29 TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does not diminish therapeutic window in human tumor xenografts

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.