Improved, one-pot synthesis of 6-[18F]fluorodopamine and quality control testing for use in patients with neuroblastoma

Article abstract of DOI:10.1002/jlcr.3685

6-[¹⁸F]Fluorodopamine ([¹⁸F]F-DA) is taken into cells via the norepinephrine transporter (NET). Recent [¹⁸F]F-DA positron emission tomography–computed tomography (PET-CT) imaging of adult neuroendocrine tumors shows a dramatic improvement in sensitivity over the standard-of-care, meta-iodobenzylguanidine (MIBG) single-photon emission computed tomography (SPECT)–CT. A new precursor (ALPdopamine) allows no-carrier-added synthesis resulting in high–molar activity [¹⁸F]F-DA. Automated synthesis of [¹⁸F]F-DA was performed in a single reactor using a two-step procedure: 1) fluorination via thermolysis of a diaryliodonium salt precursor, followed by 2) acid hydrolysis. Phase transfer agents, Kryptofix 222 and two tetraalkylammonium salts, were investigated. Optimized synthesis of [¹⁸F]F-DA was achieved in 56 to 60?minutes (26% end of synthesis [EOS], nondecay corrected). The product passed all Food and Drug Administration (FDA)–required quality control testing for human use. Accumulation of [¹⁸F]F-DA in SK-N-BE(2)-C (high NET expression) cells was significantly higher than in SH-EP (minimal NET expression) cells (P?18F]F-DA for human use. Validation of uptake by neuroblastoma (NB) cell lines supports the use of [¹⁸F]F-DA for imaging NB patients. A pediatric NB imaging trial using [¹⁸F]F-DA PET has been approved (Investigational New Drug application (IND) no. 138638) based on the methods reported here. We expect [¹⁸F]F-DA will be localized in NB tumors and that high-quality functional images will be obtained within minutes after injection.

Full text of DOI:10.1002/jlcr.3685

18
Improved, One-pot Synthesis of 6-[ F]Fluorodopamine and
Quality Control Testing for Use in Patients with Neuroblastoma
a
b,1
a
c,2
Amy L. Vāvere , Kiel D. Neumann , Elizabeth R. Butch , Bao Hu ,
c,
a
Stephen G. DiMagno , and Scott E. Snyder *
a
St. Jude Children’s Research Hospital, Division of Nuclear Medicine,
Department of Diagnostic Imaging, Memphis, TN USA;
b
Ground Fluor Pharmaceuticals, Lincoln, NE USA;
c
University of Illinois Chicago, Department of Medicinal Chemistry and Pharmacognosy,
Chicago, IL USA
*
Corresponding Author:
Scott E. Snyder, Ph.D.
Department of Diagnostic Imaging
St. Jude Children’s Research Hospital
2
62 Danny Thomas Place, Mail Stop #220
Memphis, TN 38105
Email: scott.snyder@stjude.org
P: 901-595-3347
1
2
Present address: University of Virginia, Department of Radiology and Medical Imaging,
Charlottesville, VA USA - kielneumann@virginia.edu
Present address: Weill Cornell Medicine, Department of Radiology and Citigroup
Biomedical Imaging Center, New York, NY USA – bah2017@med.cornell.edu
Disclaimers:
Financial Support: Research funding was provided by ALSAC-St. Jude Children’s Research
Hospital, The Hartwell Foundation, and NIH (R01 EB013713-01A1).
18
Abbreviated Title: Improved [ F]F-DA Synthesis and Quality Control Validation
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3685
This article is protected by copyright. All rights reserved.

ABSTRACT
18
18
6
-[ F]fluorodopamine ([ F]F-DA) is taken into cells via the norepinephrine transporter
1
8
(
NET). Recent [ F]F-DA PET-CT imaging of adult neuroendocrine tumors shows a dramatic
improvement in sensitivity over the standard-of-care, meta-iodobenzylguanidine (MIBG)
SPECT-CT. A new precursor (ALPdopamine™) allows no-carrier-added synthesis resulting
1
8
18
in high molar activity [ F]F-DA. Automated synthesis of [ F]F-DA was performed in a
single reactor using a two-step procedure; fluorination and thermolysis of a diaryliodonium
®
salt precursor followed by acid hydrolysis. Phase transfer agents, Kryptofix 222 and two
1
8
tetraalkylammonium salts, were investigated. Optimized synthesis of [ F]F-DA was
achieved in 56-60 minutes (26% EOS, non-decay corrected). The product passed all FDA-
1
8
required quality control testing for human use. Accumulation of [ F]F-DA in the SK-N-
BE(2)-C (high NET expression) cells was significantly higher than in SH-EP (minimal NET
TM
expression) cells (p < 0.0001). ALPdopamine provides an effective scaffold for the routine
18
production of [ F]F-DA for human use. Validation of uptake by neuroblastoma (NB) cell
18
lines supports the use of [ F]F-DA for imaging NB patients. A pediatric NB imaging trial
1
8
using [ F]F-DA PET has been approved (IND #138638) based on the methods reported here.
18
We expect [ F]F-DA will be localized in NB tumors and that high quality functional images
will be obtained within minutes after injection.
18
Key Words: Fluorine-18, 6-[ F]Fluorodopamine, F-DA, Neuroblastoma, PET
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1
. INTRODUCTION
Neuroendocrine tumors arise from the sympathetic nervous system and share many of the
1
same biochemical pathways and proteins as these neurons. As such, the nerve-like
phenotype of these tumor cells can be exploited for functional imaging using radiotracers
originally designed for neuronal applications. The norepinephrine transporter (NET) is highly
expressed in most neuroblastomas (NB) and pheochromocytomas (PHEO), where NET
2
, 3
actively transports norepinephrine and its analogs across the cell membrane. This uptake is
also influenced by the vesicular monoamine transporter (VMAT), which is known to
4
transport monoamines for storage in synaptic vesicles.
Currently, evaluation of NB depends on a combination of anatomical imaging using
computed tomography (CT) and functional imaging by single photon emission computed
1
23
tomography (SPECT) using meta-[ I]iodobenzylguanidine (MIBG). MIBG uptake into NB
cells is also mediated by NET, however, several experimental parameters reduce the quality
of current NB imaging, including: 1) poor image resolution of SPECT compared to PET, 2)
complex quantification, and 3) the relatively high lipophilicity of MIBG prolongs non-
specific retention resulting in higher background and poor contrast. The ~24 hour delay
required for tumor visualization presents a practical burden to patients and limits the clinical
1
8
18
utility of MIBG. 6-[ F]Fluorodopamine, ([ F]F-DA), is a catecholamine that is actively
1
8
taken into cells via NET. Recent clinical PET-CT imaging of [ F]F-DA in adults with
neuroendocrine tumors shows that this tracer possesses high specificity and improved
5
-7
18
sensitivity vs. MIBG SPECT-CT for visualization of disseminated disease. Because [ F]F-
DA is less lipophilic than MIBG, background activity clears quickly. Thus, we expect that
18
[
F]F-DA will be highly concentrated in NB tumors, and that utilizing modern PET-CT
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methods, high quality functional images will be obtained within minutes after injection,
eliminating the need for patients to return for next-day imaging as with MIBG.
18
Investigation of [ F]F-DA in pediatric NB patients has been limited by the inability to
produce usable quantities of tracer that pass the rigorous quality testing required for injection
1
8
into children. A key stumbling block has been access to [ F]F-DA that possesses high molar
1
8
activity. The pharmacokinetics and pharmacodynamics of [ F]F-DA have been extensively
6
, 8, 9
studied and validated in adults with neuroendocrine tumors
. The focus of this
investigation was to develop a facile method for nucleophilic production of this tracer in high
molar activity and sufficient radiopharmaceutical quality to allow pediatric use. Currently,
1
8
the only synthesis of [ F]F-DA for human use in the U.S. is achieved by the
18
10
fluorodestannylation of a 6-aryl trimethylstannyl precursor using [ F]F gas. Sufficient
2
yields for patient studies are attainable, but due to the target gas containing 3% F , molar
2
activities are low (50.32 GBq/mmol, 1.36 Ci/mmol). As a result, dose activities must be
limited to avoid injection of a physiologically active, high mass dose of non-radioactive
fluorodopamine present in the final product. This electrophilic synthesis, while direct,
necessitates the purchase of a dedicated F target and the appropriate equipment for handling
2
corrosive fluorine gas for targetry and chemistry. A more recent report presented an updated
method with improved molar activity of the final product while still using an electrophilic
9
method. Despite this improvement, the synthesis still requires the use of specialized
equipment for corrosive fluorine gas. Furthermore, use of tin-based precursors requires
additional testing during quality control to ensure that all residual tin has been effectively
removed.
18
The first nucleophilic preparation of [ F]F-DA, reported by Ding and Fowler in 1991,
1
8
11
was a manual, 120 min synthesis that provided [ F]F-DA in 10% yield (EOS). The impetus
1
8
for this research was to investigate no-carrier-added [ F]F-DA as a tracer for imaging
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12-14
cardiac sympathetic denervation in baboons.
. This multistep, tour-de-force synthesis was
1
5
recently automated in our lab ; however, the complexity of this synthesis limits the
robustness and feasibility for clinical studies.
1
8
The proven utility of [ F]F-DA for imaging neuroendocrine tumors has stimulated efforts
to find an efficient, reliable nucleophilic route for this tracer. For example, Preshlock and
1
8
coworkers described a synthetic approach in which aryl boronic esters react with [ F]fluoride
1
6
18
in the presence of copper ion catalyst. While isolated yields of [ F]F-DA were sufficient
(29%), deprotection reaction conditions were harsh (hydroiodic acid and temperatures > 150

°
C), and analyses of the final product for residual copper catalyst or Kryptofix 222 were not
included. Zlatopolskiy and coworkers described radiofluorination of an aryl nickel(II)
1
8
17
complex by [ F]fluoride in the presence of a hypervalent iodine oxidant. A radiochemically
pure product was achieved with a radiochemical yield of 12% (6.3% activity yield).
Radiochemical yield suffered in part due to the duration of the synthesis (100 min).
Development of a highly selective radiofluorination methodology and an appropriate
diaryliodonium salt precursor by Neumann and coworkers provided a reliable means for
1
8
18, 19
nucleophilic production of [ F]F-DA.
Since the introduction of this chemistry,
improvements in precursor synthesis and protecting group strategies have enabled milder
1
8
20
reaction conditions to be used in the production of F-labled catecholamines. The work
1
8
presented here describes an efficient, one-pot automated synthesis of [ F]F-DA that features
milder reaction conditions, fewer synthetic steps, a new, acid-labile precursor, and improved
final purification (Scheme 1). These advances have made possible the routine production of
18
[
F]F-DA, which meets USP <823> specifications and is available for pediatric human use.
18
Finally, we show preliminary in vitro data to support the accumulation of [ F]F-DA in NB
cell lines.
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[SCHEME 1.]
2
. MATERIALS AND METHODS
2
.1. General
All chemicals were purchased from Sigma Chemical Company (St. Louis, MO), unless
otherwise noted. All aqueous solutions were prepared with distilled, deionized water (Milli-Q
Integral Water Purification System, Millipore Corp.; 18.2 Μ·cm resistivity). The purified
precursor and organic solvents for radiosynthesis were stored in an anaerobic chamber (Coy
Laboratory Products, Inc.) under argon to reduce exposure to atmospheric water. Radioactive
samples were analyzed in a CRC-15R Dose Calibrator (Capintec, Inc.) for determination of
activity (expressed in megabecquerels or millicuries). Cell uptake assay samples were
counted for radioactivity (decays per minute) in a WIZARD2™ 2480 Automatic Gamma
Counter (PerkinElmer Life and Analytical Sciences). Purification of the final radiotracer was
achieved using a custom-built injector system and an Agilent 1100 Series high performance
liquid chromatography (HPLC) pump. Thin-layer chromatography (TLC) samples were
analyzed for radioactivity using an AR-2000 radio-TLC Imaging Scanner (Eckert & Ziegler -
Bioscan) equipped with WinScan 3 image analysis software. Analytical HPLC was
performed on an Agilent 1200 Series LC System using both diode array detection and a
Bioscan Flow-Count radionuclide detector (Eckert & Ziegler - Bioscan). Gas chromatography
was conducted on an Agilent 7890A GC System.
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2
.2 Synthesis of (2‐{2‐[bis(tert‐butoxycarbonyl)amino]-ethyl}‐4,5‐bis-(ethoxy-methoxy)-
phenyl)(4‐methoxyphenyl)-iodanium triflate (1)
Preparation of the iodonium salt precursor, known as ALPdopamine™, was previously
2
1, 22
reported in the literature.
Due to the high purity of precursor needed for radiochemical
syntheses, a survey of different solvents and conditions led to simple recrystallization from
methyl-tert-butyl ether (MTBE) which provided material of > 99% (by HPLC) chemical
purity.
1
8
2
.3 Synthesis of 6-[ F]fluorodopamine
1
8
Briefly, [ F]F-DA was produced in a two-step synthesis by thermolysis of a
1
8
18
diaryliodonium [ F]fluoride and subsequent deprotection of the resulting [ F]fluoroarene

(
Scheme 1). The method was adapted for preparation on a Synthera automated synthesizer
(Version 1, IBA) using an Integrated Fluidic Processor (IFP) originally designed for
18
nucleophilic production of [ F]fluorodeoxyglucose (FDG) as shown in Figure 1.
[FIGURE 1.]
18
®
18
Aqueous [ F]fluoride, produced in an IBA Cyclone 18/9 cyclotron from O-enriched
water, was passed through a QMA cartridge (46 mg; ORTG, Inc. or Myja Scientific -
pretreated with 2 mL of 1 M sodium bicarbonate and rinsed with 5 mL water) to capture the
18
18
[
F]fluoride. The [ F]fluoride was eluted from the QMA resin with 620-650 μL of one of
three phase-transfer reagent solutions: (1) 94:6 acetonitrile:water solution containing
potassium carbonate (0.62 mg) and Kryptofix 222 (6.2 mg), (2) 1.5 - 3.4 mg
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tetraethylammonium bicarbonate (TEA HCO ) dissolved in acetonitrile:water (95:5), or (3)
3
5
0-150 uL (1.5 - 3.4 mg) of tetrabutylammonium bicarbonate (TBA HCO ) solution (ABX,
3
provided as 0.075 M in water and ethanol for stabilization) diluted to 650 µL with
acetonitrile. The solvents were removed under argon flow with reduced pressure and heating
at 110 °C for 2.5 minutes. The diaryliodonium salt precursor (1, ALPdopamine™), dissolved
in 10% anhydrous acetonitrile and toluene (15 mg in 1 mL), was added to the reactor for ion-
exchange at room temperature followed by heating to 120 °C for 8 minutes to allow for
thermolysis of the fluorinated complex. The solvent was removed under reduced pressure
with argon flow (approx. 3 minutes) and deprotection of the fluorinated intermediate (2) was
achieved by addition of 1 mL of 4 M hydrochloric acid and heating to 95 °C for 20 minutes
1
8
to afford the crude 6-[ F]fluorodopamine product (3). The solution was diluted by the
addition of 4 mL of HPLC eluent (details below), then passed through a 0.45 μm filter and
into a 5 mL HPLC loop. Purification was achieved by semi-preparative HPLC utilizing a
Hamilton PRP-1 column (10 x 250 mm, 10 µm) with an eluent of 0.1% acetic acid and 0.02%
ascorbic acid at a flow rate of 3 mL/min. The desired product was collected in 3.3 mL of
eluent at a retention time of approximately 8-12 minutes. The retention time of the desired
peak was shifted 3 minutes later when using the tetra-n-alkyl ammonium salts vs. Kryptofix
2
.2.2. Peak collection was initiated based on monitoring with a radioactivity detector and
collected for 1 minute 10 seconds. Neutralization of the final product was achieved by the
addition of 2 mL of 50 mM ammonium acetate (pH 9.2), resulting in a final pH of 5.5 and an
osmolarity of 60.4 mOsm/L.
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2
.4. Quality Control
Full quality control testing was designed to comply with the published FDA
1
0
requirements for release. In addition to the detailed testing described below, tests for pH,
color and clarity, radionuclidic identity, filter integrity, bacterial endotoxins, and sterility
were performed. The final product was confirmed to be clear and colorless with no visual
evidence of cloudiness or particulate matter as per USP <823> and USP <631> Color and
1
8
achromicity. After the product [ F]F-DA was collected, the sterilizing filter was tested for
filter integrity to give an indication of likelihood of product sterility by using a bubble point
procedure, whereby the sterilizing filter was placed on a gas line with a pressure gauge and
the outlet of the filter is placed under water. The gas pressure on the inlet to the filter was
increased slowly until a steady stream of bubbles was observed at the filter outlet. The
pressure when the bubble stream began had to be ≥ 50 psi to pass filter integrity. The pH of
18
the final formulation of [ F]F-DA was tested by spotting on a narrow range pH indicator
strip and the color was compared to the color range chart provided by the manufacturer. The
radionuclidic identity was determined on a small aliquot of the final product by observing the
radioactivity half-life (t ) using a Capintec CRC-15PET dose calibrator with measurements
½
taken at intervals of every 10 minutes over a period of 20 min and the half-life was
determined.
The final drug product was tested for the presence of bacterial endotoxins utilizing the
®
Endosafe -PTS™ unit to determine the endotoxin concentration in a sample. Sterility was
tested using the direct inoculation method as is recommended by the USP, and the samples
were observed over 14 days. Positive growth is indicated by cloudiness in the culture media.
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2
.4.1. Radiochemical Purity & Molar Activity
18
Radiochemical purity of 6-[ F]fluorodopamine was confirmed by analytical HPLC using
an Agilent Zorbax SB-Aq column (4.6 x 150 mm, 5 µm) with an eluent of 10% acetonitrile
and 0.1 monosodium phosphate monohydrate, 0.27 mM disodium
ethylenediaminetetraacetic acid (EDTA) dihydrate, and 0.92 mM octanesulfonic acid (pH
.5) at a flow rate of 1 mL/min with UV monitoring at 220 nm. Initially, a range of 6-
M
3
fluorodopamine (ABX) standard concentrations was analyzed to confirm linearity of the UV
absorbance curve and to determine the lowest detectable injected mass for this system.
18
[
F]F-DA sample to confirm system suitability and to calculate the amount of F-DA mass in
the sample, if present. The area of any UV peak that corresponded to the retention time of the
standard was used to extrapolate the mass present in the sample. The radioactivity of the
sample was also measured, and together, these values were used to calculate the molar
activity.
2
.4.2. Volatile Organic Impurities


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18
[
F]F-DA sample to confirm system suitability.
2
.4.3. Residual Phase Transfer Agent

Kryptofix 222


Kryptofix 222


Residual TEA HCO was determined via ion-pair HPLC with indirect UV detection using
3
4
-aminophenol HCl and 1-heptanesulfonic acid as background UV absorbing regents based
2
5
on a method by Zou et al. A series of injections (0.05 to 5 µg, 0.1 to 10 µL ) of TEA HCO
3
were performed using an Agilent Zorbax XDB-C18 column (5 µm, 4.6 x 150 mm) and a flow
rate of 1 mL/min with 20% methanol , 0.7 mM 4-aminophenol HCL, and 0.15 mM 1-
heptanesulfonic acid as the eluent. The standards were prepared in a solution containing
HPLC purification eluent and neutralization buffer (0.1% acetic acid, 0.02% ascorbic acid, 50
mM sodium bicarbonate). The area of the negative peak near 3.2 minutes (230 nm) was
plotted vs. injected mass to create a linear curve. The equation of this line was used to
18
determine the residual TEA HCO mass in [ F]F-DA samples based on the area of the same
3
negative peak. In addition, a second experiment was performed to confirm the predictive
power of this method by injecting eight known amounts of TEA HCO (0.2 to 3.5 µg) and
3
comparing the calculated mass value to the actual mass.
Residual TBA HCO was determined by visual spot test on silica by over-spotting each
3
sample with a MeOH/NH OH solution and developing in an I chamber by adapting the
4
2
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2
6
method of Kuntzsch et al. Reference standards of 0, 50, 100, 150, and 200 µg/mL TBA
HCO were prepared in a solution of HPLC purification eluent and a neutralization buffer
3
(0.1% acetic acid, 0.02% ascorbic acid, 50 mM sodium bicarbonate). Each standard was
spotted (4 µL) on a glass-backed silica TLC plate. After drying, 10 µL of methanol/25%
ammonium hydroxide solution (9:1) was applied on top of each spot. The plate was then
placed in an iodine chamber to develop for one minute. Samples were visually compared to
reference standards for interpolation of TBA HCO concentration.
3
2
.5. NB Cell Uptake
All cell culture reagents were purchased from Invitrogen and cells were cultured in
humidified atmospheric conditions (37 °C, 5% CO ). Two neuroblastoma cell lines were
2
selected based on their differential NET expression and demonstrated uptake of
27, 28
norepinephrine. SH-EP cells - with virtually no expression of NET
and non-detectable
2
9
uptake of norepinephrine - were kindly provided by Dr. Eva L. Feldman, M.D., Ph.D.,
University of Michigan (Ann Arbor, MI) and grown in high glucose Dulbecco’s Modified
Eagle Medium (DMEM), 10% fetal bovine serum (FBS), pyruvate and 2 mM glutamate. SK-
N-BE(2)-C cells showed expression of adrenergic neuronal markers and significant uptake of
2
9
®
norepinephrine and were obtained from American Type Culture Collection (ATCC CRL-
2
268™). These cells were grown in a 1:1 mixture of Eagle’s MEM and F-12 medium
containing 10% FBS and 2 mM L-glutamine. Cells were seeded approximately 16 hours prior
5
to the experiment in 35 mm, 6-well plates (BD, Falcon) at a density of 8 x 10 cells/well.
18
Wells were treated with 0.185 MBq (5 µCi) of 6-[ F]fluorodopamine in 1 mL of media and
incubated for 2, 10 and 30 minutes at 37 °C. The reaction was terminated by aspirating the
media and rinsing the wells three times with ice-cold phosphate-buffered saline (PBS), pH
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7
.4. Cells were incubated for 5 minutes with 0.5 mL 1X cell lysis buffer (Cell Signaling)
containing 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice. Plates were scraped and
lysates were counted in a gamma counter. Each sample was normalized for protein
concentration by Pierce BCA protein assay (Thermo Scientific). Error bars denote the
standard deviation from three wells.
3
3
. RESULTS
1
8
.1. Synthesis of 6-[ F]fluorodopamine
18
As [ F]fluoride incorporation into electron-rich aromatic systems has historically been
challenging, several radiolabeling reactions were performed and halted prior to deprotection
1
8
to investigate the effect of the phase transfer agent on the conversion of precursor (1) to F-
1
8
fluorinated intermediate (2). The extent of [ F]fluoride incorporation was analyzed by TLC
on silica developed in 30:70 ethyl acetate:hexane and analyzed for radioactivity. Unreacted
fluorine-18 remained at the origin on the plate while the fluorinated intermediate (2) migrated

(
R = 0.4 - 0.45). Utilizing a Kryptofix 2.2.2 as the phase transfer agent (620 µL of a 94:6
f
1
8
acetonitrile:water solution containing 6.2 mg of K222 and 0.62 mg of K CO ), [ F]fluoride
2
3
incorporation averaged 72.2 ± 3.7% (n = 4; Figure 2).
[FIGURE 2.]
A second phase-transfer agent, tetraethylammonium bicarbonate (TEA HCO ), was
3
dissolved in 650 µL acetonitrile:water (95:5) at 1.5 - 3.4 mg per reaction. Conversion did not
proceed reliably with less than 2 mg (5.1% and 32.4% for 1.5 and 1.75 mg, respectively), but
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resulted in approximately 55% conversion for 2.0, 2.5, and 3.4 mg TEA HCO . A third
3
phase-transfer agent, tetrabutylammonium bicarbonate (TBA HCO ), was purchased as a
3
0
-
.075M solution in water and ethanol (for stabilization). Varying amounts of this solution (50
150 µL,1.5 - 3.4 mg) were diluted to 650 µL with acetonitrile. This reagent resulted in at
least 20% higher conversion of the intermediate over TEA HCO with 74.3% to 84.9% for
3
2
.0 to 3.4 mg.
The complete synthesis of [ F]F-DA was achieved using an IBA Synthera and a single
18

nucleophilic fluorination cassette (IFP™) in 56-60 minutes, including HPLC purification. For

syntheses using Kryptofix 222/K CO solution as a phase transfer agent, radiochemical
2
3
1
8
purity of the crude [ F]F-DA prior to HPLC purification was 53.1 ± 0.9% (n = 3), and the
HPLC purified product was obtained in 22.9 ± 3.7% radiochemical yield at EOS (n = 19).
Full syntheses using the tetraalkylammonium compounds resulted in similar, radiochemical
18
yields (uncorrected) of [ F]F-DA (Figure 3).
[FIGURE 3.]
18
Syntheses with TEA HCO as the phase-transfer agent resulted in a purified [ F]F-DA
3
yield of 23.0 ± 3.8% (2.5 mg TEA HCO , n = 9) and 22.5 ± 3.4% (3.4 mg TEA HCO , n =
3
3
8
). Finally, the highest yields were achieved with TBA HCO as the phase-transfer agent and
3
resulted in a yield of 25.8 ± 2.6% (2-2.5 mg, n = 12) resulting in a significant improvement

over Kryptofix 222/K CO (p = 0.025).
2
3
3
.2. Quality Control
Full quality control testing was designed to comply with FDA-approved acceptance
1
8
criteria for [ F]F-DA previously reported for the electrophilic method in preparation for
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1
0
human use (see Table 1.) Standard tests for visual appearance, pH, radionuclidic identity,
bacterial endotoxins, filter integrity, and sterility all passed without issue.
[TABLE 1.]
3
.2.1. Chemical and Radiochemical Purity
The chemical and radiochemical purity of the final product was confirmed by analytical
HPLC by comparison to a 6-fluorodopamine standard (ABX). Radiochemical purity was
determined to be > 97% in all cases. In the samples analyzed for molar activity (n = 3), a UV
peak correlating to the retention time of fluorodopamine showed an average mass
concentration of 2.4 ± 1.1 µg/mL. These measurements were calculated on typical syntheses
1
8
for the preparation of [ F]F-DA for in vitro or in vivo experiments resulting in an average
molar activity of 42.5 ± 28.7 TBq/mmol (1148 ± 776.2 Ci/mmol) with an average starting
activity of 10.8 ± 0.48 GBq (293 ± 12.9 mCi) of fluorine-18. While no mass peak matching
the specific retention time of fluorodopamine was present, a small peak within 0.2 min was
observed. To be conservative in our determination of injected mass, this peak was attributed
to fluorodopamine for the calculation of mass. Therefore, the calculated molar activity was
likely much lower than the actual value, and in either case remained well above the lower
limit permitted for injection.
3
.2.2. Volatile Organic Impurities
The amount of each volatile solvent present in the final product was calculated based on
1
8
the ratio of peak areas for the [ F]F-DA sample vs. the standard values for analysis by gas
chromatography (n = 3). All organic compounds visible on the gas chromatograph were
significantly below allowable limits established by the United States Pharmacopoeia (USP).
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Ammonium acetate was visible in the final product as a known excipient added as a diluent
and buffer solution. Toluene was not detected in any of the samples (limit: ≤ 890 ppm), while
trace amounts of ethanol (70 ± 90 ppm) and acetonitrile (10 ± 20 ppm) were seen if at all.
USP limits for ethanol and acetonitrile are ≤ 5000 ppm and ≤ 410 ppm, respectively.
3
.2.3. Residual Phase Transfer Agent
Assessment of the residual phase transfer agent was key to the optimization of this
radiotracer for human use. Initial syntheses using Kryptofix 222 resulted in levels of
residual phase-transfer agent in the final product ( > 200 µg/mL) which were significantly
®
higher than the USP limit of 50 µg/mL. Multiple attempts to remove the Kryptofix 222 from
the final product while maintaining the one-pot reaction method proved unsuccessful. As a
1
8
result, other phase transfer reagents were tested. The [ F]F-DA product using TBA HCO
3
consistently contained < 50 µg/mL residual TBA HCO when visually compared to reference
3
2
6
standards (n = 8) based on the method of Kuntzsch et al. (Figure 4.) This qualitative, visual
test was quick and easy to perform making it amenable for quality control analysis during
production for human use.
[FIGURE 4.]
Since the visual spot test was not effective for analysis of TEA HCO , an HPLC method
3
employing a linear standard curve of injected masses between 0.05 and 2.5 µg was performed
2
(
R =0.998). The equation of this line was used to determine the residual TEA HCO mass in
3
18
[
F]F-DA. By dividing the determined mass by the injected volume, the result was an
average concentration of 48±7 ug/mL (n = 8, Figure 5).
FIGURE 5.]
[
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To confirm the accuracy of the predicted values from the standard curve of TEA HCO3
injections, eight injections of known mass were performed over the range of the curve. Each
calculated value was compared to the known mass injected and resulted in an average error of
1
8
4
1
.8% (Figure 5). Of note, samples within the range of mass seen in [ F]F-DA samples (0.5 -
.5 µg injected) showed an error of only 1.3%.
[FIGURE 6.]
3
.3. NB Cell Uptake
3
Since NB tumors are known to have high expression of NET , it was hypothesized that
1
8
uptake of [ F]F-DA would be significant in these tumor models. To test this in vitro, we
selected two NB cell lines (SH-EP and SK-N-BE(2)-C) confirmed to have varying uptake of
2
9
18
norepinephrine , and [ F]F-DA accumulation was measured as a preliminary determination
of the effectiveness of this tracer in this tumor type. We assessed transporter selectivity by
comparing uptake in the SH-EP model, known as a NB cell line with minimal NET
28
expression , to that in SK-N-BE(2)-C, which has higher expression of NET. Even at two
18
minutes, accumulation of [ F]F-DA in the SK-N-BE(2)-C cell lines was significantly higher
than the control SH-EP cells, as is seen in Figure 7 (p < 0.0001). This distinction continued
over the course of 30 minutes resulting in a >4-fold difference in accumulation in SK-N-
BE(2)C vs SH-EP.
[FIGURE 7.]
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4
. DISCUSSION
Recent successes in improving the delineation of neuroendocrine tumors with PET
1
8
30-33
imaging and F-labeled catecholamines and analogs targeting NET
have created a
demand for more efficient and routine syntheses of these tracers. We previously reported a
1
8
reliable synthesis method for producing [ F]F-DA that was compatible with multiple
3
4
automated systems. However, we sought to improve upon this method by developing a
precursor and synthesis that would not require the use of corrosive acids such as HI or HBr
and that would eliminate the need for organic solvents in the purification. In addition, we

wanted to design a route that would allow a one-pot reaction on a single Synthera system
rather than a two-pot synthesis in which the synthesizers are connected in series. Further, we
wanted to validate a full quality control method suitable for pediatric imaging applications.
1
8
35
Building on our work optimizing the synthesis of [ F]MFBG , we took a methodical
18
approach to streamline and validate the synthesis of [ F]F-DA and subsequent quality
control testing.
1
8
Our previous method demonstrated that up to 50% of the [ F]fluoride radioactivity
delivered to the reactor adhered to the glass in the presence of toluene. To overcome this, the
vial was pretreated with a base wash and thoroughly rinsed prior to use. This was effective in
reducing the retention of activity by 15-20%, however, addition of this step was not easily
amenable to clinical production. Previously, we demonstrated that the reaction of
18
diaryliodonium salts with [ F]fluoride in 100% acetonitrile resulted in poor yields of
3
4
18
intermediate (2), however, acetonitrile dramatically reduced the amount of [ F]fluoride
adsorbed to the vial. Based upon these observations and on a series of fluorination
experiments performed in various solvents (see Supplemental Information), we determined
that adding a small amount of acetonitrile (5-10%) was enough to keep the fluorine
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solubilized without inhibiting the reaction. Previously, the precursor (1) was dissolved in
acetonitrile to permit ion-exchange of the diaryliodonium triflate to the diaryliodonium
18
[
F]fluoride, and this solvent was removed prior to the thermolysis reaction in toluene.
Transition to this new solvent system allowed solubilization of the precursor directly in the
thermolysis solvent thereby eliminating one reagent needed for the synthesis. The subsequent
elimination of an intermediate silica SepPak purification allowed the entire synthesis to be
performed with only four reagents - phase-transfer solution, precursor (1) in 10%
acetonitrile/toluene, 4 M HCl, and HPLC eluent for dilution - thus, allowing use of a single
®
Synthera IFP™.
1
8
In our prior work, purification of crude [ F]F-DA (3) was achieved utilizing a C-18
column and an eluent of 20% ethanol and 55 mM citric acid. While the purification was
18
effective, varying amounts of unreacted [ F]fluoride were retained in the column, which
prohibited an accurate assessment of the crude purity. In addition, the final product required
significant dilution to reduce the ethanol concentration to acceptable levels for injection into
pediatric patients. A transition to the Hamilton PRP-1 column with an aqueous eluent (0.1%
acetic acid, 0.02% ascorbic acid) eliminated the need for dilution and reduced the amount of
18
18
unreacted [ F]fluoride retained in the column, including unreacted [ F]fluoride salts in the
same amount of time.
The optimized one-pot synthesis resulted in activity yields slightly lower than that
3
4
previously reported for the two-pot process (35 ± 4%). However, the operationally simpler
one-pot process increases feasibility, maintains reproducibility, and produces quantities of the
radiotracer sufficient for clinical studies.

Phase-transfer reagent modification did require optimization; when Kryptofix 2.2.2 was
used as the phase-transfer reagent, the lack of intermediate purification gave rise to
incomplete removal of the kryptand. Quality control results showed high levels of residual
This article is protected by copyright. All rights reserved.


Kryptofix 222, significantly above the release limit established by the USP (50 µg/mL per
injection). Our investigation concluded the source of increased Kryptofix® 222 in the final
1
8

product solution was due to co-elution of [ F]F-DA with Kryptofix 222; any attempts to

remove residual Kryptofix 222 also removed the desired product. Due to the lower-toxicity
36-40
of alternate phase-transfer catalysts, TBA HCO and TEA HCO3
, we assessed what
3
effect these regents had in the thermolysis reaction to produce (#, Scheme 1). No significant
1
8
18
effect on [ F]fluoride ion reactivity or overall yield of [ F]F-DA was observed using tetra-n-
alkyl ammonium salts as phase-transfer reagents. It is worth noting that use of TBA HCO3
did result in a three-minute delay in the elution of the final product from the semi-preparative
HPLC column. This likely results from interaction of the tetraalkylammonium salts with the
non-polar polyvinyl styrene/divinyl benzene matrix of the Hamilton PRP-1column. When
tetraalkylammonium ions are used in the buffer, these cations interact with the hydrophobic
support in proportion to the length of the alkyl substituents (i.e. greater chain length, greater
column surface interaction). The interaction of the tetrabutylammonium ions with the
stationary phase likely leads to a surface modification which causes the PRP-1 column to
behave more like a standard reverse phase column and leads to greater retention of the
desired fluorodopamine product.
Methods for determining residual amounts of TEA HCO or TBA HCO in the final
3
3
product were validated and they demonstrated that approximately 0.05 mg/mL remained for
either of the agents. While the USP does not provide a recommended release limit for either
compound, the European Pharmacopoeia does have an established release limit of 2.6 mg/V
4
1
(
V = injected volume) for TBA HCO . Moving forward, syntheses will be performed with
3
no more than 2.0 mg of TBA HCO so that even if all of the reagent were to be carried
3
through to the final product, this recommended release limit could not be reached with a
maximum injection of 10 mL.
This article is protected by copyright. All rights reserved.

18
An average mass concentration of [ F]F-DA in the final product was determined to
be approximately 2.5 µg/mL which is at least 100-fold less than the average mass of 3.4-5.5
1
0
mg/dose reported via the F method currently in clinical use and more than 1000 times less
2
than the injectable mass limit of 6 mg/dose. This significant improvement in molar activity
will allow clinicians to administer doses based on dosimetry and image quality rather than
being restricted to lower activities due to a physiologically relevant mass of fluorodopamine
approaching the injectable limit.
18
[
F]F-DA has not been specifically tested in NB patients, thus, we sought
confirmation of specific accumulation in NB cell lines in vitro. Over the course of 30
1
8
minutes, [ F]F-DA uptake in the high NET expressing cells was an average of 7-fold higher
than that of the low expressing control cells. While some accumulation was observed in the
SH-EP cells, suggesting involvement of other transport mechanisms, the 7-fold higher
accumulation in NET-positive SK-N-BE(2)-C cells provides sufficient evidence to pursue
18
[
F]F-DA as a tracer for NB.
5
. CONCLUSIONS
TM
18
ALPdopamine is an effective scaffold for the clinical production of [ F]F-DA. Mild
deprotection conditions and a simplified method resulted in greater feasibility and
reproducibility in production. Improvements to the synthesis include (1) milder deprotection
®
18
with HCl, (2) use of a single Synthera , (3) less retention of [ F]fluoride to the reactor vial,
and (4) eliminating the use of organic solvents in purification. These optimizations
contributed to consistent activity yields of > 25% with the introduction of TBA HCO as the
3
1
8
phase transfer agent. Extensive quality control testing confirmed that [ F]F-DA prepared via
this method met the published release criteria for human use. This new procedure has
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subsequently been approved for use in an imaging trial of pediatric patients (IND #138638).
Preliminary in vitro studies validate uptake by NB cell lines expressing NET and suggest that
18
[
F]F-DA is viable as a PET tracer for NB patients.
Disclaimer
Acknowledgements
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SCHEME 1.
3
1
2
1
8
19
Scheme 1. Synthetic procedures for [ F]F-DA (3): previously reported method (top),
method presented in this work (bottom). Initial ion exchange at the hypervalent iodine center
of the diaryliodonium precursor (1) is conducted in 10% acetonitrile and toluene to afford the
1
8
diaryliodonium-[ F]fluoride complex followed by thermolysis to produce protected
intermediate (2). Deprotection of the radiolabeled intermediate (2) in HCl produces (3).
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FIGURE 1.
18
Figure 1. Synthetic steps for production of [ F]F-DA using an IBA Synthera and a
nucleophilic fluorination IFP.
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