Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity

Article abstract of DOI:10.1021/jm701093y

The tetrapeptide sequence His-Phe-Arg-Trp, derived from melanocyte-stimulating hormone (αMSH) and its analogs, causes a decrease in food intake and elevates energy utilization upon binding to the melanocortin-4 receptor (MC4R). To utilize this sequence as an effective agent for treating obesity, we improved its metabolic stability and intestinal permeability by synthesizing a library of backbone cyclic peptidomimetic derivatives. One analog, peptide 1 (BL3020-1), was selected according to its selectivity in activating the MC4R, its favorable transcellular penetration through enterocytes and its enhanced intestinal metabolic stability. This peptide was detected in the brain following oral administration to rats. A single oral dose of 0.5 mg/kg in mice led to reduced food consumption (up to 48% vs the control group) that lasted for 5 h. Repetitive once daily oral dosing (0.5 mg/kg/day) for 12 days reduced weight gain. Backbone cyclization was shown to produce a potential drug lead for treating obesity.

Full text of DOI:10.1021/jm701093y

1026
J. Med. Chem. 2008, 51, 1026–1034
Backbone Cyclic Peptidomimetic Melanocortin-4 Receptor Agonist as a Novel Orally
Administrated Drug Lead for Treating Obesity
Shmuel Hess,†,‡ Yaniv Linde,†,§ Oded Ovadia,‡ Eli Safrai,| Deborah E. Shalev,| Avi Swed,‡ Efrat Halbfinger, Tair Lapidot,
Ilan Winkler, Yael Gabinet, Avi Faier, Dana Yarden, Zhimin Xiang,# Federico P. Portillo,# Carrie Haskell-Luevano,#
Chaim Gilon,§ and Amnon Hoffman*,‡
Department of Pharmaceutics, School of Pharmacy, The Hebrew UniVersity of Jerusalem, Jerusalem 91120, Israel, Institute of
Chemistry, and Wolfson Centre for Applied Structural Biology, Safra Campus, GiVat Ram, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel, Bioline Rx, Jerusalem 91450, Israel, and Department of Medicinal Chemistry, UniVersity of Florida,
GainesVille, Florida 32610-0485
ReceiVed September 4, 2007
The tetrapeptide sequence His-Phe-Arg-Trp, derived from melanocyte-stimulating hormone (RMSH) and
its analogs, causes a decrease in food intake and elevates energy utilization upon binding to the melanocortin-4
receptor (MC4R). To utilize this sequence as an effective agent for treating obesity, we improved its metabolic
stability and intestinal permeability by synthesizing a library of backbone cyclic peptidomimetic derivatives.
One analog, peptide 1 (BL3020–1), was selected according to its selectivity in activating the MC4R, its
favorable transcellular penetration through enterocytes and its enhanced intestinal metabolic stability. This
peptide was detected in the brain following oral administration to rats. A single oral dose of 0.5 mg/kg in
mice led to reduced food consumption (up to 48% vs the control group) that lasted for 5 h. Repetitive once
daily oral dosing (0.5 mg/kg/day) for 12 days reduced weight gain. Backbone cyclization was shown to
produce a potential drug lead for treating obesity.
Introduction
neuropeptide melanocyte stimulating hormone (RMSHa) to the
melanocortin 4 receptor (MC4R). MC4R agonists decrease food
intake and elevate energy utilization3 such that they could be
used for treating obesity. For example, a study performed with
melatonan II (MTII), a commercially available peptide-based
MC4R agonist, administered intracerebroventricularly for four
weeks, resulted in a 40% decrease in rat weight.4
The effective role of a MC4 agonist in obesity therapy was
also demonstrated in humans. Intravenous (iv) administration
of the melanocortin sequence MSH/ACTH (4–10) to normal-
weight human subjects for six weeks decreased body fat by 1.7
kg.5 The sequence Phe-DPhe-Arg-Trp, a potent MC4R agonist,6
was used in this study as the stem peptide to achieve an effective
agent for treating obesity.7,8 MC4R peptide agonist was found
to decrease body fat in humans,9 however, a major drawback
of current MC4R agonist peptides is that they can not be taken
orally due to poor bioavailability. Oral administration is the most
convenient mode of drug intake and therefore is favored for
chronic treatment to preserve proper patient compliance. The
Obesity is a major health problem in the developed world
and is a high risk factor in many diseases, including diabetes
type II, high blood pressure, cardiovascular and respiratory
diseases, and cancer, which result in a significant reduction in
life span and quality.1
According to the NHANES (National Health and Nutrition
Examination Survey) conducted in 2003–2004, 17.1% of the
children and adolescents aged 2 to 19 years in the United States
were overweight and 32.2% of the adults aged 20 years or older
were obese.2 Although the United States has the highest
prevalence of obesity among the developed nations, it is not
alone in terms of trends. Increases in the prevalence of
overweight and obese children and adults have been observed
throughout the world.
Weight loss is always difficult to achieve through changes
in life style and currently licensed antiobesity drug treatments
such as orlistat and sibutramine, if tolerated, only achieve modest
weight loss. Therefore, there is a need to identify more potent
pharmacological targets.
The pharmaco-therapeutic approach for treating obesity has
been to regulate the biochemical pathways that control food
consumption and metabolic balance in the body. A key
endocrine system that controls these physiological processes is
the melanocortin pathway,3 which includes binding the catabolic
a Abbreviations: The abbreviations for amino acids are according to the
IUPAC-IUB Commission of Biochemical Nomenclature, http://www.chem.
qmul.ac.uk/iupac/aminoacid. The following abbreviations are used through-
out the text: ACN, acetonitrile; Alloc, allyloxycarbonyl; BBMVs, brush
border membrane vesicles; ; Boc, t-butoxycarbonyl; BTC, bis(trichloro-
methyl)carbonate; Bri, bridge unit; COSY, correlation spectroscopy; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine; DMAP, 4-dimeth-
ylaminopyridine; DMF, dimethylformamide; EtOAc, ethyl acetate; ESI,
electrospray ionization; Fmoc, 9-fluorenylmethoxycarbonyl; Fmoc-OSu,
9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide; HBTU, (2-(1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate); HOBt,
1-hydroxybenzotriazole; iv, intravenous; MALDI, matrix assisted laser
desorption ionization; MBHA, methylbenzhydrylamine; MC4R, melano-
cortin-4 receptor; MCR, melanocortin receptor; RMSH, melanocyte stimu-
lating hormone; NMP, 1-methyl-2-pyrrolidinone; NMR, nuclear magnetic
resonance; NOESY, nuclear Overhauser effect spectroscopy; Papp, perme-
ability apparent; po, per oral; ROESY, rotating frame Overhauser effect
spectroscopy; SPPS, solid phase peptide synthesis; TBTU, O-(benzotriazol-
1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate; tBu, tert-butyl; TIS,
triisopropylsilane; TLC, thin layer chromatography; TOCSY, total correla-
tion spectrometry.
* To whom correspondence should be addressed. Prof. Amnon Hoffman,
Department of Pharmaceutics, School of Pharmacy, The Hebrew University
of Jerusalem, Post Office Box 12065, Jerusalem 91120 Israel. Tel.: +972
2 6757014. Fax: + 972 2 6757246. E-mail: amnonh@ekmd.huji.ac.il.
†
These authors contributed equally.
Department of Pharmaceutics, The Hebrew University of Jerusalem.
Institute of Chemistry, The Hebrew University of Jerusalem.
Wolfson Centre for Applied Structural Biology, The Hebrew University
‡
§
|
of Jerusalem.
Bioline Rx.
#
University of Florida.
10.1021/jm701093y CCC: $40.75
2008 American Chemical Society
Published on Web 01/26/2008

MC4R Peptidomimetic Agonist for Treating Obesity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1027
Figure 1. Scheme of peptide 1 synthesis.
Table 1. Structure of Selected Members of the Peptide Librarya
generally poor oral bioavailability of peptides is due to extensive
intestinal metabolic degradation, mainly by the intestinal brush
border enzymes and poor intestinal permeability.10
In this work we assessed the effect of backbone cyclization
on the MC4R agonist as a potential method to improve oral
bioavailability while enabling brain availability and maintaining
high affinity and selectivity at the receptor level.
ring size
(number of atoms)
peptide
m
n
Backbone cyclization11,12 is a general method by which
conformational constraint is imposed on peptides.13 In backbone
cyclization, a peptidomimetic is formed by covalently intercon-
necting atoms in the backbone (N and/or C) of a target linear
peptide to form a ring. The advantage of backbone cyclization
over other modes of peptide cyclization is that cyclization is
achieved mainly using backbone atoms and not side chains that
are essential for biological activity. This method has been shown
to dramatically enhance the metabolic stability of peptides in
serum.12 In addition, this method has been shown to improve
the pharmacological selectivity of a given peptide, as demon-
strated for substance P12 and somatostatin analogs.14 It should
be noted that the specific selectivity of the melanocortin agonist
to MC4R is important to obtain the antiobesity activity while
avoiding adverse effects caused by activation of other subtypes
of MCR.
1
2
3
4
5
2
2
3
2
3
2
20
23
20
21
21
4
b
2
3
a The peptides in the library differ in their ring size (e.g., peptides 1 and
2) and their ring chemistry (e.g., peptides 4 and 5 or peptides 1 and 3).
b The succinyl spacer (m ) 2) of peptide 1 was replaced by o-phtaloyl
spacer. The structure of peptide 3 is shown in Figure 3.
The aim of this study was to generate a selective, highly
potent, MC4R agonist that is orally and brain available and can
serve as an antiobesity drug lead.
Results and Discussion
Chemistry. A library of 16 backbone cyclic pentapeptides
(called the BL3020 library15) was synthesized based on the
parent peptide Phe-D-Phe-Arg-Trp-Gly NH2, which was found
to have high potency in activating the MC4R.6 The parent
peptide, Phe-D-Phe-Arg-Trp-Gly NH2, of the BL3020 library
was cyclized by a bridge that connects the N-terminus to the
NR of the C-terminal Gly building unit by a dicarboxylic acid
spacer. The synthesis of 1 (BL3020-1) is depicted in Figure 1.
The building unit Fmoc-[ꢀ-N(Alloc)-ethyl]Gly OH, and other
building units used for the preparation of the library, were
prepared as previously described.16 All the peptides in the library
bear the parent sequence but differ from each other in their ring
size and ring chemistry (Table 1 shows selected members of
the library). The peptides were studied for MC4R binding, MC
receptor activation, and selectivity, as well as in vitro intestinal
permeability and resistance to intestinal metabolic degradation.
Metabolic Stability of the Cyclic Peptide Analogs. The
metabolic stability of members of the peptide library in the
Figure 2. Metabolic stability of cyclic peptides 1 and 2 and the linear
analog. The tested molecules were mixed with purified BBMVs and
incubated at 37 °C for 90 min. Duplicate samples were taken at time
0 and after 15, 30, 45, 60, and 90 min. The samples were diluted 1:1
with ice-cold acetonitrile, centrifuged (7500 g, 10 min, 4 °C) and
transferred to analysis, SD < 15% (peptide 1 b, peptide 2 1, linear
tetra-peptide analog O).
intestine was compared to that of the linear peptide (Figure 2).
While the concentration of the linear peptide was reduced to
nearly 40% of the initial concentration after 40 min of incubation
with the brush border membrane vesicles (BBMVs), the cyclic
peptides 1 and 2 (BL3020-15) were stable toward enzymatic
degradation and showed less than a 5% reduction in the initial

1028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Hess et al.
Table 2. Summary of Affinity (IC50) and Functional Activity (EC50) at the Mouse Melanocortin Receptorsa
stimulation/potent activation (EC50, nM)
binding to MC4R
(IC50, nM)
mMC1R
mMC3R
mMC4R
mMC5R
NDP-MSH
NTb
90
60
100
60
NT
0.008 ( 0.001
64 ( 14
0.063 ( 0.004
770 ( 175
7750 ( 1960
20500 ( 2100
1260 ( 385
1460 ( 145
0.097 ( 0.016
4.0 ( 0.6
5.0 ( 0.08
150 ( 22
4 ( 2
0.078 ( 0.008
7.0 ( 0.4
26 ( 17
390 ( 120
2 ( 1
20 ( 4
1
2
3
4
5
240 ( 41
550 ( 97
28 ( 12
120 ( 28
18 ( 4
a The affinities (half maximal inhibitory concentration IC50, nM) and receptor functional activation (EC50, nM) of the backbone cyclic peptides to the
melanocortin-4 receptor. The efficacy was 100% (maximal stimulation relative to the NDP-MSH control) for all compounds tested. b NT ) not tested.
Table 3. Permeability coefficient (Papp) of Cyclic Peptides Compared to
concentration following 90 min of enzymatic reaction (Figure
2). The capability of the peptides to withstand the enzymatic
activity of various peptidases located in the gut lumen and the
intestinal brush border is a major factor in the oral bioavailability
of peptides. Because much of the enzymatic digestion takes
place at the surface of the small intestinal epithelial cells, there
is usually a significant reduction in the ability of intact peptides
to reach the systemic circulation after reaching the intestinal
milieu. For tetra- and higher amino acid peptides, more than
90% of the proteolytic activity occurs in the brush border
membrane, whereas for tripeptides 10–60% and for dipeptides
only 10% of the proteolytic activity occurs in the brush border
membrane.17 These results are in agreement with previous
reports from our laboratory that have demonstrated significant
metabolic stability of backbone cyclic peptides in various media
such as serum and kidney homogenate.12,14
Intestinal Permeability. In vitro permeability studies of the
BL3020 library through Caco-2 monolayers resulted in perme-
ability coefficient values of the cyclic peptides that were
significantly higher than permeability coefficients (Papp) obtained
for mannitol and atenolol, markers of paracellular transport, and
only slightly smaller than Papp of propranolol, a marker for
transcellular transport (Table 3). The permeability of the linear
equivalent peptide was higher than atenolol (12 × 10-6 and
8.5 × 10-6 cm/sec, respectively); however, it was 50–60% lower
than that of the cyclic peptides. The permeability of active
compounds through Caco-2 monolayer (derived from a human
colorectal adenocarcinoma) is an established model to determine
intestinal permeability18 and was also used to predict perme-
ability of molecules through the blood-brain barrier (BBB).19
The most exciting finding achieved in this work was that the
high permeability coefficient of the tested analogs was similar
to that of propranolol, a molecule that is known for its ability
to traverse the intestinal wall via the transcellular pathway,
indicating that most of the examined library analogs are highly
permeable compounds.20 The significant superior permeability
in comparison to mannitol and atenolol is another indication
that the mechanism of intestinal permeability is by a transcellular
mechanism. It should be noted that this finding is unique in
view of the tendency of many peptides to permeate the intestinal
wall via the paracellular route.21 Furthermore, backbone cy-
clization increased permeability relative to the linear analogue
(Table 3). This is probably due to the conformational constraints
imposed by cyclization, the elevated lipophilicity, and the
reduced hydrogen-bonding potential characteristic of the cyclic
peptides.10 The transport of 1 from the apical side of the
membrane to basolateral side (A to B) was compared to the
transport from the basolateral to the apical (B to A) using Caco-2
cells. The transport from A to B was found to be 2-fold higher
than the opposite direction.
the Linear Analog and to Known Standardsa
compound
Papp (cm/sec) × 106 ( SEM
mannitol
atenolol
propranolol
linear
1
2
4
5
2.05 ( 0.8
8.5 ( 0.4
25 ( 2.3
12 ( 0.98
17.8 ( 1.37
19 ( 5.5
18.8 ( 4.39
21 ( 3.6
a Caco-2 cell monolayers were incubated with the tested molecules at
37 °C, added to the apical side, and detected at the basolateral side for 150
min; n g 3 ( SEM.
of the underlying transport mechanism of this peptide will be
performed to characterize the transport mechanism of 1.
Ligand–Receptor Interactions and Agonist Activity at
the Melanocortin Receptors. Following the stability and in
vitro permeability studies, the peptides of the library were
analyzed for biological activity in binding and activating the
melanocortin receptors (Table 2). The changes in the ring size
and chemistry led to observed changes in binding and potency
of activating of the melanocortin receptors and, hence, the
selectivity of the peptides to the different MC4s. The binding
affinity values, IC50, of the various backbone cyclic peptides to
the MC4R receptors are given in Table 2. The binding assays
indicate that most of the peptides had high affinity to the MC4R.
Peptide 2 had the highest affinity, with an IC50 value of 60 nM.
The IC50 value of peptide 1 reached 90 nM, and the IC50 value
of peptide 3 (BL3020-16) was 100 nM. Because the IC50 values
do not indicate whether the binding generates an agonist or
antagonist effect, the potency of activating various melanocortin
receptors was investigated (Table 2). The peptides were found
to have diverse potency. For example, peptide 4 (BL3020-8)
exhibited a similar potency to MC4R as that of 1 and increased
potency by 2- and 4-fold toward MC1R and MC5R, respec-
tively. The stimulation/agonist potency of MC4R was in the
range of 4.0–18.5 nM for all library peptides (except for peptide
3, which was found to be significantly less potent on all tested
melanocortin receptors). The high affinity that was detected gave
the first indication that the chemical modifications made did
not damage the biological activity of the peptides. The results
confirm that most of these peptides range from 80- to 1550-
fold selective for the MC4R versus the MC3R.
Table 2 shows the selectivity and specificity of cyclic peptides
from the library. While all the library components share the
same sequence, the size and chemistry of the ring moiety differ,
thereby affecting their binding affinity and potency in activating
the melanocortin receptors. The conformational constraint
achieved by backbone cyclization produced peptides with a
much smaller and more defined conformational space than their
linear counterparts. Moreover, the limited conformational space
of each peptide in the library is different, hence, some of the
This data suggest that there is an involvement of an active
mechanism in the transport of the peptide. Further investigation

MC4R Peptidomimetic Agonist for Treating Obesity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1029
Figure 3. Structure of the backbone cyclic peptides 1 and 3.
Table 4. NMR Characterization of Peptide 1; Proton Chemical Shifts
(ppm) of Peptide 1 Main (Top) and Minor (Bottom) Conformations
members of such libraries could become inactive because they
cannot adopt the bioactive conformation of the MCRs. However,
peptides that can conform to the bioactive structure are likely
to be very potent and have all the pharmacological advantages
of cyclic peptides.22 Backbone cyclization affected both speci-
ficity and selectivity toward the various melanocortin receptors,
allowing us to determine which peptide is a selective agonist
that activates the target receptor, MC4R, with maximal specific-
ity and minimal side effects.
Selection of the Lead Compound from the Peptide
Library. The metabolic stability, permeability, and functional
agonist activity assay of the library performed on the mouse
melanocortin receptors indicated that the backbone cyclic
peptide 1 (Figure 3) is the best candidate for a lead compound:
it selectively activated the MC4R, it was metabolically stable,
and it had high intestinal permeability. Therefore, it was selected
for further characterization and its pharmacokinetic behavior
was investigated.
NMR Analysis of 1. All protons were accounted for except
the aromatic region, which was partially unresolved due to
overlap. All the HRi-HNi+1 connectivities were clearly seen.
A strong ROE/NOE peak was observed between the Hꢀs and
Hδs of each phenylalanine, giving the connectivity of the
respective aromatic moieties. The HNBri of the bridge unit gave
a unique triplet in the amide region of the spectrum and, thus,
its identity. An interaction between HRBri and HNPhe-1, together
with the interaction of HδBri to HRTrp-4, gave the direction of
the bridge moiety. A COSY peak between HRBri and HꢀBri and
a strong ROE interaction between HꢀBri and HNBri gave the
identity and orientation of this segment of the bridge. TOCSY
and COSY peaks between HNBri, HγBri, and HδBri identified
this portion of the bridge unit. There were two conformations
evident in the spectra as is common among N-alkylated cyclic
peptides.23 The ratio of conformers was 1:2 in the 1D spectrum.
Chemical exchange during the time of the measurement was
evident from the inverted exchange peaks in the ROESY spectra.
The difference between conformations is due to the dihedral
angle between the tertiary nitrogen and the carbonyl carbon of
Trp4 (HRGly-5, NGly-5, CTrp-4, OTrp-4); this angle is 0° in the main
conformation and 180° in the minor conformation. The main
conformation is supported by ROE peaks between HRTrp-4 and
HδBri (or H9Bri in BBC12) and the lack of a ROE signal between
HRTrp-4 and HRGly-5. The HRTrp-4 of the minor conformation in
most cyclic peptides overlaps the water signal, except for the
case of BBC12, where the conformation of the minor conforma-
tion is inferred from the clear NOE interaction observed between
HRTrp-5 and HRGly-6 and the lack of a strong HRTrp-5 to H9Bri
NOE. The HRTrp-4 of the minor conformation overlaps the water
signal and, therefore, its conformation is inferred from similar
residues
HN
HR
Hꢀ
others
main conformation
Bri0*
Phe1
7.73
8.14
7.41
7.60
2.39
2.31
δCH2 2.82, 3.54
γCH2 3.14, 3.28
δCH2 7.16
ꢀCH2 URa
δCH2 7.16
ꢀCH2 UR
γCH2 1.36
δCH2 3.09
HN 7.60
4.26
4.51
4.20
2.93
D-Phe2
Arg3
2.83, 2.88
1.52, 1.73
Trp4
8.26
4.89
3.23
Hδ1 7.25
Hꢀ1 10.05
Hꢀ3 7.58
Hη2 UR
Hꢁ2 7.48
Hꢁ3 UR
NH2 6.89, 7.13
Gly5
3.75, 4.04
minor conformation
Bri0*
Phe1
7.54
7.96
8.11
7.84
2.51, 2.58
2.38, 2.46
δCH2 3.80
γCH2 3.23, 3.40
δCH2 7.14
ꢀCH2 UR
δCH2 7.09
ꢀCH2 UR
γCH2 0.96, 1.08
δCH2 2.92
HN 7.0
4.50
2.98
D-Phe2
Arg3
4.42
2.85
4.15
1.30, 1.51
Trp4
7.89
4.73
3.21
Hδ1 7.25
Hꢀ1 10.06
Hꢀ3 7.64
Hη2 UR
Hꢁ2 7.48
Hꢁ3 UR
NH2 7.57
Gly5
4.01, 4.28
a UR unresolved.
peptides in other solvents (unpublished results). The proton
chemical shift values of peptide 1 are summarized in Table 4.
Pharmacokinetic Parameters following iv and po
Administration. The PK of peptide 1 was studied following
iv and po (dissolved in water, 1 mg/kg and 10 mg/kg,
respectively) administration to rats. Analyzing the results
produced several pharmacokinetic parameters, including the
peptide half-life in the body (>105 min), the AUC value (24,980
min·ng/ml), and the volume of distribution at steady state (Vss,
2.1 L/kg). Measured Cmax values were 977 ( 97 (iv) and 202
( 39 ng/ml (po). The Tmax for iv was at 5 min and at 37 ( 10
min following po administration. The relatively high volume
of distribution provides an indirect indication of the ability of
1 to cross biological membranes such as the intestine and the
BBB.24 The pharmacokinetic findings indicate a relatively high
bioavailability of 8.5%.

1030 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Hess et al.
Figure 4. Short time effect of MC4R agonists on mice food consumption following po administration. Following fasting for 16 h, ICR:Hsd (CD-1)
male mice (n ) 24) were subjected to a single oral gavage (po, 5 mL/kg) of the peptide (100 µg/ml) or vehicle (water). Immediately after administration,
fixed food doses were added and reweighed after 1, 2, 3, 4, and 5 h. (A) Black bars, control; gray bars, peptide 1. (B) Black bars, control; gray bars,
peptide 3. Data are expressed as the mean ( SEM. Statistical analysis made by RM ANOVA with post hoc multiple comparisons using Tukey’s
test: *P < 0.05; **P < 0.01.
In Vivo Studies by Oral Administration. In vivo studies
in mice showed reduced food consumption following oral
administration of 1, as depicted in Figure 4A. Reduced food
consumption was already apparent 1 h after a single oral
administration. The decrease in food consumption reached
46–48% in comparison to the untreated (control) group. No
abnormal physiological or behavioral signs were observed during
the assay. A parallel study was performed with peptide 3,
another member of the library that was found to be nonspecific
and less potent than peptide 1 in MC4R stimulation or potent
activation (Table 2). In this study no reduction in food
consumption was observed up to 5 h postoral administration of
peptide 3 (Figure 4B).
Following the observed acute effect of peptide 1 on food
consumption, the effect of longitude repetitive once daily oral
administration was tested in order to assess the effect on weight
increase. As depicted in Figure 5, oral administration of peptide
1 resulted in inhibition of weight gain compared to the untreated
animal group. While there is a consistent rise in weight gained
by the control group, reaching over 7.5% after 10 days of
treatment, the increase in weight in mice treated with peptide 1
was inhibited, gaining only 2.8% compared to their original
weight on day 0.
addition to the requirement of intestinal metabolic stability and
bioavailability, the ability to penetrate the BBB is of interest.
The concentrations of peptide 1 in the brain determined 8 h
post iv and po administration of 10 mg/kg were 107.3 ( 14.2
ng/mg and 4.7 ( 2.5 ng/mg, respectively. This provides direct
evidence for the ability of peptide 1 to reach the brain tissue
following oral administration.
Biological Activity of Peptide 1. The main concern in the
process of improving oral bioavailability of peptides by using
structural and conformational modifications is the loss of
biological activity. Although it was demonstrated that the
backbone cyclic peptides were found to preserve their in vitro
binding and potent activation of the MC4R as agonists, further
investigation of the in vivo effect was required. The effect of
acute per oral administration on the food consumption (shown
in Figure 4) provided proof of our concept that backbone
cyclization is a practical means to obtain an active, orally
available peptide that can be used for treating obesity.
It should be noted that the effect of both doses used on food
consumption lasted for at least 5 h post oral administration. The
elimination half-life (≈100 min in rats) suggests that the effect
of the peptide on food consumption follows the indirect
pharmacodynamic concept.27
Brain Bioavailability. The site of action of RMSH and other
The effect of repetitive once daily dosing of peptide 1 on
inhibiting weight gain corroborated the concept of using the
MCR agonists is within the hypothalamus.25,26 Therefore, in

MC4R Peptidomimetic Agonist for Treating Obesity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1031
Figure 5. The effect of peptide 1 on mice weight gain after repeated po administration. Following daily weighing, mice (n ) 9) were subjected
to daily oral gavage (po, 5 mL/kg) of peptide 1 (100 µg/ml) or vehicle (water). Black bars, control; Gray bars, peptide 1. Data are expressed as the
mean ( SEM. Statistical analysis made by student’s t-test: *P < 0.05.
Japan) using a Vydac preparative RP column (C8, 22 × 250 mm,
backbone cyclic MC4R analog as a potential novel orally
available antiobesity drug.
catalog number 218TP1022). The flow rate of the mobile phase
was 9 mL/min. All preparative HPLC runs were carried out using
a gradient system with solvent A corresponding to water with 0.1%
TFA and solvent B corresponding to ACN with 0.1% TFA. MS
characterization was performed on a Voyager-DE PRO Biospec-
trometry workstation using matrix assisted laser desorption ioniza-
tion (MALDI) technology in the positive mode.
Conclusions
The results of this study indicate that by utilizing backbone
cyclization it is possible to synthesize bioactive peptides that
are stable in the intestinal milieu and cross the intestinal wall,
thus providing enhanced oral bioavailability. The current
knowledge regarding the role of MC4R in regulating appetite,
energy homeostasis and neuroendocrine function, coupled with
an advanced peptide chemistry approach has resulted in the
synthesis of a promising novel MC4R agonist lead for obesity
therapy.
Solid Phase Peptide Synthesis of Peptide 1. The synthesis was
performed in a reaction vessel equipped with a sintered glass
bottom, following general Fmoc chemistry protocols: Rink amide
methylbenzhydrilamine (MBHA) resin (1 g, 0.66 mmol/g) was
preswollen in N-methylpyrrolidone (NMP) for 2 h. Fmoc depro-
tection step was carried out with 20% piperidine in NMP (2 × 15
min), followed by washing with NMP (5 × 2 min) and DCM (2 ×
2 min). Couplings of the building unit Fmoc-[ꢀ-N(Alloc)-ethyl]Gly
OH to the resin and of Fmoc-amino-acid-OH (Fmoc-AA-OH) to
the building unit were carried out according to a modified procedure
published by Falb et al.28 Briefly, FmocAAOH or building unit (3
equiv, 1.98 mmol) and bis-(trichloromethyl) carbonate (BTC,
triphosgene) (1 equiv, 0.66 mmol) were suspended in DCM. 2,4,6-
Collidine (10 equiv, 6.6 mmol) was added to the precooled
suspension in an ice bath. After all the solids were dissolved (about
1 min), the solution was poured onto the resin and shaken for 3 h
at room temperature. This coupling cycle was repeated once more.
At the end of the second coupling cycle, the peptidyl-resin was
washed with DCM (5 × 2 min). Capping was carried out after the
anchoring of the building unit to the resin and was repeated twice
by reaction of the peptidyl-resin with a mixture of acetic anhydride
(1.1 mL, 0.5 M), diisopropyl amine (DIEA; 0.5 mL, 0.125 M),
and N-hydroxybenzotriazole (HOBT; 0.05 g, 0.015 M) in dimethyl
formamide (DMF, 25 mL). Capping was followed by resin wash
with DMF (5 × 2 min), DCM (2 × 2 min), and NMP (2 × 2 min).
Coupling of Fmoc-Trp(BOC)-OH to the building unit was carried
out as follows: the Fmoc protecting group was removed from the
peptidyl-resin with 20% piperidine in NMP (2 × 30 min) and the
resin was washed (NMP, 5 × 2 min; DCM 5 × 2 min). Fmoc-
Trp(BOC)-OH (3 equiv, 1.98 mmol) and BTC (1 equiv, 0.66 mmol)
were suspended in dicholoromethane (DCM) and cooled in an ice
bath for 30 min. 2,4,6-Collidine (10 equiv, 6.6 mmol) was added
to the precooled suspension in an ice bath and mixed to received
clear solution (1 min). Then the solution was added to the peptidyl-
resin and the vessel was shaken for 3 h at RT. Washing steps were
carried out with DCM (5 × 2 min). The cycle of Fmoc removal
and coupling was followed with Fmoc-Arg(Pbf)-OH, Fmoc-DPhe-
OH, and Fmoc-Phe-OH. All coupling steps were carried out using
Methods
Chemistry. General. Protected amino acids, 9-fluorenylmeth-
yloxycarbonyl-N-hydroxysuccinimide (Fmoc-OSu), bromo-tris-pyr-
rolidone-phosphonium hexafluorophosphate (PyBrop), Rink amide
methylbenzhydrylamine (MBHA) polystyrene resins, organic and
supports for solid phase peptide synthesis (SPPS) were purchased
from Nova Biochemicals (Laufelfingen, Switzerland). Bis(trichlo-
romethyl)carbonate (BTC) was purchased from Lancaster (Lan-
cashire, England). Glyoxylic acid, 1,2-diaminoethane, 1,3-diami-
nopropane, and 1,4-diaminobutane were purchased from Merck
(Darmstadt, Germany), and tetrakis (triphenylphosphine) pal-
ladium(0) was purchased from ACROS (Geel, Belgium).
Nuclear magnetic resonance (NMR) spectra during synthesis
were recorded on a Bruker AMX 300 MHz spectrometer. Mass
spectra were performed on a Finnigan LCQ DUO ion trap mass
spectrometer. Thin layer chromatography (TLC) was performed on
Merck F245 60 silica gel plates (Darmstadt, Germany). HPLC
analysis was performed using a Vydac analytical RP column (C18,
4.6 × 250 mm, catalog number 201TP54) and was carried out on
a Merck-Hitachi L-7100 pump and a Merck-Hitachi L-7400 variable
wavelength detector operating at 215 nm. The mobile phase
consisted of a gradient system, with solvent A corresponding to
water with 0.1% TFA and solvent B corresponding to acetonitrile
(ACN) with 0.1% TFA. The mobile phase started with 95% A from
0 to 5 min followed by a linear gradient from 5% B to 95% B
from 5 to 55 min. The gradient remained at 95% B for an additional
5 min and then was reduced to 95% A and 5% B from 60 to 65
min. The gradient remained at 95% A for additional 5 min to
achieve column equilibration. The flow rate of the mobile phase
was 1 mL/min. Peptide purification was performed by reversed
phase HPLC (RP-HPLC; on a L-6200A pump, Merck-Hitachi,

1032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Hess et al.
BTC as the coupling agent in the same way that clarified above.
The last amino acid on the peptidyl-resin (Phe) was acylated with
10 equiv of succinic anhydride in NMP, for 2 h at room temperature,
in the presence of 1 equiv DMAP. The resin was washed with NMP
(2 × 5 min) and DCM (2 × 5 min) and dried overnight in a
desiccator. Alloc protecting group was removed with tetrakis
(triphenylphosphine)Pd(0) (0.1 equiv, 0.066 mmol) in DCM
containing acetic acid (5%) and N-methyl morpholin (2.5%) under
argon. This step was carried out for 4 h with vigorous shaking in
the dark. Washing steps were carried out with chloroform (8 × 2
min) and NMP with 0.5% DIEA (3 × 2 min). The peptide was
cyclized by the addition of 6 equiv of PyBoP and 12 equiv of DIEA
in NMP for 24 h (repeated twice) and then with 3 equiv of PyBoP
and 6 equiv of DIEA in NMP. Washing steps were carried out
with NMP (5 × 2 min) and DCM (5 × 2 min). The peptidyl-resin
was dried under vacuum overnight.
Cleavage from the resin and removal of side chain protecting
groups were carried out simultaneously using a precooled mixture
of 95% TFA, 2.5% TDW, and 2.5% triisopropylsilane (TIS). After
the resin was added, the mixture was agitated for 30 min in an ice
bath, and then was shaken for 2.5 h at RT. The combined TFA
filtrates were evaporated to dryness by a stream of nitrogen. The
oily residue was triturated three times with cold ether to remove
the scavengers, and the ether was removed by centrifugation. The
dry crude peptide was dissolved in ACN/H2O (1:1) and lyophilized.
After final purification on preparative HPLC, product was obtained
as white solid in 92% purity and 35% yield MS m/z 836.4 MH+.
Additional HPLC preparative purification yielded a compound with
a purity of 98.5%, as assessed by NMR analysis.
NMR. Peptide 1 (see structure in Figure 3) was dissolved in
20% TFE-d3, pH 6.25 (5.5 mM 1, 20% TFE-d3, 80% H2O, 0.02%
wt. NaN3). Spectra were recorded at 303.0 K on a Bruker Avance
600 MHz DMX spectrometer operating at the proton frequency of
600.13 MHz using a 5 mm selective probe equipped with a self-
shielded xyz-gradient coil. To characterize the sample, TOCSY,29
COSY,30 and ROESY31,32 experiments were acquired under
identical conditions. TOCSY spectra were recorded using the
MLEV-1729 pulse scheme for the spin lock during mixing periods
of 150 ms and the residual water resonance was suppressed using
the Watergate sequence.33 ROESY was used instead of NOESY
because the latter gave a null signal for this sample at this field.
Water suppression was achieved by continuous wave saturation.
2D spectra were acquired and processed using the XWINNMR
program (Bruker Analytische Messtechnik GmbH) and the
SPARKY34 software was used for analysis. Chemical exchange
among conformers was confirmed by exchange peaks in the ROESY
spectra, which show inverted signs compared to the ROE signals.35
Assignment was done according to the sequential assignment
methodology described by Wüthrich.36
Biochemistry. Preparation of Brush Border Membrane
Vesicles. Brush border membrane vesicles (BBMVs) were prepared
from combined duodenum, jejunum, and upper ileum by a Ca2+
precipitation method.37–39 The intestines of five male Wistar rats,
200–250 gr, were rinsed with ice cold 0.9% NaCl and freed of
mucus; the mucosa was scraped off the luminal surface with glass
slides and put immediately into buffer containing 50 nM KCl and
10 mM Tris-HCl (pH 7.5, 4 °C) and then homogenated (Polytron
PT 1200, Kinematica AG, Switzerland). CaCl2 was added to a final
concentration of 10 mM. The homogenate was left shaking for 30
min at 4 °C and then centrifuged at 10000 g for 10 min. The
supernatant was then centrifuged at 48000 g for 30 min, and an
additional two purification steps were undertaken by suspending
the pellet in 300 mM mannitol and 10 mM Hepes/Tris (pH 7.5)
and centrifuging at 24000 g/hr. Purification of brush border
membranes was assayed using the brush border membrane enzyme
markers γ-glutamyl transpeptidase (GGT), leucine amino peptidase
(LAP), and alkaline phosphatase. During the course of these studies,
enrichment in brush border membrane enzymes varied between 13-
and 18-fold.
time 0 and after 15, 30, 45, 60, and 90 min. The samples were
diluted 1:1 with ice-cold acetonitrile, centrifuged (7500 g, 10 min,
4 °C), and sent to HPLC analysis.
Receptor Binding Assays. The receptor binding assays were
performed by Cerep (France). Generally, transfected CHO cells
were washed with binding buffer and distributed into 96-well plates.
The cells were incubated for 2 h at 37 °C with 0.05 mL binding
buffer in each well, containing a constant concentration of [125I]
NDP-R-MSH, and appropriate concentrations of an unlabeled
ligand. After incubation, the cells were washed with ice-cold binding
buffer and detached from the plates with 0.1 N NaOH. Radioactivity
is counted and the data were analyzed with a software package for
radio-ligand binding analyses by fitting it to formulas derived from
the law of mass-action by the method generally referred to as
computer modeling. The binding assays were performed in duplicate
wells.
Functional Bioassay. HEK-293 cells stably expressing the
melanocortin receptors were transfected with 4 µg of CRE/ꢀ-
galactosidase reporter gene, as previously described.8 Briefly,
5000–15000 post-transfection cells were plated into 96-well Primera
plates (Falcon) and incubated overnight. A total of 48 h post-
transfection, the cells were stimulated with 100 µL of peptide
(10-4-10-12 M) or forskolin (10-4 M) control in assay medium
(DMEM containing 0.1 mg/mL BSA and 0.1 mM isobutylmeth-
ylxanthine) for 6 h. The assay media was aspirated and 50 µL of
lysis buffer (250 mM Tris-HCl, pH 8.0, and 0.1% Triton X-100)
was added. The plates were stored at -80 °C overnight. The plates
containing the cell lysates were thawed the following day. Aliquots
of 10 µL were taken from each well and transferred to another
96-well plate for relative protein determination. To the cell lysate
plates, 40 µL of phosphate-buffered saline with 0.5% BSA was
added to each well. Subsequently, 150 µL of substrate buffer (60
mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM
ꢀ-mercaptoethanol, and 200 mg ONPG) was added to each well,
and the plates were incubated at 37 °C. The sample absorbance,
OD 405 nm, was measured using a 96-well plate reader (Molecular
Devices). The relative protein was determined by adding 200 µL
of 1:5 dilution BioRad G250 protein dye/water to the 10 µL cell
lysate sample taken previously, and the OD 595 nm was measured
on a 96-well plate reader (Molecular Devices). Data points were
normalized both to the relative protein content and to the nonre-
ceptor-dependent forskolin stimulation. The pA2 ((Ki) - log pA2)
values were generated using the Schild analysis method.40
In Vitro Permeability Study. Growth and Maintenance of
Cells. Caco-2 cells were obtained from ATCC, (Manassas, VA,
U.S.A.) and then grown in 75 cm2 flasks with approximately 0.5
× 106 cells/flask at 37 °C in 5% CO2 atmosphere and at a relative
humidity of 95%. The culture growth medium consisted of
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
10% heat-inactivated fetal bovine serum (FBS), 1% nonessential
amino acids, and 2 mM L-glutamine. The medium was replaced
twice weekly. All medium supplements were purchased from
Biological Industries, Beth-Haemek, Israel.
Preparation of Cells for Transport Studies. For transport
studies, cells in a passage range of 60–66 were seeded at a density
of 25 × 105 cells/cm2 on untreated culture inserts of polycarbonate
membrane with 0.4 µm pores and surface area of 1 cm2. The culture
inserts containing Caco-2 monolayers were placed in 24 transwell
plates, 12 mm, Costar. The culture medium was changed every
other day. Transport studies were performed 21–23 days after
seeding, when the cells were fully differentiated and the TEER
(trans epithelial electrical resistance) values were stable (300–500
Ωcm2).
Experimental Protocol. Transport study was initiated by
medium removal from both sides of the monolayer and replacement
with apical buffer (550 µL) and basolateral buffer (1200 µL), both
warmed to 37 °C. The cells were incubated for a 30 min period at
37 °C with shaking (100 cycles/min). After the incubation period,
the buffers were removed and replaced with 1200 µL of basolateral
buffer at the basolateral side. Test solutions were warmed previously
to 37 °C and added (600 µL) to the apical side of the monolayer.
The tested molecule was mixed with purified BBMVs and
incubated in 37 °C for 90 min. Duplicate samples were taken at

MC4R Peptidomimetic Agonist for Treating Obesity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1033
(3) Cummings, D. E.; Schwartz, M. W. Melanocortins and body weight:
A tale of two receptors. Nat. Genet. 2000, 26, 8–9.
(4) Jonsson, L.; Skarphedinsson, J. O.; Skuladottir, G. V.; Watanobe, H.;
Schioth, H. B. Food conversion is transiently affected during 4-week
chronic administration of melanocortin agonist and antagonist in rats.
J. Endocrinol. 2002, 173, 517–523.
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al. The melanocortin melanocyte-stimulating hormone/adrenocorti-
cotropin(4–10) decreases body fat in humans. J. Clin. Endocrinol.
Metab. 2001, 86, 1144–1148.
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Characterization of melanocortin NDP-MSH agonist peptide fragments
at the mouse central and peripheral melanocortin receptors. J. Med.
Chem. 2001, 44, 2247–2252.
(7) Haskell-Luevano, C.; Sawyer, T. K.; Hendrata, S.; North, C.; Panahinia,
L.; et al. Truncation studies of alpha-melanotropin peptides identify
tripeptide analogues exhibiting prolonged agonist bioactivity. Peptides
1996, 17, 995–1002.
(8) Haskell-Luevano, C.; Lim, S.; Yuan, W.; Cone, R. D.; Hruby, V. J.
Structure activity studies of the melanocortin antagonist SHU9119
modified at the 6, 7, 8, and 9 positions. Peptides 2000, 21, 49–57.
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al. The melanocortin melanocyte-stimulating hormone/adrenocorti-
cotropin(4–10) decreases body fat in humans. J. Clin. Endocrinol.
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(10) Okumu, F. W.; Pauletti, G. M.; Vander Velde, D. G.; Siahaan, T. J.;
Borchardt, R. T. Effect of restricted conformational flexibility on the
permeation of model hexapeptides across Caco-2 cell monolayers.
Pharm. Res. 1997, 14, 169–175.
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cyclization: A new method for conferring conformational constraint
on peptides. Biopolymers 1991, 31, 745–750.
(12) Byk, G.; Halle, D.; Zeltser, I.; Bitan, G.; Selinger, Z. Synthesis and
biological activity of NK-1 selective, N-backbone cyclic analogs of
the C-terminal hexapeptide of substance P. J. Med. Chem. 1996, 39,
3174–3178.
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Backbone cyclic peptide, which mimics the nuclear localization signal
of human immunodeficiency virus type 1 matrix protein, inhibits
nuclear import and virus production in nondividing cells. Biochemistry
1998, 37, 5616–5622.
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potent suppression of growth hormone but not of insulin. Endocrinol-
ogy 2001, 142, 477–486.
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peptidomimetic Melanocortin-4 receptor agonist, PCT/IL 2006/
000640, 2006.
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Backbone metal cyclization: novel Tc-99m labeled GnRH analog as
potential SPECT molecular imaging agent in cancer. Nucl. Med. Biol.
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and metabolism of peptide drugs: Implication for oral peptide drug
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Samples (50 µL) were taken from the apical side immediately at
the beginning of the experiment, resulting in a 550 µL apical volume
during the experiment. For the duration of the experiment, the cells
were kept at 37 °C with shaking. At predetermined times (30, 60,
90, 120, 150, and 180 min), 200 µL samples were taken from the
basolateral side and replaced with the same volume of fresh
basolateral buffer to maintain a constant volume.
Data Analysis. The permeability coefficient (Papp) for each
compound was calculated from the linear plot of drug accumulated
versus time, using the following equation: Papp ) dQ/dt
/
(C0×A), where dQ/dt is the steady state rate of the appearance of
the drug on the receiver side, C0 is the initial concentration of the
drug on the donor side, and A is the surface area, 1.1 cm2.
In Vivo Studies. All surgical and experimental procedures were
reviewed and approved by the Animal Experimentation Ethics
Committee of the Hebrew University Hadassah Medical Center,
Jerusalem.
Pharmacokinetic Study. Studies were performed in conscious
wistar male rats using jugular vein. An indwelling cannula was
implanted 24 h before the pharmacokinetic experiment to allow
full recovery of the animals from the surgical procedure. Animals
(n ) 5) received an iv bolus dose of 1 mg/kg of peptide 1 or 10
mg/kg for po administration (n ) 2) dissolved in water. Blood
samples (with heparin, 15 U/ml) were collected at several time
points up to 24 h after peptide 1 administration. Plasma was
separated by centrifugation (4000 g, 5 min, 4 °C) and stored at
-70 °C pending analysis. Noncompartmental pharmacokinetic
analysis was performed using WinNonlin software, standard edition
version 5.0.1 (Scientific Consulting, Inc., Cary, NC).
Pharmacodynamic Study. ICR:Hsd (CD-1) male mice, 7–8 wks
old were maintained in separate cages at 23 ( 1 °C on a 12 h
light, 12 h dark cycle (0700–1900 h light). Mice were allowed ad
libitum access to water and standard chow pellets. Upon arrival,
mice were allowed to acclimate for 1 week. Following fasting for
16 h, the animals (n ) 24) were subjected to a single oral gavage
(po, 5 mL/kg) of peptide 1 (100 µg/ml) or vehicle (water).
Immediately after administration, fixed food doses were added and
reweighed after 1, 2, 3, 4, and 5 h. For chronic administration of
peptide 1, animals (3–4 wk) were weighed daily and then subjected
to oral gavage (po, 5 mL/kg) of 1 (100 µg/ml) or vehicle (water).
Bioanalysis of Peptide 1. Brain Samples. The initial extraction
of peptide 1 from the tissue was by single protein precipitation
step using strong extraction reagent (A): 90% acetonitrile/10%
formic acid and 50 mM ammonium formate thereafter; further
purification of the analyte fraction was undertaken by adding another
extraction step using reagent (A) diluted 2-fold with water. Peptide
2 was added to the brain samples as an internal standard (IS) prior
to the extraction. Following the second extraction step, peptides 1
and 2 were analyzed successfully. Analysis of peptide 1 in rat brain
using the API-3200 LC-MS/MS was developed and shown to be
linear, accurate, reproducible, specific, and with high extraction
recovery from rat brain.
Plasma/Buffer Samples. This study was performed using a
HPLC-MS Waters Millenium instrument equipped with Micromass
ZQ detector, Waters 600 Controller gradient pump, and Waters 717
auto sampler. Nitrogen flow was 500 L/hr; source temperature was
400 °C; the cone voltage was 60 V; the column was Xterra MS
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the transcellular and paracellular pathway to the transport of passively
absorbed drugs in the Caco-2 cell culture model. Pharm. Res. 1997,
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drugs: Barriers and developments. BioDrugs 2005, 19, 165–177.
(22) Gilon, C.; Mang, C.; Lohof, E.; Friedler, A.; Kessler, H. Synthesis of
Cyclic Peptides. Houben-Weyl: Methods of Organic Chemistry,
Synthesis of Peptides and Peptidomimetics Georg Thieme Verlag:
Stuttgart, 2003;pp 461–542.
C
18 2.1 × 150 mm (Waters). The mobile phase at 0.3 mL/min was
30% acetonitrile, 0.1% formic acid, and 0.05% TFA, with a linearity
range of 0.025–1 µg/mL.
(23) Chatterjee, J.; Mierke, D.; Kessler, H. N-Methylated cyclic pentaalanine
peptides as template structures. J. Am. Chem. Soc. 2006, 128, 15164–
15172.
Acknowledgment. This work is part of Shmuel Hess and
Yaniv Linde Ph.D. dissertations. This work was funded in part
by a grant of the Israel Science Foundation.
(24) Ritschel, W. A. Handbook of Basic Pharmacokinetics, 4th edition;Drug
Intelligence Publications, Inc.: Hamilton, IL, 1992; pp 213-216.
(25) Cowley, M. A.; Smart, J. L.; Rubinstein, M.; Cordan, M. G.; Diano,
S.; et al. Leptin activates anorexigenic POMC neurons through a neural
network in the arcuate nucleus. Nature 2001, 411, 480–484.
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et al. Hypothalamic localization of the feeding effect of agouti-related
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