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 transfluorinated 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. 18F-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 18F-CDKi. Results:18F-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 18F-CDKi and 19F-CDKi is 27.4 min as demonstrated by coinjection with 19F-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 18F PET CDK4/6 imaging agent and a promising imaging agent for ER-positive, HER2-negative breast cancer.

Protein-Engineered Nanoscale Micelles for Dynamic 19F Magnetic Resonance and Therapeutic Drug Delivery.

Engineered proteins provide an interesting template for designing fluorine-19 (19F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed "fluorinated thermoresponsive assembled protein" (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting 19F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation ( T2) times and unaffected spin-lattice relaxation ( T1) times. The nearly unchanging T1 relaxation rates and linearly dependent T2 relaxation rates have allowed for detection via zero echo time 19F MRI, and the in vivo MR potential has been preliminarily explored using 19F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive 19F MRI/MRS-traceable theranostic agents.