Radioiodinated progesterone derivative for progesterone receptor targeting with enhanced nucleus uptake via phenylboronic acid conjugation

Zhang Xianzhong (Orcid ID: 0000-0001-8591-8301)

Lawrie Ken (Orcid ID: 0000-0002-8061-3696)

Radioiodinated progesterone derivative for progesterone receptor targeting with

enhanced nucleus uptake via phenylboronic acid conjugation

1

Fei Gao , Chenyu Peng , Jindian Li , Rongqiang Zhuang , Zhide Guo , Duo Xu , Xinhui

2

*

1*

Su , Xianzhong Zhang

. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for

1

Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University,

Xiamen 361102, China

2

. Department of Nuclear Medicine, Zhongshan Hospital affiliated to Xiamen University,

Xiamen 361004, China

*

For correspondence contact:

Xianzhong Zhang, PhD, Professor

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

Xinhui Su, PhD

Department of Nuclear Medicine, Zhongshan Hospital affiliated to Xiamen University,

Xiamen 361004, China

E-mail: suxinhuii@163.com

This article has been accepted for publication and undergone full peer review but has not been

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:

10.1002/jlcr.3741

ABSTRACT

Objectives: A novel ¹³¹I-radiolabeled probe with aromatic boronate motif ( I-EIPBA) was

designed to target progesterone receptor (PR) positive breast cancer with enhanced nucleus

uptake.

131

Methods: Acetylene progesterone was conjugated with pegylated phenylboronic acid via click

reaction and radiolabeled with ¹³¹I to afford ¹³¹I-EIPBA. Meanwhile, ¹³¹I-EIPB without

boronate was prepared as control agent. After determination of the lipophilicity and stability of

these tracers, in vitro cell uptake studies and in vivo biodistribution in rats were performed to

verify the enhanced nucleus uptake and PR targeting ability of ¹³¹I-EIPBA.

Results: ¹³¹I-EIPBA was obtained with moderate radiochemical yield (40.35 ± 3.52%) and

high radiochemical purity (> 98%). As expected, the high binding affinity (39.58 nM) of ¹³¹I-

EIPBA for PR was determined by cell binding assay. The internalization ratio of ¹³¹I-EIPBA

was remarkably higher than that of ¹³¹I-EIPB in PR-positive MCF-7 cells. Furthermore, the

enhanced nucleus uptake of ¹³¹I-EIPBA (0.59 ± 0.02 %) was found to be significantly higher

than that of ¹³¹I-EIPB (0.13 ± 0.01 %) in MCF-7 cells.

Conclusions: A novel ¹³¹I-EIPBA compound was developed for PR-targeting with improved

cellular nucleus uptake. Furthermore, the introduction of aromatic boronate motif provides a

worthwhile strategy for enhancing the nuclear receptor targeting of tracers.

Keywords: breast cancer, progesterone receptor, radioiodination, nuclear receptor targeting

1

. Introduction

Progesterone receptor (PR) is overexpressed in 70-80% of breast cancers and in a similar

1

-4

subset of ovarian cancers, uterine cancers, and prostate cancers . There is an abundance of

accumulating data clearly suggesting loss of PR expression in breast cancer is associated with

5

-7

more aggressive tumors and worse prognosis . Additionally, estrogen-receptor (ER)-negative,

PR-positive patients are also suitable for progesterone imaging, about comprises 5%-10% of

8

patients with breast cancer . PR levels are measured by in vitro immunohistochemistry assays

of biopsy samples, which is invasive and may lead false-positive or false-negative results.

Therefore, development of PR-targeting agent for noninvasive imaging is of interested and

could offer several advantages⁵.

So far, a number of radiolabeled PR-targeted imaging probes have been reported and

6

125

evaluated, including radioiodinated progesterone derivatives , such as (16α-[ I]iodo-19-

1

25

nortestosterone, (17α, 20E/Z)-[ I]iodovinyl derivatives of 19-nortestosterone and

125

testosterone, isomeric 7α-cyano-(17α, 20E/Z)-[ I]iodovinyl-19-nortestosterone), (17α,

1

23

18

2

0E/Z)-[ I]iodovinyl analogues, et al), radiofluorinated progesterone derivatives ([ F]FMNP,

[¹⁸

F]FENP , [ F]FPTP , [ F]FFNP , [ F]EAEF , et al), as well as other radioactive

8

18

9

18

10

18

11

progesterone derivatives¹²^,¹³. These radiotracers are highly lipophilic which leads to a relatively

high level of nonspecific binding. Only a few of these radiotracers have been tested in

preclinical and clinical trials.

1

4

15

PR is regulated by ER and belongs to the type-1 nuclear receptor subfamily . 55-70%

1

6

PR locates in the nucleus of breast cancer . Shimizu et al. reported that some malignant cells

lose the ability to transfer the cytoplasmic hormone receptor complex to nucleus. Although

some of the hormone receptors can still be measured in the cytoplasm, and they have lost their

1

7

response to endocrine therapy and have been nonfunctional receptors . Currently, the vast

majority of the development of technologies for the targeted intracellular delivery of proteins

(including antibodies, their fragments, or antibody-like molecules) use of a peptide localization

signal¹⁸^,¹⁹, and synthetic small molecule drug (triphenylphosphonium myocardial imaging

agent)²⁰^,²¹by the high potential of the mitochondrial membrane. In 2017, Tang et al. reported

a simple benzylboronate chemical motif which provides highly efficient active targeting of

2

proteins to the nucleus . Similarly, the strategy is an effective approach for small molecules

to increase the nucleus targeting.

In this study, we proposed to introduce a simple aromatic boronate motif onto ethisterone,

a natural progesterone derivative with high PR affinity, to enhance the ability of nucleus

targeting. The radiolabeling of ethisterone was achieved by using a copper (II)-mediated click

2

3

reaction of alkynyl structure, which is considered to be easy to modify and does not affect

the targeting ability. Polyethylene glycol (PEG) modification was used to link the boronate

motif and targeting group ethisterone, and to reduce lipophilicity and lower the non-specific

binding of tracer as well. Herein, a novel ¹³¹I-radiolabeled ethisterone probe with PEGylated

aromatic boronate is designed to target PR-positive breast cancer with enhanced nucleus uptake.

2

. Materials and methods

2

.1. General

All reagents and solvents were purchased from commercial sources. High performance

liquid chromatography (HPLC) was performed on Dionex Ulti-Mate 3000 (Thermo Scientific,

Waltham, MA, USA) and with a flow-counter radioactivity detector (Bioscan, Poway, CA,

USA). HPLC chromatograms analysis was performed using the C-18 reversed-phase column

1

13

(

4.6×250 mm, 5 μm, 120Å, Thermo) and monitored at 254 nm wavelength. H and C NMR

spectra were recorded on BIXI-I 400 (Wuhan Zhongke Niujin Magnetic Resonance

Technology Co., Ltd) and mass spectra were recorded on Mass Spectrometer (Waters, Xevo,

G2-XS TOF).

The HPLC method for ¹³¹I-EIPBA, A: 0.1% trifluoroacetic acid in H

O, B: 0.1%

2

trifluoroacetic acid in acetonitrile; 45% B (0–20 min), B from 45% to 80% (20-30 min). For

¹³¹I-EIPB, 50% B (0–20 min), B from 50% to 80% (20-30 min).

The human breast carcinoma MCF-7 cells were obtained from China Infrastructure of Cell

Line Resources, which cultured in DMEM medium supplemented with 10% fetal bovine serum

(Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Beyotime, Shanghai, China). All

cells were grown as a monolayer at 37 ℃ in a moist atmosphere of 5% CO and 95% air.

2

The Sprague Dawley rats (female, ~200 g) and BALB/C mice (female, ~20 g) were

obtained from the Animal Center of Xiamen University. The experimental procedures and

animal use and care protocols were approved by Xiamen University’s animal care and use

committee.

2

.2. Chemistry and radiochemistry

Radioiodinated EIPBA was synthesized according to the procedure shown in Scheme 1.

2

3

CuCl /TEA complex were prepared by using a previously reported method . The ethisterone

2

(1.0 μmol) was dissolved with anhydrous THF (40 μL) in a 1.5 mL-tube, followed by addition

of the CuCl

2

/TEA complex in anhydrous acetonitrile (40 μL). After 5 min, ¹³¹I-NaI (6.0 μL, 1

mCi) and compound 4a or compound 4b (1.0 μmol) dissolved with anhydrous acetonitrile (20

μL) were added successively to the above solution. The tube was uncapped and the reaction

was heated at 60 ℃ for 1 h.

After the reaction is completed, the mixture was diluted with acetonitrile (100 μL) and

then purified by HPLC to give the final product of ¹³¹I-EIPBA. It was filtered through a 0.22-

m filter (Millipore Co., Billerica, MA, USA) to generate the final injections for use.

2

.3. Octanol/water partition coefficient

The octanol-to-water partition coefficients of ¹³¹I-EIPBA and ¹³¹I-EIPB were measured

following the method: 100 μL tracer solution was added to a centrifuge tube that contained 3

mL of 1-octanol and 2.9 mL of phosphate-buffered saline (PBS, pH=7.4). The radiotracer was

fully mixed with vigorous vortexing for 1 min, and the tube was centrifuged at 10,000 rpm for

5

min. Take 100 μL of the organic phase from the above solution and add to a new centrifuge

tube that contained 2.9 mL of 1-octanol and 3 mL of PBS (pH=7.4) and repeat the above step

three times. Counts of 100 μL organic and 100 μL aqueous phase (n = 3) were determined by

γ-counter (Wizard 2480, Perkin Elmer, USA). The logP value was reported as mean ± SD of

triplicate samples.

2

.4. In vitro and in vivo stability studies

For in vitro stability study, radiotracer ( I-EIPBA or ¹³¹I-EIPB) was incubated in

1

31

physiological saline at room temperature or in mice serum at 37 ℃ for 4 h. After incubation,

the samples were centrifuged after adding equally acetonitrile, and the supernatants were

filtered through a 0.22 μm filter before subject to HPLC analysis.

For the metabolic stability study, BALB/C mice (female, ~20 g) were intravenously

injected with 37 MBq of ¹³¹I-EIPBA or ¹³¹I-EIPB. The mice were sacrificed at 30 min and 1 h

post-injection. Blood, urine, and liver samples were collected and treated using previously

24

published methods . The blood was immediately centrifuged at 13,000 rpm. The liver sample

was homogenized and suspended in 1 mL of acetonitrile and then centrifuged for 5 min at

1

3,000 rpm. The liver and blood supernatants and urine samples were collected and passed

through a 0.22 μm filter and analyzed by HPLC using the procedure described above.

2

.5. In vitro protein binding

The protein binding of ¹³¹I-EIPBA and ¹³¹I-EIPB to human serum albumin (HSA) were

1

31

tested using a dialysis method. Approximately 200 μL(about 7.4 MBq) radiotracer ( I-EIPBA

or ¹³¹I-EIPB) was added to 3 mL (3 mg/mL) solution of HSA and vortexed at room temperature

for 1 min and then incubated at 37 ℃ for 20 min. After measuring the total activity by CRC-

2

5R dose calibrator, it was transferred to a dialysis membrane (molecular weight cutoff

(MWCO) value of 7 kDa) and immersed in 1000 mL PBS (pH = 7.4) to separate the unbound

fraction of each tracer from the protein-bound fraction. Each radiotracer was dialyzed without

HSA to account for the nonspecific dialysis membrane binding in the process. Radioactivities

in PBS and membrane were measured respectively after 6 h dialysis. Mean corrected protein-

bound fraction were calculated with triplicated experiment data and reported as mean ± SD.

2

.6. Cellular saturation binding studies

Saturation binding experiments were performed by using PR-positive MCF-7 cells

according to the published procedures ²⁵^,²⁶. There were approximately 2 × 10 cells in each

well of a 24-well plate to which eight different concentrations of ¹³¹I-EIPBA or ¹³¹I-EIPB were

used, ranging from 1ꢀnmol/L to 150ꢀnmol/L. Followed by incubation at 37 ℃ for 1 h, the cells

were washed with 1 mL of PBS (pH = 7.4) three times and then lysed with 1 mL of NaOH (1

5

mol/L). Radioactivity was counted using a γ-counter. Bmax and K

d

(nmol/L) values were

determined using GraphPad Prism 5.0 software.

2

7

.7. Specific cell binding studies

The specificity of ¹³¹I-EIPBA for PR was tested following the literature method . MCF-

27

5

cells seeded in 24-well plates (2×10 cells per well) were incubated for 24 h. Then washed

twice with 1 mL PBS (pH = 7.4) and incubated with or without PR inhibitor (10 μL per well,

1

mg/mL) for 1 h. ¹³¹I-EIPBA in physiological saline (0.37 MBq in 500 μL per well) were

added and then the cells were incubated at 37 ℃ for 0.5, 1, 2, 3 and 4 h. After that, the cells

were washed twice with 1 mL of PBS (pH = 7.4) and lysed with 1 mL of NaOH (1 mol/L), and

the cell extract was collected and analyzed using γ-counter.

Cell binding study of ¹³¹I-EIPB was repeated following the same procedure described

above for comparison.

2

.8. Cell internalization and nucleus uptake studies

For the internalization²⁷^,²⁸, MCF-7 cells seeded in 24-well plates (2 × 10 cells per well)

5

were incubated for 24 h. Wash the cells once with binding buffer, then 450 µL binding buffer

were added to each well. The ¹³¹I-EIPBA or ¹³¹I-EIPB (0.37 MBq per well) in physiological

saline (500 μL per well) were added and incubated for 2 h at 4 ℃. After pre-incubation, cells

were washed with ice-cold binding buffer three times then incubated with previously warmed

binding buffer for different time point (5, 15, 30, 60, 120 min) at 37 ℃ for internalization. After

that, the cells were subsequently incubated twice with 0.5 mL of glycine HCl (50 mM; pH =

2

.8) for 5 min each to remove the surface-bound fraction, and the supernatant was collected.

After an additional washing step with 1 mL of ice-cold PBS, the cells were lysed with 1 mL of

NaOH (1 N) and collected, and radioactivity was measured with γ-counter.

The nucleus localization of radiotracer has been determined following the published

protocol . Approximately 150 nmol/L of ¹³¹I-EIPBA or ¹³¹I-EIPB contained in 500 μL of

medium was then added and the cells were incubated at 37 ℃ for 0.5, 1, 2, 3 and 4 h. After that,

the cells were washed twice with 1 mL of PBS (pH = 7.4) and ground 20-30 times in 0 ℃ ice

bath with pre-cooled 1 mL of lysis buffer and 50 µL of Reagent A. Then the cell homogenate

was centrifuged at 700 g for 5 minutes, the supernatant fraction was discarded, and then 0.5

mL of pre-cooled lysis buffer was added. The above solution was added to a new centrifugal

29

tube containing 0.5 mL medium buffer and centrifuged at 4 ℃ (700 g for 5 min). The

suspension recentrifuged (1000 g for 10 min) to extract the nuclei, which was then measured

by γ-counter. The nucleus uptakes were calculated as the percentage of radiotracers in the

nucleus to the total amount added to the cells.

2

.9. Biodistribution

Female Sprague Dawley rats were intravenously injected with 0.1 mL (0.37 MBq) of ¹³¹I-

EIPBA (or ¹³¹I-EIPB) for biodistribution study (n = 5 for each group). Then the rats were

sacrificed and dissected at 30 min, 1 h and 2 h after injection, respectively. All interested organs

and tissues (including heart, liver, spleen, lung, kidney, bone, muscle, stomach, intestine,

thyroid, uterus, ovary and blood) were collected, weighed and measured using a γ-counter to

calculate the percentage injected dose per gram (%ID/g, mean ± standard deviation (SD)) and

the percentage injected dose per organ (%ID, mean ± SD).

Statistical Analysis

Statistical analyses were performed by using t-test and one-way variance tests (ANOVA)

followed with the SPSS statistical software and the level of significance was set at P = 0.05

(*P <0.1, **P <0.05 and ***P <0.01).

3

. Results

3

.1. Chemistry and radiochemistry

The synthetic routes are shown in Scheme 1. All intermediates and final products of

EIPBA and EIPB were synthesized and characterized by H NMR, ¹³C NMR and mass

spectrometry (Fig. S1-S16 in the Supplementary File). The HPLC retention time of non-

radioactive ¹²⁷I-EIPBA and ¹²⁷I-EIPB were 10.04 min and 15.42 min respectively, which

1

consistent well with that of radiotracers (10.38 min and 15.80 min for ¹³¹I-EIPBA and ¹³¹I-

EIPB, respectively) (Fig. 1a and 1b). The HPLC profiles of radiotracers after purification were

recorded with UV and radiometric detector (Fig. 1c and 1d). The radiochemical yields of ¹³¹I-

EIPBA and ¹³¹I-EIPB were 40.35 ± 3.52% (n = 3) and 45.12 ± 4.59% (n=3), respectively. Both

of the radiotracers have high radiochemical purity (> 98%) after HPLC purification. The molar

activities of ¹³¹I-EIPBA and ¹³¹I-EIPB were calculated as 8.53 ± 0.87 GBq/μmol and 8.17 ±

0

.16 GBq/μmol respectively based on the standard absorbance curves of EIPBA and EIPB (Fig.

S17).

Both of radiotracers showed moderate lipophilicity with the octanol/water partition

coefficient (log P) of 1.38 ± 0.48 (n = 3) for ¹³¹I-EIPBA and 1.56 ± 0.43 (n = 3) for ¹³¹I-EIPB,

respectively.

3

.2. Stability studies and protein binding

Both of the ¹³¹I-EIPBA and ¹³¹I-EIPB displayed good stability in saline (Fig. 2a and 2c).

The stability in rat serum at 37 ℃ of ¹³¹I-EIPBA was more than 98%, higher than that of ¹³¹I-

EIPB, which remained 83% of the original product after 4 h incubation (Fig. 2b and 2d).

The results of in vivo metabolic stabilities of ¹³¹I-EIPBA and ¹³¹I-EIPB were shown in Fig.

S18 and S19. The radiometabolites in the blood, urine and liver at different time were analyzed

by HPLC. At 30 min and 1 h after injection, more than 90% of intact ¹³¹I-EIPBA was observed

in blood (main radioactivity was found at 10.35 min). At the same time, ¹³¹I-EIPB was almost

disappeared. A new peak appeared about 3.00 min in the HPLC analysis, possibly was the peak

of deiodinated ¹³¹I. After modification with phenylboronic acid, ¹³¹I-EIPBA was enriched in

blood, which can be confirmed by biodistribution study. Results from these experiments

indicated that ¹³¹I-EIPBA was much more stable than ¹³¹I-EIPB.

The protein binding of ¹³¹I-EIPBA and ¹³¹I-EIPB were measured in HSA solution. Both of

these two radiotracers have moderate protein binding levels, although ¹³¹I-EIPBA had higher

protein-bound fraction (33.08 ± 7.88%) than that of ¹³¹I-EIPB (16.01 ± 3.16%), which indicated

that ¹³¹I-EIPBA may has a higher blood uptake at the early time points after injection.

3

.3. Cellular saturation studies

The cell binding studies of ¹³¹I-EIPBA and ¹³¹I-EIPB were performed in MCF-7 cells to

give the saturated binding curves (Fig. 3). The dissociation constant (K

d

) of ¹³¹I-EIPBA was

calculated as 39.58 ± 3.47 nM (n = 3), with a maxima number of binding sites (Bmax) of 2.39

6

5

of ¹³¹I-EIPB was calculated as 46.83 ± 5.21 nM

×

10 cps per 10 cells (Fig. 3a). While the K

d

n = 3), with the Bmax of 3.51 ×10 cps per 10 cells (Fig. 3b). Both of ¹³¹I-EIPBA and ¹³¹I-

6

5

(

EIPB showed high binding affinities toward PR.

3

.4. Cell uptake studies

In cell uptake study, both of ¹³¹I-EIPBA and ¹³¹I-EIPB demonstrated increased uptakes

with time in PR-positive MCF-7 cells, as shown in Fig. 4a. The cellular uptake of ¹³¹I-EIPBA

reached maximum (8.33 ± 0.42%) after 2 h of incubation, much higher than that of ¹³¹I-EIPB

(3.22 ± 0.16%). After blocking with ethisterone (10 μL, 1 mg/mL), the cell uptakes of both

1

31

tracers were significantly inhibited and decreased to 0.24 ± 0.05% ( I-EIPBA) and 0.31 ±

1

31

0

.05% ( I-EIPB) respectively after 1 h incubation, which demonstrated the specificity binding.

The cell internalization ratio (calculated as the percentage of internalized tracer to the total

added amount) of ¹³¹I-EIPBA was 17.7 times higher than that of ¹³¹I-EIPB in MCF-7 cells (Fig.

4

b). The internalization ratios of ¹³¹I-EIPBA and ¹³¹I-EIPB at 5 min incubation were 0.64 ±

.01% and 0.04 ± 0.01%, respectively (P＜0.001). Obviously higher internalization of ¹³¹I-

0

EIPBA than that of ¹³¹I-EIPB were observed at each subsequent time point of incubation.

Enhanced nucleus accumulation of ¹³¹I-EIPBA was observed in MCF-7 cells (Fig. 4c).

Nucleus uptake at 1 h after the addition of ¹³¹I-EIPBA in MCF-7 cells was calculated as 0.59

±

0.02%, obviously higher than that of ¹³¹I-EIPB (0.13 ± 0.01%). The area under time-activity

curve of ¹³¹I-EIPBA was increased by 4.5-fold for ¹³¹I-EIPB (P＜0.05), indicating enhanced

nucleus uptake efficiency of ¹³¹I-EIPBA with phenylboronic acid motif.

3

.5. Biodistribution

The biodistribution results of ¹³¹I-EIPBA and ¹³¹I-EIPB in female Sprague Dawley rats

was shown in Table 1. High accumulation and prominent retention in liver and intestine were

observed indicating hepatoenteric metabolism of these two radiotracers. The uptakes of ¹³¹I-

EIPBA in PR-rich ovarian and uterine tissues were 1.18 ± 0.16 %ID/g and 6.90 ± 0.80 %ID/g

at 30 min after injection, which obviously higher than that of ¹³¹I-EIPB (0.61 ± 0.02 %ID/g and

1

.50 ± 0.02 %ID/g for uterus and ovary, respectively). While the high uptakes of ¹³¹I-EIPBA

decreased quickly to the nonspecific background level at the later time point, which close to

the uptakes of ¹³¹I-EIPB. ¹³¹I-EIPBA showed relatively low uptakes in nonspecific organs and

tissues, such as spleen, lung and muscle. Much higher blood uptake, and lower liver and

intestine uptakes of ¹³¹I-EIPBA than that of ¹³¹I-EIPB may due to the modification of

phenylboronic acid which leads to a higher protein binding level. In addition, both of the

radiotracers showed relatively high thyroid accumulation at the later time points indicating the

deiodination in vivo. As discussed in the literatures²³^,³⁰, the high thyroid uptake might be

reduced by increasing steric hindrance of trisubstituted triazoles or avoiding prolonged

retention in vivo.

4

. Discussion

Nowadays, most of the radioactive PR imaging agents belong to progesterone,

testosterone and 19-nortestosterone. Their moderate tumor uptake may due to the limited

binding rate with the nuclear receptors and rapid metabolism. Effective drug delivery to the

nucleus is a challenge because of various physiological barriers, which provide a broader

platform for cancer diagnosis and treatment by enhancing nuclear uptake and reducing uptake

3

1

of normal tissues . In this study, a novel radioiodinated progesterone derivative conjugated

1

31

with PEGylated phenylboronic acid ( I-EIPBA) was prepared to enhance the nucleus uptake

and the feasibility of PR targeting was demonstrated in vitro and in vivo.

Firstly, ¹³¹I-EIPBA was obtained successfully with considerable radiolabeling yield and

molar activity by using a one-pot, copper (II)-mediated click reaction. The introducing of

PEGylated aromatic boronate onto ethisterone via triazole formation not only remain

nanomolar affinity of ¹³¹I-EIPBA (K

boronate ¹³¹I-EIPB (K

= 39.58 nM) but also slightly higher than that of non-

d

= 46.83 nM). Both of two tracers have comparable binding affinities

with the previously reported radiolabeled progesterone derivatives (K = 0.3 to 60 nM).

d

In the cell uptake studies, the activity intensity enhancement in the nucleus achieved by

using the boronate targeting strategy. The tracer of ¹³¹I-EIPBA with phenylboronic acid-

modified had 4.5-fold higher uptake in the nuclei of PR-positive MCF-7 cells. The result was

consistent with the previous report, which using fluorescence to track the delivery process and

2

degree of nuclear localization .

In biodistribution, the uptakes of PR-rich organs may reflect the binding affinity of tracers.

The considerable uterine and ovarian uptakes of ¹³¹I-EIPBA in rats were 1.18 %ID/g and

6

.90 %ID/g at 30 min after injection, which obviously higher than that of ¹³¹I-EIPB

(0.61 %ID/g and 1.50 %ID/g at 30 min and for uterus and ovary, respectively). The improved

binding affinity of aromatic boronate modified radiotracer was confirmed again. Although the

latter has the better retention at 1 h and 2 h time points, the possible reason may due to the

nonspecific uptake of ¹³¹I-EIPB in uterus and ovary. While compared to the most recently

18

report PR targeting tracer, [ F]EAEF, which had the uptakes of 5.89%ID/g and 4.33 %ID/g

1

for uterus and ovary at 1 h after injection , the obviously lower uterus and ovary uptakes

0.76 %ID/g and 2.62 %ID/g at 1 h after injection) were observed for ¹³¹I-EIPBA. Thinking of

(

the high ratios of ovary to muscle of ¹³¹I-EIPBA, high contrast tumor SPECT imaging was

expected. The further evaluation should be conducted in PR-positive tumor-bearing mice and

estrogen-primed immature rat model with microSPECT imaging.

Although the introducing of aromatic boronate onto ethisterone could improve the PR

targeting ability with enhanced nucleus uptake, the obviously in vivo deiodination and

unfavorite biodistribution characteristics, such as high liver accumulation, should be optimized

in the future study. Furthermore, based on the successful enhancement in the nucleus achieved

by using the boronate targeting strategy provides a promising strategy for design radiolabeled

probes for nuclear receptor targeting.

5

. Conclusions

A radioiodinated ¹³¹I-EIPBA was prepared successfully with good radiochemical yield

and high radiochemical purity. It was designed base on progesterone structure for nuclear

receptor PR targeting with aromatic boronate motif to improve nucleus uptake. The results

showed that this radiotracer had specific cell uptake with high PR binding affinity. The

obviously enhanced nucleus accumulation of ¹³¹I-EIPBA than that of the control compound

without aromatic boronate motif indicating this is a potent strategy for nuclear receptor

targeting drug modification. The further in vivo evaluation of ¹³¹I-EIPBA for PR-targeting

breast cancer SPECT imaging is in progress and the results will be reported separately.

Acknowledgments

This study was financially supported by the National Key Basic Research Program of

China (2014CB744503) and

Natural Science Foundation of Fujian Province, China (No. 2016J05200).

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Scheme 1. Synthetic routes of unlabeled EIPBA and EIPB, and radiolabeled ¹³¹I-EIPBA and

1

31

I-EIPB. Reagents and conditions: (a) p-toluensulfonyl chloride, Et

3

N, DCM, overnight; (b)

NaN3, DMF, overnight; (c) 4-carboxyphenylboronic acid/benzoic acid, DMAP, EDCI, DMF,

overnight; (d) CuI, NIS, Et

3

N, DMF, R.T., overnight; (e) NaOI

4

, HCl, THF, water, R.T., 6 h.

1

31

(f) I, CuCl

2

/TEA, CH

3

CN, water.

Fig. 1. HPLC analysis of radiotracers and non-radioactive references with radiometric (red

1

31

127

131

line) and UV (black line) detectors. HPLC profiles of I-EIPBA and I-EIPBA (a), I-

1

27

131

EIPB and I-EIPB (b), and the injections of I-EIPBA (c) and I-EIPB (d) after

purification.

Fig. 2. In vitro stability of ¹³¹I-EIPBA (a, b) and ¹³¹I-EIPB (c, d) in saline (a, c) at room

temperature and after incubation in rat serum (b, d) at 37 ℃ for 30 min, 60 min, 2 h, 4 h.

Fig. 3. Saturation cell binding assays of ¹³¹I-EIPBA (a) and ¹³¹I-EIPB (b) in MCF-7 cells.

Fig. 4. (a) Specific cell uptakes of ¹³¹I-EIPBA and ¹³¹I-EIPB in MCF-7 cells at the indicated

time points. (b) Cell internalization of ¹³¹I-EIPBA and I-EIPB in MCF-7 cells at the

131

1

31

131

indicated time points. (c) Cell nucleus accumulations of I-EIPBA and I-EIPB in MCF-7

cells at the indicated time points.

Table 1. Biodistribution results of ¹³¹I-EIPBA and ¹³¹I-EIPB in rats.

Post-injection time (mean ± SD, n=5)

Tissues

30 min

¹³¹I-EIPBA ¹³¹I-EIPB ¹³¹I-EIPBA ¹³¹I-EIPB ¹³¹I-EIPBA ¹³¹I-EIPB

ID/g 2.22±0.49 1.55±0.54 1.20±0.09 1.16±0.69 0.40±0.06 0.25±0.04

1 h

2 h

%

Heart

Liver

%

ID 1.29±0.64 0.68±0.24 0.58±0.11 0.54±0.38 0.17±0.03 0.11±0.02

ID/g 6.39±0.06 15.4±0.32 3.42±0.42 8.97±3.49 1.18±0.22 1.27±0.14

%

ID 37.38±0.34 71.18±23.30 20.01±2.46 46.41±21.90 6.91±1.33 7.48±0.84

ID/g 0.83±0.01 0.67±0.22 0.41±0.04 0.34±0.12 0.21±0.11 0.20±0.02

Spleen

Lung

%

ID 0.31±0.07 0.16±0.04 0.18±0.04 0.10±0.04 0.07±0.02 0.06±0.01

ID/g 2.16±0.43 2.12±1.03 1.28±0.16 1.34±1.01 0.85±0.44 0.43±0.16

%

ID 1.96±0.39 1.93±0.94 1.16±0.15 1.23±0.92 0.78±0.40 0.38±0.14

ID/g 1.86±0.18 1.42±.034 1.04±0.09 1.02±0.73 0.49±0.14 0..39±0.03

Kidney

%

ID 1.38±0.18 1.38±0.33 0.94±0.24 1.08±0.82 0.56±0.21 0.39±0.02

Bone %ID/g 0.84±0.16 0.89±0.35 0.40±0.09 0.58±0.38 0..33±0.12 0.20±0.03

Muscle %ID/g 0.35±0.07 0.35±0.04 0.32±0.03 0.31±0.12 0.19±0.15 0..15±0.04

%

ID/g 0.79±0.16 1.03±0.27 0.79±0.18 3.42±0.54 0.95±0.36 2.34±0.08

ID 0.70±0.24 0.83±0.23 0.66±0.38 2.48±0.89 0.54±0.05 2.02±0.57

Intestine %ID/g 1.34±0.43 3.31±1.14 3.84±1.47 5.77±0.33 1.06±0.81 7.33±1.10

Stomach

%

ID/g 6.19±1.09 14.08±0.98 11.81±6.38 19.88±0.65 18.71±0.28 20.11±1.30

Thyroid

Blood

%

ID 0.37±0.05 1.07±0.17 0.61±0.28 1.40±0.28 1.14±0.01 1.22±0.17

ID/g 7.30±0.39 0.57±0.14 3.67±0.06 0.44±0.16 1.00±0.15 0.99±0.33

%

ID 47.45±2.07 3.71±0.74 23.86±0.32 2.86±0.85 6.50±0.80 6.45±1.75

ID/g 1.18±0.16 0.61±0.02 0.76±0.07 1.04±0.56 0.87±0.18 0.60±0.13

Uterus

Ovary

%

ID 0.27±0.01 0.14±0.02 0.24±0.01 0.21±0.12 0.15±0.01 0.06±0.01

ID/g 6.90±0.80 1.50±0.02 2.62±0.90 1.50±0.59 0.82±0.27 1.40±0.25

%

ID 0.17±0.01 0.14±0.01 0.14±0.04 0.10±0.05 0.11±0.04 0.09±0.03

Article Doi

Radioiodinated progesterone derivative for progesterone receptor targeting with enhanced nucleus uptake via phenylboronic acid conjugation

DOI: 10.1002/jlcr.3741

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Article abstract of DOI:10.1002/jlcr.3741

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