Unless otherwise noted, all reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. N-succinimidyl-4-(tributylstannyl) benzoate was purchased from Synthonix (Cambridge, UK), 3-(3-iodophenyl)propionic acid and 3-(4-iodophenyl)propionic acid from Matrix Scientific (Columbia, SC), and 4-iodophenyl acetic acid from Alfa Aesar (Cambridge, UK). Olaparib (AZD2281) was purchased from LC Laboratories (Woburn, MA). 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one was synthesized as described previously [22]. 1H-nuclear MR (NMR) spectra were recorded at room temperature on a Bruker Avance 500 instrument operating at the frequency of 500 MHz (Billerica, MA) and internally referenced to the residual solvent peaks, CDCl3 (7.26 ppm) or dimethyl sulfoxide (DMSO)-d6 (2.49 ppm). Mass spectroscopy data was recorded on a Waters Acquity Ultra Performance LC (Milford, MA). High-resolution mass data was recorded on a Waters LCT Premier XE mass spectrometer. High-performance liquid chromatography (HPLC) and radio-HPLC was performed on a Shimadzu HPLC system equipped with 2LC-10AT pumps and an SPD-M10AVP photodiode array detector (Columbia, MD). Radio-HPLC was performed using an identical Shimadzu system, additionally equipped with a Lablogic Scan-RAM Radio-TLC/HPLC detector (Brandon, FL). Analytic runs were performed on a C18 Waters Atlantis T3 column (6 × 250 mm, 5 mm). The solvent system included water (solvent A) and acetonitrile (AcN) (solvent B) for the purification and quality control of the radiotracers with a gradient of 5–95 % B between 0 and 15 min and 100 % B between 15 and 25 min. For the purification of non-radioactive precursors, water (0.1 % trifluoroacetic acid, solvent A) and acetonitrile (AcN) (0.1 % trifluoroacetic acid, solvent B) were used, all with a flow rate of 1 mL/min and a gradient of 5–95 % B between 0 and 15 min, 95 % B between 15 and 17 min, and 95–5 % B between 17 and 18 min.
Synthesis of PARP1 inhibitors
4-(4-fluoro-3-(4-(3-iodobenzoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (I1-PARPi)
To a solution of 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol), triethylamine (40 μL, 0.3 mmol) and HBTU (16 mg, 0.0413 mmol) in dimethyl formamide (DMF, 500 μL) were added to 3-iodobenzoic acid (6 mg, 0.0275 mmol). The mixture was stirred at room temperature for 20 h. The crude product was then purified by preparative HPLC and dried under vacuum, yielding a white solid (6.9 mg, 48 % yield). 1H-NMR (CDCl3) δ = 10.00 (s, 1H), 8.40–8.38 (m, 1H), 7.71–7.69 (m, 4H), 7.64–7.63 (m, 1H), 7.30–7.26 (m, 3H), 7.09 (m, 1H), 7.04–6.87 (m, 1H), 4.21 (s, 2H), 3.71–3.29 (m, 8H). LC-ESI-MS (+) m/z = 597.1 [M+H+]+. HRMS-ESI [M-H+]− m/z calculated for [C27H22FIN4O3]− 595.0642, found 595.0660.
4-(4-fluoro-3-(4-(4-iodobenzoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (I2-PARPi)
A solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol), HBTU (16 mg, 0.0413 mmol), triethylamine (40 μL, 0.3 mmol), and 4-iodobenzoic acid (6 mg, 0.0245 mmol) in DMF (500 μL) was stirred overnight at room temperature. The crude product was purified by preparative HPLC and dried under vacuum, yielding a white solid (8.8 mg, 61 % yield). 1H-NMR (CDCl3) δ = 10.48 (s, 1H), 8.40–8.39 (m, 1H), 7.74–7.66 (m, 5H), 7.27–7.26 (d, 2H), 7.09–7.07 (d, 2H), 4.22 (s, 2H), 3.73–3.14 (m, 8H). LC-ESI-MS (+) m/z = 597.1 [M+H+]+. HRMS-ESI [M-H+]− m/z calculated for [C27H22FIN4O3]− 595.0642, found 595.0640.
4-(4-fluoro-3-(4-(2-(3-iodophenyl)acetyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (I3-PARPi)
A solution of 3-iodophenyl acetic acid (6.5 mg, 0.048 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (10.5 mg, 0.055 mmol), N-hydroxy succinimide (NHS), and 600 μL DMF was stirred for 30 min at room temperature. Then, 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol) was added to the solution, and the mixture was stirred at room temperature overnight. The reaction was washed with 500 μL of H2O and extracted with 500 μL dichloromethane (DCM). The resulting organic solution was purified on silica gel, using a gradient elution from neat DCM to DCM/hexane 5:1 to obtain the desired product as a white solid (3 mg, 20 % yield). 1H-NMR (CDCl3) δ = 9,82 (s, 1H), 8.40–8.38 (m, 1H), 7.71–7.69 (m, 2H), 7.55–7.53 (m, 1H), 7.51–7.50 (m, 2H), 7.25–7.24 (m, 2H), 7.09–6.90 (m, 3H), 4.20 (s, 2H), 3.64–3.31 (m, 8H), 2.84 (s, 2H). LC-ESI-MS (+) m/z = 633.1 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C28H24FIN4O3]+ 611.0955, found 611.0948.
4-(4-fluoro-3-(4-(2-(4-iodophenyl)acetyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (I4-PARPi)
A solution of 4-iodophenyl acetic acid (6.5 mg, 0.048 mmol), EDC (10.5 mg, 0.055 mmol), NHS, and 600 μL DMF was stirred for 30 min at room temperature. After this time, the 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol) was added to the solution and the mixture was stirred at room temperature overnight. H2O (500 μL) was added, the mixture was extracted with DCM (2 × 500 μL), and the combined extracts were dried under vacuum. The crude mixture was purified by silica column chromatography (100 % DCM), and the product obtained was a white solid (8.8 mg, 61 %). 1H-NMR (CDCl3) δ = 9.82 (s, 1H), 8.40–8.38 (m, 1H), 7.83–7.81 (d, 1H), 7.77–7.75 (d, 1H), 7.70–7.69 (m, 2H), 7.63–7.56 (m, 3H), 7.00–6.89 (m, 3H), 4.20 (s, 2H), 3.63–3.11 (m, 8H), 2.84 (s, 2H). LC-ESI-MS (+) m/z = 632.9 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C28H24FIN4O3]+ 611.0955, found 611.0971.
4-(4-fluoro-3-(4-(3-(3-iodophenyl)propanoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (I5-PARPi)
A solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol), HBTU (16 mg, 0.0413 mmol), triethylamine (40 μL, 0.3 mmol), and 3-(3-iodophenyl)propionic acid (7.6 mg, 0.0275 mmol) in 400 μL of AcN was stirred overnight at room temperature. The crude product was then purified by preparative HPLC and the isolated product dried at vacuum to obtain a white solid (5.1 mg, 38 %). 1H-NMR (CDCl3) δ = 10.33 (s, 1H), 8.41–8.39 (d, 1H), 7.71–7.63 (m, 3H), 7.51–7.45 (m, 2H), 7.27–7.25 (m, 2H), 7.12–6.92 (m, 3H), 4.22 (s, 2H), 3.65–3.12 (m, 8H), 2.88–2.83 (m, 2H), 2.59–2.48 (m, 2H). LC-ESI-MS (+) m/z = 647.1 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C29H26FIN4O3]+ 625.1112, found 625.1111.
4-(4-fluoro-3-(4-(3-(4-iodophenyl)propanoyl)piperazine-1-carbonyl)benzyl) phthalazin-1(2H)-one (I6-PARPi)
4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mg, 0.0275 mmol) was mixed with HBTU (16 mg, 0.0413 mmol), triethylamine (40 μL, 0.3 mmol), and 4-iodo-3-phenyl propionic acid (7.6 mg, 0.0275 mmol) in 400 μL of AcN, and the solution was stirred overnight at room temperature. The crude product was then purified by preparative HPLC and the isolated product dried at vacuum to obtain a white solid (7.5 mg, 45 %). 1H-NMR (CDCl3) δ = 9.71 (s, 1H), 8.40–8.38 (d, 1H), 7.70–7.69 (m, 2H), 7.64–7.63 (m, 1H), 7.55–7.52 (m, 2H), 7.27–7.25 (m, 2H), 7.00–6.97 (m, 1H), 6.91–6.87 (m, 2H), 4.20 (s, 2H), 3.64–3.11 (m, 8H), 2.87–2.85 (m, 2H), 2.63–2.47 (m, 2H). LC-ESI-MS (+) m/z = 647.1 [M+Na+]+. HRMS-ESI [M+Na+]+ m/z calculated for [C29H26FIN4O3Na]+ 647.0931, found 647.0941.
Radiochemistry
[131I]-NaI was purchased at Nordion (Ottawa, ON, Canada) in NaOH solution (0.1 M) with a concentration of 0.99–2.5 mCi/μL. [124I]-NaI was produced at Memorial Sloan-Kettering Cancer Center (New York, NY) in NaOH solution (0.5 M) with a concentration of 0.20–0.40 mCi/μL.
Synthesis of [131I]-NHS-benzoate
Precursor N-succinimidyl-4-(tributylstannyl) benzoate (30 μg, 5.9 nmol) was dissolved in 30 μL of AcN, and the solution was added to a solution of methanol (40 μL), chloramine T (6 μg, 0.03 nmol) in acetic acid (2 μL), and [131I]-NaI in NaOH 0.1 M (1–2.5 mCi). After 5 min at room temperature, the reaction was purified by HLPC on a C18 Waters Atlantis T3 column (6 × 250 mm, 5 mm), using water (solvent A) and AcN (solvent B) as mobile phase, with an elution gradient from 5 to 100 % for solvent B over 15 min and then 100 % of solvent B from 15 to 25 min. The retention time of [131I]-NHS benzoate was 14.3 min, and its identity was established by co-elution with the reference cold compound. The radiochemical yield was 67 ± 6 % (n = 12), and the radiochemical purity was >98 %. The collected fraction containing [131I]-NHS benzoate was concentrated to dryness under vacuum.
The same procedure was followed for the synthesis of [124I]-NHS benzoate. In this case, the radiochemical yield was 32 ± 5 % (n = 5) and the purity was >95 %.
Synthesis of [131I]-I2-PARPi
The dried radiolabeled [131I]-NHS-benzoate precursor was dissolved in 200 μL of AcN, and an excess of HBTU (1 mg, 2.6 nmol) and 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (1 mg, 2.7 nmol) was added and allowed to react for 3 h at 32 °C. The final product was purified by HPLC, using water (solvent A) and AcN (solvent B) as solvents with a gradient elution from 5 to 100 % of solvent B over 15 min and then 100 % of B from 15 to 25 min. The retention time of [131I]-I2-PARPi was 13.1 min, and its identity was established by co-elution with the reference cold compound. The radiochemical yield was 72 ± 8 % (n = 12) and the radiochemical purity >95 %. The collected fraction containing [131I]-I2-PARPi was concentrated to dryness under reduced pressure.
The same procedure was followed for the synthesis of [124I]-I2-PARPi. In this case, the radiochemical yield was 68 ± 5 % (n = 5) and the purity >95 %.
Cell culture
The human glioblastoma cell lines U251 MG and U87 MG were generously provided by the Laboratory of Dr. Ronald Blasberg (MSKCC, New York, NY). All cell lines were grown in Eagle’s minimal essential medium (MEM) containing 10 % (v/v) heat-inactivated fetal bovine serum, 100 IU penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37 °C in a humidified 5 % CO2 atmosphere. All media was purchased from the media preparation facility at MSKCC (New York, NY).
Mouse models
Six 10-week-old female athymic nude CrTac:NCr-Fo mice from Taconic Laboratories (Hudson, NY) were used for all mouse experiments. During subcutaneous injections, mice were anesthetized using 2 % isoflurane gas in 2 L/min medical air. During orthotopic injections, mice were anesthetized using a 150 mg/kg ketamine and 15 mg/kg xylazine cocktail (10 μL/g). Before all intravenous injections, mice were gently warmed with a heat lamp and placed in a restrainer and tails were sterilized with alcohol pads. The lateral tail vein was used for all intravenous injections. All mouse experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committee of MSKCC and followed National Institutes of Health (NIH) guidelines for animal welfare.
PARP-1 IC50 determination
A commercially available colorimetric assay (Trevigen, Gaithersburg, MD) was used to measure PARP-1 activity in vitro in the presence of varying concentrations of the different iodo-PARPis. Specifically, dilutions of iodo-PARPi (final concentrations ranging from 3.3 μM to 0.1 nM) were incubated with 0.5 U of PARP1 high specific activity (HSA) enzyme for 10 min in histone-coated 96-well plates. All experiments were carried out in triplicate. Positive control samples did not contain inhibitor, and negative control samples did not contain PARP1. All reaction mixtures were adjusted to a final volume of 50 μl, and a final concentration of 1 % DMSO in assay buffer. The remainder of the assay was performed according to the manufacturer’s instructions. PARP1 activity was measured by absorbance at 450 nm in each well using a SpectraMax M5 spectrophotometer with SoftMax Pro software (Molecular Devices, Sunnyvale, CA).
Hydrophobicity index determination
Chemical hydrophobicity indices (CHIs) were measured using procedures developed previously [24]. Briefly, reverse phase HPLC was used to measure the retention times of a set of standards with known CHI. A standard curve was then created to calculate the CHIs of all iodo-PARPi based on the HPLC retention time. Log P values were derived from CHI values following the equation: Log P = 0.0566 ± CHI − 1.107.
Plasma protein fraction
The plasma protein fraction was determined using the Rapid Equilibrium Dialysis Device System (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. Membrane dialysis was performed with 10 μM of compound in mouse serum (500 μL) on one side of the membrane and PBS (750 μL) on the other side. The system was sealed with parafilm and incubated for 4 h at 37 °C on an orbital shaker set to 250 rpm. Thereafter, 400 μL of solution was taken from both sides, and samples were treated twice with an equal amount of AcN and vortexed to remove protein before HPLC analysis. After injection (100 μL), the I-PARPi peaks from each sample were then integrated and the protein bound fraction was determined. The data was analyzed using Prism 6.0c.
Immunohistochemistry
PARP1 expression in tissues
PARP1 antigen detection in glioblastoma xenografts and mouse brain was performed at MSKCC’s Molecular Cytology Core Facility using the Discovery XT processor (Ventana Medical Systems, Tucson, AZ) and detected using immunofluorescence (IF) staining. Paraffin-embedded formalin-fixed 3 μm sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer (both Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Richmond, CA). Anti-PARP1 rabbit polyclonal antibody (sc-7150, 0.2 μg/mL; Santa Cruz Biotechnology, Santa, Cruz, CA) was incubated for 5 h, followed by 1 h incubation with biotinylated goat anti-rabbit IgG (Vector labs, PK6106) at a 1:200 dilution. Detection was performed with Streptavidin-HRP D (from DABMap Kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 594 (T20935; Invitrogen, Carlsbad, CA) prepared according to the manufacturer’s instructions. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and coverslipped with Mowiol® mounting medium (Sigma-Aldrich, St. Louis, MO). H&E staining was performed on adjacent sections for morphological evaluation of tissue characteristics.
Quantification of PARP1 expression
Protein expression was quantified on digitalized PARP1-stained sections using at least ten fields of view per section. Thresholding of the blue (nuclei stained with DAPI) and red fluorescent area (nuclei stained with PARP1) was performed using MetaMorph® Software (Molecular Devices, Sunnyvale, CA). PARP1 intensity was determined by measuring the red fluorescence intensity in the area of all nuclei, and the % PARP1 positive nuclear area was calculated by dividing the PARP1 positive area by the DAPI positive area in each field of view.
In vitro blocking study
U87 MG cells were seeded into a 96-well plate in a concentration of 1 × 104 cells per well. After 24 h, the cells were incubated with either the fluorescent PARP1 inhibitor PARPi-FL (250 nM) alone or with one of the iodo-PARPi inhibitors at a 100-fold higher concentration (25 μM) for 20 min. Additionally, as a positive control, the PARP1 inhibitor Olaparib was used. All incubation solutions also included Hoechst 33342 nuclear stain (Sigma-Aldrich, St. Louis, MO). The cells were washed twice with media and once with PBS for 5 min each and imaged on an LSM 5Live confocal microscope (Zeiss, Oberkochen, Germany). All wells were imaged with the DAPI filter for the Hoechst staining and the FITC filter for the PARPi-FL staining. The DAPI and FITC channels were co-registered, and the green fluorescence in the location of the Hoechst staining was quantified for each image. The percent reduction in PARPi-FL uptake was calculated based on the level of fluorescence intensity seen in each image and normalized to the cells receiving no iodo-PARPi inhibitor. Experiments were performed in triplicate.
Blood half-life
Blood half-life was determined by measuring the activity in serial blood samplings. Specifically, healthy female nude mice (8–10 weeks old, 20–25 g in weight, n = 3) were injected via the tail vein with 50 μCi [131I]-I2-PARPi in 200 μL of solution PBS/PEG300 (10:1). The blood was sampled from the saphenous vein at 5, 15, 30, 60, 120, and 240 min post injection. The blood was weighed and radioactivity was measured on a Wizard 2470 Automatic Gamma Counter (Perkin Elmer, Waltham, MA). Measurements in counts per minute were calculated as the mean %ID/g. The blood half-life was calculated using Prism 6.0c (GraphPad Software, La Jolla, CA).
In vivo blocking study
To verify the specificity of tumor uptake of I2-PARPi in vivo, the level of blocking of the fluorescent PARP1 inhibitor on a macroscopic and microscopic scale was determined. Nude mice bearing subcutaneous U87 MG tumors were injected with either the fluorescent PARP1 inhibitor PARPi-FL alone (2.5 mg/kg, 200 μL of 19.5 % 1:1 DMAC:Kolliphor, 3.5 % DMSO, 77 % PBS), PARPi-FL 30 min after a pre-injection of a 50-fold excess of I2-PARPi (125 mg/kg, 100 μL of 10 % PEG300, 90 % PBS), or injected with saline alone. One hour post injection, the mice were sacrificed and the tumors were resected and imaged with the IVIS spectrum fluorescence imaging system (PerkinElmer, Waltham, MA) using Living Image 4.4 software. The tumors were also imaged microscopically with the 5Live fluorescent confocal microscope using the 488 nm laser for PARPi-FL excitation.
In vitro whole blood stability
The in vitro stability was assessed by incubating 6 μCi [131I]-PARPi in mouse blood for 0 to 60 min at 37 °C. At baseline, 15, 30, and 60 min, the samples were immediately placed on ice and mixed 1:1 with a solution of AcN/DMSO (250 μL) and then vigorously vortexed for 30 s to precipitate out serum protein. The sample was centrifuged at 3000 RCF for 3 min at 4 °C, and the supernatant was collected. This procedure was repeated three times, and the combined supernatants were analyzed by HPLC equipped with radioactive detector (Shimadzu, Kyoto, Japan), collecting samples every 30 s. Radioactivity of each fraction was measured on a Wizard 2470 Automatic Gamma Counter (Perkin Elmer, Waltham, MA), and the blood stability was analyzed using Prism 6.0c (GraphPad Software, La Jolla, CA).
Biodistribution studies
Biodistribution experiments were conducted on female nude mice (8–10 weeks old and 20–25 g in weight, n = 21) bearing U87 MG or U251 MG subcutaneous xenografts. The radiolabeled small molecule preparation (30–20 μCi of [131I]-I2-PARPi in 200 μL of a solution 90 % PBS 10 % PEG300) was administrated via the lateral tail vein. To determine the optimal specific activity to achieve the highest tumor to organ ratio, various specific activities were tested (5, 50, and 250 mCi/μmol) in mice bearing U87 MG tumors. The compound was allowed to circulate for 2 h post injection at which time the mice were sacrificed and organs were harvested (n = 3). After determining the optimal specific activity, the optimal time for imaging was determined by testing the drug distribution in nude mice bearing U87 MG tumors at different time points. The drug was allowed to circulate for various times (1, 2, and 6 h), after which the mice were sacrificed (n = 3). The radioactive content in the tissue of interest (blood, tumor, muscle, bone, liver, spleen, kidney, heart, lung, pancreas, brain, skin, small intestine, large intestine, stomach, tail, thyroid, and feces) was measured on a Wizard 2470 Automatic Gamma Counter and the tissue-associated activity was calculated as the mean %ID/g.
Autoradiography
U251 MG glioblastoma cells (5 × 104 in 2 μL of PBS) were orthotopically implanted in athymic nude mice, using a stereotaxic device, and the tumors were allowed to grow for approximately 4 weeks. Once tumors reached the sufficient size, the orthotopic U251 MG tumor-bearing mice were injected intravenously with 500 μCi [131I]-PARPi (in 200 μL of a solution PBS 90 % PEG300 10 %, n = 2) alone or with a pre-injection of 15 μmol Olaparib (in 100 μL of 7.5 % DMSO, 12.5 % PEG300, 80 % PBS) 30 min prior to the injection of [131I]-PARPi. Additionally, healthy mice were also injected with 500 μCi [131I]-PARPi. After 2 h of circulation time after the [131I]-PARPi injection, the mice were sacrificed. Liver, tumor, muscle, and brain tissues were excised and embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) and frozen at −20 °C, and a series of 8 μm frozen sections was cut and mounted on microscope slides. To determine radiotracer distribution, digital autoradiography was performed by placing tissue sections in a film cassette against a phosphor image plate (BASMS-2325; Fujifilm) for 48 h at −20 °C. Phosphor imaging plates were read at a pixel resolution of 25 μm with a Typhoon 7000IP plate reader (GE Healthcare, Piscataway, NJ). After autoradiographic exposure, the same frozen sections were then used for immunohistochemical staining. Areas of brain slides containing tumor tissue were identified using the H&E staining and then overlaid with the autoradiographic data. Intensity of tumor areas and non-tumor areas were then quantified using ImageJ 1.47u.
In vivo imaging
SPECT/CT was acquired in athymic nude mice (6–10 weeks old). Before administration of the radioiodinated tracer, in terms to block the thyroid, the animals were treated with an intraperitoneal injection of NaI (100 μL, 0.6 mM) 60 min previous to the injection of 450–600 μCi (145–210 mCi/μmol) [131I]-I2-PARPi in 200 μL PBS solution (10 % PEG300) via the lateral tail vein and then anesthetized with isoflurane mixed with medical air (2 % for induction and maintenance). Animals were placed in prone position, and scans were then performed 90 min after injection for 60 min using a SPECT/CT small animal imaging system (NanoSPECT/CT, Mediso, Boston, MA). SPECT Images were reconstructed using HiSPECT software, and in vivo Scope software was used for CT image reconstruction.
In the case of PET imaging, images were acquired after the injection of 200–250 μCi (110–170 mCi/μmol) [124I]-I2-PARPi in 200 μL PBS solution (10 % PEG300) via the lateral tail vein under isoflurane anesthesia (2 % for induction and 1.5 % for maintenance). Mice were also treated with an intraperitoneal injection of NaI (100 μL, 0.6 mM), 60 min previous to the administration of the radioiodinated tracer. Animals were immediately placed in prone position under isoflurane anesthesia, and scans were then performed 90 min after injection for 30 min using the Inveon PET/CT imaging system (Siemens, Knoxville, TN). PET and CT Images were reconstructed using Inveon research workplace software.
Formulation of [131/124I]-I2-PARPi for in vivo injection
For in vivo applications, the radioactive 124I/131I-I2-PARPi was injected intravenously, using hypodermic syringes with 200 μL of a solution of PBS 1× and PEG300 (9/1 v/v). We used approximately 450–600 μCi of radiotracer for PET imaging, 200–250 μCi for SPECT imaging, 500 μCi for autoradiography, and 30–20 μCi for biodistributions.