Purification of N-succinimidyl-4-[ 18 F]fluorobenzoate by SPE

[¹⁸F]SFB was diluted with 15 mL of water for injection and passed through an Oasis HLB 30 mg (1 cc) cartridge for solid phase extraction. The cartridge was washed

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with 10 mL of water and eluted with 500 µL acetonitrile to give the purified [¹⁸F]

SFB.

4.11 Radiosynthesis of [¹⁸F]FB-ML5

[¹⁸F]SFB dissolved in 500 μL acetonitrile was added to a solution of ML5 (0.50 mg, 1.08 μmol) in 500 μL 0.01 M phosphate buffer pH 8.5. The reaction was performed at 50⁰C for 30 min. After cooling, the reaction mixture was diluted with 600 µL acetonitrile and 600 µL H₂O and was purified by semi-preparative reverse phase HPLC. HPLC was performed with Elite LaChrom Merck Hitachi L-7100 pump system using a Phenomenex reversed-phase Luna C18 column (10 mm x 250 mm, 5µm), preceded of a 20 x 4.6 mm² precolumn, equipped with both UV (Elite LaChrom VWR Hitachi L-2400 UV detector set at 254 nm, AUFS = 0.5) and a Bicron radioactivity monitor. Sample injection was carried out using an injector block with a loop of 1 mL. Gradient elution was performed using a mixture of 0.01 M monosodium phosphate buffer (NaH₂PO₄) pH 6.0 (solvent A) and ACN (solvent B). The following gradient profile (overall time = 47 min) at a flow rate of 2.5 mL.min^-1 was used: 30%

of ACN in solvent A over 5 min, followed by a linear gradient from 30% to 60% of ACN in solvent A over 40 min and followed by a linear gradient from 60% to 10% of ACN in solvent A over 2 min. The retention time of [¹⁸F]FB-ML5 was about 20.3 min.

The HPLC-collected fraction was diluted with about 100 mL of water for injection and passed through an Oasis HLB 30 mg (1 cc) cartridge. The cartridge was washed with 10 mL of water for injection and eluted with 0.7 mL of EtOH. The obtained product was redissolved in saline to decrease the percentage of EtOH to less than 10% for the subsequent cell/animal experiments.

Quality control was performed as described for FB-ML5. The retention time of [¹⁸F]FB-ML5 was 37 min.

4.12 In vitro stability of [¹⁸F]FB-ML5 in human plasma and saline

Human plasma stability

The stability of [¹⁸F]FB-ML5 was evaluated in vitro in human plasma. Whole blood from a healthy donor, kept at room temperature for 15 min, was centrifuged at 3000 rpm for 5 min, subsequently the supernatant was taken. 100 µL of formulated [¹⁸F]FB-ML5 was dissolved in 1 mL human plasma and incubated at 37°C for 3 h.

After 1 h and 3 h of incubation, aliquots of 250 µL were taken. 750 µL of ACN were

added in order to deproteinize the plasma, and the mixture was centrifuged for 3 min at 3000 rpm. The supernatant was passed through a Millex Filter (0.22 µm), diluted with 600 µL ACN and 600 µL H₂O and analysed by semi-preparative HPLC using a Phenomenex reversed-phase Luna C18 column (10 mm x 250 mm, 5µm), preceded by a 20 x 4.6 mm² precolumn. Gradient elution was performed using a mixture of 0.01 M monosodium phosphate buffer (NaH₂PO₄) pH 6.0 (solvent A) and ACN (solvent B). The following gradient profile (overall time = 47 min) at a flow rate of 2.5 mL.min^-1 was used: 30% of ACN in solvent A over 5 min, followed by a linear gradient from 30% to 60% of ACN in solvent A over 40 min and followed by a linear gradient from 60% to 10% of ACN in solvent A over 2 min. Fractions of the eluate were collected every minute and radioactivity in the fractions was determined with a gammacounter (LKB Wallac, Turku, Finland).

Saline stability

The stability of [¹⁸F]FB-ML5 was evaluated in vitro in saline. Formulated [¹⁸ F]FB-ML5 was dissolved in 1 mL saline and incubated at 37°C for 3 h. After 1 h and 3 h of incubation, aliquots of 250 µL were taken and diluted with 1 mL ACN and 1 ml H₂O and analysed by semi-preparative HPLC as described above. Fractions of the eluate were collected every minute and radioactivity in the fractions was determined with a gammacounter (LKB Wallac, Turku, Finland).

4.13 Octanol/water partition coefficient of [¹⁸F]FB-ML5

About 5 kBq of formulated [¹⁸F]FB-ML5 diluted in 5 µL saline was diluted in 495 µL PBS (pH = 7.4) and 500 µL n-octanol in an Eppendorf cup. The mixture was vortexed for 5 min and the cup was centrifuged at 3000 rpm for 5 min. Radioactivity in 100 µL aliquots of the water and n-octanol layers was determined in a gammacounter (LKB Wallac, Turku, Finland). The experiment was performed in triplicate. The par-tition coefficient (log P) was calculated as: log P = log₁₀ (cpmoctanol layer / cpmaqueous layer).

4.14 In vitro evaluation of [¹⁸F]FB-ML5

Human breast cancer MCF-7 cells and human bronchial epithelium 16HBE cells were obtained from American Type Culture Collection, Manassas, USA. MCF-7 and 16HBE cells were maintained in 15 mL Eagle’s Minimum Essential Medium (EMEM) (Lonza, Walkersville, USA) supplemented with 10% fetal calf serum (FCS) in a T₇₅

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culture flask. Cells were grown in a humidified atmosphere containing 5% CO₂ and were passaged twice per week.

For 16HBE cells

16HBE cells were seeded in a 24-well plate at 50.000 cells/mL 6 days before the experiment. An equal number of cells were dispensed in each well in 0.5 mL of serum-containing medium: EMEM supplemented with 10% FCS. Cells were grown to confluency and serum-starved overnight. One day before the experiment, the medium was changed to low serum medium: EMEM supplemented with 0.5% FCS.

For MCF-7 cells

MCF-7 cells were seeded in a 12-well plate 48 h before the experiment. An equal number of cells were dispensed in each well in 1 mL of serum-containing medium:

EMEM supplemented with 10% FCS. Cells were grown to confluency and serum-starved overnight. One day before the experiment, the medium was changed to low serum medium: EMEM supplemented with 0.5% FCS.

Binding/specificity studies were performed when confluency had reached 80-90%.

For binding study with MCF-7 cells

0.5 MBq of [¹⁸F]FB-ML5 in < 50 μL of saline (containing maximum 10% of absolute ethanol) were added to each well. After 57 min of incubation, the medium was quickly removed and the monolayer was washed 3 times with PBS. Cells were then treated with 0.2 mL of trypsin. When the monolayer had detached from the bottom of the well, 0.8 mL of EMEM supplemented with 10% FCS was added to stop the proteolytic action. Cell aggregates were resolved by repeated pipetting of the tryp-sin/EMEM mixture. Radioactivity in the cell suspension (1 mL) was assessed using a gamma counter for 15 sec for [¹⁸F]FB-ML5. A sample of the suspension was mixed with trypan blue solution (1:1 v/v) and was used for cell counting. Cell numbers were determined manually, using a phase contrast microscope (Olympus, Tokyo, Japan), a Bürker bright-line chamber (depth 0.1 mm; 0.0025 mm² squares) and a hand tally counter. All experiments were performed as a quadruplicate study with at least two repeats.

For specificity study For 16HBE cells

Four different experimental conditions were examined in quadruplicate: non-stimulated cells, non-non-stimulated cells + 10 µM of non-radioactive ML5 (10 µL), stimulated cells (25 ng/mL PMA and 100 ng/mL LPS added 60 min before [¹⁸F]

FB-ML5 addition) and stimulated cells + 10 µM of ML5 (10 µL). Blocker was added 2 min before tracer addition. 0.5 MBq of [¹⁸F]FB-ML5 in < 50 μL of saline (contain-ing maximum 10% of absolute ethanol) was added to each well and incubated for 60 min. After washing 3 times with 500 μL PBS, the cells were detached with 100 μL of trypsin and transferred to test tubes. After addition of 400 μL of EMEM + 10%

FCS and resuspension, radioactivity in the cell suspension (0.5 mL) was assessed using a gamma counter. A sample of the suspension was mixed with trypan blue solution (1:1 v/v) and was used for cell counting. Cell numbers were determined manually, using a phase contrast microscope, a Bürker bright-line chamber and a hand tally counter.

For MCF-7 cells

Six different experimental conditions were examined in quadruplicate: non-stimu-lated cells, non-stimunon-stimu-lated cells + 100 nM of ML5 (10 µL), non-stimunon-stimu-lated cells + 10 μM of ML5 (10 µL), stimulated cells (100 nM PMA added 2.5 h before [¹⁸F]FB-ML5 addition), stimulated cells + 100 nM of ML5 (10 µL) and stimulated cells + 10 µM of ML5 (10 µL). Blocker was added 2 min before tracer addition. 0.5 MBq of [¹⁸F]

FB-ML5 in < 50 μL of saline (containing maximum 10% of absolute ethanol) was added to each well and incubated for 60 min. After washing 3 times with 1 mL PBS, the cells were detached with 200 μL of trypsin and transferred to test tubes after addition of 800 μL of EMEM + 10% FCS and resuspension. Radioactivity in the cell suspension (1 mL) was assessed using a gamma counter. A sample of the suspen-sion was mixed with trypan blue solution (1:1 v/v) and was used for cell counting.

Cell numbers were determined manually, using a phase contrast microscope, a Bürker bright-line chamber and a hand tally counter.

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