Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment.

AIM: Three intracellularly formed metabolites are responsible for the antineoplastic effect of capecitabine: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP), and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The objective of this study was to explore the pharmacokinetics of these intracellular metabolites during capecitabine treatment. METHODS: Serial plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 13 patients treated with capecitabine 1000 mg QD (group A) and eight patients receiving capecitabine 850 mg m(-2) BID for fourteen days, every three weeks (group B). Samples were collected on day 1 and, for four patients of group B, also on day 14. The capecitabine and 5-fluorouracil (5-FU) plasma concentrations and intracellular metabolite concentrations were determined using LC-MS/MS. Pharmacokinetic parameters were estimated using non-compartmental analysis. RESULTS: Only FUTP could be measured in the PBMC samples. The FdUTP and FdUMP concentrations were below the detection limits (LOD). No significant correlation was found between the plasma 5-FU and intracellular FUTP exposure. The FUTP concentration-time profiles demonstrated considerable inter-individual variation and accumulation of the metabolite in PBMCs. FUTP levels ranged between

Population Pharmacokinetics of Intracellular 5-Fluorouridine 5'-Triphosphate and its Relationship with Hand-and-Foot Syndrome in Patients Treated with Capecitabine.

Capecitabine is an oral pro-drug of 5-fluorouracil. Patients with solid tumours who are treated with capecitabine may develop hand-and-foot syndrome (HFS) as side effect. This might be a result of accumulation of intracellular metabolites. We characterised the pharmacokinetics (PK) of 5-fluorouridine 5'-triphosphate (FUTP) in peripheral blood mononuclear cells (PBMCs) and assessed the relationship between exposure to capecitabine or its metabolites and the development of HFS. Plasma and intracellular capecitabine PK data and ordered categorical HFS data was available. A previously developed model describing the PK of capecitabine and metabolites was extended to describe the intracellular FUTP concentrations. Subsequently, a continuous-time Markov model was developed to describe the development of HFS during treatment with capecitabine. The influences of capecitabine and metabolite concentrations on the development of HFS were evaluated. The PK of intracellular FUTP was described by an one-compartment model with first-order elimination (ke,FUTP was 0.028 h-1 (95% confidence interval 0.022-0.039)) where the FUTP influx rate was proportional to the 5-FU plasma concentrations. The predicted individual intracellular FUTP concentration was identified as a significant predictor for the development and severity of HFS. Simulations demonstrated a clear exposure-response relationship. The intracellular FUTP concentrations were successfully described and a significant relationship between these intracellular concentrations and the development and severity of HFS was identified. This model can be used to simulate future dosing regimens and thereby optimise treatment with capecitabine.

Lack of uptake, release and action of UTP at sympathetic perivascular nerve terminals in rabbit ear artery.

A possible role of uridine 5'-triphosphate (UTP) and uridine at sympathetic nerve terminals was studied in the rabbit ear artery after incubation of isolated vessels with [3H]uridine or [3H]noradrenaline. It was found that [3H]uridine was taken up by rabbit ear artery. This uptake was largely suppressed after the removal of endothelium and was inhibited by ethidium bromide and dipyridamole. Chemical denervation of the vessels with 6-hydroxydopamine did not reduce the uptake. Following pre-incubation of the isolated vessels with [3H]uridine, there was a release of radioactivity from the superfused rabbit ear artery. UTP, UDP, UMP and uridine were detected by thin layer chromatography both in the superfusate and inside the vessels. Transmural electric stimulation (30 V, 5 Hz) induced a contraction of the vessels but did not increase the release of uridine nucleotides into the superfusate. [3H]Noradrenaline was released during electric stimulation and the addition of UTP (100 microM) had no effects on this release. To conclude, this study shows that in contrast to endothelial cells, the sympathetic nerve terminals of the rabbit ear artery do not take up uridine and do not release uridine-derived nucleotides. UTP at 100 microM is also unable to modulate the evoked release of noradrenaline. These results mainly confine the role of UTP in endothelium-derived vasodilatation via P2Y2 and/or P2Y4 receptors.

Ionic interaction of amiloride and uridine 5'-triphosphate in nebulizer solutions.

Combination therapy using nebulized amiloride hydrochloride and uridine-5'-triphosphate (UTP) trisodium salt aerosols has been investigated for the treatment of cystic fibrosis (CF). Amiloride in aqueous solution precipitates in the presence of UTP, reducing drug concentrations. Interactions between these drugs and NaCl in solution were studied using phase-solubility techniques monitored by UV spectrophotometry. Elemental analyses were employed for precipitate characterization. Amiloride solubility was reduced by more than 85% in saline. Amiloride solubility decreased with increasing UTP concentration, resulting in formation of a precipitated complex. The theoretical molar ratio of complexes range from 1-3 amiloride:1 UTP. At most concentrations only 3 amiloride:1 UTP complex was observed in precipitate. This is a reflection of low Ksp for the 3:1 complex of 2.92 x 10(-11) M4 compared with 2.09 x 10(-4) M2 for amiloride alone. Equilibration over excess bulk solid resulted in higher solubility estimates and different phase solubility diagrams than solubility studies utilizing precipitation technique. This may be explained by the absence of amiloride in the solid state and its impact on complex equilibria with UTP. The solubility suppressing effects of UTP and saline were largely additive. A number of ionic interactions increase complex solubility profile of amiloride hydrochloride in the presence of UTP and NaCl.

Reversible depression of transcription during hibernation.

Mammalian hibernators downregulate processes of energy production and consumption while maintaining cellular homeostasis. Energetic costs of transcription must be balanced with demands for gene products. Data from nuclear run-on assays indicate transcriptional initiation is reduced two fold in torpid golden-mantled ground squirrels ( Spermophilus lateralis) as compared to euthermic animals between bouts of torpor. In addition, elongation rates across the temperature range experienced by hibernators indicate a virtual arrest of transcription at the low body temperatures of torpor. Finally, there is no seasonal compensation or species-specific adaptation for increased elongational capacity in the cold. Thus, it appears that hibernators are not specifically adapted to continue transcription during torpor. Taken together, these data indicate that transcription arrests during torpor because of a moderate depression of initiation and a more severe inhibition of elongation, largely due to temperature effects. Restoration of euthermic body temperatures during the interbout arousals reverses this transcriptional depression and permits gene expression.

Conditions favoring RNA polymerase I transcription in permeabilized cells.

RNA synthesis can be detected in nuclei using modified RNA precursors (Br-UTP) introduced in permeabilized cells. Surprisingly, RNA pol I transcripts are detected only after inhibition of RNA pol II or salt enhancement of RNA pol I activity. By modifying a previously reported protocol, we found that RNA pol I transcripts can be detected selectively or simultaneously with RNA pol II transcripts without any drug treatment. Removing glycerol from the permeabilization and transcription buffers and improving the permeabilization using Triton X-100 revealed RNA pol I transcription in two cell lines (mammalian and Xenopus) and in isolated mouse oocytes. The transcripts were most probably rRNA because they were detected in the nucleoli, digested by RNase, sensitive to actinomycin D, and resistant to alpha-amanitin. We found by microinjection of the Br-UTP precursors in living cells that low ionic strength allows the detection of RNA pol I transcription. Electron microscopy of mouse oocytes showed that the "looseness" of the nucleolar organization is associated with the detection of the RNA pol I transcription; this detection does not necessarily need nucleolar disorganization. The data obtained with both permeabilized cells and microinjections of RNA precursors in the absence of glycerol support the hypothesis that the degree of hydration of the cell plays a role in RNA pol I transcription.