Toshihisa Koga

The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

Purpose: Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-13C]uracil breath test (UraBT) with 96% specificity and 100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath 13CO2 metabolite formation, plasma [2-13C]dihydrouracil formation, [2-13C]uracil clearance, and DPD activity. Experimental Design: An aqueous solution of [2-13C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath 13CO2 concentrations and plasma [2-13C]dihydrouracil and [2-13C]uracil concentrations were determined over 180 minutes using IR spectroscopy and liquid chromatography-tandem mass spectrometry, respectively. Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPD activity was measured using the DPD radioassay. Results: The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath 13CO2 DOB50 significantly correlated with PBMC DPD activity (rp = 0.78), plasma [2-13C]uracil area under the curve (rp = −0.73), [2-13C]dihydrouracil appearance rate (rp = 0.76), and proportion of [2-13C]uracil metabolized to [2-13C]dihydrouracil (rp = 0.77; all Ps < 0.05). Conclusions: UraBT breath 13CO2 pharmacokinetics parallel plasma [2-13C]uracil and [2-13C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPD activity. These pharmacokinetic data further support the future use of the UraBT as a screening test to identify DPD deficiency before 5-fluorouracil-based therapy.

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against •OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H2O2), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a •OH-trapping agent after UVB exposure (305 nm) to H2O2 for 1 min in the presence of rebamipide. The signal intensity of •OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 1010, 8.16 × 109 and 1.65 × 1010 M-1 s-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the •OH generated by UVB/H2O2. Specific formation of this product explained the molecular characteristics of rebamipide as a potential •OH scavenger.

Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans

&NA; An adequate evaluation system for drug intestinal absorption is essential in the pharmaceutical industry. Previously, we established a novel prediction system of drug intestinal absorption in humans, using the mini‐Ussing chamber equipped with human intestinal tissues. In this system, the TI value was defined as the sum of drug amounts transported to the basal‐side component (Xcorr) and drug amounts accumulated in the tissue (Tcorr), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. In order to apply this system to the screening assay, it is important to understand the differences between animal and human tissues in the intestinal absorption of drugs. In this study, the transport index (TI) values of three drugs, with different levels of membrane permeability, were determined to evaluate the rank order of drug absorbability in intestinal tissues from rats, dogs, and monkeys. The TI values in small intestinal tissues in rats and dogs showed a good correlation with those in humans. On the other hand, the correlation of TI values in monkeys was lower compared to rats and dogs. The rank order of the correlation coefficient between human and investigated animal tissues was as follows: dog (r2 = 0.978), rat (r2 = 0.955), and monkey (r2 = 0.620). TI values in large intestinal tissues from rats (r2 = 0.929) and dogs (r2 = 0.808) also showed a good correlation. The obtained TI values in small intestinal tissues in rats and dogs were well correlated with the fraction of drug absorbed (Fa) in humans. From these results, the mini‐Ussing chamber, equipped with intestinal tissues in rats and dogs, would be useful as a screening tool in the drug discovery stage. In addition, the obtained TI values can be used for the prediction of the Fa in humans. Graphical abstract Figure. No caption available.

Pharmacokinetic Modelling of [2-13C]Uracil Metabolism in Normal and DPD-Deficient Dogs

A physiologically based pharmacokinetic (PBPK) model to simulate the plasma concentration and 13CO2 exhalation after [2-13C]uracil administration to DPD-suppressed dogs was developed. Simulation using this PBPK model should be useful in clinical situations where DPD-deficient patients at risk are to be detected with [2-13C]uracil as an in vivo probe.

Evaluation of intestinal metabolism and absorption using the Ussing chamber system equipped with intestinal tissue from rats and dogs

Graphical abstract Figure. No Caption available. Abstract The purpose of this study was to evaluate the intestinal metabolism and absorption in a mini‐Ussing chamber equipped with animal intestinal tissues, based on the transport index (TI). TI value was defined as the sum of drug amounts transported to the basal‐side component (Xcorr) and drug amounts accumulated in the tissue (Tcorr), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. Midazolam was used as a test compound for the evaluation of intestinal metabolism and absorption. The metabolite formulation of midazolam was observed in both rats and dogs. Ketoconazole inhibited the intestinal metabolism of midazolam in rats and improved its intestinal absorption to a statistically significant extent. Therefore, the mini‐Ussing chamber, equipped with animal intestinal tissues, showed potential to use the evaluation of the intestinal metabolism and absorption, including the assessment of species differences.