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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey (R.K.B., P.J.S., G.K.); Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, México (D.H.-R.); and Bristol-Myers Squibb Research Institute, Discovery Pharmaceutics, Princeton, New Jersey (O.S.G.)
Received March 30, 2005; accepted May 11, 2005.
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Abstract |
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). For example, human peptide transporter 1 (hPepT1) is a 708-amino acid member of the mammalian protein-coupled oligopeptide transporter superfamily that is expressed in the small intestine and present in the brush border of the absorptive epithelial cells (Freeman et al., 1995; Liang et al., 1995; Herrera-Ruiz and Knipp, 2003). The substrates reported to be transported by the proton-coupled hPepT1 are di- and tripeptides and many peptide-based compounds including 
-lactam antibiotics, angiotensin-converting enzyme inhibitors, renin inhibitors, and nonpeptide prodrug, e.g., valacyclovir (Leibach and Ganapathy, 1996; Tamai et al., 1997; Zhang et al., 2002; Herrera-Ruiz and Knipp, 2003).
To better screen, predict, and understand the different routes of transport across the intestinal epithelium, several cellular models have been reported to study the oral absorption of pharmaceuticals (Artursson, 1991; Hillgren et al., 1995). The most commonly used are "intestinal like" cell lines including Caco-2, TC-7, HT29-MTX, and 2/4/A1 (Knipp et al., 1997; Gres et al., 1998; Hilgendorf et al., 2000; Bohets et al., 2001). Caco-2 cells, a human colon carcinoma-derived cell line, has been the most widely utilized model due to the fact that it forms a microvillous apical surface upon differentiation, which exhibit morphological and functional similarities to the polarized absorptive epithelial cells of the small intestine. However, the utilization of Caco-2 cells for drug screening also offers several disadvantages, including the fact that it is labor intensive to handle the cells due to their long culturing times (2 to 3 weeks) for expressing fully differentiated functions and that they exhibit additional transporter systems that can vary across individual laboratories (Hu, 1993; Mahraoui et al., 1994; Behrens et al., 2004).
The Madin-Darby canine kidney (MDCK) cell model, derived from the distal tubular part of dog kidney, has been investigated as a possible screening tool for absorption studies. The advantages of MDCK cells in contrast to Caco-2 cells is that they form tight monolayers in a shorter culturing time and are more easily transfected to delineate the contribution of individual transporters in the absence of significant endogenous transporter "noise" (Pastan et al., 1988; Irvine et al., 1999; Herrera-Ruiz et al., 2004).
The ability to isolate, clone, and transfect the cDNAs for peptide transporters (Liang et al., 1995; Liu et al., 1995) has provided a new dimension in developing cell culture models as a screening tool. Studies conducted in either hPepT1 adenovirally transfected Caco-2 (Hsu et al., 1999) or Chinese hamster ovary/hPepT1 (Han et al., 1999) cells have demonstrated the utility of transfected cell lines for investigation of uptake of peptide transporter substrates. We have recently shown that differently expressing, stably transfected MDCK/hPepT1-V5&His clonal cell lines could be utilized to delineate the functional interaction of PepT1 substrates (Gly-Sar and Carnosine) as a function of the molar levels of hPepT1 transgene expression (Herrera-Ruiz et al., 2004). The limitation of this study was that the substrates used are considered to have high affinity for hPepT1 and lack interaction with other nonpeptide transporters.
To further validate the utility of the MDCK/hPepT1-V5&His clonal cell lines, valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were utilized as hPepT1 substrates that also have potential affinities for other transporter proteins (McLoughlin and Cantrill, 1984; Poschet et al., 1996; Doring et al., 1998; Sinko and Balimane, 1998; Guo et al., 1999; Langer et al., 1999; Sawada et al., 1999; Rud et al., 2000; Kikuchi et al., 2003). In these studies, hPepT1-mediated uptake and transport were determined in the MDCK/hPepT1-V5&His clonal cell lines, previously developed by Herrera-Ruiz et al. (2004). Time-, pH-, sodium-, buffer-, and concentration-dependent studies were performed using mock and the transfected cells. Similar experiments were also performed with Caco-2 cells, and values were compared with cloned cells. Michaelis-Menten kinetics and permeability studies were also correlated with the amount of the transgene hPepT1 protein. Thus, the present study provides a better model for determining hPepT1 affinity and capacity, while minimizing the contributions of other transporters or from endogenous factors. This model can be readily extended to the study of other transporters and enzymes as well.
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Materials and Methods |
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Cell Culture
The mock and stably transfected MDCK/hPepT1-V5&His clonal cells were cultured as described previously (Herrera-Ruiz et al., 2004). Briefly, the cells were maintained in Dulbecco's modified Eagle's medium (4.5 g/l D-glucose, 0.7 mM L-glutamine, and 110 mg/l sodium pyruvate) supplemented with 10% FBS, 1% nonessential amino acids, and 200 mM glutamine, containing 0.3 mg/ml G418 to provide selective pressure. The cells were cultured in T-75 flasks at 37°C in 5% CO2 and at 90% humidity. Cells were harvested and passaged at 80 to 90% confluency and used between passages 7 and 17 in these studies, where no changes in the expression of hPepT1-V5&His were observed (Herrera-Ruiz et al., 2004). The Caco-2 cell line was obtained from ATCC (Rockville, MD). Briefly, cells were cultured in T-75 flasks in culture medium, which consisted of Dulbecco's modified Eagle's medium with 10% FBS, 1% nonessential amino acids, 100 mg/ml penicillin, and 200 mM glutamine. Cells used in this study were between passages 30 and 45.
Uptake Studies
Transfected cells were grown under the conditions specified above and described previously (Herrera-Ruiz et al., 2004). Cells were seeded at a density of 5 x 104 cells/cm2 per well in 24-well plates. Uptake studies were performed in triplicate 2 days postseeding. On the day of the experiment, the cells were washed twice with buffered Ringer's solution (15 mM MES, pH 6.0 or 5 mM HEPES, pH 7.4). The appropriate substrate solutions (see below) were added for the measurement of uptake. The uptake was then stopped by washing the cells two times with ice-cold phosphate-buffered saline. The cells were solubilized by adding 200 µl of 1% Triton X-100 per well. A 150-µl aliquot was used for scintillation counting and a 20-µl sample was used for the protein assay. Protein concentration was determined by the bicinchoninic acid assay following the microtiter plate protocol (Pierce, Rockford, IL). For Caco-2 experiments, the same procedure was followed, except seeding cell density was 1 x 105 cells/cm2 per well in 24-well plates, and the uptake studies were performed on the 21st day.
Time Dependence Assay. HPepT1/V5&His-MDCK stably transfected cells, mock, and Caco-2 cells were seeded as specified above. Subsequently, the cells were incubated with 1 µCi/ml VACV, 5-ALA, or BENZ at 37°C for 5, 10, 20, 60, and 90 min. The complete time course was determined at pH 6.
pH Dependence Assay. HPepT1/V5&His-MDCK stably transfected cells, mock controls, and Caco-2 cells were seeded as specified above. Subsequently, the cells were incubated for 15 min, with 1 µCi/ml VACV, 5-ALA, or BENZ at 37°C at pH 6 and pH 7.4. The proton-dependent uptake of VACV at various pH values (5.5, 6, 6.5, 7, 7.5, and 8) was also studied using cloned and Caco-2 cells. To rule out the possibility of buffer component affecting VACV uptake, different buffer systems (MES, PIPES, and HEPES) were also studied.
Sodium Dependence Assay. HPepT1/V5&His-MDCK stably transfected cells, mock controls, and Caco-2 cells were incubated for 15 min at 37°C with 1 µCi/ml VACV, 5-ALA, and BENZ at pH 6 and pH 7.4 with and without Na+-containing buffer. For Na+-depleted medium, sodium chloride and sodium bicarbonate in MES and HEPES buffer at pH 6 and 7.4, respectively, were replaced by choline chloride and choline bicarbonate.
Concentration Dependence Assay. The concentration dependence of VACV, 5-ALA, and BENZ was studied at pH 6 over a concentration range of 0.01 to 50 mM. The mock, clonal MDCK/hPepT1-V5&His (low, medium, and high), and Caco-2 cells were incubated with each individual substrate for 15 min. The mock cell line kinetic values were subtracted from those observed in each hPepT1/V5&His-containing clone to account for any endogenous and nonspecific transport activity. Michaelis-Menten kinetic parameters were determined (Km and Vmax) using the following equation with GraphPad Prism version 4.02 (GraphPad Software Inc., San Diego, CA).
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To estimate the kinetic values of the saturable uptake by Caco-2 cells, the uptake rate was fit to the following equation that consists of both saturable and nonsaturable components. Modeling was performed using a nonlinear regression program of GraphPad Prism version 4.02.
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Permeability Studies
All transport experiments were run for 2 h in triplicate with the three hPepT1/V5&His-MDCK cell lines (low-, medium-, and high-expressing clones), mock, and Caco-2 cells. Studies were performed in 12-mm tissue culture collagen-coated polycarbonate membranes (0.4-µm pores, Transwells; Costar, Cambridge, MA) at a cell density of 5 x 104 cells/cm2. After seeding, the media were changed every other day, and the study was performed on the 7th day. On the day of the study, culture media were removed, and cells were washed two times with transport buffer. The cells were equilibrated in the transport buffer for 30 min prior to each study. After this time, the substrate containing buffer solution was added and cells kept on a rocker platform inside a humidified culture incubator at 37°C. VACV, 5-ALA, and BENZ at pH 6 (1 µCi/ml) were added to the apical (AP) side and transport buffer pH 7.4 to the basolateral (BL) compartment. Samples (100 µl) at 15, 30, 45, 60, 90, and 120 min were collected from the BL solution, and the volume was replaced with prewarmed Hanks' balanced salt solution. Apparent permeabilities from the AP to BL compartment were determined. BL to AP permeabilities were also evaluated, and 50-µl samples were collected from the AP side at the times specified. The monolayer integrity was tested using the paracellular marker [14C]mannitol. Transepithelial electrical resistance (TEER) was also measured to determine monolayer integrity (von Bonsdorff et al., 1985) and corrected for background. The Caco-2 transport experiments were performed in a similar manner at a cell density of 1 x 105 cells/cm2 and the experiments performed on the 21st day.
The apparent permeability coefficients (Papp) were calculated using the following equation:
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) and did not change during these studies. The values obtained in the uptake studies were normalized to the amount of protein expressed in these cells. VACV, 5-ALA, and BENZ uptake in low-, medium-, and high-expressing cells was measured for 90 min, showing a linear range of uptake over the first 20 min (data not shown). The uptake of VACV, 5-ALA, and BENZ in the transfected cells was pH-dependent (pH 6 versus pH 7.4), with significant increases observed for VACV (p < 0.001), 5-ALA (p < 0.01), and BENZ (p < 0.01) at pH 6 in the high hPepT1-V5&His-expressing cells when compared with pH 7.4 (Fig. 1a). Similar results were also obtained with Caco-2 cells, which also demonstrated higher uptake at pH 6 compared with pH 7.4 (Fig. 1b). Mock cells demonstrated no significant difference in the uptake between pH 6 and pH 7.4 (Fig. 1a).
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Concentration-dependent profiles of VACV, 5-ALA, and BENZ uptake determined in low, medium, and high hPepT1/V5&His-MDCK cell lines after correcting for mock uptake are shown in Fig. 4. Km and Vmax values for VACV, 5-ALA, and BENZ were determined in the hPepT1/V5&His-MDCK cell lines (Table 1). Concentration-dependent studies of VACV, 5-ALA, and BENZ uptake in Caco-2 are shown in Fig. 5. The active (saturable) uptake of each substrate is calculated after subtracting the passive (nonsaturable) uptake values from the total uptake. The Km and Vmax values for Caco-2 uptake were also determined and are illustrated in Table 1.
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Transport Studies. TEER and [14C]mannitol permeability were used to characterize each monolayer's integrity. The TEER values for the MDCK and Caco-2 cells that were included in the studies were found to be more than 300 and 450 
cm2, respectively. The [14C]mannitol apparent permeability coefficient (Papp) in the hPepT1/V5&His-MDCK cell line was found to be similar to the Papp values observed in the parental and control cell lines. In Caco-2 cells, the [14C]mannitol permeability coefficient (AP to BL and BL to AP) was found to be 0.9 x 10–6 cm/s. The AP to BL permeability values of VACV, 5-ALA, and BENZ using low, medium, and high hPepT1/V5&His-expressing cells, after subtracting the values from mock Papp, are shown in Table 2. The AP to BL apparent permeability coefficients of VACV, 5-ALA, and BENZ exhibited a statistically significant difference (p < 0.05) between the mock and high hPepT1/V5&His-expressing MDCK cell lines. However, the BL to AP permeability coefficients across the low, medium, and high hPepT1/V5&His-expressing cell lines for VACV, 5-ALA, and BENZ were found to be similar to the BL to AP permeability coefficient of mock cells. Furthermore, the AP to BL permeability for the transport of VACV, 5-ALA, and BENZ in Caco-2 cells are also listed in Table 2.
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Discussion |
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) stably transfected hPepT1/MDCK cells were utilized to evaluate the functional contribution of hPepT1 on the uptake and transport kinetics of three substrates (VACV, 5-ALA, and BENZ) that also presumably share affinities for other transporters. Similar experiments were performed using the Caco-2 cell culture model and compared with the results from the cloned cells. The cells studied for time dependence showed a linearity of 20 min in cloned and Caco-2 cells for VACV, 5-ALA, and BENZ, which led to our selection of 15 min of incubation time. An inwardly driven proton gradient (extracellular pH = 6) increased 5-ALA and BENZ uptake 1.84- and 2.04-fold, respectively, compared with pH 7.4 in high PepT1/MDCK-expressing cells (Fig. 1a). This is consistent with the findings of Doring et al. (1998) and Poschet et al. (1996), who demonstrated higher uptake of 5-ALA and BENZ at pH 6 versus 7.4.
Consistent with the findings of Sinko et al. (1998), a proton gradient (pH 6) increased the uptake of VACV (Fig. 2a) when compared with pH 7.4 in the different hPepT1-expressing cells (low, medium, and high). Further studies using different buffer systems showed increased uptake at pH 6 thereby confirming the role of proton gradient in increasing the uptake of VACV at pH 6 (Fig. 2b). However, other reports (Guo et al., 1999; Balimane and Sinko, 2000) demonstrated there was no proton gradient effect on the uptake of VACV. This discrepancy is most likely due to differences in the cell systems, with the possibility that VACV uptake was affected by the involvement of various transporters in different studies.
The role of Na+ on the uptake of PepT1 substrates in the high hPepT1-expressing and mock MDCK cells was also investigated. At pH 6 and 7.4, VACV and 5-ALA showed an insignificant change in the uptake with and without Na+ in both high and mock cells suggesting the inability of sodium-dependent transporters to affect their uptake. However, BENZ at pH 6 (Fig. 3a) and pH 7.4 both showed significant (p < 0.05) decreases in the uptake in Na+-depleted buffer. The decrease in the uptake could be due to the influence of the Na+-dependent exchange mechanism affecting its transport. Skowronski et al. (1999) demonstrated that the addition of amiloride and quabain, an inhibitor of Na+/H+ exchanger and Na+/K+ ATPase, respectively, reduced the BENZ transport by 90%, suggesting the role of Na+. Busch et al. (1996) reported the interaction of BENZ with a Na+-dependent phosphate cotransporter. Recently, Burckhardt et al. (2004) reported the interaction of BENZ with the Na+-dependent dicarboxylate (NaDC-3) transporters located in the basolateral membrane of renal proximal tubule cells. In Caco-2 cells at pH 6 and pH 7.4, VACV showed a significant (p < 0.05) Na+-dependent decrease in uptake (Fig. 3b). The role of other transporters affecting the uptake of VACV in Caco-2 cannot be ruled out. Hatanaka et al. (2004) have shown that the transport of VACV was demonstrable in heterologous expression systems expressing Na+- and Cl--coupled amino acid transporter ATB(0,+). Moreover, Sinko and Balimane (1998) suggested that VACV can interact with organic anion and organic cation transporters. Furthermore, the uptake of VACV was observed in human organic anion transporter 3-expressing cells (Takeda et al., 2002), and VACV had an inhibition constant Ki value of 0.22 mM for rPepT2 when blocking the uptake of glycylsarcosine (Sawada et al., 1999).
Although several different routes of active transport across cellular barriers have been characterized (Amidon and Sadee, 1999), it is still difficult to determine the contribution of a single transporter to the permeation of a solute across monolayers. The present study attempts to address the need to develop cell lines expressing a transporter of interest and to functionally evaluate its substrates by establishing and validating these cell lines. It is believed that the current work may contribute toward 1) reducing the possibility of false-positive kinetic assessments due to the presence of families of other transporters and their overlapping specificities, 2) decreasing the possible role of varying endogenous expression levels of the functional active transporter(s) responsible for solute permeation, and 3) minimizing the interlaboratory variation observed in the presence of different culturing conditions that can potentially affect substrate uptake and transport kinetics. Consistent with other studies, we did not observe an effect of the epitope tag on hPepT1 in the present and prior studies (Herrera-Ruiz et al., 2004).
The concentration-dependent uptake studies (0.01–50 mM) were performed to justify the potential of utilizing hPepT1/V5&His-MDCK cell line as a screening tool for peptide and peptide-based compounds. We have quantitatively evaluated the enhancement of the uptake activity in hPepT1/V5&His-MDCK cells after endogenous correction in all of the three clones expressing different hPepT1 levels (low, medium, and high). Michaelis-Menten Km values for VACV, 5-ALA, and BENZ uptake were obtained, and the results demonstrated that the hPepT1 affinity was similar in the three clones (Table 1). The Km values using Caco-2 cells were also calculated and compared with literature-reported values. VACV showed a Km value of 2 mM in Caco-2 cells (Cook et al., 1997), 1.64 mM in hPepT1-transfected Chinese hamster ovary cells (Guo et al., 1999), 3.4 mM in monkeys (Smith et al., 1993), and 1.2 mM in rats (Sinko and Balimane, 1998), which are comparable with our Km values in Caco-2 and cloned cells. For 5-ALA, Km values for the apical and basolateral transporters were 1.6 and 2.4 mM, respectively, suggesting that peptide transporters are the major transport route for 5-ALA in both membranes of Caco-2 cells (Irie et al., 2001). The Vmax/Km ratios for the VACV, 5-ALA, and BENZ were also calculated. VACV Vmax/Km ratios of 3.71 were obtained when comparing the high hPepT1-expressing cell line to the low hPepT1-expressing cells, respectively. Similarly, 5-ALA and BENZ showed 1.88 and 1.89 times, respectively, higher Vmax/Km ratios compared with low hPepT1-expressing cells, respectively, thereby showing the differences in hPepT1 capacity with change in hPepT1 expression. The Km values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells were compared with Caco-2 cells, showing 2.4, 3.8, and 1.9 times decrease, respectively, in the Caco-2 cells in contrast to high hPepT1-expressing MDCK cells. A similar decrease in Km values were also observed when contrasting the low and medium hPepT1-expressing cells, thereby implicating the possible role of multiple transporters in Caco-2 cells.
To obtain a better prediction of drug absorption, mock, low, medium, and high hPepT1/V5&His-MDCK cells were further evaluated for their transepithelial transport across the monolayers. The apparent permeability coefficients of VACV, 5-ALA, and BENZ after 7 days of culture was calculated and found to be significantly higher for the apical to basal permeability compared with the mock values (data not shown). Furthermore, after subtracting the mock transport values from cloned cells, AP to BL transepithelial Papp of VACV in high-expressing PepT1 cells was observed to be 5.17-fold higher than observed in the low-expressing cells, respectively. Similarly, the AP to BL transepithelial Papp of 5-ALA and BENZ was 2.10 and 2.62 times higher, respectively, in the high-expressing hPepT1 cells when compared with the low-expressing cells. This confirms the validity of using these cell lines to elucidate the hPepT1 transport capabilities toward various substrates. BL to AP permeability coefficients of VACV, 5-ALA, and BENZ in the mock cell line are similar to the BL to AP permeability coefficients of the compounds in the hPepT1-expressing cell lines, indicating no effect from the BL side. The results obtained from the TEER and [14C] mannitol permeability studies also showed that the transfection procedure did not modify the ability of MDCK cells to form tight monolayers. VACV, 5-ALA, and BENZ permeability coefficients (after endogenous correction) in the hPepT1/V5&His-MDCK clones increased with the increasing hPepT1 protein levels (correlation coefficients of r = 0.77, 0.93, and 0.99, respectively), demonstrating that changes in the observed transport levels were primarily due to changes in the hPepT1 protein expression. This is consistent with the correlation coefficient of r = 0.75, 0.95, and 0.97 for the uptake values Vmax with respect to change in the hPepT1 protein levels. The Caco-2 transport results are comparable with earlier published reports showing higher transport from the AP to BL side compared with the BL to AP direction (Irie et al., 2001). Compared with the mock-corrected low, medium, and high hPepT1/MDCK cells, Caco-2 Papp values are higher for VACV, 5-ALA, and BENZ (Table 2). The increase in Papp values suggests the potential contribution of additional transport pathways in Caco-2 cells.
The results of this functional evaluation of hPepT1 substrates, using the hPepT1/V5&His cell line, illustrates that the uptake and transport kinetics of PepT1 substrates is mainly due to hPepT1 protein activity. This approach could also be utilized to determine the effect of any transporter on drug absorption. Ultimately, this system could be used as a better tool to assess and screen various substrates and their affinity for designated transporters.
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ABBREVIATIONS: hPepT1, human peptide transporter 1; MDCK, Madin-Darby canine kidney; VACV, valacyclovir; 5-ALA, 5-aminolevulinic acid; BENZ, benzylpenicillin; FBS, fetal bovine serum; MES, 2-(4-morpholino)-ethanesulfonic acid; PIPES, 1,4-piperazine-bis(2-ethanosulfonic acid); AP, apical; BL, basolateral; TEER, transepithelial electrical resistance.
Address correspondence to: Dr. Gregory T. Knipp, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8022. E-mail: gknipp{at}rci.rutgers.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Amidon GL and Sadee W (1999) Membrane Transporters As Drug Targets. Kluwer Academic/Plenum Publishers, New York.
Artursson P (1991) Cell cultures as models for drug absorption across the intestinal mucosa. Crit Rev Ther Drug Carrier Syst 8: 305–330.[Medline]
Balimane P and Sinko P (2000) Effect of ionization on the variable uptake of valacyclovir via the human intestinal peptide transporter (hPepT1) in CHO cells. Biopharm Drug Dispos 21: 165–174.[CrossRef][Medline]
Balimane PV, Tamai I, Guo A, Nakanishi T, Kitada H, Leibach FH, Tsuji A, and Sinko PJ (1998) Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. Biochem Biophys Res Commun 250: 246 –251.[CrossRef][Medline]
Behrens I, Kamm W, Dantzig AH, and Kissel T (2004) Variation of peptide transporter (PepT1 and HPT1) expression in Caco-2 cells as a function of cell origin. J Pharm Sci 93: 1743–1754.[CrossRef][Medline]
Bohets H, Annaert P, Mannens G, Van Beijsterveldt L, Anciaux K, Verboven P, Meuldermans W, and Lavrijsen K (2001) Strategies for absorption screening in drug discovery and development. Curr Top Med Chem 1: 367–383.[CrossRef][Medline]
Burckhardt BC, Lorenz J, Burckhardt G, and Steffgen J (2004) Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent dicarboxylate transporter NaDC-3. Cell Physiol Biochem 14: 415–424.[CrossRef][Medline]
Busch AE, Schuster A, Waldegger S, Wagner CA, Zempel G, Broer S, Biber J, Murer H, and Lang F (1996) Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions. Proc Natl Acad Sci USA 93: 5347–5351.
Cook TJ, Jiansheng Y, and Sinko PJ (1997) Involvement of the organic cation and oligopeptide carrier proteins in the apical transporter of vacyclovir across Caco-2 cell monolayers. Pharm Res (NY) 14 (Suppl): S-20.
Doring F, Walter J, Will J, Focking M, Boll M, Amasheh S, Clauss W, and Daniel H (1998) Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. J Clin Investig 101: 2761–2767.[Medline]
Freeman TC, Bentsen BS, Thwaites DT, and Simmons NL (1995) H+/di-tripeptide transporter (PepT1) expression in the rabbit intestine. Pflugers Arch 430: 394–400.[CrossRef][Medline]
Gres MC, Julian B, Bourrie M, Meunier V, Roques C, Berger M, Boulenc X, Berger Y, and Fabre G (1998) Correlation between oral drug absorption in humans and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line. Pharm Res (NY) 15: 726–733.
Guo A, Hu P, Balimane PV, Leibach FH, and Sinko PJ (1999) Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line. J Pharmacol Exp Ther 289: 448–454.
Han HK, Rhie JK, Oh DM, Saito G, Hsu CP, Stewart BH, and Amidon GL (1999) CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs. J Pharm Sci 88: 347–350.[CrossRef][Medline]
Hatanaka T, Haramura M, Fei YJ, Miyauchi S, Bridges CC, Ganapathy PS, Smith SB, Ganapathy V, and Ganapathy ME (2004) Transport of amino acid-based prodrugs by the Na+- and Cl(-)-coupled amino acid transporter ATB0,+ and expression of the transporter in tissues amenable for drug delivery. J Pharmacol Exp Ther 308: 1138–1147.
Herrera-Ruiz D, Faria TN, Bhardwaj R, Timoszyk J, Gudmundsson OS, Moench P, Wall DA, Smith RL, and Knipp GT (2004) A novel hPepT1-stably transfected cell line: establishing a correlation between expression and function. Mol Pharm 1: 136–144.[CrossRef]
Herrera-Ruiz D and Knipp GT (2003) Current perspectives on established and putative mammalian oligopeptide transporters. J Pharm Sci 92: 691–714.[CrossRef][Medline]
Hilgendorf C, Spahn-Langguth H, Regardh CG, Lipka E, Amidon GL, and Langguth P (2000) Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport. J Pharm Sci 89: 63–75.[CrossRef][Medline]
Hillgren KM, Kato A, and Borchardt RT (1995) In vitro systems for studying intestinal drug absorption. Med Res Rev 15: 83–109.[CrossRef][Medline]
Hsu CP, Walter E, Merkle HP, Rothen-Rutishauser B, Wunderli-Allenspach H, Hilfinger JM, and Amidon GL (1999) Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells. AAPS PharmSci 1: E12.[CrossRef][Medline]
Hu M (1993) Comparison of uptake characteristics of thymidine and zidovudine in a human intestinal epithelial model system. J Pharm Sci 82: 829–833.[CrossRef][Medline]
Irie M, Terada T, Sawada K, Saito H, and Inui K (2001) Recognition and transport characteristics of nonpeptidic compounds by basolateral peptide transporter in Caco-2 cells. J Pharmacol Exp Ther 298: 711–717.
Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE, and Grove JR (1999) MDCK (Madin-Darby canine kidney) cells: a tool for membrane permeability screening. J Pharm Sci 88: 28–33.[CrossRef][Medline]
Kikuchi R, Kusuhara H, Sugiyama D, and Sugiyama Y (2003) Contribution of organic anion transporter 3 (Slc22a8) to the elimination of p-aminohippuric acid and benzylpenicillin across the blood-brain barrier. J Pharmacol Exp Ther 306: 51–58.
Knipp GT, Ho NF, Barsuhn CL, and Borchardt RT (1997) Paracellular diffusion in Caco-2 cell monolayers: effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J Pharm Sci 86: 1105–1110.[CrossRef][Medline]
Langer S, Abels C, Botzlar A, Pahernik S, Rick K, Szeimies RM, and Goetz AE (1999) Active and higher intracellular uptake of 5-aminolevulinic acid in tumors may be inhibited by glycine. J Investig Dermatol 112: 723–728.[CrossRef][Medline]
Leibach FH and Ganapathy V (1996) Peptide transporters in the intestine and the kidney. Annu Rev Nutr 16: 99–119.[CrossRef][Medline]
Liang R, Fei YJ, Prasad PD, Ramamoorthy S, Han H, Yang-Feng TL, Hediger MA, Ganapathy V, and Leibach FH (1995) Human intestinal H+/peptide cotransporter. Cloning, functional expression and chromosomal localization. J Biol Chem 270: 6456–6463.
Liu W, Liang R, Ramamoorthy S, Fei YJ, Ganapathy ME, Hediger MA, Ganapathy V, and Leibach FH (1995) Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney. Biochim Biophys Acta 1235: 461–466.[Medline]
Mahraoui L, Rodolosse A, Barbat A, Dussaulx E, Zweibaum A, Rousset M, and Brot-Laroche E (1994) Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption. Biochem J 298: 629–633.
McLoughlin JL and Cantrill RC (1984) The effect of 
-aminolevulinic acid on the high affinity uptake of aspartic acid by rat brain synaptosomes. Gen Pharmacol 15: 553–555.[Medline]
Pastan I, Gottesman MM, Ueda K, Lovelace E, Rutherford AV, and Willingham MC (1988) A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci USA 85: 4486–4490.
Poschet JF, Hammond SM, and Fairclough PD (1996) Characterisation of penicillin-G uptake in rabbit small-intestinal brush-border membrane vesicles. Biochim Biophys Acta 1278: 233–240.[Medline]
Rud E, Gederaas O, Hogset A, and Berg K (2000) 5-Aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters. Photochem Photobiol 71: 640–647.[CrossRef][Medline]
Sawada K, Terada T, Saito H, Hashimoto Y, and Inui KI (1999) Recognition of L-amino acid ester compounds by rat peptide transporters PEPT1 and PEPT2. J Pharmacol Exp Ther 291: 705–709.
Skowronski MT, Ishikawa Y, and Ishida H (2000) Enhancement by epinephrine of benzylpenicillin transport in rat small intestine. J Pharmacol Exp Ther 293: 128–135.
Sinko PJ and Balimane PV (1998) Carrier-mediated intestinal absorption of valacyclovir, the L-valyl ester prodrug of acyclovir: 1. Interactions with peptides, organic anions and organic cations in rats. Biopharm Drug Dispos 19: 209–217.[CrossRef][Medline]
Smith C, Klein A, and Zimmerman T (1993) Influx of valacyclovir into cynomolgus monkey intestinal brush border membranes is transporter mediated and enhanced over acyclovir, in 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy; 1993 Oct 17–20; New Orleans, LA. Abstract 1750, American Society for Microbiology, Washington, DC.
Takeda M, Khamdang S, Narikawa S, Kimura H, Kobayashi Y, Yamamoto T, Cha SH, Sekine T, and Endou H (2002) Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther 300: 918–924.
Tamai I, Nakanishi T, Hayashi K, Terao T, Sai Y, Shiraga T, Miyamoto K, Takeda E, Higashida H, and Tsuji A (1997) The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine. J Pharm Pharmacol 49: 796–801.[Medline]
Tavelin S, Milovic V, Ocklind G, Olsson S, and Artursson P (1999) A conditionally immortalized epithelial cell line for studies of intestinal drug transport. J Pharmacol Exp Ther 290: 1212–1221.
von Bonsdorff CH, Fuller SD, and Simons K (1985) Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO (Eur Mol Biol Organ) J 4: 2781–2792.[Medline]
Zhang EY, Knipp GT, Ekins S, and Swaan PW (2002) Structural biology and function of solute transporters: implications for identifying and designing substrates. Drug Metab Rev 34: 709–750.[CrossRef][Medline]
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), medium-expressing (
), and high-expressing (
) hPepT1/MDCK cells. Data are presented as mean ± S.D., n = 3.