Transport of Amino Acid-Based Prodrugs by the Na - and Cl -Coupled Amino Acid Transporter ATB and Expression of the Transporter in Tissues Amenable for Drug Delivery
نویسندگان
چکیده
We evaluated the potential of the Na and Cl -coupled amino acid transporter ATB as a delivery system for amino acidbased prodrugs. Immunofluorescence analysis indicated that ATB is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of -carboxyl derivatives of aspartate and -carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB , several of the aspartate and glutamate derivatives inhibited glycine transport via ATB . Direct evidence for ATB -mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB heterologously, to aspartate -benzyl ester as a model derivative induced inward currents in a Na and Cl dependent manner with a Na /Cl /aspartate -benzyl ester stoichiometry of 2:1:1. ATB transported not only the -carboxyl derivatives of aspartate and the -carboxyl derivatives of glutamate but also valacyclovir, which is an -carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB was demonstrable in both heterologous expression systems. This process was dependent on Na and Cl . The ability of ATB to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB has significant potential as a delivery system for amino acid-based drugs and prodrugs. ATB is a Na and Cl -coupled amino acid transporter that is energized by transmembrane gradients of Na and Cl as well as by the membrane potential (Palacin et al., 1998; Ganapathy et al., 2001, 2003). It belongs to the neurotransmitter transporter gene family (SLC6) (Chen et al., 2003). Among the currently known mammalian amino acid transporters, ATB is the only transporter with a very broad substrate specificity that is driven by a Na gradient and a Cl gradient. This transporter recognizes neutral as well as cationic amino acids as substrates. In addition to the surprisingly broad substrate specificity of ATB with regard to the naturally occurring L-amino acids, this transporter can also transport various D-amino acids (Hatanaka et al., 2002), nitric-oxide synthase inhibitors (Hatanaka et al., 2001), and carnitine and its esters (Nakanishi et al., 2001). Unlike most other amino acid transporters that exhibit a broad expression pattern, Northern blot analysis has revealed that ATB transcripts are detectable primarily in the colon, lung, and mammary gland (Sloan and Mager, 1999). The fact that ATB recognizes neutral and cationic amino acids, but not anionic amino acids as substrates, indicates that the presence of a negative charge in the side chain of amino acids prevents their recognition by the transporter as substrates. The presence of an uncharged side chain or a positively charged side chain does not interfere with the recognition as evidenced from the transport of neutral and cationic amino acids by the transporter. This is further exemplified by the findings that although aspartate and glutamate are not substrates for ATB , the amide derivatives of This work was supported by the National Institutes of Health Grant GM65344. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.057109. ABBREVIATIONS: ATB , amino acid transporter B ; Z-Glu-OBzl, N-benzyloxycarbonyl-L-glutamic acid -benzyl ester; Pd/C, palladium 10% (w/v) on activated carbon; HRPE, human retinal pigment epithelial; PEPT1, peptide transporter 1; MES, 4-morpholineethanesulfonic acid; Asp-OBzl, L-aspartate -benzyl ester; Glu-OBzl, L-glutamate -benzyl ester; Acv-Glu, acyclovir L-glutamate -ester. 0022-3565/04/3083-1138–1147$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 57109/1127386 JPET 308:1138–1147, 2004 Printed in U.S.A. 1138 at A PE T Jornals on M ay 8, 2017 jpet.asjournals.org D ow nladed from these anionic amino acids, namely, asparagine and glutamine, are excellent substrates. Therefore, we hypothesized that if the -carboxyl group of aspartate and the -carboxyl group of glutamate are modified with substitutions such that the resulting products do not have a negatively charged side chain, such modified amino acids may be recognized by ATB as substrates. If this hypothesis is correct, it might be feasible to design prodrugs in the form of side chain derivatives of aspartate and glutamate that would be recognized as substrates by ATB to facilitate the delivery of drugs into cells. We provide evidence in this article in support of the feasibility of this approach. In addition, our results show evidence in support of even a broader applicability of this strategy than originally hypothesized. Drugs that are derivatized as the -carboxyl esters of neutral amino acids are also recognized as substrates by ATB . This broadens the spectrum of amino acid-based prodrugs that can be designed for delivery into cells via this transporter. Because the utility of any transporter as a drug delivery system is influenced by its tissue distribution, we investigated whether the distribution of ATB was suitable to provide effective drug delivery. These studies have shown that ATB is expressed abundantly on the luminal surfaces of the large intestine and bronchi and in the conjunctival epithelium. This tissue distribution pattern lends support to our hypothesis that ATB is potentially very useful for the delivery of amino acid-based prodrugs in the form of, for example, oral pills and rectal suppositories, aerosol, and eye drops. Materials and Methods Materials. [2-H]Glycine (specific radioactivity, 30 Ci/mmol) and [8-H]valacyclovir (specific radioactivity, 5 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). Valacyclovir was from GlaxoSmithKline (Research Triangle Park, NC). Acyclovir, L-aspartate, L-asparagine, and various -carboxyl derivatives of aspartate were purchased from Sigma-Aldrich (St. Louis, MO). Similarly, L-glutamate, L-glutamine, and various -carboxyl derivatives of glutamate were also purchased from Sigma-Aldrich. N-Benzyloxycarbonyl-L-glutamic acid -benzyl ester (Z-Glu-OBzl) was obtained from Novabiochem (San Diego, CA). 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide, palladium 10% (w/v) on activated carbon (Pd/C), 4-dimethylaminopyridine, dimethylformamide, and CH2Cl2 were purchased from Aldrich Chemical Co. (Milwaukee, WI). Immunofluorescent Localization of ATB in Mouse Colon, Lung, and Eye. A polyclonal antibody, raised against the peptide TDHEIPTISGSTKPE corresponding to the C-terminal region of mouse ATB (amino acid position 624–637), was used for immunofluorescent detection of the protein in mouse tissues. The specificity of the antibody has been described previously (Hatanaka et al., 2002). Immunolocalization was carried out as described previously (Smith et al., 1999; Bridges et al., 2000; Ola et al., 2001). Cryosections (10 m in thickness) of the tissues, fixed with ice-cold acetone, were blocked with 10% normal goat serum. These sections were incubated with the primary antibody for 3 h at room temperature at a dilution of 1:50 followed by an overnight incubation at 4°C. Incubation with 0.1% normal rabbit serum (preimmune) or with buffer only served as negative controls. After rinsing, all sections were incubated at 4°C with a fluorescein isothiocyanate-conjugated AffiniPure goat anti-rabbit IgG antibody at a dilution of 1:100. The sections were then optically sectioned (z series) using an MRC-600 laser scanning confocal imaging system (Bio-Rad, Hercules, CA). Images were analyzed using the COSMOS software package (BioRad). Additional sections were subjected to hematoxylin-eosin staining. Functional Expression of ATB in Human Retinal Pigment Epithelial (HRPE) Cells. The mouse ATB cDNA (Hatanaka et al., 2001; Nakanishi et al., 2001) was used for functional expression in HRPE cells in the analysis of its role in the transport of amino acid-based prodrugs. The vaccinia virus expression system was used for this purpose (Hatanaka et al., 2001, 2002). This procedure involves infection of the cells with a recombinant vaccinia virus carrying the gene for T7 RNA polymerase, followed by lipofectinmediated transfection of the cells with plasmid DNA in which the cDNA insert is under the control of T7 promoter. Glycine was used as the substrate for ATB (Hatanaka et al., 2001, 2002; Nakanishi et al., 2001). Transport of 10 M glycine (radiolabeled glycine, 0.05 M; unlabeled glycine, 9.95 M) in cDNA-transfected cells was measured at 37°C for 30 min. The transport was linear under these conditions. The transport buffer was 25 mM Hepes/Tris (pH 7.5) containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. Interaction of various amino acid-derivatives and amino acid-based prodrugs with the transporter was assessed by monitoring the ability of these compounds to inhibit ATB -mediated glycine transport. Transport of 5 M valacyclovir (radiolabeled valacyclovir, 1 M; unlabeled valacyclovir, 4 M) via ATB was monitored directly by measuring its uptake. Transport measurements were made in parallel in vector-transfected cells and in ATB cDNA-transfected HRPE cells to account for endogenous valacyclovir transport activity. The ATB -specific transport of valacyclovir was determined by subtracting the transport values measured in vector-transfected cells from the transport values measured in cDNA-transfected cells. The dependence of ATB -mediated transport of valacyclovir on Na was determined by comparing the transport values measured in the presence of Na and in the absence of Na (N-methyl-D-glucamine chloride replacing NaCl isoosmotically). The dependence of the transport process on Cl was determined by comparing the transport values measured in the presence of Cl and in the absence of Cl (sodium gluconate replacing NaCl isoosmotically and also potassium gluconate and calcium gluconate replacing KCl and CaCl2, respectively, at equimolar concentrations). Transport of valacyclovir via PEPT1 was measured in HRPE cells expressing the human PEPT1 cDNA (Liang et al., 1995) heterologously. Because PEPT1 is a H -coupled transporter (Ganapathy and Leibach, 1991; Leibach and Ganapathy, 1996), the transport of 5 M valacyclovir (radiolabeled valacyclovir, 1 M; unlabeled valacyclovir, 4 M) via this transporter was monitored with the transport buffer that contained 25 mM MES/Tris (pH 6), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. Transport measurements were made at 37°C with 15-min incubation to represent initial transport rates. Transport measurements were made in parallel in vector-transfected cells and in cDNA-transfected cells to correct for endogenous transport activity in determining the PEPT1specific valacyclovir transport. Functional Expression of ATB in Xenopus laevis Oocytes. Capped cRNA from the cloned mouse ATB cDNA was synthesized using the mMESSAGE mMACHINE kit (Ambion, Austin, TX). Mature oocytes (stage IV or V) from X. laevis were isolated by treatment with collagenase A (1.6 mg/ml), manually defolliculated, and maintained at 18°C in modified Barth’s medium supplemented with 10 mg/ml gentamycin as described previously (Hatanaka et al., 2001, 2002; Nakanishi et al., 2001). On the following day, oocytes were injected with 50 ng of cRNA. Water-injected oocytes served as controls. The oocytes were used for electrophysiological studies 6 days after cRNA injection. Electrophysiological studies were performed by the two-microelectrode voltage-clamp method (Parent et al., 1992). Oocytes were perfused with a NaCl-containing buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 3 mM Hepes, 3 mM MES, and 3 mM Tris, pH 7.5), followed by the same buffer containing different amino acid derivatives. The membrane potential was clamped at 50 mV. The differences between the steady-state currents measured in the presence and absence of substrates were considered as the substrate-induced currents. In the analysis of the saturation ATB as a Drug Delivery System 1139 at A PE T Jornals on M ay 8, 2017 jpet.asjournals.org D ow nladed from kinetics of substrate-induced currents, the kinetic parameter K0.5 (i.e., the substrate concentration necessary for the induction of halfmaximal current) was calculated by fitting the values of the substrate-induced currents to Michaelis-Menten equation. The Na and Cl activation kinetics of substrate-induced currents were analyzed by measuring the substrate-specific currents in the presence of increasing concentrations of Na (the concentration of Cl kept constant at 100 mM) or in the presence of increasing concentrations of Cl (the concentration of Na kept constant at 100 mM). In these experiments, the composition of the perfusion buffer was modified to contain 2 mM potassium gluconate, 1 mM MgSO4, and 1 mM calcium gluconate in place of KCl, MgCl2, and CaCl2, respectively. The data from these experiments were analyzed by the Hill equation to determine the K0.5 values for Na and Cl (i.e., the concentrations of Na and Cl necessary for half-maximal activation) and the Hill coefficient (nH; the number of Na and Cl ions involved in the activation process). The kinetic parameters were determined using the commercially available computer program Sigma Plot, version 6.0 (SPSS Science, Inc., Chicago, IL). Data Analysis. Experiments with HRPE cells were repeated three times with three independent transfections and transport measurements were made in duplicate in each experiment. Electrophysiological measurements of substrate-induced currents were repeated at least three times with separate oocytes. The data are presented as means S.E. of these replicates. Synthesis of Acyclovir Glutamic Acid -Ester. Acyclovir (0.888 mmol) and Z-Glu-OBzl (2.22 mmol) were dissolved in dimethylformamide (6 ml); 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide (426 mmol) and 4-dimethylaminopyridine (2.22 mmol) were then added to the solution. The solution was stirred for 18 h at room temperature. Dimethylformamide was removed in vacuo, and the residue was chromatographed on NH silica gel (Chromatorex DM1020; Fuji Silysia Chemical LTD., Japan) using 1:10 to 1:5 methanolCH2Cl2 as the eluent to generate acyclovir Z-Glu-OBzl -ester. This compound was then dissolved in ethanol/methanol/CH2Cl2 [1:1:1 (v/v)], and 20 mg of Pd/C was added to the solution. The mixture was stirred under hydrogen for 3 days. The mixture was then filtered to remove the catalyst (Pd/C) followed by removal of the solvent in vacuo. The product (acyclovir Glu -ester) was a white amorphous solid (120 mg) with an overall yield of 38%. The compound was analyzed by H NMR and electrospray ionization-mass spectrometry. The purity of the final product was confirmed by reverse-phase high-performance liquid chromatography using a Dionex analytical high-performance liquid chromatography system (Dionex Corp., Sunnyvale, CA) with an analytical column (Cyclobond I 2000, 4.6 100 cm; Advanced Separation Technologies Inc., Whippany, NJ). The purity of the compound was 96%. The molecular weight of the compound was determined by electrospray ionization-mass spectrometry (Finnigan Mat LC-Q; Thermo Finnigan, San Jose, CA).
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