New 64Cu-labeled CXCR4-targeting ligands for theranostic applications

Introduction: Theranostics is a stratified approach to medicine allowing selection of patients most likely to respond to therapy and monitoring of patient treatment and response, which ultimately aims to improve patient outcomes. This approach combines a diagnostic imaging modality like positron emission tomography (PET) and endoradiotherapy, the use of molecular targeted radiopharmaceuticals to deliver cytotoxic radiation to tumor tissues. Targeted Copper Theranostics (TCTs) using the radionuclides of copper, ⁶⁴Cu (t_1/2 = 12.7 hours, β⁺ = 17.4%) for imaging and ⁶⁷Cu (t_1/2 = 61.9 h, β^- = 100%, E_max = 562 keV) for therapy, offer significant advantages in the development of next generation theranostics including: (1) centralized production and broad distribution of ready-to-use TCTs for either diagnosis or therapy due to the sufficient physical half-lives; (2) Scalable product supply for ⁶⁴Cu and ⁶⁷Cu due to favorable production methods using cyclotrons and accelerators, respectively; (3) ⁶⁴Cu can be imaged on the same day (as with current patient scheduling) but also offers the ability to collect multiple images from one hour to 48 hours for flexible patient scheduling; and (4) since ⁶⁴Cu and ⁶⁷Cu are a “true” matched pair, the radiochemical products will have the same in vitro and in vivo behavior. A theranostic agent can be developed using the sarcophagine (SAR) chelator, an ideal choice as they complex copper with fast kinetics, and form extremely stable complexes resistant to transmetalation and metabolism in vivo. To demonstrate the utility of the ⁶⁴Cu/⁶⁷Cu radionuclide pair in oncology theranostics, two ⁶⁴Cu-labeled CXCR4-targeting SAR constructs, [⁶⁴Cu]Cu-SAR-CXCR4 and its PEGylated analogue [⁶⁴Cu]Cu-SAR-PEG-CXCR4, were developed for preclinical PET imaging of CXCR4. In this case, PEGylation was utilized as a strategy to improve the pharmacokinetics of the radiotracer. These radiopharmaceuticals can be translated into ⁶⁷Cu-labeled therapeutics.

Methods: Two constructs were synthesized containing the high affinity CXCR4-targeting pentixather scaffold and the sarcophagine-derived chelator, MeCOSar. Cell binding and internalization assays were performed in SK-BR-3 adenocarcinoma cells. The metabolic and transmetalation stabilities of the tracers were evaluated in mouse serum and excess EDTA respectively, and lipophilicity determined via shake-flask assays. Small-animal PET-CT and biodistribution studies at different time-points were performed in xenografts of Daudi lymphoma in mice.

Results: The radiotracers were efficiently radiolabeled at 37 ºC with high radiochemical yields (>95%) and radiochemical purities (>98%). Both tracers demonstrated high metabolic stability in serum, with high resistance towards transchelation via EDTA challenge assays. [⁶⁴Cu]Cu-SAR-CXCR4 displayed a more efficient CXCR4-binding compared to the PEG- tracer in vitro. Interestingly, both tracers showed similar cell internalization profiles over 4 h, independent of the lipophilicities. PET-CT imaging and biodistribution study showed a higher non-specific accumulation of [⁶⁴Cu]Cu-SAR-CXCR4 in the liver and spleen compared to [⁶⁴Cu]Cu-SAR-PEG-CXCR4, which could be attributed to its enhanced lipophilicity. Although both compounds showed tumor targeting over 24 h p.i., [⁶⁴Cu]Cu-SAR-CXCR4 demonstrated retention in the tumors over 24 hours (6.1 ± 2.7% ID/g at 1 h p.i., 7.0 ± 0.7% ID/g at 24 h p.i.), leading to comparatively higher tumor-to-organ ratios at all time-points.

Conclusions: We have successfully prepared two CXCR4-targeting radiotracers showing efficient radiolabeling with ⁶⁴Cu and high stabilities in serum and with EDTA challenge. The compounds showed tumor targeting and retention resulting in high resolution PET-CT images, and may make promising tracers for future application in endoradiotherapy with ⁶⁷Cu.