Breast cancer remains a leading cause of cancer-related death worldwide, with tumor heterogeneity and absent biomarkers limiting diagnostic and therapeutic options. While PET tracers targeting estrogen receptor and HER2 have proven valuable, many breast cancers lacking these markers remain without suitable imaging agents. The oxytocin receptor, a G protein-coupled receptor classically associated with reproductive and social functions, has emerged as a potential biomarker in breast cancer. Elevated expression at protein or mRNA levels appears across multiple breast cancer subtypes, correlating with tumor growth and metastasis. Developing radiolabeled peptides capable of visualizing oxytocin receptor expression could enable noninvasive tumor profiling and guide patient selection for receptor-directed therapies.
Researchers in the Wuest Group at the University of Alberta, published in Bioconjugate Chemistry, developed three novel 68Ga-labeled oxytocin peptides as potential PET imaging agents for breast cancer. The work introduced linchpin chemistry as a strategy for simultaneous peptide cyclization and radiometal chelator incorporation, streamlining synthesis compared to conventional sequential approaches. Two linchpin reagents, LP1 and LP2, were designed to react selectively with the cysteine residues at positions 1 and 6 of oxytocin, forming stable macrocycles while installing chelating moieties for gallium-68 complexation. LP1 featured a DOTAGA-derived scaffold with dibromobenzyl functionality, while LP2 employed a dibromomaleimide group that undergoes nucleophilic addition with thiol side chains. For comparison, DOTA-Lys8-oxytocin was prepared through traditional two-step chelator conjugation followed by disulfide cyclization.
Tissue microarray analysis of 176 breast cancer patient samples confirmed consistently elevated oxytocin receptor mRNA levels compared to healthy controls, validating the receptor as a molecular target. Immunohistochemistry revealed stronger receptor staining in estrogen receptor-positive MCF-7 xenografts than in triple-negative MDA-MB-231 tumors. Binding studies using an aequorin-based calcium release assay in cells expressing human oxytocin receptor yielded EC50 values of 1.38 nM for natGa-DOTA-Lys8-oxytocin, 123 nM for natGa-LP2-oxytocin, and 376 nM for natGa-LP1-oxytocin. The reduced affinity of linchpin-cyclized peptides likely reflects conformational constraints introduced by the alternative ring geometry. Radiolabeling with generator-produced gallium-68 proceeded efficiently in HEPES buffer at 99°C, achieving radiochemical purities exceeding 98% and decay-corrected yields of 52–74% within 10 minutes. All three tracers displayed highly hydrophilic character with logD values between −3.6 and −4.2, predicting favorable renal clearance.
Dynamic PET imaging in MCF-7 tumor-bearing mice revealed that 68Ga-LP1-oxytocin and 68Ga-LP2-oxytocin reached maximum tumor uptake at 10 minutes post-injection with SUVmax values of 0.64, while 68Ga-DOTA-Lys8-oxytocin achieved comparable uptake only after 30 minutes. The linchpin-cyclized peptides demonstrated superior background clearance and faster blood pool washout. Blocking experiments confirmed receptor-mediated uptake: predosing with oxytocin reduced 68Ga-LP2-oxytocin tumor accumulation by 33% at 50 μg and 68% at 300 μg in a concentration-dependent manner. Metabolic stability varied substantially among the tracers. 68Ga-LP1-oxytocin retained greater than 50% intact peptide at 60 minutes, while 68Ga-LP2-oxytocin degraded rapidly to approximately 5% by the same timepoint, likely due to maleimide instability in the presence of endogenous thiols. Despite this difference, both linchpin peptides achieved equivalent tumor uptake, suggesting that rapid initial delivery compensates for reduced metabolic stability. These findings establish linchpin chemistry as a versatile platform for radiopharmaceutical development, enabling single-step cyclization and chelator installation while offering tunable stability and pharmacokinetic profiles for receptor-targeted imaging applications.
