Preparation and biological evaluation of 99mTcN-labeled pteroyl-lys derivative as a potential folate receptor imaging agent

Article abstract of DOI:10.1002/jlcr.3116

In order to develop a novel ^99mTc-labeled folate receptor (FR) imaging agent, a dithiocarbamate derivative, pteroyl-lys-DTC, was synthesized and radiolabeled with ^99mTc through the [^99mTcN] ²⁺ intermediate. The radiochemical purity of the corresponding ^99mTc-complex, ^99mTcN-pteroyl-lys-DTC, was over 95% as measured by reversed-phase HPLC. The ^99mTcN complex was stable under physiological conditions. ^99mTcN-pteroyl-lys-DTC exhibited specific FR binding in FR-positive KB cells in vitro. The biodistribution in tumor-bearing mice showed that the ^99mTcN-labeled radiotracer had good uptake (3.56 ± 0.09%ID/g at 2 h postinjection) in FR-positive KB tumors, as well as in the kidneys (30.34 ± 3.53%ID/g at 2 h postinjection). After coinjection with excess folic acid, the uptake in tumor and kidneys was significantly blocked. The results indicated that ^99mTcN-pteroyl-lys-DTC was able to target the FR-positive tumor cells and tissues specifically both in vitro and in vivo. Copyright

Full text of DOI:10.1002/jlcr.3116

Research Article
Received 5 March 2013,
Revised 12 August 2013,
Accepted 18 August 2013
Published online 23 September 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3116
Preparation and biological evaluation of
99m
TcN-labeled pteroyl-lys derivative as a
potential folate receptor imaging agent
*
Yuan Chen, Hongjuan Guo, Fang Xie, and Jie Lu
In order to develop a novel 99mTc-labeled folate receptor (FR) imaging agent, a dithiocarbamate derivative, pteroyl-lys-DTC,
was synthesized and radiolabeled with 99mTc through the [99mTcN]2+ intermediate. The radiochemical purity of the
corresponding 99mTc-complex, 99mTcN-pteroyl-lys-DTC, was over 95% as measured by reversed-phase HPLC. The 99mTcN
complex was stable under physiological conditions. 99mTcN-pteroyl-lys-DTC exhibited specific FR binding in FR-positive KB
cells in vitro. The biodistribution in tumor-bearing mice showed that the 99mTcN-labeled radiotracer had good uptake
(3.56 0.09%ID/g at 2 h postinjection) in FR-positive KB tumors, as well as in the kidneys (30.34 3.53%ID/g at 2 h
postinjection). After coinjection with excess folic acid, the uptake in tumor and kidneys was significantly blocked. The results
indicated that 99mTcN-pteroyl-lys-DTC was able to target the FR-positive tumor cells and tissues specifically both in vitro and
in vivo.
Keywords: 99mTc-nitrido complex; folate receptor; tumor uptake; pteroyl-lys
type (Dap-Asp-Cys for EC20) ligand19,20 have been reported for
Introduction
the preparations of these 99mTc-labeled folate conjugates. In
this study, we selected the [99mTcN] chelating system to prepare
Folate, vitamin B9, is required for the survival and proliferation of
a novel 99mTc-based FR imaging agent.
eukaryotic cells and involved as
a coenzyme in many
biosynthetic and epigenetic processes.1 Three folate transporters
have been found to contribute to the folates’ absorption
mechanisms2,3: (i) the reduced folate carrier, a high capacity
but low affinity membrane transporter; (ii) the proton-coupled
folate transporter, which is highly expressed in the duodenum
and jejunum and transports preferentially at low pH; and (iii)
the folate receptor (FR), which binds folate in high affinity
and internalizes folic acid into cells by endocytosis. The FR is
over-expressed on ovarian cancers (more than 90%) and various
epithelial tumor cells, such as breast, cervical, colorectal, renal,
Compared with 99mTcO core, the nitride ligand of [99mTc ≡ N]2+
is a more powerful π-electron donor and has a higher capacity to
stabilize the Tc (V) oxidation state.21 Reported herein are the
synthesis of the pteroyl-lys-DTC, radiolabeling, cell binding, and
preliminary in vivo evaluation of 99mTcN-pteroyl-lys-DTC.
Results and discussion
Chemistry
and nasopharyngeal cancers. Meanwhile, the FR is highly On the basis of the structure of pteroyl-lys22, in which the
restricted in most normal human tissues,4–6 which makes the glutamate (Glu) residue of folate is replaced by a lysine, the novel
FR an important molecular target to develop novel radiolabeled pteroyl-lys-DTC derivative was prepared by treatment with
probes for FR-positive tumor detection.7
carbon disulfide in basic condition (Scheme 1). The active
For the past few years, several folate-based radiopharmaceuticals ε-amino group of the lysine moiety reacted with carbon disulfide
have been investigated for single-photon emission computed in KOH solutions to form the corresponding dithiocarbamate
tomography and positron emission tomography imaging derivative. The pteroyl-lys-DTC was synthesized in a moderate
purpose.8–10 Among them, 111In-DTPA-folate (γ) has been yield of 55%, and the product was characterized by infrared
evaluated in phase I/II clinical studies and appeared to be an (IR) spectrum, NMR spectrum and electrospray ionization mass
effective probe to distinguish benign and malignant ovarian spectrometry (ESI-MS). In IR spectra, the stretching band at
masses.11–13 Compared with 111In, 99mTc is the most widely used 998cmÀ1 (characteristic of C = S) indicated the desired
radionuclide in routine nuclear medicine because of its almost dithiocarbamate derivative. IR/cmÀ1: 1510, 1441 (C―N), 998
ideal physical decay properties (T1/2 = 6.02 h, Eγ = 140 Kev) and
the availability through commercial 99Mo/99mTc generator.14
The development of new 99mTc-based FR imaging agents
with optimal characteristics has been a subject of interest in
the radiopharmaceutical field. Several bifunctional chelating
systems, such as the hydrazinonicotinic acid (HYNIC),9 DTPA-
type ligand, 15,16 99mTc(CO)3 chelating systems,17,18 and N3S-
Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of
Education; College of Chemistry, Beijing Normal University, Beijing100875, China
*Correspondence to: to Jie Lu, College of Chemistry, Beijing Normal University,
Beijing 100875, China
E-mail: ljie74@bnu.edu.cn
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Y. Chen et al.
Scheme 1. Synthetic routes of the pteroyl-lys-DTC and the corresponding 99mTc-nitrido complex.
1
Table 1. The Rf values of 99mTcO4À, 99mTcO2 · xH2O, [99mTc ≡
(C=S). H-NMR (DMSO-d6, 400 MHz) δ 1.37 (m, 2H), 1.54 (m, 2H),
N]2+ intermediate, and 99mTcN-pteroyl-lys-DTC in two thin
layer chromatography systems
1.78 (m, 2H), 2.76 (m, 2H), 4.3 (s, 1H), 4.49 (s, 2H), 6.64 (d, 2H,
J = 8.7 Hz), 7.66 (d, 2H, J = 8.7 Hz), 8.64 (s, 1H); 13C-NMR
(DMSO-d6, 400 MHz) δ 23.8, 27.3, 31.3, 45.9, 47.6, 55.5, 112.4,
128.8, 130.8, 147.4, 150.8, 155.3, 163.9, 168.2, 172.9, 175.4,
179.5, 181.3, 209.8; ESI-MS: m/z [M+] calculated for 554.1, found
554.2
System 1 (polyamide
System 2
film/saline)
(ITLC-SG/ACD)
99mTcOÀ4
0.0~0.1
0.0
0.8~1.0
0.7~1.0
0.7~1.0
0.7~1.0
99mTcO2 · xH2O
[
99mTc ≡ N]2+
Radiochemistry
intermediate
99mTcN-pteroyl-
lys-DTC
The complex 99mTcN-pteroyl-lys-DTC was prepared by a two-step
procedure, as shown in Scheme 1.
The succinic dihydrazide (SDH) kit vial, containing the
following lyophilized formulation: 0.05 mg SnCl2 · 2H2O, 5.0 mg
SDH, and 5.0 mg propylenediamine tetraacetic acid, was used
for preparing the [99mTcN]2+ intermediate. For [99mTc ≡ N]2+
labeling, SDH plays the role of an efficient donor of nitride atoms
0.0~0.1
0.0~0.1
ITLC-SG, instant TLC-silica gel; ACD, acid-citrate-dextrose.
(N3À), and SnCl2 · 2H2O behaves as reducing agent. The presence produced in high labeling yield (>95%) with the SDH kit. When
of propylenediamine tetraacetic acid is required in order to the pteroyl-lys-DTC was added, successful ligand exchange was
prevent precipitation of Sn2+ in the form of insoluble tin salts. evidenced by the disappearance of the radioactivity at Rf of
The complex, 99mTcN-pteroyl-lys-DTC, was obtained by adding 0.8~1.0 and formation of new radioactivity accumulation at
the pteroyl-lys-DTC into the [99mTcN]2+ intermediate at RT for origin (Rf 0.0~0.1). The system 2 (an instant TLC-silica gel strip
26
15 min. The final complex was purified by HPLC to remove the with acid-citrate-dextrose buffer as the mobile phase)
was
excess unlabeled pteroyl-lys-DTC. After HPLC purification, the used to distinctly separate the 99mTcO4À and 99mTcO2 · xH2O from
99mTcN-pteroyl-lys-DTC was obtained with overall radiochemical the 99mTcN-pteroyl-lys-DTC, in which, 99mTcN-pteroyl-lys-DTC
yields of 40~50% (uncorrected for decay) and a specific activity remained at the origin (Rf = 0.0~0.1), while 99mTcO4À and
of more than 1.85 GBq/nmol.
99mTcO2 · xH2O moved at Rf 0.7~1.0. Figure 1 shows the HPLC
The labeling efficiency and radiochemical purity of the chromatograms of [99mTc ≡ N]2+ intermediate and 99mTcN-
99mTc ≡ N]2+ intermediate and of 99mTcN-pteroyl-lys-DTC were pteroyl-lys-DTC. The retention time of [99mTc ≡ N]2+ intermediate
[
evaluated with thin layer chromatography (TLC) and HPLC. The was 2.3 min, while that of 99mTcN-pteroyl-lys-DTC was found to
thin-layer chromatography analysis was carried out on two be 20.5 min. The single peak suggested that only one product
different systems. The Rf values of 99mTcO4À, 99mTcO2 · xH2O, was formed. The radiochemical purity of 99mTcN-pteroyl-lys-
[
99mTc ≡ N]2+ intermediate, and 99mTcN-pteroyl-lys-DTC are listed DTC was more than 95% determined by TLC and HPLC.
in Table 1. In system 1 (a polyamide film with saline as the
mobile phase), 99mTcO4À and 99mTcO2 · xH2O remained at the room temperature. After 4 h incubation with mouse serum at
point of spotting (Rf = 0.0~0.1), while the
99mTc ≡ N]2+ 37 °C, more than 60% of the radioactivity added remained
99mTcN-pteroyl-lys-DTC exhibited a high stability in saline at
[
intermediate moved with the solvent front (Rf = 0.8~1.0). As with the precipitate fraction when 200 μL of methanol was
confirmed by TLC, the [99mTc ≡ N]2+ intermediate can be easily added to precipitate the proteins. However, the radioactivity
J. Label Compd. Radiopharm 2014, 57 12–17
Copyright © 2013 John Wiley & Sons, Ltd.
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Y. Chen et al.
Figure 2. Binding of 99mTcN-pteroyl-lys-DTC in KB cells; cell total binding,
internalization, and blocked by excess folic acid (*p < 0.05).
incubation in serum. The partition coefficient (logP) of the complex
99mTcN-pteroyl-lys-DTC was À1.40 0.01 at pH 7.0.
Cell experiment
To evaluate the ability of 99mTcN-pteroyl-lys-DTC to target FRs,
the specificity of cell uptake and internalization studies were
performed with the FR-positive KB tumor cells. The results are
shown in Figure 2. The complex 99mTcN-pteroyl-lys-DTC
displayed a high cell binding of 16.03 3.15% of the total added
radioactivity. The internalized fraction was 7.63 2.43% of total
added radioactivity. The cellular uptake of 99mTcN-pteroyl-lys-
DTC was significantly blocked by incubation of excess folic acid
(<2% of the total activity, p < 0.05). These data demonstrated
that the cellular uptake of 99mTcN-pteroyl-lys-DTC was FR
specific, and more than 40% of total cellular uptake could be
internalized in the cells.
Biodistribution
To further evaluate the ability of 99mTcN-pteroyl-lys-DTC to
target FR in vivo, the biodistribution and pharmacokinetics were
performed in athymic BALB/C nude mice bearing KB tumor. The
results are shown in Table 2. The complex 99mTcN-pteroyl-lys-
DTC exhibited significant tumor uptake of 3.56 0.09%ID/g at
2 h postinjection and a good retention (1.71 0.53%ID/g at 4 h
postinjection). Clearance from the blood was fast, leading to an
increasing tumor-to-blood ratio over time (from 1.26 at 1 h post
injection to 4.14 at 2 h postinjection). A significant accumulation
and retention of radioactivity were observed in the kidneys
(30.34 3.53%ID/g at 2 h postinjection and 18.12 1.08%ID/g at
4 h postinjection), because the kidney proximal tubule cells
express a high concentration of FR on their apical membranes.
Coinjection of excess folic acid significantly blocked the tumor
uptake (1.67 0.22%ID/g at 2 h postinjection, p < 0.05), as well
as the accumulation of radioactivity in the kidney
(18.78 1.62%ID/g at 2 h postinjection, p < 0.05), indicating the
FR-specific uptake of the radiotracer in these FR-positive tissues.
Although 99mTcN-pteroyl-lys-DTC was stable in mouse serum
Figure 1. Radioactive HPLC profiles of 99mTcOÀ4 (A), [99mTcN]2+ intermediate (B),
and 99mTcN-pteroyl-lys-DTC (C). The retention time of 99mTcO4À, [99mTcN]2+
intermediate, and 99mTcN-pteroyl-lys-DTC were 12.9, 2.3, and 20.5 min, respectively.
could disassociate when more methanol was added to wash the in vitro, the urine analysis showed that about 53% of 99mTcN-
proteins, indicating significant unspecific serum protein binding. pteroyl-lys-DTC was metabolized into pertechnetate formation
The analytical results of the supernatant displayed that no new at 1 h after injection (Figure 3), which might be responsible for
radioimpurities were detected by reversed-phase (RP)-HPLC, the high radioactivity accumulation in the stomach
and the radiochemical purity was more than 90% after 4 h of (7.66 2.52%ID/g at 1 h after injection).
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Y. Chen et al.
Table 2. Biodistribution of 99mTcN-pteroyl-lys-DTC in athymic nude mice bearing KB tumor xenograftsa
%ID/g (mean standard deviation)
1 h
2 h
2 h (blocking)b
4 h
Tissue
Heart
Liver
Lung
Kidney
Spleen
Stomach
Bone
Muscle
Intestines
Blood
1.85 0.05
9.84 0.43
4.52 0.59
18.93 0.64
2.55 0.43
7.66 2.52
1.06 0.10
1.21 0.12
2.66 0.63
1.84 0.39
4.77 0.76d
2.31 0.21
1.72 0.30
9.57 0.94
5.58 1.33
30.34 3.53
2.85 0.03
2.89 0.38
2.14 0.85
1.14 0.33
3.32 0.87
0.86 0.75
-
2.46 0.29
13.71 4.76
6.76 1.79
18.78 1.62c
3.92 1.40
12.31 8.6
3.04 0.42
2.35 1.10
4.72 2.34
1.75 0.62
-
1.96 0.08
7.37 0.61
6.45 1.13
18.12 1.08
2.85 0.33
11.51 1.15
2.20 0.58
1.09 0.36
2.43 1.05
0.89 0.03
-
Thyroid
Tumor
c
3.56 0.09
1.67 0.22
1.71 0.53
Ratios
Tumor/Blood
Tumor/Muscle
1.26
1.90
4.14
3.12
-
-
1.92
1.56
aAll data are the mean percentage (n = 5) of the injected dose of 99mTcN-pteroyl-lys-DTC per gram of tissue (%ID/g), the
standard deviation of the mean.
bCoinjection of excess folic acid.
cp < 0.05, significance comparisons on tumor and kidney uptakes between the radiotracers with or without folate blockade at 2 h
postinjection.
dThe thyroid uptake of 99mTcN-pteroyl-lys-DTC in normal nude mice (n = 3) at 1 h postinjection.
derivatization to synthesize the folate conjugates. Unfortunately,
the preparation of the defined γ-derivatives is often difficult,
because both carboxyl groups of the Glu moiety have similar
reactivities for the traditional coupling reactions. Recently, our
group has reported a new strategy to synthesize derivatives with
free α-carboxylic group based on the structure of pteroyl-lys, in
which the Glu residue of folate is replaced by a lysine.22 In this
study, a dithiocarbamate derivative of pteroyl-lys was obtained
by the reaction of the active ε-amine group of the lysine moiety
with carbon disulfide in KOH solutions. As we anticipated, the
corresponding 99mTcN-labeled complex still retained the FR
binding potential and could target the receptor specifically both
in vitro and in vivo. These results further confirmed that the Glu
moiety of folate is not critical for the FR recognition, highlighting
the potential of this class of 99mTcN-labeled pteroyl-lys
conjugates for the FR imaging.99mTcN-pteroyl-lys-DTC exhibited
good tumor uptake (3.56 0.09%ID/g at 2 h postinjection). The
tumor-to-muscle and tumor-to-blood ratios were 3.12 and 4.14
at 2 h postinjection, respectively. These results indicated the
potential of this class of 99mTcN-pteroyl-lys-DTC conjugates for
FR-positive tumor imaging.
Figure 3. Radioactive HPLC profile of the urine sample of a nude mouse at 1 h
postinjection of 99mTcN-pteroyl-lys-DTC.
Several 99mTc labeling bifunctional chelating systems, such as
The folic acid, a combination of the pteroic acid and Glu, offers
two positions for derivatization, the α-carboxylic and γ-carboxylic
acid. The role of the Glu residue of folate and the necessity of
a free α-carboxyl group to retain binding affinity to FR are
still debated in the literature.23,24 The results of recent
studies showed both γ-glutamyl-linked and α-glutamyl-linked
conjugates were able to bind to FR-positive cells in vitro.17,25
However, the γ-derivative possessed more favorable
pharmacokinetic features than the α-derivative, which indicated
the 99mTc(CO)3 chelating systems
and HYNIC-conjugated
17,18
ligands,9,22 have been used for the preparations of FR targeting
agents. Table 3 illustrates the comparison of the partition
coefficient values (logP) and biodistribution data between
99mTcN-pteroyl-lys-DTC, 99mTc(CO)3-his-folate,17 and 99mTc(HYNIC-
lys-pteroyl)(Tricine/TPPTS)22 in the same KB tumor-bearing mice
model at 1 h postinjection. The data for 99mTc(HYNIC-lys-pteroyl)
(Tricine/TPPTS) were obtained from previous studies of our group.
The tumor uptake of 99mTcN-pteroyl-lys-DTC (2.31 0.21%ID/g at
that the γ-carboxyl group should be
a better site for
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Y. Chen et al.
Table 3. Comparison of the partition coefficient value (logP) and biodistribution data between 99mTcN-pteroyl-lys-DTC, 99mTc
(CO)3-his-folate, and 99mTc(HYNIC-lys-pteroyl)(Tricine/TPPTS) in the same KB tumor-bearing mice model at 1 h postinjection
Complex
99mTcN-pteroyl-lys-DTC
99mTc(CO)3-his-folate
99mTc(HYNIC-lys-pteroyl)(Tricine/TPPTS)
logP
À1.40 0.10
2.31 0.21
9.84 0.43
2.66 0.63
Present study
-
À2.89 0.06
5.39 0.40
3.19 0.09
1.60 0.58
[22]
Tumor (%ID/g)
Liver (%ID/g)
Intestines (%ID/g)
Reference
2.63 0.48
9.73 1.32
3.84 2.23
[17]
1 h postinjection) is comparable with that of 99mTc(CO)3-
his-folate (2.63 0.48%ID/g at 1 h postinjection) but is 50% less
than that of 99mTc(HYNIC-lys-pteroyl)(Tricine/TPPTS) (5.39 0.40%
ID/g at 1 h postinjection). 99mTcN-pteroyl-lys-DTC displayed a
significant accumulation of radioactivity in liver (9.84 0.43%ID/g
at 1 h postinjection), as well as in the intestines (2.66 0.63%ID/g
at 1 h postinjection), which were similar to those of 99mTc(CO)3-
his-folate (9.73 1.32%ID/g and 3.84 2.23%ID/g at 1 h
postinjection, respectively). The results suggested these
The solution was then stirred for 3 h in the ice bath. The solvent was
removed under reduced pressure and dried in vacuum. The crude product
was recrystallized from ethanol/water, then washed with Et2O (1× 3mL)
and dried under vacuum to afford 45mg of pteroyl-lys-DTC, yield 55%.
The pteroyl-lys-DTC was characterized by IR, 1H-NMR, 13C-NMR, and
ESI-MS spectroscopy.
Radiolabeling of 99mTcN-pteroyl-lys-DTC
99mTcN-pteroyl-lys-DTC was prepared by a two-step procedure. First,
radiotracers were partially excreted via the hepatobiliary and 1.0 mL of saline containing 99mTcO4- (277 MBq) was added into a SDH
kit and kept at room temperature for 15 min to obtain the [99mTcN]2+
gastrointestinal extraction. Generally, hepatobiliary elimination
often correlates with the lipophilicity of a drug. 99mTcN-pteroyl-
intermediate. Then, 1 mg of the pteroyl-lys-DTC ligand was added to
the [99mTcN]2+ intermediate. The mixture was kept at room temperature
lys-DTC (logP = À1.40 0.10) exhibited higher lipophlicity than
99mTc(HYNIC-lys-pteroyl)(Tricine/TPPTS) (logP = À2.89 0.06). The
liver uptake of 99mTcN-pteroyl-lys-DTC was three times higher than
that of 99mTc(HYNIC-lys-pteroyl)(Tricine/TPPTS). The hepatobiliary
extraction of 99mTcN-pteroyl-lys-DTC is undesirable, because
the high abdominal accumulation will hamper the tumor
visualization in this region. Introduction of one more hydrophilic
for 15 min. The radiolabeled 99mTcN-pteroyl-lys-DTC was purified by RP-
HPLC to completely separate from its excess unlabeled pteroyl-lys-DTC.
The fraction (19.5–21.0 min, Rt = 20.5 min) was collected, and volatiles
in HPLC mobile phase were removed under vacuum. The residues
were dissolved in saline for further stability, specific binding, and
animal studies. The labeling efficiency and radiochemical purity of the
[
99mTc ≡ N]2+ intermediate and of 99mTcN-pteroyl-lys-DTC were evaluated
linker between the 99mTcN chelating moiety and targeting with TLC and HPLC.
molecule might decrease the gastrointestinal tract accumulation
of this class of 99mTcN-radiolabeled radiotracers in future studies.
In vitro stability
The in vitro stability of this 99mTcN-pteroyl-lys-DTC complex was
Experimental
evaluated by monitoring the RCP at different time points. Briefly, HPLC
purified 99mTc complex was diluted with 4–5 mL saline and kept at room
temperature for 4 h. Samples of the resulting solutions were analyzed by
Methods and materials
Pteroyl-lys22 was prepared according to literature methods. SDH kit was radio-HPLC at 0, 2, and 4 h.
obtained from Beijing Shihong Pharmaceutical Center, Beijing Normal
To 900 μL of fresh mouse serum, 100 μL of the purified 99mTcN-pteroyl-
University, China. All other chemicals were of reagent grade and used lys-DTC complex solution was added and incubated at 37 °C. At 0, 2, and
without further purification. Technetium-99m as sodium pertechnetate 4 h, 100 μL aliquots were withdrawn and treated with 200 μL methanol to
(Na99mTcO4) was obtained from commercial 99Mo/99mTc generator
precipitate the proteins. Sample was centrifuged at 3000 rpm. The
(China Institute of Atom Energy, China) just before use. IR spectrum supernatant was purged with N2 gas to remove the ethanol. The
was obtained with an AVATAR 360 FT-IR spectrometer using KBr pellets. resulting sample was mixed with 100 μL of water and analyzed by
NMR spectra were recorded on Bruker Avance-400 (400 MHz) radio-HPLC.
spectrometers. ESI-MS spectrum was recorded on a LC-MS Shimadzu
2010 series. TLC were performed on polyamide films with saline as the
mobile phase (system 1) and instant TLC-silica gel strips with acid-
Octanol/water partition coefficient
citrate-dextrose buffer (0.068 mol/L citrate, 0.074 mol/L glucose, pH 5.0) The octanol/water partition coefficient was measured by the following
as the mobile phase (system 2). RP-HPLC experiments were performed procedure: mixing 100 μL of the purified radiotracer solution with
on a SHIMAZU SCL-10AVP HPLC pump system (SHIMAZU Corporation, 1.9 mL of phosphate buffer (0.05 mol/L, pH 7.0) and 2 mL of octanol in
Japan) and BIOSCAN flow-counter, using a Venusil XBP C18 reversed- centrifuge tube. The tube was vortexed for 2 min and then centrifuged
phase column (250 × 10 mm, 5 μm), working at a flow rate of 1.0 mL/ at 4000 rpm for 5 min. the counts in 100 μL aliquots of both organic
min. 10 mM NH4HCO3 (A) and CH3OH (B) mixtures were used as mobile and inorganic layers were determined by use of a NaI well-type γ-
phase (0–10 min, B: 5–50%; 10–20 min, B: 50–50%; 20–25 min, B: counter. All experiments were performed in triplicate. The partition
50–5%). All biodistribution studies were carried out in compliance with coefficient (P) was calculated using the following equation: P = (cpm in
the national laws related to the conduct of animal experimentation
octanolÀcpm in background)/(cpm in aqueous layerÀcpm in
background). The final partition coefficient value was expressed as logP.
Synthesis of pteroyl-lys-DTC
Cell experiment
In the dark, pteroyl-lys22 (65mg, 0.148mmol) was dissolved in a solution of
KOH (10mL, 0.3mmol). A 0.3-mL portion of carbon disulfide (4.96 mmol) The specific FR binding of 99mTcN-pteroyl-lys-DTC was determined in KB
was added dropwise to the solution stirred in an ice bath at 0 °C. carcinoma cell according to our published procedure.22 Briefly, the KB
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Y. Chen et al.
was FR specific. In biodistrubution studies, 99mTcN-pteroyl-lys-
DTC exhibited a good tumor uptake, suggesting the potential
of this class of pteroyl-lys conjugates for the FR-positive tumor
imaging; however, further investigation toward novel 99mTcN-
labeled pteroyl-lys derivatives with reduced lipophilicity are
warranted.
carcinoma cell line is cultured as monolayers at 37 °C in a humidified
atmosphere containing 5% CO2, The cells were cultured in FFRPMI
medium (modified RPMI 1640, without folic acid) supplemented with
10% fetal calf serum and antibiotics (penicillin 100 IU/mL, streptomycin
100 μg/mL, and fungizone 0.25 μg/mL). Twenty-four hours prior to the
experiment, the KB cells were seeded in 24 well plates (2 × 105 cells/well)
and incubated at 37 °C to form confluent monolayer. All experiments
were performed in triplicate. After being washed once with FFRPMI
medium, the cells were incubated at 37 °C for 1 h with approximately
7.4 KBq of HPLC purified complex in 1 mL of FFRPMI medium. The
blocking studies were performed by coincubation of free folic acid
solution (100 μM) into the incubation medium. After incubation, the
reaction media were aspirated, and the cells were rinsed with 2 × 1 mL
of cold PBS (pH 7.4). Cellular internalization of the 99mTc complex was
assessed by washing the cells with 1 mL of acidic buffer (aqueous
solution of 0.1 M acetic acid and 0.15 M NaCl, pH 3). Finally, the cells were
lysed by treatment with 1 mL of 1 N NaOH for 5 min and transferred to
tubes. All the samples were counted for radioactivity using a γ-counter.
The cell binding fractions and cell internalized fractions were
calculated and expressed in relation to the total added activity (% of
total added activity).
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (20701004, 81201120) and the Fundamental
Research Funds for the Central Universities.
Conflict of Interest
The authors did not report any conflict of interest.
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