Radioiodinated estradiol dimer for estrogen receptor targeted breast cancer imaging

Article abstract of DOI:10.1111/cbdd.13754

The aim of this study was to develop a 1-(2-(2-(2-(1,2,3-triazol)ethoxy)ethoxy)ethyl)-5-[^125/131I]iodo-1,2,3-triazole-diestradiol ([^125/131I]ITE2), for estrogen receptor (ER)-expressing breast cancer imaging with single-photon emission computed tomography (SPECT). [^125/131I]ITE2 was prepared in good radiochemical yield (94.4?±?0.4%) with high radiochemical purity (>99%). [^125/131I]ITE2 had good stability in vitro and moderate molar activity (0.3?±?0.2?GBq/μmol). Higher uptake in ER-positive MCF-7 cells than that of ER-negative MDA-MB-231 cells was observed at all time points. Rats biodistribution showed that [¹³¹I]ITE2 had high uptake in ER-abundant uterine and ovarian (5.7?±?0.4 and 10.1?±?1.4%ID/g at 1?hr postinjection) and could be blocked by co-injection of estradiol (2.7?±?0.1 and 5.5?±?0.4%ID/g) obviously. In the SPECT/CT imaging study, [¹²⁵I]ITE2 showed significant higher uptake in MCF-7 tumor (3.1?±?0.4%ID/g) than that of MDA-MB-231 (0.9?±?0.1%ID/g). Furthermore, the specific uptake of [¹²⁵I]ITE2 in ER-positive MCF-7 tumor could be blocked effectively by preadministration of fulvestrant (1.2?±?0.4%ID/g). A novel radioiodinated dimeric estrogen was designed and synthesized with promising ER targeting ability and specificity. It is worthy of further investigation to validate the advantages of the dimer in ER-positive breast cancer diagnosis.

Full text of DOI:10.1111/cbdd.13754

PROFESSOR XIANZHONG ZHANG (Orcid ID : 0000-0001-8591-8301)
Article type
: Research Article
Radioiodinated Estradiol Dimer for Estrogen Receptor Targeted Breast Cancer Imaging
Duo Xu1; Chenyu Peng1; Fei Gao1; Zhide Guo1; Rongqiang Zhuang1; Xinhui Su2; Xianzhong Zhang1*
1
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and
Translational Medicine, School of Public Health, Xiamen University, Xiang’an South Rd, Xiamen 361102, China;
2 Zhongshan Hospital Affiliated to Xiamen University, Hubin South Road, Xiamen 361004, China.
*For correspondence contact: Xianzhong Zhang, PhD, Professor, Center for Molecular Imaging and Translational
Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health,
Xiamen University, Xiang’an South Rd., Xiang’an district, Xiamen 361102, China. E-mail: zhangxzh@xmu.edu.cn;
Phone: +86(592)2880645; Fax: +86(592)2880645.
First author: Duo Xu, student, Center for Molecular Imaging and Translational Medicine, State Key Laboratory of
Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiang’an South Rd.,
Xiang’an district, Xiamen 361102, China. E-mail: 13164604466@163.com; Phone: +86(592)2880641; Fax:
+86(592)2880641.
Author Contributions: The manuscript was written through the contributions of all authors. All authors have given
approval to the final version of the manuscript.
Running title: Radioiodinated estradiol dimer for breast cancer imaging
Word count text: 5770 including title page, abstract, and references
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through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13754
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Abstract
The
aim
of
this
study
is
to
develop
a
1-(2-(2-(2-(1,2,3-triazol)ethoxy)ethoxy)ethyl)-5-[125/131I]iodo-1,2,3-triazole-diestradiol ([125/131I]ITE2), for estrogen
receptor (ER)-expressing breast cancer imaging with single-photon emission computed tomography (SPECT).
[
125/131I]ITE2 was prepared in good radiochemical yield (94.4 ± 0.4%) with high radiochemical purity (> 99%).
125/131I]ITE2 had good stability in vitro and moderate molar activity (0.3 ± 0.2 GBq/µmol). Higher uptake in
[
ER-positive MCF-7 cells than that of ER-negative MDA-MB-231 cells was observed at all time points. Rats
biodistribution showed that [131I]ITE2 had high uptake in ER-abundant uterine and ovarian (5.7 ± 0.4 and 10.1 ±
1.4 %ID/g at 1 h post-injection) and could be blocked by co-injection of estradiol (2.7 ± 0.1 and 5.5 ± 0.4 %ID/g)
obviously. In the SPECT/CT imaging study, [125I]ITE2 showed significant higher uptake in MCF-7 tumor (3.1 ±
0.4 %ID/g) than that of MDA-MB-231 (0.9 ± 0.1 %ID/g). Furthermore, the specific uptake of [125I]ITE2 in
ER-positive MCF-7 tumor could be blocked effectively by pre-administration of fulvestrant (1.2 ± 0.4 %ID/g). A
novel radioiodinated dimeric estrogen was designed and synthesized with promising ER targeting ability and
specificity. It is worthy of further investigation to validate the advantages of the dimer in ER-positive breast cancer
diagnosis.
Key words: radioiodinated dimeric estrogen, estrogen receptor, breast cancer, SPECT/CT imaging, biodistribution.
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1. Introduction
The use of estrogen radiopharmaceuticals to image estrogen receptor (ER) in vivo has the potential to determine
the ER status of breast cancer. Fluorine-18, iodine-131 and other radionuclides are widely used to label estrogen
derivatives for in vivo imaging of ER density in steroid-sensitive tumors (Kiesewetter et al., 1984; Paquette et al.,
2013; VanBrocklin et al., 1993; Lim et al. and Mortimer et al., 1996; Xia et al., 2015; Nayak et al., 2008; Neto et al.,
2012; Linden et al., 2006; Hochberg, 1979; Ali et al., 1988, 1991, 1993 and 2003; Antunes et al., 2017; Huang et al.,
2018; Yan et al., 2013; Rijks et al., 1998; Symes et al., 1985; Gatley et al., 1981). Among them, the most successful
18F-labeled estrogen derivative, 16α-[18F]fluoro-17β-estradiol ([18F]FES), has a good affinity for ER+ tumors and
predicts response to tamoxifen (Linden et al., 2006; Mortimer et al., 1996). Meanwhile, a variety of
radioiodine-labeled estrogen derivatives for SPECT imaging of ER+ breast cancer have also been reported (Hochberg,
1979; Ali et al., 1988, 1991, 1993 and 2003; Yan et al., 2013; Rijks et al., 1998; Symes et al., 1985).
16α-[125I]iodoestradiol, 11β-methoxy substituent [125I]iodovinylestradiol and its analogs had been reported as potential
single photon-emitting ER imaging agents (Hochberg, 1979; Ali et al., 1988, 1991). However, their high thyroid
radioactivity uptake suggested severe deiodination occurred in vivo. Just like these probes, metabolic deiodination,
resulting in increased non-specific uptake, has become a common problem with radioiodine-labeled-estrogen tracers.
Therefore, few radioactive iodine-labeled probes have reached clinical trials so far and recent studies of radioactive
iodine-125/131 labeled estrogen ligands are almost stalled while the radioactive fluorine-18 labeled estrogen receptor
ligands have been developed rapidly (Antunes et al., 2017; Huang et al., 2018).
From previous research, we know that increasing the metabolic stability is essential for the development of
estradiol-skeleton radiotracer. A one-pot, copper(II)-mediated reaction of azides, alkynes, and [125I]iodide to yield
5-[125I]iodo-1,2,3-triazole was reported with high radiochemical yield and good metabolic stability in vivo (Yan et al.,
2013). Compared with other multistep radiolabeling methods, the one-step iodo-labeling approach is much easier and
more practical for application.
Encouraged by the advantage of the iodo-labeling method and the application of dimers in radiotracer design, the
first iodine-125/131 labeled steroidal estrogen dimers, [125/131I]ITE2, has been synthesized and labeled in this study. It
was prepared by copper-catalyzed reaction of azide, alkyne and iodine to give the metabolically stable
5-iodo-1,2,3-triazole in a single step. In addition, the in vitro and in vivo stabilities, receptor-binding affinity,
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biodistribution of the radiotracer were evaluated, and small-animal SPECT/CT imaging of tumor-bearing mice was
also performed to verify its feasibility for specific diagnosis of ER-positive breast cancer.
2. Materials and methods
2.1. General
All of the reagents and materials were commercially available. The radioiodine [125I]NaI was bought from China
Isotope & Radiation Corporation (Beijing, China) and [131I]NaI was obtained from Zhongshan Hospital Affiliated to
Xiamen University. Reversed-phase high-pressure liquid chromatography (HPLC) was performed on a system with an
UltiMate 3000 pump (DIONEX) and a B-FC-1000 flow counter (Bioscan). Hypersil GOLD-C18 reversed-phase
HPLC column (250*4.6 mm, 5-µm particle size; Thermo Scientific) was used. The mobile phase was changed from
65% of solvent A (water) and 35% solvent B (acetonitrile) to 40% solvent A and 60% solvent B at 10 min. Flow rate:
1 mL/min. Nonradioactive reference [127I]ITE2 was analyzed in the same gradient and monitored at 280 nm. 1H NMR
spectra were recorded on a Bruker (400 MHz) spectrometer. Chemical shifts are expressed in ppm (δ) and coupling
constants (J) in Hz. Mass spectra were obtained with a Bruker Esquire 3000 plus ESI-MS under electron impact
ionization conditions.
All cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai,
China). The human breast carcinoma MCF-7 cells were cultured in DMEM/high-glucose medium (Gibco, Carlsbad
CA, USA) with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Beyotime,
Shanghai, China) in it. The human breast cancer MDA-MB-231 cells were cultured in medium containing 10% fetal
bovine serum and 1‰ penicillin/streptomycin. All cells were grown as a monolayer at 37 C in a moist atmosphere of
5% CO2 and 95% air.
All procedures and animal use and care protocols were approved by Xiamen University’s animal care and use
committee (SYXK 2013-0006). The experimental procedures, involving animals, were in accordance with the ethical
standards of the institution. The immature female Sprague Dawley rats (3–4 weeks old) and female Balb/c nude mice
(18–20 g) were ordered from the Laboratory Animal Center of Xiamen University. MCF-7 cells (2 × 107 cells) or
MDA-MB-231 cells (2 × 107 cells) in 100 μL PBS, were injected subcutaneously into the right shoulders of Balb/c
nude mice. In vivo SPECT/CT imaging performed when the tumor size reached 0.8–1.2 cm in diameter.
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2.2 Chemistry and radiochemistry
The precursor, radioactive [125/131I]ITE2 and nonradioactive [127I]ITE2 were synthesized according to the
previously reported references (Moldovan et al., 2016; Cao et al., 2017; Ji et al. and Liu et al., 2014; Li et al., 2010).
The specific steps are available in scheme 1 and supplementary information. Briefly, the radioiodinated compounds
([125I]ITE2 and [131I]ITE2) were prepared by one-step labeling method and identified by co-injection with the
non-radioactive reference compound, their molar activities and radiochemical purities were determined as well using
analytical HPLC. After HPLC purification and sterile filtration, the injections were used for the studies of
octanol-to-water partition coefficient, stability, receptor-binding and biodistribution ([131I]ITE2) and small-animal
SPECT/CT imaging ([125I]ITE2).
2.3 In vivo metabolic stability
The metabolic stability studies in mice were performed in accordance with the published procedures (Mou et al.,
2012). Balb/c mice were injected with 7.4 MBq of [131I]ITE2 intravenously and sacrificed at 10, 20, 30 and 60 min
post-injection (p.i.). The blood samples were collected and immediately centrifuged at 10,000 rpm for 5 min. After the
serum was isolated and added 0.2 mL of methanol, it was centrifuged at 10,000 rpm for 5 min again. The urine
samples were collected and centrifuged at 10,000 rpm for 5 min after adding 0.1 mL of methanol. The blood and urine
supernatants were collected and passed through a Sep-Pak C18 cartridge (Waters Co., Milford, MA, USA). Then, the
cartridges were washed with water (0.5 mL) and eluted with methanol (0.5 mL). The combined aqueous and organic
solutions were passed through a 0.22-m filter (Millipore Co., Billerica, MA, USA) and analyzed by radio-HPLC.
2.4 Receptor-binding studies
Saturation binding assay was performed as described previously (Linden et al., 2006). There were about 2 × 105
MCF-7 cells in each well of a 24-well plate incubated at 37 °C for 24 h. Various concentrations (0–200 nM) of
[
131I]ITE2 were added and incubated at 37 °C for 1 h. After washing with PBS (1 mL, pH = 7.4) for three times, the
cells were lysed with 1 M NaOH (1 mL) for 5-10 min. Radioactivity was measured using a γ-counter (WIZARD 2480,
PerkinElmer, USA).
Cell uptakes of [131I]ITE2 in ER-positive MCF-7 and ER-negative MDA-MB-231 cells were studied respectively.
After 2  105 cells in each well of a 24-well plate were incubated overnight, each well was incubated with [131I]ITE2
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(0.185 MBq) at 37 °C for 5, 30, 45, 60, 120, 240 and 360 min. Then, the cells were washed with PBS (1 mL, pH = 7.4)
three times and then lysed with 1 M NaOH (1 mL) for 5-10 min. The radioactivity collections were counted using a
γ-counter.
To confirm the specificity of the ER binding, MCF-7 cells were treated with ER inhibitor 17β-estradiol (0.17 mM)
before incubating with [131I]ITE2 (0.185 MBq) at 37 °C for 5, 30, 45, 60, 120, 240 and 360 min for blocking study.
After washing with PBS (1 mL) for three times, the radioactive residues were collected and analyzed using a
γ-counter.
2.7 Biodistribution
Biodistribution studies were conducted in accordance with the approved guidelines. The biodistribution of
[
131I]ITE2 in major organs (n = 3-5/group) was evaluated in immature female SD rats by intravenous injection of 1.85
MBq of [131I]ITE2. Blocking studies were also performed in rats by intravenous co-injection of 1.85 MBq [131I]ITE2
with an excess dose of 17β-estradiol (15 µg/rat). The rats were isoflurane anesthesia, sacrificed and dissected at 1 h or
2 h after injection. Samples of blood, muscle, tumor and major organs were collected and weighted, and then the
radioactivity was measured with a γ-counter. The results were expressed as the mean ± SD percentage injected dose
per gram (%ID/g).
2.8 Small-animal SPECT/CT imaging
Small-animal SPECT/CT imaging study was performed on ER-positive MCF-7 and ER-negative MDA-MB-231
tumor-bearing nude mice. Each mouse was injected with 18.5 MBq of [125I]ITE2 via the tail vein (n = 3/group). For
the blocking study, mice bearing ER+ tumors were subcutaneously injected with the competitive ER-inhibitor
fulvestrant (0.5 mg/mouse) at 48 h prior to SPECT/CT imaging. Each mouse was anesthetized and placed on the
scanner bed in the prone position. The imaging of [125I]ITE2 was acquired at 15 min or 1 h p.i. and a
nanoScan-SPECT/CT preclinical scanner equipped with low energy high-resolution collimators was used. The
acquisition parameters: energy peak of 35 keV for 125I, window width of 20%, a matrix of 256 × 256, medium zoom,
frame: 30 s. All images were performed in the same manner and reconstructed, processed and further analyzed with
Nucline software (Mediso Medical Imaging System) by drawing appropriate Volume of Interests on tumor and
muscle.
2.9 Statistical analysis
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Differences in data between the experimental, control and inhibition groups were measured by student’s t tests
and nonparametric tests with the GraphPad prism 5.0 and when P is less than 0.05, significant statistical difference is
considered between the two groups (P >0.05, *P <0.05, **P <0.005 and ***P <0.0005).
3. Results
3.1 Chemistry and radiochemistry
The synthetic routes of [125/131I]ITE2 are shown in Scheme 1. [125I]ITE2 was obtained in a radiochemical yield of
94.4 ± 0.4% (n = 5) with a retention time of 10.75 min using radio-HPLC and the radiochemical purity was more than
99% after HPLC purification (Figure 1). The molar activity was 0.3 ± 0.2 GBq/µmol (n = 3) based on the UV standard
curve derived from HPLC analysis (Figure S1). The total radiosynthesis time of [125I]ITE2, including HPLC
purification, was about 110-120 min. The HPLC retention times of the non-radioactive compound [127I]ITE2 (Figure 1)
was 10.65 min. The octanol-to-water partition coefficient (log P) of [131I]ITE2 was calculated as 1.2 ± 0.1, suggesting
that it is a lipophilic compound (method was described in the supplementary information). TLC analysis showed that
the stability of [131I]ITE2 in physiological saline and murine plasma was maintained for four hours (Figure S2).
3.2 Metabolic stability
[
131I]ITE2 found to be metabolic stable in the blood (Figure 2a-b, peak at 10.63 min) at early time points. After
20 min p.i., however, it was metabolized gradually to form a new peak at 3.85 min (Figure 2b-c) and finally the tracer
and its metabolite were cleared from the blood at 1 h p.i. (Figure 2d). The metabolite of [131I]ITE2 in the urine was
found at 30 min p.i. (Figure 2g, 60%). Thereafter, the peak of [131I]ITE2 gradually disappeared and the metabolite
peak at 3.28 min was increased and finally became the only peak at 60 min p.i. (Figure 2h). The radioactive
metabolites at 3.85 min and 3.28 min have not been identified while the retention time of free radioiodine was about
3.76 min under the same HPLC conditions.
3.3 Receptor-binding studies
The cell-binding result of [131I]ITE2 was shown in Figure 3a. The dissociation constant (Kd) of [131I]ITE2 was
calculated as 49.7 ± 7.7 nM (n = 3). The maximal number of binding sites (Bmax) was 1.27 × 104 sites per cell.
As shown in Figure 3b, [131I]ITE2 had much higher uptake in ER-positive MCF-7 cells (from 17.9 ± 1.9 to 31.4 ±
3.4 %) than that of ER-negative MDA-MB-231 cells (from 1.1 ± 0.1 to 1.4 ± 0.2 %) at every point of time, which
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indicating that [131I]ITE2 has strong affinity to ER-positive MCF-7 cells. The specificity of ER binding was
demonstrated in Figures 3c and 3d. After blocking with 0.17 mM of estradiol, the MCF-7 cells uptake was decreased
significantly to 5.2 ± 0.1% after 1 h incubation (Figure 3c, ***P < 0.0001). Furthermore, the non-specific binding of
[
131I]ITE2 in ER-negative MDA-MB-231 cells was much lower than that of ER-positive MCF-7 cells after 1 h
incubation (***P < 0.0001).
3.4 Biodistribution
The biodistribution of [131I]ITE2 was studied in normal female SD rats at 1 and 2 h p.i. As shown in Figure 4a
and Table S1, [131I]ITE2 displayed marked uptake and retention of radioactivity in uterine (5.7 ± 0.4 %ID/g and 4.8 ±
0.7 %ID/g at 1 h and 2 h p.i., respectively) and ovarian (10.1 ± 1.4 %ID/g and 10.2 ± 1.8 %ID/g at 1 h and 2 h p.i.,
respectively), organs known to contain high ER concentrations. The uptake of [131I]ITE2 in non-specific organs and
tissue, such as the heart, spleen, lung and muscle, was 1.2 ± 0.0, 1.6 ± 0.2, 0.8 ± 0.2 and 1.5 ± 0.4 %ID/g at 1 h p.i.,
respectively. Low blood uptake was observed of only 0.7 ± 0.2 %ID/g at 1 h p.i. and the amount of radioactivity
remained nearly constant until 2 h p.i. The uptake of fat (2.4 ± 0.4 %ID/g) was lower than that in the uterus and
ovaries, but higher than the muscle uptake. Organs involved in steroid hormone metabolism and excretion, such as
liver, intestine and kidneys, had the uptakes of 1.3 ± 0.2 %ID/g, 2.3 ± 0.5 %ID/g and 1.1 ± 0.2 %ID/g, respectively.
There had no significant difference between the uptakes of thyroid and muscle (2.3 ± 0.6 %ID/g and 1.5 ± 0.4 %ID/g,
respectively, P = 0.1) of [131I]ITE2 indicating negligible deiodination of it in vivo.
The tissue uptake specificity of the [131I]ITE2 is also presented. Uterus and ovarian uptake were inhibited by the
presence of an excess estradiol (2.7 ± 0.1 %ID/g and 5.5 ± 0.4 %ID/g, ***P = 0.0004 and *P = 0.007, respectively).
Radioactivity uptake in the non-target tissues, such as heart, spleen, lung muscle and fat, was not affected by the
co-injection of unlabeled estradiol. Besides, the uptake of radioactivity in organs involved in steroid hormone
metabolism and excretion (liver, intestine and kidneys), usually no statistically significant decrease in uptake of the
[
131I]ITE2 was found. The ratios of the uterus to blood and uterus to muscle were 9.2 ± 2.4 and 4.2 ± 1.5 at 1 h p.i.
respectively, and decreased sharply to 4.1 ± 1.1 and 1.7 ± 0.2. (Figure 4b, c and Table S1, *P = 0.02, P = 0.06) after
blocking.
3.5 Small-animal SPECT/CT imaging
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SPECT/CT images of tumor-bearing mice were acquired at 15 min and 1 h (MCF-7 tumor) or 1 h
(MDA-MB-231 tumor) after injection of [125I]ITE2 (Figure 5). MCF-7 tumors were visible as early as 15 min p.i. and
tumor uptake increased at 1 h p.i., while MDA-MB-231 tumors showed low radioactive accumulation at 1 h p.i. Under
blocking conditions with excess ER-inhibitor fulvestrant, MCF-7 tumor uptake of [125I]ITE2 was reduced significantly
at 1 h p.i. As shown in Figure 5b and Figure S3, [125I]ITE2 had obviously higher MCF-7 tumor to muscle ratio (T/M =
2.3 ± 0.6) than that of MDA-MB-231 (T/M = 0.6 ± 0.1, *P = 0.01). These results confirm the specificity of [125I]ITE2
for ER-positive tumors in vivo. The MCF-7 tumor uptake of [125I]ITE2 was uneven, mainly due to the fact that
MCF-7 tumor is a highly heterogeneous tumor (Yang et al., 2017; Babayan et al., 2013).
4. Discussion
An accurate and noninvasive method of assessing ER status, which avoids unnecessary biopsies and allows for a
series of clinical evaluations during breast cancer endocrine therapy, is always necessary. Besides, it has been reported
that dimeric and polymeric may enhance the receptor binding affinity by the local enrichment of radiotracer thereby
increasing the probability of radiotracer binding to a receptor protein (Chen et al., 2004). To our knowledge,
[
125/131I]ITE2 is the first radioactive iodine-labeled estrogen dimer.
[
125/131I]ITE2 was prepared successfully with high yield (94.4 ± 0.4%) and good labeling efficiency (about 90
min). While for the classical method of iodine-125 labeled estradiol, it requires an overnight radiolabeling reaction
with only 25 to 65% yield (Hochberg, 1979). Furthermore, the formation of 5-[125/131I]iodo-1,2,3-triazole was reported
to be effective in preventing metabolic deiodination in vivo (Ali et al., 1991). However, the molar activity of
[
125/131I]ITE2 (0.3 ± 0.2 GBq/µmol) is not as high as expected (2−3 GBq/μmol, Yan et al., 2013). The reason for the
failure to obtain the theoretical molar activity may be related to the concentration of [125/131I]NaI. Nevertheless, the
iodinated estrogen, made as described, is more than adequate for use in estrogen receptor studies.
A series of modifications have been made to estrogen to enhance the specificity of steroidal radiotracers for
targeted tissues in vivo. For example, 2-F, 4-F and 7α-cyano-substituted radioiodine-labeled estradiol have been
reported. Their uterus and thyroid uptakes were 0.46 ± 0.06 %ID/g and 57.96 ± 9.47 %ID/g for 2-F-16α-[125I]IE2; 2.7
± 0.10 %ID/g and 43.73 ± 5.67 %ID/g for 4-F-16α-[125I]IE2; 4.72 ± 0.33 %ID/g and 107.92 ± 24.31 %ID/g for
2-F-11β-OMe-16α-[125I]IE2 (Ali et al., 1993); 6.35
±
0.52 %ID/g and 252
±
11.75 %ID/g for
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7-Cyano-16α-[125I]iodoestradiol and
12.77
±
0.61 %ID/g and 118
±
8.28 %ID/g for
7-Cyano(17α,20E)[125I]iodovinylestradiol (Ali et al., 2003) at 1 h p.i., respectively. [131I]ITE2 developed in this work
showed a moderate uterine uptake (5.7 ± 0.4 %ID/g) with good retention and obviously reduced thyroid uptake (2.3 ±
0.6 %ID/g) when compared to the previously reported probes (Ali et al., 1993, 2003; Rijks et al., 1996, 1998),
indicating its binding affinity and good metabolic stability. Furthermore, the low non-specific uptake was fulfilled
through the PEGylation of estrogen to reducing lipophilicity.
As we all know, the rapid clearance of the radiotracer in the body causes the relatively low uptake of target sites
(Mankoff et al., 1997). If the radiotracer can be stable in the circulation for a longer time, it will have a greater chance
of enrichment in the tumor. In this study, about 100% of [131I]ITE2 still exists in intact form after 20 min p.i., much
better than that of [18F]FES and 4FM[18F]FES, the classic ER imaging agents, which were rapidly metabolized in
blood, resulting in less than 20% of intact radiotracer after only 5 min p.i. (Paquette et al., 2013). Since the heartbeat
of mice is faster than humans, it can be predicted that [131I]ITE2 may have a longer metabolic time in the human body,
which makes it possible for [125/131I]ITE2 to become a radiotracer for imaging and even treating ER+ tumors in the
future.
Decades ago, the application of the bivalent concept to the estrogen receptor had been studied by several groups.
Among them, Shan et al. (2013) deeply studied the ER-binding affinities and cellular activities of nonsteroidal
bivalent estrogen ligands. Their findings on the nature of the ligand, the length and nature of the spacer might be
useful for further design of radiolabeled bivalent or multivalent steroidal estrogen ligands. Their study had shown that
the use of large polar groups such as carboxylic acids, amides or esters as linkers for divalent ligands will greatly
reduce the affinity of divalent molecules to receptors. While PEGylation has been proved to be an effective strategy to
enhance the affinity with the optimized distance between the divalent ligands (10.8 Å, ~ 2 PEG). Refer to previous
studies, herein we developed a radioiodinated dimeric molecule and evaluated it in vitro and in vivo for ER targeting.
However, the direct comparison of the ER binding affinity and targeting ability of this dimeric ligand with an
appropriate monovalent control compound should be made in the future to further demonstrate the success of
dimerization conception. In addition, we will conduct more in-depth research on estradiol dimers or polymers and the
linker type and length between targeting motifs in the future, and the content of the comparison will be reflected in the
subsequent research.
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So far, none of the radiolabeled steroid agents have been commercialized for routine steroid receptor
PET/SPECT imaging. This is mainly because the current clinical research on these imaging agents is not enough.
Other than to develop more new estrogen receptor imaging agents, there needs more studies to clarify the tumor
uptake threshold of breast cancer, which directly affects the clinical judgment of hormone therapy (Fowler et al., 2016;
van Kruchten et al., 2013).
5. Conclusion
A novel radioiodinated estrogen dimer [125/131I]ITE2 was prepared successfully in high radiochemical yield with a
good specific binding affinity to the estrogen receptor. The metabolic time of [131I]ITE2 in blood has been greatly
extended with almost no deiodination, which provides a basis for breast cancer typing and guides the clinical
treatment programs. The biological function of the dimeric [125/131I]ITE2 can also be optimized by changing the length
of the PEG chain to adjust the distance between two estrogen molecules.
Acknowledgments
This study was financially supported by the National Key Basic Research Program of China (2014CB744503), the
National Natural Science Foundation of China (21976150) and the Major Research plan of the National Natural
Science Foundation of China (91959122).
Conflicts of interest
The authors declare no conflict of interest.
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Figure and Legends
Scheme 1. Synthetic routes of radioiodinated compounds [125/131I]ITE2 and non-radioactive compound [127I]ITE2.
Figure 1. The HPLC chromatograms of compound [127I]ITE2 and [125I]ITE2. They were analyzed with the same
gradient HPLC method. The non-radioactive compound was measured with a UV detector while the radioactive
compound was measured radiometrically.
Figure 2. The HPLC chromatograms of haematic radio-metabolites in mice at (a) 10 min, (b) 20 min, (c) 30 min and
(d) 60 min. The HPLC chromatograms of urinary radio-metabolites in mice at (e) 10 min, (f) 20 min, (g) 30 min and
(h) 60 min. The haematic and urinary radio-metabolites were detected at 3.85 min, 3.28 min and 3.01 min, and the
intact [131I]ITE2 were detected at 10.63 min, 10.61 min and 10.50 min.
Figure 3. Cell saturation binding, uptake, blocking study of [131I]ITE2. (a) Representative results of saturation assay
using MCF-7 cells. The curve was generated by measuring the radioactivity in MCF-7 cells incubated with increasing
concentrations of [131I]ITE2 as indicated. (b) Cell uptake of [131I]ITE2 in MCF-7 and MDA-MB-231 cells; Uptake of
[
131I]ITE2 in MCF-7 cells was blocked in the presence of 0.17 mM of estradiol. (c), (d) Cell uptake values of
131I]ITE2 at 1 h from b. (***P < 0.0001). All data were determined from three independent experiments and
[
expressed as the mean ± SD.
Figure 4. (a) Tissue distribution of 131I radioactivity (%ID/g) after intravenous injection of [131I]ITE2 into immature
female rats for 1 h and 2 h. A group of animals for the 1 h-blocked experiment received 15 µg/rat of unlabeled
estradiol (ES), co-injected with [131I]ITE2 (***P = 0.0004). (b), (c) Uterus to blood ratio and uterus to muscle ratio
from a. (n = 3 - 5, *P = 0.02, P = 0.06. Data are means ± SD).
Figure 5. (a) Representative SPECT/CT images obtained at 15 min and 1 h p.i. of [125I]ITE2 in nude mice bearing
MCF-7 tumor, fulvestrant-treated MCF-7 tumor-bearing nude mice and MDA-MB-231 tumor-bearing nude mice; (b)
Tumor to muscle ratios were derived from SPECT images (means ± SD, n = 3, *P = 0.01). Tumor sites were indicated
by circles.
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