All solvents and non-radioactive reagents were obtained in reagent grade from ABCR (Karlsruhe, Germany), Sigma-Aldrich (München, Germany), Acros Organics (Geel, Belgium) or VWR (Bruchsal, Germany) and were used without further purification. Precursors carrying the chelating moiety NOTA were synthesized similarly to previously described procedures (Lindner et al. 2018; Loktev et al. 2018). 2,3,5,6-Tetrafluorophenlyl 6-trimethylaminiumnicotinate chloride salt was synthesized according to Olberg et al. (Olberg et al. 2010), using a 2 m solution of trimethylamine in tenfold excess instead of the passing through of trimethylamine gas. Further details and additional protocols are given in the supporting information.
HPLC analyses were performed on Agilent 1100 systems with VWD-detectors (Agilent Technologies Germany) equipped with Chromolith Performance RP18e columns (3 × 100 mm; Merck, Germany). UV-traces were recorded at 214 nm using the included ChemStation software. Solvents used were water and acetonitrile, each containing 0.1% trifluoroacetic acid. Unless noted otherwise a linear gradient of 0–100% acetonitrile in 5 min was chosen. In case of radioactive compounds, an equal setup equipped with a gamma detector (GABI, Elysia-Raytest, Germany) was used.
Radiolabeling of 6-trimethylammonium nicotinamides FAPI-72 and -73 was performed by the previously established methods of Richarz et al. (2014) and Basuli et al. (2016). Briefly, 1–4 GBq [18F]fluoride (ZAG Zykloron AG, Karlsruhe, Germany) in 2 mL water were trapped on an anion exchange cartridge (Waters Accel Plus QMA Light cartridge), preconditioned with 5 mL 1 m KHCO3 and 10 mL of water, washed with 3 mL acetonitrile and dried by a stream of nitrogen. Elution was performed with 0.45–0.55 mg of the individual precursor dissolved in 500 µL ethanol. After evaporation of the solvent under reduced pressure, 100 µL tert-butanol/acetonitrile 4:1 were used to dissolve the residue. The mixture was heated at 70 °C for 15 min before fractions containing 100–200 MBq of the reaction mixture were diluted with 150 µL water/acetonitrile 3:1 and purified by preparative HPLC (LaChrom L7100, Merck, Darmstadt, Germany; Chromolith Performance RP18e 100 × 4.6 mm, Merck, Darmstadt, Germany; 0–30% acetonitrile in 10 min). Subsequently, the solvents were removed and the residue dissolved in 0.9% NaCl for animal studies.
Chelations of [18F]AlF for FAPI-42, -52 and -74 to -76 were performed according to the protocol of McBride et al. (2009). 2–10 GBq [18F]fluoride (ZAG Zyklotron AG, Karlsruhe, Germany) in 4 mL water were trapped on an anion exchange cartridge (Waters Accel Plus QMA Light cartridge, preconditioned with 5 mL 0.5 m NaOAc pH 3.9 and 10 mL of water) and eluted with 0.3 mL 0.5 m NaOAc pH 3.9. The solution was incubated with 6 µL of AlCl3 in water (10 mm) and 300 µL DMSO for 5 min at room temperature before 20 µL of the respective precursor (4 mm) was added. The reaction was carried out at 95 °C for 15 min, cooled to room temperature, diluted with 5 mL water and worked up by SPE (Waters Oasis HLB Plus Light cartridge). Subsequently, the solvents (1 mL of water/ethanol 1:1) were removed and the residue dissolved in 0.9% NaCl for animal studies.
In case of clinical application, the product was eluted (1 mL of water/ethanol 1:1) into a sterile 20 mL vial with the use of a sterile syringe filter. Subsequently, the filter was rinsed with 10 mL sterile 0.9% saline and 0.5 mL sterile phosphate buffer to dilute the preparation to less than 5% ethanol content. Finally, a reference sample was drawn from the final product, which was analyzed by means of radio-HPLC (product-peak area higher than 95%) and tested for neutral pH (pH 6–8).
Determination of logD values
All experiments for the determination of logD values were performed in triplicate. For the determination by means of radioactivity, around 5 × 106 cpm in ca. 1 µL of radioactive stock solutions (water/acetonitrile 1:1) were added to 100 µL phosphate buffered saline (PBS, pH 7.4) and 100 µL of 1-octanol. The mixture was vortexed for 1 min, followed by 5 min of centrifugation for phase separation. A sample of each phase (10 or 50 µL) was measured for radioactivity per volume (Packard Cobra II Autogamma, GMI Inc., USA). In case of FAPI-72 and -73, the reference compounds [19F]-FAPI-72 and -73 were dissolved in PBS at a concentration of 1 mg/mL. 50 µL of these solutions were each mixed with 50 µL 1-octanol and processed by vortexing and centrifugation as described above. 1 µL of each phase was analyzed by HPLC and the individual content of compound was determined by peak integration. The area under the peaks of the individual runs was used to calculate the log D value.
Serum stability assay
Five MBq of purified [18F]AlF-FAPI-74 (20 GBq/µmol) were dried under reduced pressure and the residue incubated in 300 µL human serum (Sigma Aldrich Germany) at 37 °C. After different time intervals (10 min to 4 h) 20 µL samples were precipitated with 40 µL acetonitrile. The stability of the labeled peptide was monitored by radio-HPLC of the supernatant. A gradient of 0% to 30% acetonitrile in 10 min was used for enhanced separation performance.
In vitro binding experiments
The binding properties of the FAPI derivatives were evaluated using the FAP‐transfected HT‐1080‐FAP and CD26 expressing HEK-CD26 cells. All cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at 37 °C/5% carbon dioxide. Cells were seeded in 6-well plates and cultivated to a final confluence of approximately 80–90% (1.2 to 2.0 × 106 cells/well). After washing with FCS free medium, for each well ca. 3 kBq of the radiolabeled compound (10–20 GBq/µmol for [18F]AlF-labeling) was added to the cells in FCS free medium. On completion of the individual incubation time, cells were washed with PBS and treated with 1.4 mL lysis buffer (0.3 m NaOH, 0.2% SDS) before being transferred to measuring tubes. Competition experiments were performed by co-incubation with the unlabeled precursor for 60 min. Radioactivity was determined in a Wizard Gamma Counter (Perkin Elmer), normalized to 106 cells and calculated as percentage of the applied dose (%AD). Each experiment was performed in triplicate.
Eight-week-old BALB/c nu/nu mice (Charles River) were subcutaneously inoculated with 5 × 106 HT-1080-FAP cells. The subcutaneous xenografts were grown at the flanks of the mice. Xenografts were grown to a tumor diameter of 10–15 mm. Mice were anesthetized using isoflurane inhalation. 5–10 MBq of the [18F]AlF-labeled compounds/ the respective tracer (FAPI-42/52/74/75/76; approx. 0.5 nmol; Am approximately 10–20 GBq/µmol) were injected intravenously for PET imaging. In vivo competition (blocking) experiments with 68 Ga-FAPI-74 (9 MBq; 18.2 GBq/µmol) were performed by adding 30 nmol of unlabeled FAPI-74 to the injection solution. Images were acquired using a small-animal PET scanner (Inveon, Siemens). Within the first 60 min p.i., a dynamic scan was performed in list mode, followed by a static scan from 120–140 min post injection. Images were reconstructed iteratively using the 3D-OSEM + MAP method (Siemens) and were converted to standardized uptake value (SUV). For the dynamic analysis, 28 frames were reconstructed. Quantification was based on ROI analysis and expressed as SUV.
To reduce radiation exposure, the organ distribution study was performed with 1 MBq per animal. The specific activity was lowered to provide the amount of precursor injected per animal being equal to the small animal PET experiments (approximately 0.5 nmol; Am approximately 2 GBq/µmol). The animals were sacrificed 30, 60, 120 and 240 min p.i., organs of interest dissected and weighted. The radioactivity was measured using a γ-counter (Packard Cobra II Autogamma, GMI Inc., USA) and expressed as percentage of injected dose per gram of tissue (%ID/g). PET imaging of [18F]AlF-FAPI-74 and -75 as well as the biodistribution were performed in triplicate.
[18F]AlF-FAPI-74 (16.2 GBq/µmol at time of injection) was applied intravenously (20 nmol; 323 MBq) to a 68 y old patient with NSCLC. The PET/CT scans were performed 1 and 3 h post tracer administration with a Biograph mCT Flow™ PET/CT-Scanner (Siemens Medical Solutions). The parameters used were slice thickness of 5 mm, increment of 3–4 mm, soft-tissue reconstruction kernel and care dose. After CT scanning, a whole-body PET was acquired in 3D (matrix 200 × 200) in FlowMotion™ with 0.7 cm/min. The emission data were corrected for random, scatter and decay. Reconstruction was conducted with an ordered subset expectation maximization (OSEM) algorithm with 2 iterations/21 subsets and Gauss-filtered to a transaxial resolution of 5 mm at full-width half-maximum. Attenuation correction was performed using the low-dose non-enhanced CT data. The quantitative assessment of standardized uptake values (SUV) was done using a region of interest technique.