Synthesis, Transport and Pharmacokinetics of 5′-Amino Acid Ester Prodrugs of 1-β-D-Arabinofuranosylcytosine

Cytarabine (1-beta-D-arabinofuranosylcytosine, ara-C,1) suffers from low oral bioavailability due to low intestinal membrane permeability and poor metabolic stability, and intravenous infusion is usually adopted as the clinical standard dosir g administration. To develop an oral alternative for 1 and utilize the intestinal oligopeptide transporter 1 (PepT1), a series of 5'-amino acid ester derivatives of 1 was synthesized to clarify which modification was the most suitable to increase the oral bioavailability of 1. Their apical-to-basolateral permeability across Caco-2 cells and the antiproliferative activity with HL-60 cells were screened. 5'-Valyl prodrug 2 demonstrated the highest permeability and was selected for further study. Glycylsarcosine (gly-sar, a typical substrate of PepT1) uptake by Caco-2 cells can be inhibited by 2 in a concentration-dependent manner, and IC50 for 2 was 2.18 +/- 0.12 mM. The uptake of 2 was markedly increased in the long-term leptin-treated Caco-2 cells compared with the control Caco-2 cells, and was significantly inhibited by the excess of glysar, but not by L-valine. A dose-proportional pharmacokinetics was observed in rats when 5, 15, 30 mg/kg doses of 2 (calculated as 1) were orally administered. The oral absolute bioavailability of 1 was 60.0% and 21.8% after 2 and 1 were orally administered to rats 30 mg/kg, respectively. Following oral administration of 15 mg/kg, the absorption and bioactivation of 2 were extensive and rapid, over 98% of prodrug hydrolysis occurring before appearance in the portal vein. The in vivo dispositions of 1-beta-D-arabinofuranosyluracil (ara-U), a deaminated product of 1, were investigated. Oral administration of 2 resulted in an increased 1/ara-U ratio (2.76) in the blood, much higher than that (1.25) after 1 orally taken. Overall, these results demonstrated that the PepT1-mediated absorption of 2 and the increased metabolic stability resulted in a dramatic increase in the oral bioavailability of 1 in rats and further corroborated the thought that prodrug design strategy targeting intestinal PepT1 was an important and promising strategy to improve oral bioavailability of poorly absorbed drugs.