We previously evaluated the renal excretion mechanism of quinidine, which is a tertiary amine compound, using porcine kidney epithelial LLC-PK 1 cells and P-glycoprotein (P-gp)-expressed LLC-GA5-COL150 cells.1) The transepithelial transport of quinidine in the basolateral-to-apical direction in LLC-PK 1 cells was similar to that in the opposite direction. In contrast, quinidine was transported actively in the basolateral-to-apical direction in LLC-GA5-COL150 cells. The results suggested that P-gp is mainly responsible for the tubular secretion of quinidine in the kidney. 1) We also evaluated the intestinal absorption mechanism of quinidine using human intestinal epithelial Caco-2 cells.2) The temperaturedependent uptake of quinidine in Caco-2 cells grown on a plastic dish was increased by alkalization of the apical medium, and was inhibited by diphenhydramine and imipramine. The results suggested that a cation transport system was involved in the influx of quinidine at the apical membrane in intestinal epithelial cells. 2)Procainamide, another tertiary amine compound with a pK a value of 9.23, is classified as a type IA antiarrhythmic drug that works by decreasing conduction velocity, and prolonging tissue refractoriness.3) More than 80% of orally administered procainamide is absorbed from the intestine in humans.4) The Kp (octanol/buffer at pH 7.4) value of procainamide is about 0.1, and approximately half of the dose is excreted in the urine as unchanged drug.3-6) However, the mechanisms responsible for the membrane transport of procainamide in intestinal and renal epithelial cells are still unclear.In the present study, the transport characteristics of procainamide in LLC-PK 1 cells were compared with those of quinidine. In addition, we evaluated whether the transport system for quinidine is present in another intestinal cell line, LS180, as well as Caco-2. We also investigated whether the transport system of procainamide in LS180 cells is the same as that of quinidine. Cell Culture and Preparation of Monolayers LLC-PK 1 cells at passage 197 and LS180 cells at passage 38 were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.). These cells were maintained by serial passage in plastic dishes with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Biowest Inc., Nuaille, France) in an atmosphere of 5% CO 2 -95% air at 37°C. MATERIALS AND METHODS MaterialsLLC-PK 1 cells were seeded at a density of 5ϫ10 5 cells/ cm 2 on a 1.12 cm 2 porous membrane (3 mm pore size) in a polyester membrane Transwell ® -Clear insert (Costar, Cambridge, MA, U.S.A.) to evaluate the transcellular transport of cationic drugs. The seeded cells were maintained for 6 d to prepare differentiated cell monolayers. The maturity of the monolayer was judged by transepithelial electrical resistance (TEER). TEER was measured using a Millicell-ERS resistance system (Millipore, Bedford, MA, U.S.A.). LLC-PK 1 cell monolayers whose TEER was above 60 W · cm 2 were used to The aim of the present study was t...

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.

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.

We previously reported that renal function is partly responsible for the interindividual variability of the pharmacokinetics of bisoprolol. The aim of the present study was to examine the variability of bioavailability (F) of bisoprolol in routinely treated Japanese patients and intestinal absorption characteristics of the drug. We first analyzed the plasma concentration data of bisoprolol in 52 Japanese patients using a nonlinear mixed effects model. We also investigated the cellular uptake of bisoprolol using human intestinal epithelial LS180 cells. The oral clearance (CL/F) of bisoprolol in Japanese patients was positively correlated with the apparent volume of distribution (V/F), implying variable F. The uptake of bisoprolol in LS180 cells was temperature-dependent and saturable, and was significantly decreased in the presence of quinidine and diphenhydramine. In addition, the cellular uptake of bisoprolol dissolved in an acidic buffer was markedly less than that dissolved in a neutral buffer. These findings suggest that the rate/extent of the intestinal absorption of bisoprolol is another cause of the interindividual variability of the pharmacokinetics, and that the uptake of bisoprolol in intestinal epithelial cells is highly pH-dependent and also variable.

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.