Introduction

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It is widely accepted that some orally active -lactam antibiotics, such as cyclacillin (ACPC; Takahashi et al., 1998), cephradine (Okano et al., 1986), cephalexin (CEX; Dantzig and Bergin, 1990), cefixime (Tsuji et al., 1987; Inui et al., 1992), and ceftibuten (Muranushi et al., 1989; Matsumoto et al., 1995), are transported by the proton-coupled oligopeptide transporter, which has been demonstrated by using brush border membrane vesicles (BBMV), human intestinal Caco-2 cells, and transporter expression systems. It is believed that these -lactam antibiotics are recognized by the oligopeptide transporter due to their peptide-like structures. Recently, another proton-cotransport system, the monocarboxylic acid transport system(s), has been found in the enterocyte of rat and rabbit and also in Caco-2 cells (Tamai et al., 1995). It transports not only monocarboxylic acids, such as L-lactic, salicylic, and benzoic acids (Tiruppathi et al., 1988; Takanaga et al., 1994; Tsuji et al., 1994), but also some monoanionic -lactams, such as cefdinir (Tsuji et al., 1993) and phenethicillin (Itoh et al., 1998). Therefore, the multiplicity in the absorption mechanism of -lactams appears to exist.

Carbenicillin (CBPC; Fig. 1), a semisynthetic penicillin, is a mixture of two epimers because of the chirality of the side chain (Bird and Steele, 1982; Itoh and Yamada, 1995). It has been used parenterally in clinical practice due to its instability in acidic conditions. Its low lipophilicity also makes the drug relatively impermeable through the intestinal wall (Yamana et al., 1974). Carindacillin (CIPC; Fig. 1), a 5-indanyl ester of CBPC, is orally active and the absorbed CIPC is hydrolyzed rapidly to generate CBPC in the enterocyte and liver (Clayton et al., 1975). Although there is a chiral carbon in the side chain, no information is available regarding its chirality. It has been thought that the improved absorption of CIPC is due not only to its improved acid stability but also to the increased membrane permeability. Because CIPC is much more lipophilic than CBPC, it is likely that CIPC is more permeable through the intestinal membrane. On the other hand, Tanigawara et al. (1982) reported the nonlinear absorption of CIPC in humans and suggested the possibility of carrier-mediated process in CIPC absorption. Because CIPC exists as a monoanion at physiological pH, it may be transported either by the oligopeptide transporter or the monocarboxylic acid transport systems, or by both of them. According to our study using rat intestinal BBMV, CIPC was predominantly transported by the monocarboxylic acid transport system(s) (Y-H.L., T.I., H.Y., and M. Tarro, unpublished observations).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemical structures of CIPC and CBPC. *, chiral carbon in the side chain.

Caco-2 cells have been used extensively to characterize the absorption of many compounds (Inui et al., 1992; Tsuji et al., 1994; Matsumoto et al., 1995) because the cells exhibit both morphological and functional similarities to the human small intestinal epithelial cells (Hilgers et al., 1990). The oligopeptide, monocarboxylic acid, and folic acid transport systems that exist in the small intestine are also expressed in Caco-2 cells (Inui et al., 1992; Tsuji et al., 1994; Prasad et al., 1995). A good correlation between the transport through Caco-2 cells and the absorption in humans is also obtained for several compounds (Artursson and Karlsson, 1991). To clarify whether carrier-mediated transport mechanisms are involved in the absorption of CIPC, uptake, transport, and accumulation of CIPC were studied using Caco-2 cells.

	Experimental Procedures

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Materials. CIPC and ACPC were kindly donated by Pfizer (Tokyo, Japan) and Takada Pharmaceutical Co. (Tokyo Japan), respectively. CBPC (R/S = ca. 1.2), CEX, sodium salicylate, oligopeptides (Gly-Sar, Gly-Pro, and Ala-Ala), carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone (FCCP), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) were purchased from Sigma Chemical Co. (St. Louis, MO). L-Lactic acid, acetic acid, succinic acid, and sodium azide (NaN₃) were purchased from Wako Pure Chemical industries, Ltd. (Osaka, Japan). Sodium benzoate was purchased from Kanto Chemical Co. (Tokyo, Japan). L-[¹⁴C]Lactic acid sodium salt was purchased from DuPont NEN Products (Boston, MA). All other chemicals were of the highest grade available.

Cell Culture. Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD) at passage 18. Cells were cultured on flasks (150 cm²; Falcon, Becton Dickinson Co., Ltd., Oxnard, CA) and maintained at 37°C in a humidified atmosphere containing 5% of CO₂. The culture medium consisted of Dulbecco's modified Eagle's medium, 1% nonessential amino acids, 2% Gluta MAX-1, and 10% fetal bovine serum, which were all obtained from Life Technologies (Gaithersburg, MD). For the uptake study, the cells were seeded in a dish (9.6 cm²; Falcon) at a density of 1 × 10⁵ cells/ml. For the transport and accumulation study, cells were seeded on a polycarbonate filter (3-µm pores, 4.71 cm²; Costar, Cambridge, MA) at a density of 6 × 10⁴ cells/cm². The culture medium was changed every 2 or 3 days and the cells were used for the experiments between 21 and 28 days after seeding. Before the transport study, the transepithelial electrical resistance (TEER) was measured using Millicell-ERS (Millipore Co., Bedford, MA) to check the integrity of the monolayer. Monolayers with TEER above 400 cm² were used for transport studies. Cells between the 29th and 40th passages were used in the present uptake and transport studies.

Uptake and Transport Studies. Hank's balanced salt solution (137 mM NaCl, 1.0 mM CaCl₂, 5.4 mM KCl, 0.8 mM MgSO₄, 0.4 mM KH₂PO₄, and 0.4 mM Na₂HPO₄) containing 10 mM D-glucose and 10 mM 2-(N-morpholino)ethanesulfonic acid (for pH 6.0) or 10 mM HEPES (for pH >6.0) was used as the uptake and transport medium.

Stability Study. Stability of CIPC and CBPC in Caco-2 cell homogenate was measured by incubating the drug with cell homogenate. Cell homogenate was prepared by the method of Annaert et al. (1997) with some modification. Cells grown in three dishes (60 cm²) were scraped and collected in 5 ml of ice-cold transport medium (pH 7.4) and then homogenized with a glass/Teflon Potter homogenizer (Type SM-3; Shimada Co., Tokyo, Japan) at 1000 rpm for 10 strokes. The homogenate was centrifuged at 8000g for 10 min (Himac CP 70G, rotor RP-50T; Hitachi Co., Japan) at 4°C and the supernatant was used as the cell homogenate (protein concentration = 1.8 mg/ml). One milliliter of 1 mM CIPC or CBPC was added to the same volume of the cell homogenate and incubated at 37°C. At the predetermined time, a 100-µl aliquot was sampled from the mixture and 10 µl was injected onto HPLC immediately.

Analytical Methods. The HPLC system of Hewlett-Packard 1050 series (Hewlett-Packard Co., Palo Alto, CA) was used for the analyses of CIPC and CBPC. The analytical column was Excelpak SIL-C18 (4.6 × 150 mm; Yokogawa Co., Tokyo, Japan). The mobile phase was a mixture of 20 mM ammonium acetate and methanol with gradient conditions to measure CIPC and its degradation products, CBPC and benzylpenicillin (PCG), simultaneously. The methanol content was 12% for the first 17 min to elute CBPC, and then the methanol content was increased to 65% to elute PCG and CIPC. The flow rate was 0.8 ml/min and CIPC, CBPC, and PCG were detected at 220 nm. The uptake and transport of CIPC was calculated as the total of CBPC, PCG, and CIPC. Although the epimers of CBPC were resolved using the present HPLC conditions, a single peak was observed for CIPC.

Data Analysis. Apparent uptake clearance (CL_uptake) was calculated as the following equation (eq. 1)

(1)

where C₀ is the initial concentration of the drug. Uptake in 2 or 5 min was used to calculate the initial uptake rate of CIPC and CBPC, respectively.

(2)

Statistical analysis. Student's t test was used for statistical analyses with a P value of less than 0.05 being considered statistically significant.

	Results

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Stability of CIPC and CBPC in the Presence of Caco-2 Cell Homogenate. As shown in Fig. 2, CIPC was hydrolyzed and converted to CBPC when incubated with Caco-2 cell homogenate (pH 7.4). The hydrolysis occurred at a first-order rate with a half-life of approximately 18 min. In contrast, CBPC was stable under the same conditions. After 2 h of incubation, almost all CIPC converted to CBPC, whereas the disappearance of CBPC was only 5% in the same period. PCG was not detected when CIPC or CBPC was incubated with Caco-2 cell homogenate. No degradation of CIPC or CBPC was observed in the transport medium (pH 7.4) at 37°C without cell homogenate (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Stability of CIPC and CBPC when incubated with Caco-2 cell homogenate. CIPC (0.5 mM, ) or CBPC (0.5 mM, ) was incubated with cell homogenate at pH 7.4 at 37°C.  Represent CBPC generated from CIPC.

Time Courses of Uptake of CBPC and CIPC. Time courses of the uptake of CBPC and CIPC into Caco-2 cells at pH 6.0 are shown in Fig. 3. Because the uptake of CBPC was not stereoselective, the uptake of CBPC was calculated as the total of the two epimers. After 1 h of incubation, the uptake of CBPC was less than 1% of the drug administered in the uptake buffer. In contrast, the uptake of CIPC was significantly greater than that of CBPC, with the initial uptake clearance (CL_uptake) of CIPC (7.8 µl/min/mg protein) being approximately 44-fold greater than that of CBPC (0.18 µl/min/mg protein). On the other hand, after 60 min of incubation with 0.5 mM CIPC, 81 and 5% of the total uptake was measured as CBPC and PCG, respectively. When the uptake medium was sampled and analyzed after 60 min of incubation, only 2% of CIPC was converted to CBPC in the uptake medium. Because the uptake of CIPC was linear up to 5 min, the uptake in 5 min was considered as the initial uptake in the following studies.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time courses of uptake of CIPC and CBPC into Caco-2 cells. Caco-2 cells were incubated at 37°C in uptake medium (pH 6.0) containing 0.5 mM CIPC () or 0.5 mM CBPC (). Each point represents mean ± S.D. of three determinations.

Dependence of CIPC Uptake on Proton Gradient. Uptake of CIPC into Caco-2 cells in 5 min was measured at different extracellular pH and the results are shown in Fig. 4. The uptake of CIPC was significantly dependent on the extracellular pH and showed a greater uptake at lower pH. Moreover, the uptake of CIPC was inhibited significantly in the presence of FCCP, a protonophore, which dissipates the proton gradient (Table 1). In contrast, the uptake of CBPC was independent of the extracellular pH. The uptake amounts of CBPC were 1.44 ± 0.16 and 1.49 ± 0.26 nmol/mg protein/5 min at pH 5.0 and 6.0, respectively. These results suggested that the uptake of CIPC is dependent on the proton gradient, whereas the uptake of CBPC is not.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of extracellular pH on uptake of CIPC into Caco-2 cells. Caco-2 cells were incubated for 5 min in the presence of 0.5 mM CIPC at 37°C with the pH of uptake medium being varied between 5.0 and 7.4. Each point represents mean ± S.D. of three determinations.

Effects of Various Compounds on CIPC Uptake. Effects of various compounds on the initial uptake of CIPC (uptake in 5 min) are summarized in Table 1. Sodium azide (NaN₃), a metabolic inhibitor, significantly inhibited the uptake of CIPC, suggesting that the uptake of CIPC is energy dependent. Monocarboxylic acids such as L-lactic, salicylic, and benzoic acids, which are the substrates of the monocarboxylic acid transport system(s), also inhibited the uptake of CIPC significantly. On the other hand, the dipeptides (Gly-Sar and Ala-Ala) and the -lactams that are the substrates of the oligopeptide transporter (CEX and ACPC) showed no inhibitory effect. Moreover, succinic acid (a dicarboxylic acid) and DIDS (an inhibitor of the anion exchanger) exhibited no inhibitory effect on CIPC uptake.

Time Courses of Transport of CIPC and CBPC through Caco-2 cell Monolayer from Apical to Basal Side. Time courses of the transport of CIPC and CBPC through Caco-2 cell monolayer are shown in Fig. 5. The amount of the drug transported from the apical to basal side increased linearly with time after a lag time of 12 and 2 min for CIPC and CBPC, respectively. Transported amounts of CIPC and CBPC at 60 min were 5 and 0.1% of the drug administered on the apical side, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Time courses of transport of CIPC and CBPC through Caco-2 cell monolayer at 37°C. For measuring the transport from the apical to basal side, drug solution (pH 6.0) containing 0.5 mM CIPC () or 4.0 mM CBPC () was added to the apical side. For measuring transport of CIPC from the basal to apical side, the drug solution (pH 7.4) containing 0.5 mM CIPC () was added to the basal side. For both studies, pH values of the apical and basal solutions were 6.0 and 7.4, respectively. Each point represents mean ± S.D. of three determinations.

6

7

Time Course of Transport of CIPC through Caco-2 Cell Monolayer from Basal to Apical Side. Time course of CIPC transport from the basal to apical side of Caco-2 monolayer is also shown in Fig. 5. The amount of CIPC transported through the monolayer increased linearly with time after a lag time of approximately 10 min. Transported amount of CIPC at 60 min was 0.9% of the drug administered on the basal side, and the P_app value calculated according to eq. 2 was 6.0 × 10⁷ cm/s. The P_app of CIPC transport from the basal to apical side was approximately one-seventh of that from the apical to basal side.

Effects of Various Compounds on Transport of CIPC from Apical to Basal Side. Transport of CIPC in 15 min through Caco-2 cell monolayer was measured in the presence of various compounds. As shown in Table 1, the metabolic inhibitor (NaN₃) and monocarboxylic acids (L-lactic, salicylic, benzoic, and acetic acids) inhibited the transport of CIPC, but the dipeptides (Gly-Sar and Gly-Pro), succinic acid, and DIDS showed no inhibitory effect. These results were very similar to those obtained in the uptake study.

Effect of Temperature on Transport and Accumulation of CIPC from Apical to Basal Side. Transport and accumulation of CIPC were measured at 4 and 37°C (Table 2). Both transport and accumulation of CIPC were significantly lower at 4°C than at 37°C, indicating that the transport of CIPC through apical membrane of Caco-2 cells is carrier-mediated.

Effects of Monocarboxylic Acids and CIPC on Transport of L-Lactic Acid from Apical to Basal Side. Transport of L-lactic acid across Caco-2 cell monolayer was measured and a P_app value of 3.3 × 10⁶ cm/s was obtained (data not shown). When the transport of L-lactic acid was measured in the absence or presence of 0.5 mM CIPC, 10 mM benzoic acid, or salicylic acid, all these compounds significantly inhibited the transport of L-lactic acid (Table 3).

	Discussion

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Caco-2 cells, a human intestinal epithelial cell line (Hilgers et al., 1990), are reported to express the oligopeptide, monocarboxylic acid, and other transport systems that also exist in the human intestinal epithelial cells (Inui et al., 1992; Tsuji et al., 1994; Prasad et al., 1995). Existence of the oligopeptide transporter in our cultured Caco-2 cells was demonstrated by the pH-dependent uptake of Gly-Sar, which was inhibited in the presence of CEX (data not shown). Our results were similiar to those reported by Thwaites et al. (1993). Existence of the monocarboxylic acid transport system(s) in our Caco-2 cells was also demonstrated by the pH-dependent transport of L-lactic acid, which was inhibited by benzoic and salicylic acids (Table 3) (Takanaga et al., 1994; Tsuji et al., 1994; Ogihara et al., 1996). Moreover, the TEER and the permeability coefficients (P_app) of L-lactic acid and D-mannitol (data not shown) observed in the present study were similar to those reported by Ogihara et al. (1996). These results indicated that the present Caco-2 cells were well differentiated to express both the oligopeptide and monocarboxylic acid transport systems and that the formation of the tight junction was comparable with that reported by other groups (Inui et al., 1992; Duizer et al., 1997).

When the stability of CIPC and CBPC in Caco-2 cell homogenate was studied, CIPC was hydrolyzed rapidly to generate CBPC at a first-order rate with a half-life of approximately 18 min. In contrast, CBPC was stable for 2 h (Fig. 2). Because no degradation of both compounds was observed in the transport medium without cell homogenate, it is likely that CIPC is hydrolyzed by esterase(s), which exists in the cell homogenate. In the uptake and transport studies, PCG was also detected as a degradation product, which may be due to the degradation of CBPC by decarboxylase.

Degradation of CIPC by membrane-bound or secreted esterase(s) is much smaller than the intracellular degradation. In the uptake study, 86% of the drug was degraded intracellularly, which was much greater than the 2% degradation in the uptake medium. In the transport study, approximately 70% of CIPC was degraded during the passage through Caco-2 cells, whereas the degradation in the donor solution was less than 8%.

Dantzig et al. (1994) reported cefuroxime axetil, a prodrug of cefuroxime, was taken up into Caco-2 cells by passive diffusion, because the uptake of cefuroxime was neither affected by the metabolic inhibitor nor FCCP. In contrast, in the present study, the uptake of CIPC was inhibited by both the metabolic inhibitor and FCCP (Table 1), suggesting that the uptake of CIPC is different from that of cefuroxime axetil and may be carrier-mediated. The greater uptake at a lower extracellular pH indicated that the uptake of CIPC is driven by the H⁺ gradient (Fig. 4), and the temperature-dependent transport and accumulation (Table 2) further suggested the possibility of carrier-mediated transport of CIPC.

When the uptake of CIPC into Caco-2 cells was measured in the presence of various compounds, the substrates of the monocarboxylic acid transport system(s) (L-lactic, salicylic, and benzoic acids) showed significant inhibitory effects, whereas the substrates of the oligopeptide transporter (Gly-Sar, Ala-Ala, CEX, and ACPC) exhibited no inhibitory effect (Table 1). These results suggest that CIPC is transported by the monocarboxylic acid transport system(s), but not by the oligopeptide transporter. Moreover, the transport of L-lactic acid, a model substrate of the monocarboxylic acid transport system(s) (Ogihara et al., 1996), was significantly inhibited in the presence of 0.5 mM CIPC (Table 3). The mutual inhibitory effects between CIPC and L-lactic acid suggest that CIPC and L-lactic acid may share the same transport system(s). This is consistent with the results obtained in our previous study using rat intestinal BBMV, which demonstrated that CIPC competitively inhibited the uptake of L-lactic acid and that CIPC is mainly transported by the monocarboxylic acid transport system(s) (Y-H.L., T.I., H.Y., and M. Tarro, unpublished observations). Because succinic acid and DIDS had no inhibitory effect on the uptake or the transport of CIPC (Table 1), it seems that neither the dicarboxylic acid transporter nor the anion exchanger for OH are involved in the transport of CIPC (Tsuji et al., 1994). Because the monocarboxylic acid transport system(s) may be involved in the absorption of CIPC as well as cefdinir and phenethicillin (Tsuji et al., 1993; Itoh et al., 1998), it is likely that not only the oligopeptide transporter but also the monocarboxylic acid transport system(s) may contribute to the absorption of orally active -lactam antibiotics.

The uptake of CBPC was not affected either by the change in extracellular pH or the metabolic inhibitor (data not shown). Therefore, it seems that the permeation of CBPC through the apical membrane takes place mainly by passive diffusion, not by carrier-mediated transport. This agrees with the results obtained in our BBMV studies, in which CBPC did not show measurable affinity to the oligopeptide or monocarboxylic acid transport systems (Y-H.L., T.I., H.Y., and M. Tarro, unpublished observations).

Because the uptake of CIPC into Caco-2 cells was approximately 40-fold greater than that of CBPC (Fig. 3), the transport of the prodrug from the intestinal lumen to the epithelial cells is likely to be remarkably improved. Although 81 and 5% of the total uptake of CIPC at 60 min was measured as CBPC and PCG, respectively, it is probably true that CIPC itself is taken up into the cells and subsequently degraded into CBPC and PCG because only 2% of CIPC in the uptake medium was converted to CBPC after 60 min of incubation. In the transport study, the P_app of CIPC was approximately 40-fold greater than that of CBPC (Fig. 5), which is very similar to the result obtained in the uptake study. These results indicated that the improved transport of CIPC may be mainly due to the improved uptake through the apical membrane.

As reported by Artursson and Karlsson (1991), drugs with a P_app value of greater than 1 × 10⁶ cm/s in Caco-2 cell monolayer may be absorbed completely from the small intestine in humans. Although the P_app of CIPC was 4.2 × 10⁶ cm/s in the present study, only about 48 to 69% of orally administered CIPC is absorbed in humans (Tanigawara et al., 1982). This may be due to the rapid degradation of CIPC in the intestinal lumen and also in the enterocyte. On the other hand, Tsuji et al. (1982) reported that the absorption of CIPC from the rat small intestine in situ is only 9-fold greater than that of CBPC, which disagrees with the 40-fold difference observed in the present study. The possible reason may be the difference of the tightness of the tight junction between Caco-2 cell monolayer and the rat small intestine. It has been reported (Duizer et al., 1997) that Caco-2 cell monolayers have much greater TEER (>400 · cm²) than that of rat ileum (88 · cm²). Therefore, hydrophilic compounds, such as CBPC, may much more easily permeate through the small intestinal wall of the rat via a paracellular route, whereas the permeation through Caco-2 cell monolayer is restricted. Species difference in the absorption from the small intestine is another possibility.

It is reported that P-glycoprotein (Pgp) is present at the brush border membrane of the small intestinal epithelial cells, which is responsible for secretion of some neutral and basic compounds (Sharom, 1997). Because CIPC is acidic, it is unlikely that CIPC is a substrate of Pgp. Indeed, the apical to basal permeability was approximately 7-fold greater than that from the basal to apical side in the present study, indicating that CIPC is not likely to be secreted into the intestinal lumen. It is possible that transport systems other than the monocarboxylate transport system and Pgp may be involved in the absorption or secretion of CIPC in the small intestine. Further studies are necessary to clarify the involvement of other transport systems.

Because the present uptake study was conducted using Caco-2 cells grown on a plastic dish, it is possible that these cells are not as well differentiated as those grown on a microporous filter. However, the differences in permeability between CIPC and CBPC were similar between the uptake and transport studies as mentioned previously. Also, it is reported that the transepithelial electrical resistance is similar between the filter-grown Caco-2 cell layers and those grown on plastic supports (Grasset et al., 1984). These observations suggest that the Caco-2 cells grown on a plastic dish are well differentiated and may be used for absorption studies.

Takagi et al. (1998) reported that the pH-dependent uphill transport of a monocarboxylic acid, such as salicylic acid, across the lipid bilayer was observed in the absence of a carrier protein. However, there are differences between their results and our results. First, the temperature dependency appears to be much greater in our study. The uptake of salicylic acid at 25°C was only 2-fold greater than that at 4°C in their study, whereas the transport of CIPC at 37°C was more than 30-fold greater than that at 4°C in our study. Second, in their studies, the uptake of salicylic acid was not inhibited by L-lactic acid, which is a typical substrate of the monocarboxylic acid transport system. In contrast, the uptake and transport of CIPC were significantly inhibited in the presence of L-lactic acid in our study (Table 1). Further studies are necessary to clarify involvement of carrier proteins in the transport of monocarboxylates.

Uptake and transport of CIPC were measured at a fixed concentration of 0.5 mM in the present study. When we studied the concentration dependence of CIPC uptake into rat intestinal BBMV, the K_m value of 0.59 mM was obtained (Y-H.L., T.I., H.Y., and M. Tarro, unpublished observations). Therefore, the CIPC concentration in the present study is almost equal or slightly smaller than the K_m value obtained with rat intestinal BBMV.

Ganapathy et al. (1998) recently reported that the oligopeptide transporter mediates the absorption of valacyclovir, a prodrug of acyclovir, and that the parent drug (acyclovir) is not recognized by the transporter. The present study gives another example that the prodrug can be recognized by the transport system, which may lead to the improved absorption. These results should provide a new insight into the mechanism of improved absorption of prodrugs, which, in turn, should lead to the prodrug design for the improvement of oral drug absorption.