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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Transport of Angiotensin-Converting Enzyme Inhibitors by H⁺/Peptide Transporters Revisited

Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg, Halle, Germany (I.K., C.W., M.G.H., W.F., K.Z., M.B.); Molecular Nutrition Unit, Center of Life and Food Science, Technical University of Munich, Munich, Germany (G.K., H.D.); and Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle, Germany (R.H.H.N.)

Received for publication July 9, 2008
Accepted August 18, 2008.

	Abstract

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Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H⁺/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [¹⁴C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

Because transport functions of peptide transporters (but in particular the apparent affinity of substrates) depends on a variety of variables from cell type to buffer composition and pH to membrane potential, methodological differences might be responsible for the conflicting data. Therefore, we assessed in a standardized manner— based on three different approaches and using 14 compounds from which three never were studied before—the involvement of PEPT1 and PEPT2 in the transport of ACE inhibitors.

	Materials and Methods

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Materials. The human colon carcinoma cell line Caco-2 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The renal cell line SKPT-0193 Cl.2 established from isolated cells of rat proximal tubules was provided by U. Hopfer (Case Western Reserve University, Cleveland, OH). Culture media, media supplements, and trypsin solution were purchased from Invitrogen (Karlsruhe, Germany) or PAA Laboratories GmbH (Pasching, Austria). Fetal bovine serum was obtained from Biochrom (Berlin, Germany), and collagenase A was from Roche Diagnostics (Mannheim, Germany). The recombinant-modified vaccinia virus Ankara (rMVA) was a kind donation from the GSF-Institute (München, Germany). pBluescript II SK(-), pBluescript-hPEPT1, and pBluescript-hPEPT2 were kind donations from V. Ganapathy (Medical College, Augusta, GA). Dexamethasone, apotransferrin, Igepal CA-630, Ala-Ala-Ala, Ala-Pro, Gly-Sar, and captopril were obtained from Sigma-Aldrich (Deisenhofen, Germany). [Glycine-1-¹⁴C]Gly-Sar (specific radioactivity, 53 mCi/mmol) was custom-synthesized by GE Healthcare (Little Chalfont, Buckinghamshire, UK). Most ACE inhibitors were gifts from companies: benazepril (Salutas Pharma, Barleben, Germany), cilazapril (Roche Diagnostics), enalapril maleate (Berlin-Chemie, Berlin, Germany), fosinopril (Solvay, Hannover, Germany), lisinopril and ramipril (AstraZeneca, Macclesfield, UK), moexipril (Schwarz Pharma, Zwickau, Germany), perindopril (Servier, München, Germany), quinapril and quinaprilat (Pfizer Pharmaceuticals Group, Groton, CT), spirapril (AWD Pharma, Radebeul, Germany), trandolapril (Abbott, Ludwigshafen, Germany), and zofenopril (Menarini Ricerche S.p.A., Firenze, Italy). Captopril was also obtained from MP Biomedicals (Heidelberg, Germany). According to the manufacturer's high-performance liquid chromatography (HPLC) protocols, the purity of the ACE inhibitors was approximately 100%. All other chemicals were of analytical grade.

Culture of Caco-2 and SKPT Cells and Uptake Studies. Caco-2 cells were routinely cultured with minimum essential medium with Earle's salts and L-glutamine, supplemented with 10% fetal bovine serum, 1% nonessential amino acid solution, and gentamicin (45 µg/ml) (Knütter et al., 2001; Neumann et al., 2004). SKPT cells were cultured in Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture 1:1 and L-glutamine, 10% fetal bovine serum, recombinant insulin (4 µg/ml), epidermal growth factor (10 ng/ml), apotransferrin (5 µg/ml), dexamethasone (5 µg/ml), and gentamicin (45 µg/ml) as described previously (Brandsch et al., 1995; Theis et al., 2002; Neumann et al., 2004). Both cell lines were subcultured in 35-mm disposable Petri dishes (Sarstedt, Nümbrecht, Germany) at a seeding density of 0.8 x 10⁶ cells per dish. Uptake of [¹⁴C]Gly-Sar was measured 7 days (Caco-2) or 4 days (SKPT) after seeding at room temperature as described previously (Knütter et al., 2001; Theis et al., 2002; Neumann et al., 2004). The uptake buffer was 25 mM Mes/Tris, pH 6.0, containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. Uptake was initiated after washing the cells for 30 s in uptake buffer by adding 1 ml of uptake medium containing [¹⁴C]Gly-Sar (10 µM) and increasing concentrations of the test compounds (0–100 mM). If necessary, the pH of the solutions was corrected before preparing the required dilutions. After incubation for 10 min, the cells were quickly washed four times with ice-cold buffer, solubilized in 1 ml of Igepal CA-630 (0.5% v/v) in buffer [50 mM Tris/HCl (pH 9.0), 140 mM NaCl, and 1.5 mM MgSO₄], and prepared for liquid scintillation spectrometry. For each experiment, the samples for the protein measurements were prepared and measured as described earlier (Knütter et al., 2001).

Heterologous Expression of hPEPT1 and hPEPT2 in Human Retinal Pigment Epithelium Cells and Uptake Studies. Human retinal pigment epithelium (HRPE) cells (passages 12–25) were cultured in Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture 1:1 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Metzner et al., 2008). For subculturing, the cells were rinsed with phosphate-buffered saline, trypsinated, and seeded in 75-cm² flasks with a cell density of 5 to 8 x 10⁶ per flask or in 24-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) with a cell density of 0.5 x 10⁶ (hPEPT1) or 0.7 x 10⁶ (hPEPT2) per well, respectively.

For the vaccinia virus expression of hPEPT1 and hPEPT2, a modified protocol of the procedures described by Ganapathy et al. (1995) and Metzner et al. (2008) was used. First, HRPE cells were infected 24 h after seeding in 24-well plates with rMVA (50 IU/cell) encoding the T7 RNA polymerase (Sutter et al., 1995) and incubated for 30 min at 37°C. After 30-min incubation with rMVA, for PEPT1 the HRPE cells were transfected with pBluescript-hPEPT1 cDNA construct and pBluescript (1 µg/well) using Nanofectin (3.2 µl/well; PAA Laboratories GmbH, Cölbe, Germany), whereas for hPEPT2 the HRPE cells were transfected with pBluescript-hPEPT2 cDNA construct and pBluescript (1 µg/well) using Metafectene Pro (2 µl/well; Biontex, Martinsried/Planegg, Germany) according to manufacturers' protocols. HRPE cells transfected with empty plasmid served as the control. To minimize toxic effects of the infection/transfection procedure, the medium was replaced after 4 h. Twenty-four hours post-transfection, uptake of [¹⁴C]Gly-Sar was measured at room temperature. The uptake buffer was 25 mM Mes/Tris, pH 6.0, containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. Uptake was initiated after washing the cells once in uptake buffer by adding 0.3 ml of uptake medium containing [¹⁴C]Gly-Sar (20 µM for PEPT1, 30 µM for PEPT2) and increasing concentrations of the test compounds (0–100 mM). If necessary, the pH of the solutions was corrected before preparing the required dilutions. In HRPE-hPEPT1 cells, [¹⁴C]Gly-Sar uptake is linear for up to 5 min. In HRPE-hPEPT2 cells, uptake is linear for up to 20 min (data not shown). After incubation for 5 min (hPEPT1) or 10 min (hPEPT2), the cells were quickly washed four times with ice-cold buffer, solubilized in 0.5 ml of SDS (1%) in 0.2 M NaOH, and prepared for liquid scintillation spectrometry.

X. laevis Oocytes Expressing PEPT1 and PEPT2 and Electrophysiology. Female X. laevis were purchased from African Xenopus Facility (Knysna, South Africa). Surgically removed oocytes were separated by collagenase treatment and handled as described previously (Boll et al., 1996; Knütter et al., 2001; Theis et al., 2002). Individual oocytes were injected with 30 nl of RNA solution containing 30 ng of rabbit PEPT1 or rabbit PEPT2 cRNA. All electrophysiological measurements were performed after 3 to 6 days by incubation of oocytes in buffer composed of 88 mM NaCl, 1 mM KCl, 0.82 mM CaCl₂, 0.41 mM MgCl₂, 0.33 mM Ca(NO₃)₂, 2.4 mM NaHCO₃, and 10 mM MES/Tris at pH 6.5 (modified Barth-solution). The two-electrode voltage-clamp technique was applied to characterize responses in current (I) to substrate addition in oocytes expressing PEPT1 or PEPT2 (Boll et al., 1996; Knütter et al., 2001; Theis et al., 2002). In short, oocytes were placed in an open chamber with a volume of 0.5 ml and continuously superfused with modified Barthsolution or with solutions of Gly-Sar and/or the test compound. Electrodes with resistances between 0.5 and 2 M were connected to a TEC-05 amplifier (NPI Electronic GmbH, Tamm, Germany), and oocytes were clamped at -60 mV. Current-voltage (I-V_m) relationships were measured using short (100 ms) pulses separated by 200-ms pauses in the potential range from -160 to +80 mV. I-V_m measurements were made immediately before and 30 s after substrate application when current flow reached steady state. Currents evoked at -60 mV (PEPT1) or at -160 mV (PEPT2) were calculated as the difference of the currents measured in the presence and the absence of substrate.

HPLC Analysis. Benazepril, captopril, enalapril, and lisinopril (1 mM, in buffer pH 6.0) were incubated for 10 min with Caco-2 cells. Samples of the extracellular uptake medium were taken at t = 0 min and t = 10 min, and the ACE inhibitors were quantified according to the laboratory standard HPLC (La-Chrom; Merck-Hitachi, Darmstadt, Germany) with a diode array detector and a Purospher STAR RP-18 endcapped column (125-4, 5 µm; Merck, Darmstadt, Germany). The eluent was 48% acetonitrile/52% H₂O with trifluoroacetic acid pH 2.5 for captopril and enalapril, 52% acetonitrile/48% H₂O with trifluoroacetic acid pH 2.5 for benazepril, and 30% acetonitrile/70% H₂O with trifluoroacetic acid pH 2.5 for lisinopril. UV detection was done at 215 nm. Injection volume was 5 µl, and the flow rate was 0.5 ml/min.

Calculations and Statistics. All data are given as the mean ± S.E. of three to four independent experiments. The kinetic parameters were calculated by nonlinear regression methods (SigmaPlot; Systat Software GmbH, Erkrath, Germany) and confirmed by linear regression of the respective Eadie-Hofstee plots. IC₅₀ values (i.e., concentration of the unlabeled compound necessary to inhibit 50% of carrier-mediated [¹⁴C]Gly-Sar uptake) were determined by nonlinear regression using the logistical equation for an asymmetric sigmoid (allosteric Hill kinetics): Y = Min + (Max-Min)/(1 + (X/IC₅₀)^-P), where Max is the initial Y value, Min the final Y value, and the power P represents the Hill coefficient. Inhibition constants (K_i) were calculated from IC₅₀ values according to the method developed by Cheng and Prusoff (1973).

	Results

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Inhibition of Gly-Sar Uptake by ACE Inhibitors in Caco-2 and SKPT Cells. Caco-2 and SKPT cells are currently the best native cell models for transport studies on PEPT1 and PEPT2, respectively. We first studied the effect of 14 ACE inhibitors on the [¹⁴C]Gly-Sar uptake into Caco-2 cells. Carrier-mediated uptake of Gly-Sar into confluent monolayers of Caco-2 cells is solely mediated by PEPT1 (Knütter et al., 2001; Brandsch et al., 2004; Neumann et al., 2004). For all ACE inhibitors, a concentration-dependent inhibition of [¹⁴C]Gly-Sar uptake was observed (Fig. 1, A and B). From the inhibition curves, IC₅₀ values were obtained and converted into K_i values. These K_i values reflect apparent affinity of the compounds tested. As compiled in Table 1, the K_i values ranged from 0.047 to 46 mM. Most ACE inhibitors tested displayed interaction with PEPT1 with medium affinities (Table 1) (for classification of affinity constant at PEPT1, see Brandsch et al., 2004, 2008). Because most ACE inhibitors are derivatives of Ala-Pro, we also determined the K_i value of Ala-Pro and for comparison the K_i values of the prototypic PEPT1 substrates Gly-Sar and Ala-Ala-Ala (Table 1). The peptides Ala-Pro and Ala-Ala-Ala and the ACE inhibitors fosinopril and zofenopril were found to be high-affinity substrates and/or inhibitors of PEPT1 (K_i < 0.5 mM) (Brandsch et al., 2008). Medium affinity was observed for Gly-Sar, benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, and moexipril (0.5 mM < K_i < 5 mM). Quinaprilat, perindopril, and enalapril were inhibitors of the low-affinity category (5 mM < K_i < 15 mM). From the very high K_i values of lisinopril (23 mM) and captopril (46 mM), we conclude that these two compounds cannot be considered to interact with PEPT1. Because the K_i value found for captopril here is relatively high compared with values reported by others (e.g., 8.7 mM; Temple and Boyd, 1998) and captopril is considered a transported substrate of PEPT1 (Thwaites et al., 1995; Zhu et al., 2000), we determined the inhibition constant for captopril also by using two other buffer systems (Sörensen buffer, Hanks' balanced salt solution) to rule out that buffer constituents such as Tris or Mes affect affinity. Moreover, we also tested captopril obtained from different suppliers, but in all cases, measured K_i values were similar and are highly reproducible (>40 mM; data not shown). To test the stability of the drugs during the experiment, we analyzed captopril, enalapril, benazepril, and lisinopril in the extracellular uptake medium of Caco-2 cells over the incubation period of 10 min by HPLC. Ninety-four to 98% of the drug molecules were found intact after the experiment (97% captopril, 94% enalapril, 98% benazepril, 96% lisinopril; data not shown). Once inside the cell, hydrolysis-sensitive prodrugs will be hydrolyzed to their corresponding active drugs, but this would not interfere with the determination of affinity constants for extracellular binding at the transporters.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Interaction of ACE inhibitors with PEPT1 and PEPT2. Uptake of [14C]Gly-Sar (10 µM, pH 6.0, 10 min, n = 4) was measured in Caco-2 cells (A and B) and in SKPT cells (C and D) in the presence of increasing concentrations of ACE inhibitors and, for comparison, Ala-Pro (0–100 mM). Uptake rates measured in the absence of inhibitor were taken as 100%.

We next determined the K_i values of these 14 ACE inhibitors for the inhibition of [¹⁴C]Gly-Sar uptake in SKPT cells. These cells express PEPT2 but not PEPT1 (Brandsch et al., 1995; Ganapathy et al., 1995; Shu et al., 2001). The ACE inhibitors reduce [¹⁴C]Gly-Sar uptake in a dose-dependent manner (Fig. 1, C and D). The apparent K_i values (Table 1) were in a range of 13 µM to 7.9 mM. According to our classification (Luckner and Brandsch, 2005; Brandsch et al., 2008), Ala-Pro, Ala-Ala-Ala, fosinopril, and zofenopril are thereby high-affinity inhibitors of [¹⁴C]Gly-Sar uptake (K_i < 0.1 mM). Medium-affinity inhibitors (0.1 mM < K_i < 1 mM) are Gly-Sar, benazepril, quinapril, trandolapril, spirapril, ramipril, moexipril, and quinaprilat. Interaction of cilazapril, perindopril, enalapril, lisinopril, and captopril with PEPT2 was low or very low (K_i > 1 mM or >5 mM, respectively). In Table 1, we also specify the rank order of K_i values for subsequent correlation analyses (see below).

Kinetics of Inhibition of Gly-Sar Uptake into Caco-2 and SKPT Cells. Inhibition of [¹⁴C]Gly-Sar uptake by ACE inhibitors does not necessarily mean that the drugs are transported. They could represent specific inhibitors or even compounds that affect nonspecifically, for example, membrane integrity, the H⁺ gradient, or membrane voltage as the driving force of [¹⁴C]Gly-Sar uptake. Therefore, we determined the type of inhibition for selected compounds. We have chosen quinapril because of controversial reports regarding its type of inhibition and spirapril because, to our knowledge, this interesting drug has never been studied with regard to transporter interaction. First, we studied the effect of quinapril on the kinetic parameters of Gly-Sar uptake by PEPT1 and PEPT2. Gly-Sar uptake in Caco-2 and in SKPT cells was measured over a concentration range of 0.01 to 10 mM (Caco-2) or 0.01 to 5 mM (SKPT), respectively, in the absence or presence of quinapril at a concentration of 1 mM (Caco-2) or 0.5 mM (SKPT). Figure 2, A (Caco-2 cells) and B (SKPT cells), shows the relationship between the Gly-Sar uptake rates and Gly-Sar concentration. In the absence of quinapril, the Michaelis constant, K_t, for Gly-Sar uptake at Caco-2 cells was 1.1 ± 0.1 mM and the maximal velocity, V_max, was 39.4 ± 1.0 nmol x mg of protein^-1 per 10 min. These data correspond very well to values reported previously (Knütter et al., 2001; Brandsch et al., 2004). The kinetic constants obtained in the presence of 1 mM quinapril were (K_t) 2.0 ± 0.5 mM and (V_max) 25.8 ± 1.9 nmol x mg of protein^-1 per 10 min. Hence, quinapril, at a concentration close to its K_i value, increased the K_t value of Gly-Sar uptake 2-fold and decreased V_max approximately 1.5-fold. The situation is quite similar for PEPT2: nonlinear regression of the curves reveals that in the absence of quinapril, the K_t value for Gly-Sar uptake in SKPT was 0.14 ± 0.02 mM and the V_max value was 6.9 ± 0.3 nmol x mg of protein^-1 per 10 min. This too is in agreement with previously reported values (Theis et al., 2002). The corresponding kinetic constants obtained in the presence of 0.5 mM quinapril were (K_t) 0.23 ± 0.01 mM and (V_max) 5.3 ± 0.03 nmol x mg of protein^-1 per 10 min. Hence, quinapril, again at a concentration close to its K_i value, increased the K_t value of Gly-Sar uptake in SKPT cells 1.6-fold and decreased the V_max 1.3-fold. These results are in agreement with the assumption that quinapril does not represent a competitive inhibitor of PEPT1 and PEPT2, a function that is expected for a carrier substrate. In the next experiment, we determined the inhibition constant (K_i) of quinapril by measuring Gly-Sar uptake in Caco-2 cells at two different Gly-Sar concentrations (50 and 500 µM) in the presence of increasing concentrations of quinapril (0–5 mM). The results are presented as Dixon plot in Fig. 2C. They reveal linearity at both Gly-Sar concentrations with lines intersecting on the abscissa as expected for a noncompetitive inhibitor. A K_i value of 0.55 mM for quinapril at Caco-2 cells was calculated from the point of intersection. Such an analysis was also performed with spirapril at Caco-2 cells (Fig. 2D). Again, the lines in the Dixon plot were intersecting on the abscissa. A K_i value of 1.8 mM was determined. The K_i values obtained by this procedure are similar to the K_i values obtained in the competition assays described above.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Type of Gly-Sar uptake inhibition by quinapril and spirapril. A and B, effect of quinapril on the saturation kinetics of Gly-Sar uptake into Caco-2 cells (A) and SKPT cells (B). Uptake of Gly-Sar (0.01–10 mM at Caco-2 cells, 0.01–5 mM at SKPT cells) was measured at pH 6.0 for 10 min in confluent monolayer cultures. The results represent saturable uptake values after correction for the nonsaturable component. If not shown, error bars are smaller than the symbols. Inset, Eadie-Hofstee transformations of the data (v = uptake rate in nmol x 10 min-1 x mg of protein-1;S = Gly-Sar concentration in mM), n = 4. C and D, determination of the inhibition constants of quinapril (C) and spirapril (D) at Caco-2 cells in a Dixon type of experiment. Uptake of Gly-Sar was measured at pH 6.0 for 10 min at two Gly-Sar concentrations and at increasing inhibitor concentrations. The linear, nonsaturable component of [14C]Gly-Sar uptake, measured in the presence of excess an amount of Gly-Sar (30 and 20 mM, respectively), was subtracted from total uptake to calculate carrier-mediated uptake (n = 4; v = uptake rate in nmol x 10 min-1 x mg of protein-1).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Interaction of ACE inhibitors with human PEPT1 and human PEPT2 heterologously expressed in HRPE cells. Uptake of [14C]Gly-Sar at pH 6.0 (A, 20 µM, 5 min; B, 30 µM, 10 min) was measured in HRPE-hPEPT1 cells (A) and in HRPE-hPEPT2 cells (B) in the presence of increasing concentrations of ACE inhibitors and, for comparison, Gly-Sar (0–100 mM). Uptake rates measured in the absence of inhibitor were taken as 100% (n = 4).

Transport of ACE Inhibitors by PEPT1 and PEPT2 Expressed in X. laevis Oocytes. As stated above, the demonstration of [¹⁴C]Gly-Sar uptake inhibition does not imply that the respective compound is indeed transported. Therefore, the two-electrode voltage-clamp technique was applied to X. laevis oocytes expressing either rabbit PEPT1 or rabbit PEPT2. The concentrations of ACE inhibitors or peptides used to determine PEPT1-mediated transport currents was mostly 10 mM, with the exception of fosinopril (0.33 mM), zofenopril (0.1 mM), quinaprilat (3 mM), and trandolapril (5 mM). Captopril was also tested at 40 mM. For PEPT2, the drug concentration used was 2 mM, with the exception of fosinopril (0.33 mM) and zofenopril (0.1 mM). The currents elicited by the ACE inhibitors are expressed as percentage of the current induced by the dipeptide Gly-Sar applied in saturating concentration (>10 x K_t: PEPT1, 10 mM; PEPT2, 2 mM) measured in the same oocyte (Fig. 4A). For comparison, the currents induced by the peptide transporter substrates Ala-Pro and Ala-Ala-Ala were recorded as well. Importantly, in contrast to the dipeptide-induced currents, all of the ACE inhibitors generated very low currents. Because some of the inhibitors (e.g., quinapril, trandolapril, spirapril) induced membrane currents also in noninjected oocytes, the following results were corrected for the average current generated in the absence of peptide transporters. Significant current values in case of PEPT1 were recorded for quinapril (10%), lisinopril (9%), benazepril (8%), and enalapril (5%). For the 10 other drugs, the maximal currents were below or near 5% and thereby not significantly different from zero. In Fig. 4B, representative currents elicited by Gly-Sar, fosinopril, and quinapril for PEPT1 as a function of membrane potential are shown. For fosinopril, no inward currents could be recorded. It is interesting to note that for quinapril and several other ACE inhibitors, the dependence of the transport rate on membrane potential differed from that of Gly-Sar, showing an overproportionally increasing current at more negative membrane potentials (Fig. 4B). Very similar results were obtained with oocytes expressing PEPT2 (Fig. 4C). The different shapes of the I-V relations for different substrates of PEPT1 and the similar differences between PEPT1 and PEPT2 are probably caused by different rate constants during the transport cycle (Sala-Rabanal et al., 2008). Only lisinopril and spirapril with currents above 20% of those generated by Gly-Sar were measured, but due to a larger variability of currents at -160 mV membrane potential, only the current of spirapril turned out to be statistically significant (Fig. 4A). For the other ACE inhibitors, no significant currents were measurable (currents of enalapril, captopril, moexipril, and zofenopril were <2%).

View larger version (18K): [in this window] [in a new window]   Fig. 4. ACE inhibitor-induced inward currents in X. laevis oocytes expressing rabbit PEPT1 or PEPT2. A, currents induced by Ala-Ala-Ala, Ala-Pro, and 14 ACE inhibitors as the percentage of the current induced by 10 mM (PEPT1, -60 mV) or 2 mM (PEPT2, -160 mV) Gly-Sar. The concentration of ACE inhibitors or peptides at PEPT1 was generally 10 mM, with the exception of fosinopril (0.33 mM), zofenopril (0.1 mM), quinaprilat (3 mM), and trandolapril (5 mM), and at PEPT2 2 mM, with the exception of fosinopril (0.33 mM) and zofenopril (0.1 mM). If necessary, the values were corrected for the shifts of the zero line due to the presence of dimethyl sulfoxide and were corrected for the average current generated in the absence of peptide transporters. Negative values mean inhibition of the basal membrane conductance. Mean values of 2 to 8 oocytes. *, p < 0.05 and **, p < 0.01. B and C, steady-state I-V relationships were measured by the two-electrode voltage-clamp technique in oocytes expressing PEPT1 (B) or PEPT2 (C) superfused with modified Barth solution at pH 6.5 and 10 mM (PEPT1) or 2 mM (PEPT2) peptide or ACE inhibitor (with the exception of fosinopril: 0.33 mM). The membrane potential was stepped symmetrically to the test potentials shown, and substrate-dependent currents were recorded as the difference measured in the absence and the presence of substrates.

	Discussion

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In this study, we investigated systematically whether ACE inhibitors serve as substrates for PEPT1 and PEPT2. Almost every review written on drug delivery states that ACE inhibitors are substrates of intestinal and renal peptide transporters. However, published data are contradictory (for review, see Brandsch et al., 2008), and we felt that the current situation only unsatisfactory describes the role of peptide transporters in the delivery of these drugs. Moreover, the assumption that the activity of PEPT1 mediates oral availability of the drugs seems to hamper the search for other proteins that might be involved in intestinal transport of ACE inhibitors.

The inhibitory activity of 14 ACE inhibitors on [¹⁴C]Gly-Sar influx into Caco-2 cells expressing hPEPT1 and in SKPT cells expressing rPEPT2 was studied. For zofenopril and for fosinopril, a high-affinity interaction with both peptide transporters was observed. This result is in good agreement with previous reports (Lin et al., 1999; Moore et al., 2000; Shu et al., 2001). Data for the interaction of trandolapril, spirapril, and moexipril with peptide transporters are not available from the literature. For some compounds conflicting data have been reported. This may be a consequence of the different in vivo and in vitro approaches or different parameters such as buffers, pH, or membrane potential. To exemplify this, Bai and Amidon (1992) reported (based on intestinal perfusion experiments in rats) K_m values of 5.9 mM, 70 µM, and 75 µM for captopril, enalapril, and benazepril, and they concluded (based on competition studies with dipeptides) that transport occurred via the peptide transporter. Comparing the affinities with our data, it becomes obvious that the activity of the peptide transporter cannot explain these findings. Therefore, it is likely that additional transporters are involved in intestinal uptake of these compounds in the rat intestine. Likewise, Swaan et al. (1995), when defining for the intestinal peptide carrier and its substrate template, used affinity constants for enalapril, enalaprilat, and lisinopril of 0.15, 0.28, and 0.39 mM. Those data were derived from Ussing chamber experiments with the rat intestine, using a mucosal buffer pH of 7.4. In the present study, we detected only a very weak, almost negligible affinity of enalapril and lisinopril for transport by PEPT1 with a K_i of >14 mM, and this value is in good agreement with the one reported by Moore et al. (2000) with a K_i > 20 mM also obtained in Caco-2 cells. Based on these very low affinities, a major contribution of PEPT1 to the absorption of these drugs seems highly unlikely; in particular, when taking into account that based on recommended oral doses for an adult of 25 to 75 mg per day for captopril and 5 to 20 mg for enalapril and lisinopril, the mean luminal concentrations in the jejunum would be approximately 100 µM (captopril) and 10 µM (enalapril, lisinopril).

Another compound of controversy is quinapril. The K_i values measured for interaction of quinapril with PEPT1 (1.0 mM) and PEPT2 (0.39 mM) in the present analysis are in the same range as K_i values reported by other groups (Kitagawa et al., 1997; Akarawut et al., 1998; Lin et al., 1999; Moore et al., 2000; Zhu et al., 2000). However, Zhu et al. (2000) observed a noncompetitive inhibition of peptide transport and speculated that quinapril may affect the binding and/or translocation of the proton, whereas Akarawut et al. (1998) favored a different binding site for quinapril in the transporter. In contrast, Kitagawa et al. (1997) found a competitive inhibition of peptide transport by quinapril. Our data support a mixed type of inhibition for quinapril, but we do not have sufficient mechanistic information to be able to incorporate this into the kinetic 7-state models currently available for PEPT1 and PEPT2 (Sala-Rabanal et al., 2008). For this study, the important question is whether quinapril is transported at all (see below), and only when it is proven that those drugs are transporter substrates can they be included into modeling approaches for defining the pharmacophore of PEPT1 or PEPT2 substrates. It would be interesting to study in vivo whether quinapril and other competitive or noncompetitors PEPT1/2 inhibitors might interfere with the absorption of simultaneously applied drugs that are PEPT1 and PEPT2 substrates. Such drug-drug interactions with, e.g., orally available β-lactam antibiotics or valacyclovir would be a function of both their affinity constants at the transporters and their luminal concentrations.

To assess in more detail the structural elements that may determine their affinity for PEPT1 and PEPT2, we plotted the K_i values over the log D values of the compounds as obtained from the Scifinder database (Table 1). A correlation coefficient of r = 0.69 (p < 0.006) for the K_{i PEPT1} values and r = 0.80 (p < 0.0006) for the K_{i PEPT2} values of the log D clearly demonstrate that a high affinity is associated with a high hydrophobicity. Similar results were obtained by Lin et al. (1999) with nine ACE inhibitors based on inhibition of Gly-Sar uptake into rabbit renal brush border membrane vesicles.

PEPT2 represents the high-affinity H⁺/peptide cotransporter, whereas PEPT1 is the low-affinity isoform. For natural dipeptides, PEPT2 generally displays an approximately 10-fold higher affinity than PEPT1 for the same substrates; i.e., the ratios between the K_i of PEPT1 and the K_i of PEPT2 are approximately 10. In our study, the K_{i PEPT1}/K_{i PEPT2} ratios vary between 1.3 and 13.1 with an average of 6.3 (Table 1). Hence, PEPT2 recognizes the same ACE inhibitors as PEPT1 but on average with higher affinity. To study possible differences in more detail, a correlation analysis using the affinity constants obtained in Caco-2 cells and in SKPT cells was performed. From this analyses, we obtained a very high and significant correlation (r = 0.97, p < 0.0001). Because a clustering of K_i values in certain groups might lead to overestimation of the correlation coefficient, we also calculated the more robust nonparametric Spearman's rank correlation coefficient using the rank orders of K_i values (Bretschneider et al., 1999) (Table 1). This method also revealed a high and significant correlation coefficient (r_s) of 0.92 (p < 0.0001). Based on this analysis, we conclude that there are no major differences in the substrate recognition pattern of hPEPT1 and rPEPT2 with regard to the ACE inhibitors tested. Experiments in HRPE cells expressing human PEPT1 and human PEPT2, respectively, confirmed as well the K_i values and led us to conclude that differences are not due to species differences.

To assess whether the ACE inhibitors not only interfere with the substrate binding sites of the transporters but are indeed translocated by PEPT1 and PEPT2, we employed the two-electrode voltage-clamp technique to X. laevis oocytes expressing either one of the two peptide transporters. The maximal inward currents induced by the drugs were, in most cases, less than one fifth of the maximal currents elicited by the model peptides Ala-Ala-Ala and Gly-Sar. Despite the fact that there is no gold standard for the judgment on when currents may be taken as relevant and physiologically meaningful, in this study we consider currents as significant when they are as follows: 1) transporter specific, i.e., when no signals are obtained using the same substrate concentration in control oocytes not expressing transporters; 2) at least 5% of the currents elicited by reference substrates (dipeptides); and 3) statistically significantly different from zero. Our data suggest very low transport rates (<10%) of only lisinopril, enalapril, quinapril, and benazepril by PEPT1. In PEPT2, only spirapril elicited significant currents.

We conclude that the oral availability of the ACE inhibitors that were here shown to generate only very small transport currents and that displayed apparent affinity constants higher than 15 mM cannot be explained by their interaction with the intestinal peptide transporter, especially when considering the low luminal concentrations. For all compounds that failed to show significant interaction and transport by peptide transporters, one has to postulate that they may use other routes for absorption. Considering the high lipophilicity of most compounds, simple diffusion might be sufficient in many cases. Alternatively, other membrane carriers, and in particular members of the organic anion-transporting family (SLC21 and SLC22), seem to be relevant candidates to be studied in their capability for transport of ACE inhibitors. For quinapril and enalapril, the transport by organic anion transporters, e.g., OATP1B1 or OATP1B3, has already been shown (Akarawut and Smith, 1998; Pang et al., 1998; Liu et al., 2006; Chu et al., 2007).

	Acknowledgements

 
We thank Rainer Reichlmeir (Technical University Munich) for excellent technical assistance.

	Footnotes

 
This work was supported by the State Saxony-Anhalt "Life Sciences" Excellence Initiative (to M.B.) and the Deutsche Forschungsgemeinschaft, Grant KO 1605/2-4 (to G.K.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.143339.

ABBREVIATIONS: ACE, angiotensin-converting enzyme; PEPT1, H⁺/peptide transporter 1; PEPT2, H⁺/peptide transporter 2; rMVA, recombinant modified vaccinia virus Ankara; hPEPT, human PEPT; Gly-Sar, glycylsarcosine; HPLC, high-performance liquid chromatography; HRPE, human retinal pigment epithelium; rPEPT2, rat PEPT2; Mes, 4-morpholineethanesulfonic acid.

Address correspondence to: Dr. Matthias Brandsch, Biozentrum of the Martin-Luther-University Halle-Wittenberg, Membrane Transport Group, Weinbergweg 22, D-06120 Halle, Germany. E-mail: matthias.brandsch{at}biozentrum.uni-halle.de

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