N-Methyl Phenylalanine-Rich peptides as highly versatile blood-brain barrier shuttles

Article abstract of DOI:10.1021/jm901654x

Here we studied the capacity of N-MePhe-(N-MePhe)₃-CONH ₂, Cha-(N-MePhe)₃-CONH₂, and 2Nal-(N-MePhe)₃-CONH₂ to carry various drugs (cargos) in in vitro blood-brain barrier (BBB) models in or

Full text of DOI:10.1021/jm901654x

2354 J. Med. Chem. 2010, 53, 2354–2363
DOI: 10.1021/jm901654x
N-Methyl Phenylalanine-Rich Peptides as Highly Versatile Blood-Brain Barrier Shuttles
†
Morteza Malakoutikhah, Roger Prades, Meritxell Teixido, and Ernest Giralt*
†
,†,‡
ꢀ †
†Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, Baldiri Reixac 10, E-08028 Barcelona, Spain, and
‡
´
ꢁ
Department of Organic Chemistry, University of Barcelona, Martı i Franques 1-11, Barcelona, Spain
Received July 1, 2009
Here we studied the capacity of N-MePhe-(N-MePhe)3-CONH2, Cha-(N-MePhe)3-CONH2, and
2Nal-(N-MePhe)3-CONH2 to carry various drugs (cargos) in in vitro blood-brain barrier (BBB)
models in order to determine the versatility of these peptides as BBB-shuttles for drug delivery to the
brain. Using SPPS, the peptides were coupled to GABA, Nip, and ALA to examine their passive BBB
permeation by means of PAMPA and their lipophilicity by IAMC. Unaided, these nonpermeating drugs
alone did not cross the PAMPA barrier and the BBB passively; however, the peptides tested as potential
BBB shuttles transferred them by passive transfer through the PAMPA phospholipid. The permeability
of peptides that showed the highest permeability in PAMPA, and Ac-N-MePhe-(N-MePhe)3-CONH2 as
the parent peptide was also examined in bovine brain microvessel endothelial cells (BBMECs). These
peptide-based BBB shuttles open up the possibility to overcome the formidable obstacle of the BBB,
thereby achieving drug delivery to the brain.
Introduction
There are two methods, among others, to convert hydro-
philic drugs, which do not enter the brain, into drugs with
capacity to cross the BBB. The first involves the reformulation
of drug structures so that the resulting molecules become
substrates for the endogenous BBB transporters (i.e., L-dopa);6
however, knowledge of the BBB endogenous transport sys-
tems is required and, in addition, this alteration should not
influence on the biological effect of the drugs when inside the
brain. The second method comprises a peptide vector-
mediated strategy in which nontransportable drugs are linked
to peptides that have the capacity to cross the BBB. The latter
was used by Rousselle et al. and Temsamani et al. to improve
the brain uptake of several drugs, such as doxorubicin,7
penicillin,8 enkephaline analogue dalargin,9 paclitaxel,10 and
morphine-6-glucuronide.11 In some cases, this conjugation
even increased drug solubility and bypassed the P-glycopro-
tein,7,10 therefore discarding the need for solubilizers, which
could cause side effects, and for P-gp inhibitors, which limit the
clinical application of drugs. Schwarze et al.12 demonstrated
that an 11-amino acid peptide from TAT protein delivered
β-galactosidase (β-Gal) protein to several tissues, including the
brain.
Unlike many peripheral blood vessels, the endothelial cells
of brain capillaries attach to each other to form tight junctions
without fenestration, which prevents paracellular transport.
Therefore, to gain access to the brain, compounds must cross
this barrier, known as the blood-brain barrier (BBBa), via
transcellular routes, including energy-dependent transport
and passive diffusion.
Althoughnovelneuropharmaceuticaldrugsare availableto
treat some brain disorders, the presence of the BBB impedes
their reaching their site of action in the brain and consequently
they are not effective.1 In addition, the BBB can make the
treatment of noncentral nervous system (CNS) diseases diffi-
cult. For example, the human immunodeficiency virus-1
(HIV-1) enters the brain through adsorptive-mediated endo-
cytosis;2,3 however, drugs for the treatment of the acquired
immunodeficiency syndrome (AIDS) do not cross the BBB,4,5
therefore the brain can act as an HIV-1 reservoir and, despite
of drug availability, treatment is complicated. Thus new
approaches to deliver drugs to the brain should not be limited
only to the treatment of the CNS diseases.
*To whom correspondence should be addressed. Phone: (34) 93
4037125. Fax: (34) 93 4037126. E-mail: ernest.giralt@irbbarcelona.org.
a Abbreviations: ACH, R-cyano-4-hydroxy-cinnamic acid; BBB,
blood-brain barrier; CNS, central nervous system; Da, dalton; DBU,
1,8-diazabicyclo[5.4.0]-undec-7-en; DCM, dichloromethane; DIEA, dii-
sopropylethylamine; DMF, N,N-dimethylformamide; Fmoc, 9-fluore-
nylmethoxycarbonyl; HOAt, 7-aza-1-hydroxybenzotriazole; HPLC,
high performance liquid chromatography; HPLC-MS, high perfor-
mance liquid chromatography mass spectrometry; HRMS, high resolu-
tiom mass spectrometry; IAMC, immobilized artificial membrane
chromatography; MALDI-TOF, matrix-assisted laser desorption ioni-
zation-time-of-flight; MeCN, acetonitrile; MTBD, 7-methyl-1,5,7-
triazabicyclo[4.4.0]dec-5-ene; o-NBS, o-nitrobenzenesulfonyl chloride;
PAMPA, parallel artificial membrane permeability assay; PyBOP,
We have recently reported that small cyclic N-methylated
and N-MePhe-rich peptides could act as BBB shuttles and
transport L-dopa and baicalin through the PAMPA mem-
brane, an in vitro model of the BBB.13,14 PAMPA is a noncell-
based assay with an artificial membrane that was introduced
by Kansy15 for the prediction of gastrointestinal tract absorp-
tion. During the past decade, PAMPA, which presents only
nonsaturable transmembrane diffusion, has been modified to
be used as an in vitro model to study BBB and skin permea-
tion16-18 to predict the cellular activity of several hepatitis C
virus protease inhibitors19 and the passive elimination rate
constant in fish.20 It has also been applied to studies to
determine the mechanism of drugs permeation,21 the partition
benzotriazol-1-yl-oxytripyrrolidinophosphonium
sphate; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid;
hexafluoropho-
tR, retention time.
r
pubs.acs.org/jmc
Published on Web 02/19/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2355
coefficient between water and different solvents,22 and the
permeation of compounds that can not be determined by cell-
based assays because of compound instability.23 PAMPA is a
rapid, uncomplicated, and low-priced method compared to
cell-based assays. Di et al.24 illustrated that PAMPA corre-
lated with in situ brain diffusion remarkably better than
MDR1-MDCKII for a broad range of CNS drugs with
diverse physicochemical properties available on the market.
The transport of compounds across in vitro membranes is
limited by a barrier formed by the membrane and the two
unstirred water layers (UWL), one on each side of the
membrane. UWL, which is <1 μm in the BBB, is the rate
limiting factor for the transport of highly lipophilic molecules.
If its effect is not corrected with a suitable protocol, the
resulting permeability shows only properties of water instead
ofmembrane. Shakingthe solutioncanreducethe thickness of
UWL.25-28 Thus, to better mimic the BBB in vivo, we
performed all PAMPAs by placing the PAMPA plate on an
orbital shaker at 100 rpm. Given the properties of PAMPA
described above and that our peptides concern only passive
transport, PAMPA was used as a high-throughput in vitro
assay to assess peptide permeability. In all the experiments,
propranolol and carbamazepine, two CNS drugs, were used
as positive controls.
Here, we studied the efficiency of three N-MePhe-rich
peptides, N-MePhe-(N-MePhe)3-CONH2, Cha-(N-MePhe)3-
CONH2, and 2Nal-(N-MePhe)3-CONH2 (Figure 1A), to
carry a series of structurally unrelated drugs in PAMPA,
IAMC, and BBMECs with the aim to test the capacity of
thesepeptides asversatile BBB shuttles for drug delivery tothe
brain. For this purpose, 4-aminobutanoic acid (GABA),
nipecotic acid (Nip), and 5-aminolevulinic acid (ALA)
(Figure 1B) were coupled to the peptides and evaluated by
PAMPA, IAMC, and BBMECs. These peptides carried a
variety of small drugs (molecular weight <200 Da), which
cover a wide range of structures such as linear, cyclic, and
aromatic14 compounds, across the PAMPA barrier and can
therefore be considered versatile BBB shuttles for small
neurodrugs. Although differences in the permeability of each
cargo-peptide construct were detected, the peptide N-
MePhe-(N-MePhe)3-CONH2 showed the best shuttle perfor-
mance for all cargos while the peptide 2Nal-(N-MePhe)3-
CONH2 was the least effective. These findings indicate that
these peptides can be envisaged as promising BBB shuttle
candidates for drug delivery to the brain.
Figure 1. (A) Structures of three cargo-N-MePhe-rich peptides
(BBB-shuttles). (B) Structures of selected cargos GABA, ALA,
and Nip.
PAMPA phospholipid barrier (Table 1); however, the
amount of compounds that remained in the PAMPA mem-
brane (membrane retention, %R) differed between cargos.
This difference can be directly related to the different number
of H-bonds that these compounds form with water. Nip can
make four H-bonds,31 and therefore it requires less desolva-
tion energy to move from water into a phospholipid envi-
ronment. Another explanation is that Nip can be considered
a GABA or ALA analogue with less flexibility and therefore
it shows stronger attachment or greater retention. This
hypothesis is in line with an earlier study by Mahar et al.32
reporting that CNS drugs have reduced flexibility compared
to non-CNS drugs. While ALA makes one H-bond more
than GABA (6 vs 5), its retention was unexpectedly greater in
the PAMPA membrane. This observation could be attrib-
uted to the fact that the strength of H-bonds are not equal
and they do not require the same energy to be disolvated and
moved from water into membrane, as reported previously by
Barbara et al.33 An alternative explanation would be because
the additional carbonyl group in ALA structure confers this
molecule a greater tendency to attach to the membrane, as
reported previously by Fischer et al.34 These authors demon-
strated that a difference of only one C atom between two
structurally similar molecules, berberine and chelerythrine,
causes a very large difference in their permeability in PAM-
PA. Using PAMPA and MS methodology, here we show for
the first time that GABA, Nip, and ALA do not cross
artificial membranes by passive diffusion.
Results and Discussion
High-Throughput Liquid Chromatography/Mass Spectro-
metry (LC/MS) Method for PAMPA Permeability Measure-
ment. First, we determined the PAMPA permeability of three
selected cargos (GABA, Nip, and ALA). However, these
cargos lack UV-absorbing chromophore to be quantified by
UV without derivatization, thus HPLC-UV cannot be used
to measure them in PAMPA donor or acceptor wells. The use
of MS to overcome this problem has been proposed in the
literature.29,30 In our case, first we used the areas underneath
ion current profiles from HPLC-MS measurements to gen-
erate two calibration curves for each of the selected cargos,
one as acceptor curve ranging from 0.2 to 10 μM and the
other as donor curve ranging from 20 to 200 μM consisting of
seven and six points, respectively. All the curves were linear
and showed excellent coefficient correlation (Figure 2). The
cargos were then evaluated by PAMPA and analyzed
by HPLC-MS. None of the cargos permeated across the
Application of N-MePhe-Rich Peptides as BBB-Shuttles.
In a preliminary study,14 we previously showed that the
N-MePhe-rich peptides N-MePhe-(N-MePhe)3-CONH2,
Cha-(N-MePhe)3-CONH2, and 2Nal-(N-MePhe)3-CONH2
shuttled L-dopa across the artificial membrane. To explore
the capacity of the peptides to carry another drug, GABA
was chosen as a structurally diverse drug from L-dopa.

2356 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Malakoutikhah et al.
Figure 2. Calibration curves for (a) acceptor samples of GABA, (b) donor samples of GABA, (c) acceptor samples of Nip, (d) donor samples of
Nip, (e) acceptor samples of ALA, and (f) donor samples of ALA.
4-Aminobutanoic Acid (GABA). GABA, a noncoded ami-
no acid, is the major inhibitory neurotransmitter in the CNS.
Lower amounts of GABA have been observed in several
CNS disorders such as epilepsy, Huntington’s disease, and
Parkinsonism. Increased levels of GABA in the CNS have
been proposed for the treatment of these diseases. However,
GABA crosses the BBB in an energy- and temperature-
dependent manner very poorly and is pumped out from the
brain at a greater rate (16 times more) than its BBB influx.
Therefore high doses of GABA are required to increase its
uptake; however, such doses are associated with severe side
effects.35-38 To overcome this problem, several GABA
prodrugs, such as GABA-esters,39,40 GABA-amide,41 and
GABA-lactame,35 have been applied and show improved
brain uptake.
To improve GABA permeability by passive diffusion, it was
coupled to our three peptides. Peptides were evaluated by
PAMPA and IAMC (Table 1). The peptides showed good
permeability when carrying GABA through the PAMPA
membrane and were classified as compounds with medium
permeability,34 whereas PAMPA was not permeable for
GABA alone. Anderson et al.42 reported that GABA benzyl
ester with dihydropyridine caused anxiolytic activity in rats,
and its oxidized form, pyridinium salt, remained in the brain for
12 h, while GABA alone was ineffective in this case. Carelli
et al.36 also showed that dimer of GABA benzyl ester with
dihydropyridine increased the hypnosis time, a typical GABA-
mimetic effect, in rats compared with GABA alone. They
concluded that the biological activities above are because of
the pyridinium salt. Furthermore, Galzigna et al.41 demonstrated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2357
Table 1. Percentage of Transport, Effective Permeability (Pe), Membrane Retention (%R) after 4 h in the PAMPA and kIAM of GABA, Nip, ALA,
L-Dopa, X-BBB-shuttles (X = GABA, Nip, ALA, L-Dopa), and Control Compounds (Propranolol, Carbamazepine)a
Pe
( ꢀ 106) cm/sd
7.7
transport
(%) (4 h) d
membrane
retention (%R) d
d
compd
kIAM
propranolol
carbamazepine
GABA
13.9
19.3
17.2
<1
1.9
2.1
11.5
0
0
5.4 ( 1.4
<1
GABA-N-MePhe-(N-MePhe)3-CONH2
GABA-Cha-(N-MePhe)3-CONH2
GABA-2Nal-(N-MePhe)3-CONH2
Nip
0.4 ( 0.06
0.4 ( 0.1
0.2 ( 0.02
0
0.8 ( 0.1
0.8 ( 0.3
0.4 ( 0.04
0
12.5 ( 2.2
28.2 ( 6.7
30.2 ( 4.4
16.1 ( 8.3
18.4 ( 1.4
52.6 ( 1.4
4.6 ( 0.4
19.4 ( 0.7
23.6 ( 1
23 ( 0.4
14.5 ( 1.5
62 ( 4.9
4.3 ( 0.4
14.1 ( 2
52.1 ( 1.2
39.3 ( 9
Nip-N-MePhe-(N-MePhe)3-CONH2
Nip-Cha-(N-MePhe)3-CONH2
Nip-2Nal-(N-MePhe)3-CONH2
ALA
1.4 ( 0.1
1.2 ( 0.1
1.1 ( 0.06
0
2.8 ( 0.2
2.5 ( 0.2
2.4 ( 0.1
0
8.1 ( 1.3
>128b
30.4 ( 1.5
ALA-N-MePhe-(N-MePhe)3-CONH2
ALA-Cha-(N-MePhe)3-CONH2
L-dopac
1 ( 0.04
0.3 ( 0.02
0
2.1 ( 0.2
0.7 ( 0.03
0
23.1 ( 1.6
>128b
0.3
29.6 ( 1.3
c
L-dopa-N-MePhe-(N-MePhe)3-CONH2
c
L-dopa-Cha-(N-MePhe)3-CONH2
L-dopa-2Nal-(N-MePhe)3-CONH2
1.1 ( 0.1
0.7 ( 0.06
0.3 ( 0.1
2.4 ( 0.2
1.4 ( 0.1
0.7 ( 0.2
>128b
c
102 ( 6.2
a Data are expressed as the mean ( SD. b Retention time higher than 60 min in the IAMC HPLC column. c These results are from our previous study
and are included to facilitate comparison.14 d Parameters definitions and their calculations are provided in Experimental Section.
that N-benzoyl-GABA and N-pivaloyl-GABA, in contrast
to GABA, crossed the BBB in rats and were cleaved
enzymatically to release GABA. On the basis of these
observations, we hypothesize that GABA-peptides while
inside the brain either may be used as substrates for GABA
receptors or be degraded proteolytically and release GABA,
which because of its incapacity to cross the BBB passively
would be trapped the brain and therefore show a longer
retention in this organ. Eytan et al.43 reported that elimina-
tion of a drug by P-gp can be overcome by fast transmem-
berane movement rate. Hence, by greatly improving the
permeability of GABA, our peptides may also reduce GABA
efflux from the brain.
At this point, we had established that the peptides carry L-
dopa and GABA through the PAMPA phospholipid barrier.
We then decided to go further and assess the peptides as
shuttles for two more distinctive small drugs, Nip and ALA,
so as to examine the peptides as versatile BBB-shuttles.
Nipecotic Acid (Nip). Other strategies to increase the
extracellular concentration of GABA in the brain and then
prevent GABA deficiency include inhibiting its reuptake by
neurons and glial cells and direct agonist of GABA recep-
tors. Nip is one of the most potent GABA-reuptake inhibi-
tors and also a GABA receptor agonist.37,44 However, it does
not penetrate the BBB.45 In mice, Bonina et al.46 and
Manfredini et al.47 studied the anticonvulsant effect of Nip
esters from conjugation with some compounds that enter the
brain actively, such as glucose, galactose, and tyrosine46 and
ascorbic acid.47 Nipecotic esters of glucose and galactose did
not show anticonvulsant activity, and the authors concluded
that either conjugation caused carbohydrates to lose their
affinity for their BBB transporters or carbohydrates were
rapidly eliminated by the liver after injection. Conversely,
nipecotic esters of tyrosine and ascorbic acid were trans-
ported across the BBB; however, these authors could not
corroborate whether the observed anticonvulsant activity
was because of Nip was released while inside the brain or
because of the intact esters. Furthermore, Wang et al.48
studied the brain delivery of Nip and its n-butyl ester after
nasal and intravenous administration to rats. While Nip was
not detected in the brain, its n-butyl ester delivered Nip to the
brain. Andersen at el.49 also found that some N-substituted
Nip derivatives had an anticonvulsant effect in mice that was
greater than that caused by diazepam, an anticonvulsant
drug available on the market. The authors observed that
when nitrogen of Nip is substituted by an amide, the
corresponding analogue loses its activity. On the basis of
this observation, they reasoned that presence of the nitrogen
of Nip as amine group is crucial for its activity. With these
findings in mind, we coupled Nip to the peptides through its
carboxyl group to keep the feature of Nip that is required for
its activity once inside the brain. We then assessed the
capacity of the resulting Nip-peptides to carry Nip across
the PAMPA barrier. While Nip alone did not have the
capacity to cross the PAMPA phospholipid membrane, all
the peptides performed well at shuttling Nip in PAMPA and
were categorized as high permeating compounds.34 The
Nip-peptides displayed better permeability than the
GABA-peptide constructs. This improvement may be due
to the effect of the cyclic conformation of Nip.
5-Aminolevulinic Acid (ALA). ALA, precursor of photo-
sensitizer protoporphyrin IX (PpIX), has been applied for the
diagnosis and photodynamic therapy (PDT) of cancer, including
esophageal, bladder, lung, breast, and brain cancer. In cancer
therapy, PDT is an approach in which malignant cells, which
show higher expression of PpIX than healthy cells after admin-
istration of ALA or ALA prodrugs, are destroyed following
irradiation with light.50-52 Although several transport systems
have been found for ALA in a range of tissues and cell lines,
however these transporters were not observed at the BBB.53-56
Ennis et al.55 found that ALA passed the BBB poorly by passive
diffusion, while Garcia et al.57 and Terr et al.58 observed that
ALA did not enter the brain and isolated brain capillaries.
To enhance the efficiency of PDT, ALA should penetrate
malignant cells sufficiently; however, the movement of ALA
through cell membranes is limited due to its hydro-
philic character. To enhance ALA permeation, it has been
used in several forms such as esters,59 dendrimers,60 and
peptides61-63 and has been entrapped in liposome.64 ALA
hexyl ester enters the brain 7-fold more than free ALA;
however, it is toxic in the absence of light and in tumor
cultures it decreases PpIX formation.59,64

2358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Malakoutikhah et al.
The use of peptides to deliver ALA to cells is a relatively
recent approach which shows great potential. Of several
derivatives studied, Berger et al.61,62 demonstrated that the
amino acid derivative of ALA with phenylalanine, attached
through the amino group of ALA, was the best precursor for
PpIX formation in cells. They then prepared dipeptide-ALA
derivatives that were also converted to PpIX with less
toxicity in the absence of light compared to the amino
acid-ALA derivatives. Later Bourre et al.63 found that
ALA derivative with acetylated Phe is also 5 times more
efficacious than ALA in producing PpIX in cells. To evaluate
the capacity of our peptides to enhance ALA permeation
through membranes, ALA-peptides were synthesized on
solid-phase and studied by PAMPA and IAMC. Compound
ALA-2Nal-(N-MePhe)3-CONH2 was excluded from this
study because it was not obtained with sufficient purity to
be used for the assay. Using PAMPA as an in vitro model of
the BBB, two other peptides transferred ALA across the
PAMPA membrane while ALA alone did not show perme-
ability, which is consistent with an earlier study by Garcia
et al.57 reporting that ALA did not cross the BBB in rats.
ALA-Cha-(N-MePhe)3-CONH2 exhibited greater lipophili-
city and was expected to be more permeating than ALA-N-
MePhe-(N-MePhe)3-CONH2; however, this was not the
case, probably because this compound is too lipophilic and
it caused the peptide to attach to the PAMPA membrane.
For all the selected cargos, to show that the compounds in
the acceptor wells of the PAMPA plate were intact cargo-
peptides and not fragments formed during the assay, all
samples of acceptor wells were examined by both HPLC and
MALDI-TOF MS. Our peptides have been able to shuttle
these cargoes across PAMPA barrier. It has been reported in
the literature that esterification of the selected cargoes
increased their permeability.39,40,48,59 Taking this into ac-
count, it could not be excluded that the elimination of the
cargoes negative charge may, in part, play a role in their
permeability. In addition, toxicity is a key issue in this field.
From this point of view, the lack of toxicity of our shuttles14
can be advantageous when compared to other strategies.40,59
Permeability, Membrane Retention, Lipophilicity, and Hy-
drogen Binding. IAMC, in which a phospholipid is covalently
bonded to silica particle support, models the partitioning of
compounds into biological membranes. The retention time
of a compound on an IAMC column is converted to the
capacity factor (kIAM) following eq 3 (see Experimental
Section), which is applied as a predictor of membrane
partition coefficient. kIAM was determined for eight cargo-
peptide constructs (Table 1). Most of the cargo-peptide
products showed lower capacity factors in IAMC compared
to their parent peptides, which exhibited capacity factors
higher than 128.14 This observation may be explained by an
increase in H-bond number. For all cargos, the peptide with
N-MePhe as N-terminal amino acid formed fewer H-bonds
with solvent water, and therefore it was expected to show a
greater capacity factor or lipophilicity in IAMC. However,
in contrast, this peptide demonstrated the smallest capacity
factor (12.5, 8.1, and 23.1 with GABA, Nip and ALA,
respectively, Table 1). This difference may be considered
the effect of the side chains of N-terminal amino acids:
phenyl, cyclohexyl, and naphthyl. These findings show that
Cha confers greater lipophilicity to the peptide than 2Nal
and N-MePhe, while 2Nal increases peptide lipophilicity
more than N-MePhe. In addition, smaller amounts of the
cargo-N-MePhe-(N-MePhe)3-CONH2 constructs remained
in the PAMPA membrane compared with the other cargo-
peptides (1<, 4.6, and 14.5 with GABA, Nip and ALA,
respectively, Table 1). These results are consistent with those
obtained by IAMC. For our cargo-peptide constructs, there
was a good correlation (r = 0.808) between log kIAM and log
%R, thereby indicating that lipophilicity (kIAM) is one of the
factors that governs the affinity of compounds to remain in
the PAMPA membrane. This correlation also shows that the
interaction of solute with IAMC phospholipid resembles
that of PAMPA.
The logarithms of peptide permeability (log pe) did not
correlate with the logarithms of their lipophilicity from
IAMC (log kIAM). These results are in disagreement with
those reported by Adejare et al.65 and Salminen et al.66 who
found acceptable relationships (r = 0.767 and 0.576, res-
pectively) between the blood-brain partition coefficient
(log BB) and log kIAM for a set of 21 and 26 structurally
diverse compounds, respectively. However, our results are in
line with those reported by Chikhales et al.,67 who showed
poor correlation between lipophilicity from octanol-buffer
partition coefficient and permeability of N-MePhe-content
peptides across BBMECs (r = 0.389) or permeability of in
situ rat brain perfusion (r = 0.155). Chikhales et al.67 and
Conradi et al.68 demonstrated the negative effect of H-bond
number on peptide permeation in BBMECs, in situ BBB, and
Caco-2 cells. In contrast, we found no correlation between
H-bond number and permeability of our peptides in PAM-
PA. This could be attributed to the fact that the inverse
relationship between number of H-bonds and permeability is
correct only for a structurally related series of compounds
such as a family of steroid hormones69 or peptides differ only
between number of N-methylated amide bonds.67,68 How-
ever, the number of H-bonds correlated with log kIAM (r =
0.607) and log %R (r = 0.528), thereby indicating that
number of H-bonds can predict membrane retention in
PAMPA and the capacity factor of IAMC better than
PAMPA permeability for this series of compounds. Mole-
cular weight, number of H-bonds, lipophilicity, and proper-
ties of amino acids, among others, are factors that influence
the capacity of a given peptide to cross membrane phospho-
lipids. On the basis of these results, we conclude that H-bond
number or lipophilicity alone is not sufficient to interpret the
penetration of peptides into the PAMPA membrane.
Peptide Permeability across the Endothelial Cell Mono-
layer. The BBMEC model is one of the cell-based BBB
models that has been used extensively to screen drug permea-
tion to the brain67,70-73 and study the capacity of com-
pounds to carry drugs across an in vitro BBB model.74
This model shows many BBB properties, including tight
junction formation, and P-glycoprotein (P-gp) expression.70
The permeability of Nip-N-MePhe-(N-MePhe)3-CONH2,
Nip-Cha-(N-MePhe)3-CONH2, Nip-2Nal-(N-MePhe)3-
CONH2, and ALA-N-MePhe-(N-MePhe)3-CONH2, pep-
tides that showed the highest permeability in PAMPA, and
Ac-N-MePhe-(N-MePhe)3-CONH2 as the parent peptide,14
was examined in BBMECs (Table 2) and results were com-
pared with those from PAMPA to assess the correlation of
these two assays. Propranolol, carbamazepine, and caffeine
were also tested as compounds that cross the in vivo BBB
by passive diffusion. All the peptides and controls
showed higher permeability values in BBMECs than in
PAMPA. This finding could be attributed to PAMPA being
stricter than BBMECs as a result of a greater membrane
thickness in PAMPA than the in vivo BBB (125000 nm vs

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2359
Table 2. Percentage of Transport, Apparent Permeability (Papp), Membrane Retention (%R) after 2 h in BBMEC model of X-N-MePhe-(N-MePhe)3-
CONH2 (X = Ac and ALA), and Nip-Y-(N-MePhe)3-CONH2 (Y = N-MePhe, Cha 2Nal) and Control Compounds (Propranolol, Carbamazepine,
and Caffeine)a
Papp
( ꢀ 106) cm/s
transport
(%) (2 h)
membrane
retention (%R)
compd
propranolol
carbamazepine
caffeine
16.9 ( 2.3
16.5 ( 1.8
16.1 ( 2
8.3 ( 0.7
6.6 ( 0.3
5.2 ( 0.6
3.5 ( 0.04
15 ( 0.04
21.2 ( 2.7
20.0 ( 2.3
20.6 ( 2.2
9.8 ( 0.8
7.9 ( 0.4
6.2 ( 0.7
63.0 ( 3.1
67.5 ( 2.5
67.4 ( 2.2
80.2 ( 1.6
74.0 ( 0.2
71 ( 2.8
Ac-N-MePhe-(N-MePhe)3-CONH2
Nip-N-MePhe-(N-MePhe)3-CONH2
Nip-Cha-(N-MePhe)3-CONH2
Nip-2Nal-(N-MePhe)3-CONH2
ALA-N-MePhe-(N-MePhe)3-CONH2
4.1 ( 0.04
17.8 ( 0.05
83.8 ( 1.2
50.7 ( 0.8
a Data are expressed as the mean ( SD.
200-500 nm).24 ALA-N-MePhe-(N-MePhe)3-CONH2 pre-
sented the greatest permeation and was comparable to that
of positive controls. The permeability values for the three
peptides with Nip in BBMECs showed excellent correlation
(r = 0.970) with PAMPA values. Given that the BBMEC
model is predictive of BBB permeability in vivo71 and
PAMPA showed excellent correlation with BBMECs, PAM-
PA, which is simpler and cheaper than the latter, can be con-
sider an in vitro BBB model for predicting BBB permeation
by passive diffusion.
Peptide Families Containing Proline. We synthesized a
five-member peptide library with proline as the amino acid
on solid-phase [Ac-(Pro)n-CONH2 (n = 2, 4, 6, 8, 10)] to
study the permeability and lipophilicity of these compounds
in PAMPA and IAMC, respectively. We also compared
them with those from N-MePhe-rich peptides in order to
establish whether the in vitro BBB transport properties of
our versatile BBB shuttles are due to the presence of second-
ary amides. While peptides using N-MePhe as amino acid
showed great permeability in PAMPA,14 the replacement of
N-MePhe by proline dramatically decreased peptide permea-
tion and lipophilicity (Table 3). This result is attributed to
proline being a less hydrophobic amino acid compared to
N-MePhe and as such caused the peptides to be excessively
hydrophilic to interact with the phospholipids of PAMPA
and IAMC. Thus the peptides with proline did not show
permeability and membrane retention (%R) in PAMPA and
exhibited smaller retention times in IAMC than that from
citric acid (reference compound that is not retained in the
IAMC column). We postulate that the great difference
between the permeability and lipophilicity of proline family
and the N-MePhe family is the result of the phenyl group of
N-MePhe.
Table 3. Percentage of Transport, Effective Permeability (Pe) Mem-
brane Retention (%R) after 4 h in the PAMPA and kIAM of Ac-(Pro)n-
CONH2 (n = 2, 4, 6, 8, 10) and Control Compounds (Propranolol,
Carbamazepine)
membrane
retention
(%R)
Pe
( ꢀ 106) cm/s
transport
(%) (4 h)
compd
kIAM
propranolol
carbamazepine
7.9
8.1
0
14.1
14.4
22
<1
0
1.9
2.1
a
Ac-(Pro)2-CONH2
Ac-(Pro)4-CONH2
Ac-(Pro)6-CONH2
Ac-(Pro)8-CONH2
Ac-(Pro)10-CONH2
0
0
0
0
0
0
0
a
0
0
a
0
0
a
0
0
a
a Retention time in the IAMC HPLC column smaller than that of
reference compound (citric acid).
results, these three peptides could provide the means to deliver
drugs, which cannot enter the brain unaided, into the CNS by
passive-diffusion transport. The use of the BBB shuttles could
pave the way for improving brain drug delivery of small
neurodrugs that lack the capacity to reach their site of action
in the CNS.
Experimental Section
Materials and Methods. Protected amino acids and resins
were supplied by Luxembourg Industries (Tel-Aviv, Israel),
Neosystem (Strasbourg, France), Calbiochem-Novabiochem
€
AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf,
Switzerland), or Iris Biotech (Marktredwitz, Germany). PyBOP
was supplied by Calbiochem-Novabiochem AG. DIEA, ninhy-
drin, and ss-mercaptoethanol were obtained from Fluka Che-
mika (Buchs, Switzerland). HOAt was purchased from GL
Biochem Shanghai Ltd. (Shanghai, China). Solvents for peptide
synthesis and RP-HPLC were obtained from Scharlau or SDS
(Barcelona, Spain). Trifluoroacetic acid was purchased from
KaliChemie (Bad Wimpfen, Germany). Collagen type IV, Fi-
bronectine, 8-(4-chlorophenylthio(CPT))-cAMP, NaHCO3,
MEM nonessential amino acids, and HEPES were purchased
from Sigma Aldrich (USA), RO-20-1724 from Calbiochem
(USA), DMEM from Biological Industries (Israel), and fetal
bovine serum from PAA Laboratories GmbH (Austria). Other
chemicals used were purchased from Aldrich (Milwaukee, WI)
and were of the highest purity commercially available. PAMPA
plates and PAMPA system solution were from pION (Woburn,
MA). Porcine polar brain lipid extract (PBLEP) was purchased
from Avantis Polar Lipids (Alabaster, AL). IAMC column (10
mm ꢀ 4.6 mm, 12 μm, 300 A, IAM.PC.DD2 column, Regis
Technologies Inc., Morton Grove, IL). Mass spectra were
recorded on a MALDI Voyager DE RP time-of-flight (TOF)
spectrometer (PE Biosystems, Foster City, CA), using an ACH
matrix. High resolution mass spectra were recorded on a LTQ-
FT Ultra (Thermo Scientific). HPLC chromatograms were
Conclusions
Here, we evaluated the versatility of three tetrapeptides to
transport distinct drugs through an artificial membrane and
endothelial cells by passive diffusion. For this purpose, the
peptides were attached to GABA, Nip, and ALA and their
permeability and lipophilicity were assayed in PAMPA and
IAMC. The peptides allowed transport of the cargos and
notably achieved success as BBB shuttles for these drugs,
which alone did not show permeability in PAMPA. While the
peptides showed high capacity to transfer Nip across the
PAMPA membrane, they showed less capacity for GABA.
However, forall the cargos studied, the peptide withN-MePhe
as the N-terminal amino acid showed the best permeability.
Some of the peptides also were tested in BBMECs and showed
higher permeability values than in PAMPA. Given these

2360 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Malakoutikhah et al.
recorded on a Waters model Alliance 2695 with Photodiode
array detector 996 from Waters (Waters, Milford, MA) using a
Symmetry C18 column (150 mm ꢀ 4.6 mm ꢀ 5 μm, 100 A,
Waters); solvents: H2O (0.045% TFA) and MeCN (0.036%
TFA); flow: 1 mL/min and software Millenium version 4.0.
HPLC-MS [Waters model Alliance 2796, quaternary pump,
UV/vis dual absorbance detector Waters 2487, ESI-MS model
Micromass ZQ and Masslynx version 4.0 software (Waters)] was
done using a Symmetry 300 C18 column (150 mm ꢀ 3.9 mm ꢀ
5 μm, 300 A, Waters), solvents: H2O (0.1% formic acid) and
MeCN (0.07% formic acid), flow: 1 mL/min. The products were
purified in a Waters 600 with dual absorbance detector (Waters
2487 Waters), and a Symmetry C18 column (100 mm ꢀ 30 mm ꢀ
5 μm, 100 A, Waters), solvents H2O (0.1% TFA) and MeCN
(0.05% TFA), flow: 10 mL/min. Purity was checked by reverse-
phase HPLC using a Symmetry C18 column. Products showed
purity g95.
General Protocols for Solid-Phase Synthesis. Syntheses were
performed on a 100 μmol-scale/each, in all cases L-amino acids
were used. Solid-phase peptide elongation and other solid-phase
manipulations were done manually in polypropylene syringes,
each fitted with a polyethylene porous disk. Solvents and soluble
reagents were removed by suction. Washings between synthetic
steps were done with DMF (5 ꢀ 30 s) and DCM (5 ꢀ 30 s) using
5 mL of solvent/g resin each time. During couplings, the mixture
was allowed to react with intermittent manual stirring.
Identification Tests. The Kaiser colorimetric assay75 was used
for the detection of solid-phase bound primary amines, while the
De Clercq test76 was used for secondary amines bound to solid-
phase.
were added to the resin and left for 30 min, and this step was
repeated once.
C. o-NBS Removal. To remove o-NBS, ss-mercaptoethanol
(10 equiv, 70 μL) and DBU (5 equiv, 75 μL) in DMF were
added to the resin and the mixture was left to react for 10 min
under a nitrogen atmosphere. This process was repeated once
for 40 min.
Nip Coupling. Fmoc-Nip-OH (4 equiv, 140.5 mg), PyBOP
(4 equiv, 208 mg), and HOAt (12 equiv, 163.3 mg) were
sequentially added to the resin in DMF (3 mL) followed by
DIEA (12 equiv, 204 μL). The mixture was allowed to react with
intermittent manual stirring for 1.5 h. The solvent was removed
by filtration, and the resin was washed with DMF (5 ꢀ 30 s) and
DCM (5 ꢀ 30 s). The coupling was repeated two more times in
the case of coupling on N-MePhe. The extent of coupling was
checked by the De Clercq test or the Kaiser colorimetric assay.
GABA/ALA Coupling. The procedure was the same as for
Nip, except that Fmoc-GABA-OH or Fmoc-ALA-OH was
used.
Cleavage of the Peptides. Final amide peptides were cleaved
from the resin using 2% TFA in DCM (6 ꢀ 3 min).
Product Workup and RP-HPLC Purification. After cleavage
of the peptides, the solvent was evaporated by N2. The residue
was dissolved in H2O:MeCN (1:1) and then lyophilized. The
peptides were purified by reverse-phase HPLC using a symme-
try C18 column (100 mm ꢀ 30 mm ꢀ 5 μm, 100 A, Waters), at a
10 mL/min flow rate with the following solvents: A, H2O with
0.1% TFA; B, MeCN with 0.05% TFA.
Product Characterization. The identity of the compounds
synthesized was confirmed using MALDI-TOF MS, HPLC-
MS, and HRMS. Purity was checked by reverse-phase HPLC
using a symmetry C18 column. Peptides showed purity g95 (see
Supporting Information).
Initial Conditioning of Resin. The Sieber resin77 was condi-
tioned by washing with DCM (5 ꢀ 30 s) and DMF (5 ꢀ 30 s)
followed by a 20% piperidine solution in DMF (2 ꢀ 1 min and
1 ꢀ 10 min) to remove the Fmoc group. Finally, the resin was
washed with DMF (5 ꢀ 30 s).
Parallel Artificial Membrane Permeability Assay (PAMPA).
The PAMPA15 was used to determine the capacity of com-
pounds to cross the BBB by passive diffusion. The effective
permeability of the compounds was measured at an initial
concentration of 200 μM. The buffer solution was prepared
from a concentrated one, commercialized by pION, and follow-
ing the manufacturer’s instructions. pH was adjusted to 7.4
using a NaOH 0.5 M solution. The compound of interest was
dissolved in buffer solution or water (in the case of cargos alone,
evaluated by HPLC-MS) and 1-propanol (20%, cosolvent) to
the desired concentration (200 μM). The PAMPA sandwich was
separated, and the donor well was filled with 200 μL of the
compound solution of interest. The acceptor plate was placed
into the donor plate, ensuring that the underside of the mem-
brane was in contact with buffer. Four μL of the mixture of
phospholipids (20 mg/mL) in dodecane was added to the filter of
each well, and 200 μL of buffer solution was added to the each
acceptor well. The plate was covered and incubated at room
temperature in a saturated humidity atmosphere for 4 h under
orbital agitation at 100 rpm. After the 4 h, 150 μL/well from the
donor plate and 150 μL/well from the acceptor plate were
transferred to HPLC vials and 100 μL/each sample were injected
into a HPLC reverse-phase Symmetry C18 column (150 mm ꢀ
4.6 mm ꢀ 5 μm, 100 A, Waters). Transport was also confirmed
by MALDI-TOF spectrometry in order to check that the
compound had kept its integrity. For PAMPA assays evaluated
by HPLC-MS, the same procedure was used except that buffer
solution was replaced by water and after the 4 h, 100 μL/well
from the donor plate and 100 μL/well from the acceptor
plate were transferred to HPLC-MS vials and 10 μL/each donor
and 20 μL/each acceptor were injected into a HPLC-MS appa-
ratus.
Fmoc Group Removal. The Fmoc group was removed by
treating the resin with 20% piperidine in DMF (3-4 mL/g resin,
2 ꢀ 1 min and 1 ꢀ 10 min). To remove the Fmoc group from
Fmoc-N-MePhe-OH and Fmoc-Pro-OH, an additional treat-
ment with DBU, toluene, piperidine, and DMF (5%:5%:-
20%:70%) (1 ꢀ 5 min) was performed.
Coupling Methods. Method 1, Coupling of the First Amino
Acid onto the Sieber Resin. N-Protected N-methylated phenyl-
alanine (4 equiv, 160.5 mg), PyBOP (4 equiv, 208 mg), and
HOAt (12 equiv, 163.3 mg) were added sequentially to the resin
in DMF (3 mL) followed by DIEA (12 equiv, 204 μL). The
mixture was allowed to react with intermittent manual stirring
for 1.5 h. The solvent was removed by filtration; the resin was
washed with DMF (5 ꢀ 30 s) and DCM (5 ꢀ 30 s). The extent of
coupling was checked by the Kaiser colorimetric assay.
Method 2, Coupling of Second Amino Acid and the Following
Amino Acid onto the Sieber Resin. The procedure was the same
as for the first one except that N-protected phenylalanine was
used. The coupling was repeated two more times and the extent
of coupling was checked by the De Clercq test.
Method 3, Synthesis of Peptides Containing Proline. The
procedure was the same as the method 2, except that N-
protected proline was used.
Amino acid N-alkylation. The N-methylation of phenylala-
nine was performed using the method described by Miller.78
This process can be divided into three steps: (A) protection and
activation with o-nitrobenzenesulfonyl chloride (o-NBS), (B)
deprotonation and methylation, and (C) o-NBS removal.
A. Protection and Activation with o-NBS. To perform the
protection, o-NBS (3 equiv, 67 mg) and collidine (5 equiv, 66 μL)
in DCM were added to the resin. The reaction was left with
intermittent manual stirring for 1 h, and this step was repeated
once and checked by the Kaiser test.
The phospholipid mixture used was a porcine polar brain
lipid extract. Composition: phosphatidylcholine (PC) 12.6%,
phosphatidylethanolamine (PE) 33.1%, phosphatidylserine
(PS) 18.5%, phosphatidylinositol (PI) 4.1%, phosphatidic
acid 0.8%, and 30.9% of other compounds. The effective
B. Deprotonation and Methylation. Methyl p-nitrobenzensul-
fonate (4 equiv, 86.9 mg) and MTBD (3 equiv, 43 μL) in DMF

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2361
permeability after 4 h was calculated using eq 1 and also the
percentage of transport after was calculated using eq 2.
MgCl2, 6 mM NaHCO3, 2.8 mM glucose, 5 mM HEPES).
Acceptor compartments were filled with the Ringer/HEPES
solution and donor compartments with the peptides at a con-
centration of 100 μM in Ringer/HEPES solution with a final
concentration of 1% DMSO. The plate was incubated for 2 h at
37 °C and 10% CO2. After the experiments, donor and acceptor
compartments were analyzed by HPLC. All the peptides were
tested in triplicate.
ꢀ
ꢁ
-218:3
2CAðtÞ
CDðt0Þ
Pe
¼
ꢀ log 1 -
ꢀ 10-6cm=s
ð1Þ
t
CAðtÞ
T% ¼
ꢀ 100
ð2Þ
CDðt0Þ
Permeability calculations were performed following eq 474,80,81
where t is time (h); CA(t) is the compound concentration in the
acceptor well at time t and CD(t0) is the compound concentra-
tion in the donor well at 0 h. The membrane retention (%R) was
calculated from the difference between the total starting amount
and the amounts in donor and acceptor compartments at the
end of the experiment (t = 4 h).
Papp ¼ ðdQ=dtÞð1=AÞð1=C0Þðcm=sÞ
ð4Þ
Where (dQ/dt) is the amount of compound present in the acceptor
compartment in function of time (nmol/s), A is the area of the
insert (cm2), and C0 is the initial concentration of peptide placed in
the donor compartment (nmol/mL).
Immobilized Artificial Membrane Chromatography (IAMC).
Retention times were determined using an IAMC column with
phosphatidylcholine (PC), the major phospholipid in cell mem-
branes, which was covalently immobilized (10 ꢀ 4.6 mm, 12 μm,
300 A, IAM.PC.DD2 column, Regis Technologies Inc.).
The compounds were detected by UV absorption at 220 nm.
The chromatograms were obtained using an HPLC working
isocratically with a mobile phase containing 10 mM phosphate
buffer, 2.7 mM KCl and 137 mM NaCl at pH 7.4 and 20% (v/v)
MeCN. The retention times (tR) were transformed into capacity
factors (kIAM) following eq 3,
TEER Measurement. The TEER was determined by using an
ohmmeter Millicell ERS system (MERS 000 01, Millipore).
TEER measures confirmed the formation of a functionally
intact in vitro BBB by day 8 of coculture. The TEER values
represent the tightness or integrity of the in vitro BBB. The
TEER value (mean ( SD) for all wells was 141 ( 5.7 Ω/cm2.
Acknowledgment. We thank Dr. M. Vilaseca from the
Mass Spectrometry Core Facility-IRB Barcelona for high-
resolution mass spectral analyses. We also thank Prof. G.
Egea and Javier Selva from the Departamento de Biologıa
´
ꢀ
Celular y Anatomıa Patologica, Facultad de Medicina,
´
Universidad de Barcelona, for their good job in the isolation
of rat astrocytes. This work was supported by FIPSE,
MCI-FEDER (Bio2008-00799 and FIPSE), and Generalitat
de Catalunya (XRB).
kIAM ¼ ðtR -t0Þ=t0
ð3Þ
where tR is the compound retention time (min), and t0 is the citric
acid retention time (min) indicating the column dead time.
HPLC-MS Experiment for Quantification. The HPLC-MS
analysis was performed using a Waters 2795 separation module,
operating in positive ion mode with a Waters 2487 dual λ
absorbance detector. The HPLC column used was a reverse
phase symmetry column (4.6 mm ꢀ 150 mm, 5 μm, C18). The
samples were eluted at 1 mL/min using different linear gradients
of solvents A (H2O containing 0.1% formic acid (v/v)) and B
(MeCN containing 0.07% formic acid (v/v)). The detection was
performed at 220 nm. External standards at concentration of
0.2, 0.4, 1, 2, 4, 10, 20, 40, 80, 100, 120, 160, and 200 μM were
prepared by diluting the 1000 μM stock solution with 20%
1-propanol in water. Prior to the experiments, the compounds
were analyzed by HPLC-MS using scan mode to determine the
retention time of each. For the experiments, single-ion monitor-
ing (SIM) was used instead of a scan mode.
Supporting Information Available: Characterization of all
peptides. This material is available free of charge via the Internet
at http://pubs.acs.org.
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