In vitro and in vivo evaluation of 64Cu-labeled somatostatin analogues conjugated with cross-bridged phosphonate-based chelators via strain-promoted click chemistry for PET imaging

Objectives Two CB-TE1A1P-inspired chelators with aza-dibenzocyclooctyne (DBCO) for strain-promoted click chemistry were developed. They were aimed to be labeled at relatively low temperature with high specific activity (S.A.), and to be conjugated with biomolecules bioorthogonally. In vitro and in vivo evaluation of the conjugates was performed.

Methods Chelators were synthesized and clicked with N3-Y3-TATE, forming CB-TE1A1P-DBCO-Y3-TATE (AP) and CB-TE1K1P-PEG4-DBCO-Y3-TATE (KP). In vitro and in vivo experiments were performed with the conjugates in an sstr2-transfected HCT116 human colorectal carcinoma-xenografted mouse tumor model.

Results The conjugates were synthesized via strain-promoted click chemistry at 37 °C for 30 min in high yields. The AP and KP were radiolabeled in > 95% yields at 70 °C and 37 °C, respectively in high S.A., and used without purification. 64Cu-labeled AP and KP showed rapid and receptor-mediated internalization, and bound to sstr2 in high affinity (Kd = 1.3 ± 0.3 for AP, 0.8 ± 0.3, for KP). The highest tumor uptake was observed at 1 h p.i. (8.9 and 10.3 %ID/g for AP and KP, respectively). The highest tumor to blood (T/B) and tumor to muscle (T/M) ratios for AP and KP were observed at 4 h p.i. (26±5, n = 4; 22 ± 3, n = 5). Although there was no significant difference in the T/B and T/M ratios at 4 and 24 h p.i. (p > 0.05), 64Cu-KP cleared more rapidly from kidney, liver, bone, and adrenals (p < 0.01).

Conclusions New clickable cross-bridged phosphonate-based chelators were synthesized, conjugated with N3-Y3-TATE in high yield and purity. Both conjugates were radiolabeled at relatively low temperature in high S.A., and demonstrated high affinity toward sstr2. Biodistribution studies showed high tumor uptake at 1 h; this uptake cleared by 24 h. 64Cu-KP demonstrated more optimal pharmacokinetics than 64Cu-AP, and will be investigated further.

Research Support This research was supported by NCI grants R01CA093375 and R01 CA064475.