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18F-labeled ethisterone derivative for progesterone receptor targeted PET imaging of breast cancer
Nuclear Medicine and Biology 72–73 (2019) 62–69
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F-labeled ethisterone derivative for progesterone receptor targeted PET imaging of breast cancer Fei Gao, Chenyu Peng, Rongqiang Zhuang ⁎, Zhide Guo, Huanhuan Liu, Lumei Huang, Hua Li, Duo Xu, Xuejun Wen, Jianyang Fang, Xianzhong Zhang ⁎ State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
a r t i c l e
i n f o
Article history: Received 1 May 2019 Received in revised form 11 June 2019 Accepted 6 July 2019 Keywords: Breast cancer Progesterone receptor MicroPET imaging Ethisterone
a b s t r a c t Purpose: A novel radiolabeled probe 1 (17 [18F]fluoro 3,6,9,12,15 pentaoxaheptadecyl 1H 1,2,3 triazole testosterone ([18F]FPTT) was synthesized and evaluated for PET imaging of progesterone receptor (PR)-positive breast cancer. Methods: The ethinyl group of ethisterone, a PR targeting pharmacophore, was coupled with azide modified PEGOTs by click chemistry to obtain the labeling precursor. The final [18F]FPTT was synthesized by a one-step nucleophilic substitution reaction with 18F. The in vitro stabilities of [18F]FPTT in saline or rat serum were determined after 2 h incubation. Then the in vitro cell binding, ex vivo biodistribution and in vivo imaging of [18F]FPTT were further investigated to evaluate the PR targeting ability and feasibility for the diagnosis of PR-positive breast cancer with PET imaging. Results: [18F]FPTT was obtained in high decay-corrected radiochemical yield (78 ± 9%) at the end of synthesis. It had high radiochemical purity (N98%) after HPLC purification and good in vitro stability. The molar activity of [18F] FPTT was calculated as 17 GBq/μmol. The microPET imaging of [18F]FPTT in tumor-bearing mice showed much higher tumor uptake in PR-positive MCF-7 tumor (3.9 ± 0.20%ID/g) than that of PR-negative MDA-MB-231 tumor (1.3 ± 0.08%ID/g). The high MCF-7 tumor uptake could be specifically inhibited by blocking with ethisterone (1.3 ± 0.11%ID/g) or [19F]FPTT (2.20 ± 0.17%ID/g), respectively. The biodistribution in estrogenprimed female SD rats of [ 18F]FPTT showed high uterus and ovary uptakes (8.31 ± 1.74%ID/g and 3.79 ± 0.82%ID/g at 1 h post-injection). The specific uptakes of uterus and ovary in normal rats were 3.52 ± 0.29%ID/ g and 3.22 ± 0.50%ID/g respectively and could be inhibited by co-injecting of ethisterone. Conclusion: A novel [18F]FPTT probe based on ethisterone modification could be a potential diagnostic agent for PR-positive breast cancer. © 2019 Elsevier Inc. All rights reserved.
1. Introduction A large number of clinical trials have proved that the key to the prognosis of malignant tumors lies in the early detection, diagnosis and treatment of diseases [1,2]. In particular, the early diagnosis of breast cancer will help to alleviate the pain of patients and improve the prognosis of cancer [3]. Although immunohistochemical methods are currently used to assess the hormone receptor status of metastatic tumors, there are many limitations in determining the hormone receptor status of metastatic tumors [4,5]. For example, the location of the examination is not suitable for biopsy, sample processing (especially decalcification) will also produce false negative results, as well as intratumoral and tumor stroma heterogeneity. Molecular imaging has ⁎ Corresponding authors at: School of Public Health, Xiamen University, 4221-116 Xiang'an South Rd., Xiang'an District, Xiamen 361102, China. E-mail addresses: [email protected] (R. Zhuang), [email protected] (X. Zhang). https://doi.org/10.1016/j.nucmedbio.2019.07.001 0969-8051/© 2019 Elsevier Inc. All rights reserved.
been widely used to improve the specificity and quantification of screening and early diagnosis, centralized and personalized treatment, and follow-up of early treatment [6–8]. Accurate early detection of tumors requires efficient and safe molecular imaging probes [9]. According to the expression characteristics of breast cancer receptors, targeted endocrine therapy has become an attractive therapeutic option in the diagnosis and treatment of breast cancer [10,11]. Progesterone receptor (PR) is closely related to the invasiveness, prognosis and efficacy of hormone-related diseases such as breast cancer, ovarian cancer, uterine cancer and endometriosis [12,13]. In recent years, many radiolabeled PR targeted probes for PET imaging have been reported and evaluated ([ 18F]FMNP, [ 18F]FENP, [ 18F]FPTP, [ 18F]FFNP, [ 18F]EAEF, et al.) [14–19]. Most of them use modified steroid structures as probes targeting PR, such as testosterone, progesterone and 19-nortestosterone, and different modifications of these structures. This is due to the fact that most receptors have a high affinity for their cognate ligands. Ethisterone is a natural progesterone
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derivative, which has a strong affinity for PR, and its alkynyl group can be easily modified compared with other steroids [19]. Of course, besides, ethisterone is also very popular modification group on other imaging agents targeting PR [20]. 18F (t1/2 = 109.8 min) is a suitable radionuclide for PET imaging due to its advantages of high positron decay (97%), low positron energy (0.634 MeV) and smaller atomic radius. In addition, the inherent advantages of PET imaging which has high sensitivity, good spatial resolution and quantitative and dynamic monitoring has become a non-invasive method for breast cancer diagnosis [21,22]. In this study, radionuclide 18F was incorporated into ethisterone derivative to develop a novel radiotracer ([ 18F]FPTT) for PR targeting, the physicochemical properties, binding affinity, and PET imaging feasibility of [18F]FPTT for PR-positive breast cancer early diagnosis were evaluated in vivo and in vitro, respectively. 2. Materials and methods 2.1. General methods All reagents and solvents were purchased from commercial suppliers. High performance liquid chromatography (HPLC) analysis was performed on an UltiMate 3000 pump (DIONEX) and an UltiMate 3000 UV absorbance full λ detector (DIONEX) combined with a flowcounter radioactivity detector (Bioscan, USA). Characterization of intermediates and final products of FPTT including 1H NMR and 13C NMR by nuclear magnetic resonance spectrometer (Wuhan Zhongke Niujin Magnetic Resonance Technology Co., Ltd, BIXI-I 400) and mass spectrometry (Waters, Xevo, G2-XS TOF). The human breast carcinoma cell lines (MCF-7, MDA-MB-453, BT474, MDA-MB-468 and MDA-MB-231) 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 °C in a moist atmosphere of 5% CO2 and 95% air. And all animal studies were conducted in accordance with the guidelines of the Xiamen University Animal Care and Use Committee. 2.2. Chemistry 2.2.1. 3,6,9,12,15 Pentaoxaheptadecane 1, 17 diyl bis (4 methylbenzenesulfonate) (2) 3,6,9,12,15 Pentaoxaheptadecane 1, 17 diol (705.5 mg, 2.5 mmol) and triethylamine (Et3N, 607.2 mg, 6.0 mmol) were dissolved with 100 mL anhydrous dichloromethane and stirred at 0 °C. A solution of p toluenesulfonyl chloride (572.0 mg, 6.0 mmol) in anhydrous CH2Cl2 (20 mL) was added dropwise to the mixture. The reaction mixture was stirred at 0 °C for 1 h and warmed to room temperature followed by stirring of 12 h. The mixture was extracted with CH2Cl2 (1 × 150 mL), washed with water (2 × 100 mL) and dried over Na2SO4. The residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 1: 3) to produce compound 2 as a colorless oil (1.2 g, yield 81%). ESI-MS (positive) m/z: 590.1, called for C26H38O11S2: 613.1 [M + Na]+; 1 H NMR (400 MHz, CDCl3) δ = 7.79 (d, J = 8.1 Hz, 4H),7.33 (d, J = 8.1 Hz, 4H), 4.15 (t, J = 4.9 Hz, 4H), 3.68 (t, J = 4.9 Hz, 4H) 3.56 (m,16H), 2.44 (s, 6H); 13C NMR (100 MHz, CDCl3) δ = 144.90, 133.24, 129.96, 128.11, 70.89,70.76, 70.70, 70.65, 69.37, 68.82. 2.2.2. 17 Azido 3,6,9,12,15 pentaoxaheptadecyl 4 methylbenzenesulfonate (3) Sodium azide (81.3 mg, 1.3 mmol) was added to N, N dimethylformamide (DMF, 2 mL) stirring for 3 h, then compound 2 (767.3 mg, 1.3 mmol) was added to the reaction flask which stirred at room temperature overnight. The mixture was extracted with CH2Cl2 (1 × 50 mL), washed with water (3 × 25 mL) and the organic phase
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was dried over Na2SO4. The solvent was evaporated to give a crude product, which was used directly in the next reaction. ESI-MS (positive) m/z: 461.1, called for C19H31N3O8S: 484.1 [M + Na]+. 2.2.3. 17 Testosterone (3,6,9,12,15 pentaoxaheptadecyl) 1H 1,2,3 triazole pentaoxaheptadecyl 4 methylbenzenesulfonate (4, FPTT precursor) Ethisterone (259.3 mg, 0.83 mmol) and compound 3 (384.3 mg, 0.83 mmol) were dissolved in THF (5 mL). Then CuI (157.6 mg, 0.83 mmol), DIPEA (107.3 mg, 0.83 mmol) was added to the solution. The mixture was stirred at 60 °C overnight, then was transferred to a separatory funnel and washed with water (3 × 25 mL) and extracted with ethyl acetate (50 mL). The organic layer was removed under reduced pressure to give a crude product which was purified by column CH2Cl2: methanol = 20: 1 and provided 386.7 mg FPTT precursor (yield 60%). ESI-MS (positive) m/z: 773.3, called for C40H59N3O10S: 796.2 [M + Na] +; 1H NMR (400 MHz, CDCl3) δ = 7.77 (d, J = 8.1 Hz, 2H), 7.58 (s, 1H), 7.32 (d, J = 8.1 Hz, 2H), 5.68 (s, 1H), 4.51 (t, 2H), 4.13 (t, J = 5.0 Hz, 2H), 3.85 (t, J = 4.8 Hz, 2H), 3.66 (t, J = 4.9 Hz, 2H) 3.60–3.56 (m, 16H), 2.42 (s, 3H), 2.37–2.26 (m, 6H),1.95–1.34 (m, 13H), 1.16 (s, 3H), 1.04 (s, 3H); 13C NMR (100 MHz, CDCl3) δ = 199.58, 171.41, 153.57, 144.94, 133.15, 129.96, 123.89, 122.62, 82.10, 70.85,70.70, 70.66, 70.64, 70.63, 70.60, 69.63, 69.37, 68.81, 53.41, 50.35, 48.99, 46.94, 38.71, 36.40, 32.96, 32.79, 31.73, 23.82, 21.76, 20.76, 17.53, 14.39. 2.2.4. 1 (17 Fluoro 3,6,9,12,15 pentaoxaheptadecyl) 1H 1,2,3 triazole testosterone (5, [19F]FPTT) CsF (60.8 mg, 0.4 mmol) and was added in a 20-mL reaction tube under anhydrous conditions and the atmosphere of N2, followed by adding 4 mL THF. Then FPTT precursor (309.4 mg, 0.4 mmol) dissolved with 4 mL THF was added and stirred at 80 °C overnight. The mixture was transferred to a separatory funnel and washed with water (3 × 25 mL) and extract with ethyl acetate (50 mL). The organic layer was removed under reduced pressure to give a crude product which was purified by column chromatography CH2Cl2: methanol = 20: 1 and provided 50 mg [ 19F]FPTT (yield 21%). ESI-MS (positive) m/z: 621.3, called for C33H52FN3O7: 644.1 [M + Na] +; 1H NMR (400 MHz, CDCl3) δ = 7.58 (s, 1H), 5.69 (s, 1H), 4.61 (t, J = 4.2 Hz, 1H), 4.52 (t, J = 4.9 Hz, 2H), 4.49 (t, J = 4.2 Hz, 1H), 3.86 (t, J = 4.7 Hz, 2H), 3.76 (t, J = 4.6 Hz, 2H) 3.70–3.56 (m, 16H), 2.41–2.24 (m, 6H), 1.77–1.41 (m, 13H), 1.39 (s, 3H), 1.38 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ = 199.62, 171.42, 153.60, 123.93, 122.65, 84.12, 82.43, 82.13, 70.93, 70.74, 70.70, 70.67, 70.63, 70.43, 69.67, 53.44, 50.40, 49.04, 46.97, 38.73, 37.88, 36.43, 35.78, 34.05, 32.97, 32.82, 31.76, 23.84, 20.78, 17.55, 14.40; 19F NMR (376 MHz, CDCl3) δ = −220.30. 2.3. Radiochemistry [ 18F]fluoride solution unloaded from a cyclotron target was diluted and trapped on a QMA cartridge (Waters light Sep-Pak) which was activated with 5 mL of 0.5 M K2CO3 and 10 mL of H2O. Then eluted with 0.1 mL K2CO3 solution (1 mg/mL in H2O) combined with 0.9 mL Kyptofix2.2.2 solution (8 mg/mL in acetonitrile) into a reaction vial. The radioactive solution (~1.8 GBq) was dried by azeotropic distillation with acetonitrile (3 × 1 mL). Then the FPTT precursor dissolved with 200 μL anhydrous acetonitrile was added into the previous reaction vial. The mixture was heated at 90 °C for 20 min in the sealed vial. After the reaction vial was cooled to room temperature, the product (~1.4 GBq) was purified and analyzed by HPLC. The reversed-phase C18 column (4.6 × 250 mm, 5 μm, 120 Å, Thermo) was eluted with H2O (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 mL/ min and using the following gradient: 0–20 min: 35%B, 20–30 min: 35%–80% B, under ultraviolet (254 nm) and radiometric monitoring. The radioactive peak (approximately TR = 13.5–14.5 min) was collected and concentrated under a gentle stream of N2, redissolved in ethanol,
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diluted with saline, and then passed through a 0.22-μm filter (Millipore Co., Billerica, MA, USA) to give a final solution (~0.7 GBq) of 10% ethanol in saline for use. 2.4. Physicochemical properties studies The octanol-to-water partition coefficient of [ 18F]FPTT was measured according to a procedure published previously [23]. Briefly, 100 μL of [ 18F]FPTT (0.74 MBq) was mixed with 0.9 mL of phosphatebuffered saline (PBS, pH 7.4) and an equal volume of 1 octanol (1 mL). The mixture was stirred in a vortex mixer for 2 min, and the tube was centrifuged at 10,000 rpm for 5 min to partition the layers. Samples in triplicate from 1 octanol and aqueous layers were obtained and counted on a γ-counter (Wizard 2480, Perkin Elmer, USA). In vitro stability test was also performed according to the literature [24]. Briefly, the radiotracer was incubated in saline at room temperature or in rat serum at 37 °C for 2 h. After centrifugation with equal volume acetonitrile, the supernatant was obtained and then filtered through a 0.22-μm filter before subject to HPLC analysis. Metabolic stability of [18F]FPTT was measured according to the previously published procedure [25]. Briefly, BALB/c mice (female, ~20 g) were intravenously injected with 37 MBq of [ 18F]FPTT. At 1 h postinjection (p.i.), mice were sacrificed and the tissues of blood, urine and liver were collected to extract the radioactivity for HPLC analysis. All the samples were passed through a 0.22-μm filter before subject to HPLC. 2.5. Validation of cellular PR receptor expression The cells were seeded in 6-well tissue culture plates at a density of 1 × 10 6 cells/well. After cell attachment, cells were lysed with RIPA Lysis (Thermo Scientific) on ice, and total protein concentration was quantified using a BCA Protein Assay Kit (Pierce). Lysates were collected and boiled for 10 min, Equal amounts of total protein (20 μg) were loaded onto Bis-Tris gels for SDS-PAGE Western Blot electrophoresis and transferred to a polyvinylidene difluoride membrane. After transfer, membranes were blocked for 1 h at room temperature with 5% BSA in Tris-buffered saline (Sigma Chemical Co.), 0.1% Tween 20, then incubated overnight at 4 °C with antibodies specific for PR (ab131486, abcam), β-actin (12262S, Cell Signaling Technology). The membranes were incubated with antirabbit HRPconjugated secondary antibodies (A27036, Thermo Scientific) for 1 h at room temperature. Proteins were visualized on a Bio-Rad ChemiDoc XRS System.
with 1 mL of NaOH (1 mol/L), then extract was collected and analyzed using a γ-counter. The internalization was studied according to the literature [27]. MCF-7 cells (approximately 2 × 10 5 cells/well) were incubated with [ 18F]FPTT at 4 °C for 2 h. After pre-incubation, cells were washed three times with ice-cold binding buffer then incubated with 1 mL of warmed binding buffer for different time (5, 15, 30, 60, 120 min) at 37 °C for internalization. The cells were subsequently exposed to 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 being washed with 1 mL of PBS, whole cells were lysed using NaOH (1 mol/L) and collected, then counted using a γ-counter. 2.8. Animal tumor models and PET imaging The experimental procedures with animals were approved by the Xiamen University Animal Care and Use Committee. The model mice with xenograft tumor of in situ breast cancer were established according to the literature [28]. The estrogen pellets were implanted subcutaneously in the neck of BALB/c nude mice 3 d before the inoculation of tumor cells, and then the mixture of PR-positive MCF-7 cells (5 × 106 cells) and matrix gel was inoculated into the mammary fat pad. The sustained-release pellets were taken out 3 d before imaging. PRnegative MDA-MB-231 cells (1 × 106 cells) were subcutaneously inoculated into the right shoulder of nude mice for control study. After the tumor diameter reached 0.5–1.0 cm, the tumor-bearing mice were injected with [ 18F]FPTT (3.7 MBq/100 μL) via tail vein for PET scanning (Siemens Medical Solutions USA, Inc.). The blocking studies were performed on MCF-7 tumor-bearing mice by co-injecting ethisterone (100 μL, 1 mg/mL) or [ 19F]FPTT (100 μL, 1 mg/mL) at 30 min prior to the [ 18F]FPTT injection. The mice were placed in a prone position and collected for 5-min static PET images which were acquired at 0.5, 1, and 2 h p.i. under general anesthesia with 2% isoflurane, followed by scanning at low-dose CT. All PET data were reconstructed by three-dimensional ordered-subset expectation maximization (16 subsets, 2 iterations), with attenuation and scatter correction. The Inveon Research Workplace (IRW) 2.0 software was used to reconstruct and acquire PET image data, and the regions of interest (ROIs) and the average count were calculated by attenuation correction. ROI of muscle was drawn over the contralateral upper limb of tumor for reference. Assuming a tissue density of 1 g/mL, the ROIs were converted to an imaging derived percentage administered activity per gram of tissue (%ID/g) by IRW software. 2.9. Biodistribution
2.6. Cell binding assay Saturation binding study was performed in MCF-7 cells as described previously [26] to determine the binding affinity of [ 18F]FPTT for PR. Briefly, MCF-7 cells (approximately 2 × 105 cells/well) were incubated on a 24-well plate with [ 18F]FPTT, ranging from 1 nmol/L to 200 nmol/ L. To determine the non-specific uptake, excessive ethisterone (10 μL per well, 1 mg/mL) was added additionally to the cells prior to the incubation with radiotracer on another 24-well plate. Followed by incubation at 37 °C 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 mol/L). Radioactivity was measured by using a γ-counter. Bmax and Kd (nmol/L) values were calculated by GraphPad Prism 5.0 software.
The estrogen-primed immature female SD rats (3–4 weeks) were prepared by subcutaneous injection of estradiol dissolved in 20% ethanol/sunflower oil (5 μg/100 μL) on two successive days [17]. The [ 18 F]FPTT (1.85 MBq/200 μL) was injected via the tail vein of rats (n = 4 each group). After 0.5, 1 and 2 h injection, the rats were sacrificed and then the tissues of interest were dissected, weighed and counted using a γ-counter. The biodistribution results were calculated by normalizing the amount of injected radioactivity and expressed as percentage of injected dose per gram (%ID/g). Biodistributions in normal immature female SD rats with or without blocking with ethisterone (250 μL, 1 mg/mL) were studied at 1 h p.i. respectively for comparison.
2.7. Cell uptake and internalization
3. Results
For the determination of specific cellular uptake, MCF-7 cells and MDA-MB-231 cells (approximately 2 × 105 cells/well) were incubated with [ 18F]FPTT at 37 °C for 0.5, 1 and 2 h. Specific cellular uptake was determined by blocking with ethisterone (10 μL per well, 1 mg/mL). After that, the cells were washed twice with 1 mL of PBS (pH = 7.4) and lysed
3.1. Chemistry and radiochemistry Synthetic routes for nonradioactive compound [19F]FPTT and radiocompound [ 18F]FPTT are shown in Fig. 1. All the intermediates and final nonradioactive compound were analyzed by 1H NMR, 13C NMR and
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Fig. 1. Synthesis routes of non-radioactive compound [19F]FPTT and radiotracer [18F]FPTT.
mass spectrometry (Figs. S1–S7 in the Supplementary File). The radio-HPLC analysis results (Fig. S8) revealed that the retention time of [ 18F]FPTT was 14.15 min, which was consistent with that of nonradioactive reference compound [ 19 F]FPTT (T R = 13.73 min). Starting from the azeotropic of [ 18 F]fluoride (~1.8 GBq), the final injection (~0.7 GBq) was obtained within 60 min including HPLC purification, and the decay-corrected radiochemical yield at end of synthesis was about 78 ± 9% (HPLC analysis) and radiochemical purity over 98% after HPLC purification.
The specific activity was calculated as 17 GBq/μmol according to the standard curve (Fig. S9), and the log P value was 0.22 ± 0.01, indicating moderate lipophilicity. 3.2. Stability studies The good in vitro stability of [18F]FPTT was demonstrated by the high radiochemical purities after incubated in saline and rat serum (Fig. S10A and B). The results of metabolic stability showed that about 50% of the
Fig. 2. (A) The expression of PR protein in different cell lines have been evaluated by Western Blot. (B) The saturation binding assay of [18F]FPTT using MCF-7 cells. (C) Cell uptakes of [18F] FPTT in MCF-7 and MDA-MB-231 cells were measured at the indicated time points. The cell uptakes were blocked by the presence of ethisterone (250 μL, 1 mg/mL) at 0.5, 1, and 2 h time points. (D) Cell internalization of [18F]FPTT in MCF-7 cells was measured at the indicated time points. All data were determined from three independent experiments and expressed as mean ± standard deviation.
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intact compound of [ 18F]FPTT was found in the blood at 1 h p.i., while almost all of the radiotracer was metabolized into small molecules with higher polarity in urine and liver respectively from the HPLC analysis results (Fig. S10C–E).
added amount) was 6.33 ± 0.98% at 15 min, and decreased to 4.19 ± 0.02% at 30 min and then reach to a plateau (Fig. 2D), demonstrating ability for nuclear receptor targeting. 3.5. MicroPET imaging studies
3.3. Cellular PR receptor expression The expression of PR protein in different cell lines have been evaluated by Western Blot and the results were shown in Fig. 2A. The cells of BT-474 and MCF-7 have the highest PR expression among these cell lines while PR was negatively expressed in MDA-MB-231 cells. Accordingly, PR-positive MCF-7 cells and negative MDA-MB-231 cells were selected for comparison in this study. 3.4. Cell binding and internalization Saturation binding analysis can determine receptor affinity and density. The cell binding result of [ 18F]FPTT in MCF-7 cells was shown in a Scatchard plot (Fig. 2B). The dissociation constant (Kd) of [ 18F]FPTT was calculated as 29.54 ± 5.27 nM (n = 3), with a maxima number of binding sites (Bmax) of 1.39 × 10 5 cpm/10 5 cells, which indicating nanomolar binding affinity of [18F]FPTT toward PR. [ 18F]FPTT was incubated with MCF-7 and MDA-MB-231 cells respectively at different time, showing significant uptake differences (Fig. 2C). After 30 min incubation, the maximum uptake of [ 18F]FPTT in PRpositive MCF-7 cells was obtained as 9.76 ± 0.64%, which significantly higher than that of PR-negative MDA-MB-231 cells (2.09 ± 0.42%). Exposure of MCF-7 cells and MDA-MB-231 cells to ethisterone (250 μL, 1 mg/mL) resulted in significant decreased cell uptake (2.18 ± 0.30% and 1.67 ± 0.94%) indicating specificity of [18F]FPTT to PR. The cell internalization of [18F]FPTT (the percentage of internalized tracer to the total
The microPET images of tumor-bearing mice were shown in Fig. 3. From the images of PR-positive MCF-7 tumor-bearing mice (Fig. 3A), the tumor could be seen clearly at 0.5 h p.i. and the tumor uptakes were quantified as 3.40 ± 0.17, 3.90 ± 0.20 and 2.39 ± 0.41%ID/g at 0.5 h, 1 h and 2 h p.i. respectively, with the highest uptake at 1 h p.i. The tumor uptakes at earlier time points are two-fold over the background muscle uptakes indicating good image contrast. For the PRnegative MDA-MB-231 tumor-bearing mice (Fig. 3B), the tumor uptakes were 1.4 ± 0.11, 1.3 ± 0.11 and 1.3 ± 0.24%ID/g at 0.5 h, 1 h and 2 h p.i. respectively, significantly lower than that of MCF-7 tumor (P˂ 0.05). PR-specific tumor uptake of [ 18F]FPTT was demonstrated by blocking studies (Fig. 4). After co-injection of ethisterone, MCF-7 tumor uptake of [ 18F]FPTT was inhibited significantly (P ˂ 0.001), indicating it had the same binding target as ethisterone. In addition, tumor uptake of [ 18F]FPTT could also be blocked by excessive nonradioactive [ 19F]FPTT compound, revealing that the structural modification of probe had little effect on the activity of ethisterone. The above blocking studies results indicated that the MCF-7 tumor uptake of [18F]FPTT might be mediated by PR receptor specifically. 3.6. Biodistribution studies Biodistribution results of [ 18F]FPTT were shown in Fig. 5. At 1 h p.i., the uptakes in uterus and ovary of female SD rats that had been estrogen
Fig. 3. Representative PET images of [18F]FPTT in PR-positive MCF-7 (A) and PR-negative MDA-MB-231 (B) tumors in tumor-bearing mice were obtained at 0.5, 1 and 2 h p.i. Quantitative tumor uptakes (C) and tumor-to-muscle ratios (D) derived from A and B. (n = 3, red dotted lines denote tumors, ***P b 0.001).
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Fig. 4. (A) Representative PET images obtained at 1 h p.i. of [18F]FPTT in PR-positive MCF-7 tumor-bearing mice, and with excessive of ethisterone or nonradioactive compound [19F]FPTT for blocking. Quantitative tumor uptakes (B) and tumor-to-muscle ratios (C) derived from A. (n = 3, red dotted lines denote tumors, ***P b 0.001).
Fig. 5. (A) Biodistribution of [18F]FPTT in estrogen-primed immature female SD rats at different time points, and in normal immature female SD rats with or without ethisterone blocking at 1 h p.i. The ratios of uterus-to-muscle (B) and ovary-to-muscle (C) of [18F]FPTT derived from A. (n = 4, ***P b 0.001; Expressed as mean ± standard deviation).
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pretreated to induce PR expression were 8.31 ± 1.74%ID/g and 3.79 ± 0.82%ID/g, respectively. While the uptakes in uterus and ovary of normal rats (3.52 ± 0.29%ID/g and 3.22 ± 0.50%ID/g) were lower than that of estrogen-primed immature female SD rats (P b 0.05), and could be inhibited significantly by ethisterone (2.11 ± 0.18%ID/g and 1.77 ± 0.41%ID/g), indicating the specific uptakes of [ 18F]FPTT in PRrich uterus and ovary. [ 18F]FPTT had low uptakes in the heart, spleen, lung and other non-target tissues and organs, which can be a benefit to improve imaging contrast. In addition, [ 18F]FPTT showed relatively low bone accumulation at different time points, revealing no defluorination occurred in vivo. 4. Discussion Breast cancer PR expression is regulated by estrogen receptor (ER) and could be used for tumor diagnosis and to guide the antiestrogen treatment in breast cancer [29]. During the past a few decades, a variety of PR targeting radiotracers have been reported although the clinical translational study progress is limited when compared with ER imaging. 18 F-labeled steroidal progestins of [ 18F]FENP and [ 18F]FMNP were reported by Martin et al. [15] and Verhagen et al. [14], respectively. Both radiotracers had specific uptakes in the uterus and ovaries of estrogen-induced SD rats, while the first one was found had no correlated with PR levels in human breast cancer and had high accumulation in bone [30]. Recently, the first-in-human study of [ 18F]FFNP was reported by Dehdashti et al. [16], which is the most successful PR targeting PET tracer so far [29]. In 2010, a nonsteroidal PR targeting probe [ 18F] FPTP was reported by Lee et al. [17], it had high target tissue specific uptake with prolonged retention. However, as a chiral compound, the biological effects of its enantiomers remain to be studied. In recent years, our research group had focused on the development of radiotracers for breast cancer imaging [18,19,25,26,31], including two radiolabeled ethisterone derivatives [ 18F]EAEF [19] and [131I]EIPBA [18] for PR targeting. Although both of them exhibited high receptor binding affinity and PR-positive tumor uptake, however, the unfavorable liver uptake limits their potential application. In this study, a small PEG was coupled to ethinyl group of ethisterone to reduce the lipophilicity of tracer and expected to lower the liver uptake. After radiolabeled with 18 F by one-step nucleophilic substitution reaction, the [ 18F]FPTT was prepared in high radiochemical yield with good radiochemical purity. Its octanol-to-water partition coefficient (log P) was determined as 0.22 ± 0.01, which much lower than that of [ 18F]EAEF (0.53 ± 0.06) and [ 131I]EIPBA (1.38 ± 0.48), indicating moderate lipophilicity. The nanomolar binding affinity of [ 18F]FPTT to PR (Kd = 29.54 ± 5.27 nM) was determined by cell binding assay. Of course, compared with reported [ 18F]FMNP, [ 18F]FENP and [ 18F]FFNP, the Kd of [ 18F]FPTT showed relatively moderate affinity, but slightly better hydrophilicity. A more systematic, detailed and multifaceted comparison with the reported classical probes will be studied in our next study. In the cell uptake studies, [ 18F]FPTT showed higher uptake in PRpositive MCF-7 cells than in PR-negative MDA-MB-231 cells, and could be specifically inhibited. Especially, the uptake of [ 18F]FPTT in PRnegative MDA-MB-231 cells with or without PR inhibitor was not significantly different. In microPET imaging, PR-positive MCF-7 tumor could be imaged at early time point with good contrast which the highest uptake was reached at 1 h p.i. (3.90 ± 0.20%ID/g). Moreover, the specificity of [ 18F]FPTT was demonstrated by blocking and PR-negative MDA-MB231 tumor control studies, respectively. As expected, the reduced liver uptake of [ 18F]FPTT was obtained leading to a higher target to nontarget ratio. From the biodistribution study, the uterus and ovary uptakes of [18F] FPTT in estrogen-primed immature female SD rats are comparable to that of previously reported [ 18F]FPTP, [ 18F]FENP and [ 18F]FFNP. When compared with the above-mentioned tracers, much lower liver, lung, spleen and other non-specific organs uptakes, while higher muscle
and bone uptakes of [ 18F]FPTT were observed. Of course, [ 18F]FPTT also accumulated in the intestine due to its steroid metabolic pathway. The targeting ability of [ 18F]FPTT to PR had been verified through a series of in vivo and in vitro bio-evaluation and it will be further investigated to determine the safety and dose for PR-positive breast cancer imaging. 5. Conclusion A novel 18F-labeled ethisterone triazole derivative was designed and successfully synthesized by a one-step nucleophilic substitution reaction. In vitro studies showed that [ 18F]FPTT had moderate lipophilicity with nanomolar binding affinity to PR. Ex vivo biodistribution in estrogen-primed immature female SD rats resulted in high PR-rich tissues uptake. In vivo microPET imaging studies indicated that [ 18F]FPTT had specific uptake in PR-positive MCF-7 tumor with low uptakes in non-target tissues of tumor-bearing mice. The above results showed that [18F]FPTT could be a potent PET tracer for PR targeted breast cancer diagnosis and assess the efficacy of antiestrogen treatment. Acknowledgments This study was financially supported by the National Basic Research Program of China (2014CB744503) and Natural Science Foundation of Fujian Province, China (No. 2016J05200). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.nucmedbio.2019.07.001. References [1] Witten J, Hybert F, Hansen HS. Treatment of malignant tumors in the parotid glands. Cancer 2015;65:2515–20. [2] Chen W, Sun K, Zheng R, Zeng H, Zhang S, Xia C, et al. Cancer incidence and mortality in China, 2014. Chin J Cancer Res 2018;30:1–12. [3] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424. [4] Ulaner GA, Riedl CC, Dickler MN, Jhaveri K, Pandit-Taskar N, Weber W. Molecular imaging of biomarkers in breast cancer. J Nucl Med 2016;57(Suppl. 1):53S. [5] Normanno N, Di MME, De LA, De MA, Giordano A, Perrone F. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 2016;12:721–47. [6] Hoffman JM, Gambhir SS. Molecular imaging: the vision and opportunity for radiology in the future. Radiology 2015;244:39–47. [7] Massoud TF, Gambhir SS. Integrating noninvasive molecular imaging into molecular medicine: an evolving paradigm. Trends Mol Med 2007;13:183–91. [8] Pysz MA, Gambhir SS, Willmann JK, J.K. W. Molecular imaging: current status and emerging strategies. Clin Radiol 2010;65:500–16. [9] Meng Q, Li Z. Molecular imaging probes for diagnosis and therapy evaluation of breast cancer. Int J Biomed Imaging 2013;2013:1–14. [10] Oliveira MC, Neto C, Morais G, Ribeiro, Thiemann T. Steroid receptor ligands for breast cancer targeting: an insight into their potential role as PET imaging agents. Curr Med Chem 2013;20:222–45. [11] Wu Q, Li J, Zhu S, Wu J, Chen C, Liu Q, et al. Breast cancer subtypes predict the preferential site of distant metastases: a SEER based study. Oncotarget 2017;8:27990–6. [12] Diep CH, Daniel AR, Mauro LJ, Knutson TP, Lange CA. Progesterone action in breast, uterine, and ovarian cancers. J Mol Endocrinol 2015;54:R31–53. [13] Cui X, Schiff R, Arpino G, Osborne C, Adrian V. Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy. J Clin Oncol 2005;23: 7721–35. [14] Verhagen A, Luurtsema G, Pesser J, De Groot TJ, Wouda S, Oosterhuis JW, et al. Preclinical evaluation of a positron emitting progestin ([ 18F]fluoro 16 alpha methyl 19 norprogesterone) for imaging progesterone receptor positive tumours with positron emission tomography. Cancer Lett 1991;59:125–32. [15] Pomper MG, Katzenellenbogen JA, Welch MJ, Brodack JW, Mathias CJ. 21 [18F] Fluoro 16α ethyl 19 norprogesteron synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography. J Med Chem 1988;31:1360–3. [16] Dehdashti F, Laforest R, Gao F, Aft RL, Dence CS, Zhou D, et al. Assessment of progesterone receptors in breast carcinoma by PET with 21 18F fluoro 16α,17α [(R) (1′ α furylmethylidene) dioxy] 19 norpregn 4 ene 3,20 dione. J Nucl Med 2012;53:363.
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18
F-labeled ethisterone derivative for progesterone receptor targeted PET imaging of breast cancer Fei Gao, Chenyu Peng, Rongqiang Zhuang ⁎, Zhide Guo, Huanhuan Liu, Lumei Huang, Hua Li, Duo Xu, Xuejun Wen, Jianyang Fang, Xianzhong Zhang ⁎ State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
a r t i c l e
i n f o
Article history: Received 1 May 2019 Received in revised form 11 June 2019 Accepted 6 July 2019 Keywords: Breast cancer Progesterone receptor MicroPET imaging Ethisterone
a b s t r a c t Purpose: A novel radiolabeled probe 1 (17 [18F]fluoro 3,6,9,12,15 pentaoxaheptadecyl 1H 1,2,3 triazole testosterone ([18F]FPTT) was synthesized and evaluated for PET imaging of progesterone receptor (PR)-positive breast cancer. Methods: The ethinyl group of ethisterone, a PR targeting pharmacophore, was coupled with azide modified PEGOTs by click chemistry to obtain the labeling precursor. The final [18F]FPTT was synthesized by a one-step nucleophilic substitution reaction with 18F. The in vitro stabilities of [18F]FPTT in saline or rat serum were determined after 2 h incubation. Then the in vitro cell binding, ex vivo biodistribution and in vivo imaging of [18F]FPTT were further investigated to evaluate the PR targeting ability and feasibility for the diagnosis of PR-positive breast cancer with PET imaging. Results: [18F]FPTT was obtained in high decay-corrected radiochemical yield (78 ± 9%) at the end of synthesis. It had high radiochemical purity (N98%) after HPLC purification and good in vitro stability. The molar activity of [18F] FPTT was calculated as 17 GBq/μmol. The microPET imaging of [18F]FPTT in tumor-bearing mice showed much higher tumor uptake in PR-positive MCF-7 tumor (3.9 ± 0.20%ID/g) than that of PR-negative MDA-MB-231 tumor (1.3 ± 0.08%ID/g). The high MCF-7 tumor uptake could be specifically inhibited by blocking with ethisterone (1.3 ± 0.11%ID/g) or [19F]FPTT (2.20 ± 0.17%ID/g), respectively. The biodistribution in estrogenprimed female SD rats of [ 18F]FPTT showed high uterus and ovary uptakes (8.31 ± 1.74%ID/g and 3.79 ± 0.82%ID/g at 1 h post-injection). The specific uptakes of uterus and ovary in normal rats were 3.52 ± 0.29%ID/ g and 3.22 ± 0.50%ID/g respectively and could be inhibited by co-injecting of ethisterone. Conclusion: A novel [18F]FPTT probe based on ethisterone modification could be a potential diagnostic agent for PR-positive breast cancer. © 2019 Elsevier Inc. All rights reserved.
1. Introduction A large number of clinical trials have proved that the key to the prognosis of malignant tumors lies in the early detection, diagnosis and treatment of diseases [1,2]. In particular, the early diagnosis of breast cancer will help to alleviate the pain of patients and improve the prognosis of cancer [3]. Although immunohistochemical methods are currently used to assess the hormone receptor status of metastatic tumors, there are many limitations in determining the hormone receptor status of metastatic tumors [4,5]. For example, the location of the examination is not suitable for biopsy, sample processing (especially decalcification) will also produce false negative results, as well as intratumoral and tumor stroma heterogeneity. Molecular imaging has ⁎ Corresponding authors at: School of Public Health, Xiamen University, 4221-116 Xiang'an South Rd., Xiang'an District, Xiamen 361102, China. E-mail addresses: [email protected] (R. Zhuang), [email protected] (X. Zhang). https://doi.org/10.1016/j.nucmedbio.2019.07.001 0969-8051/© 2019 Elsevier Inc. All rights reserved.
been widely used to improve the specificity and quantification of screening and early diagnosis, centralized and personalized treatment, and follow-up of early treatment [6–8]. Accurate early detection of tumors requires efficient and safe molecular imaging probes [9]. According to the expression characteristics of breast cancer receptors, targeted endocrine therapy has become an attractive therapeutic option in the diagnosis and treatment of breast cancer [10,11]. Progesterone receptor (PR) is closely related to the invasiveness, prognosis and efficacy of hormone-related diseases such as breast cancer, ovarian cancer, uterine cancer and endometriosis [12,13]. In recent years, many radiolabeled PR targeted probes for PET imaging have been reported and evaluated ([ 18F]FMNP, [ 18F]FENP, [ 18F]FPTP, [ 18F]FFNP, [ 18F]EAEF, et al.) [14–19]. Most of them use modified steroid structures as probes targeting PR, such as testosterone, progesterone and 19-nortestosterone, and different modifications of these structures. This is due to the fact that most receptors have a high affinity for their cognate ligands. Ethisterone is a natural progesterone
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derivative, which has a strong affinity for PR, and its alkynyl group can be easily modified compared with other steroids [19]. Of course, besides, ethisterone is also very popular modification group on other imaging agents targeting PR [20]. 18F (t1/2 = 109.8 min) is a suitable radionuclide for PET imaging due to its advantages of high positron decay (97%), low positron energy (0.634 MeV) and smaller atomic radius. In addition, the inherent advantages of PET imaging which has high sensitivity, good spatial resolution and quantitative and dynamic monitoring has become a non-invasive method for breast cancer diagnosis [21,22]. In this study, radionuclide 18F was incorporated into ethisterone derivative to develop a novel radiotracer ([ 18F]FPTT) for PR targeting, the physicochemical properties, binding affinity, and PET imaging feasibility of [18F]FPTT for PR-positive breast cancer early diagnosis were evaluated in vivo and in vitro, respectively. 2. Materials and methods 2.1. General methods All reagents and solvents were purchased from commercial suppliers. High performance liquid chromatography (HPLC) analysis was performed on an UltiMate 3000 pump (DIONEX) and an UltiMate 3000 UV absorbance full λ detector (DIONEX) combined with a flowcounter radioactivity detector (Bioscan, USA). Characterization of intermediates and final products of FPTT including 1H NMR and 13C NMR by nuclear magnetic resonance spectrometer (Wuhan Zhongke Niujin Magnetic Resonance Technology Co., Ltd, BIXI-I 400) and mass spectrometry (Waters, Xevo, G2-XS TOF). The human breast carcinoma cell lines (MCF-7, MDA-MB-453, BT474, MDA-MB-468 and MDA-MB-231) 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 °C in a moist atmosphere of 5% CO2 and 95% air. And all animal studies were conducted in accordance with the guidelines of the Xiamen University Animal Care and Use Committee. 2.2. Chemistry 2.2.1. 3,6,9,12,15 Pentaoxaheptadecane 1, 17 diyl bis (4 methylbenzenesulfonate) (2) 3,6,9,12,15 Pentaoxaheptadecane 1, 17 diol (705.5 mg, 2.5 mmol) and triethylamine (Et3N, 607.2 mg, 6.0 mmol) were dissolved with 100 mL anhydrous dichloromethane and stirred at 0 °C. A solution of p toluenesulfonyl chloride (572.0 mg, 6.0 mmol) in anhydrous CH2Cl2 (20 mL) was added dropwise to the mixture. The reaction mixture was stirred at 0 °C for 1 h and warmed to room temperature followed by stirring of 12 h. The mixture was extracted with CH2Cl2 (1 × 150 mL), washed with water (2 × 100 mL) and dried over Na2SO4. The residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 1: 3) to produce compound 2 as a colorless oil (1.2 g, yield 81%). ESI-MS (positive) m/z: 590.1, called for C26H38O11S2: 613.1 [M + Na]+; 1 H NMR (400 MHz, CDCl3) δ = 7.79 (d, J = 8.1 Hz, 4H),7.33 (d, J = 8.1 Hz, 4H), 4.15 (t, J = 4.9 Hz, 4H), 3.68 (t, J = 4.9 Hz, 4H) 3.56 (m,16H), 2.44 (s, 6H); 13C NMR (100 MHz, CDCl3) δ = 144.90, 133.24, 129.96, 128.11, 70.89,70.76, 70.70, 70.65, 69.37, 68.82. 2.2.2. 17 Azido 3,6,9,12,15 pentaoxaheptadecyl 4 methylbenzenesulfonate (3) Sodium azide (81.3 mg, 1.3 mmol) was added to N, N dimethylformamide (DMF, 2 mL) stirring for 3 h, then compound 2 (767.3 mg, 1.3 mmol) was added to the reaction flask which stirred at room temperature overnight. The mixture was extracted with CH2Cl2 (1 × 50 mL), washed with water (3 × 25 mL) and the organic phase
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was dried over Na2SO4. The solvent was evaporated to give a crude product, which was used directly in the next reaction. ESI-MS (positive) m/z: 461.1, called for C19H31N3O8S: 484.1 [M + Na]+. 2.2.3. 17 Testosterone (3,6,9,12,15 pentaoxaheptadecyl) 1H 1,2,3 triazole pentaoxaheptadecyl 4 methylbenzenesulfonate (4, FPTT precursor) Ethisterone (259.3 mg, 0.83 mmol) and compound 3 (384.3 mg, 0.83 mmol) were dissolved in THF (5 mL). Then CuI (157.6 mg, 0.83 mmol), DIPEA (107.3 mg, 0.83 mmol) was added to the solution. The mixture was stirred at 60 °C overnight, then was transferred to a separatory funnel and washed with water (3 × 25 mL) and extracted with ethyl acetate (50 mL). The organic layer was removed under reduced pressure to give a crude product which was purified by column CH2Cl2: methanol = 20: 1 and provided 386.7 mg FPTT precursor (yield 60%). ESI-MS (positive) m/z: 773.3, called for C40H59N3O10S: 796.2 [M + Na] +; 1H NMR (400 MHz, CDCl3) δ = 7.77 (d, J = 8.1 Hz, 2H), 7.58 (s, 1H), 7.32 (d, J = 8.1 Hz, 2H), 5.68 (s, 1H), 4.51 (t, 2H), 4.13 (t, J = 5.0 Hz, 2H), 3.85 (t, J = 4.8 Hz, 2H), 3.66 (t, J = 4.9 Hz, 2H) 3.60–3.56 (m, 16H), 2.42 (s, 3H), 2.37–2.26 (m, 6H),1.95–1.34 (m, 13H), 1.16 (s, 3H), 1.04 (s, 3H); 13C NMR (100 MHz, CDCl3) δ = 199.58, 171.41, 153.57, 144.94, 133.15, 129.96, 123.89, 122.62, 82.10, 70.85,70.70, 70.66, 70.64, 70.63, 70.60, 69.63, 69.37, 68.81, 53.41, 50.35, 48.99, 46.94, 38.71, 36.40, 32.96, 32.79, 31.73, 23.82, 21.76, 20.76, 17.53, 14.39. 2.2.4. 1 (17 Fluoro 3,6,9,12,15 pentaoxaheptadecyl) 1H 1,2,3 triazole testosterone (5, [19F]FPTT) CsF (60.8 mg, 0.4 mmol) and was added in a 20-mL reaction tube under anhydrous conditions and the atmosphere of N2, followed by adding 4 mL THF. Then FPTT precursor (309.4 mg, 0.4 mmol) dissolved with 4 mL THF was added and stirred at 80 °C overnight. The mixture was transferred to a separatory funnel and washed with water (3 × 25 mL) and extract with ethyl acetate (50 mL). The organic layer was removed under reduced pressure to give a crude product which was purified by column chromatography CH2Cl2: methanol = 20: 1 and provided 50 mg [ 19F]FPTT (yield 21%). ESI-MS (positive) m/z: 621.3, called for C33H52FN3O7: 644.1 [M + Na] +; 1H NMR (400 MHz, CDCl3) δ = 7.58 (s, 1H), 5.69 (s, 1H), 4.61 (t, J = 4.2 Hz, 1H), 4.52 (t, J = 4.9 Hz, 2H), 4.49 (t, J = 4.2 Hz, 1H), 3.86 (t, J = 4.7 Hz, 2H), 3.76 (t, J = 4.6 Hz, 2H) 3.70–3.56 (m, 16H), 2.41–2.24 (m, 6H), 1.77–1.41 (m, 13H), 1.39 (s, 3H), 1.38 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ = 199.62, 171.42, 153.60, 123.93, 122.65, 84.12, 82.43, 82.13, 70.93, 70.74, 70.70, 70.67, 70.63, 70.43, 69.67, 53.44, 50.40, 49.04, 46.97, 38.73, 37.88, 36.43, 35.78, 34.05, 32.97, 32.82, 31.76, 23.84, 20.78, 17.55, 14.40; 19F NMR (376 MHz, CDCl3) δ = −220.30. 2.3. Radiochemistry [ 18F]fluoride solution unloaded from a cyclotron target was diluted and trapped on a QMA cartridge (Waters light Sep-Pak) which was activated with 5 mL of 0.5 M K2CO3 and 10 mL of H2O. Then eluted with 0.1 mL K2CO3 solution (1 mg/mL in H2O) combined with 0.9 mL Kyptofix2.2.2 solution (8 mg/mL in acetonitrile) into a reaction vial. The radioactive solution (~1.8 GBq) was dried by azeotropic distillation with acetonitrile (3 × 1 mL). Then the FPTT precursor dissolved with 200 μL anhydrous acetonitrile was added into the previous reaction vial. The mixture was heated at 90 °C for 20 min in the sealed vial. After the reaction vial was cooled to room temperature, the product (~1.4 GBq) was purified and analyzed by HPLC. The reversed-phase C18 column (4.6 × 250 mm, 5 μm, 120 Å, Thermo) was eluted with H2O (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 mL/ min and using the following gradient: 0–20 min: 35%B, 20–30 min: 35%–80% B, under ultraviolet (254 nm) and radiometric monitoring. The radioactive peak (approximately TR = 13.5–14.5 min) was collected and concentrated under a gentle stream of N2, redissolved in ethanol,
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diluted with saline, and then passed through a 0.22-μm filter (Millipore Co., Billerica, MA, USA) to give a final solution (~0.7 GBq) of 10% ethanol in saline for use. 2.4. Physicochemical properties studies The octanol-to-water partition coefficient of [ 18F]FPTT was measured according to a procedure published previously [23]. Briefly, 100 μL of [ 18F]FPTT (0.74 MBq) was mixed with 0.9 mL of phosphatebuffered saline (PBS, pH 7.4) and an equal volume of 1 octanol (1 mL). The mixture was stirred in a vortex mixer for 2 min, and the tube was centrifuged at 10,000 rpm for 5 min to partition the layers. Samples in triplicate from 1 octanol and aqueous layers were obtained and counted on a γ-counter (Wizard 2480, Perkin Elmer, USA). In vitro stability test was also performed according to the literature [24]. Briefly, the radiotracer was incubated in saline at room temperature or in rat serum at 37 °C for 2 h. After centrifugation with equal volume acetonitrile, the supernatant was obtained and then filtered through a 0.22-μm filter before subject to HPLC analysis. Metabolic stability of [18F]FPTT was measured according to the previously published procedure [25]. Briefly, BALB/c mice (female, ~20 g) were intravenously injected with 37 MBq of [ 18F]FPTT. At 1 h postinjection (p.i.), mice were sacrificed and the tissues of blood, urine and liver were collected to extract the radioactivity for HPLC analysis. All the samples were passed through a 0.22-μm filter before subject to HPLC. 2.5. Validation of cellular PR receptor expression The cells were seeded in 6-well tissue culture plates at a density of 1 × 10 6 cells/well. After cell attachment, cells were lysed with RIPA Lysis (Thermo Scientific) on ice, and total protein concentration was quantified using a BCA Protein Assay Kit (Pierce). Lysates were collected and boiled for 10 min, Equal amounts of total protein (20 μg) were loaded onto Bis-Tris gels for SDS-PAGE Western Blot electrophoresis and transferred to a polyvinylidene difluoride membrane. After transfer, membranes were blocked for 1 h at room temperature with 5% BSA in Tris-buffered saline (Sigma Chemical Co.), 0.1% Tween 20, then incubated overnight at 4 °C with antibodies specific for PR (ab131486, abcam), β-actin (12262S, Cell Signaling Technology). The membranes were incubated with antirabbit HRPconjugated secondary antibodies (A27036, Thermo Scientific) for 1 h at room temperature. Proteins were visualized on a Bio-Rad ChemiDoc XRS System.
with 1 mL of NaOH (1 mol/L), then extract was collected and analyzed using a γ-counter. The internalization was studied according to the literature [27]. MCF-7 cells (approximately 2 × 10 5 cells/well) were incubated with [ 18F]FPTT at 4 °C for 2 h. After pre-incubation, cells were washed three times with ice-cold binding buffer then incubated with 1 mL of warmed binding buffer for different time (5, 15, 30, 60, 120 min) at 37 °C for internalization. The cells were subsequently exposed to 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 being washed with 1 mL of PBS, whole cells were lysed using NaOH (1 mol/L) and collected, then counted using a γ-counter. 2.8. Animal tumor models and PET imaging The experimental procedures with animals were approved by the Xiamen University Animal Care and Use Committee. The model mice with xenograft tumor of in situ breast cancer were established according to the literature [28]. The estrogen pellets were implanted subcutaneously in the neck of BALB/c nude mice 3 d before the inoculation of tumor cells, and then the mixture of PR-positive MCF-7 cells (5 × 106 cells) and matrix gel was inoculated into the mammary fat pad. The sustained-release pellets were taken out 3 d before imaging. PRnegative MDA-MB-231 cells (1 × 106 cells) were subcutaneously inoculated into the right shoulder of nude mice for control study. After the tumor diameter reached 0.5–1.0 cm, the tumor-bearing mice were injected with [ 18F]FPTT (3.7 MBq/100 μL) via tail vein for PET scanning (Siemens Medical Solutions USA, Inc.). The blocking studies were performed on MCF-7 tumor-bearing mice by co-injecting ethisterone (100 μL, 1 mg/mL) or [ 19F]FPTT (100 μL, 1 mg/mL) at 30 min prior to the [ 18F]FPTT injection. The mice were placed in a prone position and collected for 5-min static PET images which were acquired at 0.5, 1, and 2 h p.i. under general anesthesia with 2% isoflurane, followed by scanning at low-dose CT. All PET data were reconstructed by three-dimensional ordered-subset expectation maximization (16 subsets, 2 iterations), with attenuation and scatter correction. The Inveon Research Workplace (IRW) 2.0 software was used to reconstruct and acquire PET image data, and the regions of interest (ROIs) and the average count were calculated by attenuation correction. ROI of muscle was drawn over the contralateral upper limb of tumor for reference. Assuming a tissue density of 1 g/mL, the ROIs were converted to an imaging derived percentage administered activity per gram of tissue (%ID/g) by IRW software. 2.9. Biodistribution
2.6. Cell binding assay Saturation binding study was performed in MCF-7 cells as described previously [26] to determine the binding affinity of [ 18F]FPTT for PR. Briefly, MCF-7 cells (approximately 2 × 105 cells/well) were incubated on a 24-well plate with [ 18F]FPTT, ranging from 1 nmol/L to 200 nmol/ L. To determine the non-specific uptake, excessive ethisterone (10 μL per well, 1 mg/mL) was added additionally to the cells prior to the incubation with radiotracer on another 24-well plate. Followed by incubation at 37 °C 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 mol/L). Radioactivity was measured by using a γ-counter. Bmax and Kd (nmol/L) values were calculated by GraphPad Prism 5.0 software.
The estrogen-primed immature female SD rats (3–4 weeks) were prepared by subcutaneous injection of estradiol dissolved in 20% ethanol/sunflower oil (5 μg/100 μL) on two successive days [17]. The [ 18 F]FPTT (1.85 MBq/200 μL) was injected via the tail vein of rats (n = 4 each group). After 0.5, 1 and 2 h injection, the rats were sacrificed and then the tissues of interest were dissected, weighed and counted using a γ-counter. The biodistribution results were calculated by normalizing the amount of injected radioactivity and expressed as percentage of injected dose per gram (%ID/g). Biodistributions in normal immature female SD rats with or without blocking with ethisterone (250 μL, 1 mg/mL) were studied at 1 h p.i. respectively for comparison.
2.7. Cell uptake and internalization
3. Results
For the determination of specific cellular uptake, MCF-7 cells and MDA-MB-231 cells (approximately 2 × 105 cells/well) were incubated with [ 18F]FPTT at 37 °C for 0.5, 1 and 2 h. Specific cellular uptake was determined by blocking with ethisterone (10 μL per well, 1 mg/mL). After that, the cells were washed twice with 1 mL of PBS (pH = 7.4) and lysed
3.1. Chemistry and radiochemistry Synthetic routes for nonradioactive compound [19F]FPTT and radiocompound [ 18F]FPTT are shown in Fig. 1. All the intermediates and final nonradioactive compound were analyzed by 1H NMR, 13C NMR and
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Fig. 1. Synthesis routes of non-radioactive compound [19F]FPTT and radiotracer [18F]FPTT.
mass spectrometry (Figs. S1–S7 in the Supplementary File). The radio-HPLC analysis results (Fig. S8) revealed that the retention time of [ 18F]FPTT was 14.15 min, which was consistent with that of nonradioactive reference compound [ 19 F]FPTT (T R = 13.73 min). Starting from the azeotropic of [ 18 F]fluoride (~1.8 GBq), the final injection (~0.7 GBq) was obtained within 60 min including HPLC purification, and the decay-corrected radiochemical yield at end of synthesis was about 78 ± 9% (HPLC analysis) and radiochemical purity over 98% after HPLC purification.
The specific activity was calculated as 17 GBq/μmol according to the standard curve (Fig. S9), and the log P value was 0.22 ± 0.01, indicating moderate lipophilicity. 3.2. Stability studies The good in vitro stability of [18F]FPTT was demonstrated by the high radiochemical purities after incubated in saline and rat serum (Fig. S10A and B). The results of metabolic stability showed that about 50% of the
Fig. 2. (A) The expression of PR protein in different cell lines have been evaluated by Western Blot. (B) The saturation binding assay of [18F]FPTT using MCF-7 cells. (C) Cell uptakes of [18F] FPTT in MCF-7 and MDA-MB-231 cells were measured at the indicated time points. The cell uptakes were blocked by the presence of ethisterone (250 μL, 1 mg/mL) at 0.5, 1, and 2 h time points. (D) Cell internalization of [18F]FPTT in MCF-7 cells was measured at the indicated time points. All data were determined from three independent experiments and expressed as mean ± standard deviation.
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intact compound of [ 18F]FPTT was found in the blood at 1 h p.i., while almost all of the radiotracer was metabolized into small molecules with higher polarity in urine and liver respectively from the HPLC analysis results (Fig. S10C–E).
added amount) was 6.33 ± 0.98% at 15 min, and decreased to 4.19 ± 0.02% at 30 min and then reach to a plateau (Fig. 2D), demonstrating ability for nuclear receptor targeting. 3.5. MicroPET imaging studies
3.3. Cellular PR receptor expression The expression of PR protein in different cell lines have been evaluated by Western Blot and the results were shown in Fig. 2A. The cells of BT-474 and MCF-7 have the highest PR expression among these cell lines while PR was negatively expressed in MDA-MB-231 cells. Accordingly, PR-positive MCF-7 cells and negative MDA-MB-231 cells were selected for comparison in this study. 3.4. Cell binding and internalization Saturation binding analysis can determine receptor affinity and density. The cell binding result of [ 18F]FPTT in MCF-7 cells was shown in a Scatchard plot (Fig. 2B). The dissociation constant (Kd) of [ 18F]FPTT was calculated as 29.54 ± 5.27 nM (n = 3), with a maxima number of binding sites (Bmax) of 1.39 × 10 5 cpm/10 5 cells, which indicating nanomolar binding affinity of [18F]FPTT toward PR. [ 18F]FPTT was incubated with MCF-7 and MDA-MB-231 cells respectively at different time, showing significant uptake differences (Fig. 2C). After 30 min incubation, the maximum uptake of [ 18F]FPTT in PRpositive MCF-7 cells was obtained as 9.76 ± 0.64%, which significantly higher than that of PR-negative MDA-MB-231 cells (2.09 ± 0.42%). Exposure of MCF-7 cells and MDA-MB-231 cells to ethisterone (250 μL, 1 mg/mL) resulted in significant decreased cell uptake (2.18 ± 0.30% and 1.67 ± 0.94%) indicating specificity of [18F]FPTT to PR. The cell internalization of [18F]FPTT (the percentage of internalized tracer to the total
The microPET images of tumor-bearing mice were shown in Fig. 3. From the images of PR-positive MCF-7 tumor-bearing mice (Fig. 3A), the tumor could be seen clearly at 0.5 h p.i. and the tumor uptakes were quantified as 3.40 ± 0.17, 3.90 ± 0.20 and 2.39 ± 0.41%ID/g at 0.5 h, 1 h and 2 h p.i. respectively, with the highest uptake at 1 h p.i. The tumor uptakes at earlier time points are two-fold over the background muscle uptakes indicating good image contrast. For the PRnegative MDA-MB-231 tumor-bearing mice (Fig. 3B), the tumor uptakes were 1.4 ± 0.11, 1.3 ± 0.11 and 1.3 ± 0.24%ID/g at 0.5 h, 1 h and 2 h p.i. respectively, significantly lower than that of MCF-7 tumor (P˂ 0.05). PR-specific tumor uptake of [ 18F]FPTT was demonstrated by blocking studies (Fig. 4). After co-injection of ethisterone, MCF-7 tumor uptake of [ 18F]FPTT was inhibited significantly (P ˂ 0.001), indicating it had the same binding target as ethisterone. In addition, tumor uptake of [ 18F]FPTT could also be blocked by excessive nonradioactive [ 19F]FPTT compound, revealing that the structural modification of probe had little effect on the activity of ethisterone. The above blocking studies results indicated that the MCF-7 tumor uptake of [18F]FPTT might be mediated by PR receptor specifically. 3.6. Biodistribution studies Biodistribution results of [ 18F]FPTT were shown in Fig. 5. At 1 h p.i., the uptakes in uterus and ovary of female SD rats that had been estrogen
Fig. 3. Representative PET images of [18F]FPTT in PR-positive MCF-7 (A) and PR-negative MDA-MB-231 (B) tumors in tumor-bearing mice were obtained at 0.5, 1 and 2 h p.i. Quantitative tumor uptakes (C) and tumor-to-muscle ratios (D) derived from A and B. (n = 3, red dotted lines denote tumors, ***P b 0.001).
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Fig. 4. (A) Representative PET images obtained at 1 h p.i. of [18F]FPTT in PR-positive MCF-7 tumor-bearing mice, and with excessive of ethisterone or nonradioactive compound [19F]FPTT for blocking. Quantitative tumor uptakes (B) and tumor-to-muscle ratios (C) derived from A. (n = 3, red dotted lines denote tumors, ***P b 0.001).
Fig. 5. (A) Biodistribution of [18F]FPTT in estrogen-primed immature female SD rats at different time points, and in normal immature female SD rats with or without ethisterone blocking at 1 h p.i. The ratios of uterus-to-muscle (B) and ovary-to-muscle (C) of [18F]FPTT derived from A. (n = 4, ***P b 0.001; Expressed as mean ± standard deviation).
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pretreated to induce PR expression were 8.31 ± 1.74%ID/g and 3.79 ± 0.82%ID/g, respectively. While the uptakes in uterus and ovary of normal rats (3.52 ± 0.29%ID/g and 3.22 ± 0.50%ID/g) were lower than that of estrogen-primed immature female SD rats (P b 0.05), and could be inhibited significantly by ethisterone (2.11 ± 0.18%ID/g and 1.77 ± 0.41%ID/g), indicating the specific uptakes of [ 18F]FPTT in PRrich uterus and ovary. [ 18F]FPTT had low uptakes in the heart, spleen, lung and other non-target tissues and organs, which can be a benefit to improve imaging contrast. In addition, [ 18F]FPTT showed relatively low bone accumulation at different time points, revealing no defluorination occurred in vivo. 4. Discussion Breast cancer PR expression is regulated by estrogen receptor (ER) and could be used for tumor diagnosis and to guide the antiestrogen treatment in breast cancer [29]. During the past a few decades, a variety of PR targeting radiotracers have been reported although the clinical translational study progress is limited when compared with ER imaging. 18 F-labeled steroidal progestins of [ 18F]FENP and [ 18F]FMNP were reported by Martin et al. [15] and Verhagen et al. [14], respectively. Both radiotracers had specific uptakes in the uterus and ovaries of estrogen-induced SD rats, while the first one was found had no correlated with PR levels in human breast cancer and had high accumulation in bone [30]. Recently, the first-in-human study of [ 18F]FFNP was reported by Dehdashti et al. [16], which is the most successful PR targeting PET tracer so far [29]. In 2010, a nonsteroidal PR targeting probe [ 18F] FPTP was reported by Lee et al. [17], it had high target tissue specific uptake with prolonged retention. However, as a chiral compound, the biological effects of its enantiomers remain to be studied. In recent years, our research group had focused on the development of radiotracers for breast cancer imaging [18,19,25,26,31], including two radiolabeled ethisterone derivatives [ 18F]EAEF [19] and [131I]EIPBA [18] for PR targeting. Although both of them exhibited high receptor binding affinity and PR-positive tumor uptake, however, the unfavorable liver uptake limits their potential application. In this study, a small PEG was coupled to ethinyl group of ethisterone to reduce the lipophilicity of tracer and expected to lower the liver uptake. After radiolabeled with 18 F by one-step nucleophilic substitution reaction, the [ 18F]FPTT was prepared in high radiochemical yield with good radiochemical purity. Its octanol-to-water partition coefficient (log P) was determined as 0.22 ± 0.01, which much lower than that of [ 18F]EAEF (0.53 ± 0.06) and [ 131I]EIPBA (1.38 ± 0.48), indicating moderate lipophilicity. The nanomolar binding affinity of [ 18F]FPTT to PR (Kd = 29.54 ± 5.27 nM) was determined by cell binding assay. Of course, compared with reported [ 18F]FMNP, [ 18F]FENP and [ 18F]FFNP, the Kd of [ 18F]FPTT showed relatively moderate affinity, but slightly better hydrophilicity. A more systematic, detailed and multifaceted comparison with the reported classical probes will be studied in our next study. In the cell uptake studies, [ 18F]FPTT showed higher uptake in PRpositive MCF-7 cells than in PR-negative MDA-MB-231 cells, and could be specifically inhibited. Especially, the uptake of [ 18F]FPTT in PRnegative MDA-MB-231 cells with or without PR inhibitor was not significantly different. In microPET imaging, PR-positive MCF-7 tumor could be imaged at early time point with good contrast which the highest uptake was reached at 1 h p.i. (3.90 ± 0.20%ID/g). Moreover, the specificity of [ 18F]FPTT was demonstrated by blocking and PR-negative MDA-MB231 tumor control studies, respectively. As expected, the reduced liver uptake of [ 18F]FPTT was obtained leading to a higher target to nontarget ratio. From the biodistribution study, the uterus and ovary uptakes of [18F] FPTT in estrogen-primed immature female SD rats are comparable to that of previously reported [ 18F]FPTP, [ 18F]FENP and [ 18F]FFNP. When compared with the above-mentioned tracers, much lower liver, lung, spleen and other non-specific organs uptakes, while higher muscle
and bone uptakes of [ 18F]FPTT were observed. Of course, [ 18F]FPTT also accumulated in the intestine due to its steroid metabolic pathway. The targeting ability of [ 18F]FPTT to PR had been verified through a series of in vivo and in vitro bio-evaluation and it will be further investigated to determine the safety and dose for PR-positive breast cancer imaging. 5. Conclusion A novel 18F-labeled ethisterone triazole derivative was designed and successfully synthesized by a one-step nucleophilic substitution reaction. In vitro studies showed that [ 18F]FPTT had moderate lipophilicity with nanomolar binding affinity to PR. Ex vivo biodistribution in estrogen-primed immature female SD rats resulted in high PR-rich tissues uptake. In vivo microPET imaging studies indicated that [ 18F]FPTT had specific uptake in PR-positive MCF-7 tumor with low uptakes in non-target tissues of tumor-bearing mice. The above results showed that [18F]FPTT could be a potent PET tracer for PR targeted breast cancer diagnosis and assess the efficacy of antiestrogen treatment. Acknowledgments This study was financially supported by the National Basic Research Program of China (2014CB744503) and Natural Science Foundation of Fujian Province, China (No. 2016J05200). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.nucmedbio.2019.07.001. References [1] Witten J, Hybert F, Hansen HS. Treatment of malignant tumors in the parotid glands. Cancer 2015;65:2515–20. [2] Chen W, Sun K, Zheng R, Zeng H, Zhang S, Xia C, et al. Cancer incidence and mortality in China, 2014. Chin J Cancer Res 2018;30:1–12. [3] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424. [4] Ulaner GA, Riedl CC, Dickler MN, Jhaveri K, Pandit-Taskar N, Weber W. Molecular imaging of biomarkers in breast cancer. J Nucl Med 2016;57(Suppl. 1):53S. [5] Normanno N, Di MME, De LA, De MA, Giordano A, Perrone F. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 2016;12:721–47. [6] Hoffman JM, Gambhir SS. Molecular imaging: the vision and opportunity for radiology in the future. Radiology 2015;244:39–47. [7] Massoud TF, Gambhir SS. Integrating noninvasive molecular imaging into molecular medicine: an evolving paradigm. Trends Mol Med 2007;13:183–91. [8] Pysz MA, Gambhir SS, Willmann JK, J.K. W. Molecular imaging: current status and emerging strategies. Clin Radiol 2010;65:500–16. [9] Meng Q, Li Z. Molecular imaging probes for diagnosis and therapy evaluation of breast cancer. Int J Biomed Imaging 2013;2013:1–14. [10] Oliveira MC, Neto C, Morais G, Ribeiro, Thiemann T. Steroid receptor ligands for breast cancer targeting: an insight into their potential role as PET imaging agents. Curr Med Chem 2013;20:222–45. [11] Wu Q, Li J, Zhu S, Wu J, Chen C, Liu Q, et al. Breast cancer subtypes predict the preferential site of distant metastases: a SEER based study. Oncotarget 2017;8:27990–6. [12] Diep CH, Daniel AR, Mauro LJ, Knutson TP, Lange CA. Progesterone action in breast, uterine, and ovarian cancers. J Mol Endocrinol 2015;54:R31–53. [13] Cui X, Schiff R, Arpino G, Osborne C, Adrian V. Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy. J Clin Oncol 2005;23: 7721–35. [14] Verhagen A, Luurtsema G, Pesser J, De Groot TJ, Wouda S, Oosterhuis JW, et al. Preclinical evaluation of a positron emitting progestin ([ 18F]fluoro 16 alpha methyl 19 norprogesterone) for imaging progesterone receptor positive tumours with positron emission tomography. Cancer Lett 1991;59:125–32. [15] Pomper MG, Katzenellenbogen JA, Welch MJ, Brodack JW, Mathias CJ. 21 [18F] Fluoro 16α ethyl 19 norprogesteron synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography. J Med Chem 1988;31:1360–3. [16] Dehdashti F, Laforest R, Gao F, Aft RL, Dence CS, Zhou D, et al. Assessment of progesterone receptors in breast carcinoma by PET with 21 18F fluoro 16α,17α [(R) (1′ α furylmethylidene) dioxy] 19 norpregn 4 ene 3,20 dione. J Nucl Med 2012;53:363.
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