HONG LI, SUK-JAE CHUNG, DONG-CHOOL KIM, HYUN-SOO KIM, JONG-WOOK LEE, AND CHANG-KOO SHIM

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1 /01/ $3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 1 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 44/ DMD 29:54 59, 2001 Printed in U.S.A. THE TRANSPORT OF A REVERSIBLE PROTON PUMP ANTAGONIST, 5,6-DIMETHYL-2-(4- FLUOROPHENYLAMINO)-4-(1-METHYL-1,2,3,4-TETRAHYDROISOQUINOLINE-2-YL) PYRIMIDINE HYDROCHLORIDE (YH1885), ACROSS CACO-2 CELL MONOLAYERS HONG LI, SUK-JAE CHUNG, DONG-CHOOL KIM, HYUN-SOO KIM, JONG-WOOK LEE, AND CHANG-KOO SHIM Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul, Korea (H.L., S.J.C., C.K.S.); College of Pharmacy, Chungnam National University, Taejon, Korea (D.C.K); and Yuhan Research Center, Yuhan Corporation, Kyunggi-do, Korea (H.S.K., J.W.L.) (Received March 7, 2000; accepted September 21, 2000) This paper is available online at ABSTRACT: 5,6-Dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (YH1885) is under development as a novel acid pump antagonist by Yuhan Research Center. Previous studies have suggested that the AUC and C max of orally dosed YH1885 are dose-dependent in the range of 2 to 500 mg/kg. The objective of the present study was to investigate the absorption mechanism of YH1885 using a human colon carcinoma cell line, Caco-2. The cells were grown to confluency on a permeable polycarbonate membrane insert to permit loading of YH1885 on either the apical or basolateral side of the cell monolayer. The flux across the monolayer from the apical to basolateral side was 3 to 5 times greater than that from the basolateral to apical side. The uptake of YH1885 into the Caco-2 cell monolayer was saturable and appeared to be mediated by a high-affinity transporter, 5,6-Dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (YH1885 1, 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). When administered by i.v. injection, YH1885 is cleared by hepatic mechanisms, including both catabolism and biliary elimination (Ahn This work was supported by Grant HMP-99-D from the Ministry of Health and Welfare of Korea. 1 Abbreviations used are: YH1885, 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1- methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine hydrochloride; YH957, 2-[(4- hydroxyphenyl)amino]-4-(1,2,3,4-tetrahydroisoquinoline-2-yl)quinazoline hydrochloride; YH1041, 2-methyl-4-(4,5,6,7-tetrahydrothieno[3,2-C]pyridine-5-yl)quinazoline hydrochloride; YH1070, 8-methoxy-4-(2-methoxyphenylmethyl-N-methylamino)-2- methylquinazoline hydrochloride; YH1013, 2-methyl-4-[(thiophen-2-yl)methylamino]quinazoline hydrochloride; HBSS, Hanks balanced salt solution; MES, 2-(N-morpholino)ethane sulfonic acid; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. Send reprint requests to: Chang K. Shim, Ph.D., Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul , Korea. shimck@plaza.snu.ac.kr 54 with an apparent K m of M and a V max of pmol/cm 2 /40 s. The apical to basolateral transport across the monolayer was Na -independent, H -sensitive, and energydependent. The transport was inhibited significantly by the presence of structural analogs of YH1885 (e.g., YH957, YH1070, and YH1041), some pyrimidine nucleobases (uracil and 5-methyluracil), and nucleobase transport inhibitors (e.g., papaverine, dipyridamole, and phloridzin). These results demonstrate that the apical to basolateral transport of YH1885 across the Caco-2 cell monolayer is partially mediated by a nucleobase transport system, which exhibits high-affinity and energy-dependent properties for YH1885. Saturation of this transport system, in addition to the limited solubility of YH1885 (i.e., 5.3 M), appears to contribute to the dose-dependent bioavailability of the drug. 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. [ 14 C]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. [ 14 C]YH1885, which was more than 98.7% pure, had a specific activity of 26 mci/mmol by a thin-layer chromatography. [ 14 C]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.,

2 YH1885 TRANSPORT IN CACO-2 CELLS 55 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% CO 2 and 90% relative humidity. Stock cultures were grown in 75-cm 2 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-cm 2, 0.4- m pore size; Corning Costar Corp., Cambridge, MA) in 12 Transwell plates at a density of 1 to 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 cm 2. In each experiment, the transport of [ 14 C]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 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 FIG. 1.Chemical structures of YH1885 and its analogs. * 14 C. was then measured. The incubation medium on both sides of the cell monolayers was then removed by aspiration (Augustijns et al., 1993). For the measurement of the apical to basolateral transport, 0.5 ml of the incubation medium containing [ 14 C]YH1885 ( M) and dimethylsulfoxide (1%) was added on the apical side, and 1.5 ml of the incubation medium without the drug was added on the basolateral side. The inserts were moved to wells containing fresh incubation medium every 15 min for 1 h. In each transport experiment, three inserts were used. At the end of the experiment, 20 l of 1 mm YH1885 was added to the basolateral side, and the incubation continued for 1 h at 37 C to minimize the adsorption of the transported [ 14 C]YH1885 to the basolateral side. After the incubation, the entire volume of the medium in the basolateral side was transferred into a scintillation vial containing 6 ml of scintillation cocktail (Ultima Gold, Packard, Meriden, CT) using a 200- l disposable pipet tip, and placed (along with the tip) in vials for liquid scintillation counting. To maximize the extraction recovery of [ 14 C]YH1885 from the basolateral side, 2 ml of the fresh incubation medium containing YH1885 (10 M) and dimethylsulfoxide (10%) was then added to the basolateral side, the incubation was continued overnight, and the entire volume of the side was again transferred to another scintillation vial for the liquid scintillation counting. The amount of [ 14 C]YH1885 transported was calculated from the sum of the radioactivity in the first and second vials (Augustijns et al., 1993). For the measurement of the basolateral to apical transport, 0.5 ml of the incubation medium containing [ 14 C]YH1885 ( M) and dimethylsulfoxide (1%) was added on the basolateral side, and 0.5 ml of the incubation

3 56 LI ET AL. medium without the drug was added to the apical side. In each experiment, three inserts were used. The inserts were then incubated at 37 C, and the incubation medium in the apical side was replaced by the fresh medium at 15-min intervals. The radioactivity in a 0.4-ml aliquot of each 15-min sample was determined as described above. The effect of apical ph ( ) on the transport of 0.5 M [ 14 C]YH1885 from the apical to the basolateral side was examined under constant ph of the basolateral side (7.4). The ph of the apical side was varied by substituting appropriate amounts of HEPES in the incubation medium by equimolar (25 mm) MES. In experiments to investigate the effect of various compounds on the apical to basolateral transport of 0.5 M [ 14 C]YH1885, nucleobases, nucleosides and their inhibitors, and structural analogs of YH1885 were added to the incubation medium on the apical side of the cell monolayer under the ph gradient condition (6.5/7.4 for apical/basolateral side). In experiments to investigate the effect of Na on the transport and uptake of [ 14 C]YH1885 across and into the Caco-2 cell monolayers (0.5 and 2.0 M for the transport and uptake experiments, respectively), the sodium chloride in the HBSS was replaced by equimolar amounts (140 mm) of potassium chloride or choline chloride. Cellular Uptake of YH1885. The incubation medium (0.5 ml) containing [ 14 C]YH1885 ( M) 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 M) 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 M) 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/cm 2 /min) because the surface area of the monolayer membrane (A) was1cm 2 in the present study. The transport clearance ( l/cm 2 /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. For the cellular uptake study, an apparent linear initial rate was calculated from the linear portion of the uptake versus time profiles. Kinetic parameters according to the Michaelis-Menten equation were calculated by nonlinear regression analysis (Yamaoka et al., 1981) of the rate versus concentration profiles of YH1885: V V max [S]/(K m [S]), where V is the apparent linear initial rate, [S] the initial concentration, V max the maximum uptake rate, and K m the Michaelis-Menten constant for YH1885. The initial uptake rate and Michaelis-Menten parameters were determined for each monolayer of Caco-2 cells, and three different batches were used in the determination of the means ( S.D.). The statistical significance of differences between treatments was evaluated using unpaired Student s t tests, and a value of P 0.05 was considered statistically significant. Results 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, or % of the dose, respectively, in mean S.D., n 3 for 0.28 M). 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 ( M). 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 cellmediated metabolism of YH1885 during these experiments seemed almost negligible, since most of the YH1885 ( % of dose, n 5) was recovered after the incubation with the cell monolayer for 1h. The influence of the concentration of YH1885 on its flux across the Caco-2 cell monolayers was examined (Fig. 3). The range of YH1885 concentration was 0.28 to 3.4 M in this study due to its limited solubility in the incubation medium ( 5.3 M). The apical to basolateral flux was clearly saturable, indicating the possible involvement of carrier-mediated transport. For the basolateral to apical flux, only a slight trend in terms of saturation was observed, indicating a minimal contribution of carrier-mediated transport to this flux. Assuming passive diffusion for the basolateral to apical transport, the carriermediated transport from the apical to basolateral side was calculated (apical transport basolateral transport), and a Lineweaver-Burk plot for the carrier-mediated transport yielded an apparent K m value of M and a V max value of pmol/cm 2 /min. 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 M. 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 M and pmol/cm 2 /40 s, respectively. 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 ( ) on the transport of 0.5 M 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 g/ml on the apical side, significantly inhibited (P 0.05) the uptake of 0.5 M YH1885 into the cell monolayer (Table 3). These results

4 YH1885 TRANSPORT IN CACO-2 CELLS 57 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 (F), 0.53 (E), 0.77 ( ), 1.2 (ƒ), 2.4 (f), and 3.4 M ( ). Each point represents the mean value of three experiments. 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 M) (Said et FIG. 3.Concentration dependence of YH1885 transport across Caco-2 cell monolayers. The apical to basolateral (F) and the basolateral to apical (E) fluxes obtained in Fig. 2 were plotted against the concentration of YH1885. 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. al., 1987) had no effect on the transport of 0.5 M YH1885 in the proton-gradient medium (Table 2). Effect of Metabolic Inhibitors on YH1885 Uptake. The effect of the metabolite inhibitors on the initial uptake of 0.5 M 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 com-

5 58 LI ET AL. TABLE 1 Effect of replacement of sodium chloride in the incubation medium in the apical side by equimolar (140 mm) potassium chloride or choline on the uptake and apical to basolateral transport of YH1885 into and across Caco-2 cell monolayers The concentrations of YH1885 in the uptake and transport experiments were 2.0 and 0.5 M, respectively. The experiments were performed under the ph gradient condition of 6.5/7.4 for apical/basolateral. Data represent the mean S.D. of three experiments. Cation Uptake Rate Transport Rate pmol/cm 2 /40 s pmol/cm 2 /min Na (control) K Choline 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 M) 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 (C o ). pounds 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 M 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 M, none of these compounds had any effect on the transport (data not shown). The classical nucleobase inhibitors, papaverine (100 M), dipyridamole (100 M), 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 M 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 M, 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 M) had no effect on the transport of YH1885. TABLE 2 Effect of various compounds on the apical to basolateral transport of 0.5 M YH1885 under the ph gradient condition 6.5/7.4 for apical/basolateral ph For each transport experiment, the slope of the linear portion of the plot of the total amount of YH1885 transported versus time was divided by the initial concentration of the drug in the donor chamber for the calculation of the transport clearance. Data represent mean S.D. of three (except eight for organic cations and organic anions) experiments. Compounds Concentration YH1885 Transport Clearance Control mm /cm 2 /min % Control Organic cations Brompheniramine Tetraethylammonium Choline Organic anions Benzylpenicilin Cefodizime Dipeptides Glycine-proline Glycine-leucine Folates Folic acid Methotrexate Nucleobases Uracil * Methyuracil * Fluorouracil Hypoxanthine Adenine Nucleosides Guanosine Uridine Nucleobase inhibitors Papaverine ** 81.6 Dypiridamole ** 73.1 Phloridzin ** 68.4 Structure analogs YH ** 37.3 YH ** 56.7 YH ** 61.3 YH Significantly different from the Student s t test, * P 0.05, ** P TABLE 3 Effect of an ionophore and metabolic inhibitors on the uptake of 0.5 M YH18855 After preincubation of the Caco-2 cell monolayer for 15 min with the indicated compounds on the apical side, the uptake of 0.5 M YH1885 from the apical side was measured under the ph gradient conditions (6.5/7.4 for apical/basolateral ph). In these experiments, glucose was removed from the incubation medium in the apical and basolateral sides. Data represent mean S.D. of three experiments. Ionophore or Metabolic Inhibitors Uptake Discussion 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 de- Rate Control pmol/cm 2 /40 s % Control FCCP (10 g/ml) * ,4-Dinitrophenol (1 mm) * 82.4 Sodium azide (10 mm) * 79.5 * P 0.05 from Student s t test.

6 YH1885 TRANSPORT IN CACO-2 CELLS 59 pendently (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 M in the present study) is expected to be fairly good, when evaluated based on the apical to basolateral P app value ( cm/s at M), which is approximately 10-fold higher than 10 6 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 M 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 M). 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 M) slightly exceeds the apparent K m values for the uptake (1.47 M) and transport (0.54 M) 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. 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