In vitro evaluation of N-methyl amide tripeptidomimetics as substrates for the human intestinal di-/tri-peptide transporter hPEPT1

Article abstract of DOI:10.1016/j.ejps.2006.03.007

Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The aim of the present

Full text of DOI:10.1016/j.ejps.2006.03.007

european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
available at www.sciencedirect.com
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In vitro evaluation of N-methyl amide tripeptidomimetics as
substrates for the human intestinal di-/tri-peptide
transporter hPEPT1
Rikke Andersena, Carsten Uhd Nielsena, Mikael Begtrupb, Flemming Steen Jørgensenc,
Birger Brodina, Sven Frokjaera, Bente Steffansena,∗
a
Molecular Biopharmaceutics, Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical
Sciences, 2-Universitetsparken, DK-2100 Copenhagen, Denmark
b
Medicinal Chemistry, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2-Universitetsparken,
DK-2100 Copenhagen, Denmark
c
Biostructural Research, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2-Universitetsparken,
DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide
transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degra-
dation in the GI-tract by various peptidases. The aim of the present study was to evaluate the
general application of N-methyl amide bioisosteres as peptide bond replacements in tripep-
tides in order to decrease degradation by peptidases and yet retain affinity for and transport
via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected
based on a principal component analysis of structural properties of 6859 N-methyl amide
tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as
well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2
cells. Decreased apparent degradation was observed for all tripeptidomimetics compared
to the corresponding natural tripeptides. However, affinity for and transepithelial transport
via hPEPT1 were only seen for Gly-Sar-Sar, Asnꢀ[CONCH3]Pheꢀ[CONCH3]Trp, and Gly-Sar-
Leu. This implies that tripeptidomimetics originating from tripeptides with neutral side
chains are more likely to be substrates for hPEPT1 than tripeptidomimetics with charged
side chains. The results of the present study indicate that the N-methyl amide peptide bond
replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is
not generally applicable to all tripeptides. Nevertheless, retained affinity for and transport
via hPEPT1 were shown for three of the evaluated N-methyl amide tripeptidomimetics.
© 2006 Elsevier B.V. All rights reserved.
Received 24 November 2005
Received in revised form 22
February 2006
Accepted 21 March 2006
Published on line 28 March 2006
Keywords:
hPEPT1
PEPT1
Tripeptides
Tripeptidomimetics
Transepithelial transport
N-methyl amide isostere
Abbreviations:
ꢀ, N-methyl amide isostere
(ꢀ[CONCH3]); PCA, Principal
Component Analysis; AP, apical; BL,
basolateral; Sar, sarcosine/sarcosyl
1.
Introduction
The recently discovered tripeptides Tyr-Ser-Val (YSV) and Tyr-
Ser-Leu (YSL) extracted from pig spleen may hold promising
Tripeptides with crucial physiological activities are poten-
tial lead compounds for drug discovery and development.
prospectives, since these tripeptides were reported to have an
antitumor effect on human hepatocarcinoma in nude mice
∗
Corresponding author. Tel.: +45 3530221; fax: +45 35306030.
E-mail address: bds@dfuni.dk (B. Steffansen).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2006.03.007

326
european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
(Jia et al., 2005; Yao et al., 2005). In another study Gly-Pro-Arg
was suggested to reduce neuronal cell death through inhibi-
tion of apoptosis in rat hippocampal neurons (Ioudina and
Uemura, 2003). Likewise, the tripeptide aldehyde D-Phe-Pro-
Arg-H derived from the thrombin cleavage site has been re-
cognized as a core structure for the development of thrombin
inhibitors (Lange et al., 2003).
The possibilities in using natural tripeptides as drug com-
pounds are often very limited due to their instability in body
fluids and tissues. Tripeptides are susceptible to hydrolysis in
the GI-lumen as well as in the enterocytes. To exert their phar-
macological activity, tripeptides must remain intact during the
absorption process. Therefore, stabilization of the tripeptides
is an obvious way to increase the bioavailability of such com-
pounds. The strategy for stabilization could be to introduce
unnatural amino acids, modifying the N- or C-termini or to
introduce peptide bond bioisosteres.
Structural modifications of tripeptides might, however,
influence their absorption in the small intestine. Natural
tripeptides arising from food are substrates for the human
di-/tri-peptide transporter, hPEPT1, which is localized in the
luminal membrane of enterocytes (Liang et al., 1995). There-
fore, this carrier may also play an important role in the absorp-
tion of tripeptidomimetic drugs. Thus, in the process of sta-
bilizing tripeptides, an equally important requirement is to
maintain affinity for and transport via hPEPT1.
The tripeptide-like ␤-lactam antibiotics are well-known
examples of compounds where, retrospectively, the high oral
bioavailability has been explained by a high resistance towards
enzymatic degradation in the GI tract and an absorption partly
due to transport via hPEPT1 across the intestinal epithelium
(Bretschneider et al., 1999; Dantzig, 1998). Furthermore, se-
veral stabilization strategies yielding substrates for hPEPT1
are described in the literature, although, primarily applied on
dipeptides. Transport via hPEPT1 of dipeptides containing a
ketomethylene bioisosteres as peptide bond replacement has
been shown for the aminopeptidase inhibitor arphamenine
A (an Arg-Phe mimetic) (Enjoh et al., 1996), the photosensi-
tizer ␦-aminolevulinic acid (a Gly-Gly mimetic) (Doring et al.,
1998), and a series of model dipeptidomimetica prodrugs as
well as the dipeptidomimetica of Phe-Gly (Va˚benø et al., 2004a,
2004b). The thioamide isostere, when introduced into Ala-Pro,
is accepted by hPEPT1 (Brandsch et al., 1998). Gly-Sar, which
contains a N-methyl amide isostere, is a well characterized
standard substrate for hPEPT1. The corresponding tripeptide
Gly-Sar-Sar (GꢀGꢀG) has also been suggested to be actively
transported across the intestinal epithelium via peptide trans-
porters when investigated in rings of everted hamster jejunum
and in rat renal brush border membrane vesicles (Addison et
al., 1975; Sleisenger et al., 1976; Daniel et al., 1992; Minami
et al., 1992). Despite many examples of peptide bond replace-
ments, the potential and general application of peptide bond
bioisosteres such as N-methyl amide in relation to hPEPT-
affinity and -mediated transport has not yet been systemati-
cally investigated.
Fig. 1 – The general structure of N-methyl amide
tripeptidomimetics. R1, R2, and R3 are side chains
originating from naturally occurring amino acids except
proline.
series of N-methyl amide tripeptidomimetics using Caco-2 cell
monolayers.
The evaluated tripeptidomimetics were selected in order
to represent the structural diversity among all possible N-
methyl amide tripeptidomimetics constructed from natural
amino acids (Fig. 1). Recently, we reported the successful use of
VolSurf descriptors when describing the diversity in structural
properties of tripeptides, and how these structural properties
correlated well with the affinity for hPEPT1 (Andersen et al.,
2006). Therefore, VolSurf descriptors were generated for all
possible N-methyl amide tripeptidomimetics. Based on a prin-
cipal component analysis (PCA) of the molecular descriptors,
the structural property space spanned by the 6859 tripep-
tidomimetics was created. The evaluated tripeptidomimetics
were selected to cover as much of the variation in structural
properties as possible, yet limiting the number of compounds
to be synthesized and investigated. This compound selection
approach enables a general evaluation of N-methylated tripep-
tides with regard to In vitro apparent degradation and affinity
and transport via hPEPT1.
2.
Materials and methods
2.1.
Materials and apparatus
H-Lysꢀ[CONCH3]Trpꢀ[CONCH3]Arg-OH (KꢀWꢀR) (purity
>95%),
(purity
H-Aspꢀ[CONCH3]Argꢀ[CONCH3]Glu-OH
>99%), H-Glyꢀ[CONCH3]Gluꢀ[CONCH3]Trp-OH
(DꢀRꢀE)
(GꢀEꢀW) (purity >97%), and H-Asnꢀ[CONCH3]Pheꢀ[CONCH3]
Trp-OH (NꢀFꢀW) (purity >98%) were custom synthesized
as TFA salts by BaChem (Weil am Rhein, Germany). H-
Lys-Trp-Arg-OH (KWR), H-Asp-Arg-Glu-OH (DRE), and
H-Asn-Phe-Trp-OH (NFW) were custom synthesized as
TFA salts with HPLC purity >98% at SynPep (Dublin, Cali-
fornia). H-Gly-Glu-Trp-OH (GEW) was custom synthesized
as TFA salts with HPLC purity >99% at NeoSystem (Stras-
bourg, France). H-Gly-Sar-Sar-OH (GꢀGꢀG), H-Gly-Gly-Gly-OH
(GGG) (purity >99%), H-Gly-Gly-Leu-OH (GGL) (purity >99%),
and H-Gly-Phe-Gly-OH (GFG) (purity >98%) were purchased
at BaChem AG (Weil am Rhein, Germany). Proper char-
acterization and proof of structure was provided by the
manufacturer. H-Glyꢀ[CONCH3]Phe-Sar-OH (7) (GꢀGꢀL) and
H-Gly-Sar-ꢀ[CONCH3]Leu-OH (12) (GꢀFꢀG) were prepared
and characterized as described below. Structures of H-Gly-
Sarꢀ[CONCH3]Leu-OH and H-Glyꢀ[CONCH3]Phe-Sar-OH were
verified by 1H and 13C NMR spectra recorded on a 300 MHz
instrument. Chemical shifts are given in ppm (ı-values)
In the present study our aim was to evaluate the gen-
eral application of N-methyl amide isosteres as peptide bond
replacements in tripeptides to decrease degradation by pep-
tidases and yet retain affinity for and transport via hPEPT1.
The affinity for and transport via hPEPT1 were studied for a

european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
327
plate used for cell culture experiments was a Swip KS 10 Digi
from Edmund Bu¨ hler (Hechingen, Germany).
relative to the internal standard tetramethylsilane (TMS). The
1H NMR spectra were assigned by comparison with spectra of
the individual amino acids. In many cases, marked with an
asterix, both E- and Z-conformers, due to restricted rotation
around the amide C–N bond, could be observed. Elemental
analyses were performed by Microanalytical Laboratory,
Department of Physical Chemistry, University of Vienna,
Austria.
2.2.
Synthesis of H-Gly-Sarꢀ[CONCH3]Leu-OH (12)
and H-Glyꢀ[CONCH3]Phe-Sar-OH (7)
All reactions involving air-sensitive reagents were performed
under nitrogen. All glassware was flame-dried prior to use.
N-(3-Dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochlo-
ride (EDAC) was handled under a nitrogen atmosphere as pre-
viously described (Friedrichsen et al., 2001). The crude prod-
ucts were purified by flash chromatography (Still et al., 1978).
The procedure for peptide coupling between N-methylated
amino acids is illustrated by the following procedure
and a schematic outline is presented in Scheme 1. Boc-
Glyꢀ[CONCH3]Phe-OH (4), (200 mg, 0.59 mmol), Sar-OBn, tosy-
late (5) (186 mg, 0.53 mmol), 1-hydroxybenzotriazole hydrate
(91 mg, 0.59 mmol), EDAC (170 mg. 0.89 mmol), DMF (1.5 ml),
and diisopropylethylamine (0.5 ml) were stirred under nitro-
gen for 20 h, the mixture was then evaporated to dry-
ness in vacuo, extracted with boiling ethyl acetate (25 ml),
washed with sat. aq NaCl (2 ml × 15 ml), sat. aq. NaHCO3
(2 ml × 15 ml), 10% aq. citric acid (2 ml × 20 ml), and sat. aq.
NaCl (1 ml × 15 ml), dried on MgSO4. After filtration, the ethyl
acetate was removed in vacuo and followed by flash chro-
matography (ethyl acetate–heptane 1:2). The yield was 200 mg
(76%) of Boc-Glyꢀ[CONCH3]Phe-Sar-OBn (6) as an oil, rf 0.10
(ethyl acetate–heptane 1:2). ıH (CDCl3) 7.29–7.10 (10H, m, 2 Ph),
5.68–5.58 (1H, m, CH in Phe), 5.04 (2H, m, CH2 in Bn), 4.32–.57
(4H, m, CH2 in Gly and Sar), 3.14–2.77 (2H, m, CH2 in Phe),
2.77–2.92 (6H, m, 2NCH3), 1.36 and 1.31* (9H, s, (CH3)3). *Two
conformers.
The benzyl and Boc protecting groups were removed by dis-
solving Boc-Glyꢀ[CONCH3]Phe-Sar-OBn (6) (0.18 g, 0.36 mmol)
in ethyl acetate (30 ml). 10% Palladium on carbon (300 mg)
was added, and the solution was stirred under hydrogen
for 16 h. Filtration, extraction with further 100 ml of ethyl
acetate and evaporation to dryness gave 90 mg (61%) Boc-
Glyꢀ[CONCH3]Phe-Sar-OH as an oil, rf 0.09 (ethyl acetate).
The crude product (85 mg, 0.21 mmol) was dissolved in
dichloromethane (25 ml). After addition of TFA (12.5 ml), the
solution was stirred for 3 h. The solvent was removed in vacuo.
Co-evaporation with chloroform (3 ml × 3 ml) yielded 90 mg
(100%) of semi-solid H-Glyꢀ[CONCH3]Phe-Sar-OH, trifluoroac-
etate (7). ıH (CD3OD) 7.26–7.20 (5H, m, Ph from Phe), 5.78–5.65
Caco-2 cells were obtained from the American Type
Culture Collection (Manassas, Virginia). Cell culture media
and Hanks balanced salt solution (HBSS) were obtained
from Life Technologies (Høje Taastrup, Denmark). Phosphate-
buffered saline (PBS) was purchased from BioChrom AG
(Berlin, Germany). All solvents were of analytical grade,
and chemicals used in buffer preparations were of labora-
tory grade. Trifluoroacetic acid (TFA), 1-octanesulfonic acid
(OSA), 2-[N-morpholino]ethanesulfonic acid (MES) and N-
[2-Hydroxyethyl]piperazine-Nꢀ-2-ethanesulfonic acid (HEPES),
bovine serum albumin (BSA), ␦-aminolevulinic acid, H-Gly-
Pro-OH, Triton X-100, and N,N-dimethylacetamide were from
Sigma (St. Louis, MO, USA). [14C]Gly-Sar and [3H]mannitol,
with specific activities of 49.94 and 51.50 mCi/mmol, respec-
tively, were purchased from New England Nuclear (Boston,
MA, USA). Chemicals used in the described syntheses were
purchased from Sigma (St. Louis, MO, USA) or BaChem AG
(Weil am Rhein, Germany) and used without further purifica-
tion with the exceptions of dimethyl formamide (DMF) and
dichloromethane (DCM), which were dried and stored over
˚
3 A molecular sieves. Chromatography was performed with
matrex silica, 60A/35–70 ␮m (Millipore, Copenhagen, Den-
mark).
The HPLC system for quantitative analyses was a Merck
Hitachi LaChrom Elite equipped with an autosampler (L-2300)
with integrated cooling unit, a HTA pump (L-2130), a column
own (L-2300), and a diode array detector (L-2450). The sys-
tem was operated with Merck EZChrom Elite (v3.1.3) software.
All compounds were eluted on a Waters Spherisorb S5OdS2
reversed-phase column (5 ␮m, 250 mm × 4.6 mm). Radioacti-
vity was counted on a Tri-Carb 2110TR liquid scintillation ana-
lyzer from Packard (Perkin-Elmer Life and Analytical Sciences,
Boston, MA).
The transepithelial electrical resistance (TEER) of Caco-2
cell monolayers was measured in a tissue resistance measure-
ment chamber (Endohm) with a voltohmmeter (EVOM) from
World Precision Instruments (Sarasota, FL, USA). The shaking
Scheme 1 – Synthesis of tripeptides 7 and 12. (a) Coupling conditions given in Section 2.2, (b) H2 Pd/C, (c) TFA.

328
european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
(1H, m, CH in Phe), 4.28–3.73 (4H, m, CH2 in Gly and Sar),
3.22–3.03 (2H, m, CH2 in Phe), 3.03–2.93 (6H, m, 2NCH3).
Using these procedures, coupling of Boc-Gly-OH (1)
and (N-methyl)-Phe-OBn, tosylate (2) afforded Boc-Gly
ꢀ[CONCH3]Phe-OBn (3), rf 0.35 (ethyl acetate–heptane 1:2). ıH
(CDCl3) 7.32–7.13 (10H, m, 2Ph), 5.21 (1H, m, CH in Phe), 5.10
(2H, m, CH2 in Bn), 3.86–3.65 (2H, m, CH2 in Gly), 3.35–2.98 (2H,
m, CH2 in Phe), 2.82 and 2.67* (3H, m, NCH3 in (N-methyl)-
Nielsen et al. (2001b). In brief, Caco-2 cells were equilibrated
for 15 min with 0.5 ml buffer pH 6.0 (HBSS supplemented
with 10 mM MES and 0.05% BSA) on the apical (AP) side and
1 ml buffer pH 7.4 (HBSS supplemented with 10 mM HEPES
and 0.05% BSA) on the basolateral (BL) side. After 15 min,
the buffers were removed and solutions of 20 ␮M (1 ␮Ci/ml)
[
14C]Gly-Sar with varying concentrations of GꢀGꢀG, GꢀGꢀL,
GꢀFꢀG, NꢀFꢀW, GꢀEꢀW, DꢀRꢀE or KꢀWꢀR (0–10 mM) in
buffer adjusted to pH 6.0 were added to the AP side, and
buffer pH 7.4 was added to the BL side of the monolayers.
The aqueous solubility of GꢀGꢀL and GꢀFꢀG was increased
by addition of 3% N,N-dimethylacetamide, which was shown
to have no influence on the uptake of [14C]Gly-Sar (data
not shown). After 5 min, the solutions were removed and
cells were washed with ice-cold HBSS buffer. The filter sup-
ports were cut out and the amount of [14C]Gly-Sar was mea-
sured by liquid scintillation spectrometry. Experiments with
Caco-2 cells were performed using at least four separate
monolayers.
Phe), 1.36 and 1.34* (9H, s, (CH3)3). Anal. calc. for C24
H30N2O5:
C, 67.59; H, 7.09; N, 6.57. Found: C, 67.84; H, 6.92; N, 6.48.
Debenzylation produced Boc-Glyꢀ[CONCH3]Phe-OH (4), rf
0.04 (ethyl acetate-heptane 1:2) which was used without
further purification in the next step. *Two conformers.
Similarly, coupling of Boc-Gly-OH (1) and Sar-OBn, tosylate
(5) gave Boc-Gly-Sar-OBn (8), rf 0.06 (ethyl acetate–heptane
1:3). Debenzylation produced Boc-Gly-Sar-OH (9), rf 0.08
(ethyl acetate) which was used without further purifica-
tion in the next step where coupling of Boc-Gly-Sar-OH
(9) and (N-methyl)-Leu-OBn, tosylate (10) gave Boc-Gly-
Sarꢀ[CONCH3]Leu-OBn (11), rf 0.67 (ethyl acetate), ıH (CDCl3)
7.31 (5H, m, Ph), 5.39 and 5.25* (1H, m, CH in Leu), 5.08 (2H,
m, CH2 in Bn), 4.35–3.95 (4H, m, 2 CH2 in Gly and Sar), 2.92
and 2.82* (6H, m, 2 NCH3), 1.68 (3H, m, CH and CH2 in Leu),
1.38 (9H, s, (CH3)3), 0.86 (6H, m, 2 CH3 in Leu), anal. calc. for
2.3.3. Transepithelial transport
The concentration-dependent AP to BL transepithelial trans-
port was measured in the concentration range of 0.4–15 mM
GꢀGꢀG (Gly-Sar-Sar) or 2–5 mM GꢀGꢀG in presence of 20 mM
Gly-Pro in buffer pH 6.0. Accordingly, AP to BL transepithelial
transport was measured for 2 mM GꢀGꢀG, NꢀFꢀW, GꢀGꢀL,
or GꢀFꢀG in presence of 20 mM Gly-Pro in buffer pH 6.0. 3%
N,N-dimethylacetamide were added to the solutions of GꢀGꢀL
and GꢀFꢀG. The AP to BL transepithelial transport studies
were performed by adding the various solutions of tripep-
tidomimetics to the AP side and buffer pH 7.4 to the BL side
of the cell monolayers. Before the transport experiments, the
Caco-2 cells were equilibrated for 15 min with 0.5 ml buffer pH
6.0 on the AP side and 1 ml buffer pH 7.4 on the BL side. After
15 min, buffers were removed and the AP to BL transport stud-
ies were initiated. AP donor samples (20 ␮l) were taken at 0, 60,
and 140 min. BL receiver samples (100 ␮l) were taken at 60, 80,
100, 120, and 140 min and replaced with buffer pH 7.4.
Concentration-dependent BL to AP transepithelial trans-
port of 2–10 mM GꢀGꢀG was initiated by addition of solutions
in buffer pH 7.4 to the BL side and buffer pH 7.4 to the AP
side. Before the experiments, the Caco-2 cells were equili-
brated with buffer pH 7.4 on both AP and BL side for 15 min.
AP receiver samples (25 ␮l) were taken at 60, 80, 100, 120, and
140 min and replaced with buffer pH 7.4. BL donor samples
(20 ␮l) were taken at 0, 60, and 140 min.
C24H37N3O6: C, 62.18; H, 8.04; N, 9.06. Found: C, 61.90; H, 7.77;
N, 9.34. Debenzylation produced Boc-Gly-Sarꢀ[CONCH3]Leu-
OH, rf 0.02 (ethyl acetate). Subsequent removal of the Boc-
group gave H-Gly-Sarꢀ[CONCH3]Leu-OH, trifluoroacetate (12),
ıH (CD3OD), 5.15 (1H, m, CH in Leu), 4.44–3.77 (4H, m, CH2
in Gly and Sar), 2.98–2.83* (6H, m, 2NCH3), 1.78–1.51 (3H, m,
CH and CH2 in Leu), 0.97–0.90 (6H, m, 2 CH3 in Leu). *Two
conformers.
The HPLC purity of H-Gly-Sarꢀ[CONCH3]Leu-OH (12) and
H-Glyꢀ[CONCH3]Phe-Sar-OH (7) was >96% and >94%. The
compounds will be referred to as GꢀGꢀL and GꢀFꢀG, respec-
tively.
2.3.
Biological investigations
2.3.1. Cell culture
Caco-2 cells were cultured as previously described (Nielsen et
al., 2001a). Briefly, cells were seeded in culture flasks and pas-
saged in Dulbecco’s Modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum, penicillin/streptomycin
(100 U/ml and 100 ␮g/ml, respectively), 1% l-glutamine, and 1%
nonessential amino acids. Caco-2 cells were seeded onto tis-
sue culture treated 12-well Transwell plates (1.13 cm2, 0.4 ␮m
pore size) at a density of 105 cells × cm−2. Monolayers were
grown in an atmosphere of 5% CO2–95% O2 at 37 ◦C. Growth
media were replaced every other day. TEER was measured
at room temperature before each experiment. The mono-
All samples were transferred to HPLC vials and ana-
lyzed by HPLC-UV. The integrity of the Caco-2 cell mono-
layers were evaluated after the transport studies by mea-
suring the [3H]mannitol flux. The apparent permeability
(Papp) of [3H]mannitol typically ranged from 0.1 × 10−6 to
0.6 × 10−6 cm/s. Fluxes of the tripeptidomimetics were con-
stant after 60 min.
layers used had TEER’s in the range of 400–700 ꢁ cm−2
.
Experiments were performed on day 25–27 after seed-
ing. Caco-2 cell from passages 21–48 were used for the
experiments.
2.3.4. Apical and basolateral uptake
Concentration-dependent AP uptake of GꢀGꢀG was mea-
sured at concentrations ranging from 0.5 to 10 mM. Solutions
(0.5 ml) were adjusted to pH 6.0 in buffer and added to the
AP side and 1 ml buffer pH 7.4 to the BL side. AP donor sam-
ples were taken every 20 min. The BL uptake experiments
2.3.2. Affinity measurements
Affinity for hPEPT1 of tripeptidomimetics was measured
as concentration-dependent inhibition of apical [14C]Gly-Sar
(20 ␮M) uptake in Caco-2 cell monolayers as described by

european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
329
were initiated by adding 20 mM GꢀGꢀG in absence or pres-
ence of 20 mM Gly-Pro in buffer pH 7.4 (pH adjusted) to the
BL side and buffer pH 7.4 to the AP side of Caco-2 cells. BL
donor samples were taken every 20 min. After 10 min AP or
BL uptake experiment, solutions were removed and cells were
washed with ice cold HBSS. Cells were detached from filters
by adding 100 ␮l 0.1% Triton-X in PBS and incubating at 37 ◦C
for 15 min. The cell suspensions were transferred to eppen-
dorf tubes and frozen to −18 ◦C. Before analyzing, cell sus-
pensions were thawed and centrifuged for 15 min, 4 ◦C, and
14000 rpm. The supernatants and donor samples were ana-
lyzed by HPLC-UV. The time dependent uptake AP to BL and
BL to AP of 15 mM GꢀGꢀG was linear until 10 and 15 min,
respectively.
2.5.
Data analysis
2.5.1. Transport kinetics
The AP to BL transepithelial flux, J, of 2 mM GꢀGꢀG, GꢀGꢀL,
NꢀFꢀW, and GꢀFꢀG in presence or absence of 20 mM Gly-
Pro and the basolateral uptake of 20 mM GꢀGꢀG in presence
or absence of 20 mM Gly-Pro were calculated as J = ꢂQ/ꢂt/A,
where ꢂQ/ꢂt is the accumulated amount of intact tripep-
tidomimetic on the receiver side, and A is the area of the
Caco-2 cell monolayer (1.13 cm2).
The BL to AP transepithelial transport of GꢀGꢀG and the
AP to BL transepithelial transport of GꢀGꢀG in presence of
Gly-Pro were non-saturable and apparent permeabilities (Papp
)
were determined by linear regression according to Fick’s first
law:
2.3.5. Apparent degradation
J
The apparent degradation of the tripeptidomimetics, when
exposed to extracellular peptidases, was estimated by adding
the compounds to the AP side of Caco-2 cell monolayers. To
limit uptake of the tripeptidomimetics during the experiment,
an excess of a high affinity hPEPT1 substrate (␦-aminolevulinic
acid (20 mM, Ki = 1.5 mM)) was added to the AP side. Depend-
ing on the Ki-value of the compounds, this resulted in more
than 98% inhibition of uptake. The experiments were initi-
ated by adding 0.5 ml of 0.5 mM tripeptidomimetic and 20 mM
␦-aminolevulinic acid in buffer pH 6.0 to the AP side and 1 ml
buffer pH 7.4 to the BL side of the Caco-2 cell monolayers. AP
samples (20 ␮l) were taken at different time points (0–60 min),
transferred to vials on ice and analyzed on HPLC-UV. The
degradation of tripeptidomimetics was investigated in three
separate experiments for each compound.
Papp
=
(1)
C0
where C0 is the initial concentration on the donor side.
The AP to BL transepithelial transport and apical uptake of
GꢀGꢀG at various concentrations were saturable and fitted to
a Michaelis–Menten-like expression:
Jmax[S]n
H
J =
(2)
Kmn + [S]n
H
H
where J is the flux of the tripeptidomimetic, Jmax the maxi-
mal flux, S the concentration of the tripeptidomimetic, Km the
Michaelis–Menten constant, and nH is the Hill-constant.
Apparent affinity for hPEPT1 in Caco-2 cells was deter-
mined as concentration-dependent inhibition of [14C]Gly-
Sar uptake. The IC50-values were calculated as described in
Nielsen et al. (2001b) and the conversion of IC50 to Ki as
described by Cheng and Prusoff (1973).
2.3.6. Analytical HPLC methods
All the developed HPLC methods kept a flow rate at 1 ml/min
and an auto sampler temperature at 4 ◦C. The following mobile
phases was used; GꢀGꢀG: 1% phosphoric acid, 10 mM 1-
octanesulfonic acid, and 10% acetonitril (AcN); GꢀGꢀL and
GꢀFꢀG: 0.05% TFA and 25% AcN; NꢀFꢀW: 20 mM acetate (pH
5), 0.05% TEA, and 30% AcN; KꢀWꢀR: 0.05% TFA and 10%
AcN; GꢀEꢀW: 0.05% TFA and 20% AcN; DꢀRꢀE: 1% phospho-
ric acid, 10 mM 1-octanesulfonic acid, and 30% Acn. Column
temperatures were 25–50 ◦C and the wavelength used in UV
detection was 210 or 220 nm. Retention times ranged from 6.3
to 10.4 min.
2.5.2. Apparent degradation
The apparent degradation of tripeptidomimetic was measured
as the amount of intact compound on the apical side of Caco-2
cell monolayers. The apparent degradation was considered to
be pseudo-first-order, thus calculated as
% intact tripeptidomimetic = 100% − (1 − e−k t) × 100%
(3)
obs
where kobs is the estimated pseudo-first-order rate constant
and t is the time.
2.4.
Molecular descriptors
Tripeptidomimetics were excluded from the study if less
than 90% of the compound was available as intact tripep-
tidomimetic on the AP side of the cell monolayers after 5 min
(length of affinity assay). This 90% limit was assessed due to
the influence degradation products (dipeptidomimetics) may
exert on the interpretation of measured apparent binding
affinities of the tripeptidomimetics.
VolSurf descriptors were calculated using the VolSurf (v4.2.1)
program implemented in SYBYL (v6.9.1). Cruciani et al. (2000)
have extensively described the methodology behind the Vol-
Surf descriptors. According to standard procedures, the Vol-
Surf descriptors were based on GRID 3D molecular interaction
fields (MIFs) resulting from interaction energies between the
ligand and different probes. VolSurf descriptors were gener-
ated for a data set of 6859 N-methyl amide tripeptidomimetics
(cf. Fig. 1), using the probes H2O, DRY, and O. The tripep-
tidomimetics were built in SYBYL and protonated according
to pH 6.0. The VolSurf descriptors were generated with default
settings. For a detailed description of VolSurf descriptors cf.
ref. VolSurf Manual (2005) and Cruciani et al. (2000).
2.5.3. Multivariate data analysis
The VolSurf descriptors were analyzed by PCA (Principal Com-
ponent Analysis) in SIMCA-P (v10.0). Default settings were
applied. The variables were centered and scaled to unit vari-
ance. Cross validation (seven rounds) was performed to test
the significance of the model.

330
european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
Table 1 – Kinetic parameters for the transepithelial
transport and apical uptake of GꢀGꢀG
Transport direction
AP to BL
Kinetic parametersa
Jmax = 0.55 0.02 nmol min−1 cm−2
Km = 1.7 0.2 mM
nH = 1.1 0.1
AP to BL (20 mM Gly-Pro)
BL to AP
Papp = 1.2 × 10−7 2.4 × 10−8 cm s−1
Papp = 2.0 × 10−7 1.7 × 10−8 cm s−1
AP to cytosol (uptake)
Jmax = 1.8 0.2 nmol min−1 cm−2
Km = 1.5 0.5 mM
nH = 0.9 0.2
a
Variation on kinetic parameters are given as S.E.M.
Jmax is the maximal flux, Km the Michaelis–Menten constant, nH
the Hill-constant, and Papp is the apparent permeability.
Fig. 2 – Transepithelial transport of GꢀGꢀG. Circles
represent the apical to basolateral flux of GꢀGꢀG in absence
(black) (N = 6) and presence (white) of 20 mM Gly-Pro (N = 3).
Black triangles represents the basolateral to apical flux of
GꢀGꢀG (N = 3). Data are given as mean S.E.M.
3.2.
Selection and structural properties of
tripeptidomimetics
A five principal component analysis (PCA) model was devel-
oped based on the 6859 tripeptidomimetics and their struc-
tural properties represented by the VolSurf descriptors. From
this model the PCA score plots (Fig. 4, I and II) and PCA loading
plot (Fig. 4, III) were extracted. The seven tripeptidomimetics
included in the study were selected from different areas of the
structural property space spanned both by the first and second
principal component and the second and third principal com-
ponent (Fig. 4, I and II). Hence, the compounds were selected
in order to cover the variation in the structural properties of
this type of tripeptidomimetics.
The VolSurf descriptors described the structural properties
of the tripeptidomimetics. Compounds situated in different
areas of the PCA score plots (Fig. 4, I and II) are predominantly
described by the descriptors represented in the same area of
the PCA loading plot (Fig. 4, III). Thus, the diversity in structural
properties of the tripeptidomimetics was recognized by the
differences in influential descriptors. In more details, DꢀRꢀE
(Fig. 4, I. Grey full line circle) was mainly described by descrip-
tors representing hydrophilic properties (Fig. 4, III. Grey full
2.5.4. Statistics
The statistical significance of the results was determined
using a two-tailed unpaired Students t-test. Affinity and trans-
port experiments on Caco-2 cell monolayers were performed
in multiple passages. N is the number of repetitions for each
experiment, which in all experiments were ≥3. Experiments
on apparent stability were performed in triplicates (N = 3).
p < 0.05 was considered significant.
3.
Results
3.1.
Transport mechanism of GꢀGꢀG
The transepithelial transport and apical (AP) uptake mecha-
nisms of GꢀGꢀG (Gly-Sar-Sar) were investigated to determine
whether N-methyl amide tripeptidomimetics could be sub-
strates for hPEPT1.
The AP to basolateral (BL) transport of GꢀGꢀG across Caco-
2 cell monolayers was found to be saturable (Fig. 2). Data
were fitted to Eq. (2), which gave the kinetic parameters
listed in Table 1. In the presence of Gly-Pro, a hPEPT1 sub-
strate, carrier-mediated AP to BL transport of GꢀGꢀG was
abolished, and only a minor non-saturable flux component
(Papp = 1.2 × 10−7 8.0 × 10−9 cm s−1) was observed (Fig. 2). BL
to AP transport of GꢀGꢀG was non-saturable with a Papp of
2.0 × 10−7 1.0 × 10−8 cm s−1, which was comparable to the
non-saturable AP to BL transport.
The intracellular uptake of GꢀGꢀG from the AP side
was saturable (Fig. 3, left) and data fitted nicely to the
Michaelis–Menten equation (Eq. (2)), which gave the kinetic
parameters listed in Table 1. The intracellular uptake of
20 mM GꢀGꢀG from the BL side was significantly inhib-
ited by 20 mM Gly-Pro (Fig. 3, right). This inhibitable
component was approximately 65% of the total GꢀGꢀG
uptake.
Fig. 3 – Left: Apical uptake of GꢀGꢀG (N = 6). Right:
Basolateral uptake of 20 mM GꢀGꢀG in absence and
presence of 20 mM Gly-Pro (N = 6), *significant difference
(p > 0.05). Data are given as mean S.E.M.

european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
331
line circle); KꢀWꢀR and NꢀFꢀW (Fig. 4, I, large punctuated cir-
cle) were primarily described by size, shape and hydrophobic
properties (Fig. 4, III, large punctuated circle); GꢀGꢀG, GꢀGꢀL,
and GꢀFꢀG (Fig. 4, I, black full line small circle) were mainly
described by hydrophilic properties in the sense of the bal-
ance between the centre of the mass in the molecule and the
centre of the polar region of the molecule (Fig. 4, III, black full
line small circle); GꢀEꢀW (Fig. 4, I, small punctuated circle)
was predominantly described by descriptors representing the
balance between the centre of the mass in the molecule and
the centre of the hydrophobic region of the molecule (Fig. 4,
I, small punctuated circle) (for detailed descriptor definitions
see ref. VolSurf Manual (2005) and Cruciani et al., 2000).
3.3.
Affinity and transepithelial transport of the
tripeptidomimetics
The affinities of the seven tripeptidomimetics and their cor-
responding tripeptides are given in Table 2. High apparent
affinity (Ki, app < 0.5 mM) was seen for the tripeptides GGL, GFG,
and NFW. GGG, GEW, DRE, GꢀGꢀG, GꢀGꢀL, and NꢀFꢀW had
medium apparent affinities (0.5 mM < Ki,app < 5 mM). The intro-
duction of N-methyl amide bioisosteres lowered the affinity
for hPEPT1 of all tripeptidomimetics compared to the corre-
sponding tripeptides, and it abolished the affinity in the case
of GꢀFꢀG, KꢀWꢀR, GꢀEꢀW, and DꢀRꢀE (Table 2).
The AP to BL transepithelial transport at 2 mM was deter-
mined for the three tripeptidomimetics with affinity for
hPEPT1, i.e. GꢀGꢀG, GꢀGꢀL, and NꢀFꢀW and for GꢀFꢀG with
no affinity for hPEPT1 (Fig. 5). The transport was also measured
in presence of 20 mM Gly-Pro to determine the contribution
of hPEPT1-mediated transport to the overall transepithelial
transport of the tripeptidomimetics.
The transport across Caco-2 cell monolayers of GꢀGꢀG was
significantly higher than the transport of GꢀGꢀL, NꢀFꢀW,
and GꢀFꢀG. The transepithelial transport of GꢀGꢀL was sig-
nificantly higher than the transport of NꢀFꢀW and GꢀFꢀG,
where the transport of NꢀFꢀW was significantly higher than
the transport of GꢀFꢀG.
The transport of the tripeptidomimetics with affinity for
hPEPT1 could be significantly inhibited by 20 mM Gly-Pro. This
inhibitable component accounted for approximately 96% and
35% of the total transport of GꢀGꢀG and NꢀFꢀW, respectively,
Fig. 4 – (I) PCA score plot where t[1] and t[2] is the first and
second principal component, respectively. (II) PCA score
plot where t[2] and t[3] is the second and third principal
component, respectively. The principal components were
extracted from the five principle component model. The
obtained regression coefficient R2 (goodness of model fit)
and the predictive correlation coefficient Q2 (goodness of
predictions by the model) was 0.78 and 0.75, respectively.
The large circle (black full line) is the 95% confidence
interval. The first, second, and third principal component
explained 33%, 19%, and 13%, respectively, of the structural
variation derived from VolSurf descriptors of the
tripeptidomimetics. The grey and black dots represent the
6859 N-methyl amide tripeptidomimetics. (III) PCA loading
plot of the loadings of the first (p[1]) and second (p[2])
principal component. Variables important for the
description of a tripeptidomimetic are located in the same
area of the loading plot as the tripeptidomimetic in the
score plot. Punctuated black circles and full line grey- and
black circles (I and III) enclose tripeptidomimetic and
corresponding influential descriptors.

332
european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
Table 2 – Apparent Ki-values (Ki,app) for hPEPT1 of
tripeptides/-mimetics measured as inhibition of
14C]Gly-Sar uptake in Caco-2 cells
[
Tripeptide/-mimetic
Ki,app (mM)a
R
R
H
GGG
0.77 0.04b
1.6 0.1b
CH3
GꢀGꢀG
R
R
H
GGL
0.29 0.002b
4.0 1.2
CH3
GꢀGꢀL
R
R
H
GFG
0.39 0.004b
NAc
CH3
GꢀFꢀG
Fig. 5 – Transepithelial transport of 2 mM GꢀGꢀG (N = 8),
GꢀGꢀL (N = 12), GꢀFꢀG (N = 9), and NꢀFꢀW (N = 3). Grey part
of bar is the hPEPT1-mediated flux. Black part is the
non-hPEPT1 mediated flux obtained in presence of 20 mM
Gly-Pro. ND = No detectable transport. *Significant difference
between groups (p > 0.05). Data are given as mean S.E.M.
R
R
H
NFW
0.19 0.003b
2.0 0.7
CH3
NꢀFꢀW
Table 3 – Apparent degradation of tripeptide/-mimetic
on the apical side of Caco-2 cell monolayers
% Intact tripeptide/-mimetic
after 60 mina
R
R
H
GEW
2.5 1.2b
NAc
GGGb
GꢀGꢀGb
GGLb
GꢀGꢀL
GFGb
GꢀFꢀG
NFWb
NꢀFꢀW
GEWb
GꢀEꢀW
DREb
91
94
79
87
53
84
57
89
82
92
1
2
3
3
4
3
5
6
0
6
CH3
GꢀEꢀW
R
R
H
DRE
4.2 2.5b
NAc
CH3
DꢀRꢀE
71 12
DꢀRꢀE
KWR
96
21
2
2
KꢀWꢀR
85 11
a
%Intact tripeptide/-mimetic are given as mean S.D. (n = 3).
Data from Andersen et al. (2006).
b
b,d
R
R
H
KWR
–
CH3
KꢀWꢀR
NAc
was generally significantly higher than the amount of the cor-
responding tripeptide. However, because of the initially high
stability of GGG only a slight increase in intact compound was
observed after the N-methylation of this compound.
a
Ki,app values are given as mean S.E.M.
Data from Andersen et al. (2006).
NA = No affinity.
KWR was excluded from the affinity study as less than 90% of the
compound was intact after 5 min.
b
c
d
4.
Discussion
A general evaluation of the affinity and transport of N-methyl
amide tripeptidomimetics was performed in the present
study. From all possible N-methylated tripeptides, seven struc-
turally diverse compounds were selected and their affinity
for and transepithelial transport via hPEPT1 of intact tripep-
tidomimetics were investigated and compared to the corre-
sponding tripeptides.
while the transport of GꢀGꢀL was totally inhibited by Gly-Pro
(Fig. 5).
To evaluate the effect of N-methylation on the stabiliza-
tion of the tripeptides the amount of intact tripeptide/mimetic
was measured on the AP side of the Caco-2 cells (Table 3).
The amount of intact tripeptidomimetic present after 60 min

european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
333
tion of cis/cis, trans/trans, cis/trans, and trans/cis conformations
of the peptide bonds. Thus, a change in the isomeric ratio of
the tripeptidomimetics may decrease the concentration of the
compound species adopting trans/trans conformation, which
is assumed to be the conformation that binds to hPEPT1.
Hence, the change in isomeric ratio may lead to an under-
estimation of the Ki-value of the binding compound specie.
However, in the present evaluation of N-methylation as a gen-
eral approach for stabilizing tripeptides and retain affinity for
4.1.
Transport mechanism of GꢀGꢀG
The transport mechanism of the simplest N-methylated
tripeptidomimetic, GꢀGꢀG, was investigated to initially
explore the potential of this type of tripeptidomimetic as sub-
strates for hPEPT1.
The predominant AP to BL transepithelial transport route
for GꢀGꢀG was clearly active transport via hPEPT1. Thus,
the paracellular and passive transepithelial transport could be
considered negligible in the overall transport of GꢀGꢀG in the
concentration range investigated. Furthermore, the transport
across the BL membrane was suggested to occur partly via a
yet unknown basolateral peptide transporter as the basolat-
eral uptake of GꢀGꢀG could be partially inhibited by Gly-Pro
(cf. Fig. 3) (Irie et al., 2004; Terada et al., 1999; Thwaites et al.,
1993).
and transport via hPEPT1, it is the apparent Ki-values (Ki,app
)
which should be assessed. When determining the apparent
affinity, the isomeric ratio is not taken into account. More-
over, the Ki,app-values are more representative of what may
occur in an in vivo situation than Ki-values of the binding
trans/trans conformation, assuming that the binding process
is faster than the conversion of the cis/cis, cis/trans, or trans/cis
conformations of the peptide bonds to the binding trans/trans
form.
Considering GꢀGꢀG as
a representative for the N-
methyl amide tripeptidomimetics, it was concluded that N-
methylated tripeptides might be transported by hPEPT1. Thus,
a general evaluation of N-methylated tripeptides as substrates
for hPEPT1 was initiated.
4.3.
Stability and transepithelial transport of selected
tripeptidomimetics
The N-methylations were introduced into the tripeptides to
increase their stability. The stabilizing effect, at least In vitro,
was observed by the increased amounts of intact tripep-
tidomimetics when applied to Caco-2 cells compared to the
amount of the corresponding intact tripeptides (cf. Table 3).
Our strategy was to combine both stability and transport
properties. Therefore, the transepithelial transport of GꢀGꢀG,
NꢀFꢀW, and GꢀGꢀL, which had affinity for hPEPT1, was inves-
tigated to evaluate their possible translocation via hPEPT1 and
subsequent ability to be effluxed across the basolateral mem-
brane of Caco-2 cells. Furthermore, the transport of GꢀFꢀG,
which was shown to have no affinity for hPEPT1, was investi-
gated as a negative control.
The transepithelial transport of GꢀGꢀG, NꢀFꢀW, and
GꢀGꢀL could be significantly inhibited by 20 mM Gly-Pro (cf.
Fig. 5). In accordance with the lacking affinity of GꢀFꢀG, the
transport of this compound could not be inhibited by Gly-Pro.
Thus, the tripeptidomimetics with affinity for hPEPT1
were also translocated by the transporter and subsequently
effluxed across the basolateral membrane. Surprisingly, the
flux of GꢀGꢀL with a low affinity (Ki,app = 4.0 mM) for hPEPT1
was larger than the flux seen for NꢀFꢀW (Ki,app = 2.0 mM) with
a medium affinity for hPEPT1. This may indicate that the
apparent affinities obtained as inhibition of the [14C]Gly-Sar
uptake are not predictive for the apical hPEPT1 translocation
process. Alternatively, it could imply that the rate-limiting step
in the transepithelial transport of GꢀGꢀL and NꢀFꢀW may
not be the apical translocation via hPEPT1, but presumably
the transport across the basolateral membrane.
4.2.
Affinity of selected tripeptidomimetics
The tripeptidomimetics GꢀGꢀG, NꢀFꢀW, and GꢀGꢀL showed
affinity for hPEPT1 (cf. Table 2). However, the modification of
the peptide bonds by N-methylation decreased the affinity for
hPEPT1 compared to the affinities of the corresponding nat-
ural tripeptide (GGG, NFW, and GGL). Surprisingly, when the
N-methyl amide isosteres were inserted into GFG, GEW, DRE,
and KWR, the affinity for hPEPT1 was abolished.
In a recently proposed QSAR model partly based on tripep-
tides and ␤-lactam antibiotics it was suggested that the pres-
ence of a hydrogen bond donor in the second peptide bond
of a tripeptide may have an unfavorable effect on the bind-
ing affinity for hPEPT1 (Biegel et al., 2005). Furthermore, the
first general hPEPT1 substrate template proposed by Bailey et
al. (2000) suggested that alkylation of the second amide bond
was allowed. However, our results strongly indicate that the
removal of hydrogen bond donors from the peptide bond of a
tripeptide by N-methylation has an unfavorable effect on the
affinity for hPEPT1. Still, the hPEPT1 transport protein to some
extent accepts methylation of the peptide bonds as illustrated
by GꢀGꢀG, NꢀFꢀW, and GꢀGꢀL. Yet, this acceptance is not
general but depends on the structural properties of the side
chains of the tripeptidomimetic as implied by the abolished
affinity of GꢀFꢀG, GꢀEꢀW, DꢀRꢀE, and KꢀWꢀR. Thus, the
affinities of this series of structurally very diverse compounds
indicate that tripeptides with neutral side chains are more
likely to bind to hPEPT1 after N-methylation of the peptide
bonds than tripeptides with charged side chains. This obser-
vation corresponds well with the trend seen for affinities of
natural tripeptides (Andersen et al., 2006).
In aqueous solution, tripeptides normally exist in a trans
conformation around both peptide bonds. Based on studies of
dipeptides, it has been suggested that the preferred conforma-
tion for binding to hPEPT1 is the trans conformation (Brandsch
et al., 1998, 1999; Bailey et al., 2005). The introduction of N-
methyl amide isosteres in tripeptides raises the possibility of
a changed cis and trans conformation ratio, i.e. the combina-
5.
Conclusion
The present investigation revealed that the N-methyl amide
isostere is not generally applicable as a peptide bond replace-
ment in a tripeptide, when the intension is to both increase
stability and retain affinity and transport via hPEPT1 of the
compound. Increased stability was seen for all tripeptides;

334
european journal of pharmaceutical sciences 2 8 ( 2 0 0 6 ) 325–335
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GꢀGꢀL had affinity for hPEPT1 and hPEPT1-mediated trans-
port across Caco-2 cell monolayers. Thus, it was implied that
N-methylated tripeptides with neutral side chains are more
likely to be substrates for hPEPT1 than N-methylated tripep-
tides with charged side chains.
This In vitro evaluation indicated that the N-methyl amide
isostere approach for increasing bioavailability of tripep-
tidomimetic drug candidates is only possible for a limited
number of compounds where the overall structural proper-
ties of the tripeptide facilitates retention of affinity for and
transport via hPEPT1.
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Acknowledgements
The Danish Medicinal Council supported this work (Project
Grant #22-03-0274). Zealand Pharma A/S financially supported
this work via the Drug Research Academy, Danish University of
Pharmaceutical Sciences, Copenhagen, Denmark. Partly fund-
ing was achieved from the BioSim workpackage 15 funded by
the European Commission (BioSim NoE LSH-CT-005137). The
HPLC system was co-funded by the Hørslev Foundation. The
technical assistance of Bettina Dinitzen and Susanne Nørskov
Sørensen is highly valued. Peter Elm and Karina Thorn per-
formed excellent synthesis assistance. We appreciate the sup-
port by Tripos Associates and Gabriele Cruciani regarding the
access to Sybyl and VolSurf, respectively.
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VolSurf Manual (VolSurf v4.1.3) Molecular Discovery Ltd.
http://www.moldiscovery.com/soft volsurf.php (accessed
22/11/05).