Development of Novel PSMA Ligands for Imaging and Therapy with Copper Isotopes.

Prostate-specific membrane antigen (PSMA)-binding tracers have been shown to be promising agents for the specific targeting of prostate tumors. Upon labeling with the short-lived isotopes 18F and 68Ga, excellent molecular imaging performance is achieved. This potential could be further exploited by using long-lived isotopes. Due to the favorable half-life of 64Cu, tracers labeled with this PET nuclide could solve logistic problems. Moreover, this isotope provides a theranostic pair with the therapeutic copper isotope 67Cu. Hence, novel tracers that combine dedicated copper chelators with the PSMA-specific urea-based binding motif were developed.
Methods: The precursors were obtained by solid-phase synthesis. The purity and molecular weight of the nine novel PSMA ligands were confirmed by HPLC and LC-MS. The compounds were labeled with 64Cu; the labeling resulted in radiolabeling yields > 99%. Competitive cell binding assays and internalization assays were performed with C4-2 cells, a subline of the PSMA positive cell line LNCaP (human lymph node carcinoma of the prostate). In vitro serum stability, stability of 64Cu-CA003 in the blood and the in vivo fate of neat 64Cu-chloride or 64Cu-CA003 were determined in order to prove whether the stability of the radiolabeled compounds are sufficient to ensure that there is no significant loss of copper during the targeting process. For PET imaging and biodistribution studies a C4-2 tumor-bearing mouse model was used.
Results: The radiolabeled 64Cu-PSMA ligands showed high serum stability. All PSMA ligands showed high inhibition potencies with [Ki] (equilibrium inhibition constants) values in the low nanomolar range. The compounds 64Cu-CA003 and 64Cu-CA005 showed high internalization ratios (34.6% ± 2.8 and 18.6% ± 4.4 respectively). Both the in vitro serum stability determination and the in vivo characterization of the main radiolabeled compounds confirm that except for 64Cu-PSMA-617 all compounds show high serum stability within the observation period of 24 h. Small animal PET imaging demonstrated high tumor uptake within 20 min. Organ distribution studies confirmed the high specific uptake in the tumor with 30.8 ± 12.6 %ID/g at 1 h post injection. Rapid clearance from the kidneys was observed: a decrease from 67.0 ± 20.9 %ID/g at 1 h post injection to 7.5 ± 8.51 %ID/g at 24 h after injection (in the case of CA003). The performance of CA003, the compound with the best preclinical properties, was assessed in a first patient. In line with its preclinical data, PET imaging resulted in a clear visualization of the cancer lesions with a high contrast.
Conclusion: The novel 64Cu-labeled PSMA ligands are promising agents to target PSMA and visualize PSMA positive tumor lesions as shown in the preclinical evaluation by small-animal PET studies, organ distribution and a patient application. Most importantly, the images obtained at 20 h enabled the delineation of unclear lesions - this shows that the compounds fulfill the prerequisite for dosimetry in the course of therapy planning with 67Cu. Thus, we suggest clinical use of copper labeled PSMA-CA003 for diagnostics and radiotherapy of prostate cancer.