Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands

Article abstract of DOI:10.1016/j.nucmedbio.2018.11.002

Introduction: Primary aldosteronism accounts for 6–15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([¹¹C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [¹¹C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([¹⁸F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. Methods: Two strategies for the one-step radio-synthesis of [¹⁸F]FAMTO were developed. [¹⁸F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [¹⁸F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [¹⁸F]FAMTO metabolites. Results: [¹⁸F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [¹⁸F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [¹⁸F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [¹⁸F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [¹⁸F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [¹⁸F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. Conclusions: [¹⁸F]FAMTO, a new ¹⁸F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

Full text of DOI:10.1016/j.nucmedbio.2018.11.002

NMB-08044; No of Pages 8
Nuclear Medicine and Biology xxx (xxxx) xxx
Contents lists available at ScienceDirect
Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio
Development of [18F]FAMTO: A novel fluorine-18 labelled positron
emission tomography (PET) radiotracer for imaging CYP11B1 and
CYP11B2 enzymes in adrenal glands
Salvatore Bongarzone a, Filippo Basagni a, Teresa Sementa a, Nisha Singh a,b, Caleb Gakpetor a,
Vincent Faugeras a, Jayanta Bordoloi a, Antony D. Gee a,
⁎
a
School of Imaging Sciences & Biomedical Engineering, 4th floor Lambeth Wing, St Thomas' Hospital, King's College London, London SE1 7EH, United Kingdom
Department of Neuroimaging, Institute of Psychiatry, King's College London, London SE5 8AF, United Kingdom
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2018
Received in revised form 2 November 2018
Accepted 4 November 2018
Available online xxxx
Introduction: Primary aldosteronism accounts for 6–15% of hypertension cases, the single biggest contributor to
global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas,
only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11
radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively
identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is lim-
ited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a
fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position
([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-
cyclotron-based imaging centres.
Keywords:
Metomidate
Adrenal glands
Fluorine-18 radiochemistry
Primary aldosteronism
CYP11B2
Methods: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was ob-
tained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was eval-
uated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents.
Rat plasma and urine were analysed to determine [18F]FAMTO metabolites.
Positron emission tomography
Results: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-
isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with N99% radiochemical
and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-
exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding
in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was
about 34% of that in the adrenals and all other organs were b12% of the adrenal uptake at 60 min post-injection.
Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid uri-
nary excretion. In vitro assays in human blood showed a free fraction of 37.5%.
Conclusions: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and
in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and
CYP11B2), supporting the potential of this radiotracer for human investigation.
© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
1. Introduction
in four people with hypertension globally [1–7]. PA is characterized by
an excess secretion of aldosterone, steroid hormone with mineralocor-
The most common secondary cause of hypertension is primary aldo-
steronism (PA), which is reported in approximately 6–15% of all hyper-
tensive patients. By the year 2025, PA is expected to directly impact one
ticoid activity produced by the zona glomerulosa of the adrenal cortex
where aldosterone synthase (CYP11B2) enzymes play an essential role
in aldosterone production [3,8,9]. Aldosterone increases sodium reab-
sorption and potassium excretion in the renal distal tubules and
collecting ducts of nephrons, influencing water retention and blood
pressure [3].
⁎
Corresponding author at: School of Imaging Sciences and Biomedical Engineering,
King's College London, 4th Floor, Lambeth Wing St Thomas' Hospital, London SE1 7EH,
United Kingdom.
Therefore, pathological conditions showing an overproduction of al-
dosterone frequently lead to hypertension and hypokalaemia resulting
E-mail address: antony.gee@kcl.ac.uk (A.D. Gee).
https://doi.org/10.1016/j.nucmedbio.2018.11.002
0969-8051/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

2
S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
in detrimental effects to an individual's cardiovascular system [1]. Over
90% of all PA patients have a sporadic form [10]. The two most common
subtypes of sporadic PA are unilateral aldosterone-producing adenomas
(UAPA, 40%) or bilateral adrenal hyperplasia (BAH, 60%) [5,11]. Cor-
rectly differentiating between the unique features of UAPA and BAH is
crucial in identifying an appropriate intervention. UAPA is curable
through surgical removal of the diseased adrenal whilst BAH is treated
pharmacologically with mineralocorticoid receptor antagonists (e.g.
spironolactone) [12]. The most significant obstacle for decision makers
is the need to distinguish UAPA from other causes of PA such as BAH
or non-functioning adrenal adenoma (incidentalomas) [13]. Invasive bi-
lateral adrenal vein sampling (AVS) is the current gold-standard diag-
nostic procedure adopted, with cannulation success rates varying from
8% to 95% [10,14–16].
Computed tomography (CT) and magnetic resonance imaging (MRI)
have been used for non-invasive lateralisation of aldosterone hyperse-
cretion. These show low diagnostic accuracy due to suboptimal spatial
resolution and sensitivity to detect small UAPA (b1 cm) [14,17–20]. Pos-
itron emission tomography (PET) is considered an accurate and non-
invasive alternative to AVS in the management of patients with PA
and adrenal adenoma [21].
Methyl and ethyl esters of 1-[(1R)-1-phenylethyl]-1H-imidazole-5-
carboxylic acid, such as (R)-metomidate (MTO, Fig. 1) and (R)-
etomidate (ETO), are a class of compounds that interact with the mito-
chondrial cytochrome P-450 enzymes (11β-hydroxylase (CYP11B1)
and CYP11B2) in the adrenal cortex (IC50 for CYP11B1 = 0.24
0.05 nM (MTO) and 0.5 0.2 nM (ETO), IC50 for CYP11B2 = 0.59
0.64 nM (MTO) and 1.7 0.9 nM (ETO)) [22–24]. MTO and ETO have
been used as template to develop PET radiolabelled radiotracers using
β+-emitting radionuclides such as 11C and 18F [21,25,26].
at 20 min and 40 min post-injection, with two polar metabolites present
in the plasma [30]. Although [11C]MTO has good imaging properties for
visualisation of UAPA [21], novel radiotracers with increased adrenal-
to-liver ratio (large liver uptake hampers the assessment of the right ad-
renal) and a longer half life radionuclide (e.g. 18F) are needed for a more
widespread adoption of this diagnostic approach.
An increased adrenal-to-liver PET signal was achieved by adding a
halogen atom (e.g. chlorine or bromine) in the para-position of MTO's
benzene group such as chlorine ([11C]ClMTO, Fig. 1) and bromine
([11C]BrMTO, Fig. 1). In rats [11C]MTO gives an adrenal-to-liver ratio of
3.8 at 30 min post-injection, increasing to 4.4 and 7.2 using [11C]
ClMTO and [11C]BrMTO, respectively [32]. [11C]MTO is not used in a
widespread manner, partly because of its short half-life that limits its
use to PET centres with on-site cyclotrons. A MTO analogue with a lon-
ger fluorine-18 half-life would enable the examination of more patients
per tracer production, and its distribution to PET-centres without on-
site cyclotrons.
Wadsak and Mitterhauser developed an ETO derivative
radiolabelled with fluorine-18 ([18F]FETO, Fig. 1) to enhance the
image resolution, and pharmacokinetic and pharmacodynamic proper-
ties of [11C]MTO [33]. MTO and FETO have similar binding affinities to
displace the radioligand 4-[131I]iodometomidate ([131I]IMTO) on ho-
mogenized rat adrenal membranes as an acceptable surrogate for test-
ing inhibitory affinity versus CYP11B1 and CYP11B2 (IC50 = 3.69
1.92 nM and 2.9 0.55 nM, respectively) [34]. [18F]FETO was produced
using an automated one-step protocol within 70 min (total synthesis
time) with a non-isolated radiochemical yield (RCY) of 20 3% [35].
Regional organ distribution of [18F]FETO in rat revealed an adrenal-to-
liver ratio of 14.48 at 60 min post-injection [36]. Metabolite analysis in-
dicated that [18F]FETO is degraded to 2-[18F]fluoroethanol in vitro and
in vivo [37]. In humans, [18F]FETO is rapidly metabolised in the first
10 min (91% intact [18F]FETO after 2 min; 24% after 10 min, 11% after
20 min and 3.7% after 90 min) [37]. [11C]MTO and [18F]FETO showed
rapid metabolism in vivo corresponding to de-esterification to the
Bergström et al. have developed [11C]MTO (Fig. 1) by radiolabelling
the methyl ester group with 11C. [11C]MTO possesses high specific bind-
ing to adrenal enzymes in vitro and in non-human primate studies
[25,27,28]. Blocking experiments using ETO (10 μM) confirmed the spe-
cific uptake of [11C]MTO to pig adrenal sections [25]. In humans [11C]
MTO showed high uptake in adrenal glands (SUV ratio of adrenal
gland/liver ratio 1.5 0.7, 15–45 min) and high standardised uptake
values (SUV) in aldosterone-hypersecreting adenomas [25,27,29–31].
Metabolite studies of [11C]MTO in humans revealed that unchanged
non-radioactive and inactive carboxylic acid derivative (IC50
=
0.12 mM) [34,38] and radioactive metabolites, such as [11C]methanol
and [11C]formaldehyde for [11C]MTO and [18F]fluoroethanol for [18F]
FETO, affecting the signal/noise ratio and specificity of the imaging
data acquired [30,36,37].
[
11C]MTO accounted for 40% and 28% of total blood-borne radioactivity
A para-fluorinated aromatic (R)-MTO derivative (FAMTO, Fig. 1) has
comparable affinity for adrenal enzymes (Ki = 7.3 nM measured using
[
131I]IMTO) as MTO (Ki = 4.02 1.87 nM) in rat adrenal membranes
[39]. We therefore developed a radiosynthetic strategy to produce a
18F-radiolabelled analogue of MTO ([18F]FAMTO) in order to evaluate
it as a potential radiotracer for aldosterone-producing adenomas.
2. Materials & methods
2.1. General consideration
[
18F]Fluoride ion was produced using an RDS112 cyclotron at King's
College London PET Centre by the 18O(p,n)18F reaction via proton irradi-
ation of enriched (95%) 18O water.
2.2. Trapping and releasing of [18F]fluoride ion on a Sep-Pak Accell Plus
QMA cartridge
0.5–1.5 GBq of aqueous [18F]fluoride ion was trapped in an anion-
exchange resin cartridge (Sep-Pak Accell Plus QMA cartridge,
WAT023525, Waters) pre-activated with 10 mL 1 N NaHCO3 (aq),
10 mL water (H2O) followed by 10 mL air. The trapped [18F]fluoride
ion was released with solution A, composed of 25.5 μmol Kryptofix
(K222) and 4.5 μmol KHCO3 in acetonitrile (MeCN):H2O (85:15, 1 mL)
or solution B, composed of 19.55 μmol K222 and 0.9 μmol K2CO3 in
MeCN:H2O (85:15, 1 mL). The solvent was removed by heating at
90 °C under a stream of N2 and fluoride ion was dried by azeotropic
Fig. 1. Chemical structures of ETO, MTO, [11C]MTO, [11C]BrMTO, [11C]ClMTO, [18F]FETO,
and [18F]FAMTO.
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
3
distillation with MeCN (2 × 0.5 mL). Compound 1 (5 mg) was dissolved
in anhydrous dimethyl sulfoxide (DMSO, 100–500 μL) and the obtained
solution was added to the dried [18F]fluoride ion/K222/K2CO3 mixture.
The vial was then sealed and stirred at 110–180 °C for 15–30 min,
quenched with H2O and analysed by radioTLC.
calculated by relating the amount of isolated [18F]FAMTO to the amount
of [18F]fluoride ion trapped on QMA cartridge.
The molar activities (GBq/μmol) were calculated by dividing the ra-
dioactivity of [18F]FAMTO by the amount of the unlabeled FAMTO deter-
mined from the peak area in the UV-HPLC chromatograms (λ =
254 nm, common wavelength for identifying metomidate derivative
compounds such as etomidate and iodometomidate [25,38], Fig. S1).
FAMTO concentrations were determined from a UV-absorbance calibra-
tion curve (see Appendix A).
2.3. Radioynthesis of [18F]FAMTO
2.3.1. High base procedure
The trapped [18F]fluoride ion was released with 0.5 mL of a solution
composed of K222 (16 μmol) and K2CO3 (2.4 μmol) in MeCN:H2O
(80:20). Azeotropic drying was performed by addition of MeCN
(2 × 0.5 mL) and heating at 90 °C under a stream of N2. To the
dried mixture, 500 μL of DMF was added. Aliquots of 500, 250, 125
and 87 μL of [18F]KF/K222/K2CO3, 2 (6.3 mg, 18 μmol) and tetrakis
(pyridine)copper(II) triflate (Cu(OTf)2(py)4, 3.6 mg, 5 μmol) were
added in a vial. DMF was added to achieve a final volume of 500 μL
(see Table 2). Radiofluorination was conducted at 118 °C for 20 min,
cooled to room temperature, and diluted with H2O.
2.7. Pig organ tissues
Pig tissues were purchased from Seralab, UK. Adrenal glands, kid-
neys, and liver were collected from an healthy animal (female,
9 months old). Tissues were delivered at 4 °C in Dulbecco's media.
Once collected, tissues were cut in small and medium pieces. Pieces
were snap frozen in an isopentane bath cooled to −30 °C and stored
at −80 °C.
2.3.2. Low base procedure
2.8. Autoradiography
Aqueous [18F]fluoride ion (100–1000 MBq) was loaded onto a QMA
cartridge, the cartridge was flushed with air and the 18F elution was
performed using 2 mL of K222 (1.5 μmol) and K2CO3 (0.87 μmol) in
MeCN:H2O (80:20). Azeotropic drying was performed by addition of
MeCN (2 × 1 mL) and heating at 90 °C under a stream of N2. To the
dried mixture DMF (500, 300, 200, and 100 μL), 2 (6.3 mg, 18 μmol)
and Cu(OTf)2(py)4 (3.6 mg, 5 μmol) were added (see Table 2).
Radiofluorination was conducted at 118 °C for 20 min under air, cooled
to room temperature, and diluted with H2O.
Frozen sections (20 μm) of pig adrenal glands, kidneys, and liver
were prepared in a Cryostat and put on superfrost glass slides. At the
start of the experiment, the slides warmed to room temperature and
preincubated for 10 min in TRIS buffer (50 mM, pH 7.4). Slides were
then transferred to containers containing 1 nM of [18F]FAMTO in
40 mL of TRIS buffer (50 mM, pH 7.4). In a duplicate set of containers,
1 μM of MTO was added to block specific binding. After incubation for
30 min (the slides were washed 2 × 3 min in fresh 50 mM TRIS buffer,
pH 7.4). The slides were dried and then exposed to phosphor imaging
plates (BAS-IP TR 2040, GE Healthcare, UK) for 30 min and scanned in
a Typhoon 8600 phosphorimager (GE Healthcare, UK). Images (Fig. 2)
were analysed using OptiQuant 5.0 software (PerkinElmer, UK).
2.4. Purification and analysis of [18F]FAMTO
The crude mixture was purified by semi-preparative HPLC (see
Appendix A). The purified [18F]FAMTO was subsequently diluted with
40 mL of PBS, the solution loaded onto a Sep-Pak tC18 Plus Long SPE
Cartridge 900 mg, 37–55 μm (cat. no. WAT036800, Waters) and eluted
with ethanol (2 mL). Ten fractions (0.2 mL) were collected and radioac-
tivity determined. Fractions with highest radioactivity were combined
and formulated with saline. The formulated solution (4–6 mL of 10%
ethanol in saline) containing 1–10 MBq of [18F]FAMTO was used for
in vitro and in vivo experiments. Analytical reverse-phase HPLC (see
Appendix A) was used to determine the molar activity, radiochemical
and chemical purity. Identification of the radioactive products was con-
firmed by co-elution of added non-radioactive compounds.
2.9. Animal studies
Biodistribution and metabolite studies were carried out in adult
male Sprague-Dawley rats (300–400 g). All animal studies were carried
out in accordance with the UK Home Office Animals (Scientific Proce-
dures) Act 1986. Experiments complied with UK Research Councils'
and Medical Research Charities' guidelines on responsibility in the use
of animals in bioscience research, under UK Home Office project and
personal licenses.
2.5. Determination of chiral conformation and enantiomeric excess of 2,4
and [18F]FAMTO
2.10. Ex vivo biodistribution
Nine anaesthetised rats (2–3% isoflurane/oxygen) were injected in-
travenously with 1–3 MBq/kg of formulated [18F]FAMTO and sacrificed
at 15, 30 and 60 min post-injection. The specific uptake of [18F]FAMTO
was evaluated in three anaesthetised rats (2–3% isoflurane/oxygen)
using 1 mg/kg ETO i.v. (95% saline and 5% ethanol) 15 min before [18F]
FAMTO injection (1–3 MBq/kg), and animals sacrificed 30 min post-ra-
diotracer injection. Tissues including whole brain, heart, lungs, stomach,
liver, spleen, small intestine, large intestine, kidneys, bladder, thigh
bone, adrenal glands, and testes were excised. Urine and blood were
also collected. All samples were weighed and radioactivity content mea-
sured using an automated well counter with a standard dilution of [18F]
FAMTO. Counts were decay-corrected and the %ID/g calculated (Fig. 3A
and Table S1). Data are expressed as mean standard error (SE) from
three independent replicates, unless otherwise indicated. Statistical
analysis of %ID/g in the biodistribution study was performed with IBM
SPSS Statistics (version 24). Student t-tests were used to determine sta-
tistical significance with p b 0.05 considered significant.
Circular dichroism (CD) spectra of (R)-ETO (enantiomeric excess
(e.e.) N99%, Sigma Aldrich), (R)-2, (R)-4 and (S)-8 (e.e. N97%, Sigma
Aldrich) were acquired on the Applied Photophysics Ltd. Chirascan
Plus spectrometer (Fig. S1).
Enantiomeric purity of (R)-2, (R)-4, (R)-5, and (R)-[18F]FAMTO was
performed on a chiral HPLC Lux 5 μm Cellulose-3250 × 4.6 mm coloumn
(Table S2). Commercial (R)-ETO (e.e. N99%) and (S)-8 (e.e. N97%) were
used as control.
2.6. Determination of RCY and molar activity of [18F]FAMTO
The entire quenched reaction mixture was used to determine non-
isolated and isolated RCY.
All RCYs are reported as decay corrected values [40]. Non-isolated
RCYs were determined by integrating the area under the curve in the
preparative radio-HPLC chromatogram or iTLC. Isolated RCY were
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

4
S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
Fig. 2. Frozen tissue-section autoradiography of [18F]FAMTO (1 nM) binding to pig adrenal, pig liver, pig kidney. Lower panel [18F]FAMTO (1 nM) blocking studies using 1 μM MTO.
were incubated at room temperature with 30 μL of a solution containing
18F]FAMTO (1–2 MBq). After 10 min, each solution was sampled and
2.11. Analysis of radiolabelled metabolites in rats
[
counted. The solution (200 μL) was injected into a filter device
(Millipore Centrifree tubes, YM-30) and subsequently centrifuged for
5 min at 6000 rpm. The solution passed through a centrifugal filters
unit (nominal molecular weight limit 30,000 Da) and the filtrate and
control samples were gamma-counted.
Blood and urine samples were collected at 10, 30, and 60 min post-in-
jection to measure the amounts of unchanged tracer and radioactive me-
tabolites. Blood was centrifuged at 6000 rpm for 5 min, the supernatant
was treated with MeCN (1:1) and centrifuged to precipitate plasma pro-
teins. Plasma supernatant and urine samples were then injected into an
HPLC semipreparative column (see Appendix A), and the eluate collected
in 1.5 mL test tubes and gamma-counted. Two metabolites (tR = 3 and
21 min) and [18F]FAMTO were observed in the plasma (Fig. 4). One me-
tabolite (tR = 3 min) was observed in the urine (Fig. S2). 2.5 μL aliquots
of radioactivity eluting at tR = 3 min and compound 12 (Scheme 1) were
analysed by TLC plates (Merck F-254 silica gel) eluted with dichloro-
methane:methanol:trifluoroacetic acid (TFA) (8:2:0.05). TLC plates
were scanned in a Typhoon 8600 phosphorimager (GE Healthcare, UK)
and analysed using OptiQuant 5.0 software (PerkinElmer, UK).
2.14. PET imaging
PET-CT scans were performed on a BioScan nanoPET-CT® PLUS
(Mediso, Hungary) scanner using their proprietary acquisition software
(Nucline 1.07). The rats were anaesthetised and maintained at 2.5%
isofluorane and 1 L/min flow of Oxygen (O2). A CT scan was acquired
prior to the commencement of the PET scan. Dynamic PET scans were
acquired for 60 min post-intra-venous (IV) administration by tail vein
injection of [18F]FAMTO (1.0 MBq, n = 2) or by pre-administration
ETO (1 mg/kg) 15 min prior to [18F]FAMTO injection (1.0 MBq, n = 1).
PET scans were reconstructed using Tera-Tomo 3D image reconstruc-
tion algorithm. Reconstructions were performed with the detector coin-
cidence mode set at 1:3. Corrections for decay, randoms, crystal dead
time, detector normalization and, attenuation correction was imple-
mented. A total of 6 subsets and 4 iterations were applied resulting in a
voxel size of 0.25 × 0.25 × 0.25 mm3 for CT, and 0.4 × 0.4 × 0.4 mm3
for PET. The PET and CT images were co-registered automatically.
Image analysis was performed using pre-clinical image postprocessing
software VivoQuant™.
2.12. Stability studies in plasma
Human blood (1 mL) was incubated with 0.7–1 MBq (200 μL) [18F]
FAMTO at room temperature for 10 and 20 min. After incubation,
plasma was separated by centrifugation (5 min, 4000 ×g) and the pro-
teins were precipitated by the addition of an equal volume of MeCN.
The tubes were centrifuged for 5 min at 4000 ×g. The supernatant was
then injected onto the HPLC (HPLC method 1) and serial 1.5 mL HPLC
fractions were collected and gamma-counted. [18F]FAMTO (0.7–
1 MBq, 200 μL) incubated in PBS (1 mL) was used as control.
2.13. Plasma protein binding
3. Results
The plasma protein binding of [18F]FAMTO was measured in the
plasma of blood samples. Plasma was prepared from fresh human
blood by centrifugation (5 min, 4000 ×g) at room temperature. Two
vials containing fresh human plasma (300 μL) and PBS (300 μL, control)
3.1. Chemistry
Aryl sulfonium salt 1 (Scheme 1) was synthesised following the pro-
cedure reported by Sander et al. [41]. The thioether 5 reacted with
A
B
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en
C
r
d
A
Fig. 3. (A) Organ uptake of [18F]FAMTO (i.v.) in male Sprague Dawley rats (n = 3, for each group) at 10, 30, 60 min post-injection. Rats pre-treated with ETO (1 mg/kg, i.v.) 15 min prior to
[
18F]FAMTO i.v. injection and culled after 30 min. A two-tailed paired Student's t-test was used to compare adrenal-liver uptake and adrenal uptake in rats pre-treated with ETO versus that
of control rats. (B) In vivo PET imaging of a male Sprague Dawley rats showing adrenal uptake of [18F]FAMTO after pre-treatment with ETO (1 mg/kg).
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
5
Fig. 4. Plasma metabolite analysis of [18F]FAMTO in male Sprague Dawley rats at 10, 30, 60 min post-radiotracer injection.
diaryliodonium triflate under copper(II) catalysis (125 °C, 1 h) giving 1
3.2. Radiosynthesis of [18F]FAMTO
in 53% yield. Pinacol boronate ester 2 was prepared by the following
procedure adapted from the literature [42]. The para-bromo MTO deriv-
ative (6) reacted with bis(pinacolato)diboron in the presence of potas-
sium acetate and Pd-catalyst to form 2 with a yield of 76 10% (n = 3).
Scheme 1 shows the route used to synthesise 3–6 via Mitsunobu
reaction that involves the use of both an oxidising agent such as di-
tert-butyl azodicarboxylate (DtBad) and a reducing agent such as
triphenylphosphine (PPh3) under mild conditions. Yields between 40
and 60% were obtained.
Two 18F-labeling strategies were selected to produce [18F]FAMTO,
the first via an aryl sulfonium salt (1, Scheme 1) and the second via an
aryl boronic precursor (2).
The strategy of labeling sulfonium salts with 18F fluoride ion has
been reported to be a direct, straightforward nucleophilic substitution
[41]. Following the optimized procedure developed by Sander et al., di-
methyl sulfoxide (DMSO), potassium bicarbonate (KHCO3, 30 mM),
Kryptofix (K222, 30 mM) and 1 (5 mg) in 500 μL DMSO at 110 °C for
Scheme 1. Reaction scheme for production of MTO, FAMTO, 12, and [18F]FAMTO. a: DtBAD, PPh3, THF, 0 °C, rt., 17 h; b: Ph2ITfO, Cu(C6H5COO)2 TFSA, PhCl, 125 °C, 2 h; c: (BPin)2, KOAc,
(C17H14P)2Fe·PdCl2, DMSO, 80 °C, 15 h; d: [18F]fluoride ion, DMSO, 110 °C, 14–30 min; e: [18F]fluoride ion, Cu(Py)4(OTf)2, DMF, 110 °C, 20 min; f: NaOH 2 N, MeOH, rt., 17 h.
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

6
S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
(py)4 and 2 in 500 μL DMF was added to the dried residue of [18F]fluo-
Table 1
Radiosynthesis of [18F]FAMTO from 1.
ride ion/K222/K2CO3 and the reaction mixture heated at 110 °C for
20 min. [18F]FAMTO was produced with non-isolated RCY of 32 6%
(entry 5). We next optimized the reaction with respect to solvent vol-
ume. Decreasing the volume of DMF from 500 to 300 and 200 μL (entries
6–7), the RCY of [18F]FAMTO remained the same. However a further de-
crease of the volume to 150 μL (entry 8), resulted in a non-isolated RCY
drop from 32% to 11%. The synthesis, purification/reformulation and
quality control time for the preparation of [18F]FAMTO are 75, 25 and
20 min, respectively. The radiosynthesis of [18F]FAMTO was achieved
with an overall decay-corrected RCY of isolated [18F]FAMTO of 18
2%, N99% RCP, N99% e.e. and 105 39 GBq/μmol molar activity within
120 min of work, starting from 850 to 734 MBq of 18F-fluoride.
Entry Releasing
solution
Time
(min)
Volume
(mL)
Temperature
(°C)
Non-isolated RCY
(%)c
1
2
3
4
5
6
7
8
Aa
A
A
Bb
B
B
B
B
15
15
30
30
30
30
30
30
500
250
250
500
250
100
250
250
110
110
110
110
110
110
150
180
1
0.5 (n = 3)
0 (n = 2)
0 (n = 2)
8 (n = 1)
7
2 (n = 3)
1, 4 (n = 2)
0 (n = 1)
0 (n = 1)
a
Solution A: 0.5 mL K222 (30 mM) and KHCO3 (30 mM) in MeCN:H2O (85:15).
Solution B: 1 mL of K222 (23 mM) and K2CO3 (6 mM) in MeCN:H2O (85:15).
Determined by radio-TLC (%
b
c
SEM).
3.3. In vitro autoradiography
15 min, [18F]FAMTO was obtained with very low non-isolated radio-
chemical yield (RCY, 1%, entry 1, Table 1).
The in vitro binding of [18F]FAMTO was evaluated by autoradiogra-
phy using pig adrenal, kidney and liver tissues. The incubation of adre-
nal gland sections with [18F]FAMTO showed a high specific binding
with the signal of [18F]FAMTO completely blocked with 1 μM of MTO.
Both liver and kidney organ samples were selected due to their
known role in [11C]MTO metabolism and excretion [25]. Blocking stud-
ies revealed a high specific binding in the liver and kidney, organs
known to be rich in cytochrome P450 enzymes.
Screening of solvent volume, time and bases commonly used for nu-
cleophilic substitution reactions with [18F]fluoride ion was investigated
in an attempt to improve the RCY. Increasing the concentration of pre-
cursor from 0.01 mg/mL to 0.02 mg/mL (entry 2) or increasing the reac-
tion time from 15 to 30 min (entry 3) did not give the desired product.
By substituting KHCO3 with K2CO3, [18F]FAMTO was obtained using 500
and 250 μL of DMSO with a non-isolated RCY of 8% and 7%, respectively
(entries 4–5). Decreasing further the volume of DMSO to 100 μL caused
a RCY drop (entry 6). Increasing the temperature from 110 to 150 or
180 °C, afforded no [18F]FAMTO whatsoever.
3.4. Ex vivo biodistribution, metabolite analysis and in vivo PET imaging
studies in rats
An alternative radiolabelling strategy to produce [18F]FAMTO was
subsequently investigated starting from the aryl boronic derivative 2.
Initially, the experiment was performed by mixing a solution of 2 and
Ex vivo biodistribution data (Fig. 3A and Table S1), performed in
healthy Sprague-Dawley rats, was used to assess the adrenal uptake as
well as the adrenal-to-organ ratio. [18F]FAMTO showed high liver up-
take in the first 10 min (%ID/g = 3.61 1.53) followed by a slight de-
crease at 30 min (%ID/g = 2.16 1.53) and 60 min (%ID/g = 1.68
0.18). Adrenal uptake increased slowly during the first 30 min up to %
ID/g = 6.2 0.46, followed by a slow decrease up to %ID/g = 4.92
0.70 at 60 min. Adrenal-to-liver ratio increased from 0.66 at 10 min to
2.8 at 30 min post-injection (Fig. 3A and Table S1).
Cu(OTf)2(py)4 in DMF with the dried residue of [18F]fluoride ion/K222
/
K2CO3 and heated at 110 °C for 20 min under air. The formation of
[
18F]FAMTO under these conditions was not observed (entry 1, Table 2).
Neumaier and coworkers have reported an efficient protocol to in-
crease the RCY of copper-mediated aromatic radiofluorination using a
“low amount of base” strategy [43]. Indeed using small amount
of K222/K2CO3 in DMF obtained by aliquoting the dried residue of [18F]
fluoride ion/K222/K2CO3 (K222 = 10 μmol and K2CO3 = 6 μmol), a selec-
tive 18F-incorporation was observed affording [18F]FAMTO with a non-
isolated RCY of 17% (entry 2, Table 2). As the “low base” approach
gave promising results, we further reduced the amount of K222 and
K2CO3 present in the reaction solution. Decreasing 2- and 4-times the
amount of K222 and K2CO3, [18F]FAMTO was produced with a RCY be-
tween 16 and 23% (entries 3–4, Table 2).
Subsequent experiments were performed using the same amount of
K2CO3 and K222 for [18F]fluoride ion elution and radiofluorination reac-
tion allowing an easy transferability of manual procedures to automated
synthesis modules. [18F]Fluoride ion was eluted from an anion exchange
resin with a solution of K2CO3 (0.87 μmol) and K222 (1.5 μmol) dissolved
in MeCN:H2O. Following the solvent evaporation, a solution of Cu(OTf)2
Rats that received ETO (1 mg/kg) 10 min prior to injection of [18F]
FAMTO showed a significant decrease in liver uptake (66%, p = 0.005,
see Table S1) and a moderate decrease in the adrenal glands (39%,
p = 0.03). Pre-treatment with ETO increased the ratio of %ID/g in adre-
nal to liver by 38% (p = 0.12) leading to an enhanced adrenal-to-liver
PET signal compared to the non-pre-treatment group. An in vivo PET
image of the adrenal uptake of [18F]FAMTO after pre-treatment with
ETO (1 mg/kg) is shown in Figs. 3B and S4.
Plasma metabolite analysis revealed 13%, 8% and 2% of unchanged
[
18F]FAMTO at 10, 30 and 60 min post-injection, respectively. Two hy-
drophilic metabolites at retention times 3 and 21 min were observed
(Fig. 4). The metabolite eluting at 3 min has been identified as the free
Table 2
Optimization of radiofluorination of 2 to form [18F]FAMTO.
Entrya
Releasing solution
Activity released from QMA (%)
Reaction
Non-isolated RCY (%)b
Isolated RCY (%)c
K222 (μmol)
K2CO3 (μmol)
K222 (μmol)
K2CO3 (μmol)
Volume (DMF)
1
2
3
4
5
6
7
8
20
20
20
12
12
12
95
4 (n = 8)
20
10
5
12
6
3
1.5
0.87
0.87
200
150
500
500
500
500
500
300
200
150
0 (n = 2)
n.d.
n.d.
n.d.
n.d.
14 (n = 5)
n.d.
17, 16 (n = 2)
18, 16 (n = 2)
22, 23 (n = 2)
20
12
3
1.5
1.5
1.5
1.5
0.87
0.87
0.87
0.87
82
2
1.5
1.5
0.87
0.87
32
29
32
6 (n = 5)
7 (n = 3)
2 (n = 18)
28
18
(n = 27)
2 (n = 18)
n.d.
11 (n = 1)
a
b
c
2 (18 μmol), Cu(OTf)2(py)4 (5 μmol), DMF (150–500 μL), 20 min, 110 °C.
Estimated by analytical radio-HPLC of the crude product (% SEM).
Calculated from the amount of isolated [18F]FAMTO to the initial amount of [18F]fluoride ion trapped in the Sep-Pak Accell Plus QMA cartridge (%
SEM).
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
7
acid [18F]12 (Fig. S3). Urine metabolite analysis revealed only one me-
tabolite in solution corresponding to [18F]12 (Fig. S2).
The simplicity of radiolabelling and encouraging results from in vitro
studies justified further evaluation of the new tracer in vivo. Clinically
desirable features for a PET radiotracer to be used in the identification
of adenomas include high and specific radioactivity concentration in ad-
renal glands, rapid renal excretion, and low non-specific uptake of [18F]
FAMTO in non-target tissues (e.g. liver, bone).
3.5. Stability studies in human blood and plasma free fraction
Plasma-derived metabolites can cause a non-specific signal that re-
duces the specificity and quality of PET imaging endpoints. Metabolite
studies of [11C]MTO or [18F]FETO in humans revealed that unchanged
radiotracer accounted for 40% and 11% of total blood-borne radioactivity
at 20 min post-injection, respectively and, considering the rapid in vivo
metabolism of both radiotracers [30,37], we decided to perform [18F]
FAMTO metabolite studies over a similar (20 min) timeframe for
comparison. No degradation of [18F]FAMTO was observed after in-
cubation in human blood for 20 min. The plasma free fraction of [18F]
FAMTO was determined to be 37.55 5.78% (mean SD, n = 5) by
ultrafiltration.
In vivo studies confirmed the high specificity of [18F]FAMTO uptake
in adrenal glands showing a ratio of adrenal-liver uptake of 2.81 at
30 min post-injection, which is similar to the adrenal gland-to-liver
ratio of [11C]MTO in rats (3.8 at 30 min) [32]. This ratio would be ex-
pected to be elevated in the presence of UAPA due to an overexpression
of adrenal enzymes [8,46].
Image analysis of the rats administered with [18F]FAMTO was ham-
pered by the high liver uptake masking the signal of the right adrenal
gland. The pre-treatment of ETO 15 min prior to [18F]FAMTO injection
lowered the liver uptake and increased the signal adrenal gland-to-
liver of [18F]FAMTO (Figs. 3B and S4). Because of the known rapid me-
tabolism of ETO in vivo [47–49], it is not expected that pre-treatment
with 1 mg/kg ETO will cause complete blockade. However, in vitro, in
the absence of MTO metabolism, 1 μM MTO was sufficient to fully
block [18F]FAMTO specific binding.
High radioactivity was observed in the urine (urine-blood ratio was
4.36, 24.9 and 49.1 at 10, 30 and 60 min, respectively). Conversely, no
uptake was visualised in bone, indicating an absence of [18F]FAMTO
defluorination.
4. Discussion
The development of the novel 18F radiotracer targeting adrenal
gland enzymes represents a valuable advancement to the field of PA
PET imaging. Our PET radiotracer development approach started from
the structure-activity relationship analysis of MTO (Ki = 4.02
1.87 nM), a potent inhibitor of CYP11B1 and CYP11B2 adrenal gland en-
zymes, and its derivatives bearing a halogen atom on the benzene ring
(e.g. Br, I and F). [11C]MTO is a prototype radiotracer offering a rapid
non-invasive procedure localizing aldosterone-producing adenomas
[21,44]. MTO derivatives bearing an iodine, bromine or fluorine atom
A metabolite study collecting rat blood samples after [18F]FAMTO in-
jection revealed that 13% of radioactivity after 10 min was due to [18F]
FAMTO (Fig. 4). The main metabolite in plasma and urine was identified
to be the carboxylic acid of [18F]FAMTO ([18F]12, Figs. 4, S2 and S3). In
analogy to the carboxylic acid derivative of MTO, we anticipate that
compound 12 would also have negligible affinity for adrenal gland en-
zymes [34].Low metabolic stability of MTO and FETO has been observed
in vitro and in vivo [30,37]. However, in humans [18F]FETO is
metabolised faster than [11C]MTO. At 20 min post-injection, the un-
changed [18F]FETO is 11% whereas [11C]MTO is 40% of total radioactivity
[30,37]. The methyl-ester [18F]FAMTO might have the same profile
as [11C]MTO, rather then the ethyl-ester [18F]FETO, however this hy-
in the 4-position of the benzene ring (Ki = 8.7
3.2 nM, 8.8
2.4 nM and 8.15 0.85 nM, respectively) have similar inhibitory activ-
ity to MTO, indicating that structural modification on the benzene ring
does not significantly impact the affinity to the target's binding pocket
[24,34,39]. These results encouraged us to attach a fluorine-18 atom at
para-position of the MTO benzene ring generating a novel PET radio-
tracer ([18F]FAMTO, Fig. 1).
Initial investigations, focused on using previously recognised strate-
gies to incorporate fluorine-18 into aromatic rings in a one-step reaction
using synthetically accessible precursors such as aryl sulfonium salt 1 or
an aryl boronic precursor 2 to obtain the R-enantiomer of [18F]FAMTO.
pothesis needs to be confirmed by determining
[
18F]FAMTO
radiometabolic profile in humans. Blood-borne esterases might also
produce radiometabolites to generate a non-specific background signal,
reducing the specificity and quality of PET imaging endpoints. Because
of this the stability of [18F]FAMTO was evaluated in human plasma re-
vealing that [18F]FAMTO is highly stable in human plasma and blood.
The R-configuration of MTO (IC50 = 3.69
1.92 nM) is a 130 times
more potent an inhibitor than its S-enantiomer (IC50 = 492
281 nM) [34].
Using the aryl sulfonium strategy, a benzylic 1H-imidazole deriva-
tive, ETO fragment, has been radiolabelled by Sander et al. in 31% RCY
[41]. Applying the same conditions to 1, the production of [18F]FAMTO
was not achieved. Following a procedure developed by Mu et al. [45]
that uses K2CO3 instead of KHCO3 and lowering the base concentration
from 15 to 6 μmol, [18F]FAMTO was obtained in low non-isolated RCY
(7 2%).
5. Conclusion
[
18F]FAMTO, a new 18F-labelled analogue of (R)-MTO was synthe-
sised in a one-step radiofluorination procedure in good yields. The
method utilises a synthetically accessible boronic ester precursor 2, pro-
duced in a one-step reaction. A low base protocol is crucial for successful
An alternative radiolabelling strategy to produce [18F]FAMTO was
subsequently investigated starting from the aryl boronic derivative 2.
When the dried residue of [18F]fluoride ion/K222/K2CO3 is taken up in
a solution in DMF in the presence of copper-catalyst and 2, the 18F-
incorporation was not observed. A similar trend was observed by
Neumaier et al. who demonstrated that large quantities of K2CO3 to
elute [18F]fluoride ion from ion exchange cartridges impacts negatively
on copper-mediated radiofluorination reactions, decreasing the RCY of
the desired radiotracer [43]. Indeed using small aliquots of K222/K2CO3
in DMF obtained by dissolving the dried residue of [18F]fluoride ion/
[
[
18F]FAMTO labelling. In vitro and in vivo experiments have shown that
18F]FAMTO accumulates in adrenal glands, with good adrenal-to-liver
ratios in rodents and a good radiometabolic profile with slow kinetics
in the adrenals and rapid kinetics in the liver. The simplicity of
radiolabelling and encouraging preclinical results justify progression
to toxicology safety assessment studies and translation of [18F]FAMTO
to healthy volunteers and patients with adrenal lesions of different ad-
renocortical and non-adrenocortical origin.
K
222/K2CO3, a selective 18F-incorporation was observed affording [18F]
Acknowledgment
FAMTO with an overall decay-corrected RCY of isolated [18F]FAMTO of
18%.
This work was supported by Medical Research Council (MRC, MR/
K022733/1) and Wellcome/EPSRC Centre for Medical Engineering [WT
203148/Z/16/Z]. The authors acknowledge financial support from the
Department of Health via the National Institute for Health Research
(NIHR) comprehensive Biomedical Research Centre award to Guy's
In vitro autoradiography on pig adrenal, liver and kidney sections,
initially used to characterize the binding properties of [18F]FAMTO,
demonstrated that [18F]FAMTO bound specifically to adrenal gland en-
zymes and that binding was completely blocked using 1 μM of MTO.
Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002

8
S. Bongarzone et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
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Appendix A. Supplementary data
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Please cite this article as: S. Bongarzone, F. Basagni, T. Sementa, et al., Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission
tomography (PET) radiotracer for imaging CYP11B1..., Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2018.11.002