Abstract Gemcitabine (dFdC), a cytosine analog, is currently being evaluated in combination with other chemotherapeutic agents as therapy to treat children and young adults with CNS tumors in institutional clinical trials (NCT01878617, NCT03434262, and NCT04023669). Once in cells, dFdC undergoes a complex transformation into its active metabolite, gemcitabine triphosphate (dFdCTP), which exerts cytotoxic effects by inhibiting DNA synthesis. Our overall goal is to develop a translational pharmacokinetic (PK) model to predict dFdCTP concentrations in brain tumor tissue from mice to humans. This model will provide insights to potentially adjust clinical dFdC dosages. The intracellular formation of dFdCTP is known to be limited in humans due to the saturation of deoxycytidine kinase (dCK), an enzyme involved in dFdC metabolism, which occurs at dFdC plasma concentrations ~20 µM. It is critical to include the saturation of dCK in the PK model to assure the translatability between species; however, it has not been well characterized in mice. Thus, the objective of this study was to determine the formation of dFdCTP in peripheral blood mononuclear cells (PBMCs) in mice at clinically relevant dosages to investigate the dCK saturation. CD-1 nude mice (N=4 per group) were dosed with 40, 60, and 120 mg/kg dFdC via IV bolus (<1 min infusion). dFdC and dFdCTP concentrations were measured in plasma and in PBMCs, respectively, for up to 4h post-single dose, using validated LC-MS/MS methods, and analyzed with a naïve-pooled modeling approach using ADAPT 5 (BSMR, Los Angeles, CA, USA). dFdC plasma PK data were adequately fitted with a two-compartment model, and dFdCTP PBMC data were captured with a one-compartment model linked to the dFdC model. The 40, 60, and 120 mg/kg dosages resulted in plasma dFdC exposures (area under the curve, AUC) of 20.9 ± 6.8 µM·h, 30.1 ± 8.1 µM·h, and 62.8 ± 17.0 µM·h, which were within the range of dFdC AUC values previously published for children with brain tumors (Campagne et al, J Clin Oncol, suppl.e13547). The dFdCTP formation rate decreased in a nonlinear manner as the dFdC dosage increased from 40 mg/kg (0.80 /h) to 60 mg/kg (0.21 /h) to 120 mg/kg (0.13 /h) leading to higher dFdCTP exposures after 40 mg/kg (76.1 ± 26 µM·h) compared with 60 mg/kg (31.1 ± 10.0 µM·h) or 120 mg/kg (35.2 ± 12.0 µM·h). These results suggest an inhibition of dFdCTP intracellular formation, more than a saturation, at high dFdC dosages in mice. The plasma and PBMC murine model are now being used along with the pediatric trial PK data to develop a translational model to predict dFdCTP exposure in PBMCs. This translational model will then be extended to include the murine brain tumor tissue compartment. Citation Format: Elizabeth G. Gibson, Abi Davis, Bo Zhong, Olivia Campagne, Martine F. Roussel, Clinton F. Stewart. Characterization of gemcitabine triphosphate formation in mice to aid in the development of a translational tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3031.