Rationally designed cyclic analogues of luteinizing hormone-releasing hormone: Enhanced enzymatic stability and biological properties

Article abstract of DOI:10.1016/j.ejmech.2012.09.043

This article describes the rational design, synthesis and pharmacological properties of amide-linked cyclic analogues of Luteinizing Hormone-Releasing Hormone (LHRH) with substitutions at positions 1 (Pro), 6 (d-Leu/d-Trp), 9 (Aze) and 10 (BABA/Acp). These LHRH analogues fulfil the conformational requirements that are known in the literature (bend in the 5-8 segment) to be essential for receptor recognition and activation. Although, they are characterised by an overall low binding affinity to the LHRH-I receptor, the cyclic analogues that were studied and especially the cyclo(1-10)[Pro¹, d-Leu⁶, BABA¹⁰] LHRH, exhibit a profoundly enhanced in vitro and in vivo stability and improved pharmacokinetics in comparison with their linear counterpart and leuprolide. Upon receptor binding, cyclo(1-10)[Pro¹, d-Leu⁶, BABA¹⁰] LHRH causes testosterone release in C57/B16 mice (in vivo efficacy) that is comparable to that of leuprolide. Testosterone release is an acutely dose dependent effect that is blocked by the LHRH-I receptor antagonist, cetrorelix. The pharmacokinetic advantages and efficacy of cyclo(1-10)[Pro¹, d-Leu⁶, BABA¹⁰] LHRH render this analogue a promising platform for future rational drug design studies towards the development of non-peptide LHRH mimetics.

Full text of DOI:10.1016/j.ejmech.2012.09.043

European Journal of Medicinal Chemistry 58 (2012) 237e247
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rationally designed cyclic analogues of luteinizing hormone-releasing hormone:
Enhanced enzymatic stability and biological properties
,
,
,
d,
Despina Laimou a 1, Theodora Katsila b 1, John Matsoukas a , Andrew Schally e, Kostas Gkountelias c,
*
George Liapakis c, Constantin Tamvakopoulos b, Theodore Tselios a
,
*
a Department of Chemistry, University of Patras, GR-26500 Patras, Greece
b Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation, Academy of Athens, Athens 11527, Greece
c Department of Pharmacology, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece
d Veterans Affairs Medical Center, University of Miami, Miller School of Medicine, Miami, FL 33125, USA
e Departments of Pathology and Medicine, Division of Hematology/Oncology, University of Miami, Miller School of Medicine, Miami, FL 33125, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This article describes the rational design, synthesis and pharmacological properties of amide-linked
cyclic analogues of Luteinizing Hormone-Releasing Hormone (LHRH) with substitutions at positions 1
(Pro), 6 (D-Leu/D-Trp), 9 (Aze) and 10 (BABA/Acp). These LHRH analogues fulfil the conformational
Received 28 February 2012
Received in revised form
24 September 2012
Accepted 27 September 2012
Available online 4 October 2012
requirements that are known in the literature (bend in the 5e8 segment) to be essential for receptor
recognition and activation. Although, they are characterised by an overall low binding affinity to the
LHRH-I receptor, the cyclic analogues that were studied and especially the cyclo(1-10)[Pro1, -Leu6,
D
BABA10] LHRH, exhibit a profoundly enhanced in vitro and in vivo stability and improved pharmacoki-
netics in comparison with their linear counterpart and leuprolide. Upon receptor binding, cyclo(1-10)
Keywords:
Amide linked cyclization
Hormone dependent cancer
Cyclic peptide analogues
Luteinizing hormone-releasing hormone
Medicinal chemistry
[Pro1,
D
-Leu6, BABA10] LHRH causes testosterone release in C57/B16 mice (in vivo efficacy) that is
comparable to that of leuprolide. Testosterone release is an acutely dose dependent effect that is blocked
by the LHRH-I receptor antagonist, cetrorelix. The pharmacokinetic advantages and efficacy of cyclo(1-
10)[Pro1, -Leu6, BABA10] LHRH render this analogue a promising platform for future rational drug design
D
studies towards the development of non-peptide LHRH mimetics.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
LHRH-I receptor agonists (triptorelin, leuprolide, buserelin, goser-
elin, nafarelin, histrelin) and antagonists (degarelix, cetrorelix),
which are used for the treatment of endocrine disorders and
hormone-dependent cancers. In addition, in the past few years
diverse cytotoxic analogues targeting the LHRH receptor (cytotoxic
radicals, such as doxorubicin, coupled to LHRH analogues that
function as carriers) have been synthesized and evaluated with the
intention of targeted chemotherapy [7,8]. In particular, leuprolide
Luteinizing hormone-Releasing hormone (LHRH) (pGlu1-
His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly10-NH2) regulates the
synthesis and release of luteinizing hormone (LH) and follicle-
stimulating hormone (FSH), which in turn modulates the secretion
of sex steroids. Thus, LHRH plays a vital role in reproduction and it
is implicated in various pathophysiological processes, involving
hormone-dependent cancers and endocrine disorders [1e6].
An intense systematic approach has resulted in the synthesis of
[D
-Leu6, NHEt10] LHRH, which is a synthetic altered peptide ligand
(APL) of LHRH with agonistic activity has been shown to suppress
LH and testosterone secretion in prostate cancer patients [9]. Thus,
leuprolide could be a good starting point for the rational drug
design of cyclic peptide analogues and non-peptide mimetics,
towards the next generation of potent LHRH-I receptor agonists and
antagonists that will share an improved pharmacokinetics/phar-
macodynamics profile.
LHRH receptor agonists currently in clinical use, including leu-
prolide, are superior to the native hormone in terms of potency,
because of their high receptor affinity and relatively improved
Abbreviations: Acp, aminocaproic acid; Aze, azetidine; BABA, 3-aminobutyric
acid; DTT, dithiothreitol; EPI, enhanced product ion; LHRH, luteinizing hormone-
releasing hormone; HPLC, high pressure liquid chromatography; HSA, human
serum albumin; IP, intraperitoneal; IS, internal standard; LCeMS/MS, liquid
chromatographyetandem mass spectrometry; MRM, multiple reaction moni-
toring; PBS, phosphate-buffered saline; QC, quality control; QqLIT, triple quadru-
pole hybrid linear ion trap; SD, standard deviation.
* Corresponding authors. Tel.: þ30 2610997905; fax: þ30 2610997118.
E-mail addresses: imats@chemistry.upatras.gr (J. Matsoukas), ttselios@
upatras.gr (T. Tselios).
proteolytic stability. However, they still suffer
a
poor
1
Both authors have contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.043

238
D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
pharmacokinetic profile that consists of limited absorption, low
bioavailability and noteworthy, susceptibility to the action of
proteolytic enzymes. Consequently, these peptides are adminis-
tered intramuscularly or subcutaneously and as depot formula-
tions, compromising the patient well-being and efficacy (since it is
dependent on patient compliance). Thus, increasing the stability of
analogues would be advantageous, possibly either by allowing
a reduction in dosing frequency or by allowing the use of analogues
that act in situ, with possible additive effects and subsequent
enhancements in efficacy.
The proteolysis of leuprolide is associated with the cleavage of
Trp3-Ser4 and Ser4-Tyr5 amide bonds in the presence of kidney
membrane preparations (neutral endopeptidase e EC 3.4.24.11,
angiotensin converting enzyme e EC 3.4.15.1) [10e12]. Thus, based
on our knowledge and characterisation of leuprolide, we proceeded
to the synthesis of cyclic peptides that are more resistant to
proteolysis by virtue of their structure. The advantages of using
cyclic analogues versus their linear counterparts are the following:
(i) the conformation of a cyclic analogue is locked compared to the
conformational flexibility characterizing their linear counterparts,
(ii) a cyclic analogue is more stable and it is therefore resistant to
enzymatic degradation, (iii) the synthesis of a cyclic analogue is an
important intermediate step towards the rational design and
development of a non-peptide mimetic for oral administration, (iv)
the cyclization of amino acid sequences likely results in increased
bioavailability and receptor selectivity and consequently in a better
pharmacological profile.
and activity. BABA or Acp was used as anchor for amide linked
cyclization with the Pro N-terminal residue and for their methylene
lipophilic groups as in the ethylamide of leuprolide. In analogues 3
and 4 the backbone size was increased compared to the cyclic
analogues 1 and 2 by extending the backbone length with Acp. The
rationale of our approach is that novel LHRH peptide analogues
with a profound pharmacokinetic advantage that is particularly
valuable with respect to local/direct effects can serve as therapeutic
alternatives for the treatment of endocrine disorders and hormone-
dependent cancers. Indeed, previous studies have further sup-
ported our aims [12].
2. Results and discussion
The therapeutic potential of LHRH analogues for the treatment
of hormone-dependent cancers and endocrine disorders derives
from a number of findings [8,39]. Although extensive research has
been performed in the field of hormonal therapy, LHRH peptide
analogues are still characterized by poor pharmacokinetic proper-
ties. Poor stability of LHRH analogues necessitates their subcuta-
neous administration (depot formulations) and leads to several side
effects (leukocytoblastic vasculitis, injection site granulomas). We
hypothesize that stable analogues of leuprolide (superagonist)
would be advantageous, either by allowing a reduction in dosing
frequency or by allowing the use of analogues that act in situ, with
possible additive effects and subsequent enhancements in efficacy.
Several approaches can be employed in the drug development
process aiming to rational design of novel LHRH analogues. One is
the design and synthesis of non-peptide mimetics, either agonists
or antagonists. Another is the design of constrained cyclic
analogues. Cyclic analogues of LHRH offer several advantages, such
as increased resistance to metabolic degradation and conforma-
tional restriction compared to their linear counterparts. Further-
more, a bioactive constrained cyclic peptide analogue provides
important structural information regarding the bioactive confor-
mation and the pharmacophoric groups for rational drug discovery
and development. The novel cyclic analogues reported herein were
designed based on the conformational properties of LHRH, which
adopts a U-shaped conformation, when it approaches and binds to
the LHRH-I receptor and synthesized by applying a novel cycliza-
Several modified LHRH analogues (LHRH-I receptor agonists
[13] or antagonists [14,15]) have been designed and synthesized,
using non-natural amino acids [16], cyclization and in some cases
dicyclization [17,18]. Variable substitutions have been involved in
almost all positions, especially in the N-terminal, leading to the
synthesis of antagonists [19,20]. Our experience in cyclization
procedures has been applied previously towards the development
of novel analogues of angiotensin (ANG II) [21,22], myelin basic
protein (MBP) [23e33] and thrombin receptor peptides (TRAPS)
[34]. Our interest and expertise have led us to pursue the synthesis
of rationally designed cyclic LHRH analogues as well as the design
of non-peptide mimetics to be used in drug therapy. Examples of
non-peptide mimetics are the Sartans which mimic the peptide
hormone angiotensin [35] and the mimetics of TRAPS [36].
Previously [37], we described the design, synthesis, molecular
modelling and gonadotropin gene expression of the stable cyclic
tion strategy. The introduction of a
D amino acid at position 6 (such
as -Leu) in analogues 1e4 or -Trp in 5, intended to stabilize the U-
D
D
shaped conformation of LHRH, a conformation that has been
previously shown to improve the binding affinity for the LHRH-I
receptor [40e44]. For cyclization purposes Pro at position 1
replaced pGlu, which links the carboxyl group. The ethylamide
group of leuprolide was replaced by BABA in order to conserve the
ethylamide C-terminal as well as the carboxyl group, which is
important for cyclization. Furthermore, in the case of analogues 2
and 5, the non-natural amino acid azetidine (Aze, four member
ring) at position 9 was used in order to improve binding to the
LHRH-I receptor and further stabilize conformational integrity.
Cyclic analogues 3 and 4 contain Acp as a bridge between the N-
and C-terminal, since this modification has been shown to promote
the adaptation of the correct conformation for binding to and
activation of the LHRH-I receptor. Finally, the substitution of Tyr5
with Tyr(OMe), in the case of analogues 2, 4 and 5 could possibly
create analogues that desensitize the LHRH-I receptor to a much
lower degree than LHRH as has been previously shown [38].
LHRH analogue cyclo(4-9)[Lys4,
D
-Trp6, Glu9] LHRH. This cyclic
analogue stimulated gonadotropin gene expression in the goldfish
pituitary with potency similar to the native hormone. A clustering
of the three aromatic rings (His2, Trp3 and Tyr5), considered
important for triggering receptor activation, was further shown to
be important based on modern ROE NMR [38]. The design of the
cyclic analogue was aimed at retaining a b-turn mimetic through 4,
9 residue amide cyclization and also the three aromatic rings (His2,
Trp3 and Tyr5), important for receptor activation.
This paper reports a novel cyclization strategy for the design and
synthesis of novel leuprolide analogues, cyclized at the C- and N-
terminal, retaining the basic backbone sequence of leuprolide. The
analogues synthesized were: cyclo(1-10)[Pro1,
D
-Leu6, BABA10
-Leu6, Aze9, BABA10] LHRH
-Leu6, BABA10, Acp11] LHRH (3), cyclo(1-11)
-Leu6, BABA10, Acp11] LHRH (4), cyclo(1-10)[Pro1,
-Trp6, Aze9] LHRH (5). We replaced the N-terminal
]
LHRH (1), cyclo(1-10)[Pro1, Tyr(OMe)5,
(2), cyclo(1-11)[Pro1,
[Pro1, Tyr(OMe)5,
Tyr(OMe)5,
D
D
D
D
pGlu with the cyclic residue Pro which bears the amino group
available for cyclization with the C-terminal carboxyl group of 3-
aminobutyric acid (BABA) or aminocaproic acid (Acp) as the C-
2.1. Chemistry
The synthesis of the described cyclic analogues is schematically
depicted in Fig. 1. The linear protected counterparts of cyclic
analogues were synthesized by the Fmoc/tBu methodology,
terminal amino acids in place of ethylamide. It is known that
D-Leu
in position 6 forms a -turn in the sequence necessary for binding
b

D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
239
Fig. 1. Synthetic procedure of the studied LHRH cyclic analogues: a) Fmoc-X-OH, X: BABA (1), (2) or Acp (3), (4) or Gly (5), DIPEA, DCM, 1 h, RT; b) Fmoc deprotection with 20%
piperidine in DMF, 35 min, RT; c) step by step Fmoc/tBu synthesis using DIC/HOBt and Fmoc-BABA-OH (3), (4) or Fmoc-Pro-OH (1), (3), (4) or Fmoc-Aze-OH (2), (5), Fmoc-Arg(Pbf)-
OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH (1), (2), (3), (4) or Fmoc-D-Trp-OH (5), Fmoc-Tyr(OMe)-OH (2), (4), (5) or Fmoc-Tyr(tBu)-OH (1), (3), Fmoc-Ser(tBu)-OH, Fmoc-Trp-OH, Fmoc-
His(Trt)-OH, Fmoc-Pro-OH, 4e6 h, RT; d) after each coupling, Fmoc deprotection with 20% piperidine in DMF, 35 min, RT; e) cleavage from resin with DCM/HFIP (8/2), 2 h, RT; f)
cyclization with TBTU (3ꢃ), HOAt (3ꢃ) and collidine (6ꢃ) in dry DMF, 16 h, RT; g) deprotection with 65% TFA in DCM and 3% EDT, 4 h, RT.
utilizing the 2-chlorotrityl chloride (CLTR-Cl) resin [23e26,36e
38,45e47]. The side chain of the amino acids was protected as
follows: Trt for His, Pbf for Arg, tBu for Ser and Tyr. Tyr was used as
Tyr (OMe) in the case of the cyclic analogues 2, 4 and 5. The pro-
tected peptides were cleaved from the resin using the cleavage
mixture DCM/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (8/2) in
order to conserve the Trt protection of the imidazole group of His
and avoid acetylation. The use of the CLTR-Cl resin and mild
cleaving conditions allowed peptide release from the resin as well
as the subsequent cyclization of the desired protected peptides. The
amide bond cyclization for each analogue was established using O-
benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium tetrafluoroborate
(TBTU), 1-hydroxy-7-azabenzotriazole (HOAt) and collidine (2,4,6-
trimethylpyridine) in DMF as cyclization reagents for the highest
yield reactions (w60%). The deprotection of cyclic analogues was
performed with 65% TFA in the presence of 3% 1,2-ethanedithiole
(EDT) in DCM. The free cyclic analogues were purified by semi
preparative RP-HPLC with a Waters system equipped with a 600E
controller and a Waters 996 photodiode array UV detector. The
analysis was controlled by an operating Millenium 2.1 system.
Peptide final purity was ꢀ98%. Peptide structures were confirmed
by Electrospray Ionization-Mass Spectrometry (ESI-MS). ESI-MS
experiments were performed with a TSQ 7000 spectrometer
(Electrospray Platform LC of Micromass) coupled to a MassLynx NT
2.3 data system. The novel synthetic approach described herein
resulted in high yield final cyclic products as mild conditions were
employed for coupling, cleavage from the resin, cyclization as well
as for the final deprotection.
expressing the human LHRH receptor and using [125I- Tyr6, His5]
D
LHRH as the radioligand. Leuprolide served as a control. In this
study, all analogues evaluated were bound to the human LHRH-I
receptor in a dose-dependent manner. Notably, the binding affinity
of 1 was 2-fold, 4-fold, 7-fold and 4-fold higher, than that of 2
(ꢁlog IC50 ¼ 5.82 ꢂ 0.15), 5 (ꢁlog IC50 ¼ 5.58 ꢂ 0.25), 3
(ꢁlog IC50 ¼ 5.31 ꢂ 0.02) and 4 (ꢁlog IC50 ¼ 5.54 ꢂ 0.26), respec-
tively. Analogue 1 bound to LHRH-I receptor with lower, yet bio-
logically considerable, affinity (ꢁlog IC50 ¼ 6.17 ꢂ 0.15) compared to
leuprolide (ꢁlog IC50 ¼ 9.12 ꢂ 0.19). We believe that this finding
results by the low flexibility and orientation of the right side chains
of the synthesized cyclic analogues, which are significant for the
interactions with the LHRH receptor [32]. Overall, the results
suggest that the biological activity of 1 (see below Section 4.2.6) is
mediated through binding on the LHRH-I receptor. A property of 1
that is superior to the other cyclic analogues is studied. Data are
depicted in Fig. 2 and summarized in Table 1.
2.3. Stability of novel cyclic LHRH analogues
Peptide stability was evaluated both in vitro and in vivo. In vitro
stability data were acquired following the incubation of the
analogues 1e5 as well as the native hormone (LHRH) with mouse
kidney membrane preparations on the basis that kidney is the
major site for the metabolism of LHRH and the analogues [10e12].
Leuprolide served as a control. After extraction and detection by the
novel LCeMS/MS methodology developed, the peak areas for the
analogues 1e5 and LHRH were set as 100% at t ¼ 0. Those peak
areas were subsequently compared with the peak areas derived
from samples at t ¼ 30 min, t ¼ 60 min, and t ¼ 120 min. The native
decapeptide LHRH showed an extensive in vitro degradation within
30 min of incubation. Following 30 min of incubation with mouse
kidney membranes, only 3% of leuprolide remained intact. All cyclic
analogues tested were significantly more stable compared to leu-
prolide and/or the native hormone. In particular, 90% of analogues 2
2.2. Binding of cyclic LHRH analogues to the LHRH-I receptor
The binding affinities (ꢁlog IC50) of the novel cyclic LHRH
analogues reported herein for the human LHRH-I receptor were
determined from competition experiments performed under
equilibrium conditions in membranes from HEK 293 cells stably

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D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
Fig. 2. Competition binding isotherms of LHRH analogues to human LHRH-I receptor. Competition of [125I-D-Tyr6, His5] LHRH specific binding by increasing concentrations of cyclic
analogues 1e5 or leuprolide was performed on membranes from HEK 293 cells stably expressing the human LHRH-I receptor. The mean values and S.E. are shown from 3 to 4
different experiments. The data were fit to a one-site competition model by nonlinear regression and the IC50 values were determined as described.
and 4 remained intact at t ¼ 30 min, compared to 72% and 68% of 3
and 5 (at t ¼ 30 min), respectively. In all cases, the susceptible
bonds were found to be Ser4-Tyr5 and Tyr5-Gly6 (LHRH) and/or the
substituted amino acids at the same positions, when Leuprolide or
its analogues are considered. In every case, a carboxy-terminal
tripeptide and an amino-terminal pentapeptide were yielded, with
an amino acid sequence depended on the modifications made. This
proteolysis event was not totally aborted in none of the analogues
tested, however it is deemed to affect the degradation rates ob-
tained. The susceptibility of the aforementioned amide bonds has
been also confirmed employing our in vitro system (mouse kidney
membrane preparations) towards the evaluation of linear Leupro-
lide analogues [12]. Overall, 1 showed a profound in vitro stability
compared to its cyclic counterparts, leuprolide and LHRH, since
100% of peptide was intact at t ¼ 30 min (and till the end of the
study, at t ¼ 120 min). Data are depicted in Fig. 3A and summarized
in Table 1. This in vitro system also allowed compound ranking of
selected LHRH analogues (in vitro screen).
the basis of all the pharmacokinetic parameters determined
(Table 2). Plasma peptide concentrations determined at all time
points for both peptides tested showed relatively low intersubject
variation. Early on in our screening efforts a strong linear positive
correlation was established between our in vitro (incubation of
peptides with mouse kidney membranes) and in vivo approach
(administration of peptide in mice and plasma monitoring as
a function of time) towards the evaluation of peptide stability
(R2 ¼ 0.946 at t ¼ 30 min, where R2 is the squared Pearson’s
correlation coefficient). The correlation coefficient ranges
from ꢁ1.0 to þ1.0, and represents the fraction of the variance in the
two variables that is shared.
Analogue 1 was selected among its cyclic counterparts to be
further evaluated in vivo on the basis of (i) in vitro stability data,
showing profound peptide stability upon incubation with mouse
kidney membranes (kidney is the major metabolising site for LHRH
and analogues) and (ii) binding data, as its binding affinity is
superior to that of its cyclic counterparts (Fig. 2). It should be also
considered that the experimental strategy described herein was
totally designed according to the “three Rs” (or “alternatives”) e
reduction, refinement and replacement e of animal use in life
sciences, linking scientific excellence and humane use of laboratory
animals. In this regard, the choice of cyclic analogue 1 testing
in vivo was strongly supported by in vitro data (in vitro screens),
suggesting its superiority compared to its counterparts.
A pharmacokinetic study was conducted in mice for the evalu-
ation of the in vivo stability of 1. In parallel, the pharmacokinetic
profile of leuprolide was evaluated (served as a control). A graphical
representation of peptide concentrations for both 1 and leuprolide
in mouse plasma at t ¼ 1 h, t ¼ 2 h, t ¼ 4 h, and t ¼ 6 h post-dose is
depicted in Fig. 3B. The novel cyclic analogue 1 exhibited a signifi-
cant pharmacokinetic advantage, when compared to leuprolide on
Table 1
Amino acid sequence, ꢁlog IC50 values and peptide stability data for the novel cyclic LHRH analogues reported herein.
Peptides
Amino acid sequence
ꢁlog IC50
Peptide stability
at t ¼ 30 min
LHRH
leuprolide
Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
9.12 ꢂ 0.19
Highlyunstable 3%
(Des-Gly10 -Leu6,Pro-NHEt9) LHRH
, D
(1)
(2)
(3)
(4)
(5)
Cyclo(1-10)[Pro1,
D
-Leu6, BABA10] LHRH
-Leu6, Aze9, BABA10] LHRH
6.17 ꢂ 0.15
5.82 ꢂ 0.15
5.31 ꢂ 0.02
5.54 ꢂ 0.26
5.58 ꢂ 0.25
100%
90%
72%
90%
68%
Cyclo(1-10)[Pro1, Tyr(OMe)5,
D
Cyclo(1-11)[Pro1, -Leu6, BABA10, Acp11] LHRH
D
Cyclo(1-11)[Pro1, Tyr(OMe)5,
Cyclo(1-10)[Pro1, Tyr(OMe)5,
D
-Leu6, BABA10, Acp11] LHRH
-Trp6, Aze9] LHRH
D

D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
241
Fig. 3. A. In vitro peptide stability of novel cyclic peptides in question when compared to leuprolide and LHRH following incubation with mouse kidney membranes. B. In vivo
peptide stability (pharmacokinetic study) of the cyclic analogue 1 following i.p. administration in male C57Bl/6 mice (n ¼ 5). Values are represented as the mean ꢂ SD.
The significant in vitro and in vivo peptide stability of 1 is
a critical result, suggesting that modifications at positions 5 (tyro-
sine methylation) and 9 (azetidine instead of proline) enhance
stability against proteolysis. In comparison with 1, leuprolide was
hydrolysed at a faster rate by our in vitro system and was cleared
rapidly from the plasma following i.p. dosing in mice. It should be
endopeptidase e EC 3.4.24.11 and angiotensin converting
enzyme e EC 3.4.15.1 [10e12]. Nevertheless, data from studies in
our laboratory (not shown) suggest that the replacement of proline
with azetidine at position 9 has an additive effect when it comes to
the peptide stability of 1.
noted that the enhanced peptide stability observed with
1
2.4. Functional agonism of the LHRH-I receptor (testosterone
compared to that of leuprolide is primarily due to methylation on
the tyrosine residue, presumably due to steric hindrance at the site
of cleavage (5e6 position) by endopeptidases, such as neutral
quantification in mouse plasma following IP dosing)
An efficacy study was conducted in which testosterone release
was monitored upon the intraperitoneal acute administration of 1
in C57/B16 mice (agonism of the LHRH-I receptor). Testosterone is
a key hormone that serves in males as a biomarker of efficacy of
LHRH agonists that acutely increase LH and testosterone levels,
whereas they inhibit the release of these hormones after their long-
term administration by down regulation of pituitary LHRH recep-
tors. Analogue 1 was selected among its cyclic counterparts to be
further evaluated in vivo on the basis of its binding data and
significant pharmacokinetic advantage. In this study, leuprolide
Table 2
The pharmacokinetic parameters of analogue 1, compared to those of leuprolide.
Parameters
Analogue 1
Leuprolide
Cmax
Tmax
834 ng mLꢁ1
1 h
190 ng mLꢁ1
1 h
Elimination rate constant
Half life
Area under the curve (AUC)
Clearance (CL)
1 hꢁ1
1 hꢁ1
0.5 h
0.7 h
808 ng h mLꢁ1
0.001 mL hꢁ1
138 ng h mLꢁ1
0.007 mL hꢁ1
served as
a control. Testosterone plasma concentrations as

242
D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
a function of time (t ¼ 1e6 h post-dose) are shown in Fig. 4A.
Testosterone measurements in vehicle-treated mice were low as
expected and in agreement with our previous studies [11,12]. In
comparison, testosterone concentrations in leuprolide-treated
mice were significantly higher at t ¼ 1e6 h post-dose (***, p < 0.001
vs. vehicle-treated mice). Testosterone release due to 1 dosing was
suggestive of agonism (***, p < 0.001 vs. vehicle-treated mice at
t ¼ 1 h, t ¼ 2 h, t ¼ 4 h post-dose), suggesting functional agonism of
the LHRH-I receptor. Testosterone AUC1e6 h values were equal to
244.1 and 61.1 ng h mLꢁ1 for leuprolide and peptide 1 dosing,
respectively. Values are represented as the mean ꢂ SD.
To demonstrate that testosterone release (pharmacological
response) occurs upon the specific binding to the LHRH-I receptor
and agonism by 1, this cyclic analogue was evaluated in the pres-
ence and absence of a known LHRH-I receptor antagonist, cetror-
elix. The administration of 1 in the absence of cetrorelix resulted in
a robust plasma testosterone release. Notably, no testosterone was
released when the cyclic analogue 1 was administered following
cetrorelix administration. Leuprolide served as a control (Fig. 4B).
Herein, intraperitoneal administration was selected as it is
a
mix-mode type of administration with elements of rapid
absorption [48], being a valuable alternative in cases where oral
A
vehicle-treated mice
leuprolide
cyclic analogue 1
80
70
60
50
40
30
20
10
0
1
2
4
6
Time (hr)
B
20
18
16
14
12
10
8
6
4
2
0
1 mg kg-1
1 mg kg-1
cetrorelix
1 mg kg-1
cetrorelix
vehicle
vehicle
vehicle
vehicle
cetrorelix
1 µg kg-1
100 µg kg-1
1 µg kg-1
100 µg kg-1
vehicle
leuprolide
cyclic analogue 1
leuprolide cyclic analogue 1
Dosing Regimens
Fig. 4. A. Testosterone release (functional LHRH-I receptor agonism) upon administration of the cyclic analogue 1 (1 mg kgꢁ1) when compared to vehicle- and leuprolide
(1 mg kgꢁ1) -treated mice (n ¼ 5). Plasma testosterone levels in vehicle-treated mice were low as expected and in agreement with previous studies [11,12]. Testosterone
concentration in leuprolide-treated mice was significantly higher at each time point tested (***, p < 0.001 vs. vehicle-treated mice). Testosterone release due to cyclic analogue 1
dosing was suggestive of agonistic effect (***, p < 0.001 vs. vehicle-treated mice at t ¼ 1e4 h). Values are represented as the mean ꢂ SD. B. Testosterone release upon administration
of cyclic analogue 1 occurs through the LHRH-I receptor associated pathway. Plasma testosterone concentration upon the administration of cyclic analogue 1 e and leuprolide e
dosing in mice (n ¼ 5) in the presence and absence of cetrorelix to demonstrate the specificity of the pharmacologic response. No response (absence of testosterone release) was
observed in the presence of cetrorelix. Testosterone release was observed after the administration of peptides (either leuprolide or cyclic analogue 1) in the absence of cetrorelix.
Values are represented as the mean ꢂ SD.

D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
243
bioavailability is low (such as in peptides). The mouse model
described in this study is of great value, because (i) the human
LHRH receptor is homologous to the mouse receptor [49], (ii)
in vitro and in vivo stability data can be acquired with a relatively
small amount of peptide (1.0e2.0 mg), (iii) information on the
LHRH receptor agonism (receptor specific in vivo modulation) can
be obtained by using testosterone as a marker and (iv) it can
become the basis of follow-up studies [50] evaluating further the
in vivo efficacy of our novel bioactive cyclic peptides for the
treatment of endocrine disorders and hormone-dependent cancers.
The in vitro and in vivo evaluation of the novel bioactive cyclic
LHRH analogues reported herein required a highly precise and
accurate analytical tool for peptide determination and biomarker
monitoring (testosterone) in complex matrices (mouse plasma,
mouse kidney membranes). Hence, a novel LCeMS/MS method-
ology combined with in vitro/in vivo bioassays was developed,
validated, and applied in this study, in agreement with approaches
previously described by our laboratory [11,12,51,52]. Overall, data
suggest that the enhanced in vitro and in vivo stability of the novel
cyclic analogue 1 (improved pharmacokinetics) compensates for
binding affinity differences, when compared to leuprolide, result-
ing in a highly comparable testosterone release (in vivo efficacy).
A hypothesis that is also supported in other studies [11,12,53].
detector. Human LHRH-I receptor was purchased from UMR
cDNA Resource Center. Lipofectamine, Geneticin and OPTIMEM
were purchased from Invitrogen (USA). All solvents and eluent
additives used were LCeMS grade. Acetonitrile (Riedel-de Haen,
Buchs SG, Switzerland), water (SigmaeAldrich GmbH, Munich,
Germany), ammonium acetate (Fluka, Buchs, Switzerland), for-
mic acid (Fluka), human serum albumin (HSA) (SigmaeAldrich
GmbH), dexamethasone (Riedel-de Haen), testosterone (Riedel-
de Haen), Trizma base (SigmaeAldrich GmbH), sodium chloride
(Fluka) and sodium phosphate dibasic (Riedel-de Haen) were
purchased from SigmaeAldrich GmbH. Dithiothreitol (DTT) was
purchased from AppliChem GmbH (Darmstadt, Germany).
Mouse plasma and kidneys were obtained from healthy male
C57BL/6N mice (in-house).
4.2. Synthesis of cyclic analogues: cyclo(1-10)[Pro1,
LHRH (1), cyclo(1-10)[Pro1, Tyr(OMe)5, -Leu6, Aze9, BABA10] LHRH
(2), cyclo(1-11)[Pro1, -Leu6, BABA10, Acp11] LHRH (3), cyclo(1-11)
[Pro1, Tyr(OMe)5, -Leu6, BABA10, Acp11] LHRH (4), cyclo(1-10)[Pro1,
Tyr(OMe)5, -Trp6, Aze9] LHRH (5)
D ]
-Leu6, BABA10
D
D
D
D
4.2.1. Synthesis of linear protected peptides
Each of the linear protected peptides was prepared in solid
phase using the CLTR-Cl and the Fmoc/tBu methodology. Resin (for
each peptide 1 g, 0.7 meq Clꢁ/g resin was used) in dry DCM was
stirred in a round bottom flask. Diisopropylethylamine (DIPEA)
(4.5 mmol, 0.75 mL) and Fmoc-Acp-OH (0.8 mmol) or Fmoc-BABA-
OH (0.8 mmol) or Fmoc-Gly-OH (0.8 mmol) were added and the
solution was stirred for 1 h at room temperature. A mixture of DCM/
MeOH/DIPEA (7 mL, 85/10/5) was then added and the mixture was
stirred for another 10 min at room temperature.
For every analogue, the remaining peptide chain was assembled
by sequential couplings of the following Fmoc-protected amino
acids (1.75 mmol), N,N0-diisopropylcarbodiimide (DIC) (2 mmol)
and 1-hydroxybenzotriozol (HOBt) (2.6 mmol) in dimethylforma-
mide (DMF) for 4e6 h. The completeness of each coupling was
verified by the test Kaiser and TLC. The protected amino acids
which are used in synthesis were: Fmoc-Acp-OH, Fmoc-BABA-OH,
3. Conclusions
Rationally designed cyclic analogues of Luteinizing Hormone-
Releasing Hormone (LHRH) were synthesized and found to
possess enhanced proteolytic stability and improved pharmacoki-
netic properties compared to their linear counterpart and leupro-
lide. A facile preclinical mouse model and an LCeMS/MS based
approach combined with bioassays were developed, validated and
employed herein, being valuable tools towards the evaluation of
novel bioactive peptides that could serve as a platform for future
rational drug design studies. Among the novel cyclic analogues
evaluated, cyclo(1-10) [Pro1, -Leu6, BABA10] LHRH, (analogue 1)
D
exhibits a significant pharmacokinetic advantage that greatly
compensates its low binding affinity to the LHRH-I receptor on the
basis of the in vivo efficacy data obtained. Hence, analogue 1 can
serve as a promising platform for future rational drug design
studies with the aim of pharmacokinetic advantageous analogues.
Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-
D-Leu-OH,
Fmoc- -Trp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp-
D
OH, Fmoc-His(Trt)-OH, and Fmoc-Pro-OH (Fig. 1). The Fmoc pro-
tecting group was removed by treatment with piperidine solution
(20% in DMF two times, the first for 10 min and the second for
25 min). The synthesized protected peptide on resin was dried in
vacuo and then treated with the cleavage mixture DCM/1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) (8/2) for 2 h at room temperature to
remove the peptide from the resin. The mixture was then filtered,
and the resin was washed with the splitting mixture (2 ꢃ 10 mL)
and DCM (3 ꢃ 10 mL). The solvent was removed on a rotary
evaporator, and the obtained oily product was precipitated from
cold dry diethyl either (Et2O) as a white solid.
4. Experimental section
4.1. Materials and methods
LHRH (acetate salt), [Des-Gly10
prolide acetate salt), [
-Trp6]-LHRH (triptorelin acetate salt), Ac-
2-Nal-4-chloro- -Phe- -(3-pyridyl)- -Ala-Ser-Tyr- -Cit-Leu-Arg-
Pro-
, D
-Leu6, Pro-NHEt9]-LHRH (leu-
D
D-
D
b
D
D
D
-Ala-NH2 (cetrorelix acetate salt) were purchased from
Bachem AG (Switzerland). CLTR-Cl (0.7e1.0 mmol Clꢁ/g resin,
200e400 mesh), Fmoc-Gly-OH, Fmoc-Aze-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Leu-OH, Fmoc-
D
-Leu-OH, Fmoc-
D
-Trp-OH, Fmoc-
4.2.2. Cyclization of linear protected peptides and final deprotection
To a solution of each linear protected peptide (0.1 mmol, 100e
120 mg) in dry dimethylformamide (DMF) (40 mL), collidine
(0.6 mmol, 0.079 mL) and HOAt (0.3 mmol, 40.83 mg) were added.
The resulting solution was then added dropwise to a solution of
TBTU (0.3 mmol, 96.33 mg) in dry DMF (80 mL) for 4 h and the
solution was then stirred for a period of 12 h [23e26,37,45].
Frequent controls were performed, every 3 h, by the ninhydrin test
on TLC using n-butanol/acetic acid/water (BAW, 4/1/1) as an eluent
and analytical RP-HPLC [eluents: A, 0.08% TFA/H2O, B, 0.08% TFA/
CH3CN, conditions: gradual gradient from 40% to 100% B in 30 min,
Ser(tBu)-OH, Fmoc-Trp-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH
were obtained from Chemical and Biopharmaceutical Laborato-
ries (CBL) of Patras, Greece. Fmoc-BABA-OH, Fmoc-Tyr(OMe)-OH
and Fmoc-Acp-OH were synthesized using 3-aminobutyric acid,
H-Tyr(OMe)-OH and Acp respectively and FmocOSu. All solvents
and other reagents were purchased from Merck, SigmaeAldrich
and Fluka chemical companies. DC-Alufolien Kieselgel 60
(Merck) was used for Thin Layer Chromatography (TLC) analysis
of synthetic products with the following eluent solvent: n-
butanol/acetic acid/water (BAW) 4:1:1 (v/v/v). Peptides were
purified by RP-HPLC with
a
Waters system equipped with
column: Nucleosil 100-5 C18 (5
min, detection: 214 nm, 254 nm]. The solvent was finally removed
m
m, 4 ꢃ 250 mm), flow rate: 1 mL/
a
600E controller and
a Waters 996 photodiode array UV

244
D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
from the reaction mixture under reduced pressure affording a light-
yellow oily residue. The cyclic protected peptide (w0.08 mmol,
yield w60%) was precipitated from H2O and then dried under
vacuum for 12 h.
Each dried cyclic protected peptide (100 mg) was treated with
65% TFA in DCM in the presence of 3% 1,2 ethanedithiol for 4 h at
room temperature. The final solution was concentrated under
vacuum to a small volume. The final free cyclic peptide was
precipitated as a light-yellow amorphous solid by the addition
of Et2O.
4.2.4.3. Harvesting cells and membrane preparation. HEK 293 cells
(grown in 100 mm dishes) stably expressing the human LHRH-I
receptor were washed with phosphate-buffered saline (PBS; 4.3 mM
Na2HPO4$7H2O, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2e
7.3 at room temperature), briefly treated with PBS containing 2 mM
EDTA (PBS/EDTA), and then dissociated in PBS/EDTA. Cell suspension
was centrifuged at 1000 ꢃ g for 5 min at room temperature, and the
pellet was homogenized in 1 mL of buffer O (50 mM TriseHCl
containing 0.5 mM EDTA, 10% sucrose, 10 mM MgCl2, pH 7.4 at 4 ꢄC)
using a Janke & Kunkel IKA Ultra Turrax T25 homogenizer (at setting
w20, 10e15 s, 4 ꢄC). The homogenate was centrifuged at 250 ꢃ g for
5 min at room temperature. The pellet was discarded and the
supernatant was centrifuged (16,000 ꢃ g, 10 min, 4 ꢄC). The
membrane pellet was resuspended (1 mL/10 mm dish) in buffer B
(25 mM HEPES containing 1 mM CaCl2, 10 mM MgCl2, 0.5% BSA, pH
7.4 at 4 ꢄC) and used for radioligand binding studies.
4.2.3. Purification and identification of cyclic peptides
The crude peptide products were further purified by semi
preparative RP-HPLC (column: Nucleosil 100-7, C18
, 7 mm,
10 ꢃ 250 mm), eluents: A, 0.08% TFA/H2O, B, 0.08% TFA/CH3CN,
gradual gradient: from 20% to 70% B in 45 min, flow rate:
3 mL minꢁ1, detection: 230 nm, 254 nm and were identified by ESI-
MS. The purity of the final products was assessed by analytical RP-
4.2.4.4. [125I-Tyr6, His5] LHRH binding. The [125I-Tyr6, His5] LHRH
HPLC (column: Nucleosil 100-5, C18, 5
m
m, 4 ꢃ 250 mm, eluents: A,
competition binding was performed as follows. Aliquots of diluted
0.08% TFA/H2O, B, 0.08% TFA/CH3CN, gradual gradient: from 20% to
100% B in 27 min, flow rate: 1 mL minꢁ1, detection: 214 nm or
254 nm) and the purity was ꢀ98% for each cyclic LHRH analogue.
The identification of the synthesized analogues was achieved using
ESI-MS. For analogue 1: Mcalc: 1235.43, Mfound: 1235.71; analogue
2: Mcalc: 1235.44, Mfound: 1235.65; analogue 3: Mcalc: 1348.59,
Mfound: 1349.58; analogue 4: Mcalc: 1362.62, Mfound: 1363.32;
analogue 5: Mcalc: 1280.43, Mfound: 1280.81 (Table 3, S1eS5).
membrane suspension (50
containing buffer B and 120,000e240,000 cpm [125I-Tyr6, His5]
LHRH (w3e6 L) with or without increasing concentrations of
mL) were added into low retention tubes,
m
LHRH analogues in a final volume of 0.5 mL. The mixtures were
incubated (16e19 h, 4 ꢄC) and then, filtered using a Brandel cell
harvester through Whatman GF/C glass fibre filters, presoaked for
1e2 h in 0.5% polyethylenimine at 4 ꢄC. The filters were washed 4
times with 2 mL of ice-cold 50 mM TriseHCl, pH 7.4 at 4 ꢄC. Filters
were assessed for radioactivity in a gamma counter (LKB Wallac
1275 minigamma, 80% efficiency). The amount of membrane used
was adjusted to ensure that specific binding was always equal to or
less than 10% of the total concentration of the radioligand added.
Specific [125I-Tyr6, His5] LHRH binding was defined as the total
binding less non-specific binding in the presence of 1000 nM
triptorelin. Data analysis for competition binding was performed by
nonlinear regression analysis, using GraphPad Prism 4.0 (GraphPad
Software, San Diego, CA). IC50 values were obtained by fitting the
data from competition studies to a one-site competition model and
presented as mean ꢂ standard deviation (SD).
4.2.4. LHRH-I receptor binding assay of cyclic synthesized analogues
4.2.4.1. Procedures for iodinating [125I-Tyr6, His5] LHRH. The iodin-
ation of Tyr6, His5-LHRH was performed according to the method of
Liapakis et al. (1996) with minor modifications [54,55]. In brief,
20
peptide of interest. Then, the addition of 1e2 mCi of Na125I (10e
20 L) as well as 10 L of chloramine T (1 mg/mL) followed. The
reaction taking place at room temperature was stopped after 60 s
by adding 50 L of sodium metabisulfite (1 mg/mL) and subse-
mL of sodium phosphate buffer (pH 7.4) were added to 5 mg of
m
m
m
quently, 0.5 mL of 10 mM acetic acid containing 0.1% BSA. The
iodinated species were separated by column chromatography using
a Sephadex G-25 fine column.
4.2.5. Stability of novel cyclic LHRH analogues
4.2.5.1. In vitro stability study (mouse kidney membranes).
Peptides were incubated with mouse kidney membranes as
described [11,12]. Mouse kidneys were washed with PBS, weighed,
and homogenized (0.1 g wet weight mLꢁ1) in buffer A; NaCl
(100 mM), phosphate (20 mM, pH 7.4 at room temperature), HSA
(0.1 mg mLꢁ1), and dithiothreitol (1 mM) by a Teflon-glass homog-
enizer. After centrifugation (1500 ꢃ g, 5 min), the supernatant was
centrifuged for 60 min at 100,000 ꢃ g (Sorvall Discovery 100SE
ultracentrifuge; Hitachi, Yokohama, Japan). The supernatant was
discarded and the pellet was washed by addition of buffer B; NaCl
(100 mM), phosphate (20 mM, pH 7.4 at room temperature) and HSA
(0.1 mg mLꢁ1), followed by centrifugation (100,000 ꢃ g, 60 min).
4.2.4.2. Cell culture and transfection. Human embryonic kidney
(HEK 293) cells were grown in DMEM/F12 (1:1) containing
3.15 g Lꢁ1 glucose and 10% bovine calf serum at 37 ꢄC and 5% CO2.
60 mm dishes of HEK 293 cells at 80e90% confluence were trans-
fected with 2e3 mg of human LHRH-I receptor using 9 mL of Lip-
ofectamine and 2 mL of OPTIMEM. To generate stably transfected
pools of cells expressing the receptors 5e12 h after transfection, the
medium was replaced by DMEM/F12 (1:1) containing 3.15 g Lꢁ1
glucose, 10% bovine calf serum and 700 m
g mLꢁ1 of the antibiotic,
Geneticin. The antibiotic use ensured the selection of a stably
transfected pool of cells.
Table 3
Analytical data of synthesized cyclic analogues 1e5.
a
No
Peptide
tR
ESI-MS
(min)
Mcalc (Da)
Mfound (Da)
1
2
3
4
5
Cyclo(1-10)[Pro1,
D
-Leu6, BABA10] LHRH
-Leu6, Aze9, BABA10] LHRH
14.46
12.91
12.05
12.45
14.34
1235.43
1235.44
1348.59
1362.62
1280.43
1235.71
1235.65
1349.58
1363.32
1280.81
Cyclo(1-10)[Pro1, Tyr(OMe)5,
D
Cyclo(1-11)[Pro1, -Leu6, BABA10, Acp11] LHRH
D
Cyclo(1-11)[Pro1, Tyr(OMe)5,
Cyclo(1-10)[Pro1, Tyr(OMe)5,
D
-Leu6, BABA10, Acp11] LHRH
-Trp6, Aze9] LHRH
D
a
RP-HPLC conditions. Column: Nucleosil 100-5, C18, 5
rate: 1 mL minꢁ1, detection: 214 nm and/or 254 nm.
m
m, 4 ꢃ 250 mm, eluents: A, 0.08% TFA/H2O, B, 0.08% TFA/CH3CN, gradual gradient: from 20% to 100% B in 27 min, flow

D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
245
After the supernatant was discarded, the pellet was washed by
addition of buffer C; NaCl (100 mM), phosphate (20 mM, pH 7.4 at
room temperature) and the protein content was determined by the
Bradford assay. Incubation mixtures consisted of peptide (10 nM)
and protein (4 mg/ml) (mouse kidney homogenate). Incubations
occurred at 37 ꢄC in a shaking water bath (Unitronic OR, Selecta). At
the time intervals of t ¼ 30 min, t ¼ 60 min and t ¼ 120 min (with
t ¼ 0 min as a control) samples were obtained and analysed. Addi-
tional control samples (no mouse kidney membranes) were also
obtained and analysed. The reaction was stopped by acetonitrile
addition, and samples were stored at ꢁ80 ꢄC.
humidity, and a photoperiod of 12/12 h (light starting at 07:00 h). Tap
water in drinking bottles made of glass/clear polycarbonate (PC)
complying with Title 21 FDA requirements (Techniplast, Italy) and
irradiated vacuum-packed pelleted food (HarlanTeklad diet 2018,
Oxfordshire, UK) were directly administered to the stainless steel
food hopper of each cage and provided ad libitum to the animals. All
animals were free of viral, bacterial and parasitic pathogens. Mice
were fasted overnight (12 h) prior to dosing. Leuprolide and cyclic
analogue 1 were each administered i.p. at 1 mg kgꢁ1 in saline. Blood
samples were collected by cardiac puncture under isoflurane anaes-
thesia at t ¼ 1 h, t ¼ 2 h, t ¼ 4 h and t ¼ 6 h post-dose (n ¼ 5). Blood
Sample extraction (samples left at room temperature to thaw for
20 min) was performed by the addition of acetonitrile:water 50:50,
samples (500
containing 10
m
L) were collected into heparinized Eppendorf tubes
m
L heparin (5000 IUmLꢁ1), and centrifuged (3000 rpm,
0.1% formic acid (250
addition of ice-cold acetonitrile (250
m
L). Proteins were precipitated by stepwise
10 min) for plasma preparation. Plasmawas stored in a ꢁ80 ꢄC freezer
until the analysis. All the experimental procedures were approved by
the Bioethical Committee of the Institution and the local competent
authority on the basis of the European Directive 86/609 on the
protection of animals used for experimental and other scientific
purposes. Based on quality control samples at 1 and 10 nM the intra-
and inter-assay coefficients of variation were below 10. The retention
times obtained were characteristic of the analytes; 6.69 ꢂ 0.01 min
(cyclic analogue 1), 6.22 ꢂ 0.02 min (leuprolide), 6.67 ꢂ 0.01 min
m
L and 500 L) followed by brief
m
vortexing. Samples were subsequently centrifuged (12,000 ꢃ g,
10 min) and the supernatant was evaporated in a SpeedVac
(SPD1010; Thermo Fisher Scientific, Waltham, MA) for 90 min at
50 ꢄC. An aliquot of 100
mL of mobile phase (see below) was added to
each sample, prior to LCeMS/MS analysis. An 1100 series system
(Agilent Technologies, Waldbrown, Germany) equipped with
a binary pump, autosampler, vacuum degasser, and temperature-
controlled column compartment was used for analyte separation by
HPLC. The mobile phase consisted of solvents A (10% acetonitrile, 90%
water, 2 mM ammonium acetate, and 0.1% formic acid) and B (90%
acetonitrile, 10% water, 2 mM ammonium acetate, and 0.1% formic
([Des-Gly10 -Ala6, Pro-NHEt9] LHRH, internal standard for peptide
, D
quantification).
Sample extraction and analysis by LCeMS/MS were performed
as described above. Cyclic analogue 1 (MW: 1235.4 g molꢁ1) was
monitored using the transitions of m/z 618.6 / 130.2 and
acid). A Waters AtlantisÒ dC18 3
m
m, 2.1 ꢃ 50 mm column (Waters
Corporation, Milford, MA, USA) was used at
a
flow rate of
618.6
/
1209.4 g molꢁ1), the transitions of m/z 605.5 / 249.1 and
605.5 / 221.1 were optimized by adjustments of collision energy,
159.1. For the detection of leuprolide (MW:
0.3 mL minꢁ1. A linear gradient (run time of 12 min) from 95% Ae5%
B to 50% Ae50% B over 5 min and 50% Ae50% B up to 8 min was used
for the chromatographic separation of peptides of interest. Typical
declustering potential and dwell time. [Des-Gly10 -Ala6, Pro-
, D
injection volume was 20
mL. Mass spectrometry was performed on
NHEt9] LHRH (internal standard) was monitored using the MRM
transition m/z 584.7 / 249.1. Recovery studies from mouse plasma
were also performed in two concentrations (low and high; 5 and
50 ng/mL) indicating that both peptides studied were more than
90% recovered from mouse plasma.
an API 4000 Q e TRAP LC e MS/MS system fitted with a TurboIon-
Spray source and a hybrid triple quadrupole hybrid linear ion trap
mass spectrometer (Applied Biosystems, Concord, ON, Canada). The
instrument was operated in positive ion mode under the following
conditions: Ion Spray voltage, 5500 V; source temperature, 550 ꢄC;
curtain gas (nitrogen) at 20; collision gas (nitrogen) at 5; ion source
gas 1 (air) at 40; and ion source gas 2 (air) at 45 (all arbitrary units).
Quantification of peptides was performed in the multiple-reaction
monitoring (MRM) positive mode on the basis of their characteristic
transitions, optimized by adjustments of collision energy, declus-
tering potential and dwell time.
In vitro peptide stability was determined as a function of time;
using the corresponding MRM transitions for each analyte, the peak
areas for each peptide in question (peptide concentration of 10 nM)
at t ¼ 0 were set as 100%. Those peak areas were compared to the
peak areas derived from samples at t ¼ 30, 60 and 120 min.
Respectively, the mean intra- and inter-assay coefficient of varia-
tion for the determination of each peptide in question based on
control samples (10 nM of peptide) was below 10%. Peptide
monitoring by LCeMS/MS ensured specificity since each peptide in
question had characteristic retention times (e.g., 6.22 ꢂ 0.02 min
for leuprolide) and corresponding MRM transitions. In addition,
analyses of blank samples confirmed the absence of interference
and established the selectivity of the assay.
For peptide quantification in mouse plasma, stock solutions of
leuprolide (0.5e500 ng mLꢁ1), cyclic analogue 1 (25e2500 g mLꢁ1
)
and internal standard [Des-Gly10
,
D
-Ala6, Pro-NHEt9] LHRH
(600 ng mLꢁ1) were prepared in acetonitrile:water 50:50, 0.1%
formic acid. A different set of solutions at two different concen-
trations for leuprolide (250 and 5000 ng mLꢁ1) and cyclic analogue
1 (25 and 250 ng mLꢁ1) were prepared in order to be used for
quality control samples. Calibration curves and quality control
samples were prepared daily in plasma by adding 20
and 25 L of the internal standard solutions to 20
m
L of peptide
m
mL of plasma.
Nine-point calibration curves were constructed using a linear fit
with 1/x weighting, resulting in acceptable accuracy (target
concentrations were within 15% of nominal values) in the
concentration ranges obtained; 0.5e500 ng mLꢁ1 for leuprolide,
25e2500 ng mLꢁ1 for cyclic analogue 1. On the basis of quality
control samples for leuprolide and cyclic analogue 1 analysis, the
intra-assay coefficient of variation for the assay was less than 10%
for both peptides. Assay specificity and selectivity were ensured by
using an LCeMS/MS methodology (retention time and MRM tran-
sitions characteristic of the analytes) and analysing blank samples
to test for interferences. Based on quality control samples, the
accuracy of the assays for the determination of analogue 1 and
leuprolide in mouse plasma was 99 and 96%, respectively.
4.2.5.2. In vivo stability (pharmacokinetic study). Male mice of
C57BL/6N inbred strain (Charles River, Calco, Italy) at the age of 10e12
weeks were randomly divided in groups of five and housed in H-
Temp polysulfone type III individually ventilated cages (800 cm2,
Techniplast, Milan, Italy), with 70 air changes/hour under positive
pressure. Animal handling occurred prior to the experiment, during
which labelling and weighing took place. All animals were housed in
a temperature-controlled room (22 ꢂ 1 ꢄC), with 55 ꢂ 10% relative
4.2.6. Functional agonism of the LHRH receptor e testosterone
quantification in mouse plasma following IP dosing
The ability of the cyclic analogue 1 to elicit testosterone release
(LHRH-I receptor agonism) in mice was investigated. Leuprolide
served as a control. Both peptides were administered to male

246
D. Laimou et al. / European Journal of Medicinal Chemistry 58 (2012) 237e247
C57BL/6N mice at 1 mg kgꢁ1 and blood sampling was conducted by
cardiac puncture under isoflurane anaesthesia at t ¼ 1, 2, 4, and 6 h
post-dose (n ¼ 5). The same approach was followed in the case of
vehicle (saline)- and leuprolide (1 mg kgꢁ1)-treated animals
(n ¼ 5). Sample extraction for both peptides and testosterone
(biomarker) was performed by protein precipitation as described.
To further confirm that testosterone release results from binding
of analogue 1 to the LHRH-I receptor (agonism), this cyclic analogue
was dosed i.p. to male C57BL/6N mice in the presence and absence
of cetrorelix (known LHRH receptor antagonist). Cetrorelix was
dosed (i.p.) at 1 mg kgꢁ1 and after 15 min, analogue 1 was dosed at
The pharmacokinetic parameters were determined using PK
Functions for Microsoft Excel by J. Usansky, A. Desai and D. Tang-Liu
(Department of Pharmacokinetics and Drug Metabolism, Allergan,
Irvine, CA 92606, U.S.A.). Data analysis for competition binding was
performed by nonlinear regression analysis, using GraphPad Prism
4.0 (GraphPad Software, San Diego, CA). IC50 values were obtained
by fitting the data from competition studies to a one-site compe-
tition model and presented as mean ꢂ standard deviation (SD).
Acknowledgements
100
animals were treated with vehicle (saline) and after 15 min,
analogue 1 was administered at 100
g kgꢁ1. Likewise, LHRH ago-
nism was investigated upon leuprolide dosing at 1
g kgꢁ1 (based
m
g kgꢁ1 on the basis of its binding affinity data. In parallel,
Despina Laimou was financially supported by the University of
Patras (Karatheodoris Grant). The authors would like to thank the
A.G. Leventis Foundation for funding of the Dr C. Tanvakopoulos’ lab
in the Biomedical Research Foundation of the Academy of Athens.
We would like to thank Dr N Kostomitsopoulos and Mr E Balafas for
assistance with the in vivo studies. We thank Dr Norman L. Block
(University of Miami) for the advice and assistance in the prepa-
ration of this manuscript.
m
m
on binding affinity data). Blood sampling was conducted by cardiac
puncture under isoflurane anaesthesia at t ¼ 1 h post-dose (n ¼ 5).
Stock solutions of testosterone (0.125e250 ng mLꢁ1) and dexa-
methasone (200 ng mLꢁ1, internal standard) were prepared in
acetonitrile:water 50:50, 0.1% formic acid. For testosterone
measurements, calibration curve (0.05e100 ng mLꢁ1) and quality
control samples (1 and 5 ng mLꢁ1) were prepared by adding 20
of testosterone and 20 L of dexamethasone solutions to 50 L of
plasma from female CD-1 mice.
A Waters AtlantisÒ dC18 3
Corporation, MilfordMA, USA) was used at
mL
Appendix A. Supplementary data
m
m
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.09.043.
m
m, 2.1 ꢃ 50 mm column (Waters
a
flow rate of
0.3 mL minꢁ1. A linear gradient (run time 14 min) from 75%Ae25%B
to 25%Ae75%B over 5 min, and 25%Ae75%B up to 9 min was used
for the separation of peptides and testosterone. Quantification of
peptides and testosterone was performed in the MRM positive
mode. For peptide detection, their characteristic transitions (shown
above) were optimized by adjustments of collision energy, declus-
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