New fluorine-18 radiolabeled Cdk4/6 inhibitors: potential radiotracers for tumor imaging by positron emission tomography.

Background
Cyclin-dependent kinases 4 and 6 (Cdk4/6) are important components of cell cycle activation in G1 phase and play critical roles in dysfunction of growth control during cancerogenesis. The aim of our study was the evaluation of new fluorine containing pyrido[2,3 d]pyrimidin-7-one derivatives (CKIC, CKID and CKIE) concerning their efficacy and suitability as Cdk4/6 inhibitors and, after fluorine-18 radiolabeling, as radiotracers for imaging of tumors by positron emission tomography (PET).

Materials and methods
Small molecule inhibitors CKIC, CKID and CKIE were analyzed concerning their biological and radiopharmacological properties in human tumor cell lines HT-29, FaDu and THP-1. Cell cycle distribution of cells was determined by flow cytometry DNA analysis and effects on cell growth were measured. Phosphorylation of retinoblastoma protein (pRb) at Ser780 was analyzed by Western blotting. mRNA expression of the pRb affected genes E2F-1 and PCNA was measured with quantitative RT-PCR. Stability and radiotracer uptake studies with [18F]CKIE were performed.

Results
Cell cycle analyses showed a concentration-dependent (50 nM to 10 µM) increment of percentage of tumor cells in G1 phase after 24 h of incubation with CKIC, CKID and CKIE, with CKIE to be more potent than CKIC and CKID. Cell growth studies indicated reduced tumor cell numbers after 48 h of treatment with 1 µM (< 75%) and 10 µM (< 30%) CKIE and 10 µM (< 70%) CKIC or respectively CKID. Cdk4 specific phosphorylation at pRb Ser780 is decreased in a concentration dependent manner after 24 h of incubation with CKIE. Downregulation of E2F-1 and PCNA mRNA expression could be demonstrated after treatment with CKIE. [18F]CKIE indicated high stability in physiological buffer and cell culture medium. Cellular radiotracer uptake using [18F]CKIE increased with time amounting to 46.3±11.2 %ID/mg protein in HT-29 and 46.2±13.8 %ID/mg protein in FaDu cells, respectively, after 60 min at 37°C. Uptake of [18F]CKIE could be blocked with nonradioactive CKIE dependent on concentration (e.g., 23.5±3.7 %ID/mg protein with 5 µM CKIE after 60 min at 37°C).

Conclusion
CKIE was identified as the most potent fluorine containing pyrido[2,3 d]pyrimidin-7-one derivative analyzed in our study causing arrest of tumor cells in G1 phase due to inhibition of the Cdk4/6/ pRb/ E2F pathway. In vitro radiotracer uptake studies using [18F]CKIE demonstrated tumor cell uptake, which is an important prerequisite for further PET studies in tumor-bearing mice.