General procedures and instrumentation (nuclear magnetic resonance (NMR), electrospray mass spectroscopy (ESI-MS), ultrafiltration/diafiltration, PET) have been described before [13]. [18 F]fluoride formulation for injection was prepared by adding 100 MBq of 18 F (obtained from routine cyclotron production at Klinikum rechts der Isar, Technische Universität München, München, Germany) to phosphate buffered saline (PBS) (1 mL).
Synthesis of bisphosphonate conjugates (Figure 2): TRAP·2H2O (0.3 mmol, 185 mg), diisopropylethylamide (3 mmol, 388 mg, 510 μL), and the amino-bisphosphonate (1.5 mmol); for TRAP(MDP)3, tetraethyl(aminomethylene)bisphosphonate 455 mg; and for TRAP(PDP)3, tetraethyl(1-aminopropylene)bisphosphonate 500 mg, were dissolved in DMSO (2 mL). Then, HATU (2.4 mmol, 921 mg) was added with stirring. After 25 min, the reaction mixture was diluted with water (50 mL) and subjected to diafiltration (membrane with 500 Da MWCO). After 250 mL of water had passed, the cell content was concentrated in vacuo and subjected to preparative HPLC (column: YMC C18 ec 250 × 30 mm; detection wavelength, 220 nm; eluent A, MeCN with 0.1% TFA; eluent B, water with 0.1% TFA; gradient 25% to 50% B in 20 min, t
R(dodecaethyl-TRAP(MDP)3) = 12 min, t
R(dodecaethyl-TRAP(PDP)3) = 16 min). After evaporation of the solvents, the remaining viscous oil was dissolved in HBr/glacial acetic acid (33%) and stirred for 3 days. Addition of methanol to the reaction mixture yielded the products as colorless, crystalline solids. Data for TRAP(MDP)3: yield 201 mg (61%); MW (calculated for C21H51N6O27P9) 1,098.43; ESI-MS negative m/z = 1,097 (M-H+) and 548 (M-2 H+); 1 H NMR (600 MHz, D2O) δ = 2.13 (m, 6 H), 2.67 (m, 6 H), 3.48 (d, 3
J
HH = 5.4 Hz, 6 H), 3.56 (s, broad, 12 H), and 4.71 (t, J
PH = 21.3 Hz, 3 H) ppm; 13 C NMR (151 MHz, D2O) δ = 26.11 (d, 1
J
PC = 93.18 Hz), 28.65, 52.13, 54.74 (d, 1
J
PC = 89.07 Hz), 47.45 (t, 1
J
PC = 139.28 Hz), and 174.54 (dt, 2
J
PC = 12.28 and 3
J
PC = 4.34 Hz) ppm; and 31P NMR (121 MHz, D2O) δ = 14.10 (d, 2
J
PP = 15.7 Hz) and 39.90 ppm. Data for TRAP(PDP)3: yield 195 mg (55%); MW (calculated for C27H63N6O27P9) 1,182.59; ESI-MS negative m/z = 1,181 (M-H+), 590 (M-2 H+), and 393 (M-3 H+); 1 H NMR (600 MHz, D2O) δ = 2.07 (m, 6 H), 2.10 (m, 6 H), 2.36 (tt, J
PH = 23.52 Hz, 3
J
HH = 5.97 Hz, 3 H), 2.53 (m, 6 H), 3.44 (t, 3
J
HH = 6.3 Hz, 6 H), 3.45 (t, broad, 3
J
HH = 6.6 Hz), and 3.52 (s, broad, 12 H) ppm; 13 C NMR (151 MHz, D2O) δ = 25.36 (t, 2
J
PC = 4.2 Hz), 26.29 (d, 1
J
PC = 93.5 Hz), 28.63 (d, 2
J
PC = 3.9 Hz), 35.77 (t, 1
J
PC = 128.5 Hz), 39.48 (d, 3
J
PC = 7.4 Hz), 52.14, 54.82 (d, 1
J
PC = 88.6 Hz), and 175.41 (d, 3
J
PC = 13.1 Hz) ppm; and 31P NMR (121 MHz, D2O) δ = 21.53 (d, 2
J
PP = 15.5 Hz) and 39.69 ppm.
68 Ga for labeling was obtained from a SnO2-based 68Ge/68 Ga generator (iThemba LABS, Somerset West, South Africa), eluted with 1.0 M HCl. A 1.25 mL fraction of the eluate containing ca. 80% of the total activity (ca. 1.3 GBq) was adjusted to pH 3.3 by adding a solution of 600 mg 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) in 0.5 mL water. To a 90 μL aliquot of this mixture, 10 μL of 10-4 M stock solution of the ligand was added. After heating for 5 min to 95°C, the solution was passed over a cation exchanger SPE cartridge (Chromafix HR-XC M, Macherey-Nagel, Düren, Germany) and purged with water (1 mL). This procedure removed non-complexed 68 Ga as well as HEPES, which was confirmed by processing of blank samples. Radiochemical yields, determined by measuring the activity on the cartridge and in the eluate, were > 85%. Formulation was done by adjusting the pH of the eluate to 7.4 by adding approximately 50 μL of a solution of NaOH (1 g) and NaH2PO4 (483 mg) in water (20 mL) while monitoring the pH with a pH meter. 'Free' 68 Ga formulation was prepared by addition of the generator eluate (40 μL, ca. 50 MBq) to PBS (1 mL), resulting in pH 7.2.
All animal experiments were carried out in accordance with the current animal welfare regulations in Germany. Five male Lewis rats (age 7 weeks, ca. 200 g) were kept under standard laboratory conditions (12 h light/12 h dark) and given standard diet and water ad libitum. For PET, ca. 35 MBq of tracer was injected into the tail vein under isoflurane anesthesia. Two subsequent scans of 15 min were recorded 60 min post injection, using two different axial bed positions in order to image the entire animals. Images were reconstructed using a OSEM3D algorithm without scatter and attenuation correction. For each full-body maximum intensity projection (MIP), two part-body MIPs were stitched together manually using graphics software. PET images are from representative animals reflecting the group.