Imaging of the cell cycle: Synthesis and radiopharmacological evaluation of ¹²⁴I-labeled CDK4 inhibitors

Objectives: Tumor cells are characterized by their loss of growth control resulting from alterations in regulating pathways of the cell cycle, e.g., a deregulated cyclin dependent kinase (Cdk) activity and/or expression. In 80% of tumors the cell cycle relevant Cdk4-cyclin D1/retinoblastoma (pRb) cascade is altered. Hence, appropriately radiolabeled Cdk4 inhibitors are discussed as promising molecular probes for imaging cell proliferation processes and tumor visualization by PET. This work describes the synthesis and radiopharmacological evaluation of two ¹²⁴I-labeled Cdk4 inhibitors as potential radiotracers for imaging of Cdk4 in vivo.
Methods: Reference substances and labeling precursors 1 and 2 were prepared with minor modifications according to literature procedures. Radioiodination was performed through regioselective destannylation reaction under mild conditions using [¹²⁴I]NaI and Iodogen® as the oxidizing agents (Fig. 1). ¹²⁴I-labeled radiotracers [¹²⁴I]CKIA and [¹²⁴I]CKIB were used in cell uptake studies. Biodistribution and small animal PET studies were carried out.
Results: Treatment of a solution containing labeling precursors 1 or 2 (5 mg/ml) with [¹²⁴I]NaI (29.0 to 275.0 MBq) in Iodogen® precoated tubes gave radiolabeled Cdk4 inhibitors [¹²⁴I]CKIA and [¹²⁴I]CKIB in radiochemical yields of up to 35% after removal of Boc protecting group and final HPLC purification. Both compounds were isolated in high radiochemical purity exceeding 95%. The specific radioactivity (A_s) was 25-35 GBq/μmol at the end of synthesis. The lipophilicity (logP) was determined to be 2.77±0.13 for [¹²⁴I]CKIA and 1.99±0.03 for [¹²⁴I]CKIB, respectively. Both compounds remained stable in buffer (PBS, pH 7.4) and ethanol after 24 h at 37°C. In vitro radiotracer uptake studies in human tumor cells using [¹²⁴I]CKIA showed substantial uptake in adenocarcinoma HT-29 (1264±84 %ID/mg protein) and squamous cell carcinoma FaDu cells (1429±229 %ID/mg protein) after 2 h at 37°C (A_s:25 GBq/μmol). A significantly lower uptake was detected at 4°C (HT-29: 383±38 %ID/mg protein, FaDu: 437±25 %ID/mg protein). Biodistribution studies of [¹²⁴I]CKIA and [¹²⁴I]CKIB showed rapid blood clearance in rats. [¹²⁴I]CKIA and [¹²⁴I]CKIB were mainly accumulated and metabolized by liver. Both radiotracers were administered intravenously to mouse FaDu xenograft tumor model and imaging studies were performed on a small-animal PET scanner confirming a high radioactivity concentration in the liver and intestine for both compounds. No radioactivity was found in the brain. PET showed only little uptake of both radiotracers in the tumor.
Conclusions: The radiosynthesis of two ¹²⁴I-labeled Cdk4 inhibitors has been developed. Both radiotracers were obtained in reproducible radiochemical yields and purity enabling detailed radiopharmacological characterization. Small-animal PET and autoradiography studies showed only low tumor uptake. Further studies are needed to search for more suitable derivatives of these substances as radiotracers for imaging Cdk4 by means of PET.