Automated sulfur-[18F]fluoride exchange radiolabelling of a prostate specific membrane antigen (PSMA) targeted ligand using the GE FASTlab™ cassette-based platform

Sulfur-[18F]fluoride exchange radiochemistry is a rapid and convenient method for incorporating fluorine-18 into biologically active molecules. We report a fully automated radiolabelling procedure for the synthesis of a [18F]SO3F-bearing prostate specific membrane antigen (PSMA) targeted ligand ([18F]5) using the GE FASTLab™ cassette-based platform in a 25.0 ± 2.6% radiochemical yield (decay corrected). Uptake in vitro and in vivo correlated with PSMA expression, and the radioligand exhibited favourable biodistribution and pharmacokinetic profiles.


Materials and Methods
Anhydrous solvents and reagents were purchased from Sigma Aldrich (Gillingham, UK) and used without additional purification.Flash column chromatography was performed using silica gel (Merck Kieselgel 60 F254 320-400 mesh).The production of [ 18 F]fluoride for use at Imperial College London was performed using a Siemens RDS111 cyclotron by irradiation of an enriched [ 18 O]H 2 O target, supplied by Invicro (London, UK).The production of [ 18 F]fluoride for use at the University of Hull was performed using Best ABT Inc. BG-75 cyclotron (7.5 MeV, 5 µA beam, 1 h) with a target containing 98% 18 O enriched water (Marshall Isotopes Ltd, Israel).Automated radiosynthesis was performed using the GE FASTlab™ (GE Healthcare Life Sciences, Amersham, UK).Solid phase extraction (SPE) cartridges were purchased from Waters (Hertfordshire, UK) and used according to the manufacturer's guidelines.Semipreparative reverse phase (RP) HPLC was performed using a Shimadzu LC20-AT pump attached to a custom-built system, equipped with an Phenomenex Luna C12, 4 µ (250 x 9.4 mm) column running an isocractic mobile phase of MeCN (30%) and H 2 O (70%) + 0.1% H 3 PO 4 at a flow rate of 3 mL/min.Reaction efficiency and radioactive product identity was determined by RP-HPLC using an Agilent 1200 series instrument connected to a flow-ram detector (Lablogic, Sheffield, UK).

Synthesis of ((1-carboxy-5-(4-((fluorosulfonyl)benzamido)pentyl)carbamoyl)glutamic acid (7).
To a solution of 6 (26 mg, 0.039 mmol) dissolved in DCM (1 mL) TFA (250 μL) was added dropwise and stirred overnight under N2 at RT. Reaction mixture was dried and 7 was obtained as white solid. 1 H NMR (400 MHz, Methanol-d4) δ 8.12 -8.07 (m, 2H), 8.04 (d, J = 8.3 Hz, 2H), 4.24 (dt, J = 8.3, 5.1 Hz, 2H), 3.36 (td, J = 7.0, 2.0 Hz, 2H), 2.42 -2.28 (m, 2H), 2.07 (dtt, J = 14.8, 12.7, 6.2 Hz, 1H), 1.96 -1.75 (m, 2H), 1.62 (ddt, J = 13.7,10.6, 7.2 Hz, 3H), 1.53 -1.35 (m, 2H). 13                  µmol) was then dissolved in anhydrous MeCN (1 mL) was added to the reaction vessel and allowed to react at room temperature for 5 min.HCl (1 mL, 4M) was added to the reaction vessel containing [ 18 F]3 and heated at 60 C for 15 min.After cooling, the crude was diluted to 10 mL with 0.1% H 3 PO 4 solution following a reaction vessel rinse before purification by semi-preparative HPLC (Shimadzu LC20-AT pump attached to a custom-built system, equipped with a Phenomenex Jupiter C12, 4μ (250 × 10 mm) column).The mobile phase was 70% H 2 O/30% MeCN+0.1% H 3 PO 4 at purification was performed at flow rate of 3 mL/min.[ 18 F]5 was collected as a fraction and diluted with 0.1% H 3 PO 4 (45 mL) for concentration and reformulation by tC18 plus SPE cartridge.The cartridge was washed with water and dried under a flow of nitrogen and eluted with EtOH (1 mL) for dilution into PBS (<10% EtOH v/v) for biological evaluation.The tubing used in this method was supplied in the FASTLab Developer Kit (14 cm and 42 cm).Short tubing was used to connect SPE cartridges, whereas longer tubing was used to connect to external consumables and equipment (dilution vials and HPLC loop).A tutorial account describing best practices for automating 18 F-radiochemistry is available and may assist in the implementation and adaptation of this method.).The K 222 -K[ 18 F]F complex was azeotropically dried by the addition of MeCN (3 × 0.7 mL) at 100 ºC under a gentle stream of inert gas.Precursor 4 (0.5 mg) was dissolved in 0.5 mL of MeCN and added to the dry K 222 -K[ 18 F]F complex and the reaction was stirred at RT for 20 min.The reaction mixture was passed through an activated silica cartridge and flushed with dry MeCN (500 µL).The solvent volume was reduced under a gentle stream of inert gas at 60 ºC to around 200 µL, to which HCl (200 µL of 4 M) was added to the reaction mixture and stirred for 20 min at 60 ºC.The reaction mixture was neutralized by the addition of sodium acetate (3 M, pH 4) and purified by HPLC (Merck Discovery 250 × 10 mm C18 column eluted with a gradient 40 to 60% of MeOH + 0.1% TFA / H 2 O + 0.1% TFA, 4.7 mL/min.The collected HPLC fraction was diluted with water (15 mL) and reformulated using a tC18 (145 mg) SPE cartridge preconditioned with EtOH (1 mL) and saline (5 mL).Trapped product was washed with saline (5 mL) and eluted in EtOH (200 µL) followed by sodium acetate (0.1 N, pH 6.7).

Biological Evaluation
4.1 Cell culture C4-2B, LNCaP, PC3, and PNT1A cell lines (obtained from Prof Charlotte Bevan, Imperial College London) were grown in RPMI medium (Sigma Aldrich, Gillingham, UK).The media was supplemented with 10% fetal calf serum, 1% L-glutamine and 2% penicillin-streptomycin (Life Technologies).All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 .All cells were routinely tested for mycoplasma and typically not passaged for longer than three months.

Flow Cytometry
To analyse cell surface markers, 1.5 x10 5 cells were collected and centrifuged at 1500 rpm for 5 min.The cell pellet was then resuspended in PBS, followed by the addition of Alexa Fluor® 488 anti-human PSMA (FOLH1) Antibody (342505, BioLegend) at the concentration indicated by the manufacturer.After 30 min incubation at 4 °C, cells were washed and suspended in PBS for analysis.Live/Dead fixable Near-IR dead cell stain kit (L34976, Invitrogen) was used to determine cell viability by flow cytometry.30,000 events per sample were acquired on FACS Canto flow cytometer (Becton Dickinson Immunocytometry Systems) with FACS Diva Software version 4.0.2.Data obtained were analysed using FlowJo software v7.6 (FlowJo, LLC).Unstained and isotype controls were used to define gates.

In vitro cell uptake of [ 18 F]SO 3 F-PSMA ([ 18 F]5)
PSMA positive cell lines: C4-2B, LNCaP and PSMA negative cell lines: PC3, PNT1A were plated at a seeding density of 5× 10 5 cells per well in 6-well plates.After 24 h, cells were incubated with 22.2 MBq of [ 18 F]5 for 1 h at 37 °C and 5% CO2.Cells were then washed three times with ice-cold PBS and lysed in RIPA buffer (0.5 mL per well) for 15 min on ice.Cellbound radioactivity was measured, and decay corrected using Packard Cobra II gamma counter (Perkin Elmer).The radiopharmaceutical uptake was normalised to total cellular protein as measured by the BCA assay.Uptake was expressed as % radioactivity/mg protein.
All experiments were carried out in quadruplicate or more (n = 4-6).

Animal tumour model
Male BALB/c nude mice (6 -8 weeks old) were implanted subcutaneously with LNCaP cells at 7.5 x10 6 on the right shoulder, for use with dynamic PET imaging.Inoculations were performed under anaesthesia (2% isoflurane/O 2 ).All mice were 12 weeks of age with similar weights (23.0 ± 2.4 g) and tumour volumes (212 ± 10 mm 3 ) and kept under standard conditions in individually ventilated cages with animal food provided ad libitum prior to experiments.

In vitro cell uptake of [ 18 F]SO 2 F-PSMA ([ 18 F]7)
PSMA positive cell lines: C4-2B, LNCaP, and PSMA negative cell lines: PC3, PNT1A were plated at a seeding density of 5× 10 5 cells per well in 6-well plates.After 24 h, cells were incubated with 22.2 MBq of [ 18 F]7 for 1 h at 37 °C and 5% CO2.Cells were then washed three times with ice-cold PBS and lysed in RIPA buffer (0.5 mL per well) for 15 min on ice.Cellbound radioactivity was measured, and decay corrected using Packard Cobra II gamma counter (Perkin Elmer).The radiopharmaceutical uptake was normalised to total cellular protein as measured by the BCA assay.Uptake was expressed as % radioactivity/mg protein.All experiments were carried out in quadruplicate or more (n = 4-6).The protocol for radioactive metabolite analysis outlined in ESI Section 4.5 was followed.
Radioactivity was only detected in plasma and urine, as shown in figure 22.

1
Automated method.No-carrier-added aqueous [ 18 F]fluoride in enriched 18 O water was delivered to the FASTlab™ radiosynthesis module and trapped on a Waters QMA-carbonate Sep-Pak SPE cartridge.The [ 18 F]fluoride was eluted into the reactor vial using 600 µL of eluent (6mg/mL Kryptofix 2.2.2 in 800 µL MeCN, 3.5 mg/mL K 2 CO 3 in 200 µL H 2 O) by syringe.The [ 18 F]fluoride was dried under nitrogen and vacuum at 120 C for 6 min.Compound 3 (50 µL, 5

Figure 19 .
Figure 19.Prostate cancer cell line validation.A) Cell surface expression of PSMA in C4-2B and LNCaP cells.Data from two independent experiments presented as mean ± SEM; Representative histograms of PSMA expression in B) PC3 and C) PNT1A cells; two independent experiments performed, with the dotted black line representing cells stained with isotype control antibody (n=3).

Figure 20 .4. 4
Figure 20.In vitro uptake of [ 18 F]5 in a panel of cell lines with differential PSMA expression.

Figure 25 .4. 9
Figure 25.In vitro cell uptake of [ 18 F]7 in a panel of cell lines with differential expression of PSMA.