PET Imaging of PARP Expression Using 18F-Olaparib

Poly(ADP-ribose) polymerase (PARP) inhibitors are increasingly being studied as cancer drugs, as single agents, or as a part of combination therapies. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, outcome prediction, dose optimization, genotoxic therapy evaluation, and target engagement imaging of novel PARP-targeting agents. Methods: Here, via the copper-mediated 18F-radiofluorination of aryl boronic esters, we accessed, for the first time (to our knowledge), the 18F-radiolabeled isotopolog of the Food and Drug Administration–approved PARP inhibitor olaparib. The use of the 18F-labeled equivalent of olaparib allows direct prediction of the distribution of olaparib, given its exact structural likeness to the native, nonradiolabeled drug. Results: 18F-olaparib was taken up selectively in vitro in PARP-1–expressing cells. Irradiation increased PARP-1 expression and 18F-olaparib uptake in a radiation-dose–dependent fashion. PET imaging in mice showed specific uptake of 18F-olaparib in tumors expressing PARP-1 (3.2% ± 0.36% of the injected dose per gram of tissue in PSN-1 xenografts), correlating linearly with PARP-1 expression. Two hours after irradiation of the tumor (10 Gy), uptake of 18F-olaparib increased by 70% (P = 0.025). Conclusion: Taken together, we show that 18F-olaparib has great potential for noninvasive tumor imaging and monitoring of radiation damage.


Synthesis methods
See Supplemental Figure 1 for the full synthesis scheme. For each compound relating to supplemental figure 1, where appropriate, a commentary is provided to aid the reader in translating this chemistry to other laboratories.

Dimethyl (3-oxo-1,3,-dihydroisobenzofuran-1-yl)phosphonate
Dimethylphosphite (0.92 mL, 10.0 mmol) was added dropwise to a solution of sodium (0.58 g, 10.8 mmol) in MeOH (15 mL) at 0 °C. To the solution, 2-carboxybenzaldehyde (1.00 g, 6.66 mmol) was added portion-wise while stirring. The mixture was gradually warmed to room temperature and stirred for 6 hours. Methanesulfonic acid (0.77 mL, 11.9 mmol) was added dropwise and the mixture was stirred for another 30 minutes. The solution was concentrated in vacuo to produce a white solid, to which water was added (30 mL) and the crude product was extracted into DCM (3 × 30 mL). The
The solution was then cooled to 0 °C followed by the addition of Et 3 N (0.69 mL, 4.96 mmol). The reaction mixture was allowed to warm up to room temperature and was stirred for 48 h, followed by concentration in vacuo to produce a white solid. The solid was suspended in water, collected by
The solution was cooled to 0 °C followed by the addition of Et 3 N (4.78 mL, 34.4 mmol). The reaction mixture was warmed to room temperature and was stirred for 48 h, followed by concentration in vacuo to produce a white solid. The solid was suspended in water, collected by vacuum filtration and washed with hexane (2 × 20 mL) and Et 2 O (3 × 20 mL) affording 2-Bromo-5-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)benzonitrile (5.14 g, 15.8 mmol, 92%) as a white solid in a mixture of e:z stereoisomers (10:1) and a purity of 90%. NMR spectra showed a 10:1 mixture of E and Z isomers.
The reaction was stirred at 0 °C for 30 min before warming to room temperature upon which 2-

2-Methylphthalazin-1(2H)-one
To a round bottom flask containing DMF (10 mL) under an atmosphere of argon was added, phthalazone (300 mg, 2.05 mmol). The solution was then cooled to 0 °C before sodium hydride (60% in dispersion oil, 61 mg, 2.53 mmol) was added portion wise. Finally, iodomethane (119 µL, 1.92 mmol) was added and the reaction was stirred overnight before the solvent was removed in vacuo and the crude material purified directly via flash column chromatography using n-pentane: EtOAc (10:1) The data is in accordance with known literature.(4)

2-allylphthalazin-1(2H)-one
To a round bottom flask containing DMF (30 mL) under an atmosphere of nitrogen was added, phthalazone (500 mg, 3.42 mmol), anhydrous potassium carbonate (708 mg, 5.12 mmol) and allyl bromide (442 µL, 5.12 mmol). The reaction was stirred overnight at room temperature before the solvent was removed in vacuo and the crude material purified directly via flash column chromatography using n-pentane: EtOAc (

2-((2-triethylsilyl)ethoxyl)methyl)phthalazin-1(2H)-one
To a round bottom flask containing THF (10 mL) under an atmosphere of argon was added, phthalazone (480 mg, 3.28 mmol). To solution was then cooled to 0 °C before sodium hydride (60% in dispersion oil, 97 mg, 4.04 mmol) was added portion wise. The reaction was then stirred for 30 min at 0 °C before warming to room temperature after which 2-(trimethylsilyl)ethoxymethyl chloride (0.70 mL, 3.97 mmol) was added dropwise. The reaction mixture was left to stir overnight after which the solvent was removed in vacuo. The crude mixture was then extracted with DCM (3 x 10 mL) and washed with brine (3 x 10 mL). The organic layer was collected, dried with magnesium sulfate and the excess solvent removed in vacuo. The crude material was then purified by flash column

Procedure for preparation of a solution of [ 18 F]KF/K 222 in MeCN:
A solution of Kryptofix 222 (15 mg) and K 2 CO 3 (3 mg) in 1 mL of MeCN/H 2 O, 4:1 was freshly prepared. The sealed vial was heated at 110 °C for 20 min. The reaction was quenched by addition of water (200 μL). An aliquot was removed for analysis by radioTLC and HPLC for radiochemical conversion and product identity. Analysis was performed using a Waters Nova-Pak C18 column (4 μm, 3.9 x 150 mm) at a flow rate 1 mL/min. Radio-TLC was performed on Merck Kiesegel 60 F254 plates, using nhexane/EtOAc (1:1) as eluent. Analysis was performed using a plastic scintillator/PMT detector.
The sealed vial was heated at 110 °C for 20 min. The reaction was quenched by addition of water (200 μL). An aliquot was removed for analysis by radioTLC and HPLC for radiochemical conversion and product identity. Analysis was performed using the gradient given below with a Waters Nova-Pak C18 column (4 μm, 3.9 x 150 mm) at a flow rate 1 mL/min. Radio-TLC was performed on Merck Kiesegel 60 F254 plates, using as eluent DCM/MeOH (9:1). Analysis was performed using a plastic scintillator/PMT detector.

Procedure for the Synthesis and Isolation of 18 F-Olaparib:
18 F-olaparib was obtained via the Cu-mediated 18 F-fluorodeboronation of the corresponding boronic ester precursor (Figure 1, 2 Test reactions were carried out on components of the molecule, to explore the compatibility of all chemical motifs with the Cu-assisted radiolabelling reaction (Supplemental Figure 3, 4, 5, 6).
Additional optimisation of the Cu-complex catalyst and drying methods was performed to improve activity yields (Supplemental Table 1, 2). Molar activity was calculated based on a standard series and co-injection of cold, unlabelled olaparib (Supplemental Figure 7, Supplemental Table 3).

Cells
PSN-1, MiaPaCa-2, and Capan-1 human pancreatic duct adenocarcinoma cells were originally purchased from ATCC. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Cells were grown in a 37°C environment containing 5% CO 2 and were harvested and passaged as required using Trypsin-EDTA solution. Cells were authenticated by the provider and the cumulative length of culture was less than 6 months following retrieval from liquid nitrogen storage. Cells were regularly tested to confirm the absence of mycoplasma contamination.
Capan-1 cells used in this study were subsequently found by STR profiling not to match with the ATCCheld profile. However, low PARP enzyme expression was confirmed by Western blot and immunohistochemistry.

Western Blot
Western blot probing for PARP-1 was performed after cells were exposed to external beam radiation anti-rabbit-HRP (Bio-Rad). The membrane was exposed to autoradiography film after development using an ECL western blot substrate solution (Pierce Thermo Scientific 32209). β-actin was used as the loading control. was applied to each section. Finally a coverslip was gently lowered onto each slide, and Covergrip (Biotium) used to seal the coverslips. Prepared slides were stored at 4°C in the dark. The slides were analyzed using a Leica SP8 confocal fluorescent microscope.

Cell uptake experiments
Cell uptake of 18 F-olaparib in PSN-1, MiaPaCa-2 and Capan-1 cells was determined as previously described (8)(9)(10). Aliquots of cells (1.5×10 5 cells/well) were seeded in 24-well plates in warm cell culture medium (500 µL) and the cells were allowed to adhere overnight. Cells were irradiated (10 Gy; dose rate 0.8 Gy/min) or sham-irradiated and then returned to an incubator (37°C, 5% CO 2 ) for 2-48 h. The cell culture medium was then removed and cells were washed once with fresh cell culture medium (500 µL). In 500 µL of cell culture medium (not supplemented with FBS, L-glutamine, or penicillin/streptomycin), 18 F-olaparib (50 kBq) was added to each well and the cells were then incubated at 37°C. In the blocking groups, non-radioactive olaparib, talazoparib, or rucaparib were also added in increasing concentrations (10 pM -10 µM. After 30 or 60 minutes, the cell culture medium was removed and combined with two washes (500 µL) of cell culture medium. The remaining monolayer of cells was then lysed with 0.1 M sodium hydroxide for 20 minutes at room temperature.
The amount of radioactivity contained within the cell culture medium and the cell lysate fractions was measured using a gamma counter. Protein levels from parallel plates were quantified using a Pierce

(Radiation dose dependency)
Aliquots of cells (7.5×10 4 cells/well) were seeded in 24-well plates in warm cell culture medium (500 µL). After 4 h, cells were irradiated (0, 2, 4, 6, 8, or 10 Gy; dose rate 0.8 Gy/min) and then returned to an incubator (37°C, 5% CO 2 ). After 24 or 48 h, the cell culture medium was then removed and cells were washed once with fresh cell culture medium (500 µL). In 500 µL of cell culture medium (not supplemented with FBS, L-glutamine, or penicillin/streptomycin), 18 F-olaparib (50 kBq) was added to each well and the cells were then incubated at 37°C. In the blocking groups, non-radioactive olaparib was also added to each well to achieve a concentration of 10 µM. After 30 minutes, the cell culture medium was removed and combined with two washes (500 µL) of cell culture medium. The remaining monolayer of cells was then lysed with 0.1 M sodium hydroxide for 20 minutes at room temperature.
The amount of radioactivity contained within the cell culture medium and the cell lysate fractions was measured using a gamma counter. Protein levels from parallel plates were quantified using a Pierce BCA protein assay kit (Thermo Scientific) according to the manufacturer's recommendations and bovine serum albumin was used as the protein standard. Radiotracer cell uptake levels were normalized to percent of the total added radioactivity per milligram protein.

(CaNT)
The murine adenocarcinoma NT (CaNT) was implanted subcutaneously onto the right thigh of 6-7 week-old female CBA/Carl mice. Fifty µL of a crude cell suspension, prepared by mechanical dissociation of an excized tumor from a donor animal, was injected. Tumors were selected for imaging when the geometric mean diameter reached 6-8 mm (volumes calculated as above), approximately 3 weeks after implantation (11).

(Irradiation of tumors)
Irradiation of subcutaneous tumor xenografts was performed using a Gulmay 320 kV system (2.0 Gy/min). A dose of 10 Gy was delivered to the tumor using 300 kV X-rays (Gulmay 320kV irradiator; 2 Gy/min). The radiation set-up allowed irradiation of the right hind quarter, including the tumor and right leg, only. Control mice were anesthetized and sham-irradiated for the same length of time.
Irradiation was performed 2 hours prior to administration of 18 Folaparib (see figure 3a, here repeated for clarity).

PET/CT Imaging ( 18 F-olaparib PET imaging)
Two hours prior to PET/CT imaging, animals were exposed to X-irradiation of the tumor (10 Gy or sham) (see schedule below). Radiation was delivered using a Gulmay 320 kV X-irradiator; 2.0 Gy/min.
Anesthesia was maintained at 2.5 % isoflurane throughout the duration of the irradiation, and animals were allowed to recover. To study the relationship of PARP-1 expression, 18 F-olaparib uptake and hypoxia, the same animals were administered EF5 (0.6 µg in 200 µL 0.9% saline) intraperitoneally, one hour prior to PET/CT imaging. Some animals were administered an excess of cold, unlabelled olaparib

(image analysis)
Reconstructed images were viewed and analyzed using PMOD v.3.37 (PMOD Technologies, Zurich, Switzerland). The radioactivity in each volume of interest was calculated as percent injected dose per cubic cm (%ID/mL).

Ex vivo biodistribution
After PET/CT image acquisition, animals were euthanized by cervical dislocation and selected organs, tissues and blood were removed. The amount of radioactivity in each organ was measured using a 2480 WIZARD 2 gamma counter (PerkinElmer). Counts per minute were converted into MBq using a calibration curve generated from known standards. Values were decay-corrected to the time of injection, and the percentage of the injected dose per gram (%ID/g) of each tissue was calculated.

Ex vivo analysis (Autoradiography)
After imaging and automated gammacounting, selected tissues from mice were flash-frozen with dry ice. If required, samples were stored at -80°C overnight. Frozen tissue was sectioned (8 µm) using an OTF5000 cryotome (Bright Instruments Ltd). Tissue sections were thaw-mounted onto Superfrost PLUS glass microscope slides (Menzel-Glaser, Thermo Scientific) and allowed to dry at room temperature. The slides were then exposed to a storage phosphor screen (PerkinElmer, Super Resolution, 12.5 x 25.2 cm) in a standard X-ray cassette for 15 h at 4°C or -20°C. The phosphor screen was then imaged using a Cyclone® Plus Storage Phosphor System (PerkinElmer) and images were analyzed with OptiQuant 5.0 (PerkinElmer) and ImageJ (NIH).

(Immunohistochemistry)
PARP-1 staining: PSN-1 xenografts harvested from mice were flash frozen and 8 m sections were prepared using a cryostat. Sections were stored at -80C until use. Slides were allowed to reach room temperature ( (12)). To determine the correlation between 18 F-olaparib uptake and hypoxia, tumor slices were analyzed by both autoradiography and EF5 IHC (13) (11) (14). Object-based overlap between both modalities was determined by first co-registering autoradiography and fluorescence microscopy images using a rigid transformation. Then, Manders' overlap coefficients (M1) were calculated using the JACoP plug-in for Image J (methods of Manders for spatial intensity correlation analysis with Costes method for automatic thresholding).

Statistical methods
All statistical analyses and nonlinear regression were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data were tested for normality and analyzed either by the unpaired, two-tailed Student's t-test where appropriate, or 1-way analysis of variance (ANOVA) for multiple comparisons, with Dunnet's post-tests to calculate significance of differences between groups. All data were obtained at least in triplicate and results reported as mean ± standard deviation, unless stated otherwise.

Supplemental tables: biodistribution data
Supplemental