Noninvasive PET Imaging of CDK4/6 Activation in Breast Cancer

The cell cycle is a progression of 4 distinct phases (G1, S, G2, and M), with various cycle proteins being essential in regulating this process. We aimed to develop a radiolabeled cyclin-dependent kinase 4/6 (CDK4/6) inhibitor for breast cancer imaging. Our trans fluorinated analog (18F-CDKi) was evaluated and validated as a novel PET imaging agent to quantify CDK4/6 expression in estrogen receptor (ER) positive human epidermal growth factor receptor 2 (HER2) negative breast cancer. Methods: 18F-CDKi was synthesized and assayed against CDK4/6 kinases. F-18-CDKi was prepared with a 2 -step automated synthetic strategy that yielded the final product with remarkable purity and molar activity. In vitro and in vivo biologic specificity was assessed in a MCF-7 cell line and in mice bearing MCF-7 breast tumors. Nonradioactive palbociclib was used as a blocking agent to investigate the binding specificity and selectivity of F-18-CDKi. Results: F-18-CDKi was obtained with an overall radiochemical uncorrected yield of 15% and radiochemical purity higher than 98%. The total time from the start of synthesis to the final injectable formulated tracer is 70 min. The retention time reported for F-18-CDKi and F-19-CDKi is 27.4 min as demonstrated by coinjection with F-19-CDKi in a high-pressure liquid chromatograph. In vivo blood half-life (weighted, 7.03 min) and octanol/ water phase partition coefficient (1.91 0.24) showed a mainly lipophilic behavior. 18F-CDKi is stable in vitro and in vivo (>98% at 4 h after injection) and maintained its potent targeting affinity to CDK4/6. Cellular uptake experiments performed on the MCF-7 breast cancer cell line (ER -positive and HER2-negative) demonstrated specific uptake with a maximum intracellular concentration of about 65% as early as 10 min after incubation. The tracer uptake was reduced to less than 5% when cells were coincubated with a molar excess of palbociclib. In vivo imaging and ex vivo biodistribution of ER -positive, HER2-negative MCF-7 breast cancer models showed a specific uptake of approximately 4% injected dose/g of tumor (reduced to 0.3% with a 50 -fold excess of cold palbociclib). A comprehensive biodistribution analysis also revealed a significantly lower activation of CDK4/6 in nontargeting organs. Conclusion: 18F-CDKi represents the first F-18 PET CDK4/6 imaging agent and a promising imaging agent for ER-positive, HER2negative breast cancer.