Zirconium tetraazamacrocycle complexes display extraordinary stability and provide a new strategy for zirconium-89-based radiopharmaceutical development

89Zr–Tetraazamacrocycle complexes display extraordinary stability.

In addition, radio-TLC was conducted on a Bioscan AR 2000 radio-TLC scanner equipped with a 10% methane:argon gas supply and a PC interface running Winscan v.3 analysis software (Eckert & Ziegler, Berlin, DE). Varian ITLC-SA strips and Merck C-18 TLC plates were employed using a 0.1 M EDTA (pH 5) and 1:1 MeOH:10% NH 4 Cl solution as eluents respectively, and 89 Zr-Oxalate or 89 ZrCl 4 as a standard control.
Radioactive samples were counted using a Perkin Elmer 2480 Wizard ® gamma counter (Waltham, MA) with an energy window of 500-1500 keV. PET and CT images were acquired using a GE eXplore Vista small animal PET/CT scanner (Waukesha, WI). in 40 mL of methanol. The resulting solution was refluxed for 3 h. As the reaction proceeded, a white precipitate formed. It was filtered, washed with MeOH (2 X 10 mL), and dried in an oven (604 mg, 94% yield).

Experimental Details -Crystallography
Data Collection and Structure Solution. A clear colorless needle-like specimen of C 16 H 32.72 N 4 O 12.36 Zr, approximate dimensions 0.070 mm x 0.080 mm x 0.420 mm, was used for X-ray crystallographic analysis. The X-ray intensity data were measured on a Bruker APEX CCD system equipped with a graphite monochromator and a Mo Kα sealed x-ray tube (λ = 0.71073 Å). X-rays were provided by a fine-focus sealed x-ray tube operated at 50kV and 30mA. , are rotated about the c axis by ~45˚ with respect to each other. The final model includes 6 "partial" occupancy water molecules for which 2 oxygens were refined anisotropically and 4 oxygens were refined isotropically; only 3 water hydrogens were included in the structural model. The final structural model incorporated isotropic thermal parameters for all included hydrogen atoms. The hydrogen atoms of the DOTA ligand were included in the structural model as fixed atoms (using idealized sp 3 -hybridized geometry and a C-H bond length of 0.99 Å) "riding" on their respective carbon atoms. The isotropic thermal parameters for all included hydrogen atoms were fixed at values 1.2 times the equivalent isotropic thermal parameter of the oxygen or carbon atom to which they are covalently bonded. The final anisotropic/isotropic full-matrix least-squares refinement on F 2 with 240 variables converged at R 1 = 4.32%, for the observed data and wR 2 = 11.19% for all data. The goodness-of-fit was 1.067. The largest peak in the final difference electron density synthesis was 0.863 e -/Å 3 and the largest hole was -0.816 e -/Å 3 with an RMS deviation of 0.090 e -/Å 3 . Based on the final model, the calculated density was 1.695 g/cm 3 and F(000), 1182 e -.

Refinement Details
Details of crystal data, data collection, and structure refinement are summarized in the tables below.

Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles, and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

Least-squares planes (x,y,z in crystal coordinates
The identity of the radioactive complex 89 Zr-DOTP was further confirmed by comparing its radio-HPLC elution profile to the UV-HPLC spectrum of nonradioactive Nat Zr-DOTP (Fig. S28). (10 µL, 1.0 mg/mL in water) of the ligand DOTAM with an aliquot of 89 ZrCl 4 (1.1 mCi, 40.7 MBq) diluted in 100 µL of 1 M HEPES (pH 7.2) and 10 µL of 1 % sodium dodecyl sulfate followed by 45 min incubation at 90ºC in a thermomixer (550 rpm). Formation of the 89 Zr-DOTAM complex was monitored by radio-TLC using Varian ITLC-SA strips and 0.1 M oxalic acid (pH 5) as the mobile phase. In this system, free 89 Zr formed a complex with oxalic acid and eluted with the solvent front (R f = 1), while the 89 Zr-DOTAM complex remained at the origin (R f = 0) (Fig. S29).  In vitro EDTA challenge study. In vitro EDTA challenge study was carried out by adding 10 µL of each 89 Zrlabeled complex (70 μCi, 2.59 MBq) to 500 µL of EDTA (10 mM, 50 mM and 100 mM: pH 5 and pH 7) with a 1:100, 1:500, and 1:1000 ratio of ligand/EDTA. The solutions (n=4) were incubated at 37 °C for 7 days in a thermomixer. Samples were analyzed at 30 min, 1 h, 3 h, 6 h, 1 d, 3 d, 5 d and 7 d post administration to EDTA by radio-TLC using Varian ITLC-SA strips and 0.1 M EDTA (pH 5) as the mobile phase and gamma counting using an energy window of 500-1500 keV and standard protocols .  were analyzed daily for 1 week by radio -TLC using Varian ITLC-SA strips and 0.1 M EDTA (pH 5) as the mobile phase and gamma counting using an energy window of 500-1500 keV and standard protocols . Serum samples were also analyzed after 7 days by size exclusion chromatography (SEC) using a Superdex 200   When samples of serum which contained unchelated 89 Zr were analyzed using radio-ITLC, we observed broad peaks ranging form the origin to the solvent front because unchelated 89 Zr could be bound to variety of serum protein components and migrate with different retention factors in our ITLC system. Therefore we elected to perform size exclusion chromatography to further characterize the stability of 89 Zr-complexes. Figure S35. In vitro serum stability by HPLC. UV-SE-HPLC (220 nm, black, and green) and radio-SE-HPLC chromatogram ( Table S10. Summary of in vitro serum stability of 89 Zr-complexes in human serum at 37 C for 7 days determined by adding 5 µL of each 89 Zr-labeled complex (approx. 5 µCi; 0.19 MBq) to a mixture of 500 μL of octanol and 500 μL of water. The resulting solutions (n = 5) were vigorously vortexed for 5 min at room temperature, then centrifuged for 5 min to ensure complete separation of layers. From each of the five sets, 50 μL aliquot was removed from each phase into screw tubes and counted separately in a gamma counter. Each organic phase was washed with water to remove any radioactivity remaining in the organic phase before gamma counting. The partition coefficient was calculated as a ratio of counts in the octanol fraction to counts in the water fraction. The logP values were reported in an average of five measurements.    Despite the elevated bone uptake associated with 89 Zr-DOTP, it demonstrates better clearance from the liver and kidney than 89 Zr-DFO. 89 Zr-DOTA demonstrated a superior clearance and excretion pattern when compared to all complexes studied and suggests this complex exhibits extraordinary stability in vivo.
PET/CT Imaging. PET imaging with 89 Zr-DFO or 89 Zr-DOTA was performed on healthy female NIH Swiss mice (6-8 wk old, n=6) using a small animal PET/CT scanner (eXplore VISTA model, GE Healthcare, Waukesha, WI). Before imaging, each mouse was anesthetized by inhalation of isoflurane mixed with oxygen gas (3% isoflurane for induction and 1-2% for maintenance) and a tail vein catheter was placed on the tail. The mouse was then positioned in the imaging cradle and a CT scan was performed for the subsequent attenuation correction and anatomical colocalization. After the CT scan, 89 Zr-DFO or 89 Zr-DOTA  µCi) in 100 µL saline/ mouse) was administered through the tail vein catheter, and a dynamic PET scan was simultaneously initiated and acquired for 1 hour to record the initial distribution and pharmacokinetics. Mice were re-anesthetized at 2, 4 and 24 hours, and a static PET scan was acquired for 20 min as late-phase scans. PET images were reconstructed using 2D ordered subset expectation maximization (2D-OSEM) algorithms with random, scatter, and attenuation corrections and then coregistered with the CT image. The PET system was calibrated using a Zr-89 phantom with known activity. Standard uptake value (SUV = [(nCi/mL) x (animal wt. (g))/ injected dose (nCi)]) and the measure of %ID/g was then calculated voxel-wise with the calibration factor and normalization to the injected dose and animal body weight. For dynamic scans, data were binned into 19 time frames with the following schemes: 6x10s, 2x30s, 3x60s, 5x5min, 3x10min. The tissue uptake curves were generated from the dynamic scans with regions of interest (ROIs) manually placed in heart, liver, kidney, bone, and muscle. Biodistribution data at 2, 4 and 24 hours were obtained from the PET images, with regions of interest placed in corresponding organs or tissues.  Table S16. Image-based biodistribution in selected tissues (mean %ID/g ± SD) of normal mice (n =6/cohort) receiving 89 Zr-DFO or 89 Zr-DOTA