[18F]Atorvastatin: synthesis of a potential molecular imaging tool for the assessment of statin-related mechanisms of action

Article abstract of DOI:10.1186/s13550-020-00622-4

Background: Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not respond to statin treatment, and the reason for this is still not completely understood. [¹⁸F]Atorvastatin, the ¹⁸F-labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research. Results: [¹⁸F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage ¹⁸F-deoxyfluorination. The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield. Optimization and automation of the labeling procedure reliably yielded an injectable solution of [¹⁸F]atorvastatin in 19% ± 6% (d.c.) with a molar activity of 65 ± 32 GBq·μmol^?1. Incubation of [¹⁸F]atorvastatin in human serum did not lead to decomposition. Furthermore, we have shown the ability of [¹⁸F]atorvastatin to cross the hepatic cell membrane to the cytosolic and microsomal fractions where HMG-CoA reductase is known to be highly expressed. Blocking assays using rat liver sections confirmed the specific binding to HMG-CoA reductase. Autoradiography on rat aorta stimulated to develop atherosclerotic plaques revealed that [¹⁸F]atorvastatin significantly accumulates in this tissue when compared to the healthy model. Conclusions: The improved ruthenium-mediated ¹⁸F-deoxyfluorination procedure overcomes previous hurdles such as the addition of salt additives, the drying steps, or the use of different solvent mixtures at different phases of the process, which increases its practical use, and may allow faster translation to clinical settings. Based on tissue uptake evaluations, [¹⁸F]atorvastatin showed the potential to be used as a tool for the understanding of the mechanism of action of statins. Further knowledge of the in vivo biodistribution of [¹⁸F]atorvastatin may help to better understand the origin of off-target effects and potentially allow to distinguish between statin-resistant and non-resistant patients.

Full text of DOI:10.1186/s13550-020-00622-4

Clemente et al. EJNMMI Research
(2020) 10:34
https://doi.org/10.1186/s13550-020-00622-4
ORIGINAL RESEARCH
Open Access
[18F]Atorvastatin: synthesis of a potential
molecular imaging tool for the assessment
of statin-related mechanisms of action
Gonçalo S. Clemente1, Jens Rickmeier2, Inês F. Antunes1, Tryfon Zarganes-Tzitzikas3, Alexander Dömling3,
Tobias Ritter2 and Philip H. Elsinga1*
Abstract
Background: Statins are lipid-lowering agents that inhibit cholesterol synthesis and are clinically used in the
primary and secondary prevention of cardiovascular diseases. However, a considerable group of patients does not
respond to statin treatment, and the reason for this is still not completely understood. [18F]Atorvastatin, the 18F-
labeled version of one of the most widely prescribed statins, may be a useful tool for statin-related research.
Results: [18F]Atorvastatin was synthesized via an optimized ruthenium-mediated late-stage 18F-deoxyfluorination.
The defluoro-hydroxy precursor was produced via Paal-Knorr pyrrole synthesis and was followed by coordination of
the phenol to a ruthenium complex, affording the labeling precursor in approximately 10% overall yield.
Optimization and automation of the labeling procedure reliably yielded an injectable solution of [18F]atorvastatin in
19% 6% (d.c.) with a molar activity of 65 32 GBq·μmol−1. Incubation of [18F]atorvastatin in human serum did not
lead to decomposition. Furthermore, we have shown the ability of [18F]atorvastatin to cross the hepatic cell
membrane to the cytosolic and microsomal fractions where HMG-CoA reductase is known to be highly expressed.
Blocking assays using rat liver sections confirmed the specific binding to HMG-CoA reductase. Autoradiography on
rat aorta stimulated to develop atherosclerotic plaques revealed that [18F]atorvastatin significantly accumulates in
this tissue when compared to the healthy model.
Conclusions: The improved ruthenium-mediated 18F-deoxyfluorination procedure overcomes previous hurdles
such as the addition of salt additives, the drying steps, or the use of different solvent mixtures at different phases of
the process, which increases its practical use, and may allow faster translation to clinical settings. Based on tissue
uptake evaluations, [18F]atorvastatin showed the potential to be used as a tool for the understanding of the
mechanism of action of statins. Further knowledge of the in vivo biodistribution of [18F]atorvastatin may help to
better understand the origin of off-target effects and potentially allow to distinguish between statin-resistant and
non-resistant patients.
Keywords: Statins, Atorvastatin, HMG-CoA, 18F-deoxyfluorination, Fluorine-18, Positron emission tomography
* Correspondence: p.h.elsinga@umcg.nl
1Department of Nuclear Medicine and Molecular Imaging – University
Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713, GZ,
Groningen, The Netherlands
Full list of author information is available at the end of the article
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which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
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permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Background
neurodegenerative [49–53]). The exact off-target mecha-
Cardiovascular diseases represent one of the leading nisms of statins, along with the reason why many pa-
causes of death globally [1]. Atherosclerosis, a chronic tients show resistance to this class of drugs, are still
inflammatory pathology characterized by deposition of unknown. Therefore, an increase of specific knowledge
plaques (a collection of fat, cholesterol, calcium, fibrin, of the subcellular mechanisms affected by statins and
and cellular waste products) in the walls of arteries, is the development of a more sensitive tool to investigate
the dominant cause of cardiovascular diseases [2]. The this subject are currently challenging hot topics in medi-
partial rupture and detachment of these plaques increase cinal chemistry [54, 55]. High-resolution molecular im-
the risk of clogging the blood flow, which, ultimately, aging modalities, such as positron emission tomography
can lead to highly incapacitating or even mortal condi- (PET), together with sensitive nuclear analytical tech-
tions such as myocardial infarction or cerebrovascular niques relying on radiolabeled molecules, can help in a
accidents [3]. The exact cause of atherosclerosis remains better understanding by mapping the biodistribution
a subject of discussion and has been connected to sev- profile over time within complex living organisms. To
eral distinct mechanisms, hypotheses, and theories [4– this purpose, we report the 18F-labeling of atorvastatin
16]. Nonetheless, despite its complexity, atherosclerosis (12), one of the most widely used statins in the preven-
is generally correlated to the levels of cholesterol in tion of cardiovascular risk factors and one of the best-
plasma [17–22]. HMG-CoA (3-hydroxy-3-methyl-gluta- selling drugs in pharmaceutical history [56]. Exchanging
ryl-coenzyme A) reductase is involved in the biosyn- the aryl fluoride with its 18F radioisotope enables the
thesis of cholesterol and is subject to feedback radiolabeling of atorvastatin without changing its
regulation by end-products of this same pathway [23– pharmacological properties. However, in the case of
26]. Thus, inhibition of HMG-CoA reductase with sta- atorvastatin, the 18F-fluorination of electron-rich arenes
tins became an essential strategy for the primary preven- is a classical struggle in radiochemistry and has chal-
tion
of
atherosclerosis.
Statins
contain
a
lenges associated with synthesis automation [57]. The
dihydroxycarboxylic acid moiety that mimics and out- fluorine-containing aromatic ring within the atorvastatin
competes the natural substrate molecule HMG-CoA, structure is electron-rich, and therefore, we use the
preventing its reduction to mevalonate and further chol- ruthenium-mediated radiodeoxyfluorination strategy
esterol synthesis [27, 28] (Fig. 1a).
[58–60] to synthesize [18F]atorvastatin ([18F]12). This
In addition to the cholesterol-lowering action, the suc- radiotracer may have the potential to become an attract-
cess of statins became increasingly connected with ive tool for statin-related research, enabling the under-
broader pleiotropic effects [29–32]. Statins are increas- standing of cellular and subcellular mechanisms, the
ingly being associated with potential protective effects identification of off-target activity and, ultimately, allow-
on pathologies beyond cardiovascular diseases (e.g., re- ing to select between statin-resistant and non-resistant
spiratory [33–37], carcinogenic [38–43], viral [44–48], patients for targeted therapy.
Fig. 1 Role of HMG-CoA reductase in the synthesis of cholesterol (a) and structure of some of the most clinically used statins (b)

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Scheme 1 Synthesis of ketal and tert-butyl ester side-chain protected benzyl ether pyrrole intermediate 6 and atorvastatin precursor 11
Results
palladium on carbon (Pd/C)-catalyzed hydrogenolysis
(Scheme 2). Coordination of 7 to ruthenium (II) de-
Synthesis of precursors and 18F-fluorination strategy
The synthesis of the benzyl ether pyrrole intermediate 6, creases its π-electron density, providing 8, and activates
as well as the atorvastatin precursor 11, was performed the precursor towards radiodeoxyfluorination [58–60].
by the treatment of the corresponding 1,4-diketone in-
termediates 4 and 10, respectively, with the commer- Radiochemistry optimization: improvement and
cially available primary amine 5 in a pivalic acid- simplification of the Ru-mediated 18F-deoxyfluorination
catalyzed Paal-Knorr condensation reaction (Scheme 1). Synthesis of [18F]atorvastatin ([18F]12) started with con-
The 1,4-diketone intermediates 4 and 10 were synthe- ventional trapping of the aqueous [18F]fluoride, pro-
sized via a Stetter reaction [61, 62]. In summary, the α,β- duced by a cyclotron using the 18O(p,n)18F nuclear
unsaturated ketone 1 was combined with benzaldehydes reaction, on an anion exchange cartridge (Chromafix 45-
2 and 9, respectively, in the presence of triethylamine PS-HCO3 ). The [18F]fluoride was then eluted from the
−
and the thiazolium bromide catalyst 3 to afford the cor- cartridge with a solution of the labeling precursor 8
responding 1,4-diketone intermediates 4 and 10, re- (2.5 μmol), N,N-bis(2,6-diisopropylphenyl-1-chloroimi-
spectively. Atorvastatin (12) was obtained from its dazolium chloride (13, 6.0 equiv.), and bis(trimethylneo-
precursor 11 after near quantitative removal of the pentylammonium) oxalate in a mixture of ethanol:
ketal-ester protecting groups in the side-chain with se- pivalonitrile:veratrole (400 μL, 1:4:4 v:v:v) directly into a
quential hydrochloric acid and sodium hydroxide reaction vial [60]. The reaction mixture was heated
treatment.
under vigorous stirring at 140 °C for 30 min to afford the
For the radiofluorination of atorvastatin, the benzyl key intermediate [18F]11. A drawback of this literature
ether pyrrole intermediate 6 was synthesized and subse- procedure is the reduced elution efficiency from the
quently converted to the corresponding phenol 7 by anion exchange cartridge. Besides, there is no
Scheme 2 [18F]Atorvastatin ([18F]12) synthesis approach used in this work

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commercial supplier of the oxalate salt. This may hinder systems (without the use of eluent additives) to increase
good manufacturing/radiopharmaceutical/clinical prac- the elution efficiency (Table 2).
tice (GMP/GRPP/GCP) applications with the synthe-
By changing the solvents used to dissolve the labeling
sized radiotracer as additional analytical evaluations, precursor 8 and the chloroimidazolium chloride 13,
toxicity tests, and reliable and established production of while keeping all other reaction conditions unchanged,
the additive may be needed to comply with potential we were able to improve not only the elution efficiency
clinical trials in human patients [63]. As mentioned in a but also the yield of the 18F-deoxyfluorination to achieve
previous report [58], salt additives tend to reduce the the intermediate product [18F]11. Using a mixture of
18F-deoxyfluorination yield even though the elution effi- methanol:veratrole (1:3 v:v) or methanol:DMSO (1:3 v:v)
ciency is increased. Thus, in this work, several readily provided the best results. However, the mixture with
available additives were evaluated in order to achieve a veratrole quickly builds up high pressure in the reaction
better elution alternative that can increase the elution ef- vial at 140 °C (with some associated radioactivity escape).
ficiency while minimally affecting the 18F-deoxyfluorina- Thus, replacing veratrole by dimethylsulfoxide (DMSO)
tion yield (Table 1).
is the safer choice of solvent. This optimized method
As expected, most of the tested additives have a nega- boosted and simplified the 18F-deoxyfluorination tech-
tive effect on the 18F-deoxyfluorination yield. The use of nique by avoiding the addition of salt additives, skipping
bis(trimethylneopentylammonium) oxalate [58–60] gave any need for washing the trapped [18F]fluoride, circum-
the best overall yields of [18F]11. This addition still de- venting time-consuming azeotropic drying (with the
creases the 18F-deoxyfluorination conversion by approxi- consequent radioactivity losses by natural decay, evapor-
mately 10% when compared to the experiments without ation, and unspecific adsorption to the reactor surface)
an eluent additive. An acceptable alternative to the oxal- and the use of different solvent mixtures for the various
ate seems to be the use of the commercially available steps of the process (elution and 18F-fluorination). The
tetrabutylammonium chloride, which, despite lowering amount of Ru-coordinated precursor 8 was also reduced
the elution efficiency, led to a similar [18F]11 yield.
in relation to the previously reported 18F-deoxyfluorina-
Further experiments showed that in the absence of an tion works (2.5 μmol vs. 5.0 μmol) [58–60]. During the
eluent additive, the elution efficiency can be more than optimization work, it became evident that some proced-
doubled by reversely loading and eluting the 45-PS- ure deviations such as air bubbling instead of stirring,
−
HCO3 cartridge or by replacing this cartridge to a short absence of mechanical mixing, increased solvent vol-
1/16″ PTFE tubing filled with approximately 10 mg of a umes, larger reaction vials, or flat-shaped bottom ones
Biorad MP-1 resin (Fig. 2). However, reversing the cart- can decrease the final 18F-deoxyfluorination yield. In
ridge cannot easily be implemented in most automated order to enhance the radiolabeling efficiency, these are
modules. Also, since the MP-1 mini-cartridge is not features that should be considered when choosing the
commercially available and should, therefore, be manu- most suitable radiosynthesis module.
ally prepared, it might result in significant elution differ-
Additionally, an exciting finding is the relative toler-
ences from batch to batch, which will affect the activity ance of 18F-deoxyfluorination to the presence of water,
yield. This led us to the evaluation of different solvent which allowed direct fluorination using aqueous
[18F]fluoride from the cyclotron target without the need
Table 1 Influence of eluent additives in the synthesis of the intermediate product [18F]11
Eluent additive
Elution efficiencya
TLC conversion
83%
HPLC purity
80%
[
18F]11 yieldb
Bis(trimethylneopentylammonium) oxalate
Kryptofix 222, K2C2O4
Tetraethylammonium bicarbonate
Tetrabutylammonium chloride
Sodium acetate
75%
86%
54%
62%
12%
67%
65%
54%
42%
50%
24%
9%
59%
48%
34%
50%
91%
82%
46%
0.2%
16%
16%
20%
32%
10%
15%
Silver acetate
29%
82%
Silver triflate
69%
36%
Sodium oxalate
52%
71%
None
85%
90%
aCalculated by the ratio between the [18F]fluoride trapped in the anion exchange cartridge (45-PS-HCO3−) and the radioactivity recovered (without reversing the
cartridge) in the reaction vial
bNon-isolated 18F-deoxyfluorination yield based on radio-TLC and radio-HPLC analysis of the crude product and having in consideration the elution efficiency
resulting from the salt additive used in relation to the starting radioactivity. The final percentage of [18F]11 yield was determined by multiplying the elution
efficiency with radio-TLC conversion of the starting [18F]fluoride and with radio-HPLC purity (n ≥ 2)

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−
Fig. 2 Anion exchange cartridge alternatives tested in this work. a Sep-Pak Accell Plus QMA Plus Light Cartridge (Waters). b 45-PS-HCO3
(Chromafix). c Reversed 45-PS-HCO3− (Chromafix). d Handmade 1/16″ PTFE tubing with MP-1 resin (Biorad)
Table 2 Influence of the solvent system in the synthesis (without eluent additives) of the intermediate product [18F]11
Solvent (400 μL)
Elution efficiencyc
TLC conversion
85%
HPLC purity
90%
[
18F]11 yieldd
Ethanol:pivalonitrile:veratrole (1:4:4)
Ethanol:acetonitrile:DMSO (1:4:4)
Veratrole
42%
32%
41%
16%
18%
25%
40%
75%
64%
32%
2%
69%
75%
80%
30%
85%
61%
Pivalonitrile
35%
80%
63%
Dimethyl sulfoxide (DMSO)
Methanol:DMSO (1:2)
Methanol:DMSO (1:3)
Methanol:DMSO (1:3.5)
Methanol:DMSO (1:7)
Butanol:DMSO (1:2)
57%
68%
64%
86%
64%
72%
86%
95%
92%
85%
86%
87%
65%
55%
90%
50%
20%
23%
Butanol:DMSO (1:3)
54%
80%
79%
34%
43%
25%
84%
82%
74% (45%)
Ethanol:DMSO (1:3.5)
70%
78%
78%
Water:DMSO (1:17)
36%
85%
83%
Aqueous [18F]fluoride:DMSO (3:97)a
Methanol:veratrole (1:3)
Methanol + veratrole:pivalonitrile (1:1)b
100% (no cartridge)
90%
92%
91%
94%
97%
93% (56%)
85%
94%
a30 μL of aqueous [18F]fluoride was directly added (no elution cartridge needed) to the DMSO solution containing 8 and 13 and left to react
b300 μL of methanol was used to dissolve 8 and 13 to elute the cartridge. Methanol was then evaporated, and the solvent exchanged to 400 μL of
veratrole:pivalonitrile (1:1 v:v) for the reaction. This method, despite having high elution efficiency (93%), showed significant losses (up to 45%) of the eluted
[
18F]fluoride during the evaporation of methanol. Thus, the real efficiency and [18F]11 yield is shown in brackets
cCalculated by the ratio between the [18F]fluoride trapped in the anion exchange cartridge (45-PS-HCO3−) and the radioactivity recovered (without reversing the
cartridge) in the reaction vial
dNon-isolated 18F-deoxyfluorination yield based on radio-TLC and radio-HPLC analysis of the crude product and having in consideration the elution efficiency
resulting from the solvent mixture used in relation to the starting radioactivity. The final percentage of [18F]11 yield was determined by multiplying the elution
efficiency with radio-TLC conversion of the starting [18F]fluoride and with radio-HPLC purity (n ≥ 2)

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Fig. 3 Overall scheme of the full Synthra RNplus setup used
for anion exchange cartridges. The drawback is the low the intermediate [18F]11 (18F-deoxyfluorination yield es-
volume of aqueous [18F]fluoride that can be used, which timated by multiplying radio-TLC conversion of
limits the amount of radioactivity in the reaction. Never- [18F]fluoride with radio-HPLC purity, 87 9%; n = 10).
theless, this is potentially a suitable method for small- The ketal and tert-butyl protecting groups were removed
scale productions aimed, for example, for in vitro by sequentially treating the reaction mixture with 1 mL
evaluations.
of methanol:HCl 6 M (49:1 v:v) at 60 °C for 5 min,
followed by addition of 0.5 mL of methanol:aqueous
NaOH 50% (9:1 v:v) and stirring at 60 °C for another 5
Automated synthesis of [18F]atorvastatin ([18F]12)
The optimized procedures were translated to the Syn- min. HPLC analysis revealed the complete absence of
thra RNplus radiosynthesizer. Minimal modifications the intermediate [18F]11 from the reaction mixture. The
were made to the commercial configuration of the mod- content of the reaction vial was automatically loaded
ule (Fig. 3). All unused loading ports to the reaction vial onto the built-in preparative HPLC (with UV and radio-
were closed with perfluoroalkoxy plugs, except one that activity detector) to collect the [18F]atorvastatin
was left open with an ethylene tetrafluoroethylene fe- ([18F]12) fraction. Final solid-phase extraction (SPE) en-
male luer-to-male fitting for connection to an exhaust abled the solvent exchange, and [18F]12 was then refor-
alumina N cartridge (Waters). In summary, aqueous mulated in a physiological and injectable calcium acetate
[18F]fluoride was trapped on a Chromafix 45-PS-HCO3− solution to mimic the atorvastatin (12) calcium prepar-
anion exchange cartridge preactivated by sequentially ation in which the therapeutic dose of this drug is gener-
passing 3 mL of K2C2O4 (10 mg/mL) and 2 mL of water ally administered. The radiochemical yield of
and dried with a flow of argon (trapping efficiency, 94% [18F]atorvastatin ([18F]12) was 19
6%, n = 10 (d.c.),
4%; n = 10). After drying the resin in the cartridge and the molar activity was 65 32 GBq·μmol−1 (n = 10)
under helium flow for approximately 1 min, the trapped at the end of synthesis. The activity yield achieved for
[18F]fluoride was eluted by pushing a solution of the Ru- [18F]12 was 1.3 0.4 GBq (n.d.c.) when starting with 9.6
coordinated labeling precursor 8 (2.5 μmol) and N,N-
1.9 GBq of aqueous [18F]fluoride produced in cyclo-
bis(2,6-diisopropylphenyl-1-chloroimidazolium chloride tron (n = 10).
(13, 6.0 equiv.) in methanol:DMSO (1:3 v:v, 0.4 mL) over
2 min through the cartridge, directly to a reaction vial
Stability tests and characterization of [18F]atorvastatin
preheated at 110 °C (elution efficiency, 88 5%; n = 10). ([18F]12)
This preheating seems to avoid the significant radio- The final purified and formulated radiotracer was com-
activity losses that were seen when rapidly increasing pared with the corresponding non-radioactive reference
from room temperature to the reaction temperature or standard by analytical radio-HPLC to confirm identity
when the reaction vial was already set at 140 °C. The re- (Fig. 4). [18F]Atorvastatin ([18F]12) showed a radiochem-
action was left to stir for 30 min at 140 °C to produce ical purity always higher than 95%, and no sign of

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Fig. 4 Analytical HPLC profiles (red: γ detector, blue: UV detector) of [18F]atorvastatin ([18F]12), standard atorvastatin (12), radiofluorinated
intermediate [18F]11, non-radioactive intermediate 11, and Ru-coordinated labeling precursor 8
Fig. 5 In vitro autoradiography with [18F]atorvastatin ([18F]12) on rat liver tissue counterparts without (control) and with (blocked) standard
atorvastatin (12) pre-treatment

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decomposition was observed for at least 4 h in solution showed an average 60% 8% decrease in the binding of
at room temperature. Incubation of the radiotracer ei- the radiotracer after blocking (p < 0.0001).
ther in human (collected off the clot from healthy volun-
The potential of [18F]atorvastatin ([18F]12) to be used
teers) or in Wistar rat serum at 37 °C for up to 4 h as a PET tracer for atherosclerosis imaging was also pre-
indicated an in vitro stability of > 95%. Log D was mea- liminarily evaluated by autoradiography of the aorta
sured to confirm the lipophilicity of the radiotracer with from a normal and an atherosclerotic rat model. Due to
the one described for atorvastatin (12). An experimental a reduction of lipoprotein clearance, apolipoprotein E-
log D of 1.61 0.12 (pH 7.4, n = 4) was measured, being deficient rats fed with high-cholesterol diet tend to de-
in accordance with the reported values (log D approx. velop elevated plasma levels of cholesterol and to form
1.5 [64]).
atherosclerotic plaques [69, 70]. Incubation of the aorta
excised from a validated rat model for atherosclerosis
[71] with the radiotracer showed high [18F]atorvastatin
([18F]12) uptake in the atherosclerotic aorta (Fig. 6). The
Evaluation of [18F]atorvastatin ([18F]12) in rat liver
homogenates and tissue autoradiography
HMG-CoA reductase is highly expressed in the liver, radiotracer uptake is reduced when this aorta is pre-
where it is subject to hormonal, dietary, and pharmaco- treated with standard atorvastatin (12) and in the aorta
logical regulation [65–67]. Evaluation of [18F]atorvastatin of a normal rat. Average uptake in the atherosclerotic
([18F]12) in rat liver homogenates revealed that 87%
aorta is approximately two times higher than the uptake
4% (n = 4) of the radioactivity is found in the micro- in the normal aorta (where baseline levels of the HMG-
somal and cytosolic fractions, which is in accordance CoA reductase are still present due to its ubiquitous
with known enzyme distribution [68]. Further radio- cytoplasmatic expression [72]), being the difference be-
TLC assessment showed the absence of degradation and tween these groups statistically significant (p = 0.004). In
18F-defluorination. Blocking assays using rat liver sec- the normal aorta, the uptake between control and ator-
tions with and without standard atorvastatin (12) pre- vastatin (12) treated groups showed an average 49%
treatment were performed to evaluate [18F]atorvastatin 13% decrease in the binding efficiency of the radiotracer
([18F]12) binding selectivity to the HMG-CoA reductase (p = 0.0002). When comparing the uptake results be-
(Fig. 5). The comparison of [18F]atorvastatin ([18F]12) tween non-treated and treated atherosclerotic aorta, an
uptake between non-treated and treated liver sections
Fig. 6 In vitro autoradiography with [18F]atorvastatin ([18F]12) on the aorta counterparts of a normal and atherosclerotic rat model without
(control) and with (blocked) standard atorvastatin (12) pre-treatment

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average decrease of 56% 16% was seen in the binding uptake was observed when the hepatic tissue was pre-
efficiency of the radiotracer (p = 0.0006).
treated with non-radioactive atorvastatin (12).
HMG-CoA reductase is known to be overexpressed in
atherosclerotic plaques, and its levels are correlated to
plaque instability and contribute to the risk of partial
rupture and detachment [75]. To evaluate the potential
of [18F]atorvastatin ([18F]12) as a PET tracer for athero-
sclerosis, an autoradiography imaging study was per-
formed on an excised aorta from an apolipoprotein E-
deficient rat model designed to develop atherosclerotic
plaques [71]. The uptake of the radiotracer was statisti-
cally significant higher in atherosclerotic aorta when
compared to a healthy rat aorta. Furthermore, the pre-
treatment with the reference compound 12 blocked
[18F]atorvastatin ([18F]12) binding, which supported the
potential of this PET tracer to be used for the specific
detection of atherosclerotic plaques. As a result of sev-
eral decades in clinical use, the toxicological profile of
atorvastatin (12) is widely known. Thus, first in human
studies that are already being envisaged at our Nuclear
Medicine Department are not being hampered by tox-
icity issues.
Discussion
In this work, we explored the late-stage 18F-deoxyfluori-
nation of phenols via ruthenium π-complexes, to im-
prove our previous attempt to synthesize the β+ emitter
version of atorvastatin following the boronic acid pinacol
ester cross-coupling late-stage [18F]fluorination strategy
[73]. This widely clinically prescribed statin has evident
benefits in cardiovascular disease prevention and may
play a role in other conditions that still require further
elucidation. The adjustments used in this work for the
Ru-mediated 18F-deoxyfluorination strategy proved to be
an improvement compared to the previously reported
procedures [58–60], including halving the initial labeling
precursor amount, avoiding the use of different solvent
mixtures and salt additives, and omitting [18F]fluoride
washing and solvent removal procedures. This, together
with the simplicity of the process, the tolerance to a var-
iety of solvents (including aqueous solutions), and the
low reaction volumes used, might result in the Ru-
mediated deoxyfluorination strategy being suitable for
implementation in emerging techniques such as
microfluidics-lab-on-a-chip radiochemistry or microdro-
plet radiosynthesizers. By automating the optimized pro-
cedures, this radiofluorination strategy reliably produced
[18F]atorvastatin ([18F]12) in isolated radiochemical
yields of 19% 6% (d.c.). Within a synthesis time of ap-
proximately 80 min, the final injectable physiological so-
lution of the radiotracer was obtained with suitable
in vitro stability (at least upon 4 h), radiochemical purity
Conclusions
We have optimized the late-stage Ru-mediated 18F-deox-
yfluorination strategy performing the synthesis in an au-
tomated module (n = 10) with suitable radiochemical
purity (> 95%) and molar activity (65 32 GBq·μmol−1).
The affinity of [18F]atorvastatin ([18F]12) to HMG-CoA
reductase and its potential to be used as a PET tracer for
atherosclerosis was confirmed in rat liver samples and
by comparison of the radiotracer uptake in the aorta of a
normal and atherosclerotic rat model. This radiotracer
can be a useful tool for the in vitro assessment of statin-
related mechanisms of action. Future clinical trials may
also be facilitated, as its toxicological profile is already
widely characterized due to the extensive clinical use of
the non-radioactive analog.
(> 95%), and molar activity (65
evaluate its potential to be used as a PET imaging agent
for atherosclerosis.
32 GBq·μmol−1) to
The access to a radiolabeled analog of atorvastatin al-
lows the mapping and quantification of the radiotracer
uptake either in cellular and subcellular compartments
or in living complex organisms through high sensitive
nuclear analytical and imaging techniques. [18F]Atorva-
statin-PET might become a relevant research tool for
the assessment of statin-related mechanisms of action
and to discriminate between responsive and non-
responsive patients. Standard atorvastatin (12) is known
to occupy the binding site of HMG-CoA reductase with
high affinity (Ki approx. 14 nM [27]) and effectiveness
(IC50 approx. 8 nM [74]). Thus, the pre-treatment of the
liver sections with atorvastatin (12) hinders the access of
additional substrate to the active site. By incubating rat
liver sections, widely known to highly express HMG-
CoA reductase [65–67], with [18F]atorvastatin ([18F]12),
the expected selective binding to this enzyme was con-
firmed since a statistically significant decrease of the
Methods
Chemical synthesis
The syntheses of atorvastatin (12), labeling precursor 8,
and all intermediate compounds are described and char-
acterized in the Supporting Information file.
General procedure for the radiosynthesis of atorvastatin
([18F]12) by Ru-mediated deoxyfluorination
Cyclotron-produced aqueous [18F]fluoride was loaded
onto
a polystyrene-divinylbenzene copolymers-based
−
HCO3 anion exchange cartridge (Chromafix 45-PS-
HCO3−) preconditioned by sequentially pushing 3 mL of
K2C2O4 (10 mg/mL) and 2 mL of water and dried with a
flow of Ar. Ru-coordinated labeling precursor
8
(2.5 μmol) and N,N-bis(2,6-diisopropylphenyl-1-

Clemente et al. EJNMMI Research
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Page 10 of 13
chloroimidazolium chloride (13, 6.0 equiv.), with or octanol in a microcentrifuge tube. This mixture was
without a salt additive (see Table 1), were dissolved in strongly vortexed at room temperature and then centri-
400 μL of a solvent (see Table 2). This solution was used fuged at 3000 rpm for 5 min. Triplicate samples from
to elute the [18F]fluoride directly to a 3-mL glass Whea- both organic and aqueous phases were collected and
ton reaction V-vial or a 5-mL glass reactor from a Syn- measured on a ɣ-counter. The log D value was reported
thra RNplus radiosynthesizer (containing a stirring bar). as the average ratio between the number of counts in
The reaction mixture was left under vigorous stirring at the n-octanol (upper layer) and PBS (lower layer) ob-
140 °C for 30 min. The intermediate species [18F]11 was tained in 4 independent measurements.
analyzed by radio-TLC (TLC-SG developed with hexane:
For the in vitro stability tests, final reformulated
ethyl acetate (1:1 v:v), Rf ([18F]F-) = 0.0–0.1 and Rf [18F]12 was left at room temperature and analyzed by
([18F]11) = 0.8–0–9) and radio-HPLC (SymmetryPrepTM radio-HPLC and radio-TLC at distinct time points up to
C18 7 μm 7.8 × 300 mm; A, sodium acetate 0.05 M pH
4 h. The in vitro stability assays were performed by incu-
4.7; B, acetonitrile; 0–4 min, 90% A; 4–15 min, 90% A to bating 30 μL of [18F]12 (approx. 1.5 MBq) in 0.3 mL of
20% A; 15–25 min, 20% A to 5% A; 25–33 min, 5% A; both human and Wistar rat serum, at 37 °C, up to 4 h
33–34 min, 5% A to 90% A; 34–35 min, 90% A; flow, 6
and analyzed by radio-TLC and radio-HPLC (in the lat-
mL/min; Rt ([18F]11) ≈ 24 min). To produce the final ter case after protein precipitation with acetonitrile) at
product [18F]12, 1 mL of a methanol:HCl 6 M (49:1 v:v) various time points. All experiments were done in
solution was added to the reaction mixture and left to triplicate.
stir at 60 °C for 5 min. Subsequently, 0.5 mL of a metha-
nol:aqueous NaOH 50% (9:1 v:v) solution was added to Liver homogenate assay
the reaction mixture and left to stir at 60 °C for 5 min. Livers from Wistar rats were harvested, immediately fro-
Production of [18F]12 was assessed by radio-TLC (TLC- zen in liquid nitrogen, and conserved at − 80 °C. For the
SG developed with ethanol:sodium phosphate 0.1 M pH
binding assay, tris-HCl (0.05 M, pH 7.4) was added to
7.4 (65:35 v:v), Rf ([18F]F-) = 0.0–0.1, Rf ([18F]11) = 0.5– the thawed rat livers to obtain a final concentration of
0.6, and Rf ([18F]12) = 0.8–0.9) and radio-HPLC (Rt 20 mg/mL. The mixture was homogenized, using a Hei-
([18F]12) ≈ 16 min). Final reformulated product was ob- dolph DIAX 600 homogenizer, while being cooled in an
tained after collecting the [18F]12 fraction by radio- ice bath. Liver homogenate batches were stored at −
HPLC (SymmetryPrepTM C18 7 μm 7.8 × 300 mm; A, 80 °C. For the binding assay, 300 μL of rat liver hom-
sodium acetate 0.05 M pH 4.7; B, acetonitrile; 0–4 min, ogenate or Tris-HCl solution (for evaluation of the non-
90% A; 4–15 min, 90% A to 20% A; 15–25 min, 20% A specific binding (NSB) fraction of the radiotracer to the
to 5% A; 25–33 min, 5% A; 33–34 min, 5% A to 90% A; tube walls) was added to each centrifuge tube. Tris-HCl
34–35 min, 90% A; flow, 6 mL/min; Rt ([18F]12) ≈ 16
solution containing 0.3% human serum albumin was
min; Rt ([18F]11) ≈ 24 min) and diluting it in 45 mL of added to reach a final volume of 475 μL and left for pre-
water. This bulk solution was then passed through an incubation for 15 min at room temperature. Then, 25 μL
Oasis HLB 1 cc cartridge (30 mg sorbent, Waters) to effi- of [18F]atorvastatin ([18F]12, approx. 1 MBq) was added
ciently trap [18F]12. The final radiotracer was then to each assay tube, briefly vortexed, and then incubated
washed with 10 mL of water and recovered with 0.5 mL for 60 min under gentle shaking at room temperature.
of ethanol. Final dilution in 5 mL of calcium acetate Incubation was terminated by centrifuging (Rotanta
0.05 M pH 7.0 resulted in an isotonic and injectable 46RS Hettich zentrifugen) the assay tubes at 4 °C for 15
[18F]atorvastatin ([18F]12) solution. For the quality con- min (7500 rpm). The supernatant (S9 fraction containing
trol (QC) of the final radiotracer and assessment of the cytosol and microsomes) of each tube was transferred to
molar activity,
a
radio-UPLC system was used new tubes leaving the pellet (cell debris) in the original
(ACQUITY HSS T3 1.8 μm 3.0 × 50 mm column; A, so- tube. The content of NSB assay tubes was also pipetted
dium acetate 0.01 M in H2O:MeOH:ACN 9:0.6:0.4 v:v:v; out to new tubes. All assay tubes were measured on a
B, sodium acetate 0.01 M in H2O:MeOH:ACN 1:5.4:3.6 Wallac Wizard 1480 ɣ-counter to calculate the percent-
v:v:v; 0–2 min, 100% A; 2–5 min, 100% A to 40% A; 5–6
age of radioactivity in each phase (S9 or pellet) with
min, 40% to 0% A; 6–9 min, 0% A; 9–10 min, 0 to 100% NSB percentage being subtracted to the pellet results.
A; flow, 0.8 mL/min; UV 244 nm; Rt ([18F]12) ≈ 6.2 min).
Autoradiographic imaging of rat liver
Partition coefficient and stability evaluation
Harvested livers from four Wistar rats (n = 4) were sec-
The log D was measured to determine the lipophilicity tioned in three thin (1–2 mm) sagittal slices (1–2 mm).
of [18F]atorvastatin ([18F]12). The final reformulated ra- To reduce the intervariability, each liver slice was di-
diotracer (100 μL, in about 10% ethanol) was dissolved vided into two counterparts and distributed equally be-
in a mixture of 410 μL PBS (pH 7.4) and 490 μL n- tween the control and blocking groups (n = 4). The

Clemente et al. EJNMMI Research
(2020) 10:34
Page 11 of 13
sections of each group were impregnated in 3.3 mL of to quantify the radiotracer uptake in digital lumines-
phosphate-buffered saline (PBS) enriched with glucose cence units (DLU).
(5.6 mM), MgCl2 (0.49 mM), and CaCl2 (0.68 mM) previ-
ously warmed at 37 °C. To the control group, 100 μL of
DMSO:polyethylene glycol sorbitan monolaurate (9:1 v:
v) was added. To the blocking group, 100 μL of atorva-
statin (2 mg in DMSO:polyethylene glycol sorbitan
monolaurate 9:1 v:v) was added to reach a total concen-
tration of 1 mM with a final volume of 3.5 mL. All liver
sections were left to incubate for 30 min. After the incu-
bation time, 100 μL of [18F]atorvastatin ([18F]12, approx.
4 MBq) was added to each group and left to incubate for
1 h. Finally, the total volume of liquid in all groups was
carefully pipetted out and the liver slices were washed 3
times with 3 mL of cold PBS followed by 3 mL of iced
water. The slices were then carefully dried and imaged
with a GE Healthcare Amersham Typhoon autoradiog-
raphy system. The acquired data were analyzed with the
OptiQuant 03.00 software to quantify the radiotracer up-
take in digital luminescence units (DLU).
Statistics
Data are expressed as the mean
standard deviation
(SD). Unpaired two-tailed t tests were used for statistical
evaluations. A p < 0.01 was considered statistically sig-
nificant. Statistical analyses of data were performed
using GraphPad Prism version 6.01 for Windows
(GraphPad Software, La Jolla, CA, USA).
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s13550-020-00622-4.
Additional file 1:. Supporting Information file.
Abbreviations
d.c.: Decay corrected; DLU: Digital luminescence units;
DMSO: Dimethylsulfoxide; GCP: Good clinical practice; GMP: Good
manufacturing practice; GRPP: Good radiopharmacy practice; HMG-CoA: 3-
Hydroxy-3-methyl-glutaryl-coenzyme A; HPLC: High-performance liquid
chromatography; IC50: Half-maximal inhibitory concentration; Ki: Inhibitory
constant; LD50: Median lethal dose; LDL: Low-density lipoprotein; MP-
1: Macroporous equivalent of analytical grade 1 resin; n.d.c.: Non-decay
corrected; NSB: Non-specific binding; PBS: Phosphate-buffered saline (pH 7.4);
PET: Positron emission tomography; PTFE: Polytetrafluoroethylene;
QC: Quality control; Rf: Retention factor; Rt: Retention time; SPE: Solid-phase
extraction; TLC: Thin-layer chromatography; TLC-SG: Thin-layer
chromatography paper impregnated with a silica gel; UPLC: Ultra-
performance liquid chromatography; UV: Ultraviolet
Autoradiographic imaging of rat normal and
atherosclerotic aorta
Harvested aortas from a healthy Wistar rat and from an
apolipoprotein E-deficient Wistar rat model for athero-
sclerosis [71] were transversely sectioned in 7 segments
each from the aortic arch to the suprarenal abdominal
aorta end. To reduce sample intervariability, since it is
not likely that the distribution of atherosclerotic plaque
is homogeneous throughout the total aortic length, each
segment was then opened and divided with a sagittal cut
in two counterparts. Each counterpart of the same seg-
ment was then distributed between two study groups:
non-blocked (control) and blocked with atorvastatin.
The exposed lumen was finally washed with PBS to
guarantee that any potential trace of blood was cleaned
out. Then, each aorta section was impregnated in 180 μL
of PBS enriched with glucose (5.6 mM), MgCl2 (0.49
mM), and CaCl2 (0.68 mM) previously warmed at 37 °C.
To the control group, 10 μL of DMSO:polyethylene gly-
col sorbitan monolaurate (9:1 v:v) was added. To the
blocking group, 10 μL of atorvastatin (0.1 mg in DMSO:
polyethylene glycol sorbitan monolaurate 9:1 v:v) was
added to reach a total concentration of 1 mM when in
the final 200 μL. All aorta sections were left to incubate
for 30 min. After the incubation time, 10 μL of [18F]ator-
vastatin ([18F]12, approx. 0.4 MBq) was added to each
group and left to incubate for 1 h. Finally, the total vol-
ume of liquid was carefully pipetted out and each aorta
section was washed 3 times with 250 μL of cold PBS
followed by 250 μL of iced water. The slices were then
carefully dried and imaged with a GE Healthcare Amer-
sham Typhoon autoradiography system. The acquired
data were analyzed with the OptiQuant 03.00 software
Acknowledgements
The authors thank Luiza Reali Nazario for kindly supplying the Wistar rat
livers. We also gratefully thank Jürgen Sijbesma and Nafiseh Ghazanfari for
providing the atherosclerotic and normal Wistar rat aortas. G.S.C. would like
to thank the Open Technologieprogramma from NWO STW (project no.
13547), The Netherlands, for the scholarship.
Authors’ contributions
Methodology: GSC, JR, and IFA; chemical synthesis, purification, and
analytical data: JR, GSC, and TT-Z; radiochemistry: GSC and JR; automation:
GSC and IFA; biological in vitro assays: GSC and IFA; original draft writing:
GSC; supervision: PHE, IFA, TR, and AD. All authors have made substantial in-
tellectual contributions, and read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
Samples of the compounds, and datasets used and/or analyzed during the
current study are available from the authors on reasonable request. The data
that support the findings of this study are included in this published article
and its supplementary information files
Ethics approval and consent to participate
All studies were carried out in compliance with the local ethical guidelines
for animal experiments. The protocol was approved by the Animal Ethics
Committee of the University of Groningen.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.

Clemente et al. EJNMMI Research
(2020) 10:34
Page 12 of 13
Author details
controversial therapeutic management. Arch Med Res. 2016;47:491–5.
https://doi.org/10.1016/j.arcmed.2016.11.009.
1Department of Nuclear Medicine and Molecular Imaging – University
Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713, GZ,
Groningen, The Netherlands. 2Max-Planck-Institut für Kohlenforschung,
Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany. 3Department
of Drug Design, University of Groningen, Antonius Deusinglaan 1, 9713, AV,
Groningen, The Netherlands.
21. Peters SAE, Singhateh Y, Mackay D, Huxley RR, Woodward M. Total
cholesterol as a risk factor for coronary heart disease and stroke in women
compared with men: a systematic review and meta-analysis. Atherosclerosis.
2016;248:123–31. https://doi.org/10.1016/j.atherosclerosis.2016.03.016.
22. Borén J, Williams KJ. The central role of arterial retention of cholesterol-rich
apolipoprotein-B-containing lipoproteins in the pathogenesis of
atherosclerosis: a triumph of simplicity. Curr Opin Lipidol. 2016;27:473–83.
https://doi.org/10.1097/MOL.0000000000000330.
Received: 18 February 2020 Accepted: 26 March 2020
23. Honda A, Salen G, Honda M, Batta AK, Tint GS, Xu G, et al. 3-Hydroxy-3-
methylglutaryl-coenzyme A reductase activity is inhibited by cholesterol
and up-regulated by sitosterol in sitosterolemic fibroblasts. J Lab Clin Med.
2000;135:174–9. https://doi.org/10.1067/mlc.2000.104459.
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