Enantiopure bifunctional chelators for copper radiopharmaceuticals - Does chirality matter in radiotracer design?

It is well recognized that carbon chirality plays a critical role in the design of drug molecules. However, very little information is available regarding the effect of stereoisomerism of macrocyclic bifunctional chelators (BFC) on biological behaviors of the corresponding radiopharmaceuticals. To evaluate such effects, three enantiopure stereoisomers of a copper radiopharmaceutical BFC bearing two chiral carbon atoms were synthesized in forms of R,R-, S,S-, and R,S-. Their corresponding peptide conjugates were prepared by coupling with a model peptide sequence, c(RGDyK), which targets the α_vβ₃ integrin for in vitro and in vivo evaluation of their biological behaviors as compared to the racemic conjugate. Despite the chirality differences, all the conjugates showed a similar in vitro binding affinity profile to the α_vβ₃ integrin (106, 108, 85 and 100 nM for rac-H₂-1, RR-H₂-1, SS-H ₂-1, and RS-H₂-1 respectively with all p values > 0.05) and a similar level of in vivo tumor uptake (2.72 ± 0.45, 2.60 ± 0.52, 2.45 ± 0.48 and 2.88 ± 0.59 for rac-⁶⁴Cu-1, RR-⁶⁴Cu-1, SS-⁶⁴Cu-1, and RS-⁶⁴Cu-1 at 1 h p.i. respectively). Furthermore, they demonstrated a nearly identical biodistribution pattern in major organs (e.g. 2.07 ± 0.21, 2.13 ± 0.58, 1.70 ± 0.20 and 1.90 ± 0.46 %ID/g at 24 h p.i. in liver for rac-⁶⁴Cu-1, RR-⁶⁴Cu-1, SS-⁶⁴Cu-1, and RS- ⁶⁴Cu-1 respectively; 1.80 ± 0.46, 2.30 ± 1.49, 1.73 ± 0.31 and 2.23 ± 0.71 at 24 h p.i. in kidneys for rac- ⁶⁴Cu-1, RR-⁶⁴Cu-1, SS-⁶⁴Cu-1, and RS- ⁶⁴Cu-1 respectively). Therefore we conclude that the chirality of BFC plays a negligible role in α_vβ₃-targeted copper radiopharmaceuticals. However, we believe it is still worthwhile to consider the chirality effects of BFCs on other targeted imaging or therapeutic agents.