Introduction

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5,6-Dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (YH1885¹, Fig. 1) is under development as a new reversible acid pump antagonist by Yuhan Research Center (Seoul, Korea). Unlike irreversible proton pump inhibitors such as omeprazole and lanzoprazole, YH1885 reversibly inhibits H⁺,K⁺-ATPase by binding to the K⁺-binding site of the pump (Hwang et al., 1998), thereby causing fewer side effects, compared with the irreversible proton pump inhibitors (McTavish et al., 1991).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structures of YH1885 and its analogs. *14C.

When administered by i.v. injection, YH1885 is cleared by hepatic mechanisms, including both catabolism and biliary elimination (Ahn et al., 1997; Han et al., 1998). Intact YH1885 is not detectable in the urine after either i.v. or oral administration of the drug in rats and dogs. After intraportal administration at a dose of 5 mg/kg of rat, about 30% of the administered YH1885 undergoes hepatic first-pass metabolism (Han et al., 1998). The oral bioavailability of YH1885 in rats and dogs was found to vary over the range of 47 to 17%, showing a dose-dependent decrease for the dose range of 2 to 500 mg/kg (Han et al., 1998; Kim et al., 1998). However, the issue of whether the observed low and dose-dependent bioavailability is due to absorption is not clear, based on currently available data. Thus, we studied the transepithelial flux of YH1885 across a human colonic cell line monolayer, Caco-2. This cell line is a well established model for human intestinal absorption (Hidalgo et al., 1989; Hilgers et al., 1990; Cogburn et al., 1991). The uptake of YH1885 into the cells was also studied.

Experimental Procedures

Materials. [¹⁴C]YH1885, unlabeled YH1885, and its structural analogs (YH957, YH1070, YH1013, and YH1041) were synthesized at Yuhan Research Center. Their structures and the position of labeling are shown in Fig. 1. [¹⁴C]YH1885, which was more than 98.7% pure, had a specific activity of 26 mCi/mmol by a thin-layer chromatography. [¹⁴C]Mannitol (50 mCi/mmol, New England Nuclear, Boston, MA), fetal bovine serum (Hyclone Laboratories, Logan, UT), trypsin-EDTA (Life Technologies, Inc., Gaithersburg, MD), Dulbecco's modified Eagle's medium, nonessential amino acid solution, penicillin-streptomycin, HBSS, HEPES, and MES (all from Sigma Chemical Co., St. Louis, MO) were used as purchased. All other reagents were of analytical grade.

Caco-2 Cell Culture. The human colon adenocarcinoma cell line, Caco-2 (American Type Culture Collection, Rockville, MD), was grown as monolayers, in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% nonessential amino acid solution, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in an atmosphere of 5% CO₂ and 90% relative humidity. Stock cultures were grown in 75-cm² tissue culture flasks and were split 1:3 at 80 to 90% confluency using 0.02% EDTA and 0.05% trypsin. The Caco-2 cells from passage numbers of 36 to 55 were seeded on the permeable polycarbonate inserts (1-cm², 0.4-µm pore size; Corning Costar Corp., Cambridge, MA) in 12 Transwell plates at a density of 1 to 1.5 × 10⁵ cells/insert. The inserts were fed by complete media every 2 days for the first week and then daily until they were used for experiments 18 to 25 days after the seeding (Augustijns et al., 1993). The integrity of the cell monolayers was evaluated by measuring transepithelial electrical resistance values with a EVOM epithelial volt/ohmmeter (World Precision Instruments, Sarasota, FL). The cell inserts were used for experiments when the resistance reached 300 to 600  cm². In each experiment, the transport of [¹⁴C]mannitol was measured in two inserts. The cell monolayers were considered tight when the mannitol transport was <0.35% of the dose/h, corresponding to a P_app value of 4.8 × 10⁸ cm/s.

Transepithelial Transport. Before the transport experiments, the cell monolayers were washed twice with the incubation medium (pH 7.4, HBSS containing 25 mM HEPES and 25 mM glucose). After each wash, the plates were incubated for 30 min at 37°C, and the transepithelial electrical resistance was then measured. The incubation medium on both sides of the cell monolayers was then removed by aspiration (Augustijns et al., 1993).

Cellular Uptake of YH1885. The incubation medium (0.5 ml) containing [¹⁴C]YH1885 (0.2-5.0 µM) and dimethylsulfoxide (1%) and 1.5 ml of the incubation medium without the drug were added to the apical and basolateral sides, respectively, of the grown Caco-2 cell monolayers in the Transwell insert, and the insert was incubated at 37°C for 60 s. At 20, 40, and 60 s, the incubation medium in both sides was removed by aspiration. Both sides of the monolayer were then washed rapidly twice with 0.5 (for apical side) and 1.5 ml (for basolateral side) of an ice-cold incubation buffer containing cold YH1885 (50 µM) and dimethylsulfoxide (4%). The monolayer was then detached from the insert and transferred to a scintillation vial, which contained 0.5 ml of cell digestive solution, i.e., 0.1% (w/v) Triton X-100 in 0.3 N NaOH. After an overnight digestion at room temperature, 4 ml of the scintillation cocktail was added to the vial, and the radioactivity was measured by liquid scintillation counting.

Stability of YH1885. The chemical stability of YH1885 in Caco-2 cells during the transport experiments was examined. After preincubation of the monolayers for 30 min, the incubation medium was removed and the filters with or without cell monolayers were detached from the inserts. The incubation medium (0.5 ml) containing YH1885 (5 µM) was added to the filters and the incubation continued for 1 h. At the end of the incubation, methanol (0.5 ml) was added (Inui et al., 1992) and the suspension was vortexed for 1 h at room temperature. Aliquots of the resultant solution were analyzed by high performance liquid chromatography (Han et al., 1997) after centrifugation at 10,000 rpm for 15 min to determine the remaining YH1885.

Calculations. For each transport experiment, the slope of the linear portion of the plot of the total amount of YH1885 transported versus time (i.e., 15-, 30-, 45-, and 60-min time points for a monolayer) was obtained by a linear least regression, and the slope was regarded as a transport rate (pmol/cm²/min) because the surface area of the monolayer membrane (A) was 1 cm² in the present study. The transport clearance (µl/cm²/min) of YH1885 was calculated by dividing the transport rate ( Q/ t) by the initial concentration of the drug in the donor chamber (C_o). The rate and clearance were determined for each monolayer of Caco-2 cells. The apparent permeability values (P_app) of YH1885 across the Caco-2 cell monolayer, expressed in centimeters per second, were calculated as  Q/ t × 1/A ×1/C_o. Three to eight monolayers were used in the determination of mean (±S.D.) for the rate, clearance, and P_app.

	Results

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Transepithelial Transport of YH1885. The present study was designed to minimize the adsorption of YH1885 to a Transwell chamber. Under this design, adsorption to the apical or basolateral side of the Transwell chamber was minimal (for example, 6.3 ± 0.4 or 11.4 ± 4.9% of the dose, respectively, in mean ± S.D., n = 3 for 0.28 µM). A representative flux of YH1885 across the Caco-2 cell monolayers, when the drug was loaded on either the apical or basolateral side of the cells, is shown in Fig. 2. As can be seen, the flux was essentially linear for periods of up to 60 min for all YH1885 concentrations studied (0.28-3.4 µM). The flux from the apical to the basolateral side (Fig. 2A) was 3- to 5-fold greater than that from the basolateral to the apical side (Fig. 2B). The decomposition and cell-mediated metabolism of YH1885 during these experiments seemed almost negligible, since most of the YH1885 (94.21 ± 8.35% of dose, n = 5) was recovered after the incubation with the cell monolayer for 1 h.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time course for the transepithelial transport of YH1885 by Caco-2 cell monolayers. For the apical to basolateral flux (A), 0.5 ml of incubation medium (pH 7.4) containing dimethylsulfoxide (1%) and various concentrations of YH1885 was added to the apical side, and 1.5 ml of the incubation medium (pH 7.4) without the drug was added to the basolateral side. For the basolateral to apical flux (B), 1.5 ml of incubation medium (pH 7.4) containing dimethylsulfoxide (1%) and various concentrations of YH1885 were added on the basolateral side, and 0.5 ml of the incubation medium (pH 7.4) without the drug was added on the apical side. The concentrations of YH1885 in the transport studies were 0.28 (), 0.53 (), 0.77 (), 1.2 (), 2.4 (), and 3.4 µM (). Each point represents the mean value of three experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of YH1885 transport across Caco-2 cell monolayers. The apical to basolateral () and the basolateral to apical () fluxes obtained in Fig. 2 were plotted against the concentration of YH1885.

Uptake of YH1885 into Caco-2 Cell Monolayers. The uptake of YH1885 into the Caco-2 cell monolayers from the apical side was examined for the concentration range of 0.2 to 5.0 µM. The uptake was nearly linear up to 60 s, irrespective of the YH1885 concentration examined (data not shown). Thus, the uptakes for various concentrations of YH1885 were determined from the respective uptake values at a 40-s time point after the incubation. Figure 4 shows the effect of concentration of YH1885 on its apparent uptake rate into the Caco-2 cell monolayers from the apical side. As can be seen from the figure, the uptake rate was clearly saturable for the concentration range examined, indicating a possible involvement of carrier systems in the uptake process. The apparent K_m and V_max values, as evaluated by fitting the data to the Michaelis-Menten equation, were 1.47 ± 0.21 µM and 25.14 ± 1.16 pmol/cm²/40 s, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of YH1885 uptake by Caco-2 cell monolayers. An incubation medium (0.5 ml, pH 7.4) containing dimethylsulfoxide (1%) and varying concentrations of YH1885 was added on the apical side of the monolayer, and incubation medium (1.5 ml, pH 7.4) without the drug was added on the basolateral side of the Transwell insert, followed by incubation at 37°C. The initial uptakes at 40 s after the incubation start were then plotted against the corresponding YH1885 concentrations.

Effect of Na⁺ on YH1885 Transport and Uptake. When extracellular sodium chloride was replaced by equimolar amounts (140 mM) of potassium chloride or choline chloride, no significant change was observed for either the transport or uptake of YH1885 (Table 1), indicating that the transport of YH1885 across the Caco-2 cell monolayer is independent of extracellular Na⁺. Thus, in the subsequent experiments, HBSS buffer, which contains sodium chloride (140 mM), was used in the preparation of the incubation medium.

Effect of Proton on YH1885 Transport. The effect of apical pH (5.5-7.4) on the transport of 0.5 µM YH1885 from the apical to basolateral side was examined under the constant pH of the basolateral side (7.4). As shown in Fig. 5, the transport was influenced by the apical pH and showed a maximal transport at an apical pH of 6.5. On the other hand, a proton-ionophore, FCCP, at a concentration of 10 µg/ml on the apical side, significantly inhibited (P < 0.05) the uptake of 0.5 µM YH1885 into the cell monolayer (Table 3). These results appear to indicate that the transport system for YH1885 is H⁺-dependent. Thus, subsequent experiments were performed under the conditions of this pH gradient (6.5/7.4 for apical/basolateral pH). However, substrates for the H⁺-dependent transporters, including organic cations (brompheniramine, tetraethylammonium, and choline, at 5 mM) (Mizuuchi et al., 1999), organic anions (benzylpenicillin and cefodizime, at 5 mM) (Nohjoh et al., 1989; Hirohashi et al., 2000), dipeptides (glycine-proline and glycine-leucine, at 5 mM) (Inui et al., 1992), and folates (folic acid and methotrexate, at 100 µM) (Said et al., 1987) had no effect on the transport of 0.5 µM YH1885 in the proton-gradient medium (Table 2).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of pH in the apical side on the apical to basolateral transport clearance of YH1885 across Caco-2 cell monolayers. An incubation medium (0.5 ml) at various pH values and containing YH1885 (0.5 µM) and dimethylsulfoxide (1%) was added to the apical side, and the incubation medium (1.5 ml, pH 7.4) without the drug was added to the basolateral side. The apical to basolateral transport of YH1885 across the monolayers at 37°C was measured for a 60-min period, and the transport clearance was calculated from the flux divided by the initial YH1885 concentration in the apical side (Co).

Effect of Metabolic Inhibitors on YH1885 Uptake. The effect of the metabolite inhibitors on the initial uptake of 0.5 µM YH1885 by the cell monolayers from the apical side was measured under the pH gradient defined above (6.5/7.4 for apical/basolateral side). In these experiments, the glucose was removed from the incubation medium. The uptake was decreased significantly (P < 0.05) by the presence of both 2,4-dinitrophenol (1 mM) and sodium azide (10 mM) on the apical side (Table 3), suggesting involvement of energy-dependent transport on the uptake of YH1885.

Effect of Nucleobases, Nucleosides, and Transport Inhibitors on YH1885 Transport. Because YH1885 has a pyrimidine moiety in its chemical structure, a possible relation with nucleobase-related compounds in the transport of YH1885 was examined. Table 2 shows the effects of various nucleobases, nucleosides, and their transport inhibitors on the apical to basolateral transport of 0.5 µM YH1885 under the pH gradient conditions (6.5/7.4 for apical/basolateral pH). At concentrations of 4 mM, uracil and 5-methyluracil (pyrimidine nucleobases) significantly inhibited (P < 0.05) the transport of YH1885, whereas 5-fluorouracil (pyrimidine nucleobase), hypoxanthine and adenine (purine nucleobases), and uridine and guanosine (nucleosides) had no effect on the transport. At concentrations below 100 µM, none of these compounds had any effect on the transport (data not shown). The classical nucleobase inhibitors, papaverine (100 µM), dipyridamole (100 µM), and phloridzin (1 mM) significantly inhibited the transport of YH1885 (P < 0.01).

Effect of Structural Analogs on YH1885 Transport. The effect of four structural analogs of YH1885 on the transport of 0.5 µM YH1885 from the apical to basolateral side across the Caco-2 cell monolayers was examined under the pH gradient condition (6.5/7.4 for apical/basolateral pH). As shown in Table 2, most of the analogs inhibited the transport of YH1885 at a concentration of 100 µM, with YH957 exhibiting the most potent inhibition (41% of the control), followed by YH1070 and YH1041 (P < 0.01 for all). On the other hand, YH1013 (100 µM) had no effect on the transport of YH1885.

	Discussion

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The transepithelial transport of YH1885, a reversible proton pump inhibitor, across Caco-2 cells was investigated to characterize the intestinal absorption of the drug at the cellular level. Possible involvement of carriers in the absorption of YH1885 was suggested from the saturable apical to basolateral transport (Fig. 3) and apical uptake (Fig. 4) and from the inhibited transport by structural analogs (Table 2). Additional data indicate that YH1885 is transported energy dependently (Table 3) from the apical side to the basolateral side probably via a H⁺-dependent (Fig. 3) and Na⁺-independent (Table 1) carrier system that has a high affinity for YH1885, but not for organic cations, organic anions, dipeptides, folates, nucleosides, and purine nucleobases (Table 2). The carrier system appears to be involved for the transport of pyrimidine nucleobases (Griffith and Jarvis, 1996; Shayeghi et al., 1999) such as uracil and 5-methyluracil (Table 2). The possible involvement of the nucleobase transport system(s) in the absorption of YH1885 is consistent with the facts that YH1885 is a pyrimidine analog and intestinal enterocytes use nucleobases derived from extracellular sources (Griffith and Jarvis, 1996). These transport systems are of considerable pharmacological interest since transport inhibitors for these systems can enhance the effectiveness of various substances used in the chemotherapy of tumors and viral infections, for example, by modulating drug influx and efflux or by inhibiting salvage pathways (Isono, 1991; Griffith and Jarvis, 1996).

Although the mechanisms responsible for the transport and uptake of YH1885 cannot be fully characterized, based on the present study, the intestinal absorption of YH1885 at its low concentration (e.g., 3.4 µM in the present study) is expected to be fairly good, when evaluated based on the apical to basolateral P_app value (9.1 × 10⁶ cm/s at 1.2-3.4 µM), which is approximately 10-fold higher than 10⁶ cm/s, a critical P_app value for acceptable absorption (Artursson and Karlsson, 1991). The absorption of YH1885 has a pH optimum (i.e., 6.5, Fig. 5), and the drug inhibits the secretion of proton (Hwang et al., 1998). However, YH1885 is not likely to limit significantly the absorption of the drug itself because the optimal pH is not so acidic but lies around the neutral region.

The oral bioavailability of YH1885 in rats decreased as the dose of the drug increased in the range of 2 to 500 mg/kg (Han et al., 1998; Kim et al., 1998). Assuming complete dissolution in the intestinal fluid and intestinal fluid volume of 45 ml/kg of rat (Davies and Morris, 1993), the resulting initial concentration of YH1885 in the fluid is calculated to be 111 µM-28 mM. However, the actual concentration of YH1885 in the intestinal fluid would remain below several micromolar even in higher doses due to its limited solubility in the water (i.e., ~5.3 µM). Thus, in the dose range of 2 to 500 mg/kg, a substantial fraction of the dose might remain undissolved in the intestine, leading to poor bioavailability. In addition, the solubility of YH1885 itself (~5.3 µM) slightly exceeds the apparent K_m values for the uptake (1.47 µM) and transport (0.54 µM) in the present study (Figs. 3 and 4), possibly leading to saturation of the intestinal transport systems that are responsible for the absorption of the drug. Thus, primarily the limited solubility of the YH1885 and secondarily the saturation of the intestinal absorption system appear to be responsible for the dose-dependent decrease in the bioavailability of YH1885 in the dose range of 2 to 500 mg/kg of rat.

When administered orally to human subjects at high doses, YH1885 is likely to be dissolved slowly to the intestinal fluid, thereby resulting in an extended absorption of the drug. Extended absorption of YH1885 at its high doses is consistent with the fact that the time to reach the peak plasma drug concentration (T_max) following oral administration of the drug to rats increased as the dose increased (from 1.0 h to 3.2 h for 2 and 300 mg/kg dose, respectively; Kim et al., 1998).

YH1885 undergoes a considerable first-pass effect (i.e., 30% for i.p. dose of 5 mg/kg; Han et al., 1998). For such drugs, oral bioavailability generally increases as the dose increases due to the saturation of the metabolism. However, for drugs with extremely limited solubility, the bioavailability of the drug would decrease as the dose increases, as exemplified in the present study for YH1885, primarily due to incomplete dissolution of the drug at the absorption site. Thus, solubility and transport issues of a drug should not be underestimated with respect to bioavailability in cases in which they are likely to overwhelm the first-pass metabolism of the drug.