Initial experience in synthesis of (2S,4R)-4-[18F]fluoroglutamine for clinical application

Article abstract of DOI:10.1002/jlcr.3719

We report initial experience in synthesis of (2S,4R)-4-[¹⁸F]fluoroglutamine, [¹⁸F]FGln, which has been used as a tool for monitoring glutamine metabolism in cancer patients. [¹⁸F]FGln was prepared by a fully automated PET-

Full text of DOI:10.1002/jlcr.3719

kung hank (Orcid ID: 0000-0003-3254-8049)
Lawrie Ken (Orcid ID: 0000-0002-8061-3696)
Dannals Robert (Orcid ID: 0000-0002-5483-5914)
Initial experience in synthesis of (2S,4R)-4-[18F]fluoroglutamine for clinical
application
Yan Zhanga, Lifang Zhanga, Jianhua Yangb, Zehui Wuc, Karl Ploessld, Zhihao Zhac,
Fei Liub, Xiaoxia Xub, Hua Zhub, Zhi Yangb, Lin Zhua,c*, Hank F. Kungc,d*
aCollege of Chemistry, Beijing Normal University, Key Laboratory of
Radiopharmaceuticals, Ministry of Education, Beijing, 100875, China
bKey Laboratory of Carcinogenesis and Translational Research (Ministry of
Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital &
Institute, Beijing, 100142, China
cBeijing Institute of Brain Disorders, Capital Medical University, Beijing, 100069,
China
dDepartment of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
Total pages: 10, Schemes: 1, Figures:3, Tables: 2
*Corresponding author contact information:
Hank F. Kung, PhD, Department of Radiology, University of Pennsylvania, 3700
Market Street, Room 305, Philadelphia, PA 19104, USA
Tel.: +1 215 662 3989; fax: +1 215 349 5035; email: Kunghf@gmail.com
Lin Zhu, PhD, Key Laboratory of Radiopharmaceuticals (Beijing Normal University),
Ministry of Education, No. 19, XinJieKouWai Street, Haidian District, Beijing, 100875, P. R.
China
Tel.: +86 10 62207789; email: zhulin@bnu.edu.cn
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doi: 10.1002/jlcr.3719
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Abstract
We report initial experience in synthesis of (2S,4R)-4-[18F]fluoroglutamine,
[18F]FGln, which has been used as a tool for monitoring glutamine metabolism in cancer
patients. [18F]FGln was prepared by a fully-automated PET-MF-2V-IT-I synthesizer under
GMP-compliant conditions for routine clinical studies. The total radiosynthesis time was
about 65 min, the decay-corrected radiochemical yield was 18.0 ± 4.2% (n = 59; failure n =
15) and the radiochemical purity was greater than 90%. In some situations, the yields were
low (< 5%) and the most likely cause of this problem is the initial fluorination step, the
fluoride ion might not have been fully activated. In other occasions, low final radiochemical
purity was often associated with the failure of second step - removal of protection groups by
anhydrous trifluoroacetic acid. A trace amount of water led to production of undesired
4-[18F]fluoroglutamic acid. Knowledge learned from the successes and failures of synthesis
may be helpful to identify critical steps and pitfalls for preparation of this clinically useful
metabolic probe, [18F]FGln, for imaging glutamine utilization in tumor of cancer patients.
Keywords: Cancer metabolism, PET imaging, tumor, glutamine and radiolabelling
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1. Introduction
Position emission tomography (PET) is a widely used clinical imaging device for
cancer diagnosis and monitoring. In conjunction with 2-[18F]fluoro-2-deoxy-glucose
([18F]FDG), it is a common imaging procedure for mapping cellular glucose metabolism in
different tissues and organs. The uptake of [18F]FDG, especially in tumors with higher rates
of glycolysis, provides an accurate assessment of cellular glucose metabolism with high
signal to noise ratios. Despite the tremendous promise of using [18F]FDG-PET to detect and
monitor tumor metabolism, in many cases tumors are [18F]FDG-negative; the tumors may
have cleverly changed their nutrient sources from glucose to glutamine, thus escaping
detection by PET imaging.1 To improve cancer diagnosis based on energy metabolism we
have prepared and tested a series of novel fluorinated glutamine derivatives mimicking
glutamine, which is fueling the tumor growth.2,3 Four different isomers (2S,4S; 2S,4R; 2R,4S;
2R,4R) of 4-[18F]fluoroglutamine were tested by in vitro cell uptake assays and in vivo PET
imaging in rats.4 Only the 2S isomers, the naturally occurring glutamine configuration,
showed a significant glutamine-specific uptake. Using product with correct optical purity is
essential for the success of glutamine imaging. Cell uptake in SF188 and 9L glioblastoma
tumor cells displayed a higher rate of uptake, which was higher than that of [18F]FDG.5
Dynamic small animal PET imaging studies of (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln)
were conducted in 9L tumor-cell-xenografts produced in F344 rats. Biodistribution and PET
imaging studies showed that [18F]FGln localized in tumors with a higher uptake than in
surrounding muscle and liver tissues.5 In addition, at 30 and 60 min after an iv injection into
tumor bearing rats, [18F]FGln displayed a high incorporation (30% and 70%, respectively)
into macromolecules suggesting a rapid intracellular metabolism.
Recent reports suggested that [18F]FGln may be useful for detecting and monitoring
tumor metabolism based on glutamine utilization.2,6-10 After an iv injection [18F]FGln
demonstrated good uptake and a high tumor-background ratio in patients. Results of human
PET imaging with [18F]FGln suggested that it is an attractive alternative tracer, other than
commonly used [18F]FDG, for clinical diagnosis and therapy evaluation. 2,6-12
In order to perform further studies using [18F]FGln, we report herein the experience,
successes as well as failures, in the past one year and five months. This was about using
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PET-MF-2V-IT-I modular synthesizer for routine preparation of [18F]FGln in the
PET/Cyclotron facility of Peking University Cancer Hospital for clinical application.
2. Materials and methods
The
tosylate
precursor
of
[18F]FGln,
(2S,4S)-tert-butyl-2-(tert-
butoxycarbonylamino)-5-oxo-4-(tosyloxy)-5-(2,4,6-trimethoxybenzyl-amino)pentano-ate
([α]25 = −56.01 (c = 0.1, Methanol)) and non-radiolabeled reference standard,
D
(2S,4R)-4-fluoroglutamine ([19F]FGln), were prepared as described previously.4 All other
reagents and solvents were commercial products and were used without further purification
unless otherwise indicated. Solid-phase extraction cartridges (Oasis HLB 3cc cartridge, QMA
light cartridge) were obtained from Waters (Milford, MA, USA). AG 11 A8 Resin was
obtained from Bio-Rad (Hercules, CA, USA). Sterile Millex-GS filter (SLGSV255F) was
obtained from EMD Millipore (Billerica, MA, USA). The synthesis of [18F]FGln was
performed by a PET-MF-2V-IT-I synthesizer (PET Co. Ltd., Beijing, China) with two
reaction vessels (Fig. 1). High-performance liquid chromatography was performed on an
Agilent 1260 Infinity system with a chiral column (Chirex 3126 (D)-penicillamine, 250 ×
4.6mm, Phenomenex). Gas chromatography was performed on an Agilent 6890 system with a
FID detector. Radioactivity was measured by a CRC-25PET radioisotope dose calibrator
(Capintec, Inc., Ramsey, NJ, USA). The clinical research study was approved by the Peking
University Cancer Hospital &Institute Ethics Committee (Permission No. 2017KT38).
2.1 Preparation before radiosynthesis
To initiate the synthesis of [18F]FGln on the synthesizer (Fig.1), reagent vials were
added with desired reagents: 1.44 mg KHCO3 and 8 mg 18-crown-6 in 1 mL methanol and
0.18 mL water were added to Vial 1;13 2 mL anhydrous acetonitrile was added to Vial 2; 5
mg precursor in 0.7 mL anhydrous acetonitrile was added to Vial 3; 30 mL sterile water was
added to Vial 4; 10 mL sterile water/ethanol (8/2, v/v) was added to Vial 5; 1.5 mL
anhydrous acetonitrile was added to Vial 6; 1 mL trifluoroacetic acid (TFA) and 10 μL
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anisole were added to Vial 11; 2 mL sterile water for injection was added to Vial 12; 3 mL
sterile water for injection was added to Vial 13.
Preconditioning is necessary for several cartridges before radiosynthesis. QMA light
cartridge was treated with 10 mL 0.5 M NaHCO3 solution and 10 mL water. Oasis HLB 3cc
cartridge was preconditioned with 10 mL ethanol and followed by rinse with 10 mL water.
Weighted 2 g AG 11 A8 resin, was loaded before the HLB 3cc cartridge, after which the top
was closed with a sleeve stopper. HLB-AG 11 A8 column was washed with 10 mL water. All
conditioned cartridges were kept wet unless otherwise specified. Load QMA light cartridge to
a specific location. Load Oasis HLB 3 cc cartridge between Valve 7 and Valve 10. Load
HLB-AG 11 A8 column between Valve 15 and Valve 17. Valve 17 was connected through a
sterile Millex-GS filter to a 10 mL sterile vial (final product vial).
2.2 Automated radiosynthesis of [18F]FGln
[18F]FGln was prepared using the two-step radiochemical reaction described in
Scheme 1. [18F]fluoride (37–148 GBq, 1–4 Ci), produced by a cyclotron using the 18O(p,n)18F
18
reaction, was trapped on a Sep-Pak Light QMA cartridge. The F-activity was eluted with
phase transfer catalyst solution (8 mg 18-crown-6 and 1.44 mg KHCO3 in 1 mL methanol and
0.18 mL water, Vial 1) from the cartridge into reactor 1 (H1), and evaporated under a stream
of N2 at 90 °C. 1 mL anhydrous acetonitrile in Vial 2 was added and the activity was
azeotropically dried at 90 °C under a stream of N2. 1 mL anhydrous acetonitrile in Vial 2 was
added again and the activity was further dried at 90 °C under a stream of N2. R1 was cooled
to 50 °C, the tosylate precursor in Vial 3 was added to the dried activity. The resulting
solution was stirred and heated at 90 °C for 15 min. R1 was cooled to 45 °C, the reaction
mixture was diluted with 10 mL water (Vial 4), and passed through an Oasis HLB 3cc
cartridge. The cartridge was washed twice with water (2 × 10 mL, Vial 4) and water/ethanol
(8/2, v/v, 10 mL, Vial 5) . The intermediate was eluted from the cartridge with 1.5 mL
acetonitrile (Vial 6) into reactor 2 (H2).
The eluted intermediate was heated at 60 °C under a stream of N2 to remove
acetonitrile. TFA/anisole mixture (1 mL/10 μL, Vial 11) was added to the dried residue. The
resulting solution was heated at 60 °C for 5 min. The volatiles were removed at room
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temperature under a stream of N2. 2 mL sterile water (Vial 12) was added into R2. The
content was transferred through HLB-AG 11 A8 column and sterile filter to the product vial.
R2 was rinsed with 3 mL sterile water (Vial 13) and the solution was transferred to the
product vial. The final product, [18F]FGln, was diluted with appropriate volume of sterile
saline.
Scheme 1 The synthetic scheme of [18F]FGln
Fig.1 Graphical display of the monitor of [18F]FGln radiosynthesis on the PET-MF-2V-IT-I synthesizer
2.3 Quality control
Quality control tests (requirements of Chinese Pharmacopeia), including appearance,
pH, radiochemical identity, radiochemical purity, optical purity, radionuclide identity,
residual solvent (acetonitrile, methanol), sterility and bacterial endotoxin, were carried out.
Analysis of radiochemical identity and radiochemical purity was performed on an Agilent
1260 Infinity HPLC system equipped with radio and UV detection (254 nm) in series. A
chiral column (Chirex 3126 (D)-penicillamine, 250 × 4.6 mm, Phenomenex) was used, with
an isocratic method of 1 mM CuSO4, and flow rate 1 mL/min. The optical purity was
determined by the same chiral HPLC. The retention time of [18F]FGln was 14.7 min (Fig.2).
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Fig. 2 HPLC profiles of [18F]FGln (Radiotrace), coinjected with[19F]FGln (UV trace) on chiral column
(Chirex 3126 (D)-penicillamine, 1mM CuSO4 solution, 1 mL/min)
In addition to the analytical HPLC, quality control tests were performed in accordance
with the Chinese Pharmacopoeia for radiopharmaceuticals. Details of the tests performed and
the release criteria are given in Table 1.
Table 1 Quality control tests of [18F]FGln
Test
Appearance
pH
Release Criteria
Method
Visual inspection
pH indicator strip
HPLC
Clear, colorless, and particle-free
5.0–8.0
Radiochemical purity
Optical purity
> 90%
> 95%
HPLC
Retention time difference of
radioactivity and reference peak ≤
10 %
Radiochemical
identity
HPLC
HPLC
Chemical impurities
(not included
[19F]FGln,
< 5 μg/mL
by UV at 254 nm)
Radionuclide identity
Residual
18F (t1/2 = 105–115 min)
Measured by dose calibrator
GC
< 0.041% w/w
acetonitrile
Residual
< 0.3% w/w
GC
methanol
Sterility
Sterile
Chinese Pharmacopeia
Chinese Pharmacopeia
Endotoxin Level
< 5 EU/mL
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2.4 Human PET/CT imaging study
This study was approved by the Ethic Committee of Peking University Cancer
Hospital (Permission No. 2017KT38) and conducted according to the latest guidelines of the
Declaration of Helsinki. Written informed consent was obtained from each participant prior
to PET/CT studies. Patients with specific primary malignant tumor and suspected cerebral
metastasis were enrolled in this study. Pathologic analyses of the primary lesions in all
patients were performed prior to this clinical trial. One renal cell carcinoma patient recruited
for this study was injected with a dose of [18F]FGln (3.7 MBq per kilogram). This patient
underwent a static whole-body PET/CT using a Biograph mCT Flow 64 scanner (Siemens,
Erlangen, Germany) at 60 min after intravenous injection and the acquisition time was 3 min.
PET/CT image of this patient study was presented in Figure3.
Fig. 3 An axial PET/CT fusion image of [18F]FGln in a 47-y-old man with brain metastasis in renal cell
carcinoma (SUVmax=5) after iv injection of [18F]FGln. The image was acquired at 60 min (a sum of 3
min). Similar studies of using [18F]FGln to study metastatic brain tumor has been previously reported.6
3. Results and discussion
The two-step automated radiosynthesis of [18F]FGln was performed on a
PET-MF-2V-IT-I synthesizer (PET Co. Ltd., Beijing, China, see Fig. 1). The process was
validated for each batch of new precursors. The total radiosynthesis time was about 65 min,
the decay-corrected radiochemical yield was 18.0 ± 4.2% (n = 59), the radiochemical purity
was greater than 90% and the optical purity was greater than 95%. When comparing with
previously reported HPLC purification method,13,14 there was a significant increase in
efficiency associated with using the SPE method - by reducing preparation time in synthesis
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and reducing product loss. In the past one year and five months, we have performed the
procedure for 74 times. There were successful preparations (n = 59), while there were also
many failures (n = 15). As shown in Table 2.
Table 2 Synthesis results in the past two years
Year
2017
2018
Total
Total Successes
Failures
RCY(%)
14.8–21.9
10.2–19.0
18.0 ± 4.2
RCP(%)
> 90%
Synthesis time
65 ± 5 min
45
29
74
37
22
59
8
7
> 90%
65 ± 5 min
15
RCY, radiochemical yield (decay-corrected); RCP, radiochemical purity.
In quality control tests, the radiochemical purity of the final product was greater than
90%, the optical purity was greater than 95%, and the radiochemical identity was confirmed
by chiral HPLC, which was eluted at about 14.7 min. Residual acetonitrile and methanol
were assayed by GC, and results were lower than 0.041% and 0.3%. Half-life of the final
product was 109 ± 1 min. Chemical impurities were lower than 5 μg/mL. All the necessary
parameters such as appearance, sterility, endotoxin content and the pH value of the solution
meet the requirements for GMP synthesis. The final product, [18F]FGln, which meets release
criteria is transferred to nuclear medicine clinic for patient injection.
Because of water contamination in TFA and instability of the precursor (optical
rotation changed), there were often unsuccessful radiosynthesis, the radiochemical purity was
lower than 90% and the optical purity was lower than 95%. Failed synthesis also caused by
hydrolysis of [18F]FGln to the side product, (2S,4R)-4-[18F]fluoroglutamic acid ([18F]FGlu).5
This often happened on days with high humidity. PET/cyclotron facility in the basement of
the hospital was damp and soggy, and the moisture was particularly unforgiving. The failures
led to abandoning the clinical scans, causing not only inconvenience to patients but also
disappointment to clinicians. Careful planning and prior validations will be essential to
ensure the success of preparation of [18F]FGln for clinical application.
4. Conclusion
We report successes and failures of synthesizing (2S,4R)-4-[18F]fluoroglutamine,
[18F]FGln, on a fully-automated PET-MF-2V-IT-I synthesizer under GMP-compliant
conditions. The experience helps to identify critical steps and pitfalls for preparation of this
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clinically useful metabolic probe. This more streamlined procedure may facilitate using
[18F]FGln in clinical studies for measuring glutamine metabolism.
Acknowledgments
This work was financially supported in part by the Beijing Natural Science Foundation
(7171002) and the Beijing Science and Technology Project (Z151100003915116).
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