Enhanced oral absorption and anti-colorectal cancer efficacy of trifluridine by interfering with both TPase and UPase.

Oral nucleoside anticancer drugs are frequently limited by rapid enzymatic degradation, leading to inadequate systemic exposure and poor therapeutic efficacy. Trifluridine (FTD), a thymidine analogue used for metastatic colorectal cancer (CRC), is rapidly inactivated by thymidine phosphorylase (TPase) and uridine phosphorylase (UPase). Although the approved trifluridine/tipiracil (FTD/TPI) therapy protects FTD from TPase-mediated degradation, UPase remains insufficiently controlled. Uridine triacetate (TAU), a uridine prodrug, is used to sustain systemic uridine exposure and attenuate UPase-mediated FTD degradation. Here, we developed a dual-enzyme metabolic protection strategy for enhancing FTD exposure through TAU-mediated UPase and TPI-mediated TPase. The results indicated that uridine and FTD bound to the same UPase site, and elevated uridine exposure significantly increased oral FTD absorption. Among different TAU particle sizes, 200 μm slowed gastrointestinal release, generated maximal uridine exposure, and increased FTD absorption by 4.0-fold and 1.6-fold compared with FTD alone and FTD/TPI, respectively. Further, the combination of TPI and TAU achieved dual protection against UPase and TPase, maximized FTD exposure, and demonstrated favorable short-term safety. Notably, the half-dose FTD in combination with TAU and TPI achieved a systemic exposure comparable to that of full-dose FTD/TPI, indicating a potential dose-sparing effect. In colorectal cancer models, the combination markedly promoted anti-tumor efficacy through suppression of cell proliferation and induction of cell apoptosis. These findings establish a prodrug-enabled metabolic protection strategy for improving oral pharmacokinetics and therapeutic efficacy of metabolically vulnerable nucleoside anticancer drugs.