Evaluation of 4'-[methyl-14C]thiothymidine for in vivo DNA synthesis imaging

We evaluated 4'-[methyl-11C]thiothymidine ([methyl-11C]S-dThd) to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and that might follow the same biochemistry as its surrogate.

[Methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethylstannyl-4'-thio-2'-deoxyuridine via a palladium-mediated Stille coupling reaction with 14C-methyl iodide. Degradation of [methyl-14C]S-dThd, when incubated in human blood, was analyzed by high-performance liquid chromatography (HPLC). The in vivo potential of [methyl-14C]S-dThd was evaluated by studying its distribution in EMT-6 mammary carcinoma-bearing mice. 2-Fluoro-2'-deoxycytidine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA.

[Methyl-14C]S-dThd was obtained in a 31%-41% radiochemical yield (calculated from 14C-methyl iodide) at 130 degrees C, 5-min reaction in N,N-dimethylformamide followed by semipreparative HPLC purification. The radiochemical purity of [methyl-14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of 14C-methyl iodide). [2-14C]Thymidine, when incubated with human blood, demonstrated rapid degradation. In contrast, [methyl-14C]S-dThd was stable with <3% degradation at 60 min. An in vivo distribution study showed the accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum, and tumor). On the other hand, the radioactivity of nonproliferating tissues (lung, liver, kidney, and muscle) was rapidly cleared in parallel with the clearance of blood radioactivity. The tumor uptake of [methyl-14C]S-dThd was high (8.8 percentage injected dose per gram at 60 min) and selective (tumor-to-blood ratio, 12.2 at 60 min). 2-Fluoro-2'-deoxycytidine pretreatment significantly reduced the tumor uptake of [methyl-14C]S-dThd. Relative blood flow as measured by the uptake of 4-[N-methyl-14C]iodoantipyrine was similar between the treated and untreated groups. Tissue extraction studies showed that most of the total tissue radioactivity of rapidly proliferating tissues was recovered in the DNA fraction at 60 min after [methyl-14C]S-dThd injection.

The labeling procedure is rapid and suitable for 11C labeling. Positron-labeled 4'-thiothymidine should be useful for imaging DNA synthesis by PET.