Drug transport via the intestinal peptide transporter PepT1.

The focus of this review is on the pharmaceutical relevance of the intestinal peptide transporter PepT1. The review is limited to the progress made in the field over the past two years. Much of this progress is being driven by the prevailing view that PepT1 can be used for drug delivery purposes. Studies have indeed shown that several drugs, prodrugs and drug candidates gain entry into the systemic circulation via PepT1. Very recent examples are prodrugs of zanamivir, oseltamivir and didanosine.

Inhibition of human intestinal α-glucosidases by calystegines.

Calystegines are polyhydroxylated nortropane alkaloids found in Convolvulaceae, Solanaceae, and other plant families. These plants produce common fruits and vegetables. The calystegine structures resemble sugars and suggest interaction with enzymes of carbohydrate metabolism. Maltase and sucrase are α-glucosidases contributing to human carbohydrate degradation in the small intestine. Inhibition of these enzymes by orally administered drugs is one option for treatment of diabetes mellitus type 2. In this study, inhibition of maltase and sucrase by calystegines A3 and B2 purified from potatoes was investigated. In silico docking studies confirmed binding of both calystegines to the active sites of the enzymes. Calystegine A3 showed low in vitro enzyme inhibition; calystegine B2 inhibited mainly sucrose activity. Both compounds were not transported by Caco-2 cells indicating low systemic availability. Vegetables rich in calystegine B2 should be further investigated as possible components of a diet preventing a steep increase in blood glucose after a carbohydrate-rich meal.

Transport of the areca nut alkaloid arecaidine by the human proton-coupled amino acid transporter 1 (hPAT1).

OBJECTIVES: The pyridine alkaloid arecaidine is an ingredient of areca nut preparations. It is responsible for many physiological effects observed during areca nut chewing. However, the mechanism underlying its oral bioavailability has not yet been studied. We investigated whether the H⁺-coupled amino acid transporter 1 (PAT1, SLC36A1), which is expressed in the intestinal epithelium, accepts arecaidine, arecoline, isoguvacine and other derivatives as substrates. METHODS: Inhibition of L-[³H]proline uptake by arecaidine and derivatives was determined in Caco-2 cells expressing hPAT1 constitutively and in HeLa cells transiently transfected with hPAT1-cDNA. Transmembrane transport of arecaidine and derivatives was measured electrophysiologically in Xenopus laevis oocytes. KEY FINDINGS: Arecaidine, guvacine and isoguvacine but not arecoline strongly inhibited the uptake of L-[³H]proline into Caco-2 cells. Kinetic analyses revealed the competitive manner of L-proline uptake inhibition by arecaidine. In HeLa cells transfected with hPAT1-cDNA an affinity constant of 3.8 mm was obtained for arecaidine. Electrophysiological measurements at hPAT1-expressing X. laevis oocytes demonstrated that arecaidine, guvacine and isoguvacine are transported by hPAT1 in an electrogenic manner. CONCLUSION: We conclude that hPAT1 transports arecaidine, guvacine and isoguvacine across the apical membrane of enterocytes and that hPAT1 might be responsible for the intestinal absorption of these drug candidates.

Transport of L-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells.

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 µM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate L-[(3)H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H(+) gradient-stimulated uptake of L-[(3)H]proline. Uptake was rapid, independent of a Na(+) gradient but stimulated by an inwardly directed H(+) gradient with maximal uptake occurring at pH 6.0. L-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K(t)) of 130 ± 10 µM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min(-1 )mg of protein(-1). Glycine, L-alanine, and L-tryptophan strongly inhibited L-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H(+)-amino acid cotransport system PAT2.

Protocol for effective differentiation of 3T3-L1 cells to adipocytes.

In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 µM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.

Three-dimensional quantitative structure-activity relationship analyses of substrates of the human proton-coupled amino acid transporter 1 (hPAT1).

The proton-coupled amino acid transporter hPAT1 has recently gained much interest due to its ability to transport small drugs thereby allowing their oral administration. A three-dimensional quantitative structure-activity relationship (3D QSAR) study has been performed on its natural and synthetic substrates employing comparative molecular similarity indices analysis (CoMSIA) to investigate the structural requirements for substrates and to derive a predictive model that may be used for the design of new prodrugs. The cross-validated CoMSIA models have been derived from a training set of 40 compounds and the predictive ability of the resulting models has been evaluated against a test set of 10 compounds. Despite the relatively narrow range of binding affinities (K(i) values) reliable statistical models with good predictive power have been obtained. The best CoMSIA model in terms of a proper balance of all statistical terms and the overall contribution of individual properties has been obtained by considering steric, hydrophobic, hydrogen bond donor and acceptor descriptors (q(cv)(2)=0.683, r(2)=0.958 and r(PRED)(2)=0.666). The 3D QSAR model provides insight in the interactions between substrates and hPAT1 on the molecular level and allows the prediction of affinity constants of new compounds. A pharmacophore model has been generated from the training set by means of the MOE (molecular operating environment) program. This model has been used as a query for virtual screening to retrieve potential new substrates from the small-molecule, 'lead-like' databases of MOE. The affinities of the compounds were predicted and 11 compounds were identified as possible high-affinity substrates. Two selected compounds strongly inhibited the hPAT mediated l-[(3)H]proline uptake into Caco-2 cells constitutively expressing the transport protein.

Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form.

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[(3)H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.

Uptake of codeine into intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells.

Orally administered codeine has to permeate both the intestinal and the blood-brain barrier in order to act as analgesic and cough suppressant. In this study we characterized the uptake of codeine at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells. At both cell types, uptake of [(3)H]codeine was independent of an inwardly directed Na(+) gradient. Uptake was, however, strongly stimulated by an outwardly directed H(+) gradient and inhibited by the protonophore FCCP. [(3)H]Codeine uptake into Caco-2 cells was strongly temperature dependent. In the presence of excess amounts of unlabeled codeine, the uptake was inhibited by up to 87% (Caco-2) or 94% (RBE4), respectively. Synthetic opioids and some non-opioid organic cations like propranolol, pyrilamine and quinidine potently inhibited [(3)H]codeine uptake. Several prototype substrates of known transporters for amino acids, neurotransmitters and organic cations were ineffective. Our data are consistent with a hypothetic saturable, H(+)-dependent (antiport) mechanism not yet identified on a molecular level. The pH dependence of codeine uptake and its intracellular accumulation can partially also be explained by a model comprising diffusional membrane permeation of unionized species of codeine followed by codeine sequestration into acidic vesicles and distribution into cellular lipids.

Transport of the advanced glycation end products alanylpyrraline and pyrralylalanine by the human proton-coupled peptide transporter hPEPT1.

The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in urine after consumption of pyrraline-containing food. We hypothesized that the absorption of pyrraline occurs in the form of dipeptides rather than the free amino acid. The human intestinal peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells but not in cells transfected with empty vector, the uptake of [(14)C]glycylsarcosine was strongly inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala generated significant inward directed currents. In a third approach, uptake of the dipeptides into hPEPT1-transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an electrogenic manner into intestinal cells.

The bioactive dipeptide anserine is transported by human proton-coupled peptide transporters.

The bioactive dipeptide derivative anserine (beta-alanyl-1-N-methyl-L-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H(+)/peptide cotransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hypertensive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anserine inhibited [(14)C]Gly-Sar uptake with K(i) values of 1.55 mM (Caco-2) and 0.033 mM (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, K(i) values of 0.65 mM (hPEPT1) and 0.18 mM (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [(14)C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H(+) symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally.

Transport of phenylethylamine at intestinal epithelial (Caco-2) cells: mechanism and substrate specificity.

This study was performed to characterize the intestinal transport of beta-phenylethylamine (PEA). Uptake of [(14)C]PEA into Caco-2 cells was Na(+)-independent but strongly stimulated by an outside directed H(+) gradient. At extracellular pH 7.5, the concentration-dependent uptake of PEA was saturable with kinetic parameters of 2.6mM (K(t)) and 96.2nmol/min per mg of protein (V(max)). Several biogenic amines such as harmaline and N-methylphenylethylamine as well as cationic drugs such as phenelzine, tranylcypromine, d,l-amphetamine, methadone, chlorphenamine, diphenhydramine and promethazine strongly inhibited the [(14)C]PEA uptake with K(i) values around 1mM. Tetraethylammonium, N-methyl-4-phenylpyridinium and choline had no effect. We also studied the bidirectional transepithelial transport of [(14)C]PEA at cell monolayers cultured on permeable filters. Net transepithelial flux of [(14)C]PEA from apical-to-basolateral side exceeded basolateral-to-apical flux 5-fold. We conclude that PEA is transported into Caco-2 cells by a highly active, saturable, H(+)-dependent (antiport) process. The transport characteristics do not correspond to those of the known carriers for organic cations of the SLC22, SLC44, SLC47 and other families.

Reducing stress on cells with apoferritin-encapsulated platinum nanoparticles.

The great potential for medical applications of inorganic nanoparticles in living organisms is severely restricted by the concern that nanoparticles can harmfully interact with biological systems, such as lipid membranes or cell proteins. To enable an uptake of such nanoparticles by cells without harming their membranes, platinum nanoparticles were synthesized within cavities of hollow protein nanospheres (apoferritin). In vitro, the protein-platinum nanoparticles show good catalytic efficiency and long-term stability. Subsequently the particles were tested after ferritin-receptor-mediated incorporation in human intestinal Caco-2 cells. Upon externally induced stress, for example, with hydrogen peroxide, the oxygen species in the cells decreased and the viability of the cells increased.

Transport of drugs by proton-coupled peptide transporters: pearls and pitfalls.

The pharmaceutical relevance of proton-coupled peptide transporters is currently under intense investigation in many laboratories. Studies have shown that these membrane proteins, expressed in intestine, kidney, choroid plexus and other tissues, accept many peptidomimetic drugs and prodrugs as substrates. The focus of this review is on the interaction of beta-lactam antibiotics, angiotensin-converting enzyme inhibitors, sartans and other drugs with PEPT1 and PEPT2. The article highlights progress made in recent years and the most expedient techniques that have been or are being employed. It also emphasizes the opportunities in rational drug design that are of highest priority and the pitfalls that must be avoided. Finally, an instructional flowchart that might be used to identify a peptide transporter substrate is proposed.

Identification of a disulfide bridge essential for transport function of the human proton-coupled amino acid transporter hPAT1.

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG(11)-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.

Transport of free and peptide-bound pyrraline at intestinal and renal epithelial cells.

Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in foods and can be found in urine after consumption of pyrraline-containing food items. The purpose of this study was to investigate the transport of pyrraline and its dipeptide derivatives alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala) at intestinal and renal cell lines. Pyrraline inhibited the l-[(3)H]lysine uptake with IC(50) values of 0.3 mM (Caco-2 cells) and 3.5 mM (OK cells), respectively, but not the uptake of [(14)C]Gly-Sar (Caco-2 and SKPT cells). In contrast, Ala-Pyrr strongly inhibited the uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells with IC(50) values of 0.19 and 0.017 mM, respectively. Pyrr-Ala inhibited the carrier-mediated uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells by 50% at concentrations of 0.03 and 0.008 mM, respectively. The transepithelial flux of peptide-bound pyrraline across Caco-2 cell monolayers was up to 15-fold higher compared to the flux of free pyrraline. We conclude that free pyrraline is not a substrate for the intestinal lysine transporter and that the absorption of dietary pyrraline occurs most likely in the form of dipeptides rather than as the free amino acid.

The role of N-glycosylation in transport function and surface targeting of the human solute carrier PAT1.

In the present study we show in the Xenopus laevis expression system that the proton-coupled amino acid transporter 1 (PAT1, SLC36A1) is glycosylated at asparagine residues N174, N183 and N470. To determine the functional role of N-glycosylation, glycosylation-deficient mutants were analyzed by two-electrode voltage-clamp measurements after expression in X. laevis oocytes. Single replacements of asparagine residues had no effect on transport activity. However, multiple substitutions resulted in a decreased transport rate, leaving K(t) unchanged. Immunofluorescence localisation revealed a diminished plasma membrane expression of glycosylation-defective mutants. This indicates that N-glycans are not required for transport function, but are important for membrane targeting.

The orally active antihyperglycemic drug beta-guanidinopropionic acid is transported by the human proton-coupled amino acid transporter hPAT1.

The orally administered creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a beta-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of beta-GPA is well established, the underlying uptake mechanism has not yet been studied. We investigated whether the H(+)-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Uptake of l-[(3)H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by beta-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants of about 1.5 mM. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that beta-GPA, GAA and 4-GBA are effectively transported by this transport system in an electrogenic manner. We conclude that hPAT1 might be responsible for the intestinal absorption of beta-GPA thereby allowing its oral administration. Moreover, with beta-GPA we identified a new high affinity hPAT1 substrate that might be an interesting starting point for future drug design-drug delivery strategies.