The in vitro transport of model thiodipeptide prodrugs designed to target the intestinal oligopeptide transporter, PepT1

Article abstract of DOI:10.1039/b909221h

A thiodipeptide carrier system is shown to be effective at enabling a range of covalently bound molecules, including benzyl, benzoyl and ibuprofen conjugates, to be transported via the intestinal peptide transporter PepT1, demonstrating its potential as a rational drug delivery target.

Full text of DOI:10.1039/b909221h

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The in vitro transport of model thiodipeptide prodrugs designed to target the
intestinal oligopeptide transporter, PepT1†
David Foley,a,b Myrtani Pieri,c Rachel Pettecrew,a Richard Price,a Stephen Miles,a Ho Kam Lam,a
Patrick Bailey*b and David Meredithc
Received 11th May 2009, Accepted 7th July 2009
First published as an Advance Article on the web 20th July 2009
DOI: 10.1039/b909221h
A thiodipeptide carrier system is shown to be effective at
enabling a range of covalently bound molecules, including
benzyl, benzoyl and ibuprofen conjugates, to be transported
via the intestinal peptide transporter PepT1, demonstrating
its potential as a rational drug delivery target.
other potential PepT1 “carrier” compounds, although on a more
limited scale.3,6 We focussed on glycol spacers, since our previous
work on nabumetone prodrugs had indicated that the PepT1
transporter could accommodate hydrophobic groups placed a
considerable distance (e.g. triethylene glycol spacer) from the
peptide backbone.8 This work also indicated that the length of
the glycol spacer affected the rate of PepT1 mediated transport.8
Therefore we elected to synthesise compounds 4–11 to probe
both of these effects in more detail. We also anticipated that
polyethylene glycol spacers may prove more useful generally in
modifying other physicochemical properties important to oral
delivery.14
PepT1 is a membrane transporter located principally on the
luminal cell membrane of the intestine. The broad substrate
capacity of the transporter, including most di- and tripeptides,
b-lactam antibiotics and antivirals, has made it a promising target
for improving oral drug delivery.1
Several examples of drugs and prodrugs that are transported by
PepT1 in vitro and in vivo have been reported.2 In most of these
cases, the increase in oral bioavailability of the various prodrugs
was first observed, with PepT1 mediated transport proposed
as an explanation at a later date. While the idea of rationally
targeting drugs towards transport by PepT1 has been investigated
by various groups and methods,3-7 it is only recently that we have
demonstrated the designed transport by PepT1 of a prodrug of
the non-steroidal anti-inflammatory drug (NSAID) nabumetone,8
using our thiodipeptide “carrier” prodrug approach.9 Amidon’s
recent work on floxuridine prodrugs represents the only other
example we could find of rational drug delivery via PepT1.10
Additionally, despite a wealth of empirical and modelling data
on substrate affinity for PepT1,7,11 very little is known about the
substrate structural features important to transport.12 This field
has been particularly hampered by the lack of a three dimensional
structure of the transporter, although recent homology models
offer the promise of improvements in this area in the near future.13
Following on from our work with nabumetone prodrugs,8 we
wished to investigate the limits of capacity of the transporter,
in relation to its ability to bind and transport thiodipeptide
prodrugs in particular. Our initial study involved the synthesis
and in vitro testing of a range of thiodipeptides (1–11) for
PepT1 affinity and transport. Benzyl alcohol or benzoic acid
were chosen as simple probes for potential drugs in these studies,
as this type of system has been used in the investigations of
aSchool of Chemistry, University of Manchester, Manchester, M13 9PL,
U.K.
The synthesis of the fully protected thiodipeptides 24–26 has
been reported previously9 and is included in the ESI (Scheme 1).†
Target compounds 1–3 were isolated as their TFA salts following
standard acidolysis of the Boc carbamate and OBut ester using
TFA in DCM. A dissolving metal reduction of intermediates 24
and 25 yielded the protected serine and aspartate thiodipeptide
“carriers” 27 and 28 respectively, to which we envisioned a variety
of drugs could be attached. The five step route to 27 and 28 has now
bFaculty of Natural Sciences, Keele University, Keele, Staffordshire, ST5
5BG, U.K.. E-mail: p.bailey@natsci.keele.ac.uk; Fax: +44 (0)1782 584593;
Tel: +44 (0)1782 584583
cSchool of Life Sciences, Oxford Brookes University, Headington, Oxford
OX3 0BP, U.K.
† Electronic supplementary information (ESI) available: Full experimental
details for the synthesis of all compounds, with characterisation data. Full
details of in vitro experiments, with affinity and trans-stimulation data. See
DOI: 10.1039/b909221h
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an a,b-unsaturated ester, and/or significant racemisation. The
procedure reported below, employing a key aziridine ring-opening
step, gives reliable access to this important type of compound,
and may be of value to those wishing to access other seryl ethers
(Scheme 3). A known literature route was used to form aziridine
43.19 For the crucial ring-opening step, we initially employed
the conditions reported by Ho et al.20 which in their hands
gave yields of 64% with glycol nucleophiles. In our hands, this
procedure resulted in the formation of compound 44, but in only
18% yield. A study of the effects of temperature, concentration
and catalyst loading resulted in an optimised method with an
improved yield of 50%. Following removal of the Cbz group by
catalytic hydrogenation, coupling of the serine ether to Boc-Ala-
OH was readily achieved in high yield using diphenylphosphoryl
azide as a coupling reagent.21 Lawesson’s reagent22 was used to
selectively effect an oxygen–sulfur exchange at the amide carbonyl.
Hydrolysis of the methyl ester with lithium hydroxide in aqueous
THF, followed by TFA mediated acidolysis of the Boc group
yielded ether 4 as a TFA salt.
Scheme 1 Synthesis of target compounds 1–3 and thiodipeptide “car-
riers” 27 and 28. (i) tert-Butyl-2,2,2-trichloroacetimidate, DCM/Et2O.
(ii) TBAF in THF. (iii) Boc-Ala-OH, DPPA, TEA, DMF. (iv) Lawesson’s
reagent, refluxing toluene. (v) 33% TFA in DCM. (vi) Na/NH3(l) in THF,
-78 ◦C.
been optimised in our laboratory so that multi-gram quantities can
be brought through in less than two weeks.
The synthesis of 5–11 was carried out using similar methods to
those we employed for the synthesis of thiodipeptide prodrugs of
nabumetone (Scheme 2).8 In brief, mono-, di- or triethylene glycols
were mono-functionalised with benzyl bromide using standard
sodium hydride methods or the method of Bouzide.15 Di- and
triethylene glycols were mono-functionalised with benzoic acid
using concentrated Mitsunobu conditions and sonication.16 These
mono-etherified or esterified glycols were then coupled to the
aspartate carrier 28 using standard coupling conditions (typically
HBTU, DIPEA in DMF). These same glycols were subjected
to Swern17 oxidation followed by silver oxide oxidation of the
crude aldehyde18 and the resultant glycolic acids coupled to the
serine carrier 27. TFA deprotection of these conjugates yielded
the desired compounds 5–11 as TFA salts.
Scheme 3 Synthesis of serine ether 4. (i) TrCl, TEA, CHCl3, 0 ◦C, 24 h.
(ii) TsCl, pyr, 0 ◦C, 48 h. (iii) TEA, THF, 75 ◦C, 48 h. (iv) TFA, Et3SiH,
DCM, -5 ◦C, 1 h then DIPEA, -5 ◦C, 20 min, concentrate under vacuum
and add benzylchloroformate, DIPEA, 0 ◦C, 3 h. (v) 34, BF3·OEt2, DCM,
20 h. (vi) H2, 10% Pd-C, EtOH, 3 h. (vii) Boc-Ala-OH, DPPA, TEA,
DMF, 0 ◦C–rt, 18 h. (viii) Lawesson’s reagent, refluxing toluene, 4 h.
(ix) LiOH·H2O, THF/H2O, 18 h. (x) 33% TFA in DCM, 5 h.
The PepT1 binding affinity of compounds 1–11 was determined
by measuring the concentration at which they inhibited uptake
of radiolabelled D-Phe-L-Gln in Xenopus laevis oocytes expressing
rabbit PepT1 (Table 1). Inhibition constants were calculated from
standard Michaelis–Menten kinetics.23,24
As binding studies only show affinity for PepT1 and do not
provide information as to whether the compound is a substrate
or an inhibitor, further transport assays were undertaken. Trans-
stimulation of radiolabelled D-Phe-L-Gln efflux from rabbit PepT1
expressing oocytes in the presence of 10 mM Gly-L-Gln (a
standard PepT1 substrate) and 10 mM of test compound 1–11
were compared (Table 1).24 Trans-stimulation is a consequence of
having PepT1 substrates on both sides of the membrane, removing
Scheme 2 Synthesis of target compounds 5–11. (i) NaH, KI, BnBr.
(ii) Ag2O, BnBr. (iii) Benzoic acid, DIAD, Ph3P, THF, sonication.
(iv) 27 or 28, HBTU, DIPEA, DMF. (v) 33% TFA in DCM.
(vi) (COCl)2, DMSO, TEA, -60 ◦C. (vii) Ag2O, NaOH, H2O, reflux.
We wished to synthesise an extremely hydrolysis resistant
compound, such as 4, to reduce any effect that metabolism of the
prodrugs may have on the transport results. The synthesis of the
serine ether 4 was complicated by the fact that standard methods
of etherification resulted in either elimination from 27, to form
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Table 1 Results of affinity, trans-stimulated efflux and Caco-2 monolayer studies
Overall Papp
(normalised)
PepT1 Papp
(normalised)
Compound
Ki (mM)
trans-stimulation
Overall Papp (¥ 10-6 cm s-1)
PepT1 Papp (¥ 10-6 cm s-1)
a
a
a
a
1
0.25 0.04
0.22 0.04
0.03 0.005
0.20 0.03
0.79 0.44
0.59 0.10
0.10 0.01
0.09 0.02
0.30 0.14
0.84 0.43
0.04 0.01
Yes
No
No
Yes
No
No
Yes
Yes
No
No
Yes
3.57 0.74
11.91 4.45
4.15 0.51
4.01 1.28
0.95 0.11
0.42 0.12
4.09 0.87
0.92 0.18
3.17 0.01
1.08 0.16
1.02 0.16
2.81 0.58
0.52 0.19
1.29 0.16
1.25 0.40
0.34 0.04
0.42 0.11
1.44 0.31
0.33 0.07
2.05 0.01
0.70 0.10
0.37 0.06
2
3
2.61 0.56
2.70 1.34
1.02 0.20
1.06 0.53
4
5
0.36 0.11
0.47 0.14
a
a
6
7
1.99 0.78
0.34 0.06
1.06 0.01
0.75 0.20
0.81 0.16
1.01 0.40
0.44 0.08
3.26 0.04
2.29 0.62
1.04 0.21
8
9
10
11
a The PepT1 mediated component of transport was statistically insignificant (p > 0.05, n = 3).
the need for the re-orientation of an empty PepT1 transporter
(the rate-limiting step in the transport cycle) and therefore in
this case increasing the efflux rate of the radiolabelled dipeptide
injected into the cell compared to when no substrate is present
extracellularly.25 Since trans-stimulation of efflux can only occur if
the test compound is a substrate, this simple assay can be used to
demonstrate PepT1 transport. However, it can give false negatives
and is only a qualitative assay.
of thiodipeptides and clearly indicate that these limits should not
adversely affect the rational delivery of small molecule drugs via a
thiodipeptide approach. With the exceptions of compounds 1, 2,
and 6 all of our model prodrugs were well transported by PepT1,
with PepT1 mediated transport from 0.3–2.7 times FSA (entries 4
and 8, Table 1), an orally absorbed thiodipeptide.9,28
Compound 1 induced significant trans-stimulated efflux in our
oocyte assay. Surprisingly, this did not translate into significant
PepT1 mediated transport across Caco-2 monolayers. We believe
that the high overall transport of 1 (2.8 times that of FSA)
means that other routes of absorption, not accounted for in the
relatively simple oocyte system, are masking the PepT1 mediated
component of transport. We have demonstrated that 1 is absorbed
intact into rat bloodstreams upon oral administration (data not
shown). Our in vitro data shows that whilst 1 is a substrate of
PepT1, PepT1 mediated transport is not the major pathway of
absorption of this compound. Compounds 2 and 6, however,
appear not to be PepT1 substrates, and it appears that they are
binding to the transporter in a subtly different way than similar
high affinity substrates (3, 4 and 10), preventing transport whilst
retaining affinity. The lack of a three-dimensional structure for the
transporter prevents a more detailed explanation of this effect.
Prior to this study, we had concerns that the aspartate
thiodipeptide may prove less useful generally as a PepT1 carrier,
based on poor trans-stimulation efflux results, compared to
analogous serine derivatives (Rachel Pettecrew and Richard Price,
unpublished results). With the exception of 2 and 6 it is clear
from our results that this concern was unfounded and that PepT1
can accommodate a variety of aspartate thiodipeptides of various
sizes. Indeed our results clearly show that PepT1 can accommodate
an aromatic “drug” separated from both serine and aspartate
thiodipeptides by a variety of ethylene glycol linkers. This offers a
significant range where the choice of linker between thiodipeptide
and drug could be modified to optimise diverse properties such as
affinity, rate of transport, lipophilicity, solubility and stability.
For the set of monoesters (compounds 3, 5–8 and 11) the rate of
PepT1 mediated transport drops as the spacer length is increased,
and becomes insignificant once a triethylene glycol spacer (6) is
employed. However the precise distance of the aromatic from the
backbone and its conformation appears crucial to transport, as
demonstrated by the fact that PepT1 mediated transport can be
restored for triethylene glycol spaced diether (4) and diester (10).
To overcome both of these limitations, assays in Caco-2
monolayers were performed. Caco-2 cells were chosen as they
are widely regarded as a good model of absorption from the
human intestine.26 They also have the advantage of being high-
throughput, although it has recently been suggested that Caco-
2 cells may underestimate the in vivo trans-epithelial rate of
transport.27 Apical to basolateral transport of 2 mM 1–11, applied
to the apical side, was monitored by high performance liquid
chromatography (HPLC) after one hour. This allowed us to rapidly
determine if the intact compounds were crossing the membranes.
We compared the extent of transport of compounds 1–11 to that
of a known hydrolysis resistant PepT1 substrate, PheW[CS-NH]-
Ala (FSA),9,28 by normalisation (Table 1). This normalisation
allowed direct comparison between different batches of Caco-2
monolayers and gives an indication as to how well the compounds
were crossing the monolayer, relative to a compound known to be
transported in vivo.9,28 The rate of PepT1 mediated transport was
determined by comparing the difference in apical to basolateral
transport of 2 mM 1–11 with excess (20 mM) Gly-Gln. This level
of excess Gly-Gln completely saturates the PepT1 transporter,
so the reduction in overall transport (after one hour) of the test
compounds corresponds to the PepT1 mediated component of
transport. This was again compared to the PepT1 mediated rate
of FSA transport by normalisation.
PepT1 is regarded as a low affinity, high capacity transporter
and compounds with an affinity < 1 mM are generally classed
as high affinity substrates.1 Therefore all of our compounds have
high affinity for PepT1, validating the use of thiodipeptides to
target drugs generally towards absorption by PepT1. Whilst it is
known that peptides ranging in size from Gly-Gly to Trp-Trp-
Trp interact with the transporter, it has recently been shown that
not all di- and tripeptides are substrates, indicating that there
are limits to the capacity of the PepT1 transporter.12 Our results
show the unprecedented capacity of the PepT1 as a transporter
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from Keele University. The Wellcome Trust is thanked for generous
financial support.
Notes and references
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To probe this effect further and demonstrate the capacity of
the PepT1 transporter to accommodate real drugs, we synthesised
and tested for affinity and transport in oocytes a set of ibuprofen
thiodipeptide prodrugs, 48–53. The synthetic route employed was
similar to that discussed for 5–11 and is given in the ESI.†
Ibuprofen is an orally active NSAID, however there is considerable
interest in the development of drugs of this class with reduced
gastrointestinal toxicity.29 A PepT1 targeted prodrug may allow
retention of the high oral bioavailability of these drugs, whilst
reducing interaction of the drug with cyclooxygenase enzymes in
the GI tract, especially the stomach, thus reducing toxicity.
With the exception of 48, the compounds are all diester
prodrugs similar to model compounds 9 and 10. These compounds
all induced trans-stimulated efflux in our oocyte assay, and to
a greater extent than the positive control Gly-Gln (see ESI),
suggesting that 48–53 are transported by PepT1 as well as, if
not better than, Gly-Gln. Whilst this assay can overestimate
the importance of PepT1 transport due to the simplicity of the
oocyte system (as discussed for 1), this assay confirms that these
compounds are accepted by PepT1 as substrates, even when a
pentaethylene glycol spacer (53) is employed, giving even more
scope for optimisation of parameters important to PepT1 targeted
delivery specifically and oral drug delivery in general. We have
also carried out some additional experiments with the ibuprofen
prodrug 48, demonstrating significant PepT1 mediated transport
in Caco-2 monolayers (PepT1 Papp = 2.10 0.27 ¥ 10-6 cm s-1;
1.07 0.29 times FSA), and preliminary in vivo testing in rats
indicating rapid transport of the conjugate 48 into the blood
plasma (unpublished data).
We report here a systematic investigation of the potential of
targeting the intestinal PepT1 transporter to improve oral drug
delivery. Whilst the majority of our data were collected using
compounds with benzyl alcohol or benzoic acid in place of actual
drugs, we have also demonstrated the PepT1 mediated transport of
actual prodrugs, giving the data an immediate application to real
medicinal chemistry problems. As expected, the broad substrate
capacity of the transporter resulted in it being able to transport the
majority of compounds tested. Despite the wealth of information
in the literature allowing prediction of a compound’s affinity for
PepT1, little is known about its transport capacity. Although we
could not determine any specific rules governing PepT1 transport,
as has been described for affinity,7 we believe the general trends
and observations reported here will be of benefit in the design
of rational drugs or prodrugs targeting this transporter. To this
end we have specifically demonstrated in vitro the ability of PepT1
to transport thiodipeptide prodrugs of ibuprofen, consistent with
our previous work on nabumetone.8
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The authors wish to acknowledge the University of Oxford,
where some of the biological testing described was carried out
by DM and MP, and helpful discussions with Dr Theresa Phillips
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