Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors

Abstract

Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors.

Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed.

Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h.

Conclusion: The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor-positive tumors.

FIGURE 1

Structure of unlabeled peptide chelator conjugate CB-TE2A-sst₂-ANT.

FIGURE 2

In vitro binding affinity of ⁶⁴Cu-CB-TE2A-sst₂-ANT for SSTR harvested from AR42J rat pancreatic tumor cell membranes was determined using saturation binding assay. K_d of radiopharmaceutical was determined to be 26 ± 2.4 nM, and B_max was determined to be 23,000 fmol/mg.

FIGURE 3

Results of internalization studies performed with ⁶⁴Cu-CB-TE2A-Y3-TATE (■) and ⁶⁴Cu-CB-TE2A-sst₂-ANT (▲) using SSTR-positive AR42J pancreatic tumor cells. Blocking studies were also performed using cold Y3-TATE (□) or cold sst₂-ANT (∆), respectively. (A) Internalization results demonstrate that ⁶⁴Cu-CB-TE2A-Y3-TATE is internalized with greater efficiency than is ⁶⁴Cu-CB-TE2A-sst₂-ANT. Addition of blockade at each time point significantly reduces amount of radiopharmaceutical internalized and indicates that internalization is receptor-mediated process. (B) Amount of surface-bound activity removed from cells at all time points was also greater for ⁶⁴Cu-CB-TE2A-sst₂-ANT (▲) than for ⁶⁴Cu-CB-TE2A-Y3-TATE (■), indicating that less ⁶⁴Cu-CB-TE2A-sst₂-ANT was internalized over time course experiment. CPM = counts per minute.

FIGURE 4

Biodistribution comparing clearance properties of ⁶⁴Cu-CB-TE2A-sst₂-ANT (▲) and ⁶⁴Cu-CB-TE2A-Y3-TATE (■) from blood (A), liver (B), kidney (C), tumor (D), adrenal glands (E), and pituitary gland (F). All data (n = 5; bars, ±SE) were decay-corrected. Note differences in y-axis scales. %ID = percentage injected dose.

FIGURE 5

Tumor and SSTR-positive tissue labeling at 4 h after injection using ⁶⁴Cu-CB-TE2A-sst₂-ANT without blockade and at 4 h after injection when coinjected with sst₂-ANT as blocking agent. Decrease in affinity by radiopharmaceutical for tumor and SSTR-positive tissues is evident, suggesting that interaction of radiotracer with these tissues is receptor-mediated process. %ID = percentage injected dose.

FIGURE 6

(A) Representative small-animal PET image at 4 h of rat injected with ⁶⁴Cu-CB-TE2A-sst₂-ANT. Left image is representative slice from small-animal PET/CT fusion image and right image is small-animal PET projection view of same animal. Calculated SUV for the tumor in left hind limb was determined to be 2.7 and SUV for tumor in right hind limb was determined to be 2.8. (B) Representative small-animal PET image at 4 h of rat injected with ⁶⁴Cu-CB-TE2A-sst₂-ANT and sst₂-ANT as blocking agent. Left image is representative slice from small-animal PET/CT fusion image and right image is small-animal PET projection view of same animal. In animal receiving blockade, SUV for tumor in left hind limb was calculated to be 0.74 and SUV for tumor in right hind limb was calculated to be 0.51. (C) Graphical plot of change in average SUV over time. Even after 24 h, SUV remains high, suggesting enhanced binding of ⁶⁴Cu-CB-TE2A-sst₂-ANT for SST₂ receptor. (D) Graphical representation that demonstrates change in observed SUV when excess cold sst₂-ANT is coinjected with radiopharmaceutical, indicating that binding of radiopharmaceutical to SSTR-positive tumor is receptor-mediated.