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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