Download Development of a 99mTc-labeled lactam bridge-cyclized alpha

Ann Nucl Med (2015) 29:709–720
DOI 10.1007/s12149-015-0998-y
ORIGINAL ARTICLE
Development of a 99mTc-labeled lactam bridge-cyclized alphaMSH derivative peptide as a possible single photon imaging agent
for melanoma tumors
Danial Shamshirian1 • Mostafa Erfani2 • Davood Beiki3 • Babak Fallahi3
Mohammad Shafiei2
•
Received: 22 November 2014 / Accepted: 17 June 2015 / Published online: 8 July 2015
Ó The Japanese Society of Nuclear Medicine 2015
Abstract
Objective Melanocortin-1 (MC1) receptor is an attractive
melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam
bridge a-MSH analog was labeled with 99mTc via HYNIC
and EDDA/tricine as coligands including gamma
aminobutyric acid (GABA) as a three carbon chain spacer
between HYNIC and the N-terminus of the cyclic peptide.
Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution
were thoroughly investigated.
Methods HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer
chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was
studied in MC1 receptor expressing B16/F10 cells.
Biodistribution of radiopeptide was studied in nude mice
bearing B16/F10 tumor.
Results Labeling yield of [98 % (n = 3) was obtained
corresponding to a specific activity of 81 MBq/nmol.
Peptide conjugate showed efficient stability in the presence
of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In
& Mostafa Erfani
[email protected]
1
Department of Radiopharmacy, School of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
2
Radiation Application Research School, Nuclear Science and
Technology Research Institute (NSTRI),
P.O.Box: 14395-836, Tehran, Iran
3
Research Center for Nuclear Medicine, Tehran University of
Medical Sciences, Tehran, Iran
biodistribution studies, a receptor-specific uptake was
observed in MC1 receptor-positive organs so that after 2 h
the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while
low accumulation in the kidney uptake was observed
(4.58 ± 0.68 % ID/g at 2 h after injection).
Conclusions The obtained results show that the presented
new designed labeled peptide conjugate may be a suitable
candidate for diagnosis of malignant tumors.
Keywords
imaging
a-MSH 99m
Tc B16/F10 cells Tumor
Introduction
The molecular basis for the use of radiopeptides was found
to be that peptide receptors are over-expressed by certain
tumors [1–3]. Up to now, several types of peptides have
been introduced for tumor targeting, and many types of
cancer cells have demonstrated overexpression of various
peptide receptors [4, 5]. Alpha-Melanocyte stimulating
hormone (a-MSH) is a tridecapeptide (Ac-Ser-Tyr-SerMet-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2)
produced in pituitary gland that binds to Melanocortin-1
(MC1) receptor on the surface of normal melanocytes, for
the purpose of regulating melanin production involved with
skin pigmentation. The involvement of MC1 receptor
during the proliferation of melanoma cells, suggests that aMSH and its analogs may be candidates for peptide-based
diagnosis or therapy of this tumor type [6–8]. Over 80 % of
human metastatic melanomas were reported to over-express MC1 receptors, and this disease causes greater than
75 % of skin cancer deaths [9–11]. The need for new
radiopharmaceuticals able to diagnose tumor in the early
stages is evident. Native a-MSH has an extremely short
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biological half-life because it is easily hydrolyzed by proteases and also the methionine residue in a-MSH can be
oxidized [12]. Newer generation agents have been designed
to enhance their potency toward MC1 receptor, including
peptides with linear structures [13–16] and cyclic structures [17–19]. However, development of new analogs with
higher MC1 receptor affinity, stability, and prolonged
activity of the a-MSH analog which resulted in greater
tumor uptake and lower kidney uptake is promising.
Previous studies on the a-MSH peptide agonists for the
MC1 receptor revealed that the lactam bridge-cyclized aMSH peptide with a 12-amino acid in the peptide ring
[Lys-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Arg-Pro-Val-Asp]
exhibiting high MC1 receptor-mediated tumor uptake [18,
20]. Also utilization of the concept of pseudoisosteric
cyclization by replacing Met4 and Gly10 by a Cys4, Cys10
disulfide bridge, to give a cyclic disulfide containing
7-aminoacid peptide ring analog, [Cys-Glu-His-Phe-ArgTrp-Cys] (CycMSHhept), which was found to possess
superpotent bioactivity in the frog skin bioassay [21, 22]. A
correlation between ring size and biological potency in the
frog skin bioassay system was found in 7-aminoacid peptide ring CycMSHhept disulfide containing melanotropin
analogs [23, 24]. In evaluation of the effect of ring size on
the potencies of the cyclic lactam analogs, it has been
reported that potency and prolongation decreased with
reduction of the ring size in 6-amino acid in the peptide
ring [Asp-His-D-Phe-Arg-Trp-Lys] (CycMSHhex) from 23
to 20 membered ring compounds [25].
Different amino acids and hydrocarbon linkers displayed profound favorable effects on the receptor-binding
affinities and pharmacokinetics of radiolabeled RGD [26],
a-MSH peptides [18, 20, 27], and bombesin [28]. Miao
et al. [18] reported that introduction of a negatively
charged linker (Gly-Glu) between the DOTA and CycMSH
peptide sequence decreased the renal uptake by 44 %
without affecting the tumor uptake at 4 h post injection.
Guo et al. [27] also reported that introduction of a neutral
Gly–Gly linker between the DOTA and Nle-CycMSHhex
maintained high melanoma uptake, while reducing kidney
and liver uptake of 111In-DOTA-GGNle-CycMSHhex.
a-MSH lactam bridge-cyclic analogs have been radiolabeled with different radionuclides including 111In [18],
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Ga [29], 64Cu [30], and 99mTc [31, 32]. Guo et al. [31]
recently evaluated the radiochemical and biological
behavior of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex
and concluded that the HYNIC moiety in the radioactive
molecule promoted its stability as well as enhancing renal
excretion. Our efforts to pursue a 99mTc-labeled peptide for
tumor targeting, based on the previously reported unique
structure of DOTA-Nle-CycMSHhex, have led us to prepare
a HYNIC-conjugated Nle-CycMSH peptide. Also to
examine the exhaustive effects of the amino acid linker on
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Ann Nucl Med (2015) 29:709–720
the melanoma targeting and pharmacokinetic properties of
peptide, gamma aminobutyric acid (GABA) as the three
carbon chain spacer was inserted between the HYNIC and
the Nle-CycMSH peptide to generate HYNIC-GABA-NleCycMSH. Furthermore, based on the explanation above by
which reduction of the ring size in cyclic MSHhex potency
of peptide reduced, the size of the ring was increased by
introduction of a Gly10 from the main sequence of a-MSH
peptide between Trp9 and Lys11 to yield HYNIC-GABANle-CycMSHhept (Asp-His-DPhe-Arg-Trp-Gly-Lys). This
study presents the synthesis of HYNIC-GABA-NleCycMSHhept, the optimum radiolabeling conditions with
99m
Tc via tricine and EDDA as coligands, and further
characterization of the 99mTc-HYNIC-GABA-NleCycMSHhept using an in vivo C57 mice bearing B16/F10
tumor model.
Experimental
Materials
Sieber amide resin and all of the Fmoc-protected amino
acids were commercially available. The pro-chelator
HYNIC-Boc was synthesized according to Abrams et al.
[33]. Other reagents were purchased from Sigma-Aldrich
(Munich, Germany) and used without further purification.
The reactive side chains of the amino acids protecting
agents were masked with one of the following groups: Trp,
tert-butoxycarbonyl (Boc); Lys, N0 -methyltrityl (Mtt); Arg,
2,2,4,6,7 pentamethyldihydrobenzofurane-5-sulfonyl (pbf);
His, N-T-Trityl (Trt); and Asp, 2-phenylisopropylester (o2-phipr). The cell culture medium was Roswell Park
Memorial Institute (RPMI-1640) supplemented with 10 %
fetal bovine serum (FBS), amino acids, vitamins, and
penicillin/streptomycin (Gibco, Eggenstein, Germany).
Sodium pertechnetate (Na99mTcO4) was obtained from a
commercial 99Mo/99mTc-generator (Radioisotope Division,
NSTRI, Tehran, Iran). Analytical reverse phase high performance liquid chromatography (RP-HPLC) was performed on a HPLC system (Sykam S7131, Eresing,
Germany) equipped with a multiwavelength UV detector
set at k = 280 nm, a flow-through gamma-detector
(Raytest-Gabi, Straubenhardt, Germany), and a C18 analytical column (CC 250/4.6 Reprosil-pur ODS-3.5 lm).
The mobile phase consisted of 0.1 % trifluoroacetic acid/
water (Solvent A) and acetonitrile (Solvent B), and the
gradient system used a flow rate of 1 mL/min at an
A:B ratio of: 95:5 at 0 min, 95:5 at 5 min, 0:100 at 25 min,
0:100 at 27 min, 95:5 at 30 min, and 95:5 at 35 min. A
mass spectrometer (1100/Bruker Daltonic; Agilent, Bremen, Germany) with a VL instrument (LC/MS) was also
used. Quantitative gamma counting was performed on an
Ann Nucl Med (2015) 29:709–720
EG&G/ORTEC (Model 4001M, Jackson, USA) Mini Bin
and Power Supply counter.
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Radiochemical analysis
99m
Synthesis
The peptide was synthesized by standard Fmoc solid-phase
synthesis on Siber amide resin with substitution,
0.65 mmol/g [34]. Coupling of each amino acid was performed in the presence of 3 mol excess of Fmoc-amino
acid, N-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) and 5 mol
excess of diisopropylethylamine (DIPEA) in DMF. The
Kaiser test determined the end of a coupling reaction.
Fmoc groups were removed by adding 20 % (v/v) piperidine in DMF (10 mL). The protecting groups Mtt and
2-phenylisopropyl were removed by 1 % TFA in advance
of the peptide cyclization reaction, and protected peptide
was cleaved from the resin by treatment with a mixture of
2.5 % TFA and 5 % of triisopropylsilane in dichloromethane (DCM) (10 mL). Each protected peptide was
cyclized by coupling the carboxylic group from aspartic
acid with the epsilon amino group on lysine. The cyclization reaction was achieved by an overnight reaction with
peptide in DMF (2 mL) using benzotriazole-1-yl-oxytrispyrrolidino-phosphonium-hexafluorophosphate (PyBOP)
as a coupling agent in the presence of DIPEA. HYNIC-Boc
(1.2 mol) is coupled with O-(7-azabenzotriazol-1-yl)1,1,3,3,tetramethyluronium hexafluorophosphate (HATU)
(1.2 mmol) and DIPEA (3.6 mmol) in DMF (5 mL) to the
N-terminus of the peptide (1 mmol). The amino acid side
chains of the peptide HYNIC conjugate were also deprotected by treatment with a cocktail of TFA, thioanisole,
water, and triisopropylsilane (92.5:2.5:2.5:2.5) for 30 min
at 25 °C. After removing the organic solvents in a vacuum,
the crude product was precipitated by adding a solution of
cold petroleum ether and diisopropyl ether (50:50).
99m
Tc-labeling of HYNIC-peptide
Labeling was performed in accordance with a previous
report [35, 36]. Briefly, a stock solution was prepared by
dissolving the HYNIC-peptide in distilled water. Radiolabeling of peptide was performed by adding to water
(0.5 mL), the stock solution (20 lg of peptide; 20 mL),
tricine (15 mg), and EDDA (5 mg). To this mixture was
added nitrogen-purged stannous chloride dihydrate solution
(in 0.1 M HCl; 2 mg/mL; 20 lL). Finally, 99mTc-pertechnetate (370-1110 MBq) in saline (0.5 mL) was added to
the solution in an inert atmosphere and incubated for
10 min at 95 °C. After the contents were cooled to the
ambient temperature, quality control tests were performed
as described below.
Tc-HYNIC-peptide was assayed by analytical RPHPLC in the above-mentioned analytical condition. Instant
thin layer chromatography (ITLC) strips (silica gel 60;
Merck) were loaded with sample and developed using three
different mobile phases: (1) methylethylketone for 99mTcpertechnetate (Rf = 1.0); (2) aqueous sodium citrate
(0.1 M; pH 5) to determine the non-peptide bound 99mTccoligand and 99mTc-pertechnetate (Rf = 1.0); and (3) [1:1]
methanol: aqueous ammonium acetate (1 M) for 99mTccolloid (Rf = 0.0). The radioactivity was quantified by
cutting a strip (10 cm) into 1 cm pieces and then counting
each piece in a gamma counter.
Stability
In vitro stability was evaluated in NaCl 0.9 % (W/V) and
in human serum. Aliquots were taken out at 1, 4, 6, 12, and
24 h post labeling at room temperature and analyzed by
HPLC and ITLC. The level of protein-bound radioactivity
versus non-bound radioactivity was evaluated using a
precipitation method. To a freshly prepared human serum
(1 mL), was added 99mTc-HYNIC-peptide (86 MBq;
0.1 mL), and the mixture was incubated at 37 °C for 24 h.
Samples of 100 ll aliquots were removed and treated with
ethanol (100 lL). Each sample was centrifuged for 5 min
at 3000 rpm to separate the precipitated serum protein.
Supernatant was removed with a syringe, and radioactivity
in the supernatant (containing soluble 99mTc-ligands) was
compared with that in sediment (containing insoluble
99m
Tc, HYNIC-peptide) to determine the percentage of
radiopeptide and/or 99mTc-proteins. The supernatant was
analyzed with HPLC to determine the stability of labeled
compound at 24 h.
The evaluation of in vivo stability of radiolabeled peptide was conducted in normal balb/c mice by injecting
20 MBq (100 lL) 99mTc-HYNIC-peptide intravenously
through the tail vein. After an hour, a fraction of urine was
collected and centrifuged at 15,000 rpm for 10 min and
analyzed by HPLC.
Partition coefficient (hydrophilicity)
To determine the partition coefficient value, 99mTcHYNIC-peptide in phosphate-buffered saline (PBS)
(0.5 mL) was mixed with n-octanol (0.5 mL) in a 2 ml
microtube. The tube was vigorously vortexed over a period
of 2 min and centrifuged at 40009g for 5 min. From each
layer, three samples were taken (100 mL) and then counted
in the gamma counter. The average radioactivity values
from each of the aqueous and octanol layers were calculated and then were used to compute the octanol-to-water
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partition coefficient (Po/w) by dividing the radiolabeled
peptide value (octanol phase) by the aqueous phase value.
Cell culture
The B16/F10 murine melanoma cells were cultured in
RPMI-1640 supplemented with 10 % (v/v) FBS, glutamine
(2 mM), penicillin (50 U/ml), and streptomycin (50 lg/ml).
Cells were maintained in a humidified 5 % CO2/air atmosphere at 37 °C. For all cell experiments, the cells were
seeded (1 million cells per well) in six well plates and then
incubated overnight with internalization medium (RPMI1640 with 1 % FBS).
Ann Nucl Med (2015) 29:709–720
MSH) peptide (100 mg) in saline (0.9 %; 50 mL) as a coinjection as a single dose in a one syringe. The aims are to
(1) determine the distribution of radiotracer in non-target
tissues that contain MC1 receptors and (2) broadly understand the MC1 receptor density in target versus non-target
tissues, by challenging receptor binding of 99mTc-HYNICpeptide with a-MSH peptide.
After 1, 2, 4, and 24 h, the groups of mice were sacrificed, after which the organs of interest were excised,
weighed, and counted in the gamma counter. The percentage of the injected dose per gram (% ID/g) was calculated for each tissue.
Scintigraphy studies
Binding and internalization study
Medium was removed by pipette from each of the six well
plates containing adhered B16/F10 cells, with a density of
1 million cells per well and then the cells were washed
once with 2 ml of internalization medium (RPMI-1640
with 1 % FBS; 2 mL). Furthermore, a 1.5 mL sample of
internalization medium was added to each well, the plates
were incubated at 37 °C for 1 h, and then 99mTc-HYNICpeptide (150 KBq; 2.5 pmol total peptide per well) was
added. The cells were incubated at 37 °C for various time
periods (0.5–4 h). To determine non-specific membrane
binding and internalization, we also incubated cells with
the radioligand in the presence of a-MSH peptide (150 lL;
1 lmol/L). In separate experiments, the cellular binding
and uptake reaction was stopped at 0.5, 1, 2, and 4 h by
removing medium from the adhered cells, washing them
twice with cold PBS (*4 °C; 1 mL), and then an acid
wash with a duration of 10 min was performed twice with
cold glycine buffer (pH 2.8; *4 °C; 1 mL). The last step
was designed to distinguish between membrane-bound
(acid releasable) and internalized (acid resistant) radioligand. Finally, the cells were treated with NaOH (1 M;
3 mL). All the fractions (reaction medium; PBS washes;
acid washes; cells) at the different time points were
counted in a gamma counter.
To better understand the whole body localization, the
behavior of 99mTc-HYNIC-peptide was evaluated by the
static images of the mice, each of which received (20 MBq,
100 lL) of labeled peptide via a tail vein. Also for blocking
study mice were injected with (a-MSH) peptide (100 lg)
in saline (0.9 %; 50 lL) using a co-injection as a single
dose in a one syringe. Before the imaging mice were
anesthetized with 0.05 mL ketamine 10 % (3.3 mg) and
0.05 mL xylazine 2 % (1.33 mg) intraperitoneally. After
about 5 min, the animal was fixed on a board by covering it
with pieces of cloth for immobilization during the
scanning.
Scintigraphy imaging study was obtained using a single
head gamma camera (small area mobile, 140 keV, Siemens, Germany) equipped with high sensitivity parallel
whole collimator. Whole body image was obtained using a
256 9 256 matrix size with 5000 k counts at 1 h post
injection. For image acquisition, a 10 % acceptance window around the 140 keV photo peak was used.
Statistical methods
The data are reported as mean ± standard deviation.
A Student t test was used to determine the statistical significance, which was defined as P \ 0.05.
Biodistribution assay
Results and discussion
Animal experiments were performed in compliance with
the regulations of NSTRI institution and with generally
accepted guidelines governing such work. Groups of three
C57 nu/nu mice (8–10-weeks old, Pasteur Institute, Amol,
Iran) were used in each experiment. A suspension of B16/
F10 cells (1x107) in PBS (0.1 mL) was subcutaneously
injected in the right flank of each mouse. Seven to 10 days
after inoculation, the tumors developed and then 99mTcHYNIC-peptide (20 MBq, 100 lL) was injected via a tail
vein. Also a group of three mice were injected with (a-
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Synthesis and labeling
MC1 receptors are known to be over-expressed in human
skin melanomas [6–8]. The aim of this study was to target
the MC1 receptor in vitro using a tumor cell line and
in vivo using a mouse tumor model, using an analog of aMSH containing a HYNIC-coupled lactam bridge-cyclic
structure. HYNIC chelator was covalently attached to
GABA-bonded onto the N-terminus of Nle-CycMSHhept,
Ann Nucl Med (2015) 29:709–720
713
the desired conjugating group for the chosen radionuclide
99m
Tc.
[HYNIC-GABA-Nle]-CycMSHhept was synthesized by
Fmoc strategy, and it was obtained in an overall yield of
35 % based on the removal of the first Fmoc group. The
composition and structural identity of HYNIC-peptide
were verified by analytical RP-HPLC in the above-mentioned analytical condition and LC–MS (Table 1). ESIMass analysis was consistent with the calculated molecular
weight for the [HYNIC-GABA-Nle]-CycMSHhept. Calculated mass for this new derivative is 1258.65 g/mol, and
LC–MS analysis confirmed a [M ? H]? molecular ion of
1259.399.
A variety of bifunctional chelating agents (BFCA) have
been used to label proteins, peptides, and other biologically
active molecules with 99mTc [36, 37]. More recently,
HYNIC has become a more popular BFCA because in the
presence of various coligands having an effect on the
hydrophilicity and pharmacokinetics of radiopeptides, high
specific activity products can be prepared [38]. Of the
various coligands, tricine achieves the best radiolabeling
efficiency. It has been reported that the composition for the
99m
Tc-HYNIC-tricine complex most likely is, [99mTc(HYNIC)(tricine)2], with the complex unstable in solution and found to exist in multiple forms that could be
attributed to coordination isomerism in its structure [38].
Various isomers can result from different bonding modalities of the hydrazine functionality of the HYNIC and the
two tricine coligands [38]. Both Tc-hydrazido and Tc-diazenido bonds have been previously reported and characterized by X-ray crystallography [39, 40]. These different
bonding modalities from the tricine and hydrazine ligands
are expected to produce numerous structural and
stereoisomers. [38]. The coligand EDDA is also of particular interest because of being a potentially tetradentate
ligand. It is expected to form a more symmetrical and
stable complex with technetium when compared to the
tricine [38]. It has been shown that using both coligands
together to produce 99mTc-HYNIC-peptide via a transmetalation type of reaction produces very good results [41].
In this study, 20 lg of HYNIC-peptide was used with tricine and EDDA together as a coligand in amounts of
15 mg and 5 mg, respectively, in a final volume of the
labeled solution. The structure of synthesized ligand and
Table 1 Analytical data of
[HYNIC-GABA-Nle]CycMSHhept
Compound
HYNIC-peptide
the proposed structure after preparation of 99mTc/Tricine/
EDDA/HYNIC-GABA-Nle-CycMSHhept have been shown
(Fig. 1).
Quality control
The radiochemical purity of 99mTc-HYNIC-peptide 99mTcHYNIC-peptide was evaluated by RP-HPLC using the
gradient systems consisting of 0.1 % trifluoroacetic acid
(TFA) in water (Solvent A) and acetonitrile (Solvent B).
The radiolabeling yield was [98 % (n = 3), acquired via
HPLC and also ITLC with a small amount of 99mTcpertechnetate (\0.5 %), 99mTc-radiocolloid (\0.2 %), and
99m
Tc-coligands (\1.0 %), and the specific activity was
determined to be 81 MBq/nmol. The HPLC elution times
were 3.34 min for free pertechnetate and 16 min for
labeled peptide (Fig. 2). In comparison to reported data in
the literature regarding 99mTc/tricine-HYNIC complex
instability [38] due to the exchange labeling method, a
single major peak was observed without any impurities due
to isomeric forms of the 99mTc-HYNIC-peptide.
Stability and hydrophilicity
The 99mTc-HYNIC-peptide was stable up to 24 h post
labeling at the ambient temperature. High labeling yield
and stability may be due to optimization of formulation in
the amount of materials, and also in exchanging the
labeling method.
In serum protein binding studies, 17.9 ± 1.56 % of the
activity was associated with the precipitate obtained after
ethanol addition, indicating the low binding of complex
with serum proteins due to a specific adsorption or transchelation. The ethanol fraction was characterized by
HPLC where a single peak was observed at the same time
(90 %) as that of the complex (both peaks having the same
retention time). Radiochemical purity yield started at 98 %,
and then declined to 90 % at 24 h, indicating 8 %
decomposition over this time period (Table 2). 99mTcHYNIC-peptide showed metabolic stability in human
serum up to 24 h after labeling and incubation (Fig. 3). In
contrast to the high stability for cyclic lactam analog
achieved in our work, Raposinho et al. [42] reported a
relatively low metabolic stability for the linear derivative.
Mass spectrum
RP-HPLC
Calculated mass (g/mol)
Observed mass (g/mol)
Retention time (min)
Purity (%)
1258.65
1259.399 [M?H]?
13.98
[98
Analytical HPLC performed on a C18 column using a gradient of 0.1 % aqueous TFA (Solvent A) in
acetonitrile (Solvent B) in 30 min at 1 mL/min. The following gradient was used. A:B ratio of: 95:5 at
0 min, 95:5 at 5 min, 0:100 at 25 min, 0:100 at 27 min, 95:5 at 30 min, and 95:5 at 35 min
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Ann Nucl Med (2015) 29:709–720
Fig. 1 The structure of
synthesized ligand and the
proposed structure after
preparation of 99mTc/Tricine/
EDDA/HYNIC-GABA-NleCycMSHhept
H2N
C
HN
NH
HN
HN
O
O H
C N
H
N
O
C
H
O N
C
HN
HN
C
N
TcO4-
O
tricine
C
NH2
C N
OH
C
NH
OO C
EDDA
95°C, 15 min
CH3
HN
HN
O
C
O
NH
O
O
C
HN
O
C
HN
O C
HN
O C
HN
O
C NH
2
N
H
NH
C O
HN
NH
C
H2N
N
H
NH
NH2
N
C O
O
NH
C
HN
N
H3C
H
N
HN
N
Tc
N
HOOC
O
O
99m
N
O
O
H
N
O
N
OH
OH
O OH
C
O
N
HN
The high stability of this lactam bridge-cyclic 99mTcHYNIC-peptide could be attributed to its stable secondary
structure such as a beta-turn with an intramolecular
hydrogen bound. Due to this structural peculiarity, the
cyclic peptide has less conformational freedom and higher
stabilities than the linear peptide [21, 25, 43, 44].
The partition coefficient for 99mTc-HYNIC-peptide was
calculated and found to be (log P) -1.31 ± 0.12 which is a
good indicator of its hydrophilicity.
Internalization and binding
Figure 4 shows the results related to time dependency and
specific internalization of 99mTc-HYNIC-peptide into B16/
F10 cells. High rate of internalization was observed in this
99m
Tc-HYNIC-peptide (12.45 ± 1.1 % up to 4 h) which
was not unexpected since CycMSH sequence offers agonistic property to the compound [25, 36]. Previous studies
in a series of 99mTc-labeled CycMSHhex demonstrate
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internalization and receptor-mediated trapping of labeled
compounds [18, 19, 31, 39]. As it shows the significant
differences of uptake between blocked and unblocked cells
in various time periods are very noticeable (P \ 0.05).
Garcia et al. [32] recently evaluated a 99mTc labeled
HYNIC-MSH analogs, while HYNIC without spacer is
connected directly to Nle in the peptide structure. They
reported at 60 min, only 1.3 % of 99mTc-HYNIC-cycMSH/EDDA was internalized in the B16/F1 melanoma
cells (0.5 9 106). In comparison to this we obtained a
7.1 % of internalization after 1 h in B16/F10 melanoma
cells (1 9 106). Since the cell line and the method are not
similar, it is difficult to compare the results with each other,
nevertheless this high rate of internalization may be due to
the structural modification of the 99mTc-HYNIC-peptide.
This can be explained by the fact that by placing a
bifunctional chelating agent (HYNIC) farther from the
receptor-binding region of peptide with the use of a three
carbon chain spacer (GABA), the negative effect of
Ann Nucl Med (2015) 29:709–720
715
Fig. 2 Radioactive HPLC
profiles of the 99mTc/Tricine/
EDDA/HYNIC-GABA-NleCycMSHhept
Table 2 In vitro stability of 99mTc/Tricine/EDDA/[HYNIC-GABANle]-CycMSHhept in human plasma (37 °C)
Time (h)
Radiochemical purity (%)
1
98.3 ± 0.2
4
97.4 ± 0.3
6
96.7 ± 0.2
12
94.6 ± 0.4
24
90.5 ± 0.3
The data are expressed as mean ± standard deviation (n = 3)
chelator on the receptor binding is reduced. So, the more
specific receptor binding of the compound, the higher the
radioactivity internalization.
It has been shown that peptide with positive charge in
their sequence tends to interact faster and more effectively
with proteins which are not targeted to a specific receptor
[42]. Also a somewhat different amphiphilic-like structure
has been recognized in a series of enkephalin analogs
previously [45]. Considering to reach a fast and receptorspecific internalization which is also an indication of the
balance of lipophilicity for the complex may explain the
insertion of a GABA linker as a hydrocarbon spacer in the
structure of a MSH conjugate.
Biodistribution
Results from biodistribution studies using the 99mTcHYNIC-peptide are presented in Table 3 as the percentage
of injected dose per gram of tissue (% ID/g). 99mTcHYNIC-peptide displayed rapid blood clearance with
0.62 ± 0.19 % ID/g at 1 h. Clearance from the blood circulation was fast with 0.18 % ID/g remaining in the blood
at 4 h, and the whole body clearance proceeded via the
urinary system. The HPLC analysis of urine at 1 h post
injection was performed to identify the urine metabolites.
The radiochromatogram of urine analysis is shown in
Fig. 5. The profile shows that 99mTc-HYNIC-peptide has
remained intact in urine at 1 h post injection which confirms the in vivo stability of complex.
Clearance from MC1 receptor-negative tissues was also
rapid. In normal tissues, the uptake of 99mTc-HYNICpeptide was lower than 1.00 % ID/g except for kidneys, at
1, 2, 4, and 24 h post injection. The uptake value of 99mTcHYNIC-peptide for skin was 0.84 ± 0.14 % ID/g at 1 h,
while this value decreased to 0.43 ± 0.09 % ID/g at 2 h
post injection which shows the rapid clearance of the
99m
Tc-HYNIC-peptide.
At 1 h after injection, the kidney uptake was
5.72 ± 0.40 % ID/g which decreased to 1.47 ± 0.30 %
ID/g at 24 h after injection. In an in vivo blocking study by
co-injection of cold peptide at 1 h post injection, the renal
uptake activity did not reduce, indicating that the renal
uptake was not MC1 receptor mediated and the kidney is
the major excretion pathway for the 99mTc-HYNICpeptide.
The tumor uptake was 4.90 ± 0.47 % ID/g at 1 h post
injection and reached its peak value of 5.10 ± 0.08 % ID/g
at 2 h post injection. High tumor/blood (7.90) and high
123
716
Ann Nucl Med (2015) 29:709–720
Fig. 3 Radioactive HPLC
profiles of 99mTc/Tricine/
EDDA/[HYNIC-GABA-Nle]CycMSHhept after incubation in
human serum at 24 h post
incubation (37 °C)
unblocked
blocked
% specific internalized/ID
14
12
10
8
6
4
2
0
0
50
100
150
200
250
time (min)
Fig. 4 Internalization rate of 99mTc/Tricine/EDDA/[HYNIC-GABANle]-CycMSHhept into B16/F10 cells. Data are from three independent experiments with triplicates in each experiment and are
expressed as specific internalization
tumor/skin (5.83) uptake ratios were achieved as early as
1 h post injection. In the blocking study at 1 h post injection, tumor uptake was significantly reduced from 4.90 %
ID/g in the control group to 1.63 % ID/g in the blocked
group which shows that the tumor uptake was MC1
receptor mediated. On the other hand, the uptake reduction
in normal tissues due to blocking was not significant. In
blocked skin, activity reduction was not significant
(0.84 ± 0.14 % ID/g versus 0.62 ± 0.12 % ID/g) which
proves non-specific targeting in this organ.
Uptake values compared to those of a previously reported
99m
compound
Tc(EDDA)-HYNIC-GGNle-CycMSHhex
123
[31], with the value lower than 1.02 % ID/g except for the
kidneys at 2, 4, and 24 h post injection, for this 99mTc99m
HYNIC-peptide,
Tc/Tricine/EDDA/HYNIC-GABANle-CycMSHhept, the uptake of radioactivity in normal tissues was lower than 0.51 % ID/g except for the kidneys at 2,
4, and 24 h post injection. Interestingly, the introduction of a
GABA linker reduced kidney uptake of HYNIC-GABANle-CycMSHhept, compared to HYNIC-GGNle-CycMSHhex
(4.58 ± 0.68 % ID/g versus 7.52 ± 0.69 % ID/g at 2 h post
injection, respectively). It has been reported that different
pathways may play roles in the mechanism of the renal
uptake of radiolabeled peptides such as the electrostatic
interaction between the positively charged peptides and
negatively charged tubule cells, [46], endocytosis in tubular
cells [47], and transmembrane glycoproteins such as megalin
[48]. The introduction of a negatively charged glutamic acid
as a linker has reduced the renal uptakes of radiolabeled
cyclized a-MSH peptides [49]. The reduction in kidney
uptake might be attributed to shielding of the electrostatic
interaction between positively charged Arg in the moiety of
cyclized a-MSH peptides and negatively charged surface of
tubule cells or could be explained by one of the above
mechanisms. Further investigation for better understanding
of the mechanism is required.
The tumor uptake of 99mTc/Tricine/EDDA/HYNICGABA-Nle-CycMSHhept in B16/F10 melanoma-bearing
mice was lower than that reported for 99mTc(EDDA)HYNIC-GGNle-CycMSHhex [31] which was studied in
B16/F1 melanoma-bearing mice (5.10 ± 0.08 % ID/g
versus 14.14 ± 4.90 % ID/g at 2 h post injection, respectively). As it shows the comparison of uptake values in
Ann Nucl Med (2015) 29:709–720
Table 3 Biodistribution of
99m
Tc/Tricine/EDDA/[HYNICGABA-Nle]-CycMSHhept in
B16/F10 tumor-bearing nude
mice (% injected dose/g
organ ± SD, n = 3)
717
Organ
1h
1 h block
2h
4h
24 h
Blood
0.62 ± 0.19
0.88 ± 0.16
0.29 ± 0.08
0.18 ± 0.07
0.09 ± 0.02
Heart
0.29 ± 0.05
0.48 ± 0.18
0.14 ± 0.02
0.10 ± 0.03
0.02 ± 0.04
Lung
0.64 ± 0.08
0.56 ± 0.26
0.34 ± 0.09
0.19 ± 0.11
0.02 ± 0.05
Liver
0.65 ± 0.11
0.56 ± 0.10
0.51 ± 0.07
0.41 ± 0.18
0.22 ± 0.06
Spleen
0.25 ± 0.03
0.61 ± 0.12
0.16 ± 0.02
0.12 ± 0.02
0.01 ± 0.02
Stomach
0.86 ± 0.14
0.75 ± 0.22
0.49 ± 0.14
0.33 ± 0.09
0.17 ± 0.05
Intestines
0.61 ± 0.10
0.85 ± 0.28
0.48 ± 0.10
0.31 ± 0.15
0.13 ± 0.05
1.47 ± 0.30
Kidneys
5.72 ± 0.40
6.49 ± 0.56
4.58 ± 0.68
4.06 ± 0.33
Muscle
0.14 ± 0.03
0.11 ± 0.04
0.07 ± 0.02
0.06 ± 0.01
0.04 ± 0.01
Skin
0.84 ± 0.14
0.62 ± 0.12
0.43 ± 0.09
0.39 ± 0.09
0.12 ± 0.02
Tumor
4.90 ± 0.47
1.63 ± 0.46
5.10 ± 0.08
4.27 ± 0.09
1.03 ± 0.05
Fig. 5 Radioactive HPLC
profiles of urine analysis of
mice 1 h post injection of
99m
Tc/Tricine/EDDA/[HYNICGABA-Nle]-CycMSHhept
different cell lines, it is not conclusive and in order to reach
a better conclusion, further studies in similar cell lines are
necessary.
The tumor uptake of 99mTc/Tricine/EDDA/HYNICGABA-Nle-CycMSHhept was more than two fold than that
reported for 111In-DOTA-GlyGlu-CycMSH [20] which was
studied in B16/F10 metastatic melanoma-bearing lung
mice, (5.10 ± 0.08 % versus 2.00 ± 0.74 % ID/g at 2 h
post injection, respectively).
The tumor accumulation of 99mTc/Tricine/EDDA/
HYNIC-GABA-Nle-CycMSHhept
and
its
efficient
pharmacokinetic behavior such as low tendency to accumulate in liver and kidney followed by fast clearance due to
low lipophilicity and high stability are the major advantages
of 99mTc-HYNIC-peptide for melanoma detection.
Imaging
Since the highest tumor uptake and highest urinary clearance occurred at 1-2 h post injection for 99mTc-HYNICpeptide, in order to visualize tumor localization, the planer
gamma imaging studies also were performed at 1 h post
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Ann Nucl Med (2015) 29:709–720
Conclusion
In this study, the synthesis, radiolabeling, and biodistribution of [HYNIC-GABA-Nle]-CycMSHhept were shown.
The radiolabeling step was completed within a very short
time (10 min) in high specific activity, and no significant
impurities were detected by HPLC. Furthermore, 99mTcHYNIC-peptide was prepared by labeling of peptide with a
GABA linker, HYNIC, and tricine/EDDA as coligands.
The 99mTc-HYNIC-peptide demonstrated an excellent
radiochemical stability even up to 24 h post labeling.
Presented 99mTc-HYNIC-peptide had a specific cell binding and internalization followed by a stable behavior in
human serum at 37 °C for at least 24 h. The 99mTcHYNIC-peptide showed low accumulation in the kidney,
high accumulation in tumor as a positive MC1 receptortargeted tissue followed by excretion via the kidney. These
promising characteristics make this 99mTc-HYNIC-peptide
a suitable candidate for the diagnosis of malignant tumors.
Acknowledgments This research has been part of a PhD thesis and
supported by Tehran University of Medical Sciences and AEOI,
Grant No. 24215, Tehran, Iran. There is no conflict of interest.
References
Fig. 6 Posterior whole body scan image of C57 nu/nu mice bearing
B16/F10 tumor 1 h after injection of 99mTc/Tricine/EDDA/[HYNICGABA-Nle]-CycMSHhept. a Unblock. b Block
injection. Through scintigraphy it was observed that the
99m
Tc-HYNIC-peptide mainly was accumulated in the
tumor and kidney. This confirms its specific uptake by the
tumor and its excretion through the urinary tract (Fig. 6).
Scintigraphy images also supported the results obtained by
biodistribution studies at 1 h post injection in blocked and
unblocked animals.
123
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