Multiple Applications of a Novel Biarsenical Imaging Probe in Fluorescence and PET Imaging of Melanoma

A new fluorescent biarsenical peptide labeling probe was synthesized and labeled with the radioactive isotopes 11C and 18F. The utility of this probe was demonstrated by installing each of these isotopes into a melanocortin 1 receptor (MC1R) binding peptide, which targets melanoma tumors. Its applicability was further showcased by subsequent in vitro imaging in cells as well as in vivo imaging in melanoma xenograft mice by fluorescence and positron emission tomography.

2D FT NMR spectra on a Bruker Avance III 400 MHz instrument. Solvent peaks in 13 C-and 1 H-NMR were used as chemical shift references.
High performance liquid chromatography analysis (HPLC) was performed using a Hitachi L-6200 gradient pump and a Hitachi L-4000 variable wavelength UV-detector in a series with a Bioscan β+flow detector. The products containing the xanthenone core of compound 1 could be conveniently observed at a wavelength of 380 or 465nm. Analytical HPLC analysis was performed using a reverse phase column (XBridge, C18, 5 μm, 4.6 х150 mm), unless otherwise noted.

Bis(2,4-dimethoxyphenyl)-4-fluorophenylmethanol
The 9-(4-fluorophenyl)-6-hydroxy-3-fluorone-2,5-dimercuric trifluoroacetate (5.0 g, 5.37 mmol) was dissolved in 53 mL of dry N-methylpyrrolidone under argon atmosphere, AsCl3 (9.0 mL, 107 mmol) and N,N-diisopropylethylamine (7.5 mL, 43.0 mmol) were added via syringe. 32 mg of palladium (II) acetate was added and the mixture was stirred at 55 °C for two hours and then at room temperature overnight. The reaction mixture was transferred into a 500-mL Erlenmeyer flask containing wellstirred 320 mL of 1:1 vol/vol 0.3 M potassium phosphate buffer at pH 6.9 and acetone. 21 mL of 1,2ethanedithiol was added to the solution and stirred for 30 minutes, and then 100 mL of chloroform was added and stirred for additional 20 minutes. The organic phase was separated, and the water was extracted 3 times with chloroform, the combined organic layers were washed with water and dried over Na2SO4, filtered and concentrated. Methanol was added, large amount of white precipitate was formed, also a small amount of dark red liquid at the bottom of the flask. The precipitate was decanted, the remaining red liquid was dissolved in chloroform and collected. This procedure was repeated three times. The combined crude product was concentrated and purified by column chromatography using neat toluene to toluene:acetone 50:4 to yield a orange-red solid (1.0 g, 31%). 1 H NMR (400 MHz, CDCl3) δ 10.57 (s, 1H), 7.41 -7.21 (m, 2H), 7.08 -6.97 (m, 1H), 6.62 (broad s, 1H), 3.88 -3.27 (broad m, 1H).

Synthesis of [ 12 C]CH3-1
To a suspension of 1 (27 mg, 42 µmol) and potassium carbonate (11mg, 84µmol, 2 eq) in 3 mL of dry acetone, methyl nosylate (9.1mg, 42µmol, 1 eq) was added and the mixture was heated to 60°C under microwave irradiation conditions for 60 min. The solvent was evaporated, and the residue was purified by HPLC (C8) column chromatography using 75% acetonitrile in 25 mM ammonium acetate at 2.0 mL/min. The resulting HPLC fraction was evaporated by flushing with a stream of nitrogen, to produce 11mg of brown solid, which was re-purified on silica using toluene:ethyl acetate (10:1). The isolated fraction was evaporated to produce 4.5mg (16%) of [ 12 C]CH3-1 as a red powder, which was pure according to LC-MS (m/z 653 [M+H] + ).

Synthesis of [ 19 F]F-C2H4-1
To a suspension of 1 (30 mg, 48 µmol) and potassium carbonate (13.2mg, 96µmol, 2 eq) in 3 mL of dry acetone, 2-fluoro-ethyl tosylate (21mg, 96µmol, 2 eq) was added and the mixture was heated to 120°C under microwave irradiation conditions for 10 min. The solvent was evaporated and the residue was purified by column chromatography on silica using toluene:ethyl acetate (10:1). The isolated fraction was evaporated to result in 10mg of red powder. The product could be re-purified on C8 HPLC column with ACN-NH4OAc(25mM in water) 70:30. The isolated fraction was evaporated by flushing with a stream of nitrogen, redissolved in acetonitrile and evaporated again. [ 19 F]F-C2H4-1 was obtained as a red powder (8mg, 24%) and was pure according to LC-MS (m/z 685 [M+H] + ).
The reaction mixture was injected on a preparative short C18 column, which was eluted at a flow 5.0mL/min with a mixture of ACN-NH4OAc(50mM in water) 45:55. The first major peak observed at 465nm was collected. Isolated fraction was concentrated in a stream of nitrogen and reinjected on Kromasil C18, 7µm 250x10mm with the same eluent, flow 2.0mL/min. Isolated fraction was evaporated by flushing with a stream of nitrogen, redissolved in acetonitrile and evaporated again. This resulted in 3.5mg (50%) of the product. MS calculated [M+2H] 2+ 1640.67, observed 1640.58.

General radiochemistry
All experiments were performed in accordance with local and national rules and laws that govern the work with open sources of radiation. Radioisotopes were produced on a GEMS PETtrace cyclotron (General Electrics Medical Systems, Uppsala, Sweden). No-carrier-added [ 11 C]CH4 was produced using 16.5 MeV protons in the 14 N(p,a) 11 C nuclear reaction on a mixture of nitrogen and hydrogen gas (10% hydrogen). [ 11 C]CH4 was converted to [ 11 C]CH3I by radical iodination in a gas-phase recirculation system and swept in a stream of helium through a heated glass column containing silver triflate impregnated on graph-pac to produce [ 11 C]CH3OTf. [ 18 F]-fluoroethyl triflate was produced according to a published procedure. 2 The identities of the radiolabelled products were established by coelution with non-radiolabelled reference standards. All reported yields are non-decay corrected, unless noted otherwise. The syntheses times are calculated from the moment of starting the delivery of radionuclides from the cyclotron to synthetic modules.

Preparation of [ 18 F]F-C2H4-1.
[ 18 F]-fluoroethyl triflate was synthesized according to a published procedure 2 and trapped in a mixture of 1 (0.3-0.5mg) and anhydrous potassium carbonate (3-5mg) in 400µL of anhydrous acetonitrile at room temperature. The mixture was then heated at 80°C for 5 minutes, cooled to 40°C, diluted with a solution of 15mg of sodium ascorbate in 0.6mL of acetonitrile and 1.0mL of water and injected on a preparative ACE 5 C18-HL (250x10mm) column. The column was eluted with ACN-H2O 75-25 mixture, containing 4g/L of sodium ascorbate at a flow of 7.0mL/min. The product was collected at 6-7min in 40% RCY (based on the total activity distilled to the reactor during [ 18 F]-fluoroethyl triflate production). Total synthesis time 60min.

PET-Imaging with mice
All mice were housed at the animal department of Karolinska University Hospital in a temperature (± 21°C) and humidity (± 40%) controlled environment on a 12h light/dark cycle (lights on 7:00 AM) with access to food and water ad libitum. Animals were allowed at least one week to habituate to the animal department before the start of the imaging sessions. All experiments were conducted during the light phase of the cycle. All experiments were performed in accordance with the guidelines of the Swedish National Board of Laboratory Animals under protocols approved by the Animal Ethics Review Board of Northern Stockholm, Sweden (N175/15).

Imaging Instrumentation
For the PET measurements, the Mediso nanoScan® PET-MRI and the nanoScan® PET-CT pre-clinical small animal imaging systems were used. 3,4 The two systems have identical PET performance 3 and were calibrated to provide consistent results. Two animals were measured at the same time in the two systems.

In vivo imaging
Male C57BL/6J mice have been injected subcutaneously with A375 or B16/F10 melanoma cells in the neck region. Ten days after inoculation the animals were anesthetized through inhalation of isoflurane (4-5% isoflurane in 100% oxygen). After induction the isoflurane concentration was lowered to 1.5-2% (50/50 air/oxygen) and the animals were positioned in the scanner in a designated mouse bed. A cannula was inserted in the tail vein through which the radioligand was administered. A 93-minute dynamic PET scan was initiated immediately upon intravenous injection of the radioligand.

Image-and Statistical Analysis
The acquired list mode data was reconstructed into 25 timeframes (93 min scan = 4x10 s, 4x20 s, 4x60 s, 7x180 s, 11x360 s). The image reconstruction was made with a fully 3-dimensional maximumlikelihood expectation maximization algorithm (MLEM) with 20 iterations, without scatter and attenuation correction. The reconstructed dynamic PET images were used to delineate VOIs for muscle and tumor regions in PMOD (PMOD Technologies Ltd., Zurich, Switzerland) to generate decay corrected time activity curves (TAC). The regional uptake values were expressed as standard uptake value (SUV), which normalizes for injected radioactivity and body weight.

PET imaging in a non-human primate
The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency (Dnr N452/11) and was performed according to the "Guidelines for planning, conducting and documenting experimental research" (Dnr 4820/06-600) at the Karolinska Institutet, the "Guide for the Care and Use of Laboratory Animals"), the AstraZeneca bioethics policy and the EU Directive 2010/63/EU.
A cynomolgus monkey (body weight 4.7kg) was used in the PET imaging. The NHP was housed in the Astrid Fagraeus Laboratory (AFL) of the Swedish Institute for Infectious Disease Control (AFL), Solna.
Anesthesia was induced by intramuscular injection of ketamine hydrochloride (approximately 10 mg/kg) at AFL and maintained by the administration of a mixture of isoflurane (1.5-2.0%), oxygen, and medical air with endotracheal intubation at the PET center.
PET measurement was conducted using a High-Resolution Research Tomograph (Siemens Molecular Imaging). The position of the NHP was arranged in order to put the body part as much as possible in the gantry.
A transmission scan of 6 minutes using a single 137 Cs source was performed before the [ 11 C]3 injection. List mode data were acquired continuously for 123 minutes immediately after intravenous injection of the radioligand. Images were reconstructed with a series of 34 frames (20 seconds × 3, 1 minute × 3, 3 minutes × 5, 6 minutes × 17) using the ordinary Poisson-3D-ordered subset expectation maximization (OP-3D-OSEM) algorithm.
Regions of interest (ROIs) were manually delineated on the liver, the lungs, the heart and the brain on the summation image. The time activity curves of each organ were generated by applying the ROIs to the dynamic PET data.

Fluorescence staining data
Cell cultivation and the in vivo fluorescent measurement was done according to a previously published procedure. 5 Figure S7. Excitation/emission spectrum of fluorescent probe 3.