⁶⁸ Ga-Labeled Peptides Targeting Oxytocin Receptor in Breast Cancer Using Linchpin Chemistry for Tandem Peptide Cyclization and Radiometal Chelator Incorporation

Breast cancer remains a leading cause of cancer-related death worldwide, partly due to disease heterogeneity and the lack of reliable biomarkers. The G protein-coupled oxytocin receptor (OTR) has emerged as a potential biomarker and therapeutic target in breast cancer, as its overexpression correlates with tumor growth and metastasis. OTR thus presents new opportunities for molecular imaging and targeted therapy in breast cancer. This study explores three novel ⁶⁸Ga-labeled peptides as potential OTR-specific imaging agents. Their preclinical evaluation includes in vitro assays and positron emission tomography (PET) studies in breast cancer models. The work also introduces the application of linchpin chemistry with <b>LP1</b> and <b>LP2</b> as a novel strategy for attaching bifunctional chelating agents. This tandem approach not only enables efficient peptide cyclization but also facilitates radiometal incorporation, representing a versatile platform for the design of next-generation radiopharmaceuticals. Binding studies using an aequorin-based assay in CHO cells expressing human OTR revealed the following EC₅₀ values: ^<b>nat</b><b>Ga-LP1-oxytocin</b> (376 nM), ^<b>nat</b><b>Ga-DOTA-Lys</b>^<b>8</b><b>-oxytocin</b> (1.38 nM), and ^<b>nat</b><b>Ga-LP2-oxytocin</b> (123 nM). Radiolabeling with ⁶⁸Ga was efficient and reproducible, consistently yielding high decay-corrected radiochemical yields of 52-74% and high radiochemical purity >98%. PET imaging demonstrated maximum MCF-7 tumor uptake for ^<b>68</b><b>Ga-LP1-oxytocin</b> (SUV_max 0.64 ± 0.10; <i>n</i> = 3) and ^<b>68</b><b>Ga-LP2-oxytocin</b> (SUV_max 0.64 ± 0.05; <i>n</i> = 7) at 10 min postinjection, whereas ^<b>68</b><b>Ga-DOTA-Lys</b>^<b>8</b><b>-oxytocin</b> reached comparable uptake (SUV_max 0.64 ± 0.12; <i>n</i> = 3) at 30 min. Notably, ^<b>68</b><b>Ga-LP2-oxytocin</b> showed superior background clearance and faster blood pool washout. Tumor uptake specificity was verified through competitive inhibition studies: predosing with oxytocin reduced tracer accumulation in a concentration-dependent manner at 10 min postinjection, with decreases of 33% at 50 μM and 68% at 300 μM, confirming selective OTR-mediated binding in vivo. Among the evaluated tracers, the novel ^<b>68</b><b>Ga-LP2-oxytocin</b> peptide demonstrated efficient radiolabeling, strong binding potency, and favorable in vivo characteristics, including uptake in estrogen receptor-positive MCF-7 tumors and superior background and clearance profiles. With further structural optimization, ^<b>68</b><b>Ga-LP2-oxytocin</b> holds promise as a PET radioligand for targeting OTR in breast cancer.