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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Transport Characteristics of a Novel Peptide Transporter 1 Substrate, Antihypotensive Drug Midodrine, and Its Amino Acid Derivatives

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine (M.T., T.T., M.I., T.K., K.I.), and Graduate School of Pharmaceutical Science (A.N., K.T., N.F.), Kyoto University, Kyoto, Japan

Received February 12, 2006; accepted April 4, 2006.

	Abstract

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Midodrine is an oral drug for orthostatic hypotension. This drug is almost completely absorbed after oral administration and converted into its active form, 1-(2',5'-dimethoxyphenyl)-2-aminoethanol) (DMAE), by the cleavage of a glycine residue. The intestinal H⁺-coupled peptide transporter 1 (PEPT1) transports various peptide-like drugs and has been used as a target molecule for improving the intestinal absorption of poorly absorbed drugs through amino acid modifications. Because midodrine meets these requirements, we examined whether midodrine can be a substrate for PEPT1. The uptake of midodrine, but not DMAE, was markedly increased in PEPT1-expressing oocytes compared with water-injected oocytes. Midodrine uptake by Caco-2 cells was saturable and was inhibited by various PEPT1 substrates. Midodrine absorption from the rat intestine was very rapid and was significantly inhibited by the high-affinity PEPT1 substrate cyclacillin, assessed by the alteration of the area under the blood concentration-time curve for 30 min and the maximal concentration. Some amino acid derivatives of DMAE were transported by PEPT1, and their transport was dependent on the amino acids modified. In contrast to neutral substrates, cationic midodrine was taken up extensively at alkaline pH, and this pH profile was reproduced by a 14-state model of PEPT1, which we recently reported. These findings indicate that PEPT1 can transport midodrine and contributes to the high bioavailability of this drug and that Gly modification of DMAE is desirable for a prodrug of DMAE.

Midodrine is a selective 1-receptor agonist to treat orthostatic hypotension and is a prodrug of 1-(2',5'-dimethoxyphenyl)-2-aminoethanol (DMAE) to combine the glycine by a peptide bond. After oral administration, the bioavailability of midodrine is reported to be approximately 100%, whereas that of DMAE is approximately 50%. However, mechanisms of improving intestinal absorption of midodrine have not been elucidated.

Midodrine resembles a dipeptide in structure, and improving the intestinal absorption of DMAE by modifying an amino acid is similar to the relationship between acyclovir and valacyclovir. Based on this background, in the present study, we examined whether midodrine can be a substrate for PEPT1 in vitro and in vivo. Furthermore, we synthesized various DMAE amino acid derivatives to clarify which amino acid modification by a peptide bond is the most suitable for interaction with PEPT1 using DMAE as a model compound. We do not have enough information regarding this issue, although there are various reports to examine the interaction of amino acid esterification of poorly absorbed drugs with PEPT1 (Han et al., 1998; Sawada et al., 1999). Finally, we simulated pH profiles of midodrine (cationic) and a glutamate derivative of DMAE (neutral) using a 14-state model of PEPT1, which we constructed recently (Irie et al., 2005).

	Materials and Methods

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Materials. Midodrine and DMAE were gifts from Taisho Pharmaceutical (Tokyo, Japan). [³H]Glycylsarcosine (4 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA). Glycylsarcosine was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of the highest purity available.

DMAE Amino Acid Derivatives Synthesis. Figure 1 shows the structures of DMAE amino acid derivatives. The derivatives were obtained as follows. Diisopropylethylamine (1 Eq), N-Boc-protected amino acid (1 Eq) [side chain protection: Tyr (t-Bu); Cys (Trt); Lys (Boc); and Glu (t-Bu)], HOBt·H₂O (1 Eq), and WSCDI (1 Eq) were added to a solution of DMAE hydrochloride in dimethylformamide at 0°C. The mixture was stirred for 12 h at room temperature and extracted with ethyl acetate. The extract was washed with saturated citric acid, saturated NaHCO₃, and brine and dried over MgSO₄. Concentration under reduced pressure followed by flash chromatography over silica gel gave the amide compound. Deprotection of the resulting amide with 4 M HCl-dioxane for 2 h at room temperature followed by purification through reversed-phase-HPLC (H₂O-CH₃CN containing 0.1% TFA) gave the desired midodrine derivative (1:1 mixture of diastereomers) as TFA salts (two steps, 50 -80% yield). All compounds were characterized by ¹H NMR and electrospray ionization-mass spectrometry.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Structures of midodrine, DMAE, and DMAE amino acid derivatives.

Cell Culture. Caco-2 cells obtained from the American Type Culture Collection (ATCC HTB37) were maintained by serial passage in plastic culture dishes as described previously (Irie et al., 2001). To measure the uptake of midodrine from the apical side, Caco-2 cells were seeded on 35-mm plastic dishes (2 x 10⁵ cells/dish, 2 ml of culture medium). The cell monolayers were given fresh culture medium every 2 to 4 days and were used on the 14th or 15th day for experiments.

Uptake Experiments with Cell Monolayers. The uptake of [³H]glycylsarcosine was measured as described previously (Terada et al., 1997). In experiments using midodrine, DMAE, and DMAE amino acid derivatives, the extraction solution (water/acetonitrile, 50:50) was added to the cells after the uptake period. On standing for 1 h at room temperature, the solutions were centrifuged and the supernatants were filtered through a Millipore filter (SGJVL, 0.22 µm; Millipore Corporation, Billerica, MA). The filtrates were analyzed by HPLC. Because midodrine and DMAE amino acid derivatives were partially metabolized to DMAE and each amino acid within Caco-2 cells and oocytes, the amounts of midodrine and DMAE amino acid derivatives taken up were calculated as the sum of the amounts of the unchanged form and DMAE.

Simulation. A 14-state model of PEPT1, which we recently constructed (Irie et al., 2005), was used to simulate the pH profiles of midodrine and glutamate derivative of DMAE.

Uptake Experiments with Xenopus Oocytes. The synthesis and injection of cRNA were performed as described previously (Terada et al., 1996). The uptake of midodrine and DMAE was examined as reported with slight modifications (Saito et al., 1995). In brief, the uptake reaction was initiated in a 24-well plate by incubating the oocytes in 500 µl of uptake buffer, pH 6.0 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM MES, pH 6.0, or HEPES, pH 7.4) containing 1 mM midodrine or DMAE for 1 h at room temperature. At the end of the uptake period, oocytes were washed five times in 1.5 ml of ice-cold uptake buffer, pH 7.4, and were transferred to a 1.5-ml tube. The oocytes were homogenized in 0.2 ml of extraction solution (water/acetonitrile, 50:50). The homogenates were centrifuged at 10,000 rpm for 5 min, and supernatants were filtered through a Millipore filter SGJVL (0.45 µM). The filtrates were analyzed by HPLC.

In Vivo Experiments. Experiments in vivo were performed as described previously (Pan et al., 2003). In brief, rats were anesthetized with sodium pentobarbital (50 mg/kg). The femoral artery was cannulated with a polyethylene tube (SP-31; Natsume Seisakusho, Tokyo, Japan) for blood sampling. For the intraintestinal administration of midodrine, the abdominal cavity of rats was opened via a midline incision, and the upper site of the duodenum was exposed to administer the drug. Midodrine was injected into the lumen of the duodenum at a dose of 2.91 mg/kg. Blood samples were collected from the femoral artery at 1, 5, 10, 15, 30, 45, 60, 90, and 120 min after the end of the injection. The blood samples were centrifuged at 14,000 rpm for 3 min, and 100 µl of plasma was deproteinized by adding 200 µl of methanol. The samples were centrifuged at 14,000 rpm for 3 min, and supernatants were filtered through a Millipore filter (SGJVL, 0.45 µm). The filtrates were analyzed by HPLC.

Analytical Methods. The uptake of midodrine and DMAE by Caco-2 cells and oocytes was measured simultaneously using high-performance liquid chromatograph LC-10AD_SP (Shimadzu Co., Kyoto, Japan) equipped with an SPD-10A_VP variable wavelength UV detector (Shimadzu Co.) and an integrator (Chromatopac C-R8A; Shimadzu Co.) under the following conditions: column, TSK-gel ODS 80TM (4.6 mm i.d. x 150; Tosoh Co., Tokyo, Japan); mobile phase, 1% SDS/phospholic acid/acetonitrile, 600:1:400; flow rate, 0.8 ml/min; wavelength, 290 nm; injection volume, 50 µl; and temperature, 50°C for midodrine, DMAE, and DMAE amino acid derivatives, except for a lysyl derivative of DMAE: mobile phase, water, and acetonitrile, both containing 0.1% (v/v) TFA, linear gradient of acetonitrile in water 10 to 40% over 30 min; flow rate, 1.0 ml/min; wavelength, 220 nm; injection volume, 50 µl; and temperature, 40°C for a lysyl derivative of DMAE.

Data Analysis. Each experimental point represents the mean ± S.E. of three to nine measurements from one to three separate experiments. Data from uptake experiments were analyzed statistically by a one-way analysis of variance followed by Sheffé's test. Kinetic parameters of the DMAE plasma concentration were statistically compared using a nonpaired t test.

	Results

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Effect of Midodrine on [³H]Glycylsarcosine Uptake by Caco-2 Cells. To assess the interaction of midodrine with PEPT1, we examined the inhibitory effect of midodrine on [³H]glycylsarcosine uptake by Caco-2 cells. As shown in Fig. 2A, [³H]glycylsarcosine uptake was inhibited by midodrine in a concentration dependent manner, and the IC₅₀ value was calculated at 2.8 ± 0.1 mM.

View larger version (13K): [in this window] [in a new window]   Fig. 2. A, effect of midodrine on [3H]glycylsarcosine uptake by Caco-2 cells. The cell monolayers were incubated at 37°C for 15 min with 25 µM [3H]glycylsarcosine, pH 6.0, in the presence of various concentrations of midodrine. After the incubation, the radioactivity of dissolved cells was measured. Each point represents the mean ± S.E. of three independent monolayers. The IC50 value was calculated from three separate experiments. B, uptake of midodrine and DMAE by oocytes expressing PEPT1. Water-injected (open column) or PEPT1-expressing (hatched column) oocytes were incubated at 37°C for 1 h with 1 mM midodrine or DMAE, pH 6.0. The amounts of midodrine and DMAE in the solubilized oocytes were measured by HPLC. Each column represents the mean ± S.E. of three experiments. Each experiment was performed with eight oocytes.

Uptake of Midodrine and DMAE by Oocytes Expressing PEPT1. To confirm the midodrine transport by PEPT1, we measured the uptake of midodrine and DMAE by PEPT1-expressing oocytes. As shown in Fig. 2B, the uptake of midodrine was markedly increased in PEPT1-expressing oocytes compared with water-injected oocytes. In contrast, no PEPT1-mediated transport of DMAE was observed.

Uptake of Midodrine and DMAE by Caco-2 Cells. To investigate the transport and metabolism of midodrine in the intestinal epithelial cells, uptake experiments were performed using Caco-2 cells. Figure 3A shows the time course of midodrine and DMAE uptake by Caco-2 cells. As shown in Fig. 3A, more midodrine than DMAE was taken up at each time point. After 1 h of incubation in Caco-2 cells, 35% midodrine was metabolized to DMAE (Fig. 3B)

View larger version (16K): [in this window] [in a new window]   Fig. 3. A, time course of midodrine and DMAE uptake by Caco-2 cells. The cell monolayers were incubated at 37°C for the periods indicated with 0.5 mM midodrine or DMAE, pH 6.0. After the incubation, the amounts of midodrine () and DMAE () extracted from the cell monolayers were measured by HPLC. Each point represents the mean ± S.E. of three independent monolayers. B, metabolism of midodrine in Caco-2 cells after 1-h uptake. Open and hatched columns represent midodrine and DMAE, respectively. Each column represents the mean ± S.E. of three independent monolayers.

View larger version (20K): [in this window] [in a new window]   Fig. 4. A, effect of various compounds on midodrine uptake by Caco-2 cells. The cell monolayers were incubated at 37°C for 5 min with 0.5 mM midodrine, pH 6.0, in the absence or presence of each inhibitor (10 mM). After the incubation, the amounts of midodrine and DMAE extracted from the cell monolayers were measured by HPLC. Each column represents the mean ± S.E. of three independent monolayers. *, P < 0.05, significantly different from the control. B, concentration dependence of midodrine uptake by Caco-2 cells. Nonspecific uptake was evaluated by measuring midodrine uptake in the presence of 50 mM glycylleucine, and the results are shown after correction for the nonsaturable component. The cell monolayers were incubated at 37°C for 5 min with various concentration of midodrine, pH 6.0. After the incubation, the amounts of midodrine and DMAE extracted from the cell monolayers were measured by HPLC. Each point represents the mean ± S.E. of three independent monolayers. The Km value was calculated from three separate experiments.

Absorption of Midodrine from Rat Intestine. To confirm the involvement of PEPT1 in the transport of midodrine in vivo, we performed a pharmacokinetic analysis. Figure 5 shows the time course of DMAE plasma concentration after the intraintestinal administration of midodrine in rats. The concentration peaked 5 min after the intraintestinal administration, indicating that the absorption of midodrine was very rapid. Midodrine absorption was inhibited in the presence of cyclacillin, a high-affinity substrate of PEPT1, until 30 min postadministration. The estimated pharmacokinetic parameters are summarized in Table 1. The area under the blood concentration-time curve for 30 min (AUC_0-30) and the maximal concentration in the presence of cyclacillin were reduced significantly compared with the control values, and the time to maximal concentration (t_max) was significantly longer than the control.

View larger version (17K): [in this window] [in a new window]   Fig. 5. DMAE plasma concentration after intraintestinal administration in the absence or presence of cyclacillin. After intraintestinal administration at a dose of 2.91 mg/kg midodrine and in the absence () or presence () of 6.85 mg/kg cyclacillin, blood samples were collected at the times indicated. The blood samples were determined by HPLC. Each point represents the mean ± S.E. of six rats.

Uptake of DMAE Amino Acid Derivatives in Caco-2 Cells. To examine which amino acid modification of midodrine is suitable for interaction with PEPT1, we synthesized various DMAE-L-amino acid derivatives DMAE-X (X: -Val, -Ile, -Phe, -Tyr, -Cys, -Pro, -Glu, and -Lys), in which the glycine residue is replaced by other amino acids (Fig. 1). We measured the uptake of these derivatives in Caco-2 cells in the absence or presence of the excess glycylsarcosine (Fig. 6). DMAE-Phe uptake was greatest but was not inhibited by the glycylsarcosine. DMAE-Val, -Ile, -Tyr, -Cys, -Glu, and -Lys uptake was roughly equal to midodrine uptake, but the excess glycylsarcosine did not have a significant inhibitory effect in the case of DMAE-Ile and -Lys. DMAE-Pro uptake by Caco-2 cells was low and not inhibited by the excess glycylsarcosine. Furthermore, using in vitro everted sacs of the rat small intestine, we examined the stability of three kinds of derivatives (DMAE-Gly, -Val, and -Phe) at the mucosal surface. The ratio of unchanged form at the mucosal side after 1 h of incubation was as follows: DMAE-Gly (95%), -Val (47%), and -Phe (0.7%), suggesting that DMAE-Gly (midodrine) is the most stable derivative among the tested compounds.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Uptake of DMAE amino acid derivatives in Caco-2 cells. The cell monolayers were incubated at 37°C for 30 min with 0.5 mM of each DMAE amino acid derivative in the absence (opened column) or presence (hatched column) of 10 mM glycylsarcosine. After the incubation, the amounts of DMAE amino acid derivatives extracted from the cell monolayers were measured by HPLC. Each column represents the mean ± S.E. of three independent monolayers. *, P < 0.05; **, P < 0.01, significantly different from the control.

pH Dependence of Midodrine and DMAE-Glu Uptake by Caco-2 Cells. Figure 7A shows the pH dependence of midodrine and DMAE-Glu uptake by Caco-2 cells. In contrast to neutral substrates, the cationic midodrine was taken up extensively at alkaline pH. This pH profile was similar to that of the cationic dipeptides transported by PEPT1 (Mackenzie et al., 1996). On the other hand, the pH dependence of DMAE-Glu (neutral) uptake was high at acidic pH. Recently, we constructed a 14-state model of PEPT1 and reproduced the pH profiles of various charged substrates (Irie et al., 2005). The pH profile of midodrine as well as DMAE-Glu was reproduced by our 14-state model of PEPT1 (Fig. 7B). In the simulation, the uptake of midodrine and DMAE-Glu was calculated every 0.01 pH units. Four dissociation constants of midodrine and DMAE-Glu on the exterior side (K_{d, Soc1}, K_{d, Soc2}, K_{d, Son}, and K_{d, Soa}) were recalculated based on our 14-state model (Irie et al., 2005).

View larger version (11K): [in this window] [in a new window]   Fig. 7. A, pH dependence of midodrine () and DMAE-Glu () uptake by Caco-2 cells. The cell monolayers were incubated at 37°C for 15 min with 0.5 mM midodrine or DMAE-Glu, pH 6.0. After the incubation, the amounts of midodrine, DMAE-Glu, and DMAE extracted from the cell monolayers were measured by HPLC. Each point represents the mean ± S.E. of three independent monolayers. B, simulation of pH dependence of midodrine () and DMAE-Glu () uptake by Caco-2 cells. The rates of uptake value at pH 6.0 were derived from the uptake values in Fig. 6A. The pH profiles of midodrine and DMAE-Glu uptake were delineated by simulation (curve). Four dissociation constants (in micromolars) of midodrine and DMAE-Glu on the exterior side (Kd, Soc1, Kd, Soc2, Kd, Son, and Kd, Soa) were defined as follows: Kd, Soc1 = 1500, Kd, Soc2 = 120, Kd, Son = 2000, Kd, Soa = 2000, Kd, Soc1 = 500, Kd, Soc2 = 500, Kd, Son = 300, and Kd, Soa = 2000, respectively.

	Discussion

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Midodrine is an oral drug that acts as a selective 1-receptor agonist and the medication of choice for treating orthostatic hypotension in the elderly (Mukai and Lipsitz, 2002). Furthermore, this drug is beneficial in preventing or ameliorating the symptoms of intradialytic hypotension (Prakash et al., 2004). There are many reports regarding the pharmacological and clinical effects of midodrine, but its pharmacokinetic properties, especially the mechanism behind its intestinal absorption, have not been elucidated. In the present study, we demonstrated for the first time that midodrine was transported by PEPT1 in vitro and in vivo. Because PEPT1 can transport various peptide-like drugs, such as -lactam antibiotics and antiviral drugs, and also mediates the cellular uptake of dipeptides and tripeptides derived from ingested proteins, there is potential for drug/ drug interaction and/or drug/food interaction during therapy with midodrine.

Previously, Beauchamp et al. (1992) evaluated the bioavailability of 18 ester compounds of acyclovir and found that the L-valyl ester derivative had the best bioavailability followed by the L-isoleucyl, L-alanyl, and glycyl ester derivatives. Thereafter, these amino acid preferences were demonstrated to be related to the affinity for PEPT1 (Sawada et al., 1999). Thus, for amino acid ester modification, L-valine may be a suitable target for converting a poorly absorbed drug into a substrate of PEPT1. On the other hand, for amino acid modification of a peptide bond, there is little information available on which amino acids provide for high-affinity interaction with PEPT1 and resistance against enzymatic degradation. In the present study, we demonstrated that the derivatives with -Gly, -Val, -Tyr, -Cys, and -Glu were suggested to be interacted with PEPT1, but those with -Ile, -Phe, -Pro, and -Lys did not (the lack of significant difference in the case of -Lys may be caused by the unusually high scatter). Large amounts of DMAE-Phe were accumulated in Caco-2 cells, but this derivative showed extremely low stability in the rat small intestine. These findings suggest that DMAE-Phe may not be appropriate for a prodrug for targeting PEPT1 and improving the intestinal absorption in vivo. On the other hand, DMAE-Gly (midodrine) was high stability and can be recognized by PEPT1. Although we did not perform a detailed analysis, overall, our results suggest that Gly modification of DMAE by a peptide bond is appropriate for the preparation of a prodrug for DMAE. Further studies are needed to define the amino acids suitable for targeting PEPT1 in peptide bond-based modifications.

It has been reported that pH dependences of neutral (Inui et al., 1992; Saito and Inui, 1993; Terada et al., 1999; Kennedy et al., 2002) and anionic (Matsumoto et al., 1995) substrates of PEPT1 show different profiles. The pH dependence of cationic substrates determined by measuring evoked current is also different from neutral and anionic substrates (Mackenzie et al., 1996). Midodrine almost exists as a cation at pH 5.0 to 7.4, because its dissociation constant (pKa) is 7.96. As shown in Fig. 7A, midodrine uptake by PEPT1 gradually increased as the pH rose from 5.0 to 7.4. This pH profile clearly differs from that of a neutral substrate, such as glycylsarcosine, or an anionic substrate, such as ceftibuten. In contrast, the pH profile of DMAE-Glu, which mainly exists as a neutral substrate from pH 5.0 to 7.4, was similar to that of glycylsarcosine. Recently, based on the presumed recognition patterns of PEPT1 for neutral and charged substrates, we constructed a 14-state model of PEPT1 and reproduced the pH profiles of various charged substrates (Irie et al., 2005). In this model, we hypothesized two mechanisms for the transport of cationic substrates, namely, that the transport of cationic substrate occurs with or without H⁺. Therefore, the transport of cationic substrates is assumed to be altered by the degree of contribution of the two pathways. In the previous study, we could not simulate the pH profile of cationic substrates, because there is little known regarding the transport characteristics of cationic substrates of PEPT1. The present simulation revealed that the pH profile of midodrine as well as DMAE-Glu was reproduced by our 14-state model of PEPT1 (Fig. 7B), suggesting that our model can be applied to cationic as well as neutral substrates.

In conclusion, we have demonstrated that midodrine, but not DMAE, is recognized by PEPT1 and that this recognition improves the oral bioavailability of DMAE. In addition, the transport of DMAE amino acid derivatives via PEPT1 depends on the amino acids modified. These findings suggested that modifying not only the L-valyl ester but also the glycyl peptide of poorly absorbed drugs, which are targeted to intestinal PEPT1, is useful for improving of the intestinal absorption of drugs.

	Footnotes

 
This work was supported by the 21st Century Center of Excellence Program "Knowledge Information Infrastructure for Genome Science," the Leading Project for Biosimulation, a grant-in-aid for Scientific Research from the Ministry of Education, Culture and Sports of Japan, and a grant-in-aid for Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare of Japan.

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

doi:10.1124/jpet.106.102830.

ABBREVIATIONS: PEPT1, H⁺-coupled peptide transporter 1; DMAE, 1-(2',5'-dimethoxyphenyl)-2-aminoethanol; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid; AUC, area under the curve.

Address correspondence to: Professor Ken-ichi Inui, Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: inui{at}kuhp.kyoto-u.ac.jp

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