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Abstract

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

Abstract: The apical membrane of small intestinal enterocytes possess an uptake system for di- and tripeptides. The physiological function of the system is to transport small peptides resulting from digestion of dietary protein. Moreover, due to the broad substrate specificity of the system, it is also capable of transporting a number of orally administered peptidomimetic drugs. Absorbed peptides may be hydrolysed in the cells due to the high peptidase activity present in the cytosol. Peptidomimetic drugs may, if resistant to the cellular enzyme activity, pass the basolateral membrane via a basolateral peptide transport mechanism and enter the systemic circulation. As the number of new peptide and peptidomimetic drugs are rapidly increasing, the peptide transport system has gained increasing attention as a possible drug delivery system for small peptides and peptide-like compounds. In this paper we give an updated introduction to the transport system and discuss the substrate characteristics of the di/tri-peptide transporter system with special emphasis on chemically modified substrates and prodrugs.

The classical view of protein absorption was that dietary proteins were completely broken down in the intestinal lumen to their constituent amino acids, and absorbed as such. This dogma was challenged in the beginning of the 1960's when it was shown that an intact peptide could be transported in an in vitro preparation of rat intestine (Newey & Smyth 1962). Additional studies on di/tri-peptide transport revealed that a large-scale absorption of dipeptides took place in the human intestine (Adibi & Morse 1971). During the 1970's and 1980's the intestinal di/tri-peptide transport system was characterised functionally in in vivo and in vitro studies and in 1994, an intestinal di/tri-peptide transporter, PepT1, was cloned (Boll et al. 1994; Fei et al. 1994). Hybrid depletion studies using RNA from rabbit (Fei et al. 1994) and rat (Tamai et al. 1997) enterocytes indicated that di/tri-peptide transport in the intestine was solely mediated via PepT1.

In parallel with the studies on dietary peptides, it was realised that a number of peptide-like drugs, such as β-lactam antibiotics and angiotensin converting enzyme (ACE) inhibitors, crossed the intestinal epithelium via a carrier-mediated mechanism. Following the cloning of the di/tri-peptide transporter it was possible to demonstrate that PepT1 was responsible for the carrier-mediated uptake of these drugs (Boll et al. 1994; Tamai et al. 1997; Terada et al. 1997; Zhu et al. 2000). This has spurred great interest in exploiting the di/tri-peptide transporter in drug delivery, and a wealth of data on different substrates transported by PepT1 have now accumulated. Extensive reviews have earlier been given on the history of di/tri-peptide transport (Matthews 1975), its physiological significance (Adibi 1997a), molecular biology (Daniel 1996; Fei et al. 1998; Meredith & Boyd 2000) and the ability of the transport system to transport drug compounds (Yang et al. 1999). The purpose of the present review is to give an updated introduction to the intestinal di/tri-peptide transport system with focus on the relationship between substrate structure and affinity.

Structure and function of the intestinal di/tri-peptide transporter, PepT1

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

PepT1 is a member of a family of transport proteins, termed POT (Proton-dependent Oligopeptide Transporters) (Fei et al. 1998) which currently consists of ∼70 cloned cDNA's derived from a variety of organisms (Takahashi et al. 1998; Meredith & Boyd 2000). Genomic analysis of the human di/tri-peptide transporter, hPepT1, have shown that hPepT1 is encoded by a gene located on chromosome 13, consisting of 24 exons (Liang et al. 1995; Urrti et al. 2001). This gene produces both hPepT1 (23 exons) and its splice variant, the regulatory factor hPepT1-RF (6 exons). The hPepT1 cDNA contains 3105 base pairs and the open reading frame translates into 708 amino acids which are organised into 12 putative membrane spanning domains and has a core molecular mass of ∼79 kDa (Liang et al. 1995). The predicted protein structure has a large extracellular loop between the 9th and 10th membrane spanning segment. Site-directed mutagenesis studies have indicated an absolutely essential role for His57 (Terada et al. 1996) in substrate recognition. Mutations of His121, Tyr167 , Trp294 and Glu595 also decreases di/tri-peptide transport (Bolger et al. 1998; Yeung et al. 1998; Chen et al. 2000). Two potential sites for protein kinase C phosphorylation and seven putative N-glycosylation sites have been identified (Liang et al. 1995).

In humans only two members of the POT family has been shown to possess transport activity, namely hPepT1 and hPepT2. PepT1 is expressed in the small intestine (Liang et al. 1995), in the proximal tubules of the kidney (Smith et al. 1998) and in the lysosomes of liver cells (Thamotharan et al. 1997) pancreatic cells (Bockman et al. 1997) and renal cells (Zhou et al. 2000). PepT2 is situated on the luminal membrane of the distal part of the proximal tubule (Boll et al. 1996) in brain (Dringen et al. 1998; Wang et al. 1998; Berger & Hediger 1999; Novotny et al. 2000) and lung tissue (Döring et al. 1998a). So far PepT1 remains the only intestinal di/tri-peptide transporter cloned. PepT1, has until now been cloned from human (Liang et al. 1995), rat (Saito et al. 1995), mouse (Fei et al. 2000) rabbit (Boll et al. 1994; Fei et al. 1994), sheep (Pan et al. 2001) and the human colon cell line Caco-2 (Walker et al. 1998). Furthermore, the presence of PepT1 mRNA have been detected in the small intestine of pig, cow, chicken and eel (Chen et al. 1999; Winckler et al. 1999; Verri et al. 2000), the human pancreatic carcinoma cell lines AsPc-1 and Capan-2 (Gonzalez et al. 1998) and a number of other human cancer cell lines (Nakanishi et al. 1997 & 2000). Functional studies have indicated the presence of a di/tri-peptide transport system in the human intestinal cell line HT-29 (Dantzig & Bergin 1988), the small intestine of monkey (Radhakrishnan 1977), hamster (Addison et al. 1975) guinea-pig (Himukai & Hoshi 1980), chicken (Backwell et al. 1995), toad (Abe et al. 1987), frog (Cheeseman & Parsons 1974) and salamander (Boyd & Ward 1982). The transport system thus appears to be a general feature of vertebrate protein assimilation.

PepT1 is a H+/peptide cotransporter (Ganapathy & Leibach 1991) which utilises the proton gradient from the intestinal lumen (∼pH 5.5–6.0 (Lucas et al. 1975)) into the cells (∼pH 7 (Kurtin & Charney 1984)) (Addison et al. 1972; Dantzig & Bergin 1990). The proton gradient is generally believed to be created and maintained by the activity of a Na+/H+ exchanger in the apical membrane of the enterocytes (Thwaites et al. 1999). However, from pure thermodynamic considerations, the pH gradient generated by the Na+/H+ exchanger does not generate enough driving force under some of the experimental conditions used and thus the involvement of a base loader (i.e. a Na+/HCO3 cotransporter) has been suggested (Amasheh et al. 1997). Once inside the cells, most naturally occurring peptides will be hydrolysed to their constituent amino acids and enter the blood via basolateral amino acid transporters. Non-hydrolysable substrates may pass the basolateral membrane of the enterocytes via a not yet fully characterised basolateral peptide transport mechanism (Dyer et al. 1990; Saito & Inui 1993; Thwaites et al. 1993a; Terada et al. 1999), see fig. 1. The operational mode of hPepT1 is not yet fully clarified. However, a number of studies have shown that transport is electrogenic (Thwaites et al. 1993b; Mackenzie et al. 1996; Wenzel et al. 1996) with one charge moving per substrate molecule translocated. Moreover, both anionic, cationic and neutral substrates are being translocated with fairly identical rheogenic properties (but different pH optima), implying that the number of additional charges moving (i.e. protons) differs depending on the charge of the substrates.

image

Figure 1. The PepT1 peptide transport pathway. Peptides (and peptidomimetic substrates) are transported from the intestinal lumen into the cells via the apical, proton-dependent peptide transporter PepT1. Once inside the cells, peptides can be hydrolysed by cellular enzymes and cross the basolateral membrane as amino acids or they can be effluxed into the blood via a basolateral peptide transport system. A proton gradient across the apical membrane is maintained by the activity of an apical Na + /H+ exchanger, which in turn is energised by the basolateral Na + /K + -ATPase. The apical proton gradient enhance the uptake of peptide substrates.

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Regulation of PepT1

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

Knowledge about the regulation of PepT1 activity is limited. A number of studies have shown that a dietary protein load causes an increase in di/tri-peptide transport in rat (Erickson et al. 1995) and mice small intestine (Ferraris et al. 1988). This has been shown to be accompanied by an increase in PepT1 mRNA (Erickson et al. 1995). Upregulation of PepT1 by dipeptides has also been demonstrated in cell cultures. Walker et al. (1998) and Thamotharan et al. (1998) showed an increase in hPepT1 mRNA and Gly-Sar transport in Caco-2 cells , and Shiraga et al. (1999) isolated the promoter region of rat PepT1 and showed that specific dipeptides and amino acids were able to initiate transcription in Caco-2 cells transfected with a construct of the promoter and a luciferase gene. Recently, Ogihara et al. (1999), Thamotharan et al. (1999a), and Ihara et al. (2000) presented evidence that PepT1 in rat intestine is upregulated after a short fast via an increase in gene expression. These effects could thus be due both to the lack of peptides/amino acids which could stimulate transcription, but also to some endogenous factors, i.e. changes in the blood levels of hormones such as glucagon.

Direct regulation of hPepT1 by intracellular second messengers is still controversial. A number of agonists have been reported to influence hPepT1 activity, however the signalling pathways involved have not been elucidated. It has been shown that di/tri-peptide transporter activity is down-regulated in Caco-2 cells exposed to phorbol esters (Brandsch et al. 1994), however, direct phosphorylation of the PKC consensus sites has not been demonstrated. Di/tri-peptide transporter activity is also decreased by agents which interferes with cellular cAMP levels and thereby the PKA signalling pathway, but the phosphorylation targets have not been identified (Muller et al. 1996). The hPepT1 protein itself does not posses a PKA phosphorylation site (Liang et al. 1995). Insulin has been reported to increase glycylglutamine uptake, presumably via an insertion of preformed transport molecules form a vesicular pool (Thamotharan et al. 1999b), long term treatment with EGF causes a decrease in hPepT1 via a decrease in mRNA (Nielsen et al. 2001a), the σ-receptor ligand (+) pentazocine cause an increase in hPepT1 activity and mRNA (Fujita et al. 1999) and the α2-receptor ligand clonidine has been shown to increase peptide uptake in a Caco-2 cell line engineered to express α2-receptors (Berlioz et al. 2000). In conclusion, although a number of signalling pathways have been shown to interfere with PepT1 activity, no direct regulation of the transport molecule have yet been demonstrated.

An interesting aspect of regulation of PepT1 expression is that PepT1 in the rat small intestine is resistant to tissue damage induced by 5-flourouracil, whereas other markers such as sucrase activity, D-glucose uptake and glycine uptake was significantly decreased (Tanaka et al. 1998). This suggests that expression of PepT1 is robust towards cellular damage, that might occur during states of disease, underscoring the potential of the intestinal oligopeptide transport system for therapeutic applications such as oral drug delivery mediated by the hPepT1 transport pathway and intestinal supply of glutamine containing dipeptides (Adibi 1997a).

Substrates transported by hPepT1

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

There are 20 different dietary amino acids. This implies that hundreds of different dipeptides and thousands of tripeptides may be possible. Only one di/tri-peptide transporter has been demonstrated in human enterocytes implying an extremely broad substrate specificity. A number of drugs and drug candidates have been reported to be recognised by the transporter as well, including β-lactam antibiotics such as penicillins and cephalosporins (Bretschneider et al. 1999), ACE-inhibitors (Friedman & Amidon 1989a & b; Moore et al. 2000), renin inhibitors (Kramer et al. 1990), thrombin inhibitors (Walter et al. 1995) and the dipeptide-like antineoplastic drug bestatin (Inui et al. 1992). Prodrugs of acyclovir, ganciclovir and L-Dopa are also recognised and transported by PepT1 (Hu et al. 1989; Balimane et al. 1998; Sugawara et al. 2000), as well as prodrugs of pamidronate (Ezra et al. 2000) and tetrahydrofuranyl-carbapenems (Weiss et al. 1999). The hydroxylprolylserine derivative JBP923, a peptide with antihepatitis activity (Liu et al. 2000) also appears to be taken up by hPepT1. The NMDA-antagonist PD 158473 is transported both by PepT1 and the large neutral amino acid transporter (Surendran et al. 1999).

Besides the natural dietary di/tri-peptides, drug and pro-drug compounds, a number of modified dipeptides have been shown to have an affinity for PepT1. Among these, Gly-Sar (Gly-Gly with a methylated peptide bond) and carnosine (β-Ala-His) are well characterised substrates which only undergo moderate hydrolysis and are therefore often used as radiolabelled standard compounds for affinity and transport studies.

Structural requirements for transport via PepT1

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

The three-dimensional structure of the transport protein has not yet been elucidated, and therefore information about structural requirements for transport has been obtained from uptake/transport studies on experimental models such as Caco-2 cells, hPepT1 mRNA injected Xenopus oocytes, transgenic yeast (Pichia pastoris) and perfused intestinal segments. The overall goal of this type of research has been to develop a general model for determinants of affinity/transport, thus allowing for rational drug design. In the following sections we discuss the importance of molecular structure, size and stereochemistry in order for a compound to be accepted by PepT1.

Molecular size.

It would seem plausible that transport via PepT1 would be constricted by an upper limit for the size of substrates. However, to our knowledge, no systematic investigations have shown a relationship between affinity/transport and molecular weight. The maximal size of possible substrates is still controversial. However, the peptide or peptide-like backbone of the substrate must be equivalent to that of a di-or tripeptide, with larger structures impeding transport. Sawada et al. (1999) investigated the characteristics of a series of esterfied L-Valine compounds in LLC-PK1 cells transfected with rat-PepT1 showing that as little as a methylester is recognized by the transporter, also benzyl alcohol esterfied L-Valine is recognized by PepT1. However, whether this interaction was followed by translocation of the ester was not demonstrated. Based on conformational substrate analysis of cephalosporins Li & Hidalgo (1996) hypothesised an optimal length of 5.5 Å between the N-terminal amino group and the C-teminal carboxy-group. These studies were performed on renal brush border membrane vesicles, and therefore probably represents a mixture of PepT1 and PepT2. Döring et al. (1998b) studied transport of a series of ω-amino fatty acids in Xenopus Oocytes transfected with hPepT1 and concluded that the two ionized head groups should be seperated by a distance of at least 5 Å.

Stereochemical specificity.

Transport of di/tripeptides is markedly stereochemically specific. Natural amino acids are predominantly of the L-form and peptides composed of L-enantiomers also display the highest affinity to the transporter (Wenzel et al. 1995; Tamura et al. 1996). β-Lactams also have a chiral center and therefore exist both as D- and L-forms. Stereoselective uptake of cephalexin and loracarbef has been demonstrated, with the L-enantiomers displaying the highest affinity to the transport system (Wenzel et al. 1995).

Two studies have used diastereomers of Val-Val to investigate the stereochemical specificity. Hidalgo et al. showed that whereas introducing two D-configured amino acids in D-Val-D-Val abolish affinity completely, both D-Val-L-Val and L-Val-D-Val retain an affinity comparable to the L,L-configured Val-Val (Hidalgo et al. 1995). This was later questioned by Tamura et al. (1996), who showed that the rank of affinity for hPepT1 in Caco-2 cells was L-Val-L-Val>D-Val-L-Val>L-Val-D-Val>>D-Val-D-Val. This relationship for affinity does seem to translate into transport across the apical membrane, because it has been shown that the diastereomers are taken up by Caco-2 cells in the following order D-Val-L-Val>L-Val-D-Val>>D-Val-D-Val, where the D,D-form was neither internalized nor transported across Caco-2 cell monolayers by a carrier mediated process (Tamura et al. 1996). It is noteworthy however, that a D-configured peptide with side-chain modifications can display a marked increase in affinity and transport, as compared to the parent compound (Taub et al. 1997a; Knütter et al. 2001).

Terminal groups.

It has been hypothesised that for optimal di/tri-peptide transport, both amino-terminal and carboxyl-terminal groups should bee free (Matthews 1987). However, some modifications are allowed, as depicted in table 1. In general, the N-terminal α-amino group is necessary for binding and translocation, and substitution of this group will decrease transport. However, affinity and transport have been demonstrated for a number of substrates with lacking or modified N-terminal amino groups. Hu & Amidon (1988) showed that captopril, which has a thiol group instead of a terminal amino group was accepted by the transporter. Using a series of dipeptide analogues without the N-terminal alpha-amino group, Bai et al. (1991) showed carrier-mediated uptake in perfused rat intestinal segments. This is also seen for Sar-Gly, imido-dipeptides with Proline as N-terminal amino acid, enalapril, lisinopril, SQ 29852, cefixime and ceftibuten (Hu & Amidon 1988; Friedman & Amidon 1989a & b). In addition studies investigating the uptake into rabbit intestinal brush-broder membrane vesicles of Phe-Gly modified with butyric acid (C4-Phe-Gly) and caproic acid (C6-Phe-Gly) in the amino terminal of Phe-Gly have shown that a free α amino group is not necessarily vital. Interestingly, these studies showed that modifying the N-terminus of Phe-Gly with fatty acids enhanced the affinity for an intestinal oligopeptide carrier (Fujita et al. 1997). Acetylation of the N-terminus of Phe-Tyr to give an amide highly decreased, but did not abolish affinity for PepT1 (Meredith et al. 2000).

The C-terminus has also been shown not to be absolutely essential for affinity and transport. Modification of Phe-Tyr to give the corresponding amide in the C-terminal retain an affinity although lowered for PepT1 (Meredith et al. 2000). Single amino acids modified with piperidide, thiazolidide, anilide, 4-nitroanilide, 4-chloroanilide or 4-phenylanilide also have affinity for PepT1 (Borner et al. 1998; Brandsch et al. 1999).

Peptide bonds.

The presence of a peptide bond was originally thought to be a prerequisite for transport via PepT1 (Matthews 1987). The first indication of peptide transport of substrates without a peptide bond came from studies on renal di/tri-peptide transport. The apical membrane of kidney tubules express both PepT1 and PepT2, with PepT1 expressed in the luminal membrane of early proximal tubule and PepT2 expressed in the more distal regions. (Smith et al. 1998). However, PepT2 and PepT1 have great structural similarity and the same structure specificty (with exception of certain β-lactams) although PepT2 generally shows higher substrate affinity. Using kidney brush border membrane vesicles, Daniel & Adibi (1994) showed binding of amino acid 4-nitroanilides, and that substitution of the peptide bond by a ketomethylene bond was tolerated by the transporters present in the kidney. A number of substrates without peptide bonds have now been shown to have an affinity to PepT1. Temple et al. (1998) demonstrated PepT1-mediated transport of the peptide mimic 4-aminophenylacetic acid (4-APAA). 4-APAA was designed to mimic the spatial configuration of a dipeptide, being planar and with the terminal amino and carboxyl groups in trans configuration, but lacking a peptide bond. Döring et al. (1998a) showed that δ-aminolevulinic acid, which has a ketomethylene group instead of a peptide bond, as well as ω-amino fatty acids (Döring et al. 1998b) were bound and translocated by the transporter. Recently, Brandsch et al. (1998) showed that Ala-Ψ[CS-N]-Pro, a modification of Ala-Pro where the peptide carbonyl oxygen atom was replaced by sulfur, was accepted by PepT1. An additional and very interesting finding from the study by Brandsch et al. (1998) was that PepT1 selectively transport dipeptides in the trans conformation. This was later confirmed in a study by Brandsch et al. (1999), using dipeptides containing proline as the carboxy-terminal amino acid.

Side chains.

Studies on modifications of the side-chain residue in di- or tripeptides are limited, however, Taub et al. (1997a,b & 1998) have shown that dipeptides containing asparagyl, glutamyl and serinyl residues modified with benzyl alcohol retain affinity for PepT1 (table 2). Using a series of modified benzyl alcohols, Nielsen et al. (2001b) suggested that the affinity of D-Asp-Ala and D-Glu-Ala esters of these benzyl alcohols was primarily determined by the lipophilicity of the benzyl alcohols suggesting a hydrophobic interaction with the transporter. Secondarily, the affinity was determined by the molecular weight of the dipeptide ester, which is indicating a spatial interaction with the transporter. Circumstantial evidence suggested that the benzyl ester of Asp-Sar was a competitive inhibitor (Taub et al. 1997b). Recently, Knütter et al. (2001) provided evidence that the 4-nitrobenzyloxycarbonyl carbamate of Lys-Pro, acts as a competitive inhibitor of PepT1 with a Ki value in the μM range. Since N-methylation does not abolish hPepT1 mediated transport as clearly seen with Gly-Sar, it is surprising that D-Asp(BnO)-Sar is an inhibitor, whereas D-Asp(BnO)-Ala is transported in a hPepT1 mediated fashion. The reason for inhibitory nature of the Lys-Pro carbamate of 4-nitrobenzyloxycarbonyl might be a structural feature of the linkage between the Lys-Pro dipeptide and the 4-nitrobenzyloxycarbonyl, which is a carbamate contrary to the ester and ether bonds of the remaining compounds in table 2.

Table 2.  Published modifications of the side chain of the N-terminal amino acid of dipeptides. Transported=The compound in question undergoes carrier-mediated transepithelial transport. Affinity=The compound in question displays an affinity for hPepT1 (transport has not been investigated). Inhibitor=The compound binds to hPepT1 but is not translocated.
Dipeptide (R)StructureLinkageCommentRef
D-Asp-Alainline imageEsterTransported(Taub et al. 1997a)
D-Asp-Ala D-Glu-Alainline imageEsterTransported(Taub et al. 1997a & b; Nielsen et al. 2001)
D-Ser-Alainline imageEtherTransported(Taub et al. 1998)
D-Asp-Sarinline imageEsterInhibitor?(Taub et al. 1997b)
D-Asp-Alainline imageEsterAffinity(Nielsen et al. 2001)
D-Glu-Ala D-Asp-Alainline imageEsterAffinity(Nielsen et al. 2001)
D-Glu-Alainline imageEsterAffinity(Nielsen et al. 2001)
Lys-Proinline imageCarbamateInhibitor(Knütter et al. 2001)
Lys-Proinline imageCarbamateInhibitor(Knütter et al. 2001)

Molecular modelling.

Considering the broad substrate affinity of PepT1, predictive models of structure-affinity relationships is of great value from a drug delivery viewpoint. This allows for modification of pharmacologically active compounds with poor intestinal permeability so as to fit the PepT1 binding site. Swaan & Tukker (1997) performed conformational analysis and created a pharmacophore model, using transport and binding data obtained in a varity of model systems and with penicillins, cephalosporins and ACE-inhibitors as substrates. In a later study Swaan et al. (1998) performed a 3D QSAR analysis on penicillins, cephalosporins and ACE-inhibitors (based on transport data from rat small intestine). Döring et al. (1998b) investigated minimal requirements for binding to the transporter using the transgenic yeast (Pichia pastoris) expression system developed by Hannelore Daniels group (Döring et al. 1997) and ω-amino fatty acids as substrates. A comprehensive model (including quite diverse substrate groups) was published recently by Bailey et al. (2000), who performed a computational study on native peptides, prodrugs and drugs (using affinity data from a number of experimental models), and suggested a substrate template for binding to PepT1. Although apparent substrate affinities obtained in different cell systems might vary due to experimental conditions (and in some cases as in renal vesicle preparations, presence of both PepT1 and PepT2), this model remains so far the most comprehensive. The 3D model describes 10 features of substrate interaction which each may contribute to the overall affinity for hPepT1 of a given compound. The key features: 1) A strong binding site for the N-terminal amino group; 2) Preference for L-configuration; 3) A planar backbone from the N-terminal Cα atom to the side chain of the second peptide (R2); 4) A hydrophobic pocket which can accomodate bulky side chains on the second peptide; 5) C-terminal carboxylate binding sites. The model thus predicts, as would also be intuitively expected, that the affinity is a result of a number of factors and not readily deduced from any single parameter, but rather “a sum of a number of discrete interactions” (Meredith & Boyd 2000). Although comprehensive, the model does not readily account for the changes in affinity observed when structural modifications are made on the side chain of the the N-terminal amino acid (table 2).The hydrophobicity of the side chain in this position is apparently of importance for substrate binding as well (Nielsen et al. 2001b). The interaction between the side chain of the N-terminal amino acid and the transporter thus deserves further investigation in order to clarify the possibilities for conjugating drug candidates on peptide side chains. A model incorporating the interactions of the amino terminal side-chain residue is currently being developed (Brandsch et al., personal communication; Gebauer et al. 2001).

Prodrugs utilising the hPepT1 transport pathway

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

Given the broad substrate specificity of hPepT1 it is an attractive idea to modify a drug compound with a low intrinsic bioavailability with a pro-moiety so as to make it acceptable for the transport system and thereby increase its transport across the intestinal wall. However, in order to increase transepithelial transport, the prodrug in question must both have an affinity for the apical and the basolateral transport system. Or, alternatively, the produg in question must be taken up apically, hydrolysed in the intestinal cells and a substantial amount of the parent drug compound must pass the basolatoral membrane. In order to be classified as a prodrug utilising the intestinal di/tri-peptide transport pathway, a significant saturable transcellular transport component must thus be demonstrated in vitro, in situ or, ideally, in vivo.

Prodrugs which are transported via the intestinal di/tri-peptide transporter falls into two categories; 1) Prodrugs which originally was developed in order to improve the solubility, lipophilicity or some other intrinsic parameter of the parent drug compound. 2) Prodrugs designed to resemble peptides in order to make them acceptable for the di/tri-peptide transport system. The first group includes certain β-lactams such as cefixime and cefdinir (Bretschneider et al. 1999) as well as prodrugs of aciclovir (Beauchamp et al. 1992; Ganapathy et al. 1998; Han et al. 1998; Guo et al. 1999). These prodrugs have been designed and tested, and then, retrospectively, been found to be good substrates for the di/tri-peptide transport system. Prodrugs designed with the specific aim to have an affinity for hPepT1 includes L-α-methyldopa-L-Phe (Tsuji et al. 1990), L-Val-AZT (Han et al. 1998) and L-Pro-L-Phe conjugated alendronate and pamidronate. A group of carbapenems (Tanaka et al. 1997; Weiss et al. 1999) have also shown increased bioavailability after coupling to amino acid pro-moities, however, transepithelial kinetic data are not yet available. Table 3 summarises the prodrugs which have been shown to undergo transcellular transport via the hPepT1 transport pathway. The linkages of the prodrugs shown in table 3 are bioreversible, i.e. either ester, peptide or amide bonds. The pro-moieties are either a single amino acid or a dipeptide for the non-peptidomimetic drug compounds.

A number of ACE inhibitor prodrugs such as enalapril (prodrug of enalaprilate) and alacepril (prodrug of captopril) compromise a separate category of compounds with an affinity for the di/tri-peptide transporter. A number of ACE inhibitor prodrugs, as well as their parent compounds, have been shown to bind to the di/tri-peptide transporter and to be taken up by intestinal cells, although the reported Ki values vary tremendously (for references, Moore et al. 2000). However, in the case of the prodrug enalapril the transepithelial transport is primarily dominated by passive, non-saturable transport in the therapeutic relevant dosage range (Morrison et al. 1996). Similar results have been obtained for the non-prodrug ceranopril in a study by Nicklin et al. (1996), where the authors suggest that the phenomenon is due to retardation of the drug in the cells due to a low affinity to basolateral transport mechanisms. This indicates that the increased bioavailability of some ACE-inhibitors and their prodrugs might be caused solely by their better passive permeation properties, and not by increased transport via the di/tri-peptide transport pathway. This emphasises the need for combining inhibition and uptake studies with the time-demanding transepithelial transport studies when investigating absorption kinetics.

Pharmacokinetics

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

The pharmacokinetics of peptidomimetic drugs with an affinity to the intestinal di/tri-peptide transporter must be considered in the overall evaluation of the therapeutic potential of these drugs and to obtain a basis for a rational design of new drugs utilising this transporter.

After entering the systemic circulation, the pharmacokinetics of peptidomimetics may be highly dependent on the affinity to di/tri-peptide transporters present in various organs. The kidney express PepT1 and PepT2 sequentially in the proximal tubule, thereby allowing for bulk absorption by PepT1, which is the high capacity but low affinity system, followed by absorption via the high affinity (but low capacity) transporter PepT2 (Smith et al. 1998). The kidney is the tissue with the highest dipeptidase activity and thus assimilates and hydrolyse peptides present in the plasma filtrate (Adibi 1997b). There is evidence for a basolateral peptide transport in kidney tubule cells, allowing for reentry of absorbed, nonhydrolysable compounds into the bloodstream (Arvidsson et al. 1979; Silbernagl et al. 1987; Barfuss et al. 1988; Terada et al. 2000. The kidney is thus likely to be a major target for any peptidomimetic which has entered the systemic circulation. This can be a major drawback if the compound in question is cytotoxic, however reabsortion of hydrolysis-stable peptidomimetics from the filtrate could also potentially prolong the time in which such a compound remains in circulation, given that it has an affinity for the basolateral transport system. Reabsorption from the ultrafiltrate into kidney cells has indeed been demonstrated for some β-lactams (Arvidsson et al. 1979; Garrigues et al. 1991) and the nonhydrolysable peptides Gly-Sar and Gly-Sar-Sar (Minami et al. 1992). Renal clearance of a given peptide or peptidomimetic from the ultrafiltrate is thus likely to show the same characteristics as the clearance from the intestinal lumen, since uptake via PepT1 probably is dominant in the kidney.

Renal handling of peptidomimetics transported by hPepT1 and/or hPepT2 thus plays an important role in the evaluation of these drugs as they may be eliminated by excretion in the urine. However, by optimizing the stability in the cells, reabsorption of these drugs from the glomerular filtrate may reduce their loss in the urine and thus prolong plasma half-life, increasing the possiblities for reaching their target sites.

Future perspectives

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References

During the last decade, studies on transport of peptidomimetics by the intestinal di/tri-peptide transporter has moved from simple transport studies to advanced molecular biology and molecular modelling. Rational design of peptidomimetic drugs/prodrugs has been performed, exemplified by the studies by Wang (Wang et al. 1996), Weiss et al. (1999) and Ezra et al. (2000). However, the 3D structure of the substrate binding site of the transporter remains to be elucidated, as well as the nature of the proton-binding site. The pursuit of these questions is likely to yield radical new knowledge, since the transporter is quite unique in its ability to accomodate a broad spectrum of substrates and in its differential proton-coupling modes. Investigations of the nature of the basolateral transport pathway for peptides and peptidomimetics in intestinal cells will also bring forward valubale knowledge about rate-limiting steps in the transport pathway, and affinitity values for transepithelial transport, lending more credibility to in vivoin vitro correlations. The retrospective modelling studies will have to prove their worth by being able to predict affinity of new untested substrates and further studies employing model substrates will have to be performed in order to resolve determinants in substrate-binding site interactions.

References

  1. Top of page
  2. Abstract
  3. Structure and function of the intestinal di/tri-peptide transporter, PepT1
  4. Regulation of PepT1
  5. Substrates transported by hPepT1
  6. Structural requirements for transport via PepT1
  7. Prodrugs utilising the hPepT1 transport pathway
  8. Pharmacokinetics
  9. Future perspectives
  10. References
  • Abe, M., T. Hoshi & A. Tajima: Characteristics of transmural potential changes associated with the proton-peptide co-transport in toad small intestine. J. Physiol. Lond. 1987, 394, 481499.
  • Addison, J. M., D. Burston, J. A. Dalrymple, D. M. Matthews, J. W. Payne, M. H. Sleisenger & S. Wilkinson: A common mechanism for transport of di- and tri-peptides by hamster jejunum in vitro. Clin. Sci. Mol. Med. 1975, 49, 313322.
  • Addison, J. M., D. Burston & D. M. Matthews: Evidence for active transport of the dipeptide glycylsarcosine by hamster jejunum in vitro. Clin. Sci. 1972, 43, 907911.
  • Adibi, S. A.: The oligopeptide transporter (Pept-1) in human intestine: Biology and function. Gastroenterology 1997a, 113, 332340.
  • Adibi, S. A.: Renal assimilation of oligopeptides: Physiological mechanisms and metabolic importance. Amer. J. Physiol. 1997b, 272, E723E736.
  • Adibi, S. A. & E. L. Morse: Intestinal transport of dipeptides in man; relative importance of hydrolysis and intact absorption. J. Clin. Invest. 1971, 50, 22662275.
  • Amasheh, S., U. Wenzel, W. M. Weber, W. Clauss & H. Daniel: Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes. J. Physiol. (Lond). 1997, 504(1), 169174.
  • Arvidsson, A., O. Borgå & G. Alván: Renal excretion of cephapirin and cephaloridine: evidence for saturable tubular reabsorption. Clin. Pharmacol. Ther. 1979, 25, 870876.
  • Backwell, F. R., D. Wilson & A. Schweizer: Evidence for a glycyl-proline transport system in ovine enterocyte brush-border membrane vesicles. Biochem. Biophys. Res. Commun. 1995, 215, 561565.
  • Bai, P. F., P. Subramanian, H. I. Mosberg & G. L. Amidon: Structural requirements for the intestinal mucosal-cell peptide transporter: the need for N-terminal alpha-amino group. Pharm. Res. 1991, 8, 593599.
  • Bailey, P. D., C.-A. R. Boyd, J. R. Bronk, I. D. Collier, D. Meredith, K. M. Morgan & C. S. Temple: How to make drugs orally active: a substrate template for peptide transporter PepT1. Angew. Chem. Int. 2000, 39, 505508.
  • Balimane, P. V., I. Tamai, A. Guo, T. Nakanishi, H. Kitada, F. H. Leibach, A. Tsuji & P. J. Sinko: Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. Biochem. Biophys. Res. Commun. 1998, 250, 246251.
  • Barfuss, D. W., V. Ganapathy & F. H. Leibach: Evidence for active dipeptide transport in isolated proximal straight tubules. Amer. J. Physiol 1988, 255, F177F181.
  • Beauchamp, L. M., G. F. Orr, P. De Miranda, T. Burnette & T. A. Krenitsky: Amino acid ester prodrugs of acyclovir. Antiviral Chemistry & Chemotherapy 1992, 3, 157164.
  • Berger, U. V. & M. A. Hediger: Distribution of peptide transporter PEPT2 mRNA in the rat nervous system. Anat. Embryol. (Berl.) 1999, 199, 439449.
  • Berlioz, F., J. J. Maoret, H. Paris, M. Laburthe, R. Farinotti & C. Roze: alpha(2)-adrenergic receptors stimulate oligopeptide transport in a human intestinal cell line. J. Pharmacol. Exp. Therap. 2000, 294, 466472.
  • Bockman, D. E., V. Ganapathy, T. G. Oblak & F. H. Leibach: Localization of peptide transporter in nuclei and lysosomes of the pancreas. Int. J. Pancreatol. 1997, 22, 221225.
  • Bolger, M. B., I. S. Haworth, A. K. Yeung, D. Ann, H. Von Grafenstein, S. Hamm-Alvarez, C. T. Okamoto, K. J. Kim, S. K. Basu, S. Wu & V. H. Lee: Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1. J. Pharm. Sci. 1998, 87, 12861291.DOI: 10.1021/js980090u
  • Boll, M., M. Herget, M. Wagener, W. M. Weber, D. Markovich, J. Biber, W. Clauss, H. Murer & H. Daniel: Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 284289.
  • Boll, M., D. Markovich, W. M. Weber, H. Korte, H. Daniel & H. Murer: Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors. Pflügers Arch. 1994, 429, 146149.
  • Borner, V., Y. J. Fei, B. Hartrodt, V. Ganapathy, F. H. Leibach, K. Neubert & M. Brandsch: Transport of amino acid aryl amides by the intestinal H+/peptide cotransport system, PEPT1. Eur. J. Biochem. 1998, 255, 698702.
  • Boyd, C. A. & M. R. Ward: A micro-electrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus. J. Physiol. (Lond.) 1982, 324, 411428.
  • Brandsch, M., I. Knütter, F. Thunecke, B. Hartrodt, I. Born, V. Borner, F. Hirche, G. Fischer & K. Neubert: Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter. Eur. J. Biochem. 1999, 266, 502508.
  • Brandsch, M., Y. Miyamoto, V. Ganapathy & F. H. Leibach: Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line. Biochem. J. 1994, 299, 253260.
  • Brandsch, M., F. Thunecke, G. Kullertz, M. Schutkowski, G. Fischer & K. Neubert: Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1. J. Biol. Chem. 1998, 273, 38613864.
  • Bretschneider, B., M. Brandsch & R. Neubert: Intestinal transport of β-lactam antibiotics: analysis of the affinity at the H+/peptide symporter (PEPT1), the uptake into caco-2 cell monolayers and the transepithelial flux. Pharm. Res. 1999, 16, 5561.
  • Cheeseman, C. I. & D. S. Parsons: Intestinal absorption of peptides. Peptide uptake by small intestine of Rana pipiens. Biochim. Biophys. Acta 1974, 373, 523526.
  • Chen, H., E. A. Wong & K. E. Webb, Jr.: Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens. J. Anim Sci. 1999, 77, 12771283.
  • Chen, X. Z., A. Steel & M. A. Hediger: Functional roles of histidine and tyrosine residues in the H(+)-peptide transporter PepT1. Biochem. Biophys. Res. Commun. 2000, 272, 726730.
  • Daniel, H.: Function and molecular structure of brush border membrane peptide/H+ symporters. J. Membr. Biol. 1996, 154, 197203.
  • Daniel, H. & S. A. Adibi: Functional separation of dipeptide transport and hydrolysis in kidney brush border membrane vesicles. FASEB J. 1994, 8, 753759.
  • The Danish Catalogue of Drugs. Dansk Lægemiddel Information A/S, Denmark, 2001. Eds.: Kristensen, M. B., Friis, H. & Rassing, M. R. Eleanders Publishing AS.
  • Dantzig, A. H. & L. Bergin: Carrier-mediated uptake of cephalexin in human intestinal cells. Biochem. Biophys. Res. Commun. 1988, 155, 10821087.
  • Dantzig, A. H. & L. Bergin: Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, caco-2. Biochim. Biophys. Acta 1990, 1027, 211217.
  • Döring, F., S. Theis & H. Daniel: Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotrophic yeast Pichia pastoris. Biochem. Biophys. Res. Commun. 1997, 232, 656662.
  • Döring, F., J. Walter, J. Will, M. Focking, M. Boll, S. Amasheh, W. Clauss & H. Daniel: Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. J. Clin. Invest 1998a, 101, 27612767.
  • Döring, F., J. Will, S. Amasheh, W. Clauss, H. Ahlbrecht & H. Daniel: Minimal molecular determinants of substrates for recognition by the intestinal peptide transporter. J. Biol. Chem. 1998b, 273, 2321123218.
  • Dringen, R., B. Hamprecht & S. Broer: The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures. J. Neurochem. 1998, 71, 388393.
  • Dyer, J., R. B. Beechey, J. P. Gorvel, R. T. Smith, R. Wootton & B. S. Shirazi: Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles. Biochem. J. 1990, 269, 565571.
  • Erickson, R. H., J. Gum-JR, M. M. Lindstrom, D. McKean & Y. S. Kim: Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs. Biochem. Biophys. Res. Commun. 1995, 216, 249257.DOI: 10.1006/bbrc.1995.2617
  • Ezra, A., A. Hoffman, E. Breuer, I. S. Alferiev, J. Monkkonen, N. Hanany-Rozen, G. Weiss, D. Stepensky, I. Gati, H. Cohen, S. Tormalehto, G. L. Amidon & G. Golomb: A peptide prodrug approach for improving bisphosphonate oral absorption. J. Med. Chem. 2000, 43, 36413652.DOI: 10.1021/jm980645y
  • Fei, Y. J., V. Ganapathy & F. H. Leibach: Molecular and structural features of the proton-coupled oligopeptide transporter superfamily. Prog. Nucl. Acid Res. Mol. Biol. 1998, 58, 239261.
  • Fei, Y. J., Y. Kanai, S. Nussberger, V. Ganapathy, F. H. Leibach, M. F. Romero, S. K. Singh, W. F. Boron & M. A. Hediger: Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 1994, 368, 563566.
  • Fei, Y. J., M. Sugawara, W. Liu, H. W. Li, V. Ganapathy, M. E. Ganapathy & F. H. Leibach: cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1. Biochim. Biophys. Acta 2000, 1492, 145154.
  • Ferraris, R. P., J. Diamond & W. W. Kwan: Dietary regulation of intestinal transport of the dipeptide carnosine. Amer. J. Physiol 1988, 255, G143G150.
  • Friedman, D. I. & G. L. Amidon: Intestinal absorption mechanism of dipeptide angiotensin converting enzyme inhibitors of the lysyl-proline type: lisinopril and SQ 29,852. J. Pharm. Sci. 1989b, 78, 995998.
  • Friedman, D. I. & G. L. Amidon: Passive and carrier-mediated intestinal absorption components of two angiotensin converting enzyme (ACE) inhibitor prodrugs in rats: enalapril and fosinopril. Pharm. Res. 1989a, 6, 10431047.
  • Fujita, T., Y. Majikawa, S. Umehisha, N. Okada, A. Yamamoto, V. Ganapathy & F. H. Laibach: Receptor ligand-induced up-regulation of the H+/peptide transporter PepT1 in the human intestinal cell line Caco-2. Biochem. Biophys. Res. Commun. 1999, 261, 242246.
  • Fujita, T., Y. Morishita, H. Ito, D. Kuribayashi, A. Yamamoto & S. Muranishi: Enhancement of the small intestinal uptake of phenylalanylglycine via a H+/oligopeptide transport system by chemical modification with fatty acids. Life Sci. 1997, 61, 24552465.
  • Ganapathy, M. E., W. Huang, H. Wang, V. Ganapathy & F. H. Leibach: Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2. Biochem. Biophys. Res. Commun. 1998, 246, 470475.DOI: 10.1006/bbrc.1998.8628
  • Ganapathy, V. & F. H. Leibach: Proton-coupled solute transport in the animal cell plasma membrane. Curr. Opin. Cell Biol. 1991, 3, 695701.
  • Garrigues, T. M., U. Martin, J. E. Peris-Ribera & L. F. Prescott: Dose-dependent absorption and elimination of cefadroxil in man. Eur. J. Clin. Pharmacol. 1991, 41, 179183.
  • Gebauer, S., I. Thondorf, I. Knütter, M. Brandsch & K. Neubert: Determining the pharmacophore structure of substrates of the mammalian peptide transporter (PEPT1) by molecular modelling investigations. EUFEPS World Conference on Drug Absorption and Drug Delivery 2001, 15.
  • Gonzalez, D. E., K. M. Covitz, W. Sadee & R. J. Mrsny: An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2. Cancer Res. 1998, 58, 519525.
  • Guo, A., P. Hu, P. V. Balimane, F. H. Laibach & P. J. Sinko: Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPepT1) expressed in a mammalian cell line. J. Pharmacol. Exp. Therap. 1999, 289, 447454.
  • Han, H., R. L. A. Vrueh, J. K. Rhie, K.-M. Y. Covitz, P. L. Smith, C. P. Lee, D. Oh, W. Sadee & G. L. Amidon: 5'-Amino acid esters of anti-viral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter. Pharm. Res. 1998, 15, 11541159.
  • Hidalgo, I. J., P. Bhatnagar, C. P. Lee, J. Miller, G. Cucullino & P. L. Smith: Structural requirements for interaction with the oligopeptide transporter in Caco-2 cells. Pharm. Res. 1995, 12, 317319.
  • Himukai, M. & T. Hoshi: Mechanisms of glycyl-L-leucine uptake by guinea-pig small intestine: relative importance of intact-peptide transport. J. Physiol. (Lond.) 1980, 302, 155169.
  • Hu, M. & G. L. Amidon: Passive and carrier-mediated intestinal absorption components of captopril. J. Pharm. Sci. 1988, 77, 10071011.
  • Hu, M., P. Subramanian, H. I. Mosberg & G. L. Amidon: Use of the peptide carrier system to improve the intestinal absorption of L-alpha-methyldopa: carrier kinetics, intestinal permeabilities, and in vitro hydrolysis of dipeptidyl derivatives of L-alpha-methyldopa. Pharm. Res. 1989, 6, 6670.
  • Jung, D. & A. Dorr: Single-dose pharmacokinetics of valganciclovir in HIV- and CMV- seropositive subjects. J. Clin. Pharmacol. 1999, 39, 800804.
  • Ihara, T., T. Tsujikawa, Y. Fujiyama & T. Bamba: Regulation of PepT1 peptide transporter expression in the rat small intestine under malnourished conditions. Digestion 2000, 61, 5967.
  • Inui, K., Y. Tomita, T. Katsura, T. Okano, M. Takano & R. Hori: H+ coupled active transport of betastatin via the dipeptide transport system in rabbit intestinal brush-border membranes. J. Pharmacol. Exp. Therap. 1992, 260 (2), 48486.
  • Knütter, I., S. Theis, B. Hartrodt, I. Born, M. Brandsch, H. Daniel & K. Neubert: A novel inhibitor of the mammalian peptide transporter PepT1. Biochemistry 2001, 40, 44544458.
  • Kramer, W., F. Girbig, U. Gutjahr, H. W. Kleemann, I. Leipe, H. Urbach & A. Wagner: Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics. Biochim. Biophys. Acta 1990, 1027, 2530.
  • Kurtin, P. & A. N. Charney: Intestinal ion transport and intracellular pH during acute respiratory alkalosis and acidosis. Amer. J. Physiol 1984, 247, G2-G31.
  • Li, J. & I. J. Hidalgo: Molecular modeling study of structural requirements for the oligopeptide transporter. J. Drug Target 1996, 4, 917.
  • Liang, R., Y. J. Fei, P. D. Prasad, S. Ramamoorthy, H. Han, F. T. Yang, M. A. Hediger, V. Ganapathy & F. H. Leibach: Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J. Biol. Chem. 1995, 270, 64566463.
  • Liu, K., Y. Kato, T. Kaku, T. Santa, K. Imai, A. Yagi, T. Ishizu & Y. Sugiyama: Hydroxyprolylserine derivatives JBP923 and JNP485 exhibit the antihepatitis activities after gastrointestinal absorption in rats. J. Pharmacol. Exp. Therap. 2000, 294, 510515.
  • Lucas, M. L., W. Schneider, F. J. Haberich & J. A. Blair: Direct measurement by pH-microelectrode of the pH microclimate in rat proximal jejunum. Proc. R. Soc. Lond. B. 1975, 192, 3948.
  • Mackenzie, B., Y. J. Fei, V. Ganapathy & F. H. Leibach: The human intestinal H+/oligopeptide cotransporter hPEPT1 transports differently-charged dipeptides with identical electrogenic properties. Biochim. Biophys. Acta 1996, 1284, 125128.
  • Matthews, D.: M.: Intestinal absorption of peptides. Physiol. Rev. 1975, 55, 537608.
  • Matthews, D. M.: Mechanisms of peptide transport. Beitr. Infusionther. Klin. Ernahr. 1987, 17, 653.
  • Meredith, D. & C. A. Boyd: Structure and function of eukaryotic peptide transporters. Cell. Mol. Life Sci. 2000, 57, 754778.
  • Meredith, D., C. S. Temple, N. Guha, C. J. Sword, C. A. Boyd, I. D. Collier, K. M. Morgan & P. D. Bailey: Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1. Eur. J. Biochem. 2000, 267, 37233728.
  • Minami, H., H. Daniel, E. L. Morse & S. A. Adibi: Oligopeptides: mechanism of renal clearance depends on molecular structure. Amer. J. Physiol 1992, 263, F109F115.
  • Moore, V. A., W. J. Irwin, P. Timmins, P. A. Lambert, S. Chong, S. A. Dando & R. A. Morrison: A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 2: affinities of ACE inhibitors. Int. J. Pharm. 2000, 210, 2944.
  • Morrison, R. A., S. Chong, A. M. Marino, M. A. Wasserman, P. Timmins, V. A. Moore & W. J. Irwin: Suitability of enalapril as a probe of the dipeptide transporter system: in vitro and in vivo studies. Pharm. Res. 1996, 13, 10781082.
  • Muller, U., M. Brandsch, P. D. Prasad, Y. J. Fei, V. Ganapathy & F. H. Leibach: Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP. Biochem. Biophys. Res. Commun. 1996, 218, 461465.DOI: 10.1006/bbrc.1996.0082
  • Nakanishi, T., I. Tamai, Y. Sai, T. Sasaki & A. Tsuji: Carrier-mediated transport of oligopeptides in the human fibrosarcoma cell line HT1080. Cancer Res. 1997, 57, 41184122.
  • Nakanishi, T., I. Tamai, A. Takaki & A. Tsuji: Cancer cell-targeted drug delivery utilizing oligopeptide transport activity. Int. J. Cancer 2000, 88, 274280.DOI: 10.1002/1097-0215(20001015)88:2<274::aid-ijc20>3.0.co;2-5
  • Newey, H. & D. H. Smyth: Cellular mechanisms in intestinal transport of amino acids. J. Physiol. Lond. 1962, 164, 527551.
  • Nicklin, P. L., W. J. Irwin, P. Timmins & R. A. Morrison: Uptake and transport of the ACE-inhibitor ceranopril (SQ 29852) by monolayers of human intestinal absorptive Caco-2 cells in vitro. Int. J. Pharm. 1996, 140, 175183.
  • Nielsen, C. U., J. Amstrup, B. Steffansen, S. Frokjaer & B. Brodin: Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line. Amer. J. Physiol 2001a, 281, G191G199.
  • Nielsen, C. U., R. Andersen, B. Brodin, S. Froekjaer, M. E. Taub & B. Steffansen: Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity to and transport via hPepT1 in the human intestinal Caco-2 cell line. J.Control Rel. 2001b, 76, 129138.
  • Novotny, A., J. Xiang, W. Stummer, N. S. Teuscher, D. E. Smith & R. F. Keep: Mechanisms of 5-aminolevulinic acid uptake at the choroid plexus. J. Neurochem. 2000, 75, 321328.DOI: 10.1046/j.1471-4159.2000.0750321.x
  • Ogihara, H., T. Suzuki, Y. Nagamachi, K. Inui & K. Takata: Peptide transporter in the rat small intestine: ultrastructural localization and the effect of starvation and administration of amino acids. Histochem. J. 1999, 31, 169174.
  • Pan, Y., E. A. Wong, J. R. Bloomquist & K. E. Webb, Jr.: Expression of a cloned ovine gastrointestinal peptide transporter (oPepT1) in Xenopus oocytes induces uptake of oligopeptides in vitro. J. Nutr. 2001, 131, 12641270.
  • Radhakrishnan, A. N.: Intestinal dipeptidases and the dipeptide transport in the monkey and in man. Ciba. Found. Symp. 1977, , 3759.
  • Saito, H. & K. Inui: Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line caco-2. Amer. J. Physiol. 1993, 265, G289G294.
  • Saito, H., M. Okuda, T. Terada, S. Sasaki & K. Inui: Cloning and characterization of a rat H+/peptide cotransporter mediating absorption of beta-lactam antibiotics in the intestine and kidney. J. Pharmacol. Exp. Therap. 1995, 275, 16311637.
  • Sawada, K., T. Terada, H. Saito, Y. Hashimoto & K. I. Inui: Recognition of L-amino acid ester compounds by rat peptide transporters PEPT1 and PEPT2. J. Pharmacol. Exp. Therap. 1999, 291, 705709.
  • Shiraga, T., K. Miyamoto, H. Tanaka, A. Yamamoto, Y. Taketani, K. Morita, I. Tamai, A. Tsuji & E. Takeda: Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1. Gastroenterology 1999, 116, 354362.
  • Silbernagl, S., V. Ganapathy & F. H. Leibach: H+ gradient-driven dipeptide reabsorption in proximal tubule of rat kidney. Studies in vivo and in vitro. Amer. J. Physiol 1987, 253, F448F457.
  • Smith, D. E., A. Pavlova, U. V. Berger, M. A. Hediger, T. Yang, Y. G. Huang & J. B. Schnermann: Tubular localization and tissue distribution of peptide transporters in rat kidney. Pharm. Res. 1998, 15, 12441249.
  • Sugawara, M., W. Huang, Y. J. Fei, F. H. Leibach, V. Ganapathy & M. E. Ganapathy: Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2. J. Pharm. Sci. 2000, 89, 781789.
  • Surendran, N., K.-M. Y. Covitz, H. Han, W. Sadee, D. Oh, G. L. Amidon, R. M. Williamson, C. F. Bigge & B. H. Stewart: Evidence for overlapping substrate specificity between large neutral amino acid (LNAA) and dipeptide (hPEPT!) transporters for PD 158473, an NMDA antagonist. Pharm. Res. 1999, 16(3), 391395.
  • Swaan, P. W., B. C. Koops, E. E. Moret & J. J. Tukker: Mapping the binding site of the small intestinal peptide carrier (PepT1) using comparative molecular field analysis. Receptors Channels 1998, 6, 189200.
  • Swaan, P. W. & J. J. Tukker: Molecular determinants of recognition for the intestinal peptide carrier. J. Pharm. Sci. 1997, 86, 596602.
  • Takahashi, K., N. Nakamura, T. Terada, T. Okano, T. Futami, H. Saito & K. Inui: Interaction of beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes. J. Pharmacol. Exp. Therap. 1998, 286, 10371042.
  • Tamai, I., T. Nakanishi, K. Hayashi, T. Terao, Y. Sai, T. Shiraga, K. Miyamoto, E. Takeda, H. Higashida & A. Tsuji: The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine. J. Pharm. Pharmacol. 1997, 49, 796801.
  • Tamai, I., T. Nakanishi, H. Nakahara, Y. Sai, V. Ganapathy, F. H. Leibach & A. Tsuji: Improvement of L-dopa absorption by dipeptidyl derivation, utilizing peptide transporter PepT1. J. Pharm. Sci. 1998, 87, 15421546.DOI: 10.1021/js980186o
  • Tamura, K., C. P. Lee, P. L. Smith & R. T. Borchardt: Metabolism, uptake, and transepithelial transport of the stereoisomers of Val-Val-Val in the human intestinal cell line, Caco-2. Pharm. Res. 1996, 113, 16631667.
  • Tanaka, H., K. I. Miyamoto, K. Morita, H. Haga, H. Segawa, T. Shiraga, A. Fujioka, T. Kouda, Y. Taketani, S. Hisano, Y. Fukui, K. Kitagawa & E. Takeda: Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury. Gastroenterology 1998, 114, 714723.
  • Tanaka, M., M. Hohmura, T. Nishi, K. Sato & I. Hayakawa: Antimicrobial activity of DU-6681a, a parent compound of novel oral carbapenem DZ-2640. Antimicrob. Agents Chemother. 1997, 41, 12601268.
  • Taub, M. E., B. D. Larsen, B. Steffansen & S. Frokjaer: Beta-Carboxylic acid esterified D-Asp-Ala retains a high affinity for the oligopeptide transporter in Caco-2 monolayers. Int. J. Pharm. 1997a, 146, 205212.
  • Taub, M. E., B. A. Moss, B. Steffansen & S. Frokjaer: Influence of oligopeptide transporter binding affinity upon uptake and transport of D-Asp(OBzl)-Ala and Asp(OBzl)-Sar in filter-grown Caco-2 monolayers. Int. J. Pharm. 1997b, 156, 219228.
  • Taub, M. E., B. A. Moss, B. Steffansen & S. Frokjaer: Oligopeptide transporter mediated uptake and transport of d-asp(obzl)-ala, d-glu(obzl)-ala, and d-ser(bzl)-ala in filter-grown caco-2 monolayers. Int. J. Pharm. 1998, 174, 223232.
  • Temple, C. S., A. K. Stewart, D. Meredith, N. A. Lister, K. M. Morgan, I. D. Collier, J. R. Vaughan, C. R. Boyd, P. D. Bailey & J. R. Bronk: Peptide mimics as substrates for the intestinal peptide transporter. J. Biol. Chem. 1998, 273, 2022.
  • Terada, T., H. Saito, M. Mukai & K. Inui: Recognition of beta-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Amer. J. Physiol. 1997, 273, F706F711.
  • Terada, T., H. Saito, M. Mukai & K. I. Inui: Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1. FEBS Lett. 1996, 394, 196200.
  • Terada, T., K. Sawada, T. Ito, H. Saito, Y. Hashimoto & K. I. Inui: Functional expression of novel peptide transporter in renal basolateral membranes [In Process Citation]. Amer. J. Physiol. Renal Physiol. 2000, 279, F851F857.
  • Terada, T., K. Sawada, H. Saito, Y. Hashimoto & K. Inui: Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2. Amer. J. Physiol 1999, 276, g1435g1441.
  • Thamotharan, M., Lombardo Y. B., Bawani S. Z. & S. A. Adibi: An active mechanism for completion of the final stage of protein degradation in the liver, lysosomal transport of dipeptides. J. Biol. Chem. 1997, 272, 1178611790.
  • Thamotharan, M., S. Z. Bawani, X. Zhou & S. A. Adibi: Mechanism of dipeptide stimulation of its own transport in a human intestinal cell line. Proc. Assoc. Amer. Phys. 1998, 110, 361368.
  • Thamotharan, M., S. Z. Bawani, X. Zhou & S. A. Adibi: Functional and molecular expression of intestinal oligopeptide transporter (Pept-1) after a brief fast. Metabolism 1999a, 48, 681684.
  • Thamotharan, M., S. Z. Bawani, X. Zhou & S. A. Adibi: Hormonal regulation of oligopeptide transporter pept-1 in a human intestinal cell line. Amer. J. Physiol 1999b, 276, C821C826.
  • Thwaites, D. T., C. D. Brown, B. H. Hirst & N. L. Simmons: Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H(+)-coupled carriers at both apical and basal membranes. J. Biol. Chem. 1993a, 268, 76407642.
  • Thwaites, D. T., D. Ford, M. Glanville & N. L. Simmons: H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells. J. Clin. Invest 1999, 104, 629635.
  • Thwaites, D. T., G. T. McEwan, B. H. Hirst & N. L. Simmons: Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic. Pflugers Arch. 1993b, 425, 178180.
  • Tsuji, A., I. Tamai, M. Nakanishi & G. L. Amidon: Mechanism of absorption of the dipeptide alpha-methyldopa-phe in intestinal brush-border membrane vesicles. Pharm. Res. 1990, 7, 308309.
  • Urrti, A., S. J. Johns & W. Sadée: Genomic structure of proton-coupled oligopeptide transporter hPepT1 and pH-sensing regulatory splice variant. AAPS Pharmsci. 2001 2001, Article 6.
  • Verri, T., M. Maffia, A. Danieli, M. Herget, U. Wenzel, H. Daniel & C. Storelli: Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes. J. Exp. Biol. 2000, 203(19), 29913001.
  • Walker, D., D. T. Thwaites, N. L. Simmons, H. J. Gilbert & B. H. Hirst: Substrate upregulation of the human small intestinal peptide transporter, hPepT1. J. Physiol. (Lond.) 1998, 507, 697706.
  • Walter, E., T. Kissel, M. Reers, G. Dickneite, D. Hoffmann & W. Stuber: Transepithelial transport properties of peptidomimetic thrombin inhibitors in monolayers of a human intestinal cell line (Caco-2) and their correlation to in vivo data. Pharm. Res. 1995, 12, 360365.
  • Wang, H., Y. J. Fei, V. Ganapathy & F. H. Leibach: Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain. Amer. J. Physiol. 1998, 275, C967C975.
  • Wang, H. P., H. H. Lu, J. S. Lee, C. Y. Cheng, J. R. Mah, C. Y. Ku, W. Hsu, C. F. Yen, C. J. Lin & H. S. Kuo: Intestinal absorption studies on peptide mimetic alpha-methyldopa prodrugs. J. Pharm. Pharmacol. 1996, 48, 270276.
  • Weiss, W. J., S. M. Mikels, P. J. Petersen, N. V. Jacobus, P. Bitha, Y. I. Lin & R. T. Testa: In vivo activities of peptidic prodrugs of novel aminomethyl tetrahydrofuranyl-1 beta-methylcarbapenems. Antimicrob. Agents Chemother. 1999, 43, 460464.
  • Wenzel, U., I. Gebert, H. Weintraut, W. M. Weber, W. Clauss & H. Daniel: Transport characteristics of differently charged cephalosporin antibiotics in oocytes expressing the cloned intestinal peptide transporter PepT1 and in human intestinal Caco-2 cells. J. Pharmacol. Exp. Therap. 1996, 277, 831839.
  • Wenzel, U., D. T. Thwaites & H. Daniel: Stereoselective uptake of beta-lactam antibiotics by the intestinal peptide transporter. Brit. J. Pharmacol. 1995, 116, 30213027.
  • Winckler, C., G. Breves, M. Boll & H. Daniel: Characteristics of dipeptide transport in pig jejunum in vitro. J. Comp. Physiol. B 1999, 169, 495500.
  • Yang, C. Y., A. H. Dantzig & C. Pidgeon: Intestinal peptide transport systems and oral drug availability. Pharm. Res. 1999, 16, 13311343.
  • Yeung, A. K., S. K. Basu, S. K. Wu, C. Chu, C. T. Okamoto, S. F. Hamm-Alvarez, H. Von Grafenstein, W. C. Shen, K. J. Kim, M. B. Bolger, I. S. Haworth, D. K. Ann & V. H. Lee: Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton-coupled dipeptide transporter (hPepT1). Biochem. Biophys. Res. Commun. 1998, 250, 103107.
  • Zhou, X., Thamotharan M., Gangopadhya A., Serdikoff C. & S. A. Adibi: Characterisation of an oligopeptide transporter in renal lysosomes. Biochim. Biophys. Acta 2000, 1466, 372378.
  • Zhu, T., X.-Z. Chen, A. Steel, M. A. Hediger & D. E. Smith: Differential recognitionof ACE inhibitors in Xenopus Laevis oocytes expressing rat PepT1 and PepT2. Pharm. Res. 2000, 17, 526532.