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Radiolabeled derivatives of Tyr 3 -octreotide and Tyr 3 -octreotate, synthetic analogues of the peptide hormone somatostatin, can be used for positron emission tomography (PET) imaging of somatostatin receptor expression in neuroendocrine tumors. In this work, a squaramide ester derivative of desferrioxamine B (H 3 DFOSq) was used attach either Tyr 3 -octreotide or Tyr 3 -octreotate to the metal binding ligand to give H 3 DFOSq-TIDE and H 3 DFOSq-TATE. These new peptide-H 3 DFOSq conjugates form stable complexes with either of the positron-emitting radionuclides gallium-68 ( t 1/2 = 68 min) or zirconium-89 ( t 1/2 = 3.3 days). The new complexes were evaluated in an AR42J xenograft model that has endogenous expression of SSTR2. All four agents displayed good tumor uptake and produced high-quality PET images. For both radionuclides, the complexes formed with H 3 DFOSq-TATE performed better, with higher tumor uptake and retention than the complexes formed with H 3 DFOSq-TIDE. The versatile ligands presented here can be radiolabeled with either gallium-68 or zirconium-89 at room temperature. The long radioactive half-life of zirconium-89 makes distribution of pre-synthesized tracers produced to certified standards feasible and could increase the number of clinical centers that can perform diagnostic PET imaging of neuroendocrine tumors.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability, and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with (64)Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p-SCN-Bn-Oxo-DO3A, p-SCN-NOTA, and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A″-DTPA, and ITC-2B3M-DTPA), and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)sar where sar = sarcophagine = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37 °C, and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 ± 0.9 chelators per antibody molecule. Sar-CO2H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of (64)Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the (64)Cu-NOTA-rituximab conjugate demonstrated the highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to that of other conjugates, retained high labeling efficiency (98%) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6% dissociation of (64)Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. While all the macrocyclic bifunctional chelators are suitable for molecular imaging using (64)Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions, resulting in high specific activity.

Tumor-targeting peptides radiolabeled with positron-emitting (68)Ga are promising candidates as new noninvasive diagnostic agents for positron emission tomography (PET). The targeting peptides are tethered to a chelator that forms a stable coordination complex with Ga(3+) that is inert to dissociation of Ga(3+)in vivo. Metal complexes of macrobicyclic hexaamine "sarcophagine" (sar = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) ligands exhibit remarkable stability as a result of the encapsulating nature of the cage amine ligand. A Ga(3+) sarcophagine complex, [Ga-(1-NH(3)-8-NH(2)-sar)](4+), has been characterized using X-ray crystallography, demonstrating that Ga(3+) is coordinated to six nitrogen atoms in a distorted octahedral complex. A bifunctional derivative of (NH(2))(2)sar, possessing two aliphatic linkers with carboxylic acid functional groups has been attached to two cyclic-RGD peptides that target the α(v)β(3) integrin receptor that is overexpressed in some types of tumor tissue. This dimeric species can be radiolabeled with (68)Ga(3+) in >98% radiochemical yield and (68)Ga(3+) does not dissociate from the ligand in the presence of transferrin, an endogenous protein with high affinity for Ga(3+). Biodistribution and micro-PET imaging studies in tumor-bearing mice indicate that the tracer accumulates specifically in tumors with high integrin expression. The high tumor uptake is coupled with low nonspecific uptake and clearance predominantly through the kidneys resulting in high-quality PET images in animal models.