Structural requirements for a lipoamino acid in modulating the anticonvulsant activities of systemically active galanin analogues

Article abstract of DOI:10.1021/jm801397w

Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogues yielded systemically active anticonvulsant compounds. To study the relationship between the LAA structure and anticonvulsant activity, orthogonally p

Full text of DOI:10.1021/jm801397w

1
310
J. Med. Chem. 2009, 52, 1310–1316
Structural Requirements for a Lipoamino Acid in Modulating the Anticonvulsant Activities of
Systemically Active Galanin Analogues
†
†
†
‡
‡
,†
Liuyin Zhang, Charles R. Robertson, Brad R. Green, Timothy H. Pruess, H. Steve White, and Grzegorz Bulaj*
Department of Medicinal Chemistry and Department of Pharmacology and Toxicology, College of Pharmacy, UniVersity of Utah,
Salt Lake City, Utah, 84108
ReceiVed NoVember 5, 2008
Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogues
yielded systemically active anticonvulsant compounds. To study the relationship between the LAA structure
and anticonvulsant activity, orthogonally protected LAAs were synthesized in which the Lys side chain was
coupled to fatty acids varying in length from C
glycol, PEG . Galanin receptor affinity, serum stability, lipophilicity (log D), and activity in the 6 Hz mouse
model of epilepsy of each of the newly synthesized analogues were determined following systemic
administration. The presence of various LAAs or Lys(MPEG ) did not affect the receptor binding properties
8
to C18 or was coupled to a monodispersed polyethylene
4
4
of the modified peptides, but their anticonvulsant activities varied substantially and were generally correlated
with their lipophilicity. Our results suggest that varying the length or polarity of the LAA residue adjacent
to positively charged amino acid residues may effectively modulate the antiepileptic activity of the galanin
analogues.
Introduction
When delivered directly into the brain, many neuropeptides,
including galanin, neuropeptide Y, somatostatin, neurotensin,
or opioid peptides can modulate excitatory or inhibitory circuits
1
-4
and suppress seizures and/or pain sensation.
Despite a
a
considerable interest in generating blood-brain barrier (BBB )
permeable analogues of neuropeptides, only a few successful
4
-12
examples have been reported to date.
To improve central
nervous system (CNS) bioavailability of neuroactive peptides,
various strategies have been explored, including lipidization,
13-15
cationization, and glycosylation.
Banks and Kastin showed
that the lipophilicity of peptides improved their permeability
16
through the BBB. Despite the finding that only small amounts
of the peptides entered the brain, the authors noted a direct
correlation between the log D values and the blood-to-brain ratio.
Reversible lipidization of opioid peptides also improved cen-
Figure 1. Structures of Gal-B2 and LAAs used in this SAR study.
Note that MPEG has an identical number of atoms as the C16
palmitoyl) but is significantly less lipophilic.
4
(
1
7
trally mediated analgesic effects. It is also important to note
that oral bioavailability of the somatostatin analogue TT-232
was achieved by coupling various LAAs to either N- or
protein binding, systemic elimination, and intracellular seques-
tration, thus hampering their efficient penetration into the CNS.”
Indeed, when just the lipidization strategy was applied to
galanin, the truncated galanin analogue containing a Lys-
palmitoyl residue was inactive as an anticonvulsant, despite
displaying high affinity toward galanin receptors and a high
18
C-terminal components of the analogue. Although lipophilicity
has been acknowledged to play an important role in structure-
16,19
bioavailability relationships,
very few examples are available
where lipidization of neuropeptides has been found to improve
2
2
1
7,20,21
log D.
their activity in the brain.
As pointed out in a review by
1
3
In contrast, lipidization appeared very effective in improving
the CNS bioavailability of the truncated galanin analogues when
the Lys(palmitoyl) residue was introduced in concert with
Witt et al., “lipidization of peptides may increase plasma
*
To whom correspondence should be addressed. Address: Department
2
2
of Medicinal Chemistry, College of Pharmacy, University of Utah, 421
Wakara Way, Suite 360, Salt Lake City, Utah 84108. Phone: (801) 581-
cationization. The most active analogue, Gal-B2, contained
the C-terminal “-Lys-Lys-Lys(palmitoyl)-Lys-NH ” motif (Fig-
2
4
629. Fax: (801) 581-7087. E-mail: bulaj@pharm.utah.edu.
†
ure 1). The combined lipidization and cationization resulted in
very potent antiepileptic compounds; e.g., the anticonvulsant
ED50 of Gal-B2 was found to be 0.8 mg/kg when tested
intraperitoneally (ip) in the 6 Hz pharmacoresistant model of
epilepsy. Since galanin suppresses seizures by activating GalR1
and GalR2 located in the hippocampus and other limbic
structures, our data suggest that the combination of cationization
and lipidization is effective in improving the BBB penetration
of Gal-B2 without negatively affecting receptor affinity. Fur-
thermore, our results suggest that the sequence position of LAAs
Department of Medicinal Chemistry.
Department of Pharmacology and Toxicology.
‡
a
Abbreviations: AUC, area under the curve; BBB, blood-brain barrier;
CD, circular dichroism; CNS, central nervous system; DCC, N,N′-
dicyclohexylcarbodiimide; DIPEA, N,N-diisopropylethylamine; Fmoc, N-(9-
fluorenyl)methoxycarbonyl; GalR1, galanin receptors subtype 1; GalR2,
galanin receptors subtype 2; GPCRs, G-protein-coupled receptors; ip,
intraperitoneally; LAA, lipoamino acid; MPEG
4
, monodispersed polyeth-
ylene glycol PEG ; PyBop, (benzotriazol-1-yloxy)tripyrrolidinophosphonium
4
hexafluorophosphate; SAR, structure-activity relationship; SPPS, solid
phase peptide synthesis; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroet-
hanol.
1
0.1021/jm801397w CCC: $40.75

2009 American Chemical Society
Published on Web 02/06/2009

Lipoamino Acid SAR of Galanin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1311
Table 1. Structures of LAAs Used for SPPS of the Systemically Active
Galanin Analogues
using reagent K (trifluoroacetic acid (TFA)/water/thioanisole/
phenol/ethanedithiol, 90/5/5/7.5/2.5 v/v), followed by precipita-
tion and washing with ice cold methyl tert-butyl ether (MTBE).
Crude peptides were purified by preparative reversed-phase
HPLC using a diphenyl column with a linear gradient of water/
acetonitrile (both buffered with 0.1% TFA). The purities of
peptides were greater than 95% by analytical HPLC analyses
(
Supporting Information Figure S1 and Table S1). Purified
analogues were quantified by UV absorbance (λ ) 279.8 nm,
-1
-1
ε ) 7000 cm M ), and their monoisotopic mass was
confirmed by MALDI-TOF mass spectrometry (Table 2).
Physicochemical and Structural Properties. To determine
the lipophilicity of the analogues, log D values were calculated
using the combination of the HPLC retention times and the log D
determined from the classical shake-flask method, as previously
2
2
described. Calculations of log D values are provided in
Supporting Information Table S2. For the LAA containing
is important for the systemic activity of Gal-B2, suggesting that
the lipidization/cationization motif was not simply producing
the additive effect by combining hydrophobicity and positive
charges.
In order to define the structural requirements of the LAA
residue for improving the anticonvulsant activity of Gal-B2, we
synthesized a series of LAAs in which the Lys side chain was
analogues, the log D values ranged from 0.78 for Gal-B2-C
.35 for Gal-B2-C18. As shown in Figure 2, the log D values
linearly correlated with the carbon chain length of the LAA
8
to
1
2
(
4
linear fit yielded R ) 0.98). Since the log D values increased
-fold by replacing one Lys residue with Lys-stearoyl (compare
2
2
log D of Gal-(K)4 (0.34) with that of Gal-B2-C18 (1.35)) and
almost doubled by changing the LAA from C to C18, these
results emphasize the effectiveness of the LAAs to increase the
lipophilicity of short peptides. The analogue with the MPEG
8
either coupled to fatty acids, varying in length from C
or to a monodispersed PEG (MPEG ). These LAAs were used
to replace the Lys-palmitoyl residue in position 16 of Gal-B2
Figure 1). The binding affinity, octanol/water partitioning
8
to C18,
4
4
4
-
amino acid isostere had approximately 2-fold lower log D (0.57),
compared to the Lys-palmitoyl residue (1.24).
(
coefficient, and ability to suppress seizures in the in vivo 6 Hz
model of epilepsy was determined following ip administration.
Our findings suggest that the length and polarity of the LAAs
are important for maintaining antiepileptic activity of the galanin
analogues.
Gal-B2 is largely unstructured in water but contains 23%
R-helical content in the presence of 50% trifluoroethanol
2
2
22
(
TFE). In our previous work, we hypothesized that the
presence of LAA could stabilize the helical conformation of
the systemically active galanin analogues by hydrophobic
interactions with Tyr9 and/or Trp2. To test this hypothesis, the
conformational properties of the Gal-B2 analogues were inves-
tigated by circular dichroism (CD). In the presence of 5 mM
potassium phosphate containing 150 mM NaF (pH 7.5), the
helical content varied among the analogues from 1% to 7% (only
Results
Design and Chemical Synthesis. To study the role of Lys-
palmitoyl, residue 16, in the activity of Gal-B2, we designed a
series of analogues where we systematically changed the length
of the fatty acid or replaced the palmitoyl motif with a MPEG
Figure 1). The length of the LAAs varied from C to C18 by
two carbon atom increments (Table 1). The MPEG -containing
Gal-B2 analogue was designed to dissect the lipophilic contribu-
tion of the Lys-palmitoyl residue, since MPEG is significantly
4
8
Gal-B2-C had 15%). The helical content in 50% TFE increased
(
8
for most analogues compared to that determined in “buffer only”
conditions (Table 2). No apparent correlations were observed
between the length of the LAA and percentage of helical content
in the analogues.
4
4
more polar than C16 despite having an identical number of atoms.
To synthesize these new structure-activity relationship (SAR)
analogues of Gal-B2, we applied a direct coupling of the
Metabolic Stability. To investigate how the length of the
LAA residue might affect resistance of the galanin analogues
to proteolytic degradation, we employed the in vitro metabolic
stability assay. The half-life of the analogues was determined
in buffered 25% rat blood serum incubated at 37 °C. The amount
of each analogue remaining after an appropriate time interval
was quantified by HPLC, and the time-course of disappearance
of the analogues is shown in Figure 3A. Calculated half-lives
for the analogues are summarized in Figure 3B. All analogues
exhibited pronounced resistance to proteolytic degradation (for
comparison, the unmodified Gal(1-16) analogue had 7.8 min
ε
ε
orthrogonally protected N -lipolysine intermediates (N -palmi-
R
toyl-N -Fmoc-lysine is commercially available). For the syn-
ε
thesis of N -octanoyl 1 (C
8
), -decanoyl 2 (C10), -lauryl 3 (C12),
R
-
myristoyl 4 (C14), and -stearoyl 5 (C18) substituted N -Fmoc-
2 3
lysine, a convenient method was applied using Na CO as a
coupling reagent with a corresponding acid halide in dioxane/
water solvent system (Scheme 1). Synthesis of Fmoc-Lys-
(
MPEG
perfluorophenol activated MPEG
was synthesized from the substitution reaction of MPEG
with ethyl bromoacetate followed by ester hydrolysis (Scheme
). The modified amino acids were purified by flash chroma-
4
)-OH 6 was achieved by coupling Fmoc-Lys-OH with
-acetic acid 7. Compound 7
-OH
4
2
2
4
half-time under these conditions). Interestingly, the galanin
analogues containing the shortest or the longest aliphatic chain
were the least stable whereas the analogue containing C14 had
the longest half-life of 12 h (Figure 3B). This correlation
suggested a rather complex mechanism by which the LAAs
might increase the stability of galanin analogues in plasma. We
hypothesized that the metabolic stability of the galanin analogues
might be correlated with their serum albumin binding properties
(e.g., binding properties of long-chain carboxylic acids to
2
tography and used for solid-phase peptide synthesis (SPPS).
The SPPS was carried out using an automated peptide
synthesizer at 25 µmol scale on preloaded Fmoc-Lys(Boc)-Rink
amide AM resin. The 5-fold Fmoc-amino acids/PyBop/DIPEA
(
1:1:2) were used in peptide synthesis. A 2-fold of the Fmoc
protected LAAs or MPEG -lysine was used in manual coupling
synthesis. The synthesized peptides were cleaved from the resin
4
2
3
albumin are dependent on aliphatic chain length). However,

1
312 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Zhang et al.
a
Scheme 1. Synthesis of the Fmoc-Protected LAAs
a
Reagents and conditions: (a) Na
2 3
CO , H
2
O/dioxane, room temp, overnight, 64-87%.
a
4
Scheme 2. Synthesis of Fmoc-Lys(MPEG )-OH
a
Reagents and conditions: (a) NaH, 0 °C, 1 h; ethyl bromoacetate, 50 °C, 16 h; (b) 1 M LiOH/CH
DCC, overnight; (d) Fmoc-L-lysine, DIPEA, 0 °C, 60% (from two steps).
3
OH, 6 h, 59% (from two steps); (c) perfluorophenol,
a
Table 2. Structure and Properties of the Galanin Analogues
b
c
+
analog
structure
HPLC retention time
R-helix (%)
MS (MALDI, MH ) calcd/found
d
Gal-(K)4
(Sar)WTLNSAGYLLGPKKKK
15.13(0.42
20.13(0.17
21.49(0.05
22.51(0.03
23.76(0.18
25.84(0.10
26.73(0.18
17.52(0.03
14
9
6
16
16
23
5
1873.09/1873.14
2001.21/2001.17
2029.24/2029.45
2057.28/2057.26
2085.31/2085.33
2112.31/2112.33
2141.33/2141.48
2123.23/2123.29
Gal-B2-C
8
(Sar)WTLNSAGYLLGPKK(Lys-octanoyl)K
(Sar)WTLNSAGYLLGPKK(Lys-decanoyl)K
(Sar)WTLNSAGYLLGPKK(Lys-lauroyl)K
(Sar)WTLNSAGYLLGPKK(Lys-myristoyl)K
(Sar)WTLNSAGYLLGPKK(Lys-palmitoyl)K
(Sar)WTLNSAGYLLGPKK(Lys-stearoyl)K
Gal-B2-C10
Gal-B2-C12
Gal-B2-C14
d
Gal-B2
Gal-B2-C18
Gal-B2-MPEG
4
(Sar)WTLNSAGYLLGPKK(Lys-MPEG
4
)K
18
a
b
All analogues are amidated at the C-terminus. A linear gradient of water/90% acetonitrile (buffered with 0.1% TFA) on a Vydac diphenyl column,
c
d
starting from 20% acetonitrile to 100% acetonitrile in 40 min. Determined in the presence of 50% v/v 2,2,2-trifluoroethanol. Data from ref 22.
shown in Table 3, when compared to each other, none of the
analogues studied here exhibited significantly different affinities
toward either receptor subtype. This suggested that the relatively
large LAA moiety did not affect the affinity of the analogues
toward the galanin receptors. All analogues maintained several-
fold preference in binding to GalR1 subtype, compared to
2
2
GalR2, similar to that of unmodified Gal(1-16).
The anticonvulsant activity of the analogues was studied in
the 6 Hz (32 mA) model of pharmacoresistant epilepsy following
ip administration of a bolus dose of 4 mg/kg. The analogues
were evaluated for their ability to suppress seizures at various
times (0.25-4 h) after peptide administration. The time-
response curves were then integrated to provide a qualitative
measurement of efficacy, i.e., area under the curve (AUC) values
Figure 2. Relationships between the aliphatic chain length of the LAAs
in Gal-B2 analogues and the octanol/water partition coefficient (log D).
Linear fit yielded R ) 0.9839, P < 0.0001.
2
(Table 3, Figure 4). The analogues differed significantly in their
our efforts in determining the protein binding properties of Gal-
B2 by microdialysis failed because of possible micelle forma-
ability to protect mice from seizures. On the basis of the AUC
values, the most active galanin analogues contained Gal-B2-
22
4
tion. The Gal-B2-MPEG analogue exhibited a very long half-
C
16 and Gal-B2-C18, whereas the least active analogues included
and C10. Interestingly, the Lys-MPEG -containing analogue
life (12.4 ( 0.65 h), suggesting that the extended amino acid
residue might provide steric hindrance for access of proteinases.
In Vitro and in Vivo Pharmacology. Since two galanin
receptors, GalR1 and GalR2, are known to be involved in
C
8
4
was also active as an anticonvulsant, albeit significantly less
when compared to Gal-B2.
2
4,25
controlling seizures in the brain,
we investigated the
Discussion
interactions of the galanin analogues with both receptor subtypes
using a competitive binding assay. As described in our previous
To study the structural requirements for the LAA residue 16
in mediating the antiepileptic activity of the systemically active
i
work, K values were determined using a time-resolved fluo-
2
2
22
rescence binding assay with a europium-labeled galanin. The
GPCR membrane preparations used in the assay were com-
mercially available (Perkin-Elmer or Millipore) and were derived
from recombinant human GalR1 or GalR2 gene sequences. As
galanin analogues, we synthesized and characterized a new
series of analogues that differed in their length/polarity of the
fatty acid moiety coupled to the Lys16 residue. The major
finding of this work was that the six galanin analogues (despite

Lipoamino Acid SAR of Galanin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1313
2
2
important for the anticonvulsant activity of Gal-B2, thus
implying that lipophilicity alone (which would be expected
to increase passive diffusion) is not the sole important factor
for improving seizure suppression by this analogue. Although
2
the “-Lys-Lys-Lys(palmitoyl)-Lys-NH ” motif appeared to be
the most effective in increasing the anticonvulsant activity
of the galanin analogues following ip administration, clearly,
more SAR studies, or perhaps even a combinatorial approach,
will be needed to further improve the potency of the Gal-B2
2
2
related analogues.
Introduction of the LAA to the galanin analogues significantly
improved their in vitro metabolic stability; this effect may be,
at least in part, accounted for by steric effects of the long side
chain in position 16 of the analogues. This finding is not
surprising in light of previous reports with lipopeptides, in which
2
9
prolonged in vitro and/or in vivo half-lives were observed,
3
0
including gonadotropin-releasing hormone analogues, gluca-
3
1
32
20
gon-like peptide 1 analogues, insulin, and octreotide. The
metabolic stability of lipopeptides may also be affected by their
3
2
interactions with fatty acid binding sites in serum albumin.
As shown in Figure 3B, the longest serum half-lives were
observed for Gal-B2-C14 and Gal-B2-C16 whereas the shortest
were observed for Gal-B2-C
8 8
. Interestingly, the C (octanoic
acid) moiety binds with 11-fold higher affinity to human serum
2
3
albumin compared to the C14 (myristic acid).
Another aspect of this work is our less conventional approach
of using orthogonally protected LAAs and MPEG -amino acids
4
as building blocks for SPPS. Introduction of LAA into peptides
have been reported using three distinct strategies: (1) on-resin
R
33,34
ε
35
fatty acid acylation of N -amino acids
or N -lysine, (2)
3
6
esterification of fatty acids, and (3) conjugation at the
1
8,37
C-terminus of peptide.
Our strategy of using the Fmoc
protected LAAs directly coupled during the solid-phase peptide
synthesis offers apparent advantages in applications such as
routine synthesis of lipopeptide analogues for SAR studies or
synthesis of peptide-based combinatorial libraries directed
toward optimization of the chain length of the LAAs in the
cationic/lipidic motif.
Figure 3. (A) In vitro serum stability assay for the galanin analogues.
Representative plots showing a time-course of degradation for C
8
- (b),
C
14- (9), and MPEG ([)-containing galanin analogues in the presence
4
of 25% rat serum at 37 °C. Degradation of analogues was monitored
by analytical HPLC. Data points were obtained at 0, 0.5, 1, 2, 4, and
To what extent the systemically active galanin analogues may
have improved the BBB permeability remains unanswered.
However, since some of the modified galanin analogues possess
pronounced antiepileptic activity believed to be mediated by
the galanin receptors located in the limbic system and, thus,
behind the blood-brain barrier, these data suggest that sufficient
quantities of the analogues must penetrate into the brain. As
8
h from the average of at least three independent experiments. (B)
Relationships between the length of the LAAs and the in vitro metabolic
stability of the galanin analogues. The half-lives were determined by
incubating the peptides in 25% diluted rat serum at 37 °C. The
concentrations of the remaining analogues were determined by HPLC.
Data were obtained from at least three independent experiments.
8
ANOVA single factor analysis yielded a P-value of 0.004 for C -, C14-,
and C16-containing species (*).
22
we have suggested previously, lipidization may improve CNS
bioavailability of Gal-B2 via a combination of adsorptive-
having comparable affinities toward the galanin receptors and
differing in the length of the fatty acid from C to C18) exhibited
3
8,39
mediated endocytosis and passive diffusion.
More mecha-
8
nistic studies assessing the mechanism of transport of galanin
analogues across the cell-based models of BBB are underway.
Regardless of the mechanism by which these analogues
penetrate into the brain, our data suggest that changing the length
and/or polarity of the LAA residue in the context of cationization
may offer a useful strategy to modulate CNS bioavailability of
galanin analogues. In summary, we believe that the variations
in lipidization and cationization appear to provide a useful
strategy for modulating CNS bioavailability of not only the
galanin analogues described here but perhaps even other
neuropeptides.
pronounced differences in their anticonvulsant activities fol-
lowing ip administration. The analogues containing shorter fatty
acids were less active compared to those with longer chains.
These results suggest that the increased lipophilicity provided
by the longer fatty acids is an important factor in improving
the anticonvulsant activity of galanin analogues following
systemic administration. Further work will be required to
determine whether the resulting compounds are agonists, partial
agonists, or antagonists. Interestingly, both Gal-B2 and Gal-
8
B2-C showed comparable analgesic activities in a mouse pain
assay following ip administration (E. Adkins-Scholl, H. S.
White, G. Bulaj, unpublished results), suggesting that systemi-
cally active galanin analogues varying in the length of a LAA
may control seizures or pain via galanin receptors in the CNS
Experimental Section
General Synthetic Procedures. Reagent chemicals were ob-
tained from Aldrich Chemical Corporation and were used without
2
6-28
or peripheral nerves, respectively.
Our previous data
indicated that the position of the LAA residue appeared to be
2
prior purification. Reactions were performed under N atmosphere,

1
314 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Zhang et al.
Table 3. In Vitro and in Vivo Pharmacological Properties of the Galanin Analogues
in vitro assay (receptor binding), K (nM) in vivo assay (anticonvulsant activity) (6 Hz, 32 mA, 4 mg/kg)
i
analogue
GalR1
GalR2
15 min
30 min
1 h
2 h
4 h
a
Gal-(K)4
0.4(0.1
0.7(0.1
1.3(0.7
1.4(0.4
2.6(0.1
3.5(1.0
4.0(2.0
0.5(0.1
24.0(9.9
14.9(0.6
14.4(0.6
16.1(8.5
18.2(1.6
51.5(34.4
15.0(1.0
20.5(6.5
0/4
0/4
2/4
1/4
2/4
3/4
4/4
1/4
3/4
0/4
2/4
3/4
3/4
4/4
3/4
1/4
1/3
0/4
0/4
2/4
2/4
4/4
4/4
1/4
0/3
0/4
0/4
0/4
0/4
4/4
2/4
2/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
Gal-B2-C
8
Gal-B2-C10
Gal-B2-C12
Gal-B2-C14
a
Gal-B2
Gal-B2-C18
Gal-B2-MPEG
4
a
Data from ref 22.
The solvents were extracted with CH
saturated NaHCO (20 mL), 1 M HCl (2 mL), and brine (20 mL),
dried over Na SO , and concentrated. Flash chromatography
CH Cl /CH OH, 6:1) gave a white amorphous solid 1 (0.86 g,
7%). TLC R OH, 10:1). [R] +10.2° (c 1,
2 2
Cl (250 mL), washed with
3
2
4
(
8
2
2
3
2
0
f
) 0.29 (CH
). H NMR (CDCl ): δ (ppm) 7.68 (d, J ) 7.6 Hz, 2H),
.53 (m, 2H), 7.32 (m, 2H), 7.23 (m, 2H), 5.67 (m, 1H), 4.29 (m,
H), 4.13 (m, 1H), 3.19 (m, 2H), 2.10 (tm, 2H), 1.84-1.17 (m,
6H), 0.78 (m, 3H). C NMR (CDCl ): δ (ppm) 174.40, 143.95,
41.40, 127.90, 127.29, 125.39, 120.12, 67.15, 47.30, 39.42, 36.84,
2
Cl
2
/CH
3
D
1
CHCl
3
3
7
2
1
1
3
1
3
3
1.94, 29.52, 29.27, 26.07, 22.84, 14.31. HRMS (MALDI) (m/z)
MNa ): found 517.2673. Calcd for C29
+
(
H
38
N
2
O
5
Na 517.2678.
Fmoc-Lys(decanoyl)-OH (2). Compound 2 was submitted to
the same procedure described above for the preparation of 1 and
gave a white amorphous solid (yield, 78%). TLC R ) 0.40 (CH Cl
). H NMR (CDCl ): δ
ppm) 7.67 (d, J ) 7.6 Hz, 2H), 7.52 (m, 2H), 7.30 (m, 2H), 7.21
m, 2H), 5.78 (d, J ) 7.6 Hz,1H), 4.27 (m, 2H), 4.12 (m, 1H),
.17 (m, 1H), 2.10 (tm, 2H), 1.83-1.14 (m, 20H), 0.78 (t, J ) 7.2
f
2
2
/
2
0
1
CH
(
(
3
OH, 8:1). [R]
D
+12.0° (c 1, CHCl
3
3
3
1
3
Hz, 3H). C NMR (CDCl
3
): δ (ppm) 174.69, 156.64, 144.14,
1
6
2
43.91, 141.45, 127.94, 127.31, 125.38, 120.17, 98.99, 67.27, 66.36,
1.17, 54.04, 47.31, 39.41, 36.87, 32.26, 32.09, 29.72, 29.60, 29.53,
+
9.04, 26.08, 22.90, 22.54, 14.36. HRMS (MALDI) (m/z) (MNa ):
42 2 5
found 545.2978. Calcd for C31H N O Na 545.2991.
Fmoc-Lys(lauroyl)-OH (3). Compound 3 was submitted to the
same procedure described above for the preparation of 1 and gave
Figure 4. Anticonvulsant activity of the galanin analogues. The
analogues were administered ip as a bolus dose of 4 mg/kg: (A)
time-response studies ranging from 15 min to 4 h were used to
calculate area under the curve values (AUC); (B) relationships between
log D and the anticonvulsant activity (AUC values) of the galanin
analogues in the 6 Hz model of epilepsy.
a white solid (yield, 64%), mp 126.0-128.0 °C. TLC R
f
) 0.76
20
1
(
(
2
(
CH
CDCl
H), 7.21 (m, 2H), 5.82-5.77 (m, 2H), 4.26-4.39 (m, 2H), 4.12
t, J ) 7.2 Hz, 1H), 3.17 (m, 2H), 2.09 (m, 2H), 1.83-1.35 (m,
2
Cl
2
/CH
3
OH, 4:1). [R]
D
3
+14.3° (c 1, CHCl ). H NMR
3
): δ (ppm) 7.67 (d, J ) 7.6 Hz, 2H), 7.52 (m, 2H), 7.30 (m,
1
3
6
3
H), 1.48 (m, 17H), 0.79 (t, J ) 7.2 Hz, 3H). C NMR (CDCl ):
unless otherwise indicated. Chromatography refers to flash chro-
matography on silica gel (Whatman 230-400 mesh ASTM silica
gel). Analytical thin layer chromatography was performed using
EMD Aluminium TLC silica gel 60 PF254. Preparative HPLC was
performed on a Waters 600 pump system equipped with a Waters
δ (ppm) 175.11, 174.81, 156.56, 143.93, 141.49, 127.93, 127.31,
1
2
(
25.38, 120.18, 67.32, 53.76, 47.34, 39.36, 36.94, 32.13, 29.84,
9.72, 29.56, 29.14, 26.03, 22.91, 22.34, 14.35. HRMS (MALDI)
+
m/z) (MNa ): found 573.3299. Calcd for C33
46 2 5
H N O Na 573.3304.
Fmoc-Lys(myristoyl)-OH (4). Compound 4 was submitted to
the same procedure described above for the preparation of 1 and
gave a white solid (yield, 64%), mp 132.0-134.0 °C. TLC R
2
487 dual wavelength detector (λ
1
) 220 nm, λ ) 280 nm) and
2
a preparative Vydac diphenyl column (219TP101522). Analytical
HPLC used an analytical Vydac diphenyl column (219TP54). The
HPLC mobile phases are buffer A, 100% water (0.1% TFA), and
buffer B, 90% acetonitrile (0.1% TFA). Metabolic stability used a
Waters Alliance 2695 system equipped with an autosampler, dual
f
)
2
0
1
0
(
2
4
.81 (CH
2
Cl
2
/CH
3
OH, 3:1). [R]
D
3
+13.4° (c 1, CHCl ). H NMR
CDCl
3
): δ (ppm) 7.73 (d, J ) 7.6 Hz, 2H), 7.59 (m, 2H), 7.37 (m,
H), 7.27 (m, 2H), 5.97 (br, 1H), 5.79 (d, J ) 7.6 Hz, 1H),
.41-4.33 (m, 2H), 4.18 (t, J ) 7.6 Hz, 1H), 3.23 (m, 2H), 2.15
wavelength detector, and a Waters YMC ODS-A diphenyl column
1
(m, 2H), 1.89-1.41 (m, 6H), 1.21 (m, 22H), 0.87 (t, J ) 6.8 Hz,
(
1
AA125052503WT). NMR spectra were recorded at 400 MHz ( H),
1
3
1
3
3
1
H). C NMR (CDCl
3
): δ (ppm) 175.27, 174.86, 156.58, 144.11,
01 MHz ( C) at 25 °C. Proton and carbon chemical shifts are
43.92, 141.48, 127.94, 127.32, 125.39, 120.18, 67.37, 53.78, 47.33,
given in ppm relative to TMS internal standard. MALDI-TOF MS
was conducted at the University of Utah Core Facility. CD spectra
were obtained on an Aviv 62DS CD spectropolarimeter at room
temperature. Optical rotations were measured on a Perkin-Elmer
polarimeter (model 343) using a 1 mL capacity quartz cell with a
39.39, 36.91, 32.16, 29.93, 29.89, 29.75, 29.59, 29.53, 29.08, 26.06,
+
22.93, 22.40, 14.36. HRMS (MALDI) (m/z) (MNa ): found
50 2 5
601.3623. Calcd for C35H N O Na 601.3617.
Fmoc-Lys(stearoyl)-OH (5). Compound 5 was submitted to the
same procedure described above for the preparation of 1 and gave
1
0 cm path length.
Fmoc-Lys(octanoyl)-OH (1). Fmoc-L-lysine (0.737 g, 2.0 mmol)
a white solid (yield, 68%), mp 133.0-135.0 °C. TLC R ) 0.62
f
2
0
1
was added to dioxane (10 mL), and then a solution of Na
CO (0.636 g, 6.0 mmol) in H O (12.6 mL) was added dropwise at
°C. After 5 min, a solution of octanoyl chloride (0.325 g, 2.0
2
-
(CH
2
Cl
2
/CH
3
OH, 5:1). [R]
D
3
+11.8 ° (c 1, CHCl ). H NMR
): δ (ppm) 7.68 (d, J ) 7.6 Hz, 2H), 7.52 (m, 2H), 7.31 (m,
(CDCl
3
3
2
0
2H), 7.22 (m, 2H), 5.73 (d, J ) 7.2, Hz, 1H), 4.28 (m, 2H), 4.12
(m, 1H), 3.19 (m, 2H), 2.09 (m, 2H), 1.84-1.18 (m, 37H), 0.80
mmol) in dioxane (10 mL) was added to the mixture. The solution
was allowed to warm to room temperature and stirred overnight.
1
3
(m, 3H). C NMR (CDCl ): δ (ppm) 175.27, 174.69, 156.54,
3

Lipoamino Acid SAR of Galanin Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1315
1
5
2
6
44.13, 143.94, 141.41, 127.94, 127.31, 125.40, 120.19, 67.32,
The peptides were scanned from 250 to 200 nm, with a 1 nm step
size and 1.0 s dwell time. Data were averaged from five scans.
Metabolic Stability Assay. Peptide stability was assessed in a
buffered 25% rat blood serum assay as previously described. To
200 µL of buffered serum, 2.5 µg of peptide was added and the
solution was incubated at 37 °C. Reactions were quenched at select
time points upon the addition of “quenching solution” (15%
trichloroacetic acid in 40% isopropanol), and the remaining amount
of intact peptide was determined by HPLC.
3.75, 47.35, 39.29, 36.97, 32.15, 29.93, 29.58, 29.10, 29.19, 26.03,
+
2.92, 22.33, 14.35. HRMS (MALDI) (m/z) (MNa ): found
2
2
57.4240. Calcd for C39
MPEG -acetic Acid (7). To a solution of MPEG
0.6 mmol) in THF (20 mL) was added NaH (60%, 0.635 g, 15.9
H
58
N
2
O
5
Na 657.4243.
4
4
-OH (2.20 g,
1
mmol) at 0 °C. The solution was stirred for 1 h at room temperature,
and then ethyl bromoacetate (1.4 mL, 12.6 mmol) was added
dropwise. After 16 h at 50 °C, the solution was concentrated in
vacuo. The residue was hydrolyzed with 1 M LiOH (8 mL) and
Pharmacological Characterization. Galanin receptor binding
assay and the anticonvulsant screening were carried out as previ-
3
CH OH (8 mL) for 6 h at room temperature. The solution was
2
2
ously described in detail. Briefly, the competitive binding assay
was performed using human GalR1 and GalR2 receptor membrane
preparations and europium-labeled galanin (Perkin-Elmer). Samples
were incubated at room temperature for 90 min, followed by four
washings with wash buffer (50 mM Tris-HCl, pH 7.5, and 5 mM
acidified with 1 M HCl to pH 1, and then the solvents were removed
under reduced pressure. The residue was purified by chromatog-
raphy (CH
colorless oil. TLC R
CDCl
2 2 3
Cl /CH OH, 10:1) to afford 7 (1.66 g, 59%) as a
1
f
) 0.15 (CH
2 2 3
Cl /CH OH, 4:1). H NMR
(
3
): δ (ppm) 9.10 (br, 1H), 4.03 (s, 2H), 73.57-3.44 (m, 16H),
1
3
MgCl ) using a vacuum manifold. Enhancement solution (200 µL)
2
3
.25 (s, 3H). C NMR (CDCl
3
): δ (ppm) 172.94, 71.93, 71.09,
was added, and the plates were incubated at room temperature for
7
0.64, 70.58, 70.55, 70.51, 70.45, 68.61, 61.61, 59.02. HRMS
3
+
30 min. The plates were read on a VICTOR spectrofluorometer.
(
MALDI) (m/z) (MNa ): found 289.1248. Calcd for C11
H
22
O
7
Na
Competition binding curves were analyzed using GraphPad Prism
2
89.1263.
software, using a nonlinear regression, sigmoidal dose-response
Fmoc-Lys(MPEG )-OH (6). To a solution of MPEG -acetic acid
4
4
(
variable slope) curve with no constraints or weights, seen below:
7
(0.266 g, 1.0 mmol) and perfluorophenol (0.202 g, 1.1 mmol) in
Cl (5 mL) was added DCC (0.247 g, 1.2 mmol) at room
CH
2
2
[Eu-galanin]
Top - Bottom
logKi 1+
(
)
]
temperature. After being stirred overnight, the mixture was diluted
with acetone (50 mL) and filtered. The residue was dried in vacuo
to give the activated ester, which was used directly in the next step.
The crude oil in DMF (5 mL) was added to a solution of Fmoc-
L-lysine (0.442 g, 1.2 mmol) and DIPEA (0.7 mL, 4 mmol) in DMF
y ) Bottom +
;
x-logEC50)
EC50 ) 10
[
Kd
(
1
+ 10
Top and Bottom are the plateaus on the y-axis. With this model,
we hold the K and concentration of europium-labeled galanin
d
constant (4.3 and 2 nM, respectively) and assume one binding site
with reversible binding at equilibrium. Anticonvulsant efficacy was
assessed following ip administration to five groups of CF-1 mice
(
10 mL) at 0 °C. The solution was stirred overnight at room
temperature and then poured into brine (50 mL), extracted with
CH Cl (150 mL), and washed with 1 M HCl, brine, dried, and
purified by flash chromatography (CH Cl /CH OH, 5:1) to afford
an amorphous solid 6 (0.37 g, 60%). TLC R
2
2
(
0
n ) 4 mice) at a dose of 4 mg/kg. At various times (i.e., 0.25,
.5, 1, 2, and 4 h) after administration, mice were challenged with
2
2
3
f
) 0.21 (CH
3
). H NMR (CDCl
2
Cl
2
/
2
0
1
a 6 Hz corneal stimulation (32 mA, 3 s, 6 Hz). Mice not displaying
a characteristic limbic seizure were considered protected. The
percent of animals protected at each time point was plotted against
time, and the AUC values were calculated with GraphPad Prism.
CH
3
OH, 5:1). [R]
D
+19.5 ° (c 3, CHCl
3
): δ
(
(
(
ppm) 7.67 (d, J ) 7.2 Hz, 2H), 7.52 (m, 2H), 7.29 (m, 2H), 7.21
m, 2H), 5.78 (d, J ) 6.8 Hz, 1H), 4.29 (d, J ) 6.8 Hz, 2H), 4.11
m, 1H), 3.91 (s, 2H), 3.54-3.52 (m, 14H), 3.45-3.43 (m, 2H),
1
3
3
1
1
4
.27 (s, 3H), 1.81-1.18 (m, 6H). C NMR (CDCl ): δ (ppm)
3
71.04, 156.36, 144.18, 144.03, 141.48, 127.90, 127.30, 125.39,
20.15, 72.07, 71.09, 70.69, 70.61, 70.27, 67.05, 59.15, 53.92,
Acknowledgment. This work was supported in part by the
Epilepsy Therapy Grants Program from the Epilepsy Research
Foundation, the University of Utah Startup Funds, and the NIH
Grant R21 NS059669. We thank the Anticonvulsant Screening
Program (ASP) Project Officer James Stables and his group at
the NIH/NINDS for their support with screening galanin
analogues. Technical assistance of Dan McDougle is greatly
appreciated. GB and HSW are scientific cofounders of Neuro-
Adjuvants, Inc.
7.38, 38.54, 31.78, 29.91, 29.00, 22.27. HRMS (MALDI) (m/z)
+
(
MNa ): found 639.2917. Calcd for C32
H
44
N
2
O10Na 639.2893.
Peptide Synthesis. All galanin analogues were synthesized on
a Symphony peptide synthesizer (Protein Technologies Inc.) using
22
Fmoc-based coupling protocols as previously described. Cleaved
peptides were purified by reversed-phase HPLC using preparative
HPLC column (Vydac diphenyl, 219TP1011522) and eluted with
a linear gradient of acetonitrile (0.1% TFA). The flow rate was 10
mL/min, and the elution was monitored by UV detection at 220
and 280 nm. Buffer A (0.1% TFA in water) and buffer B (0.1%
TFA, v/v, in 90% aqueous acetonitrile) were used to produce a
linear gradient from 20% to 100% of buffer B over 40 min. Purified
analogues were quantified by measuring UV absorbance at 279.8
Supporting Information Available: Tables S1 and S2 listing
the purity and log D calculations, respectively, and Figure S1
showing the HPLC chromatograms of the galanin analogues. This
material is available free of charge via the Internet at http://
pubs.acs.org.
nm (molar absorbance coefficient ε ) 7000 cm- M ).
Partitioning Coefficient, log D. The log D values were deter-
mined on the basis of calculated capacity factors derived from
1
-1
References
22
(1) Tallent, M. K.; Qiu, C. Somatostatin: an endogenous antiepileptic.
HPLC retention times as previously described. The log D for five
galanin analogues were determined using conventional shake-flask
methods, using 400 µg of peptide reconstituted in 1 mL of
phosphate buffered saline. The aqueous peptide solution was shaken
with an equal volume of water-saturated octanol for 24 h.
Concentration of the aqueous layer was determined by HPLC.
Retention times for the analogues were determine on a linear
gradient of 20-100% buffer B in 40 min, with immediate return
to initial conditions and a 20 min re-equilibration. A linear
correlation between log D and HPLC retention times gave calculated
log D values.
Mol. Cell. Endocrinol. 2008, 286, 96–103.
(
2) Lerner, J. T.; Sankar, R.; Mazarati, A. M. Galanin and epilepsy. Cell.
Mol. Life. Sci. 2008, 65, 1864–1871.
(3) Vezzani, A.; Sperk, G.; Colmers, W. F. Neuropeptide Y: emerging
evidence for a functional role in seizure modulation. Trends Neurosci.
1
999, 22, 25–30.
(
4) Boules, M.; Fredrickson, P.; Richelson, E. Bioactive analogs of
neurotensin: focus on CNS effects. Peptides 2006, 27, 2523–2533.
5) Polt, R.; Dhanasekaran, M.; Keyari, C. M. Glycosylated neuropeptides:
(
a new vista for neuropsychopharmacology. Med. Res. ReV. 2005, 25,
5
57–585.
(
6) Rousselle, C.; Clair, P.; Smirnova, M.; Kolesnikov, Y.; Pasternak,
G. W.; Gac-Breton, S.; Rees, A. R.; Scherrmann, J. M.; Temsamani,
J. Improved brain uptake and pharmacological activity of dalargin
using a peptide-vector-mediated strategy. J. Pharmacol. Exp. Ther.
2003, 306, 371–376.
Circular Dichroism. R-Helical conformation of each analogue
2
2
was studied using CD as previously described. Peptides were
reconstituted in NaF buffer to a concentration of 0.1 mg/mL.

1
316 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Zhang et al.
(
7) Prokai-Tatrai, K.; Prokai, L.; Bodor, N. Brain-targeted delivery of a
albumin studied by site-directed mutagenesis. J. Mol. Biol. 2006, 363,
702–712.
leucine-enkephalin analogue by retrometabolic design. J. Med. Chem.
1
996, 39, 4775–4782.
(24) Mazarati, A.; Lundstrom, L.; Sollenberg, U.; Shin, D.; Langel, U.;
Sankar, R. Regulation of kindling epileptogenesis by hippocampal
galanin type 1 and type 2 receptors: the effects of subtype-selective
agonists and the role of G-protein-mediated signaling. J. Pharmacol.
Exp. Ther. 2006, 318, 700–708.
(25) Mazarati, A. M. Galanin and galanin receptors in epilepsy. Neuropep-
tides 2004, 38, 331–343.
(
8) Elmagbari, N. O.; Egleton, R. D.; Palian, M. M.; Lowery, J. J.; Schmid,
W. R.; Davis, P.; Navratilova, E.; Dhanasekaran, M.; Keyari, C. M.;
Yamamura, H. I.; Porreca, F.; Hruby, V. J.; Polt, R.; Bilsky, E. J.
Antinociceptive structure-activity studies with enkephalin-based
opioid glycopeptides. J. Pharmacol. Exp. Ther. 2004, 311, 290–297.
(
9) Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.; Stropova,
D.; Polt, R.; Yamamura, H. I.; Hruby, V. J.; Davis, T. P. Improved
bioavailability to the brain of glycosylated Met-enkephalin analogs.
Brain Res. 2000, 881, 37–46.
10) Banks, W. A.; Schally, A. V.; Barrera, C. M.; Fasold, M. B.; Durham,
D. A.; Csernus, V. J.; Groot, K.; Kastin, A. J. Permeability of the
murine blood-brain barrier to some octapeptide analogs of soma-
tostatin. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6762–6766.
11) Kokko, K. P.; Hadden, M. K.; Price, K. L.; Orwig, K. S.; See, R. E.;
Dix, T. A. In vivo behavioral effects of stable, receptor-selective
neurotensin[8-13] analogues that cross the blood-brain barrier.
Neuropharmacology 2005, 48, 417–425.
(26) Liu, H. X.; Hokfelt, T. The participation of galanin in pain processing
at the spinal level. Trends Pharmacol. Sci. 2002, 23, 468–474.
(
27) Flatters, S. J.; Fox, A. J.; Dickenson, A. H. In vivo and in vitro effects
of peripheral galanin on nociceptive transmission in naive and
neuropathic states. Neuroscience 2003, 116, 1005–1012.
(
(
28) Wynick, D.; Thompson, S. W.; McMahon, S. B. The role of galanin
as a multi-functional neuropeptide in the nervous system. Curr. Opin.
Pharmacol. 2001, 1, 73–77.
(
(
29) Toth, I. A novel chemical approach to drug delivery: lipidic amino
acid conjugates. J. Drug Targeting 1994, 2, 217–239.
(
30) Blanchfield, J. T.; Lew, R. A.; Smith, A. I.; Toth, I. The stability of
lipidic analogues of GnRH in plasma and kidney preparations: the
stereoselective release of the parent peptide. Bioorg. Med. Chem. Lett.
(
12) Banks, W. A.; Wustrow, D. J.; Cody, W. L.; Davis, M. D.; Kastin,
A. J. Permeability of the blood-brain barrier to the neurotensin8-13
analog NT1. Brain Res. 1995, 695, 59–63.
2
005, 15, 1609–1612.
(
13) Witt, K. A.; Gillespie, T. J.; Huber, J. D.; Egleton, R. D.; Davis, T. P.
Peptide drug modifications to enhance bioavailability and blood-brain
barrier permeability. Peptides 2001, 22, 2329–2343.
(
31) Knudsen, L. B.; Nielsen, P. F.; Huusfeldt, P. O.; Johansen, N. L.;
Madsen, K.; Pedersen, F. Z.; Thogersen, H.; Wilken, M.; Agerso, H.
Potent derivatives of glucagon-like peptide-1 with pharmacokinetic
properties suitable for once daily administration. J. Med. Chem. 2000,
(
(
(
14) Egleton, R. D.; Davis, T. P. Development of neuropeptide drugs that
cross the blood-brain barrier. NeuroRx 2005, 2, 44–53.
4
3, 1664–1669.
15) Banks, W. A. Delivery of peptides to the brain: emphasis on therapeutic
development. Biopolymers 2008, 90, 589–594.
(
32) Havelund, S.; Plum, A.; Ribel, U.; Jonassen, I.; Volund, A.; Markussen,
J.; Kurtzhals, P. The mechanism of protraction of insulin detemir, a
long-acting, acylated analog of human insulin. Pharm. Res. 2004, 21,
16) Banks, W. A.; Kastin, A. J. Peptides and the blood-brain barrier:
lipophilicity as a predictor of permeability. Brain Res. Bull. 1985, 15,
1
498–1504.
2
87–292.
(
33) Oh, H. S.; Kim, S.; Cho, H.; Lee, K. H. Development of novel lipid-
peptide hybrid compounds with antibacterial activity from natural
cationic antibacterial peptides. Bioorg. Med. Chem. Lett. 2004, 14,
(
17) Wang, J.; Hogenkamp, D. J.; Tran, M.; Li, W. Y.; Yoshimura, R. F.;
Johnstone, T. B.; Shen, W. C.; Gee, K. W. Reversible lipidization for
the oral delivery of leu-enkephalin. J. Drug Targeting 2006, 14, 127–
1
109–1113.
1
36.
(
18) Toth, I.; Malkinson, J. P.; Flinn, N. S.; Drouillat, B.; Horvath, A.;
Erchegyi, J.; Idei, M.; Venetianer, A.; Artursson, P.; Lazorova, L.;
Szende, B.; Keri, G. Novel lipoamino acid- and liposaccharide-based
system for peptide delivery: application for oral administration of
tumor-selective somatostatin analogues. J. Med. Chem. 1999, 42, 4010–
(34) Rivett, D. E.; Hewish, D.; Kirkpatrick, A.; Werkmeister, J. Inhibition
of membrane-active peptides by fatty acid-peptide hybrids. J. Protein
Chem. 1999, 18, 291–295.
(35) Chicharro, C.; Granata, C.; Lozano, R.; Andreu, D.; Rivas, L.
N-Terminal fatty acid substitution increases the leishmanicidal activity
of CA(1-7)M(2-9), a cecropin-melittin hybrid peptide. Antimicrob.
Agents Chemother. 2001, 45, 2441–2449.
(36) Pignatello, R.; Puglisi, G. Lipophilicity evaluation by RP-HPLC of
two homologous series of methotrexate derivatives. Pharm. Acta HelV.
2000, 74, 405–410.
(37) Horvath, A.; Olive, C.; Wong, A.; Clair, T.; Yarwood, P.; Good, M.;
Toth, I. Lipoamino acid-based adjuvant carrier system: enhanced
immunogenicity of group a streptococcal peptide epitopes. J. Med.
Chem. 2002, 45, 1387–1390.
4
013.
(
19) Hitchcock, S. A.; Pennington, L. D. Structure-brain exposure relation-
ships. J. Med. Chem. 2006, 49, 7559–7583.
(
20) Yuan, L.; Wang, J.; Shen, W. C. Reversible lipidization prolongs the
pharmacological effect, plasma duration, and liver retention of
octreotide. Pharm. Res. 2005, 22, 220–227.
(
(
(
21) Weber, S. J.; Abbruscato, T. J.; Brownson, E. A.; Lipkowski, A. W.;
Polt, R.; Misicka, A.; Haaseth, R. C.; Bartosz, H.; Hruby, V. J.; Davis,
T. P. Assessment of an in vitro blood-brain barrier model using
several [Met5]enkephalin opioid analogs. J. Pharmacol. Exp. Ther.
(38) Tamai, I.; Sai, Y.; Kobayashi, H.; Kamata, M.; Wakamiya, T.; Tsuji,
A. Structure-internalization relationship for adsorptive-mediated
endocytosis of basic peptides at the blood-brain barrier. J. Pharmacol.
Exp. Ther. 1997, 280, 410–415.
1
993, 266, 1649–1655.
22) Bulaj, G.; Green, B. R.; Lee, H. K.; Robertson, C. R.; White, K.;
Zhang, L.; Sochanska, M.; Flynn, S. P.; Scholl, E. A.; Pruess, T. H.;
Smith, M. D.; White, H. S. Design, synthesis, and characterization of
high-affinity, systemically-active galanin analogues with potent anti-
convulsant activities. J. Med. Chem. 2008, 51, 8038–8047.
(
39) Drin, G.; Cottin, S.; Blanc, E.; Rees, A. R.; Temsamani, J. Studies on
the internalization mechanism of cationic cell-penetrating peptides.
J. Biol. Chem. 2003, 278, 31192–31201.
23) Kragh-Hansen, U.; Watanabe, H.; Nakajou, K.; Iwao, Y.; Otagiri, M.
Chain length-dependent binding of fatty acid anions to human serum
JM801397W