Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation

Article abstract of DOI:10.1002/psc.2769

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH₂], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

Full text of DOI:10.1002/psc.2769

Special Issue Article
Received: 22 December 2014
Revised: 9 February 2015
Accepted: 10 February 2015
Published online in Wiley Online Library: 8 April 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2769
Gonadotropin-releasing hormone receptor-
targeted paclitaxel–degarelix conjugate:
synthesis and in vitro evaluation‡
Chenhong Wang,§ Yongtao Ma,§ Siliang Feng, Keliang Liu and Ning Zhou*
To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed.
Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone
antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX–degarelix conjugates were synthesized, in which
PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX–degarelix conjugates
exhibited a half-life greater than 10h determined in human serum. A fluorometric imaging plate reader assay showed that the
conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the
conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one or-
der of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and
HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix re-
duced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that
has great potential for targeting chemotherapy drugs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: targeted drug delivery; gonadotropin-releasing hormone antagonist; paclitaxel–degarelix conjugates; in vitro stability
release of a cytotoxic drug, thereby resulting in reduced uptake
and efficacy in cancer cells and widespread toxicity for rapidly pro-
Introduction
While chemotherapy has been the main treatment modality for
metastatic diseases, its efficacy has been greatly limited by intrinsic
or acquired multidrug resistance and nonspecific toxicity to normal
cells [1–3]. Through the use of specific cell-surface markers,
targeted therapy can improve the therapeutic index of antitumor
drugs [4–7]. For instance, the gonadotropin-releasing hormone
receptor (GnRH-R) is a member of the G protein-coupled receptor
superfamily [8] and is overexpressed in the anterior pituitary and
also on the surface of tumor cells from the ovary [8], prostate [9],
breast [10], and endometrium [11,12]. In contrast, expression of
the GnRH-R is not detected in normal organs [10,13–15]. Thus,
gonadotropin-releasing hormone (GnRH) analogs have the poten-
tial to target agents that will enhance the uptake of antitumor drugs
by GnRH-R-positive cancerous cells, while sparing normal cells.
The concept of developing targeted chemotherapeutics based
on human GnRH derivatives was first introduced by A. V. Schally
in the late 1980s. Since then, several conjugates employing GnRH
peptide analogs as targeting moieties have been synthesized and
characterized [16–18]. To date, the reported GnRH-targeted
antitumor conjugates generally contain GnRH agonists conjugated
to antitumor drug molecules. For instance, the conjugate
Dox-[Lys6]-GnRH (AN-152), which contains an ester bond coupling,
is currently in phase III clinical trials and has been shown to induce
apoptosis independent of multiple-drug resistance gene-1 (MDR-1)
in GnRH-R-positive human ovarian and endometrial cancer cell
lines [19]. However, the stability of AN-152 still needs to be
improved, and the half-life of ester bonds in the presence of
carboxylesterases is 2 h in human serum and 20 min in mouse
serum [20]. This rapid degradation can lead to the premature
liferating cells [21,22]. Therefore, many anticancer drug–GnRH ago-
nists’ conjugates with higher stability in human serum were
developed, and the anticancer drug was attached to the GnRH pep-
tide through various spacers/linkers, such as oxime bond and
hydrazone bond [23,24]. It is well known that GnRH antagonists ex-
hibit a similar affinity for the GnRH-R as GnRH agonists yet they
have enhanced enzymatic stability. Therefore, it is hypothesized
that if GnRH antagonists are replaced with GnRH agonists, the
resulting conjugates would not only retain their affinity for the
GnRH-R but would also be more stable. Of the GnRH antagonists
*
Correspondence to: Ning Zhou, State Key Laboratory of Toxicology and Medical
Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing
100850, China. E-mail: zhouning6818@163.com
‡
Special issue of contributions presented at the 13th Chinese International Peptide
Symposium, Peptides: Treasure of Chemistry and Biology, June 30 - July 4, 2014 in
Datong, Shanxi, China.
§
Both authors contributed equally to this work.
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing
Institute of Pharmacology and Toxicology, Beijing 100850, China
Abbreviations: PTX, paclitaxel; GSH, glutathione; DIC, N,N′-diisopropylcarbodiimide;
HOBt, 1-hydroxybenzotriazole; DMAP, 4-dimethylaminopyridine; DIEA, N,N′-
diisopropylethylamine; TFA, trifluoroacetic acid; 4Aph, 4-aminophenylalanine; Cbm,
carbamyl; 4Cpa, 4-chlorophenylalanine; Hor, L-hydroorotyl; Ilys, Nε-isopropyl lysine;
DCM, dichloromethane; Pal, 3-pyridylalanine; MBHA, p-methylbenzhydrylamine; HBSS,
Hank’s balanced salt solution; OD, optical density; FLIPR, fluorometric imaging plate
reader.
J. Pept. Sci. 2015; 27: 569–576
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

WANG ET AL.
developed to date, degarelix is the most recently approved by Food
and Drug Administration (FDA) [25]. Degarelix has a high affinity for
the GnRH-R, is enzymatically stable, and has been used for the treat-
ment of advanced hormone-dependent prostate cancer in the USA
and the European Union [26]. Therefore, the aim of the present
study was to develop and define the antiproliferative effects of var-
ious conjugates that contain the chemotherapeutic agent, PTX, and
the targeting moiety, degarelix.
PTX is a widely used antitumor drug that interferes with the
dissociation of microtubules during cell division [27]. It has been
used to treat patients with lung, ovarian, breast, head, or neck
cancers, as well as for patients with advanced forms of Kaposi’s
sarcoma [28,29]. Here, PTX was conjugated to a degarelix analog
via disulfide bonds, which are stable under normal physiological
conditions yet are reduced to free thiols in the presence of reducing
agents, such as GSH. GSH is an ideal reducer because it is abundant
in the cell cytoplasm (~10 mM) yet is present at very low levels in
blood plasma (~0.002 mM) [30]. It is hypothesized that when the
conjugates are exposed to the reductive environment of tumor
cells, their disulfide bonds will be cleaved, thereby resulting in the
release of conjugated drug. Five PTX–degarelix conjugates were
synthesized and characterized. In vitro stability of the conjugates
were assessed in human serum by liquid chromatography, and
their in vitro cytostatic effects were evaluated using (3-(4,5-dimeth-
ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium) (MTS) assays with human breast cancer, human
colon cancer, and mouse embryonic fibroblast cell lines.
brine (50ml), and then was dried over Na2SO4. The solvent was
evaporated under vacuum, and the residual product was purified
on a silica gel column and eluted with DCM/CH3OH (100 : 1). A
white powder of 2-pyridyl disulfide PTX carbonate was obtained
(0.971g, 89% yield), with an ESI-MS mass-to-charge ratio (m/z) of
1
1068. 9 [M + H]+1 (calculated for C55H58N2O16S2, 1067.3), H NMR
(400 MHz, DMSO-d6) δ: 9.32 (d, 1H, J = 8 Hz, –NH), 8.45–7.19 (m,
19H, H-Ph, H-Py), 6.29 (s, 2H, H-10), 5.83 (m, 1H, H-13), 5.55 (t, 1H,
J= 8 Hz, H-3′), 5.41 (d, 1H, J = 8 Hz, H-2), 5.37 (d, 1H, J = 8 Hz, H-2′),
4.93 (m, 1H, H5), 4.65 (s, 1H, OH-1), 4.39 (m, 2H, –CO–CH2–CH2–S–S–),
4.11–4.01 (m, 3H, H-7, H-20α, H-20β), 3.59 (d, 1H, J = 7.2 Hz, H-3),
3.14 (t, 2H, J = 8 Hz, –CO–CH2–CH2–S–S–), 2.33 (m, 1H, H-6α),
2.26 (s, 3H, CH3CO-4), 2.12 (s, 3H, CH3CO-10), 1.78 (m, 1H,
H-14α), 1.75 (s, 3H, H-18), 1.64 (m, 1H, H-6β), 1.54 (m, 1H, H-14β),
1.50 (s, 3H, H-19), 1.03 (s, 3H, H-16), and 1.00 (s, 3H, H-17).
Preparation of Degarelix Derivatives
The five degarelix derivatives were prepared manually by solid-
phase peptide synthesis according to Boc/Fmoc strategy on MBHA
resin (1.08mmol/g coupling capacity). The following Boc-protected
amino acid derivatives were used: Boc-D-Ala-OH, Boc-Pro-OH,
Boc-ILys(Z)-OH, Boc-Leu-OH, Boc-D-Nal-OH, Boc-D-Cpa-OH, Boc-
Leu-OH, Boc-D-Pal-OH, Boc-Ser(Bzl)-OH, Boc-Aph(Fmoc)-OH, and
Boc-D-Aph(Cbm)-OH.
The derivatives were synthesized as follows: (1) 10% DIEA/DCM
(two times, 10 min each), (2) N,N,-dimethylformamide (DMF)
washing (three times, 2 min each), CH3OH washing (three times,
2 min each), DCM washing (three times, 2 min each), (3) coupling
of two equivalent Boc-protected amino acid derivatives : DIC: HOBt
(1 : 2 : 1) in DMF (4h), (4) DMF washing (three times, 2 min each),
CH3OH washing (three times, 2 min each), DCM washing (three
times, 2 min each), (5) Boc deprotection with 50% TFA/DCM
(25 min), and (6) DMF washing (three times, 2 min each), CH3OH
washing (three times, 2 min each), DCM washing (three times,
2 min each). Upon completion of the coupling of 5Aph, the Fmoc-
protecting group was removed from the ω-NH2 groups of 5D-Aph
with 50% pyridine in DMF (25min). The L-hydroorotic acid (L-Hor)
residue was then coupled to the ω-amino group of 5Aph according
to the previously mentioned protocol.
For the synthesis of Mer-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-
D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2 and Mer-Gln-Arg-D-Nal-D-
Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2,
Trt-protecting mercaptopropionic acid (Trt-Mpa-OH) was coupled
to the N-terminus of the peptide according to the previously
mentioned protocol.
To synthesize Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Mer)-
Leu-ILys-Pro-D-Ala-NH2, Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph
(-Arg-Gln-Mer)-Leu-ILys-Pro-D-Ala-NH2, and Mer-Gln-Arg-D-Nal-D-
Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(-Arg-Gln-Mer)-Leu-ILys-Pro-D-
Ala-NH2, the Fmoc-protecting group was removed from the
ω-NH2 groups of 6D-Aph with 50% pyridine in DMF (25 min).
Then, Trt-Mpa-OH was attached to the free ω-NH2 groups of
6D-Aph according to the previously mentioned protocol. For
Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(-Arg-Gln-Mer)-Leu-ILys-
Pro-D-Ala-NH2 and Mer-Gln-Arg-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-
D-Aph(-Arg-Gln-Mer)-Leu-ILys-Pro-D-Ala-NH2, prior to the coupling
of Trt-Mpa-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gln-OH were serially
coupled to the free ω-NH2 groups of 6D-Aph. The peptide
Materials and Methods
Materials
PTX was purchased from Beijing Zhongshuo Pharmaceutical
Technology Development Co., Ltd (Beijing, China). DIC, HOBt, and
all of the Boc-protected amino acids were obtained from GL
Biochem Shanghai Ltd (Shanghai, China). The reagents, DMAP,
DIEA, and TFA were purchased from J&K Chemical (Beijing, China).
As previously described [31], 2-pyridyl disulfide p-nitrophenyl
carbonate was prepared. Most amino acid derivatives were
obtained from Chengnuo Ltd (Chengdu, China), including
Boc-D-Ala-OH, Boc-D-2-naphthylalanine-OH (Boc-D-Nal-OH),
Boc-D-4Cpa-OH (Boc-D-Cpa-OH), Boc-Nε-isopropyl lysine(Z)-OH
[Boc-ILys(Z)-OH], Boc-Leu-OH, Boc-Pal-OH (Boc-D-Pal-OH),
Boc-Pro-OH, and Boc-Ser(Bzl)-OH. Boc-L-4Aph(Fmoc)-OH [Boc-L-
Aph(Fmoc)-OH] and Boc-D-Aph(Fmoc)-OH were synthesized from
Boc-L-4-nitro-Phe-OH and Boc-D-4-nitro-Phe-OH (Innochem, Beijing,
China), respectively, as previously published [32]. Boc-D-4Aph
(Cbm)-OH [Boc-D-Aph(Cbm)-OH] was synthesized from Boc-D-Aph-
OH as previously published [33]. The MBHA resin was obtained from
NanKai HeCheng (Tianjin, China). Human serum was obtained from
the 307 Hospital (Beijing, China). CellTiter 96® AQueous One Solution
Reagent was purchased from Promega Corporation (Madison, WI,
USA). All reagents and solvents were of analytical grade and used
without further purification.
Preparation of PTX Derivatives
PTX (0.8671g, 1.02 mmol), 2-pyridyl disulfide p-nitrophenyl carbon-
ate (0.3878 g, 1.1mmol), and DMAP (0.1406 g, 1.15 mmol) were
mixed with 15 ml of DCM. After the solution was stirred in the dark
for 24 h at room temperature (RT), it was diluted with 50 ml of DCM,
was washed once with a saturated solution of NaHCO3 (50 ml) and
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GnRH RECEPTOR-TARGETED PACLITAXEL–DEGARELIX CONJUGATES
sequences were then prolonged using the previously mentioned
protocol. An acetyl group was introduced by capping the peptide
with acetic anhydride and DIEA after deprotection of the Boc
group of Boc-D-Nal-OH in the peptide terminus.
The degarelix derivatives were cleaved from the resin using an-
hydrous hydrogen fluoride (HF) in the presence of a scavenger
(2% anisole) in an ice bath. After 1 h, the derivatives were precipi-
tated with ice-cold diethyl ether, were washed three times with
diethyl ether, and were solubilized in 20% acetic acid prior to freeze
drying. The crude peptides were purified by preparative RP-HPLC
and were analyzed by MALDI-TOF MS.
and were maintained in Dulbecco’s modified Eagle’s medium
(Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine
serum (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin.
CHO-K1/Gα15 cells stably expressing the GnRH-R were purchased
from GenScript (Nanjing, China) and were maintained in Ham’s
F12 medium containing 10% fetal bovine serum, 400 μg/ml G418,
and 100 μg/ml hygromycin B. Cell cultures were maintained at
37°C in a humidified atmosphere with 5% CO2.
Antagonism Activity Assay Using FLIPR
CHO-K1/Gα15 cells stably expressing the GnRH-R were plated in
384-well tissue culture-treated, black polystyrene plates with a clear,
flat bottom (BD Biosciences, San Jose, CA, USA). Briefly, 20μl of
8 × 105 cells/ml were plated and maintained at 37 °C in a humidified
atmosphere containing 5% CO2. After ~24 h, the medium was re-
moved, and 20 μl of Ca3 dye (Molecular Devices Corporation, Sunny-
vale, CA, USA) containing 2.5 mM freshly prepared probenecid was
added according to the manufacturer’s protocol. Then, 20 μl of the
antagonist, AG045572 (TOCRIS), or buffer was added into each well
with 20 mM HEPES buffer. Each compound was assayed in duplicate
at eight concentrations that descended in half-log increments. Dye-
loading solution (20 μl) was then added, and the CHO-K1 cells were
placed in a 37 °C incubator. After 1 h, followed by 15 min incubation
at RT, changes in intracellular Ca2+ were monitored using a FLIPR
(Molecular Devices Corporation). The fluorescence measurements
were read simultaneously from all of the wells at an excitation wave-
length of 480 nm and an emission wavelength of 520 nm. A baseline
fluorescence signal was measured for the first 10 s, after which 20 μl
of the eight descending concentrations of conjugates in half-log
increments were added to the cell plate. Changes in fluorescence in-
tensity were measured every second for the first minute and then
were measured every 5 s for an additional minute.
Synthesis of PTX–Degarelix Conjugates
Degarelix derivatives and 2-pyridyl disulfide PTX carbonate were
dissolved in DMSO; the latter was added in a 20% excess compared
with the degarelix derivative. The reaction mixture was stirred for
4 h at RT, and the reaction was monitored by RP-HPLC. The resulting
conjugates were separated by preparative RP-HPLC and were ana-
lyzed by MALDI-TOF MS.
HPLC
The crude products were purified on a Prep LC 4000 system (Waters
Corporation, Milford, MA, USA) using a preparative X-Bridge C8 col-
umn (195 mm × 250 mm) with 5 μm silica. A linear gradient elution
was performed with eluent A (0.1% TFA/H2O) and eluent B [0.1%
TFA in acetonitrile–water (70 : 30, v/v)] (0min 40% B; 5 min 55% B;
25 min 80% B; 35 min 40% B) at a flow rate of 16 ml/min. Peaks were
detected at 220 nm.
Analytical RP-HPLC was performed on an LC-10AT VP Plus liquid
chromatograph (Shimadzu Corporation, Kyoto, Japan) with an SPD-
10A VP Plus UV–Vis detector operating at 210 nm using a Zorbax
XDB-C8 column (4.6 mm × 150 mm) with silica (5 μm) as a stationary
phase. Linear gradient elution (0 min 30% B; 11 min 100% B; 14 min
100% B; 17 min 30% B; 21 min 30% B) was performed at a flow rate
of 1 ml/min. Peaks were detected at 210nm.
Percent inhibition was calculated as follows:
% inhibition = [1 À (ΔRFUCompound À ΔRFUBackground)/
(ΔRFUAntagonist control À ΔRFUBackground)] × 100
Drug Release of PTX–Degarelix Conjugates in Dithiothreitol
(DTT) Solution
The percent inhibition values were then plotted as a function of
the log of the cumulative doses of the compounds. Relative fluores-
cence unit (ΔRFU) intensity values were calculated according to the
maximal fluorescence units by subtracting the average baseline
value. The 50% inhibitory concentration (IC50) values were derived
by nonlinear regression to a four-parameter logistic equation
using GraphPad Prism curve fitting software (GraphPad Software,
Inc., San Diego, CA, USA). The geometric mean of the IC50
values from at least two independent experiments is reported for
each compound.
The LK-MY-10 and tenfold DTT were dissolved in a mixture of phos-
phate buffer solution (pH = 7.4, 0.1mM) with acetonitrile (2: 1) and
incubated at 37 °C. The reaction was monitored by RP-HPLC at dif-
ferent time.
Stability of PTX–Degarelix Conjugates in Human Serum
Samples were initially dissolved in DMSO, and then, human serum
was added to a final peptide concentration of 100 μM and a final
DMSO concentration of 2.5%. The mixtures were incubated at 37 °C,
and 200 μl aliquots were removed at different time intervals. The reac-
tions were quenched by adding 600 μl acetonitrile. Following centrifu-
gation, 450 μl from each supernatant was analyzed by RP-HPLC
(Agilent 1200, Agilent Technologies, Santa Clara, CA, USA). The residual
ratio for each conjugate was examined by comparing the peak areas
detected by RP-HPLC before and after treatment with human serum.
In Vitro Cytotoxicity of the PTX–Degarelix Conjugates Using an
MTS Assay
MCF-7, HT29, and 3T3 cells were each seeded in 96-well cell-culture
plates (Corning Life Sciences, Tewksbury, MA, USA) (5× 103
cells/well). After 24 h, the cells were washed twice with HBSS and
were incubated with 100 μl of PTX or the conjugates at the indi-
cated concentrations at 37°C. After 72 h, the cells were washed with
HBSS again. To each well, 80μl of HBSS and 20 μl of CellTiter 96®
AQueous One Solution Reagent (Promega Corporation) were
added. After 2 h, the OD for each well was measured at 490 nm
using a SpectraMax® M5 microplate reader (Molecular Devices
Cell Culture
MCF-7 human breast cancer cells, HT-29 human colon cancer cells,
and 3T3 mouse embryonic fibroblast cells were obtained from the
Cell Resource Center of Peking Union Medical College (Beijing, China)
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WANG ET AL.
Corporation). HBSS alone was used as a negative control. The per-
centage of cytotoxicity was calculated using the following equation:
based on its widespread use and established efficacy against a variety
of solid tumors. Targeting moieties were attached to PTX via the 2-
hydroxyl group on one of its side chains. In order to release the
chemotherapeutic agents within cancer cells, a disulfide bond was se-
lected for the linker between the chemotherapeutic agents and
targeting moieties to undergo reduction by GSH and release the
conjugated agent. To conjugate PTX to the peptide, 2-pyridyl disulfide
p-nitrophenyl carbonate was appended to PTX with thiopyridine
termination to give 2-pyridyl disulfide PTX carbonate (Figure 1).
Here, degarelix, a GnRH-R antagonist with high GnRH-R affinity
and enzymatic stability, was investigated as a targeting moiety con-
jugate for PTX. Previous studies of the structure–activity
relationship of GnRH analogs [35–37] demonstrated that the
N-terminus and position 6 of degarelix are not essential for re-
ceptor binding and, thus, are good candidates for the attachment
of PTX. To enhance the solubility of these peptides, in the peptide
sequences, MY-3, MY-4, and MY-5, the Arg-Gln chain was intro-
duced. In total, five degarelix derivatives were designed and
synthesized using solid-phase peptide synthesis on MBHA resin
with the Boc strategy as previously described [38]. Therefore, five
PTX–degarelix derivatives were generated after coupling the PTX
derivates (Figure 2). All compounds were purified by preparative
HPLC and were analyzed by analytical RP-HPLC and MS (Table 1).
To assess the release of PTX from the conjugates, LK-MY-10 was
incubated in the presence of a tenfold molar excess of the disulfide-
reducing agent, DTT, in the mixture of 0.1 mM phosphate buffer
solution (pH = 7.4) and acetonitrile. The thiol resulting from DTT
reduction was expected to cyclize into the proximate carbonyl
group of the linker, leading subsequently to the release of free
PTX (Figure 3). The HPLC profile showed (Figure 4) that the PTX
was quickly released when LK-MY-10 encountered the DTT
(T = 0 min) and the disulfide bond was completely cleaved within
1 h with concomitant release of PTX from the conjugates (T = 1 h).
Cytotoxicity % = [1À (ODtreated / ODcontrol)] × 100
where ODtreated and ODcontrol correspond to the OD values of the
treated and control cells, respectively. The percentage of cytotoxic-
ity was plotted as a function of concentration, and these points
were fitted to a sigmoid curve. The IC50 values were determined
from these curves. The experiment was repeated five times for each
concentration tested.
Receptor Saturation Tests
MCF-7 cells were seeded in 96-well cell-culture plates
(5 × 103 cells/well). After 24 h, the cells were treated with or with-
out 10 μM degarelix at 37 °C. After 12 h, the MCF-7 cells were washed
twice with HBSS and were incubated with PTX or LK-MY-10 (100 μl)
at the indicated concentrations. After 48 h, cell viability was then
measured by MTS assay, as described earlier.
Statistical Analysis
Data were analyzed using Student’s t-test for comparisons. A value
of p < 0.05 was considered statistically significant in all cases.
Results and Discussion
Synthesis of PTX–Degarelix Derivatives
The GnRH-R, also known as a luteinizing hormone-releasing hor-
mone receptor, is a member of the seven-transmembrane G
protein-coupled receptor family. Both GnRH and its receptor have
been detected in extrapituitary tissues and have also been shown
to have a role in the progression of some cancers, including breast,
prostate, endometrial, ovarian, and pancreatic and hepatocellular
carcinomas [34]. Therefore, drug molecules with a high affinity for
the GnRH-R may exhibit excellent tumor-targeting ability. In this
study, PTX was selected as the chemotherapy agent to be tested
Stability of the PTX–Degarelix Conjugates in Human Serum
The proteolytic stability of each of the conjugates in human serum
for different time was evaluated using RP-HPLC, and the half-life of
the conjugates were calculated and listed in Table 2. As expected, all
Figure 1. Synthesis of the PTX derivatives.
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J. Pept. Sci. 2015; 27: 569–576

GnRH RECEPTOR-TARGETED PACLITAXEL–DEGARELIX CONJUGATES
Figure 2. Synthesis of the PTX–degarelix derivatives.
compared with the AN-152. Among the peptide sequences of the
five conjugates, the introduction of Arg-Gln has decreased the
stability of the peptides in human serum from the half-lives data.
It may be that the Arg-Gln enhances the polarity of the peptides
and decreases the binding ability with human serum protein,
reducing the stability of the peptides. Furthermore, these data
demonstrate that these degarelix conjugates are stable enough
to allow sufficient time for cancer cell targeting.
Table 1. Chemical characteristics of the degarelix derivative conjugates
Compound
MALDI-TOF MS
RP-HPLC
Rt (min)a
Purity
(%)a
Yield
(%)
MWcal/MWexp
MYT-1
1677.36/1677.80
1678.35/1678.00
1961.68/1961.84
1962.67/1963.03
2292.08/2292.52
2634.10/2634.37
2632.79/2633.38
2917.61/2917.70
2919.10/2918.68
4202.85/4204.12
8.86
9.06
85.6
86.8
84.1
92.4
91.1
96.0
96.8
92.2
92.3
97.3
26.0
41.8
25.1
30.2
29.5
54.5
38.2
61.5
55.7
45.5
MYT-2
MYT-3
7.99
MYT-4
8.45
MYT-5
7.56
Antagonism Activity
LK-MY-7
LK-MY-8
LK-MY-9
LK-MY-10
LK-MY-11
13.10
13.24
11.86
11.60
12.46
The antagonism activity of the conjugates toward the GnRH-R were
determined in vitro using the FLIPR assay with CHO-K1 cells ex-
pressing the human GnRH-R and Gα15 protein [39]. Agonist or
antagonism-evoked changes in the levels of intracellular calcium
were detected by calcium-sensitive dyes. Table 3 shows the IC50
values for the antagonism activity of the conjugates that were
tested, and these values ranged from 1.4 to 131.0nM. In compari-
son, the biological effects of a native GnRH peptide and degarelix
were evaluated on CHO-K1/GnRH-R/Gα15 cells, and the EC50 ago-
nist activity and IC50 antagonism activity values were 41.4 and
0.2nM, respectively. These data demonstrate that all of the conju-
gates exhibited high levels of antagonism and some of the conju-
gates had IC50 values close to those of degarelix (Table 3). Taken
together, these results indicate that these conjugates would likely
gain entry into tumor cells via receptor-mediated endocytosis.
aRP-HPLC: Column: Zorbax XDB-C8 (4.6 mm × 150 mm) with 5 μm
silica; gradient: 0 min 30% B; 11 min 100% B; 14 min 100% B;
17 min 30% B; 21 min 30% B; eluents 0.1% TFA in water (A) and
0.1% TFA in acetonitrile–water (70 : 30, v/v) (B); flow rate: 1 ml/min;
detection: λ = 210 nm.
of the conjugates were relatively stable, and all had a t1/2 > 10 h,
and the t1/2 of AN-152 was 2 h in human serum [20]. Thus,
PTX–degarelix conjugates have at least six times longer t1/2
J. Pept. Sci. 2015; 27: 569–576
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WANG ET AL.
Figure 3. Disulfide-mediated release of PTX from the PTX–degarelix analog carbonate linker.
Table 2. Stability of the PTX–degarelix conjugates in human serum
Compound
Half-life in human serum(t1/2, h)
AN-152
2.10a
52.03
64.86
13.91
16.23
13.02
LK-MY-7
LK-MY-8
LK-MY-9
LK-MY-10
LK-MY-11
aThe data were from Reference [20].
Table 3. GnRH-R antagonism activity of the conjugates tested
Compound
Antagonism activity(IC50, nM)
Degarelix
LK-MY-7
LK-MY-8
LK-MY-9
LK-MY-10
LK-MY-11
0.2
89.2
102.0
1.4
6.4
131.0
Table 4. The in vitro cytostatic effects of the conjugates on MCF-7, HT-29,
and 3T3 cells
Compound
MCF-7(IC50, nM)
HT-29(IC50, nM)
3T3(IC50, nM)
PTX
7.1 2.0
>1 000 000
17.3 3.7
14.4 2.3
49.1 14.7
59.1 14.2
33.7 5.3
15.4 1.3
>1 000 000
44.2 7.0
15.7 6.8
Degarelix
LK-MY-7
LK-MY-8
LK-MY-9
LK-MY-10
LK-MY-11
>1 000 000
960.8 112.0
261.8 28.3
142.5 45.7
369.2 12.8
257.2 43.4
54.9 5.7
55.3 11.2
69.7 10.6
82.8 13.5
Figure 4. Triggered release of PTX from LK-MY-10 by disulfide reduction.
LK-MY-10 and a tenfold excess of DTT were analyzed using RP-HPLC
(Abs = 210 nm) in different time.
alone did not have any effect on the cells, consistent with previous
reports [40]. With MCF-7 and HT-29 tumor cells, both of which
express the GnRH-R, the IC50 cytostatic values of the conjugates
were several times higher than those of PTX. However, no signifi-
cant differences were observed between the cytostatic effects of
the conjugates with different coupling sites on degarelix in the
same cell lines. On 3T3 cells, which do not express the GnRH-R,
the IC50 values of the conjugates were one order of magnitude
In Vitro Cytostatic Effects of the PTX–Degarelix Conjugates
Using MCF-7 human breast cancer, HT-29 human colon cancer, and
3T3 mouse embryonic fibroblast cells, MTS assays were used to
evaluate the in vitro cytostatic effects of the five PTX–degarelix
conjugates. The calculated IC50 values are presented in Table 4. Free
PTX exhibited a similar cytostatic effect as the conjugates in the
lower nanomolar range on all tested cell lines. In contrast, degarelix
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 27: 569–576

GnRH RECEPTOR-TARGETED PACLITAXEL–DEGARELIX CONJUGATES
not eliminated by its conjugation to PTX, and these results are
consistent with the binding affinity data described earlier.
Conclusions
In this study, five PTX–degarelix conjugates were synthesized and
analyzed. All conjugates were found to be at least six times more
resistant to proteolytic degradation in human serum compared
with AN-152 and were also more cytostatic to cancer cells than to
normal cells. These results demonstrate that degarelix conjugates
are stable and efficacious targeting moieties that could be further
optimized for use in targeted chemotherapy.
Acknowledgements
This work was supported by the National Key Technologies R&D
Program for New Drugs of China (2012ZX09301003) and the
National Natural Science Foundation of China (81172925).
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