Quinidine, a tertiary amine compound with a site of ionization with a pK a value of pH 8.8, is classified as a type IA antiarrhythmic drug, and has been used for the management of ventricular arrhythmias.1,2) The drug is absorbed rapidly and almost entirely after oral administration, and is detected in the plasma within 15 min.3) Quinidine binds to both albumin and a 1 -acid glycoprotein, and the proportion that binds to plasma protein is 70 to 95%. 4) However, its apparent volume of distribution is quite large (2.0 to 3.5 liters/kg), because the drug is highly lipophilic.4) It is metabolized via 3-hydroxylation and N-oxygenation by cytochrome P450 3A4, and these metabolites are less electrophysiologically active than the parent drug. 5,6) The hepatic extraction ratio of quinidine is about 30%, and the bioavailability after oral administration is about 70%. 7) In addition, quinidine is partly excreted in the urine (15 to 40% of dose), suggesting that reabsorption at the distal tubules in the nephron is not complete. 4)The mechanism of intestinal absorption of lipophilic organic cations has been explained as passive diffusion of unionized compounds according to the pH-partition theory. On the other hand, Mizuuchi et al. investigated the mechanisms responsible for the transcellular transport of diphenhydramine in Caco-2 cells. 8,9) This cell line forms confluent monolayers of well differentiated enterocyte-like cells with functional properties of transporting epithelia, and is widely used as a model to study the absorption of drugs and other xenobiotics.10-12) The uptake of diphenhydramine at the apical membrane in Caco-2 cells was pH-and temperature-dependent, but was not inhibited by tetraethylammonium, biological amines, or neurotransmitters.8) On the other hand, the uptake was inhibited by chlorpheniramine, procainamide, and imipramine, and was trans-stimulated by the preloading of chlorpheniramine, dimethylaminochloride, and triethylamine.8,9) From these results, Mizuuchi et al. concluded that the uptake of diphenhydramine at the apical membrane in Caco-2 cells is mediated by a specific transport system, and that this system recognizes the N-dimethyl or N-diethyl moieties of compounds. 8,9) However, at present, it is unclear whether the transport system for diphenhydramine is involved in the intestinal absorption of other tertiary amine compounds, such as quinidine.During drug absorption in the intestine, therapeutic compounds or nutrients first enter intestinal epithelial cells from their apical side, then pass through the epithelia to the basolateral side, and finally appear in the blood stream. Therefore, to investigate intestinal drug absorption, it is important to separately assess these sequential processes. However, in many cases, drug transport on the apical and basolateral sides of the monolayer in Caco-2 cells was not separately examined, and the influx and efflux clearance rates were not evaluated.13) For the characterization of transcellular drug transport, a pharmacokinetic approach is useful. Tr...

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...

To characterize the membrane transport responsible for the renal excretion and intestinal absorption of levofloxacin, we performed pharmacokinetic analysis of transcellular transport across LLC-PK 1 and Caco-2 cell monolayers. Transcellular transport of levofloxacin in LLC-PK 1 cells was greater in the basolateral-to-apical direction than in the opposite direction. Pharmacokinetic analysis indicated that basolateral uptake was the direction-determining step for the transcellular transport of levofloxacin in LLC-PK 1 cells. The apical efflux clearance of levofloxacin in LLC-PK 1 cells was increased at the medium pH 6 as compared with at pH 8, suggesting that membrane transport characteristics of levofloxacin are apparently similar to those of a prototypical organic cation, tetraethylammonium. On the other hand, transcellular transport of levofloxacin in Caco-2 cells was only slightly greater in the basolateral-to-apical direction than in the opposite direction. The apical efflux clearance of levofloxacin in Caco-2 cells was greater than basolateral efflux clearance, and apical influx clearance was greater than any other membrane transport clearance. In addition, the apical uptake of levofloxacin as well as quinidine in Caco-2 cells was inhibited significantly by nicotine and imipramine. The findings indicated that some transporters are responsible not only for the efflux but also for the influx of levofloxacin at the apical membrane of Caco-2 cells.

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To evaluate the mechanism responsible for the tubular secretion of bisoprolol, we compared transcellular transport of bisoprolol with that of tetraethylammonium (TEA), cimetidine, and quinidine across LLC-PK1 cell monolayers grown on porous membrane filters. TEA and cimetidine were actively transported in the basolateral-to-apical direction by the specific transport system. Pharmacokinetic analysis indicated that basolateral influx and apical efflux were cooperatively responsible for the directional transport of TEA and cimetidine. Lipophilic cationic drugs, quinidine, S-nicotine, and bisoprolol, significantly diminished basolateral influx and apical efflux clearance of cimetidine. However, transcellular transport of quinidine in the basolateral-to-apical direction was similar to that in the opposite direction in LLC-PK1 cells. In contrast, quinidine was transported actively in the basolateral-to-apical direction in P-glycoprotein-expressed LLC-GA5-COL150 cells. Pharmacokinetic analysis indicated that P-glycoprotein increased the apical efflux of quinidine and also decreased the apical influx of the drug. Basolateral-to-apical transport of bisoprolol was also similar to apical-to-basolateral transport in LLC-PK1 cells, whereas the drug was directionally transported from the basolateral to the apical side in LLC-GA5-COL150 cells. These results suggested that bisoprolol was not significantly transported via transport systems involved in the directional transport of TEA and cimetidine, but that P-glycoprotein was responsible for the directional transport of bisoprolol as well as quinidine in renal epithelial cells.