Yukiya Hashimoto

Effect of the VKORC1 Genotype on Warfarin Dose Requirements in Japanese Pediatric Patients

The primary aim of the present study was to evaluate the effect of the genotype of vitamin K epoxide reductase complex 1 (VKORC1) on warfarin dose requirements in Japanese pediatric patients. Forty-eight pediatric patients (0.42-19.25 years old) in whom stable anticoagulation was achieved by warfarin were enrolled in this study, and the polymorphic alleles of VKORC1 and CYP2C9 were determined for each subject. The relative impact of covariates on the anticoagulant effect of warfarin was evaluated by multiple regression analysis. It was found that VKORC1 genotype and age were major factors affecting the relationship between the weight-normalized warfarin dose and the therapeutic prothrombin time-international normalized ratio (PT-INR). Because only one patient had the CYP2C9*3 allele, we could not evaluate the effect of CYP2C9 polymorphisms on the anticoagulant effect of warfarin. In contrast, the anticoagulant effect of warfarin in patients with the VKORC1 1173CT or 1173CC genotype was 52.3% of that in patients with the 1173TT genotype. In addition, the anticoagulant effect of warfarin was shown to increase by 10.5% per year in Japanese pediatric patients. In conclusion, genotyping of VKORC1 will be useful in establishing individual anticoagulant therapy with warfarin, and it should be noted that a higher weight-normalized dose of warfarin is required in younger pediatric patients.

Pharmacokinetics of Bosentan in Routinely Treated Japanese Pediatric Patients with Pulmonary Arterial Hypertension

We evaluated the pharmacokinetics of routinely administered bosentan in 46 Japanese pediatric patients with pulmonary arterial hypertension. Plasma samples were taken twice at times corresponding to the peak and trough concentrations following repetitive oral administration. The population pharmacokinetic parameters of bosentan were estimated by use of the NONMEM program, in which a one-compartment model with repetitive bolus dosing was parameterized in terms of the oral clearance (CL/F) and elimination rate constant (k). Polymorphisms of CYP3A5, SLCO1B1, SLCO1B3, and SLCO2B1 had no significant effect on the disposition of bosentan. In addition, the pharmacokinetics of bosentan was not altered by heart failure or coadministration of sildenafil. In contrast, weight (WT)-normalized values of CL/F were correlated negatively with age (AGE). The final population mean values of CL/F and k were estimated to be 0.409 · (1 - 0.0377 · (AGE - 3.81)) · WT L/h and 0.175 h(-1), respectively.

Contribution of Na+-independent nucleoside transport to ribavirin uptake in the rat intestine and human epithelial LS180 cells.

The aim of the present study was to characterize the intestinal absorption of ribavirin (1-beta-d-ribofuranosyl-1, 2, 4-trizole-3-carboxamide). We evaluated the contribution of Na(+)-dependent and -independent transport to ribavirin absorption in the rat intestine using an in situ closed loop method. In addition, we performed pharmacokinetic analysis of the uptake of ribavirin in human intestinal epithelial LS180 cells, and also evaluated the effect of extracellular Na(+) concentration and an inhibitor of the Na(+)-independent equilibrative nucleoside transporter, nitrobenzylmercaptopurine ribonucleoside (NBMPR), on the uptake of ribavirin in the cells. In the presence and also absence of Na(+) in rat intestinal loops, more than 80% of the administered dose (50 microg at a concentration of 100 microg/ml=409 microM) of ribavirin was absorbed in 40 min. The absorption of ribavirin in the rat intestine was significantly reduced by coadministration of 10 mg/ml (=37.3 mM) inosine. In LS180 cells, 100 microM ribavirin was taken up time-dependently, and the influx clearance of the drug was similar to the efflux clearance. Five mM inosine and mizoribine reduced the uptake of 100 microM ribavirin in LS180 cells. The absence of extracellular Na(+) decreased the uptake of 100 microM ribavirin only weakly in the cells, whereas the uptake of 100 microM-2 mM ribavirin was markedly decreased by 100 microM NBMPR. These findings suggested that Na(+)-independent nucleoside transport contributes significantly to intestinal absorption of ribavirin at relatively high concentrations (>or=100 microM).

Mechanisms for membrane transport of metformin in human intestinal epithelial Caco-2 cells.

The aim of the present study was to investigate the mechanisms for membrane transport of metformin in human intestinal epithelial Caco‐2 cells. The mRNA of not only organic cation transporter (OCT) 3, but also OCT1 and OCT2, was expressed in Caco‐2 cells. The uptake of 100 µm metformin at the apical membrane of Caco‐2 cells grown on porous filter membrane was significantly greater than that at the basolateral membrane. The apical uptake of 100 µm metformin in Caco‐2 cells grown on plastic dishes was inhibited significantly by 1 mm unlabeled metformin, quinidine and pyrilamine, indicating that a specific transport system is involved in the apical uptake of metformin in Caco‐2 cells. The apical uptake of 100 µm metformin in Caco‐2 cells was decreased by acidification of the medium, but not increased by alkalization. In addition, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (a protonophore) had no effect on the apical uptake of metformin in Caco‐2 cells at apical medium pH 8.4. These findings suggested that the apical uptake of metformin in Caco‐2 cells is mediated at least partly by OCTs, but that the postulated H+/tertiary amine antiport system is not responsible for the apical uptake of metformin. Copyright

Pharmacokinetic variability of flecainide in younger Japanese patients and mechanisms for renal excretion and intestinal absorption.

The aims of the present study were to evaluate the variability of pharmacokinetics of flecainide in young Japanese patients and to investigate the mechanisms of renal excretion and intestinal absorption of the drug using cultured epithelial cells. First the plasma concentration data of flecainide was analysed in 16 Japanese patients aged between 0.07 and 18.30 years using a one‐compartment model. Considerable interindividual variability was observed in the oral clearance (CL/F) and the apparent volume of distribution (V/F) of flecainide in the young patients. Flecainide was transported selectively in the basolateral‐to‐apical direction in P‐glycoprotein‐expressing renal epithelial LLC‐GA5‐COL150 cell monolayers. The uptake of flecainide into intestinal epithelial LS180 cells was decreased significantly by acidification of the extracellular medium, and was inhibited by tertiary amines, such as diphenhydramine and quinidine. These findings in the present study suggest that flecainide is excreted by P‐glycoprotein in the renal tubule and is taken up by the postulated H+/tertiary amine antiporter in the intestine, and that functional variability of not only the hepatic drug‐metabolizing enzymes, but also the transporters in the kidney and intestine, may be responsible for the interindividual variability of systemic clearance (CL) and/or the bioavailability (F) of flecainide. Copyright

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter‐like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high‐affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium‐3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na+‐independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H+ gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K+ concentration. In addition, the Na+‐independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential‐dependent, but not outward H+ gradient‐dependent. Copyright

Mechanisms responsible for the altered pharmacokinetics of bosentan: analysis utilizing rats with bile duct ligation-induced liver dysfunction.

The purpose of this study was to evaluate the mechanisms responsible for the pharmacokinetic variability of bosentan utilizing rats with liver dysfunction induced by 7‐day bile duct ligation (BDL). Bosentan was administered intravenously at a constant infusion rate (I) of 24, 40 or 60 µg/min/kg. The blood bosentan concentration (BBC) following infusion was measured by HPLC, and apparent clearance (CL) of the drug was estimated as I/BBC. The CL values in normal rats were 30.5 and 19.3 ml/min/kg at infusion rates of 24 and 60 µg/min/kg, respectively, suggesting non‐linear pharmacokinetics of bosentan. The BBC in BDL rats was much higher than that in normal rats, and the CL values in BDL rats were 3.80 and 3.08 ml/min/kg at infusion rates of 24 and 60 µg/min/kg, respectively. The CL value of bosentan at an infusion rate of 40 µg/min/kg in normal rats was decreased significantly by the coadministration of taurocholic acid or bilirubin. In addition, the hepatic mRNA expression of CYP2C6, CYP3A2, Oatp1a1, Oatp1a4 and Oatp1b2 in BDL rats decreased to 77.6%, 34.0%, 65.4%, 84.8% and 44.2% of that in normal rats, respectively. These results suggested that bile acids and/or bilirubin accumulated in BDL rat plasma inhibited the hepatic uptake of bosentan, and that the decreased bosentan clearance in BDL rats was caused at least partly by the impaired expression of hepatic drug‐metabolizing enzymes and uptake transporters. Moreover, because the pharmacokinetics of bosentan was non‐linear at the tested doses, the increased BBC in BDL rats might further induce the saturation of hepatic uptake and/or metabolism of bosentan. Copyright

Effects of probenecid on the pharmacokinetics of mizoribine and co-administration of the two drugs in patients with nephrotic syndrome.

Probenecid (PRB) is an agent that reduces the systemic level of uric acid, and has the ability to inhibit the renal tubular secretion of agents that are co-administered with it. In this study, we evaluated the effects of PRB co-administered with mizoribine (MZR) on the pharmacokinetics (PK) of MZR in 12 patients with nephrotic syndrome. The elimination rate constant (kel) was used as an indicator of changes in the PK of MZR when the secretion of MZR was inhibited by co-administration of PRB, in order to determine the extent to which MZR was influenced by PRB. In 4 of the 12 patients studied, kel decreased and the biological half-life (t1/2) of MZR was prolonged when co-administered with PRB, in comparison with the values when MZR was used alone, thus revealing that the PK of MZR was influenced by PRB. Co-administration of PRB with MZR appears to be effective in prolonging the biological half-life of MZR and enhancing its effect in patients with nephrotic syndrome, although further studies will be required to determine the optimal dosage of PRB and renoprotective effects.

Temperature‐dependent specific transport of levofloxacin in human intestinal epithelial LS180 cells

It was reported previously that specific levofloxacin uptake in Caco‐2 cells was inhibited by nicotine, enalapril, L‐carnitine and fexofenadine. The aim of the present study was to characterize the cellular uptake of levofloxacin using another human intestinal cell line, LS180. Levofloxacin uptake in LS180 cells was temperature‐dependent and optimal at neutral pH, but was Na+‐independent. The rank order of inhibitory effects of the four compounds on [14C] levofloxacin uptake in LS180 cells was nicotine>enalapril>L‐carnitine>fexofenadine, which is consistent with that in Caco‐2 cells. The mRNA levels of OATP1A2, 1B1, 1B3 and 2B1 in LS180 cells were markedly different from those in Caco‐2 cells, and OATP substrates/inhibitors had no systematic effect on the levofloxacin uptake. The mRNA levels of OCTN1 and 2 in LS180 cells were similar to those in Caco‐2 cells. However, the inhibitory effect of nicotine on L‐[3H]carnitine uptake was much less potent than that of unlabeled L‐carnitine. These results indicate that the specific uptake system for levofloxacin in LS180 cells is identical/similar to that in Caco‐2 cells, but that OATPs and OCTNs contribute little to levofloxacin uptake in the human intestinal epithelial cells. Copyright

Functional characteristics of a renal H + /lipophilic cation antiport system in porcine LLC-PK 1 cells and rats

We have recently found an H+/quinidine (a lipophilic cation, QND) antiport system in Madin-Darby canine kidney (MDCK) cells. The primary aim of the present study was to evaluate whether the H+/lipophilic cation antiport system is expressed in porcine LLC-PK1 cells. That is, we investigated uptake and/or efflux of QND and another cation, bisoprolol, in LLC-PK1 cells. In addition, we studied the renal clearance of bisoprolol in rats. Uptake of QND into LLC-PK1 cells was decreased by acidification of the extracellular pH or alkalization of the intracellular pH. Cellular uptake of QND from the apical side was much greater than from the basolateral side. In addition, apical efflux of QND from LLC-PK1 cells was increased by acidification of the extracellular pH. Furthermore, lipophilic cationic drugs significantly reduced uptake of bisoprolol in LLC-PK1 cells. Renal clearance of bisoprolol in rats was approximately 7-fold higher than that of creatinine, and was markedly decreased by alkalization of the urine pH. The present study suggests that the H+/lipophilic cation antiport system is expressed in the apical membrane of LLC-PK1 cells. Moreover, the H+/lipophilic cation antiport system may be responsible for renal tubular secretion of bisoprolol in rats.