Communications
the finding that there was no observed difference in the
permeation rate of the peptide when measured from apical to
basolateral side and vice versa (see Supporting Information).
To eliminate the possibility of model-dependent results,
[
29]
additional in vitro methods including MDCK cells
side-by-side diffusion chamber
and
[
30]
were used to verify the
transport characteristics of 8. The permeability coefficient
found in these models was in the range of paracellular
transport (data not shown).
Analogues 1 and 8 were evaluated for their oral bioavail-
ability in vivo following i.v. and p.o. administration to rats
(
Figure 5). While the Veber–Hirschmann peptide was not
orally available, the absolute oral bioavailability of the N-
methylated peptide 8 was about 10% of the administered
dose. In addition, changes were also found in additional
pharmacokinetic parameters. The enhanced volume of dis-
Figure 6. Stereoview of cyclo(-PFMewMeKTMeF-) (8).determined by
NMR spectroscopy and molecular dynamics calculations (see
Supporting Information); O red, N blue.
À1
tribution of 8 compared to 1 (3.7 and 0.3 Lkg , respectively)
suggests that while the distribution of 1 is limited to the blood
and the interstitial fluid, 8 can interact with biological
membranes. A difference was also found in the plasma half-
life of 1 and 8 which may have resulted from reduction of
proteolytic digestion or hepatic and/or renal clearance. The
transcellular transport includes an interaction of the molecule
with the hydrophobic membrane followed by crossing the
membranes (i.e., the apical and basolateral membranes) to
reach the blood circulation. Indeed, an increase in the
interaction of the N-methylated peptides with a model of
the cell membrane was observed for the N-methylated
peptides (Figure 4). Yet, this liposomal model is limited to
evaluate the interaction with the membrane, this interaction is
a mandatory but not exclusive condition to cross the
membrane. The enhanced interaction of 8, observed in the
membrane vesicle liposome model may clarify the discrep-
ancy between the in vitro permeability models which show
limited absorption and the enhanced volume of distribution,
compared to 1. The fact that peptides with identical numbers
of N-methyl groups hold different degree of interaction with
the liposomal membrane model suggest that there are
additional factors, including conformation, that affect the
interaction.
1
1
analogue is less active than MePhe . This result gives an
indication of the importance of the spatial orientation of
1
1
Phe . Although there is a loss in the bent conformation by N-
1
1
methylation of Phe which results in the deep burying of the
phenyl ring, it retains an activity comparable to the stem
peptide. These results suggest that multiple N-methylation
can also be useful in elucidating fine details of the bioactive
[
12]
conformation.
1
1
To determine the importance of the Phe for bioactivity,
we have synthesized the epimeric analogue of 8, cyclo-
(-PFMewMeKTMef-) in which the MePhe is substituted by
the enantiomeric d-MePhe . In contrast to 8 this peptide
exhibits a trans peptide bond resulting in a bII turn instead of
a bVIturn about Phe and Pro , causing the loss in deep
burying of the phenyl ring and consequently the loss of
activity. The membrane permeability of this peptide was also
greatly reduced when compared with that of peptide 8. From
this result we conclude that Phe and its surroundings are
important, not only in maintaining the activity of the peptide,
but also in maintaining the permeability profile of a peptide.
In summary we have characterized the effect of multiple
N-methylation on the intestinal permeability and enzymatic
stability of somatostatin analogues. Improving these param-
eters is a key factor in enhancing the oral bioavailability of
peptides. We show that multiple N-methylation of a cyclic
peptide improved its oral bioavailability without modifying its
biological activity and selectivity. This finding is a step
towards the development of peptide based therapeutics.
Thus, multiple N-methylation could be a simple way to
achieve oral bioavailability of peptidic drugs.
1
1
1
1
1
1
6
1
1
It is interesting to note that in all of these seven bioactive
analogues of the sublibrary, the bII’ and the bVIturn are
conserved even in the tris N-methyl compound 8 (Figure 6),
which corroborates with the earlier results that these two
[17]
turns maintain the peptide in the bioactive conformation.
In general, we observe an enhancement in the binding
9
affinity when the molecule contains MeLys and a reduction
8
11
with Me-d-Trp or MePhe , this subtle modulation in the
activity could be understood by analyzing the conformations
of these analogues. Goodman et al. suggested the bent
conformation of the peptide as the bioactive conformation,
7
10 [31]
which is stabilized by the two g turns about Phe and Thr .
Received: December 18, 2007
Published online: February 22, 2008
This bent conformation results in the deep insertion of the
9
8
11
9
Lys , d-Trp , and Phe in the receptor; N-methylation of Lys
Keywords: bioavailability · cyclic peptides · N methylation ·
enhances the stability of the bent conformation by reducing
.
peptide drugs · somatostatin
flexibility about the bII’ turn tuning the peptide into a more
8
11
potent analogue. N-Methylation at either d-Trp or Phe
decreases the activity owing to the loss of the stabilizing
8
g turns; however, it is interesting to note that the Me-d-Trp
[1] J. F. Woodley, Crit. Rev. Ther. Drug Carrier Syst. 1994, 11, 61 – 95.
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598
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 2595 –2599
Biron, Eric
