Copper-64 labeled somatostatin analogs as imaging and therapeutic agents

The goal of this research is to develop 64Cu radiolabeled somatostatin-receptor ligands for position emission tomography (PET) imaging and radiotherapy of cancer. Copper-64 radiopharmaceuticals have been used as PET imaging agents for various diseases because of the favorable decay characteristics of 64Cu (t1/2 = 12.7 h; beta + (17.8%); beta- (38.4%)). Additionally, 64Cu radiopharmaceuticals have demonstrated tumor growth inhibition with a relatively low radiation dose. Many common tumors, such as those of the lung and pancreas, are not controlled by current cancer treatment and the efficacy is reduced because of severe cytotoxic effects. As a result, radiolabeled somatostatin analogs which have high affinity for somatostatin receptor subtype 2 (SSTr2) have been investigated to control neuroendocrine tumors. The goals of this project are 1) to determine the role of p53 in the delivery of 64Cu to tumor cell nuclei from 64Cu-labeled somatostatin agonists and an antagonist; 2) to compare the DOTA vs. CB-TE2A chelator-conjugates of the somatostatin agonist Y3-TATE with respect to release of 64Cu in tumor cells and subsequent localization in the cell nucleus; and 3) to perform in vivo imaging of three 64Cu-labeled somatostatin analogs in HCT116 +/+ and -/- tumor-bearing mice.;HCT116 colorectal cancer cell clones that were either positive (HCT116+/+) or negative (HCT116-/-) for the tumor suppressor protein p53 were prepared in the laboratory of Prof. Buck Rogers (Radiation Oncology) and screened for somatostatin receptor subtype 2 expression by flow cytometry (FACS) analysis. Clones with the highest expression of SSTr2 from 42 p53 +/+ clones and 35 clones of HCT116 p53 -/- were chosen for subsequent experiments. Receptor binding, internalization, nuclear localization, microPET, and biodistribution studies were performed with 64Cu-labeled somatostatin agonists (64Cu-CB-TE2A-Y3-TATE and 64Cu-DOTA-Y3-TATE) and an antagonist (64Cu-CB-TE2A-sst2-ANT) in SSTr2-transfected HCT116+/+l and HCT116-/- cells. Two different 64Cu agonist chelators with different chelate stability, CB-TE2A and DOTA, were evaluated. 64Cu-CB-TE2A-Y3-TATE showed similar binding affinity and 64 Cu nuclear localization, but faster internalization kinetics and improved in vivo stability compared to 64Cu-DOTA-Y3-TATE. As a result, 64Cu-CB-TE2A-Y3-TATE is the better choice for PET imaging and therapeutic agent. The somatostatin antagonist, 64Cu-CBTE2A- sst2-ANT, demonstrated binding to 8 to 9-fold more SSTr2 sites than the agonists, but showed lower levels of internalization and 64Cu nuclear localization. 64Cu-labeled SSTr2 antagonist showed similar tumor uptake but higher non-specific organs uptake compared to its agonist and therefore is not an improved PET imaging agent. Additionally, higher 64Cu nuclear localization was observed with the two somatostatin agonists (64Cu-DOTA-Y3-TATE and 64Cu-CB-TE2A-Y3-TATE) in the p53 positive cell line compared to the p53 negative cell line, indicating that the tumor suppressor protein p53 played a role in 64Cu trafficking into tumor cell nuclei. However, there was no difference in 64Cu nuclear localization of the antagonist 64Cu-CB-TE2A-sst2-ANT between p53 postive cell line and p53 negative cell lines, suggesting that small structural modifications of the somatostatin analogs might affect nuclear localization of 64 Cu.;Receptor-targeted 64Cu-radiolabeled ligands are promising imaging and radiotherapeutic agents. Our studies investigating the intracellular trafficking of 64Cu provide vital information in the further development of these agents.