course as an internal assay con- Results
trol. Inhibitory activity was measured as intracellular Molecular docking
Ca2+ ([Ca2+]i) flux variation from baseline while in Compound EFB as well as A-804598 and its benzyl-
the presence of 100% activating BzATP (100 μM for desmethylated derivative were docked at the hP2X7
hP2X7 and rP2X7, 200 μM for mP2X7). Inhibition binding site, and the docking conformations of the three
was measured on both peak and plateau phases. The compounds are shown in Fig. 3.
final value was chosen as the most accurate of the
two measurements, or alternatively from the plateau. only marginally affected the strength of ligand–receptor
A-740003 was purchased from TOCRIS Bioscience. docking scores due to the decreased interaction with the
The removal of the benzyl methyl group of A-804598

Fantoni et al. EJNMMI Research (2017) 7:31
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Fig. 3 Molecular docking of compounds A-804598 (left), its benzyl-desmethylated derivative (middle) and EFB (right) into the hP2X7 receptor
ATP-binding pocket
hydrophobic pocket between Y288 and F218 (Table 2). Optimisation of the reaction conditions
Nevertheless, it did not alter the three-dimensional con- Firstly, in order to minimise the cold carrier reactants in
formation of the substrate if not by reducing the struc- the reaction pot as a means to achieve high molar radio-
tural constraint on the external phenyl group to sit activities, the procedure for the synthesis of [18F]5 was
between F218 and K66, leading to minor losses in dock- modified to suit 1–2 mg of precursor (3.2–6.4 μmol). Se-
ing scores. These were not substantially modified either quential modification of the time and temperature of re-
by the insertion of a para-fluorine atom useful as a ra- action enabled to conclude that 6.4 μmol precursor
diolabel, which appeared to comfortably sit at the outer reacted with [18F]fluoride at 110 °C for 15 min afford a
opening of the pocket.
maximal RCY of 75.9 4.7%, superior to that described
elsewhere [19] (Table 3).
Chemistry
The nitrile reduction of [18F]5 to [18F]3 was monitored
The synthesis and characterisation of precursors 4 and 6 by TLC ([18F]3 Rf 0.7) at three time points while main-
as well as of the reference compound EFB (Fig. 4) were taining the reagent proportions indicated in Koslowsky
successfully carried out. The synthesis of 6 and EFB in- [19]. As indicated in Table 4, maximal RCYs were ob-
volved two steps starting from 5-aminoquinoline and was tained with 3 min of exposure of the reagents to a re-
achieved in 43.4 and 5.6% overall yields, respectively.
ductive environment.
In our hands, the isolation of [18F]3 by Waters C18 plus
Sep-Pak solid phase extraction in basic media [19] gave
Radiochemistry
The radiofluorination procedure loosely followed methods poorly consistent product recoveries of <50%. Instead,
previously reported by Koslowsky [19] and Turkman [24]. much higher reliability and up to 60% recoveries were
The protocol, described in the materials and methods, achieved with a Phenomenex Strata-X-CW weak cation
was modified to increase the radiosynthetic reliability by exchange cartridge as described in Additional file 1.
iterative reaction parameter modifications and alterations
to the purification strategies.
For the synthesis of [18F]EFB, iterative variation of the
reaction conditions was performed while maintaining
11 μmol of reagent 6 in a 1 mL solution of compound
[18F]3 in MeCN + 5% TFA in the presence of 2 eq. of
triethylamine with respect to TFA. Despite the lack of a
statistically significant variation in yield, maximal
product formation and near-total reactant consump-
tion was achieved at 160 °C in 15 min, as outlined in
Table 5 and Fig. 5.
Table 2 Post-docking affinity analysis
Compound
A-804598
Binding_energy (kcal/mol)
Dock_pKi
5.14
−6.31
−5.92
−5.72
Desmethyl-A-804598
EFB
4.87
4.90

Fantoni et al. EJNMMI Research (2017) 7:31
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Fig. 4 Synthetic route to EFB
Purification and quality control of [18F]EFB
Preliminary in vivo evaluation
The product was purified by semi-preparative chro- LPS model validation
matography using method A (Table 1, Fig. 6a) and LPS injections are known to induce a 5–10% weight loss
identified by co-elution with an authentic reference within 24 h, which is known to indicate activation of the
compound with method B (Table 1, Fig. 6c). Quality inflammatory process [27, 28]. In this work, the valid-
control was performed after purification and reformu- ation of the LPS rat model was restricted to recording a
lation, typically yielding an HPLC chromatogram significant weight loss for all treated rats after 24 h
similar to that shown in Fig. 6b and pH within a (Additional file 1: Figure S3).
physiological range. The desired product [18F]EFB had
a retention time of 19.35 min.
[18F]EFB evaluation
Evaluation of [18F]EFB as an inflammation-dependent
tracer was performed by comparison of control and
0.5 mg/kg LPS-pre-treated Sprague–Dawley rats. The
visual PET image assessment indicates low brain up-
take with slightly increased retention in the LPS rat’s
temporal lobe (Fig. 7, also displaying elevated retinal
uptake). Rat longitudinal uptake profiles (Fig. 8) re-
vealed a relatively stable blood concentration around
0.05–0.1%ID/g. The CNS uptake stayed between
0.05–0.1%ID/g for the spinal cord, while it reached
0.02–0.07%ID/g in the whole brain, the cortex, the ol-
factory bulb, the cerebellum and the basal ganglia.
These percentages closely correlate with the terminal
biodistribution (Fig. 9). Additionally, LPS-treated rats
demonstrated a statistically significant increase in
mean tracer uptake compared to untreated rats, pos-
sibly reflective of increased P2X7 expression.
In vitro affinity
The affinity of EFB for P2X7 was determined by cal-
cium influx assays in transfected HEK293 and B16
cells and compared with A-740003 affinities (Table 6).
For EFB, the assays showed 5.74 pIC50 for the human
receptor isoform, 5.14 pIC50 for rat and 5.22 IC50 for
mouse P2X7. Assuming competitive inhibition as per
A-804598 [8], the values were transformed to their
respective inhibition constants (Ki) by means of the
Cheng–Prusoff equation [25]. Here, pEC50 values for
100% P2X7-activating BzATP (6.8 0.14 in human,
6.3 0.13 in rat and 4.7 0.07 in mouse) were calcu-
lated with respect to the YoPro dye uptake response
in P2X7-HEK293 cells as reported in Hibell [26]. This
resulted in 2.88 nM Ki in human P2X7, 36.1 nM in
rat and 547 nM in mouse. Similar calculations were
performed for the known antagonist A-740003, for
which literature values useful as internal reference are
provided.
Discussion
The development of the novel radioligand [18F]EFB rep-
resents a valuable advancement to the field of P2X7 PET
imaging. Molecular docking analysis of the benzyl-
desmethylated version of A-804598 and of EFB indicated
that subtle structural modifications in the periphery of
the A-804598 scaffold are not predicted to impact the
affinity to the target’s ATP-binding pocket [8], thus
Table 3 Iterative n.i. RCY optimisation of compound [18F]5. Values
derived from TLC chromatogram analysis
Entry Scale (μmol Time (min) Temperature (°C) RCY (% SEM)
precursor)
1
2
3
4
5
6
3.2
3.2
3.2
6.4
6.4
6.4
15
95
63.0 4.1 (n = 8)
65.2 7.4 (n = 3)
42.9 3.7 (n = 2)
68.0 17 (n = 2)
75.9 4.7 (n = 4)
66.9 3.2 (n = 2)
Table 4 N.i. RCY TLC monitoring at different time points for the
synthesis of [18F]3
20–25
15
95
110
95
Entry
Time (min)
Temperature (°C)
RCY (% SEM)
15
1
2
3
1
3
5
25
25
25
67.1 3.0 (n = 3)
90.1 4.1 (n = 7)
82.5 5.5 (n = 14)
15
110
110
20

Fantoni et al. EJNMMI Research (2017) 7:31
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Table 5 HPLC monitoring of the n.i RCY of [18F]EFB
Entry Temp
(°C)
[
18F]EFB RCY
Unreacted
compound 3
RCY (% SEM)
Major by-product
RCY (% SEM)
(% SEM)
1
2
3
4
5
6
120
125
130
140
150
160
41.4 8.2 (n = 2)
7.0 0.8 (n = 2)
13.7 4.5 (n = 2)
39.0 7.9 (n = 2) 15.3 10.6 (n = 2) 19.6 14.9 (n = 2)
43.5 8.9 (n = 2) 10.1 2.2 (n = 2)
41.4 8.2 (n = 2)
47.6 3.3 (n = 2)
38.5 2.5 (n = 3)
38.6 2.9 (n = 2)
35.9 2.4 (n = 2)
41.1 2.3 (n = 3)
43.8 0.8 (n = 2)
6.2 2.2 (n = 2)
4.5 1.9 (n = 3)
3.5 1.8 (n = 2)
offering the possibility of attaching a fluorine-18 label to
the externally oriented phenolic ring of A-804598.
The synthesis of EFB was achieved via mercury sulfide
precipitation-driven stepwise amine addition in moder-
ate yields, possibly due to interaction of the transition
metal with the guanidine moiety. Radiolabelling was
achieved in three steps starting from compound 4,
achieving an overall DC RCY of 3–5%, >99% RCP and
>300 GBq/μmol molar activity. The RCY was mostly
limited by the inherently high unknown by-product for-
mation in the final step of the process. This could con-
sist in a cyclised or truncated form of [18F]EFB, which
could be limited by the replacement of precursor 6 with
an analogue with a more labile leaving group, such as
sulfonyl methane [29] or sulfonyl chloride [30]. However,
some preliminary attempts at oxidising the thiomethyl
group with 2 eq. oxone at room temperature or m-
CPBA at −20 °C resulted in over-oxidation of compound
6. The RCY was found to be sensitive to variations due
to limited SPE recoveries unless large crude product di-
lutions were carried out.
Fig. 6 a Semi-preparative purification of the product [18F]EFB with HPLC
method A (Table 1). b Final [18F]EFB analytical chromatogram (RCP >99%,
high CP) with HPLC method B. c Identification of [18F]EFB by co-elution
with the reference standard with HPLC method B. Green chromatograms,
UV; blue/red chromatograms, radioactivity. Only the region marked by the
dashed red lines around 51 min was subject to collection
In vitro affinities were derived from calcium influx
binding assays in the presence of the calcium-sensitive
dye Fura-2/AM in stably P2X7-transfected cells. The
typical ATP-mediated Ca2+ flow response is a sharp peak
followed by a sustained plateau phase [31]. Given the
high P2X7 expression, despite the values reported repre-
sent the former, the inhibition constants were in a simi-
lar range for both the peak and plateau phases. The
variation in affinity between the receptor isoforms
60
40
20
0
Table 6 EFB and A-740003 calcium influx inhibition of P2X7
receptor subtypes
P2X7
isoform
Cell line
[BzATP] pIC50 Lit. pIC50 Ki (nM) pKi
(μM)
Human P2X7-HEK293 100
5.74
5.14
5.22
7.18
5.76
5.97
n/a
2.88 8.54
Rat
P2X7-HEK293 100
P2X7-B16 200
n/a
36.1
547
7.44
6.26
Mouse
n/a
120
130
140
Temp
150
160
Human P2X7-HEK293 100
7.36 [32]
7.75 [32]
6.57 [34]
0.10 9.98
8.67 8.06
97.20 7.01
Fig. 5 [18F]EFB n.i. RCY changes with temperature. Black, [18F]EFB; blue,
Rat
P2X7-HEK293 100
P2X7-B16 200
unknown by-product; red, unreacted [18F]3
Mouse

Fantoni et al. EJNMMI Research (2017) 7:31
Page 8 of 12
Fig. 7 Selected coronal and sagittal tile views of PET/CT scans at 22.5 min in control (ctrl) and LPS male Sprague–Dawley rats injected with comparable
doses of [18F]EFB. Tile numbers nominally indicate the distance of the slice from the front (coronal) and left side (sagittal) of the brain, but do not
necessarily correspond between different animals. The grey scale in Hounsfield units (HU) refers to the CT data, while the colour scales in Bq/mL refer to
the control rat (left) and LPS rat (right) PET data
reflects the trends observed for analogous ligands in parallel to those of the known antagonist A-740003,
[32, 33] and indicated a stronger interaction with the which were compared to literature values arising from simi-
human form. This could lead to the underestimation lar assay formats [32, 34]. In our in vitro assays, A-740003
of the sensitivity of this ligand upon clinical translation. appeared less tightly P2X7 binding than apparent from the
The EFB affinity values arising from this assay were found literature, suggesting that also EFB IC50s may result

Fantoni et al. EJNMMI Research (2017) 7:31
Page 9 of 12
Brain
LPS
WT
Spinal cord
Olfactory bulb
LPS
WT
LPS
WT
0.06
0.04
0.02
0.00
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
*
***
***
**
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time (min)
Time (min)
Time (min)
LPS
WT
Cerebellum
Basal ganglia
Blood
LPS
WT
LPS
WT
0.06
0.04
0.02
0.00
0.08
0.06
0.04
0.02
0.00
0.20
0.15
0.10
0.05
0.00
**
**
***
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time (min)
Time (min)
Time (min)
Heart
LPS
Control
Bone
LPS
Control
Cortex
LPS
Control
0.3
0.25
0.20
0.15
0.10
0.05
0.00
0.06
0.04
0.02
0.00
0.2
0.1
0.0
*
***
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time (min)
Time (min)
Time (min)
Muscle
LPS
Control
Lungs
LPS
Control
0.25
0.20
0.20
0.15
0.10
0.05
0.00
0.15
0.10
0.05
0.00
*
***
0
20
40
Time (min)
60
80
0
20
40
60
80
Time (min)
Fig. 8 In vivo %ID/g of [18F]EFB in 24 h post-LPS injection and control rats (n = 3). Matched time and treatment variation significances representative
of overall variations in the uptake are reported for each organ. Statistical analysis was carried out by a parametric two-way repeated measures ANOVA
with column matching and with Bonferroni post-test. The asterisk indicates P < 0.01; **P < 0.005; ***P < 0.001
somewhat stronger in different experimental conditions biodistribution and P2X7 binding and to explore its abil-
[26]. Moreover, it remains important to clarify if its selectiv- ity to penetrate the BBB. The use of the bacterial endo-
ity to P2X7 differs from its cyanoguanidine analogue A- toxin lipopolysaccharide (LPS)-inflamed rat model was
804598 [8] and to further elucidate whether EFB is truly a decided to enable comparison between the tracer uptake
competitive inhibitor, as suggested by Donnelly-Roberts [8] under low and high P2X7 expression levels. This is
and Honore [14] for analogous structures, but recently known to induce increased P2X7 expression as demon-
questioned by Karasawa [35]. A binding modality other strated by immunohistochemical staining of rat brains
than competitive would preclude the use of the Cheng– 24 h after an intraperitoneal 0.5 mg/kg LPS injection
Prusoff equation in its basic form [36].
[28]. Additionally, LPS-injected rats display protracted
A preliminary preclinical evaluation of [18F]EFB was microglial activation [37, 38] and raised levels of a range
carried out with the intention of clarifying its in vivo of blood inflammatory markers, including TNF-α, NF-κβ

Fantoni et al. EJNMMI Research (2017) 7:31
Page 10 of 12
***
15
0.25
10
3.5
0.20
0.15
3.0
0.10
2.5
0.05
2.0
0.00
n
b
lum
ctory bul
nglia
Bone
Brai
Heart
l cord
Blood
1.5
1.0
0.5
0.0
a
Muscle
n
i
a
Olf
Cerebel
Sp
Basal ga
t
r
r
a
s
ng
s
le
Sp
s
il
Ta
h
d
d
lb
es
es
Ey
ne
Bo
en
c
dy
Brain
ar
He
de
ea
ey
ec
ngli
Live
Pa
Urine
Fa
Bloo
y bu
Lu
testine
bellum
ncr
al cor
Muscle
Blad
e
n
n
i
Kidn
ole bo
r
Stoma
l i
sal ga
actor
Ce
Ba
Olf
Sp
ge Intestine
Wh
r
Smal
La
Fig. 9 Biodistribution of [18F]EFB at 90 min post-injection in LPS (blue bars) vs. control (red bars) rats. The insert is a blow-up of some regions of
interest with low tracer accumulation
and IL-1β [39]. Moreover, brain cortex expression of IL- or that its lipophilicity may benefit from a reduction by
1β, IL-6, toll-like receptors (TLRs) and glial fibrillary scaffold modifications (EFB: LogP 2.72 by miLogP [46]).
acidic protein (GFAP) are upregulated within 4 h, sug- However, the higher affinity of EFB for the human P2X7
gesting that glial activation and neuronal inflammation isoform may result in enhanced CNS uptake and reten-
quickly follow systemic inflammatory episodes [40, 41], tion in a clinical setting.
often outlasting them [42]. These biochemical changes
The mean longitudinal uptake of [18F]EFB appears sig-
are concomitant with an exaggerated weight loss [43, 44], nificantly higher in LPS compared to that in untreated
and this measure was employed in this work to demon- rats, although individual time points did not reach
strate effectiveness of the LPS treatment. A full validation significance (Fig. 8). This suggests that the tracer has po-
of the process linking LPS to P2X7 upregulation is on- tential to discern between high and low P2X7 expression
going. The 0.5 mg/kg LPS dose was preferred to higher in vivo.
doses based on reports that below 3 mg/kg, the endotoxin
does not cause BBB disruption [45].
The difference in blood tracer quantification values
noticeable in Fig. 8 can be a result of partial volume ef-
[18F]EFB displays a desirable in vivo distribution and fects arising from the inherently limited dimensions of
excretion profile in that target tissue retention is quanti- the blood ROI (the coronary artery lumen), whereas the
fiable and that fast removal of the radioactivity predom- blood concentration of the tracer resulting from the
inantly via the urinary route can be observed. The brain 90 min biodistribution appears substantially unvaried in
uptake of the tracer is quantifiable but low, suggesting the two groups. Furthermore, the 90 min biodistribution
that [18F]EFB could be a substrate of BBB efflux pumps data shows an increased, albeit non-significant, tracer

Fantoni et al. EJNMMI Research (2017) 7:31
Page 11 of 12
uptake in the LPS rats’ upper digestive organs (stomach,
pancreas), possibly due to the relative abundance of
P2X7 receptors on the epithelial lining of the digestive
organs.
Authors’ contributions
ERF planned and performed the synthesis, radiosynthesis and preclinical
experiments and wrote the manuscript. DDB performed the molecular
modelling experiments. SF and FDV performed the in vitro assays. AG and SL
took part in the conception, design and funding of the study. All authors
read and approved the manuscript.
The preclinical evaluation was intended as a prelimin-
ary assessment of the BBB permeability and overall dis-
tribution of the tracer in LPS and control rats. While it
was not possible to perform metabolite and kinetic ana-
lysis at this stage, further work aiming to explore EFB
scaffold variants could benefit from such data. Moreover,
the injected activity was modest for some rats, and
[18F]EFB required extensive synthesis time. This process
may benefit from automation or a revised synthetic
protocol, such as one including a better precursor leav-
ing group for the final step of the radiosynthetic proced-
ure (step c, Fig. 2).
Competing interests
The authors declare that they have no competing interests.
Ethics approval
All procedures performed on animals were approved by a King’s College
London research ethics committee and in accordance with the Animals
(Scientific Procedures) Act 1986, Amendment Regulations 2012 under PPL
70/8230 (Reza Razavi), PPL 70/8480 (Diana Cash) and PIL 70/3001 (Enrico
Fantoni).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1Department of Imaging Sciences and Biomedical Engineering, King’s
College London, St Thomas’ Hospital, 4th floor Lambeth Wing, SE1 7EH
London, UK. 2School of Pharmacy, Medicinal Chemistry Unit, University of
Camerino, via S. Agostino 1, 62032 Camerino, MC, Italy. 3Prof Francesco Di
Virgilio and Dr Simonetta Falzoni, Dipartimento di Morfologia, Chirurgia e
Medicina Sperimentale, Sezione di Patologia, Oncologia e Biologia
Sperimentale, University of Ferrara, Ferrara, Italy. 4Department of Psychiatry,
Warneford Hospital, University of Oxford, Warneford Lane, Oxford OX3 7JX,
UK.
Conclusions
The work presented here constitutes an important step
towards the development of a fluorine-18-labelled PET
tracer useful for imaging P2X7 in vivo. The synthesis
and radiosynthesis of [18F]EFB have been successfully
carried out, affording the tracer in >99% RCP and
>300 GBq/μmol molar activity. In vitro calcium influx
binding assays revealed a Ki of 2.88 nM in human P2X7,
36.1 nM in rat and 547 nM in mouse.
Received: 14 November 2016 Accepted: 8 March 2017
A preliminary in vivo evaluation indicated a low BBB
uptake indicative of limited suitability of the molecule for
P2X7 brain PET imaging. Taken together with previous
work by Janssen [6] on a different radiotracer targeting
P2X7, this data is suggestive of limited compatibility of
the cyanoguanidine moiety with BBB entry in rats. Never-
theless, the altered mean longitudinal uptake in LPS ver-
sus control rats indicates that the tracer may have
potential as a systemic P2X7 PET imaging agent.
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