Macrocycle Chelator Conjugation on the C3 Hydroxyl Group of 17β-Estradiol reduces Estrogen Receptor Binding

Tumor heterogeneity in breast cancer complicates accurate molecular characterization when using standard-of-care imaging modalities. In this study, gallium-68 labeled estradiol-derivatives were synthesized and evaluated as candidate tracers to be used for multiplexed PET imaging with the goal of improving the molecular characterization of breast cancer.

17β-estradiol was conjugated to DOTA and NODAGA macrocycles and radiolabeled with gallium-68. The selectivity of the resulting radiotracers, [⁶⁸ Ga]Ga-DOTA-estradiol and [⁶⁸ Ga]Ga-NODAGA-estradiol, were assessed in vitro using recombinant human estrogen receptor (ER) protein and cell lines. In vivo PET/CT imaging was performed in mice bearing ER-positive and ER-negative tumors using [⁶⁸Ga]Ga-NODAGA-estradiol.

Both radiotracers were obtained with high radiochemical purity. Neither [⁶⁸ Ga]Ga-DOTA-estradiol nor [⁶⁸ Ga]Ga-NODAGA-estradiol displayed selective binding to the recombinant human ER and no uptake difference could be identified between ER-positive and ER-negative cells. Competition blocking studies using non-radioactive [^NatGa]Ga-DOTA-estradiol and [^NatGa]Ga-NODAGA-estradiol significantly decreased the uptake of [¹⁸F]FES within ER-positive cells. In vivo imaging of [⁶⁸ Ga]Ga-NODAGA-estradiol revealed negligible tumor uptake and predominant hepatobiliary clearance.

Two estradiol-derived radiotracers were successfully synthesized and tested, however neither showed selective binding towards ER in vitro or in vivo. The conjugation site of the cyclic macrocycle on 17β-estradiol is likely a key parameter to retain strong binding towards ER. These findings identify an important limitation for chelator-based ER tracer design.