460
Tsuda et al.
drug interaction and/or drug/food interaction during therapy model of PEPT1 (Fig. 7B), suggesting that our model can be
applied to cationic as well as neutral substrates.
with midodrine.
In conclusion, we have demonstrated that midodrine, but
not DMAE, is recognized by PEPT1 and that this recognition
improves the oral bioavailability of DMAE. In addition, the
transport of DMAE amino acid derivatives via PEPT1 de-
pends on the amino acids modified. These findings suggested
that modifying not only the L-valyl ester but also the glycyl
peptide of poorly absorbed drugs, which are targeted to in-
testinal PEPT1, is useful for improving of the intestinal
absorption of drugs.
Previously, Beauchamp et al. (1992) evaluated the bio-
availability of 18 ester compounds of acyclovir and found that
the L-valyl ester derivative had the best bioavailability fol-
lowed by the L-isoleucyl, L-alanyl, and glycyl ester deriva-
tives. Thereafter, these amino acid preferences were demon-
strated to be related to the affinity for PEPT1 (Sawada et al.,
1999). Thus, for amino acid ester modification, L-valine may
be a suitable target for converting a poorly absorbed drug
into a substrate of PEPT1. On the other hand, for amino acid
modification of a peptide bond, there is little information
available on which amino acids provide for high-affinity in-
teraction with PEPT1 and resistance against enzymatic deg-
radation. In the present study, we demonstrated that the
derivatives with -Gly, -Val, -Tyr, -Cys, and -Glu were sug-
gested to be interacted with PEPT1, but those with -Ile, -Phe,
-Pro, and -Lys did not (the lack of significant difference in the
case of -Lys may be caused by the unusually high scatter).
Large amounts of DMAE-Phe were accumulated in Caco-2
cells, but this derivative showed extremely low stability in
the rat small intestine. These findings suggest that DMAE-
Phe may not be appropriate for a prodrug for targeting
PEPT1 and improving the intestinal absorption in vivo. On
the other hand, DMAE-Gly (midodrine) was high stability
and can be recognized by PEPT1. Although we did not per-
form a detailed analysis, overall, our results suggest that Gly
modification of DMAE by a peptide bond is appropriate for
the preparation of a prodrug for DMAE. Further studies are
needed to define the amino acids suitable for targeting
PEPT1 in peptide bond-based modifications.
It has been reported that pH dependences of neutral (Inui
et al., 1992; Saito and Inui, 1993; Terada et al., 1999;
Kennedy et al., 2002) and anionic (Matsumoto et al., 1995)
substrates of PEPT1 show different profiles. The pH depen-
dence of cationic substrates determined by measuring evoked
current is also different from neutral and anionic substrates
(Mackenzie et al., 1996). Midodrine almost exists as a cation
at pH 5.0 to 7.4, because its dissociation constant (pKa) is
7.96. As shown in Fig. 7A, midodrine uptake by PEPT1
gradually increased as the pH rose from 5.0 to 7.4. This pH
profile clearly differs from that of a neutral substrate, such as
glycylsarcosine, or an anionic substrate, such as ceftibuten.
In contrast, the pH profile of DMAE-Glu, which mainly exists
as a neutral substrate from pH 5.0 to 7.4, was similar to that
of glycylsarcosine. Recently, based on the presumed recogni-
tion patterns of PEPT1 for neutral and charged substrates,
we constructed a 14-state model of PEPT1 and reproduced
the pH profiles of various charged substrates (Irie et al.,
2005). In this model, we hypothesized two mechanisms for
the transport of cationic substrates, namely, that the trans-
port of cationic substrate occurs with or without Hϩ. There-
fore, the transport of cationic substrates is assumed to be
altered by the degree of contribution of the two pathways. In
the previous study, we could not simulate the pH profile of
cationic substrates, because there is little known regarding
the transport characteristics of cationic substrates of PEPT1.
The present simulation revealed that the pH profile of mido-
drine as well as DMAE-Glu was reproduced by our 14-state
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Address correspondence to: Professor Ken-ichi Inui, Department of Phar-
macy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: